Blue Light Green World

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A splendid chronicle of life, light, and vision on microbial bioluminescence and how we can use this good blue light to keep ourselves healthy, our food and water safe to eat and drink and our world clean

Edward A. Quinto (CoolBioluminescence)

Copyright November 2013, Manila; The Philippines

Specks of Life create Light Light flashes a new Vision Vision of Life United and One

Dedicated to the

Grand Mentors and Academic Pillars

of Philippine Microbiology:

Maam Lydia Joson Sir Ireneo Dogma Maam Priscilla Sanchez Maam Asuncion Raymundo
for their selfless devotion to excellence in teaching and research which served as a Beacon of Distinction that steered me and countless others on our rewarding expedition of a lifetime to discover, explore and enjoy the

Wonderful World of the Microbes

Prologue
In the spring of 93, my life changed after witnessing an awe-inspiring first sight of cool bioluminescence in Germany. This unique encounter has since fascinated me to a wonderful microbe and its redeeming heavenly message. Thus in the summer of 2005, several months before the movie An Inconvenient Truth was shown in Philippine cinema, I started writing a book narrating up, close, and personal a distinctive encounter that I had with the Microbes of Light. Bioluminescence, the good blue light produced by the marine luminous microbes, can be harnessed to the fullest to make our world greener by providing safe water, clean foods, and a healthy environment. Having written only technical articles for science journals in the past has made it difficult for me to reprogram my brain to compose something far less esoteric and pleasurable to read. I really felt like a fish out of water writing this book. As challenging as it was with the occasional bouts of doldrums, I confronted my limitations and finally completed this book with much elation and pride. This books tripartite prose dealt on an enriching interaction of life, light and vision distilling everything I have learned, loved, and cherished about the marine luminous microbes. The first chapter - of Life, is a brief memoir of my first encounter with the marine luminous microbes, recounting the wonderful beginning in the Saarland in Germany. It

also enumerates the blessings that many and I received in the course of my scientific inquiry on this special group of microbes. The next chapter - of Light, is a simplified discourse based on existing literatures on the what, where, how and why of microbial bioluminescence. Lastly, of Vision describes how our dark-adapted eyes can discern the luminous message that the microbes live to tell us when we used them as biological sensing tool for toxicity testing. The good blue light can uplift the quality of life by ensuring safe drinking water and a clean environment. Complementing the content of this book are pictures of the marine luminous microbes in the dark. Using long exposure digital photography, I was able to capture the apparitional manifestation of the living light, their life energy, or their aura so to speak. Through this book, I want to introduce a new world viewed in the different light of bioluminescence. Today as global warming looms forebodingly on the horizon, bioluminescence the good blue light can embody our concerted effort to fight it as well as to empower us by using it for our greener day-to-day living. Bioluminescence in addition to solar, geothermal, wind, wave and natural gas can usher in a new green and healthy way of living for all of us. So please join me now, in my personal advocacy to start a bioluminescence revolution in the world. The task ahead is so overwhelming and daunting but nothing should deter us in

upholding the publics health and to fight global warming. In my own little way, this book is it! Lastly, I am encouraging our eager beavers to embark on their own journey of discovery and exploration. To motivate the younger generations to become men and women of science is to find fulfillment in life, knowing that I have taken part in nurturing and cultivating the seeds of science. These precious seeds will germinate someday into the next batch of scientists, which will carry on with the noblest of quests of moving the frontier of knowledge forward.

Table of Contents
Acknowledgments Prologue

Chapter I - Of Life
The light-bearer in the world of the microbes From the very small comes the high and mighty Ich bin ein Berliner Having known John, a Rockefeller, in Freiburg The Saarland, a small jewel nestled in the Franco-German border Winter wonderland in the Saarland EQ in Paris Up, close and personal with the marine luminous microbes An archipelago of the marine luminous microbes Blessings through the microbes of light Winning the gold in the Asia-pacific Young Inventors Award (YIA) The awarding ceremony at the top of the world PIBiT the invention The winners of the YIA through the years Go west where the skies are blue Sojourn in the famous Silicon Valley Meeting the winners of the 2001 U.S. Collegiate Inventors Contest The Hewlett Packard experience Bioluminescence projects shining brightly in science fair contests Winning in the Intel science and engineering fairs

Chapter II - Of Light
The nature and habitats of the marine luminous microbes How does Vibrio fischeri look like? Where is Vibrio fischeri found? Cultivation of the luminous microbes on solid culture medium Cultivation of the luminous microbes in liquid culture medium Microbes that can talk with one another The identification of the marine luminous microbes The chemistry behind bioluminescence

Chapter III - Of Vision

The Eye The standard microbial bioluminescence toxicity test The bioluminescence oxygen demand (BiOD) for water pollution The tube bioluminescence extinction technology (TuBET) Preparation of small paper-discs How to make the paper-discs bioluminous Tales of light from the bioluminous paper-discs Background information behind bioluminosity and immobilization Paper-disc Immobilized Bioluminescence Technology (PIBiT) Clean and safe drinking water PIBiTs applications and its resource sustainability EQ phone NASA Future plans for PIBiT Appendix About the Author

Acknowledgment Tendering credit for an achievement gained is like sailing in ones boat of inadequacy but buoyed up by the great ocean of recognition and appreciation. Indeed, my own personal limitations have become amazing capabilities by the assistance of others. Without these people and institutions, my fortuitous encounter with the Microbes of Light will not have happened; the wonderful opportunity to contribute to a greener world will have been missed, and this book will not have been printed. In all candors, I am extending my sincere appreciation to the following whose exceptional influence and distinctive inspiration left me permanently honed to the service of science particularly in microbiology for the common good. The Helmholtz Institute (HI) formerly known as the Gesellschaft fur Biotechnologische Forschung (GBF) in Braunschweig, Germany, for my first wonderful experience of a sophisticated and international microbiology. The venerable Deutscher Akademischer Austausch Dienst (DAAD) or the German Academic Exchange Service for the superb and enriching personal experience of a postgraduate study.

 To Prof. Dr. Paul Mueller, Dr. Schaefer (Dozent), Prof. Dr. Heinrich Kaltwasser and Dr. Christoph Siersdorfer for making it possible for me to study in the University of Saarland. Lastly, to the gracious and good people of Germany and particularly to Frau Walburga Westphal of GBF, Frau Heidi Schlenther and her family and Prof. Dr. Karsten Krohn and his wife Odille whose memories of friendship, camaraderie, generosity, and kindness I will never forget and to which I will be eternally grateful..

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Chapter I: Of Life
Du bist das Licht in meinem Lebenund die Welt ist wunderschn wenn wir zusammen sind (You are the light in my lifeand the world is wonderful when we are together) - Starlight Express in Bochum, Deustche Aufnahme)

The light-bearer in the world of the microbes Do we humans want this planet to be exclusively ours? The emphatic and resounding answer I am sure is a Big No. It will be a place quite dreary to live in if we humans are planet alone. A world that lacks biodiversity will be big, boring, and barren. We love this world the way it has always been, teeming with so much life of which are own is just one of the multitudes of species of animals, plants, and microbes. A plethora of life forms nurtured and sustained by a blue planet we call mother earth.

Lichens: a symbiotic partnership between a fungus and an alga

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Surely, something binds all living organisms together on this planet into a single super family. Among all of the hundreds of millions of known species of living organisms existing today, only a few have been endeared and cherished by man. These much loved species are the higher plants and animals. Our overbearing birthright of dominion over all creations resulted in the domestication of many plants and animals. Chosen members of kingdom Animalia, the realm of those that can move, were held in captivity for livelihood, companionship, entertainment, worship, and friendship. This seemingly inadvertent intrusion into the process of natural selection has forever change the course of the evolution of the wild. Many plants and animals even suffered the tragic fate of extinction from our gung-ho incursion into nature. On the opposite end of this multifaceted relationship, is our subservience to animals to the point of venerating them as gods. This practice reached its peak in the ancient civilizations of Egypt, Babylon, and Persia where dogs, cats, cattle, birds, and snakes were accorded the status of deity and received acts of worship. However, the rise to power of the monotheistic religions put an end to this practice of animal worship. Nevertheless, our fascination with animals continues unabated even to this day. Walt Disneys enduring success, and worldwide popularity,

transcending cultures and races, are proofs of our undying love for animals.

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Disneys wonderful and colorful world of animation created the amiable characters of Mickey, a mouse; Donald, a duck; Bambi, a deer; Dumbo, an elephant and Goofy, a dog. My generation grew up so fixated with the lovable animal cartoons of Disney. Although we are now wiser, many including myself still watch the movies of Disney through a childs eye as something truly magical. For a long time now, dogs got the esteemed title as mans best friend, but cats recently stole the limelight in Broadway. The common household cat: Felix domestica, received widespread accolade on both sides of the Atlantic from Andrew Lloyd Webbers musical Cats. Cats, a deeply lovable music theater production with its origins in London, portrayed so perfectly the exquisite charming attributes and the humorous mundane lifestyles of the different breeds of the domesticated cat. Sly and stealthy feline characters; slithery and beguiling huggable furry mammals with whiskers and paws; acted out their unique roles and sing their lives in songs so emotive. The overall effect is an artistic work of melodies and music woven together into a seamless tale of the anguish and joy of our inherent human nature. Cats captured the minds and hearts of millions and went on purring its way into historical success as it broke box office records in major cities all over the world.

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Fishes and honeybees are members of Kingdom Animalia

However, to some people, the members of Kingdom Plantae, the realm of the green, like algae, ferns, roses, bromeliads, orchids, and trees, afforded the kind of personal relationship that gave lasting fulfillment in life. Indeed, many poets were inspired to immortalized flowers and trees in their poetry. The sight and distinct smell of plants with their colorful flowers unloads us of our anxieties and provides welcome relief from the stresses of everyday life. Beautiful flowers that come in a wide variety of forms and colors instill in us feelings of euphoria as well as impart deeper meaning to many of our lifes special events. In fact, love is express so naturally with flowers. In addition, fragrant essential oils produced by plants were used since ancient times to bring about the scents and aroma needed to set the right mood to our social occasions. Today, the cosmetics and food industries

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have commercialize these special plant extracts into the multi-billion dollar business of flavors and fragrances.

The members of Kingdom Plantae found in Caleruega, Batangas

My case is so completely different.

A personal encounter with an

unusual organism in the spring of 1993 in Germany made an overwhelming difference in my life. Surprisingly, the organism was neither an animal nor a plant; it was a lowly microbe, a speck of life. Microbes belong to the kingdom

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Eubacteria, the realm of those that are too small to be seen. Although it may at first seemed so trivial since the organism was a tiny and humble microbe, it was however, a one of a kind microbe endowed with a beautiful celestial gift. A microbe is a one-celled organism not visible to the human eye and considered primitive by biologists in terms of its cellular structure and function. You will need a good microscope to be able to see them one by one. Indeed multitudes of these imperceptible creatures have already taken permanent residence in our mouth, tummy, and respiratory tract as well as on our teeth and skin without us ever knowing it. They are so tiny that millions of them can easily be crammed together in a space the size of a pinhead. But what makes this microbe that I met in the spring of 93 special and distinct from the other species of its kind I met previously, is its innate ability of eerily generating light in the dark with a cool delightfully bluishgreen hue. In fact, this tiny microbe has something in common with giant celestial bodies called stars. This microbe and stars produce light copiously. However, stars generate heat while the bioluminescent microbes do not. In the realm of the microscopic, this light-bearing microbe must have been truly heaven sent. Not only is it heavenly, it is also smart; a microbe that knows how to talk to one another and to us through the cool light that it makes. Biological illumination for this microbe is achieve only by relating itself in

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unison with others of its kind and collectively as a whole attains bright luminosity. It is a perfect example of what we call togetherness or cooperation. It was from the study of this clever microbe that microbiologists learned that they though considered the lowest forms of life do actually talk to one another. Unlike what happened to the Tower of Babel, microbes because of this exceptional communication skill can initially build small communities composed of streets that gradually develop into colossal mega cities with skyscrapers and freeways. In microbiology, these microbial mega cities are simply called Biofilm.

Stacked plate cultures of the luminous microbes shining with a cool bluish-green light in darkness

My chanced encounter with this intelligent luminous microbe must have been made in heaven because it lit up my life and changed it so wondrously.
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It also made a lasting impact in the lives of many of the younger generations of Filipino scientists. Hopefully, it can also make a lasting global impact in safeguarding our health and that of our beloved planet.

From the very small comes the strong and mighty

Microorganisms or simply microbes are one of the least understood groups of living organisms existing today. Primarily because of their miniscule size we know little of them. Most of us have not yet even seen a microbe in our lifetime. Since size does matter, it is indeed not often that we see them everyday as plants and animals. Even Disney, Columbia, Pixar, Warner Brothers, and the other movie studios have yet to venture into animating microbes into lovable characters that can be popularized into famous characters like Mickey, Nemo, and Chicken Little. And why not? The lowly microbes have so much to offer to keep us incessantly amused and entertained. With their diverse and amazing ways of life, extraordinary qualities and bizarre attributes, new and inspiring movies full of enduring virtues can be produced with microbes as protagonist. Theirs is a completely new universe to explore and discover that will surely broaden our culture of science and knowledge of microbiology. To know them well is to give us a

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fair and a better understanding of this much misunderstood and maligned group of living organism. The popular medias perception of microbes as harbingers of sickness and pestilence worsened even further our ignorance of these infinitesimal organisms. This unfounded fear of the invisible must have imprinted in our psyche the various waves of deadly plagues that scourged our forefathers since time immemorial. Indeed infectious diseases like cholera, dysentery, tuberculosis and pneumonia continue to claim millions of lives today. However, to microbiologists, this is a grave misconception of the microbes since the disease-causing microbes, the bad microbes, are just a minority in the realm of the small. The vast majority of microbes, the good microbes, live freely in the soil, on our skin, inside our gut, in seawater, and other habitats where they carry out the tasks meant for them by nature. Microbes are actively involved in the building up and breaking down of substances around them. They play a major role in the recycling of the various essential chemical substances that make up the biosphere. In short, they keep our planet clean and healthy. Microbial species though miniscule in size have in eons past, acting ever so slowly but surely, achieved something enormous. Microbial activity is solely credited for transforming our atmosphere into what it has become today. From an anaerobic and reducing atmosphere in the earliest periods of earths history

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to what it has recently turned into: aerobic and oxidizing! An oxidizing atmosphere is rich in oxygen causing iron to rust rapidly. Unfortunately, for higher life forms it also speeds up a biological process called ageing. However, an oxygen-rich atmosphere creates and sustains fire allowing its manifestation on earth as the process of burning. Burning is a chemical reaction in which elements like carbon combines with oxygen to produce radiance and heat. In the absence of the diligently working microbes and their pervasive and collective activities on this planet, transforming earths atmosphere from zero percent oxygen in the past to twenty-one percent oxygen in the present must have never happened. Moreover, without a substantial amount of oxygen in the atmosphere, the emergence and existence of plants, animals, and humans would not have been possible.

Cyanobacteria like this Oscillatoria gave earth an oxygen-rich atmosphere (1,000X)

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Many of these microbes possess strange and amazing characteristics. For example, a microbe called Thiobacillus thiooxidans produces highly corrosive sulfuric acid in practically the same manner as we humans make and excrete urine. Another microbe called Aquaspirillum magnetotacticum swims along defined paths in its watery world guided by the earths lines of magnetic field. It is able to do this because of a string of spherical iron oxide magnets found within its elongated spiral body. Moreover, there is a very ancient microbe called Pyrolobus fumarii thriving in its oxygen-free world at temperatures way above the boiling point of water; hot and anaerobic conditions believed to be prevalent in the early periods of earths history. It surely is not cook at this high a temperature. Instead, it even grows at its best making the rest of its own kind to populate and prosper in their nasty niche! For many years now, microbes in the group called Actinomycetes have been seek out out by scientists in many soil samples collected all over the world. This undertaking that gained massive support from the large pharmaceutical companies led to the discovery and subsequent industrial production of wonder drugs called antibiotics. An example is the multi-billion dollar annually sold antibiotic known as erythromycin produced by the microbe Saccharopolyspora erythraea formerly called Streptomyces

erythaeus. This microbe was isolated from a soil sample in the Philippines

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by an Eli Lily chemist to produce their companys banner antibiotic product. The use of many antibiotics produced by the members of the group Streptomyces ushered in the golden era of chemotherapy in medicine.

Photomicrograph (1000 X) of an indigenous streptomycete with its long filamentous cellular structure. Most commercial antibiotics come from this group of soil microbes.

Antibiotics saved millions of lives throughout the world from the ravages of infectious diseases. My first ever research project dealt with the cultivation of Streptomyces levoris and the production of its antibiotic: Levorin using indigenous waste agricultural substances. I was under the able guidance of the late Dr. Patrocinio Santos, famed pharmacist, microbiologist and NRCP Achievement Awardee in the Philippines. Aside from the streptomyces, there is also Lactobacillus acidophilus that produces lactic acid and other edible

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products derived from the fermentation of milk and other foods. This microbe is just one of many lactic acid microbes like Lactobacillus casei and Lactobacillus plantarum employed as probiotics or good microbes in many functional foods.

Photomicrograph of a Lactobacillus acidophilus USTCMS strain (1000X) showing long rod-shaped cells

They are responsible for the traditional production of fermented dairy products like cheese, butter, yoghurt, and sour milk as well as fermented vegetables like suaerkraut and pickles. Incidentally, my work on the antimicrobial activities of the fermented medium of Lactobacillus acidophilus, a brief foray from my main work on the marine luminous microbes, earned me recently the Tom Bergan Memorial Award. The International Society for

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Chemotherapy (ISC) based in London gave me this award on its 24th Biennial International Chemotherapy Congress held in Manila in June 2004.

The 2005 Tom Bergan Memorial Award

Finally we now come to my favorite and friend, a smart and heavenly microbe scientifically named Vibrio fischeri, which like the angel Lucifer when he was still in Gods favor was the bearer of light among the angels. The creator has indeed endowed Vibrio fischeri with a celestial gift of being able to produce bright bluish light on its own initiative. Bioluminescence is a biological phenomenon in which living organisms produce cold light from

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their own life processes. Light production from inanimate systems like bulbs and incandescent lamps heats up after some time but this is not so with bioluminescence. Among the bioluminescent organisms like fireflies, lantern fishes, glowworms, dinoflagellates and the luminous microbes, almost all of the allotted biochemical fuel found inside their cells intended for bioluminescence is transformed almost entirely into light energy. Only a small fraction of the transformed energy, when converted from chemical to radiant, is wasted as heat. The conversion ratio is high that it renders bioluminescence amazingly cold to the senses. The bluish-green hue of the luminous microbes is much like the beautifully shining green beads of glowin-the-dark rosaries and wristwatch hands. Ich bin ein Berliner Ever since I was held spellbound by my first sight of brightly shining liquids of luminous microbes in a dark laboratory years ago in Germany, my devotion to these microbes has never waned and has even become an integral part of my academic life. It was candidly true love at first sight! So awe-inspiring and thrilling indeed were my visions of the luminous microbes that it reminded me of the time back in high school when I first laid telescopic eyes on the moons of Jupiter and the rings of Saturn. I was in ecstasy to see them real; it was just awesome. Indeed, all budding scientists

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are astronomers and space travelers at heart. Today, as someone who finished a career in chemistry and microbiology and has become an honest to goodness servant of the life science, I affectionately describe bioluminescence as the Light of Life or simply to coin a new word Lifelight. My unexpected encounter with the wonderful world of the marine luminous microbes started in 1992. It was an event that I believed was meant to be. In that year, the venerable DAAD (Deutscher Akademischer Austausch Dienst) or the German Academic Exchange Service gave me a one and a half-year scholarship grant. The grant was for a one-year certificate course in biogeography and environmental assessment. DAAD has been very generous to the higher educational system of the Philippines and to other developing countries. DAAD offered numerous training courses as well as opportunities to pursue research and postgraduate studies in German academic and research institutions. Countless Filipinos were recipients of DAAD scholarships and have now come back to make their outstanding marks and significant contributions in the local academe, industry and various government agencies. My scholarship grant initially provides for a six-month German language course in Freiburg im Breisgau, a charming city nestled in the nurturing embrace of the enchanting Black

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Forest. A German language course is required of the one-year certificate course in the university to enable one to live an independent life in Germany. The course on Biogeography and Environmental Studies is tenable at the University of Saarlands Department of Social and Environmental Studies. Prof. Dr. Paul Mueller, an intelligent, well respected, highly admirable, and able-bodied German ecologist and educator, who can easily climb several hills as if it were just a light stroll in the park, headed the department. It was actually my third time to be in Germany, a land I consider my second home and a country that I always regard with deep affection and joyful yearning. This nation, an economic power located in the very heart of Europe, is the great and venerable homeland of Johann Sebastian Bach, Ludwig van Beethoven, Johannes Brahms, Albert Einstein, Pope Benedict XVI, Willy Brandt, Helmut Kohl, Steffi Scholl, Steffi Graf, and other famous persons who change the direction of world affair. Germany is known, recognized, and respected far and wide for his eminent position in philosophy, the arts, sciences, and technology. For me, Germany is Gods own country of heart captivating landscapes painted in living green and freezing white; peaceful and idyllic pastoral villages; impressive and mighty cities and most of all the country of a good, noble, and lovely people. I first visited Germany for six weeks in 1988 as a participant to the international training program (ITP) in

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biotechnology. Germanys prestigious and venerable National Center for Biotechnology sponsored and held this training course. To many who went through the portals of this venerable research institution, it is simply and amiably called GBF. GBF stands for Gesellschaft fr Biotechnologische Forschung. Recently, GBF has been renamed as the Helmholtz Zentrum fr Infektionsforschung translated literally as Helmholtz Center for Infection Research. GBF has mow become HZ. The change highlights a redirection of Germanys biotechnological research thrust into a multi-disciplinary fight against infectious diseases. Visiting Germany for the first time in 1988 was like venturing into another world of endless wonder. The view, the ambiance, the places, the people, the climate, the culture, and the very air that I breathe were altogether such a fantastic first-time experience. I found it so thrilling talking a lot and exhaling my lungs out because of the thick vapor that comes out of my mouth. It happened to be my first foreign travel; an exciting journey that took me through the charming German cities of Braunschweig, Hannover, and Berlin. It was an unforgettable six-week sojourn in the summer of 88 that I consider the best thing that ever happened in my life. Having visited divided Berlin in 1988 and unified Berlin in 1990 gave me a deep and profound perspective of Germanys identity, history, and role in the modern world. Remember it was just in the last

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century when Germany went through periods of revolutions, redemption, and resurrection. The mighty German Empire ruled by Prussia disintegrated after the First World War. A fleetingly bright moment followed the collapse of the empire with the birth of the Weimar Republic in 1919. This beautiful republic, bearing the promise of freedom, peace, and prosperity for all Germans, unfortunately died in its infancy. What followed was the darkest and most chilling period in European history: the rise to influence and power of Hiltlers National Socialist Party. Because of the NAZI, millions perished in Europe from the war and from the gas chambers. Germany suffered

widespread destruction and almost complete annihilation. Though it seemed that everything was lost, Germany made contrite reparations for his mortal misgivings and underwent an immensely remarkable transformation marked by sincere reconciliation and rapid industrial development. The Fatherland rebuilds himself from the smoldering black cauldron of the last world war to rise meteorically into a proud global economic power as a free, united, and flourishing German nation. Germanys international capital, the vibrant city Berlin, once divided into two rival philosophies, now breathes a sophisticated culture and a vagabond urbanity. When Pres. John F. Kennedy bravely visited Berlin and spoke the famous words Ich bin ein Berliner, a city instantly captured the hearts and

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minds of all people on earth. Suddenly everyone was a Berliner who knew fully well to which side of the divided city he belongs. It highlighted the great divide of a world partitioned by the iron curtain; of us against them of the free against the bound. Everyone in the 60s lived precariously on the angst of a third world war kept by dtente of the superpowers. When I visited historical Berlin for the first time in 1988, I cannot help but savor and marvel at the ambiance of an occupied city; a city torn between west and east, democracy and communism, freedom and tyranny, heaven and hell. A city hallowed by the Nordic gods of Valhalla with beauty, power, and nobility. Conspicuously flying high above the city is the golden statue of the Angel of Victory symbolizing for the Berliners the blessings of success and achievement. In 1988, my first time to be in Berlin, the massive wall of shame stood arrogantly between the famed Gate of Brandenburg and me. All I can do was to gaze at it from afar inside the comfort and security of West Berlin. However, in 1990, my second time to be in Berlin, I was able to walk freely through the famed Brandenburg gate passing by the monumental colonnade of the chariot-triumphant goddess of dawn - Aurora. I entered the gate with overwhelming sentiments of harmony, happiness and prayers of thanksgiving. Indeed, the mighty Westerly Winds of Freedom knocked

down decisively the infamous Wall of Berlin and blew on farther east

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destroying the fortress of the once powerful Union of Soviet Socialist Republic (USSR). What was once thought impossible, the unification of two German nations put asunder by conflicting global powers, happened overnight so fortuitously and miraculously? A divine will must have intervened, for the special turn of events leading to the fall of communism took us all by surprise. At first, events seemed poised to trigger the third world war but instead took a complete turnaround and ended so diplomatically for the US and Russia. Indeed, we are forever grateful to the foresight of Pres. Ronald Reagan, to Pres. Mikhail Gorbachevs Perestroika and most of all to the guidance of the beloved Man of the Twentieth Century his holiness Pope John Paul II. His papacy steered the world so peacefully during the dying days of soviet communism as we move on into a new order. With enthusiastic and happy feet, I tread through the popular and spacious boulevard called the Unter den Linden Strasse extending behind the Brandenburg Gate right into the very heart of East Berlin. My strides, full of fervor, took me all the way to the imposing monumental bronze statue of Emperor Frederick the Great. I enjoyed every moment of it as I glanced all around me and savored every beautiful feelings in that historical afternoon in East Berlin in September of 1990. Soon it was darkening and I have to go back to my hotel. Tony Bennet left his heart in San Francisco but in Berlin I

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left not only my heart but my soul as well. It was indeed so great to be young then in a cosmopolitan Germany caught up in such a momentous event in world history. Coming from a developing country, the experience totally changed my outlook in life. It gave me a genuine appreciation and openmindedness of other peoples way of life. That my way of living and beliefs are not necessarily the best but there are other beliefs and ways of life equally deserving as mine. In the 1988 training course on industrial biotechnology, there were 20 participants coming from countries in South America, Africa, and Asia. My second trip to Germany in 1990 was made possible by a two-week Carl Duisberg Gesellschaft (CDG) travel grant. This travel grant enabled me to attend the Biotechnica 1990 convention in Hannover, a study visit to the GBF for one week and the second memorable visit to Berlin. Hannover is just a stones throw away from Braunschweig where GBF is located. The BIOTECHNICA is a biennial international showcase highlighting all the recent breakthroughs and up-dates in the field of biotechnology. It was so far the best international symposium, convention, and trade fair all rolled into one that I have ever attended. On my third journey in 1992, I embarked on my longest sojourn of one and a half-year in Germany. I and Sunday Duque a friend of mine from the Department of Biological Sciences of the University of Santo Tomas (UST)

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arrived in the charming city of Freiburg im Breisgau for the language course at the Goethe Institut in April of 1992. DAAD awarded us both the same scholarship program in January of 1992. When we arrived in Germany, beautiful spring was in the air as it began to lay hold of the seasons. It was still indeed quite cold for us, the people from the tropics but numerous juvenile leaves were beginning to sprout and cover the bald brown branches of trees. Bright yellow Dandelions were popping up everywhere on the bare lifeless ground around us. The sleepy hills and the groggy mountains of southern Germany were waking up from a deep winter slumber by donning various shades of green. Ah yes! I said to myself, Greek mythological goddess Proserpina has once again arrived in Europe.

Having known John, a Rockefeller, in Freiburg

John, a Rockefeller, also made my stay in Freiburg unforgettable. In the second cycle of my language course, during the blue summer skies of June and July, two graduates from the prestigious Yale University came to study German at the Goethe Institut in Freiburg. Both were assigned to the boarding house where I was staying. It was located in the outskirts of tranquil Freiburg around 15 to 20 minutes bus ride away from the city center.

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The new kids on the block were Ching Chiu, a Korean-American and of course a John D. Rockefeller V, first-born son of Sen. John D. Rockefeller IV (Democrat West Virginia). A cause that bound the three of us neatly was our desire to read the International Herald Tribune (IHT). To realize this need we would contribute one German Mark each to enable us to buy the IHT. I had been reading the Tribune since I first arrived in Freiburg in April. It kept me busy while waiting in the bus stop for my ride home after my German class. The IHT fulfilled a yearning in me to read something international and American and in some instances snippets of news about the Philippines. John and Ching Chiu saw me carrying the IHT one sunny afternoon on my way home as I entered our boarding house and asked me where I bought the papers. That question started a fruitful conversation and a friendship in the duration of the language course in Freiburg. For the next two months, I shared the cost of the IHT with John and Ching Chiu and most of all a wonderful company. We would usually go together to a nearby large mall whenever we ran out of foods and other provisions. I even remembered teaching these Yale Graduates how to cook rice properly by simply using the top line of the middle finger to measure the required level of water. Ching Chiu and I saw an impressive production of the opera Carmen in the Freiburg Theater one evening. The night was beautifully mild and warm as

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we traveled back home by bus. Though John is a Rockefeller, he did not show it in his ways. He is far from being a very spoiled brat. One time John and his friend Charles stayed over in my place for another week when his rent ended in July. My place, which is the most expensive in the boarding house for being the biggest happened to have a large ante room adjoining a bedroom. Ching Chiu left ahead of John for Berlin. Staying with me, John was able to save a weeks rent of pay from the owner of our boarding house: the Webbers. John is a tall and a delightful all American guy, very down to earth, so unassuming and charmingly friendly with always the best of intentions. Several times a week I would catch him talking by phone to his parents and friends since our phone is located in the corridor at the corner of the stairway leading down and out of the house. He is ever ready to defend the American Way of life. I remembered amusingly, John getting into an intense argument one early morning with a German while we were waiting for our bus ride to the Goethe Institut. John was complaining loudly not knowing that the only other person with us in the bus stop, a German, understands and speaks good English. Their argument centered on why the shopping malls and stores in Germany are closed on Saturdays and Sundays while in the U.S., and in the Philippines they are open. After the first week of August, John left and traveled to his next destination. Before

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John left for Bamberg, he wrote me a short funny note on a bond paper. He left it so conspicuously on my big old wooden table, which I managed to keep to this very day. The following months of August and September, the third and last cycle of my language course, were personally difficult as I went through one of the most trying times of my life. A new student from Nepal called Kedar moved into our boarding house. Kedar was a nice and friendly guy who always carried a plastic bag full of curry powder during meals. He liked to garnish all his dishes and foods with curry. It was I believe typical of the people in his homeland to eat foods containing curry much like some Filipinos use of patis (fish sauce) in every meal. After six months of stay in the city of the enchanted Black Forest, at the end of September, the three cycles of the German language course at the Goethe Institut were finally over. Sunday and I once again packed up our things, bade Freiburg farewell and traveled by train to our next destination: Saarbrcken, the heart of the Saarland.

The Saarland, a small jewel nestled in the Franco-German border The green summer in Freiburg had given way to the orange and colder season of autumn in Saarbrcken, the Saarlands capital city. Sunday and I traveled to Saarbrcken in early October of 1992 to start our two semesters
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course in biogeography and environmental studies at the University of Saarland. My recollections of the long train journey from Freiburg to Saarbrcken as I gazed out were picturesque fleeting views of a lovely emerald countryside bathing intermittently in rain and sunshine. When we arrived, Sunday and I were assigned to different accommodations located far apart from each other. The universitys Auslandsamt (Office for International Students) is in-charge of housing the international students. Once again, I was placed in a big room in the 2nd floor of a large two-storey boarding house with eight rooms and a cold basement located in the suburbs of Saarbrcken called Dudweiler. The charming house stood alone besides a big forest but it also stood strategically along a major highway with bus stops nearby. Outside this big house is a connected wide spaced area with ceilings that served as party area for the student occupants. I shared the big house with Erasmus European students studying in the university. However, the Auslandsamt assigned Sunday in a big student dormitory located close to the city center. There were seven of us DAAD scholars pursuing the biogeography program in the University of Saarland: two from Nigeria, one from Senegal, one from Indonesia, one from Pakistan and Sunday and I from the Philippines.

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Winter wonderland in the Saarland

My December in Saarbrcken was marked by a very memorable shortlived white Christmas. The winter was unusually mild in 1992, which was probably good for my nasopharynx and upper respiratory system. I experienced only a week of a half foot-deep snow-covered streets. The night the white flakes of snow first fell, I recalled dashing out of our boarding house to savor the cool, nice sensation of the slowly falling ice particles melting on my face and hands. I got so excited that evening that I aroused the curiosity of all my friends: Cyrill from France, Pawel from Poland, Marianne and Helena from Denmark, Sabina from Spain, Althea from Canada and Kirstie and Anthony from England. They too went out as well of our boarding house and shared in my excitement and my innocence of everything that is winter. There was a time one late evening when I had a hard time entering our boarding house. From the main road, the door is some way off around the house with the pathway sloping up a little. The pathway was covered by ice and so to prevent me from slipping down I have to hold on tightly to the walls and windows of the house to get to the door. In a blink of an eye, the never-ending renewal of the seasons has brought back spring. Sunday and I had spent a year in Germany.

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At the start of our last semester at the University of Saarland, we were required to complete an Aufbaustudium which is very similar to an American version of a thesis. I had always wanted then to do a thesis in the field of environmental microbiology in the Department of Applied Microbiology. So one morning, claiming only to a brief stint at the GBF way back in 1988, I went directly to the office of Prof. Dr. Heinrich Kaltwasser and applied personally for an Aufbaustudium in environmental microbiology. Prof. Dr. Kaltwasser, a soft-spoken, kind, and learned microbiologist, was the head of the Department of Applied Microbiology at the University of Saarland. Without any hesitation, Prof. Dr. Kaltwasser accepted me immediately and assigned me to the supervision of Herrn Christoph Siersdorfer. Christoph Siersdorfer was at that time a Ph.D. student doing his dissertation on the use of different microbial systems for the toxicity testing of the treated wastewaters of the coal processing plants in the industrial region of the Saar. I believed that my training at the GBF was my magic ticket in getting into the department of applied microbiology so fast and without hassles. Prof. Dr. Paul Mueller gave me his permission to do my Aufbaustudium in another department. Sunday remained and did her Aufbaustudium in the department of social and environmental studies.

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EQ in Paris

In June of 1993, a student club in the University organized an excursion to Paris, France. Oh my God, Paris! The trip was simply too precious for us to passed up and so we hurriedly secure a visa to join the trip. I vividly remember waking up very early and while it was still quite dark trekked all the way to the campus. We got up to a beautiful big tourist bus and before we knew it, we were on our way and crossed the border. For all I know, our bus made a beeline to Paris from Saarbruecken. After awhile, artwork of geometric figures began appearing along the road. This got us all so excited for we knew we are approaching the great city of the arts and fashion. As we raced to Paris, we soon entered into a long tunnel lined up with flashing visions of ceiling lights and columns that eventually opened up to a grand view of the city of Paris. The bus soon stopped near the city center and we got out of the bus at around 10:00 or 11:00 am. All the students broke up into small groups so that no one will be left behind. The organizer told us that the return trip to Saarbruecken would be at around midnight. That leaved us with just 12 hours to see all the major tourist spots in Paris! How the heck do you get to know Paris in 12 hours but we have to do it. Our group composed of Sunday and I (Phil), Bob (U.S), Vitz (Czech), Gulfraz

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(Pakistan) and Fagamou (Senegal) were at a loss as to how we will do it. All I can remember was when we took the subway train in a hurry.

From left to right, Bob, Sunday and Vitz at the Sacre Coure.

As I recall, we definitely started our city tour at the Sacre Coure (Sacred Heart) and then on to the Parisian business District where the Arch De La Defense, Arche Triomphe, the Panthenon, Notre Dame, the Eifel Tower and lastly Champ Elysee are located. It was a whirlwind sojourn in Paris that was filled with so much fun and amazement as we crisscrossed the city to see the famous places in half a days time. By the end of our stay in the city, we

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were back in the bus to begin our trip back home. Though exhausted it was well worth the trip. We will always remember the summer of 93 in Paris where fond memories were formed and friendships strengthened. It only occurred to me now that I was wearing a green polo shirt and a green United Colors of Benetton wristwatch. It was a memorable gift given to me by Benjie Jornales, a close friend going back to my college days in UST, who has now settled in Melbourne, Australia. Back then, I never knew that I will be promoting a green world through the intervention of a blue light. Only the meteorologists and geologists probably knew of climate change and global warming in the early 90s. Global warming was definitely not the issue back then. It was mainly through the movie An Inconvenient Truth by former Vice Pres. Al Gore that the world had known the perils of global warming and climate change. After that movie, global warming was transported out of the journals and conferences of scientists and has moved to the center stage of the world news. Now that we are all beginning to feel and see the ill effects of a changing climate, everyone has finally awaken and taken a pro-active stance against global warming.

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Paris in the summer of 93, a gem in my treasure trove of memories

Up, close and personal with the marine luminous microbes Working on his dissertation, Christoph used several microbial test systems to evaluate the toxicity of various untreated and treated wastewater

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samples. The toxicity tests he employed included the bioluminescence toxicity test, a standard bioassay using the marine luminous microbes. Bioluminescence from these organisms responds sensitively to the presence of minute amounts of toxicants in the water samples where they are added and suspended. Christoph Siersdorfer is a true Saarlander, he was born and raised in this small, beautiful and richly blessed land sandwiched between France and Germanys state of Rheinland Palatinate. He is a nice, blonde, gray-eyed, and handsome chap. He is very friendly, always ready to give assistance and easily accommodate requests from others students in the laboratory. Christoph is strongly committed to his profession in microbiology. He directly supervised me with my aufbaustudium on the utilization of freshly cultivated and harvested marine luminous microbes for the bioluminescence toxicity testing of wastewater samples from the coal processing plants in the Saarland. Giant industries and the coal processing plants in the Saarland generate large volumes of toxic wastewater. Ammonia, sulfides, cyanides, and the polycyclic aromatic hydrocarbons (PAH) are just some of the harmful and poisonous chemicals found in the wastewater. Before this toxic wastewater is released into rivers and lakes, it must first undergo a detoxification process through series of physical and chemical treatments. The wastewater

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treatment processes, undertaken by each industrial plant, should remove most of the dissolved toxicants and improve the physico-chemical quality of the wastewater. Though the physical and chemical treatment employed by industry to clean up their own wastewater is expensive, the people of Germany willingly shouldered the cost to live in a healthy and clean environment. Germans now enjoy longer and quality life, the product of a healthy living and a clean environment free of harmful pollutants. Each industrial company has the responsibility to transform its own dirty wastewater into clean treated water; from a wastewater that is heavily polluted to a non-polluted water that can be released safely into rivers and lakes. Protection of the environment and ecological awareness through the populaces religious practice of reducing, re-using and recycling have made Germany one of the cleanest if not the cleanest country in the world. In the Philippines, it is so sad that our neglect of the environment has caused the demise of the once beautiful and viable Pasig River of lore. The river has now become for all practical purposes dead due to so much pollution. For the river to once again sustain plant and animal life is now just probably a thing of the past. To bring Pasig River back to its former beauty and pristine life will require a gargantuan and disciplined effort on the part of industries

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located along its banks as well as from the residents of Metro Manila not to pollute it.

Bioluminescent flask and plate side by side

Christoph employed the microbial bioluminescence toxicity test based on a method from the German Standards for Water Toxicity Measurements or DIN. The standard method requires the use of a complex and expensive light-measuring instrument known as a luminometer. The luminometer measures the intensity of the light output or luminance produced by the luminous microbes when they are added to water samples, which may or may not contain toxic agents. Since the effect of toxicants on the luminance
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of the treated luminous microbes is measurable using a luminometer, it has made the process of water toxicity detection quantitative. The

bioluminescence toxicity test using a microbe is just one of several other toxicity tests utilizing lower animals like worms, insects, and fishes. These lower animal-based tests can also determine the degree of toxicity of the treated industrial wastewater after it has gone through several physical and chemical processes. Once the wastewater is rendered clean and non-toxic by the wastewater treatment processes as evaluated by the

bioluminescence toxicity test and the other animal-based toxicity tests it can then be released safely into natural bodies of water. This treated wastewater is so clean that it does not affect or alter the state of the natural environment. The protection of the environment is of paramount importance in Germany and industrial companies strictly observe its proper

implementation In addition, the bioluminescence toxicity test is one of two tests required by the German government to check the drinking water samples for potability to complement the standard coliform test. The Coliform test detects the presence of disease-causing microbes in water while the bioluminescence toxicity test using the luminous microbes detects the presence of harmful chemicals in drinking water. A water sample that

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passes both tests is surely clean and safe to drink; it is free of harmful microbes and free of toxic chemicals. By the way, my second award from the professional category of the 1oth PCHRD DOST annual scientific poster competition was on the development of a simple do-it-yourself way of detecting the presence of the fecal coliform bacterium: Escherichia coli in water samples. My research poster bore the title A Simple (Do It Yourself) and Rapid Culture Media Color Reaction Test for the Detection of Fecal and Non-fecal Coliform Bacteria in Various Types of Water Sample.

My PCHRD DOST award winning invention called EcoliTest. The picture to the left is before the addition of the water sample to be tested and the right is the result of a contaminated water sample containing fecal coliform bacteria.

In the applied microbiology laboratory, two kinds of luminous microbe were used for the toxicity testing: Vibrio fischeri from Germanys Dr. Lange Company and Photobacterium phosphoreum from the United States Microbics Company. A test kit purchased from either Dr. Lange or Microbics

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contains the luminous microbe supplied as freeze-dried preparation contained in small test tubes. Special solution included in the test kit is added to the freeze-dried luminous microbes to bring the frozen cells back to life again. Once revived, the cell suspension begins to shine bluish-green in the dark. The luminous liquid suspension, teeming with millions of shining microbial cells, is mix with a water sample whose toxicity is to be tested. Mixing is done in a small glass test tube, which is then inserted into an equipment called a luminometer. The luminometer measures the light output of the interaction between the mixed luminous microbial suspension and the water sample tested. Two results are possible, either the luminance of the mixture is sustained brightly over time or it fades out rapidly. If the bright luminance of the mixture is sustained, the water sample does not contain anything toxic. However, if the luminance fades out completely after a period, let us say half to an hour, then something toxic is present in the water. If the water sample tested is to be used domestically, then the water sample is not safe and therefore should not be used at all. The luminous microbes act as a biological sensing device whose luminance determines the presence or absence of toxic chemicals in the water samples. Since these microbes are of marine origin, the toxicity test has to be conducted in water samples that have been made saline through the addition of table salt

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(sodium chloride). In the bioluminescence toxicity test, the luminous microbes, smart as they are, are talking to us by sending light signals; telling us if the water contains something toxic or if the water is clean and safe. That was how I got introduced and trained in the nooks and corners of the proper handling and cultivation of the marine luminous microbes for the bioluminescence toxicity test. Day in, day out, I would grow and cultivate these microbes in nutrient broths or soups contained in glass flasks and in solid nutrient agar contained in plastic plates called Petri dish. In the broth, the liquid inside the flask glows brightly bluish-green specially when shaken due to the aeration effect on the large numbers of swimming, shining, and healthy cells. However, in nutrient agar plates the luminous microbes give a different appearance. They produce small, numerous, round objects known as colonies that shine brightly bluish-green in the dark. Colonies are large masses of growing microbial cells piled together and rendered visible to the unaided human eye appearing on the surface of the solid nutrient agar medium. They say that familiarity breeds contempt. However, with the marine luminous microbes my familiarity with them bred a deeper understanding and respect for this humble speck of life. Whenever, I am alone with these luminous microbes in the dark holding them in flasks and Petri plates with

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my bare hands I can feel their life energy flowing to me through the cold light that they produce. It still kept me enthralled in awe and wonder whenever I am alone with them in the dark. There is something in the cool bluish-green light emanating from them that pierces the darkness so beautifully. It is a living light shining so brightly in the surrounding darkness. These microbes can actually talk to one another and to us as well! When they are happy, enjoying a state of well being, they shine long and bright but when they are sad due to harms inflicted to them by, something toxic in their watery environment their light dims rapidly and in some instances fades out completely. The luminous cells will black out instantly when they are killed by exposure to intense heat, strong acidity, toxic chemicals, and potent disinfectants. This innate ability to communicate using light is so common with many organisms found and living in the sea. Deep in the dark vastness and great depths of the oceans of the world, inhabitants great and small have evolve the means to use bioluminescence to communicate with and attract other creatures. Others have used the bright radiance of bioluminescence to evade their predators by temporarily blinding them with a squirt of brightly shining fluid containing millions of luminous microbial cells. Set against a pitch-black world squirts of bioluminescence generate

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brief moments of intense luminosity that must have been briefly blinding to killer eyes.

My Home Laboratory in the university with a great view of the campus

At the end of the second semester, I wrote a paper on the results of my research work with the marine luminous microbes and completed my Aufbaustudium with the highest grade of Sehr Gut given by Prof. Dr. Kaltwasser. I sincerely thanked and bid Prof. Dr. Kaltwasser and Christoph farewell. On the day of my departure, Christoph fetch me at my boarding house in Dudweiler located at the outskirts of Saarbrcken. He took me to the Main Train Station in the center of the city. From Saarbrcken I was to

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travel to Frankfurt to take my flight back home to Manila. He said good-bye, gave me a bear hug, and walked away. That was the last time I saw Christoph. Christoph and I remained in contact for two years by mail and email after I arrived back in the Philippines in1993. It was at the University of Saarland that Sunday and I was introduced to the earliest semblance of what was to become the hallmark of the computer age: electronic mail and the internet. We log on to the internet then using a black and white monitor mainly to write letters to our friends the U.S. using their own university computers. Yahoo and Google were nowhere to be found in 1993. Now that the internet has evolved into far more than just sending electronic mails; it has come to symbolize everything that is modern in the advancement of information storage, retrieval and exchange. After a year, I was even able to send Christoph a box full of various kinds of Philippine shells, which he enjoyed very much. He told me that he was even proud to show it off to his friends. The last time I heard from him was when he sent me a post card from Ireland, the emerald isle, where he and his girlfriend Miriam were spending a wonderful vacation together. It would surely be nice to see him again someday to thanked and tell him of all the unimaginable nice things that happened to me for having learned from him the wonders of microbial bioluminescence.

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They say that all good things must end, and so, in September of 1993, when the red and orange autumn leaves have once again reappeared and blanketed the trees in Saarbrcken, Sunday and I bade Germany aufwiedersehen. I flew eastward directly back to the Philippines while my friend Sunday flew westward and stayed in Texas for a month before returning to the Philippines. With much trepidation, I recalled trying desperately to secure a visa at the U.S. embassy in Bonn several days before I was to depart for Manila. My friends doing their graduate studies at the Southern Illinois University in Carbondale located close to St. Louis invited me to stay with them for a month. Unfortunately, my visa application in Bonn was denied and I left the American embassy so broken hearted. It was so unforgettable that I still vividly remember the afternoon I left the embassy. It was dark and gloomy; indeed the rainy weather was with me, empathizing in my hours of unhappiness. To comfort myself, I just entertained the thought that it was not yet probably the time for me to visit the U.S. and wished profoundly that someday I will get there too. While in Bonn, I really cannot miss paying my homage to Germanys towering figure of classical music Ludwig van Beethoven. I stood for a few minutes in front of his bronze statue in the city square. In my heart, I am profoundly thankful

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to him for his musical compositions that evoke timeless elegance and power. Even today, Beethovens music continues to stir up raw human emotions of love and hate, fear and anger and above all the right for boundless joy. It is innate joy to be experienced in life even in the midst of adversity and sufferings. On my way back to Manila, flying via Lufthansa, I remembered hearing with much nostalgia the song Top of the World by the Carpenters for that was where I surely was then way above in the sky. Peeking through the window, I saw giant columns of bright white clouds billowing so majestically below. Back in the Philippines in November of 1993, Sunday and I resumed our academic responsibilities in teaching, research, and community service at the University of Santo Tomas (UST) with much vigor and with so much to share on what we learned in Germany. Every now and then, I still get

deeply nostalgic about my German experience bringing back fond memories of sights and sounds as well as feelings and even of smell. Whenever I hear over the radio the song Fields of Gold by Sting, it summons up sentiments of pleasure of a sunny late afternoon bus ride through the streets of Saarbrcken. While, Vanessa Williams Save the Best for Last conjures sensation of cool balmy and lingering summer dusks in Freiburg. Sunday became the chairperson of the Department of Biological Sciences at the

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University of Santo Tomas. Soon after that, she married Robert Phillips an American whom she met in her Sunday service congregation in Germany. Sunday introduced me to Bob in Paris during a brief trip to the City of Lights organized by a student club of the University of Saarland. Rob is a good-looking guy, very friendly, and articulate. Sunday got a marriage proposal from Rob when she was already back in Manila. Rob must have really missed Sunday very much. They soon got married in Manila and they have now settled permanently in Texas, Bobs home state, with their three pretty daughters.

An archipelago of the marine luminous microbes

Back in UST while planning for my research proposal, I became restless as a fruit fly. I wanted to work once again with the marine luminous microbes. So many possibilities can be achieved in improving the quality of life of many people with the use of the marine luminous microbes in a developing country like the Philippines. Besides, no one was doing any research in the Philippines on the marine luminous microbes at that time and to start such an endeavor was indeed a pioneering undertaking. To start my research by buying the strains of luminous microbes from foreign companies

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is simply out of the question due to the high cost involved. In UST, we work on a limited research grant of only 600 US Dollars per annum. Microbiology tells me that these marine luminous microbes should occur abundantly in the Philippines, which is blessedly rich with so much natural resources. The country ranks at the top for biodiversity in the world. Equip with my years of training in microbiology, I embark on an exploratory work to isolate the indigenous, Philippine strains, our very own species of marine luminous microbes from many local sources. At first, I thought it would be difficult to find and isolate them but I soon found out that it was actually as easy as ABC. Jumpin Jellyfish! To my surprise, I found out that our local wet markets are gold mines as sources of the marine luminous microbes. The luminous microbes are ubiquitous occurring in squids, all saltwater fishes, crustaceans, shellfishes and even in seawater itself. Before I knew it, I had achieved the isolation of many species of marine luminous microbes that I stored in a laboratory in UST. Various strains of luminous microbes, isolated from the fresh surfaces and juices of Pusit (squid) and from the intestinal contents of saltwater fishes known locally as Sapsap, Galunggong, Espada, Besugo, Matang Baka, etc. and also from the seawater of Manila Bay, were maintained and preserved in a special ultra-low freezer located in the third floor of the Thomas Aquinas Research Complex (TARC). The ultra-low

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freezer facilitated and made possible the long-term preservation of these microbes.

Fishes, squids, crustaceans, mollusks and even seawater are good sources of luminous microbes.

In 1995, when I beheld and held once again in my hands the brightly shining nutrient agar plates of my luminous microbes in a dark room in the UST Charity Hospital, feelings of euphoria overwhelmed me; it was like having found a long lost and cherished friend. It was really so good to see them again, strongly shining bluish-green in a dark room in USTs old research center with strongly sustained radiance like the ones I worked with in Germany. On many occasions, while I am all alone with them in a dark room in the process of adapting my vision to the dark for several minutes. I would talk to them in my mind, asking them as if they were my friends to be kind to me by revealing to me their lifes secrets. Lessons in Green that will

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help me better understand them so that I can employ the light they generously produce to safeguard the water we drink and to protect the environment we live in. In all candors, my Microbes of Light did not leave me wanting. The secrets they revealed brought numerous blessings to me and to many others. It has inspired the younger generation of Filipino students to investigate problems in biology using microbial bioluminescence. Teachers from high schools, colleges and even graduate schools have used them to dazzle their students with awe and bewilderment. Happy of my achievement for having isolated Philippine strains of marine luminous microbes, I began showing them to my friends, to my students and colleagues in UST and they all marveled at what they saw. Very soon I was using and demonstrating the marine luminous microbes in science seminarworkshops for secondary and tertiary level teachers conducted at the Far Eastern University, Philippine Normal University, Technological University of the Philippines, Ateneo de Manila University, Manila Central University, La Consolacion College and many times in UST. I also presented my study on the marine luminous microbes in the annual conventions of the Biology Teachers Association (BIOTA), Philippine Society for Microbiology (PSM) and the Natural Products Society of the Philippines (NPSP) as well as in the

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Philippine Association of American Scientists and Engineers (PAASE) and the Asian Association for Biology Education (AABE).

With much pride, I can also say that in 2003, the luminous microbial species that I had isolated and collected served as the initial microbial holdings that subsequently evolved into an important repository of microorganisms in UST called the University of Santo Tomas Collection of Microbial Strains (USTCMS). Like a devoted microbiologist fresh from my success with the marine luminous microbes, I also undertook the isolation of other indigenous species of bacteria, yeasts and fungi that were deposited in the USTCMS. The USTCMS served to provide microbes like bacteria, yeasts and filamentous fungi to students and faculty members of UST for their research and in their teaching of general microbiology and other microbiology subjects. Students and teachers from the neighboring universities and schools availed of this service.

Blessings through the microbes of light

Indeed, what followed were highly rewarding and exciting years of study with my luminous microbes that went far, far beyond my wildest

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expectations! My life with the Microbes of Light as I often call them now has showered me with so many blessings. Foremost in my list of blessings was finishing my Mater of Science degree in Microbiology at the UST Graduate School in 1998. My masteral thesis dealt with the isolation and identification of indigenous marine luminous microbes from various marine animals and seawater, which were then individually screened for their sensitivity to various toxic chemicals using a luminometer. The most toxicant sensitive strain of luminous microbe was selected and tested against standard toxicants like phenol, cyanide, mercury, and the detergent: sodium dodecylsulfate. This toxicant sensitive strain was identified as Vibrio fischeri with the assigned accession number of 1063 in the USTCMS. The output of my thesis work, aside from a paper presentation and a published paper in the proceedings of the Philippine Society for Microbiology (PSM), were the various species of marine luminous microbes like Vibrio fischeri, Photobacterium leiognathi, and Vibrio harveyi deposited and preserved in the USTCMS. In addition, I also received national and international awards. I was able to publish articles in local and international journals. I was also able to actively participate in numerous poster and paper presentations in local and international science conventions and symposia. I received speaking

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engagements in scientific gatherings and would you believe appearances in TV (ABS-CBN) and Isla Cable TV and also radio interviews. The Philippine Charity Sweepstakes (PCSO) announced my achievement over the radio. I also got news clippings in all the major Philippine broad sheets from the Manila Bulletin to the Inquirer and from Philippine Star to the Manila Times and also a substantial amount of entries in the internets major search engines. All of these venues were used to the best of my ability to spread the knowledge I obtained with microbial bioluminescence. The clean and useful radiant biological energy produced by these uniquely smart microbes that can be harnessed for the promotion of the well being of Filipinos by safeguarding drinking waters cleanliness and protecting the environment from pollution. In addition, it has also inspired students from near and far to pursue careers in science as well as pioneered new methods of teaching biological concepts. My first national award on the use of my luminous microbe came in July of 1997. Dr. Fortunato Sevilla, my masteral thesis adviser, and I presented a research poster in the professional category of the eight annual scientific research poster competition sponsored by the Department of Science and Technologys (DOST) agency the Philippine Council for Health Research and Development (PCHRD). The poster entitled: The Development of a

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Biosensor for the Rapid Measurement of Water Toxicity Based on Indigenous Marine Luminous Bacteria won grand slam for the first time in the annually held competition of the PCHRD by bagging the first, second and third prizes of the contest. My adviser and I won a total cash price of 45,000 pesos. I was so happy to have won in the DOST PCHRD contest for it was the first time that I won something big, prestigious and of national in scope in my budding professional career. Dr. Fortunato Sevilla attended the awarding ceremonies for the annual competition and received the cash award. We decided to split the cash prize at 25,000 for me and 20,000 for Sir Fortune Sevilla. During the contest, I remembered standing with the other contestants in front of our posters in the lobby of the Philippine General Hospital explaining to the five judges as well as to the interested public and onlookers what our entries were all about. In school, I served as the undergraduate thesis adviser of students majoring in microbiology and biology whose research studies dealt with the use of microbial bioluminescence. There is a wealth of problems in biology and chemistry that students can study using bioluminescence as a tool in shedding light to their investigative problem. Many groups of students from the BS Microbiology program and one group from the BS Biology Accelerated Program worked on microbial bioluminescence research. One

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group from the BS Microbiology won first place in the Best Poster Contest of the College of Sciences Annual Science Fair and Exhibits. In addition, a research study on the luminous microbes also won first prize in a poster competition from a group of students supervised by Mr. Arthur Alipao of UST for the analytical chemistry division in the 2001 Philippine Chemistry Congress. In 2002, something GREAT happened that was way beyond my wildest expectations. It really swept me off my feet into what I consider as the second best thing that ever happened to me in my life.

Winning the gold in the Asia-pacific Young Inventors Award (YIA)

If the year 1992 was for Her Majesty Queen Elizabeth II an annus horribilis or horrible year, the year 2002 will go down for me as my annus mirabilis or my wonderful year. What transpired was truly a shining moment in my career and the opportunity of a lifetime. In that year, UST and I got the coveted recognition of winning the Gold in the 2001 Young Inventors Awards (YIA) sponsored by the prestigious Far Eastern Economic Review (FEER) of Dow Jones Company and Hewlett Packard (HP) Invent Asia Pacific. This prestigious international contest for innovation ran for five straight years with the objective of uplifting the quality of life for many people in the region

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through original ideas hatched by students from the Asia-Pacific institutions of higher learning. The awarding ceremony was held in Hong Kong in the years 2000 to 2002 while in the years 2003 and 2004 it was held in Singapore. It is with the fondest of memory that I will always looked back to the beautiful sunny and cool morning of January 18, 2002 at the Department of Biological Sciences in UST. While USTs Varsitarian science news reporter Stephen Rojas-Chua was eagerly interviewing me on what it is like to be one of the finalists in the Young Inventors Awards, I received the most memorable phone call of my life! It was Dada from the Deans Office of the College of Science informing me with much elation that HP Philippines in Makati just disclosed the news that I won the Gold in the 2001 Asia-Pacific Young Inventors Award Competition. I was speechless and petrified holding the phone! My weeks of eager anticipation were finally over and what a magnificent ending it truly was. After regaining my senses, I immediately conveyed the good news to my friends and colleagues in the department and it opened up the floodgate of congratulatory remarks from so many people coming from near and far; and from the past and present. Fr. Victor Badillo, S.J. former President of the Philippine Astronomical Society (PAS) and former director of Ateneos Manila Observatory also congratulated me. I

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was really so happy to hear from Fr. Badillo, a Jesuit mentor and close friend whom I have known way back when I was still a member of the Philippine Astronomical Society (PAS) during my late high school and college days. Fr. Badillo an NRCP achievement awardee in Physics together with Engineer Jose Caburian of Marsman Company are the Fathers of Philippine Amateur Astronomy. They had inspired several generations of Filipinos to become dedicated amateur astronomers like my friends Edwin Aguirre and Imelda Joson, famous amateur astronomers, now based in the U.S. Edwin and Imelda became very popular recently when a new comet they discovered, Edwelda, was named after them. Many of us including my brother Johnny and friend Michael Nealega have been in many ways influenced by Fr. Badillo and Engineer Caburian. Prof. Fabian Dayrit, Ph.D., Dean of Ateneos School of Science and Engineering, a highly esteemed professor and a towering figure in Philippine chemistry education and research also congratulated me by email. I also received notes of congratulations from Filipinos living in Singapore and Australia. My winning the contest also got featured in a news-magazine devoted to Filipino activities and concerns based in Singapore.

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Winning the gold was for me truly unbelievable due to the sheer chance of winning first prize in a contest of 220 entries from different universities all over the Asia Pacific region. Universities from Pakistan to New Zealand and from Japan to Australia joined the contest in 2001. The entries were received and organized by the Far Eastern Economic Review in 2001, were subsequently narrowed down to fourteen finalists. My entry was short listed as one of the finalists and all were featured in the December 17, 2001 issue of the Far Eastern Economic Review magazine. I told myself then that I would be very happy even if I just get one of the two slots for honorable mention. It was indeed humbling to compete against such high Asiaweek ranking universities as the National University of Singapore (NUS), the Hong Kong University of Science and Technology (HKUST), South Koreas Pohang University, New Zealands Massey University, Taiwans National Cheung Kung University and other venerable institutions of higher learning. In fact among the fourteen finalists no university is ranked lower than 32 in the Asiaweek 2000 survey of Best Universities in Asia and the pacific except for UST, which was ranked 74. But hope indeed springs eternal and my pioneering work on the marine luminous microbes in the Philippines has once again brought with it a brilliant outcome.

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The December 2001 issue of FEER featuring the Asia-Pacific Young Inventors Awards

The awarding ceremony at the top of the world The weeks that followed were very stressful, as I have to make haste to secure a new passport to enable me to fly to Hong Kong. My missing

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passport had to be replaced as soon as possible. I got my new passport in the nick of time so to speak a mere three days just before I was to depart for Hong Kong to attend the awarding ceremony. I finally arrived on an all expense paid trip to Hong Kong in the afternoon of February 27, 2002 and I was amazed at the level of advancement and prosperity that this small former British colony has achieved since gaining independence. Indeed the influence of the British in the prosperity of Hong Kong, Malaysia, and Singapore is something that these countries can be proud of. Hong Kongs large spacious airport built on an isolated island is truly a national pride and an infrastructure par excellence. The awarding ceremony was held at the Apex of the posh Central Plaza, Hong Kongs then tallest building. The Apex is truly a marvel of modern architectural design. To reach it from Central Plazas impressive spacious lobby with long crisscrossing seemingly aerially suspended escalators you have to go through three changes of elevator ride walking passed by glistening floors with huge artistic pots containing beautiful plants and colorful flowers of different kinds. The last elevator trip opens up to the Apex a marvelous and opulent all glass and steel hall perched on the seventy-fourth floor and boasting of a 360 degrees panoramic view of the whole of Hong Kong. The blue surrounding sea seem to stretch infinitely far out into the edge of the hazy gray horizon. What an

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awesome breath-taking sight it was! Being in the Apex was certainly the closest place to heaven that I can ever get to here on earth. A long spire protrudes high into the great blue yonder from the center of the Apex. Central Plaza towering majestically for years against the Victoria Harbor skyline is an icon of prosperity and cosmopolitanism that was Hong Kong.

The towering and posh Central Plaza set against the glittering Hong Kong Skyline and the historical Victoria Harbor

The silver awardee Bini Thumbarathy, an Indian national, studying at the National University of Singapore and Chen-chi, the bronze awardee from Taiwans National Cheung Kung University were already in the Apex when I

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arrived. We were all very happy and pleased to have finally met one another. Both were Ph.D. students who were so nice, very friendly and so unassuming. The famous cable TV business newscaster Bernie Lo of CNBC interviewed us and we spent the whole afternoon with him going through the hectic rehearsals for the evenings awarding gala ceremony. We shared ideas and insights and Bernie gave us a brief history of Hong Kong and its famous airport. Bernie noticed the clothes I wore and he said that he saw President Joseph Estrada wearing something similar as well. I explained to Bernie that our beloved President was wearing the Philippines national costume for Filipino men, called the Barong Tagalog and we wear it for very special occasions. We then took our position before our posters after the rehearsals to explain, discuss and answer queries from the visitors as to what our inventions were simply all about. Students from the prestigious and venerable Hong Kong University of Science and Technology (HKUST) were also around for winning the honorable mentions of the 2001 Young Inventors Awards.

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A page inside the Far Eastern Economic Review (FEER)

The VIPs of the prestigious Dow Jones Company arrived from their various offices located in the same building below; the men were in their

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formal black suits and the women in their cocktail dresses. Mr. Phil Revzin publisher of the FEER, his personal guest Mr. Paul Saffo, headed the Dow Jones contingent. Mr. Paul Saffo is the director of the Institute for the Future based in California. Mr. Michael Vatikiotis, chief editor of the FEER and their associate editors and reporters also arrived. Dow Jones Company, a name synonymous with the best in global business news, is the publisher of the popular Wall Street Journal in the US, the Asian Wall Street Journal and the Far Eastern Economic Review. The representatives of HewlettPackard (HP) Invent Asia-Pacific, Ms. Cecilia Pang, and Ms. Yee Foong, advertising managers of HP based in Singapore and Mr. Raymond del Val president of HP Philippines were also in attendance. The representatives from Polycom donor of the VIACOM videoconferencing units were also present to demonstrate their latest high-tech gadget. Guest of Honor was Mr. Chau Tak Hay, secretary of Commerce & Industry of the Government of Hong Kong Semi Autonomous Region (HKSAR). Deans and academic officials from various Hong Kong universities were likewise in attendance, as well as the consul general of the Philippines to Hong Kong Mrs. Zenaida Angara-Collinson, and her British husband. Mang Nards of the Philippines Department of Trade and Industry (DTI) also graced the occasions. I am so happy to meet in person the associate editors of the FEER: Helen

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Pryzidowski whom I got to know very well through our several email correspondences during the submission of my entry and in the screening of the finalists. Helen was the one who gave me that seemingly far away glimmer of hope of ever winning in this contest when she informed me in October 2001 that my entry has been shortlisted as one of the finalists. Special mention also goes to Sofia McFarland, the freelance Swedish reporter who grilled me for several days in UST on my invention. She was the one who wrote the beautiful article on A Luminous Vision found in the January 18, 2002 issue of the FEER and in its website. Sofia is a lovely, soft-spoken, well-organized person and a freelance writer par excellence. She is a mild mannered writer who is strongly devoted to her profession. She lives permanently in New York with her children and husband - Jeffrey. Jeffrey, a tall, handsome, self-assured, and intelligent physician and epidemiologist works at the World Health Organization (WHO) for the pacific region based in Manila. To cap it all up, I was so happy almost at the verge of tears to see the arrival in the Apex of Asst. Prof. Milagros del Callar whom we fondly call Maam del. She is the cherished chairperson and endeared academic mother of USTs Department of Biological Sciences. Her husband the esteemed Dr. Achilles del Callar, professor of mathematics and physics at

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USTs Graduate School, accompanied Maam del in their trip to Hong Kong. At last, I said to myself, I have someone really close to me from the Philippines with whom I can share my happiness and this momentous event with. It was truly unfortunate that the UST administration was not able to send any representative to graze the occasion and to receive the beautiful heavy trophy and symbolic check of 7,500 US Dollars from the FEER. Evening finally came and Hong Kong unfurled a spectacular view! The city of Hong Kong literally exploded with lights of various colors as seen from our vantage point high up in the Apex with all the neighboring skyscrapers towering brightly and magnificently around us. I am simply at a loss with words to describe the sophisticated ambiance, the terrific place, and the gathering of notable academics and business people at the Apex of the Central Plaza that evening. The event was so magical and memorable rendering it almost dream-like; it was the kind of stylish sights and sounds that I had only seen in Hollywood movies. The awarding ceremony started at 6 pm with a snappy video presentation of what the Young Inventors Awards is. I remember being seated between Mr. Raymond del Val of HP Philippines and Chen-chi. Chen-chi from Taiwans National Cheng Kung University was the first to be

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called up the stage by Bernie to receive the bronze award and the trophy and computer prizes from HP. Bini Thumbarathy from the National University of Singapore was next to receive the silver award and prizes . My turn finally came to receive the gold award. I felt so happy to go up on stage and beaming with pride I delivered a brief and concise answer to Bernies question. His honor Mr. Chau Tak Hay gave me the heavy trophy and then posed with him for the picture taking. I went back to my seat carrying the heavy blue and yellow trophy of the gold award for UST.

Icon of the young inventors awards (YIA)

Lastly, it was Maam Dels turn to represent the UST administration. She went up on the stage and stood proudly during the climax of the program.

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With much applause, Maam del received the large symbolic check of 7,500 U.S. Dollars in-behalf of UST although she and her husband went to Hong Kong on their own private initiative. The ceremony took place on a formal candle-lit dinner, drapes of red and white cloths covered the long tables, catered with sumptuous foods and lavish drinks, and of course, to cap it all up, the charming and articulate Bernard Lo acted as master of ceremonies. The awarding ceremony went like clockwork; so smoothly and precisely as we had rehearsed it in the previous afternoon. At 8:00 pm the awarding ceremony full of wonderful moments finally came to pass. We all went down the Central Plaza building in the elevator in batches. The attendees all received souvenir items and a small handsome powerful binocular from HP contained in a small black leather bag. Some of us walked back to the Luk Kwok Hotel where the other winners and I were booked. The hotel is just a few blocks away from the base of Central Plaza. Somewhat tired but still very much in high spirits, I carried with me the heavy trophy for UST and of course my HP Omnibook 6100 laptop. The Omnibook 6100 is a top of the line multi-functional computer product of HP, which has been of much use to me in my teaching and research work in UST. In fact, I used this laptop, still functioning perfectly after 6 years, writing this book. Maam Del and her husband Dr. Achilles del Callar took the impressive and clean subway of

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Hong Kong to travel back to a hotel in Kowloon where they stayed. I am truly grateful to Anna Lam, a pretty and petite woman and Jonathan Hardy both of the FEER for having managed and attended to our needs during the rehearsals and the awarding ceremonies. In the Luk Kwok hotel in Wanchai as I reflected back on the exceptional events that transpired, I cannot help but wonder and be amazed at the seemingly meant-to-be turn of events. It is indeed true that it is better to have tried and failed than never to have tried at all. What started out for me as just a late evening curious internet visit to the website: www.feer.com using an old computer with a black and white monitor in the UST Research Center in the charity hospital turned out to be the chance of a lifetime. My research work on the light generating indigenous marine microbes has been for me a very rewarding endeavour ever since I learned of it in my aufbaustudium at the University of Saarland in Germany in 1993. I surely will never forget the awarding ceremonies in Hong Kong for it was for me the closest ever of winning a major science award like perhaps a Nobel Prize! Winning the Gold is indeed a fitting tribute to start-off the universitys countdown towards its Quadricentennial founding celebration in the year 2011. Someday people will look back at the list of universities, which have made their mark in this prestigious competition of innovation in the Asia-

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pacific region. Thomasians will surely take pride in knowing that in the second year of the Young Inventors Awards (YIA) sponsored by FEER and HP that ran into a full five- year course, UST took the highest honors. It is the first for a Philippine university and the first for a developing country like the Philippines to be in the list of winners. Back in UST, Maam Del and I paid a courtesy call to the Father Rector, Very Rev. Fr. Tamerlane Lana, O.P., in the imposing Rectors Office and gave him personally the Gold winning cheque worth 7,500 USD. The Polycom videoconferencing units were delivered to me in Manila. One unit was for UST and one for me, which Maam del and I decided to donate to UST so that they can have a pair of it. The cash prize was converted to 384,000 pesos, which was fully utilized to purchase much needed laboratory equipment for the Pure and Applied Microbiology Lab. of the Research Center for the Natural Sciences as well as for the Department of Biological Sciences of the College of Science. Digital balances, distillation apparatus, magnetic stirrers, refrigerators, and microwave ovens were bought using the cash prize. New computer units were also made available to the Biology and Microbiology Student Organizations of the College of Science. The laboratory equipment and computers purchased were thought out carefully to maximize the benefits to the biggest number of graduate and

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undergraduate students, faculty members, and researchers alike. Indeed the equipment purchased continues to serve the needs of students,

researchers, and faculty members up to now. The distillation unit still supplies distilled water to four laboratories in the 3rd floor of the Thomas Aquinas Research Complex. The last time I saw the FEER-HP trophy for the university was during the birthday of the Vice Rector Fr. Juan Ponce, O.P. in 2003 when the academic community paid him a courtesy call in his office. His office connects to the Rectors Office in the second floor of the universitys historical main building. The Rectors office is the universitys sanctum sanctorum and it is not often that one gets to enter it. So we took the rare opportunity to enter the Rectors Office and viewed a large table bearing the memorials of success and victory that the university has earned through the years. There I saw the trophy once again standing side by side with the others. What immediately came to my mind was how heavy and solid it was! Carrying it from the top of Hong Kongs tallest building during the awarding ceremonies back to UST in Manila was a big effort on my part but well worth the trip. I remember so well, Bernie Lo jokingly told us winners that we can use the heavy trophy as weights to keep us fit. Well he can surely say that again. I will forever be grateful to the very Rev. Fr. Tamerlane Lana, O.P. for

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it was during his rectorship that researches in the arts and sciences flourish and peak in UST earning for the university numerous recognition at the national and international arenas.

News from the Phil. Daily Inquirer. Many thanks to Prof. Dr. Abercio Rotor of the UST Graduate School for framing this news clipping for me.

Some scientists in the U.S., England, Germany, Poland and Croatia likewise took note of my novel method of immobilizing marine luminous microbes in small paper discs through reprint request of my first international article published in the Journal of Biological Education (JBE) in 2001. The JBE, an Institute for Scientific Information (ISI) listed journal, is the official publication of the Institute of Biology (IOB) the biggest organization of
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biologists in the British Isles. My articles title was used as well by the IOB in its advertisement subscription form. The JBE has the biggest worldwide circulation of a peer-reviewed journal devoted to biological education. Dr. Baby Angtuaco of Ateneos Department of Biology, a professor active in teaching and research, subscribed to the JBE and congratulated me when she saw my article in the Spring 2001 issue of the journal. The acceptance of my first article in an internationally ISI (Institute for Scientific Information) listed journal and Expanded Science Citation Indexed journal based in London has indeed given me the confidence to submit publications in other journals devoted to microbiology, water and the environment. Like any other scientist, I should be able to convey my research findings clearly to other scientists in my field by publishing them in special science publications called journals.

PIBiT the invention

I look forward to the day when the vast potentials of this simple bioluminescence invention, with the proposed name Paper-disc

Immobilized Bioluminescence Technology and having the acronym PIBiT, will have been fully realized. It may take a decade, a lifetime, or even a

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century but I am sure it will come. PIBiTs main use is to safeguard our drinking water from toxicants and protect the environment from pollution. The more the world needs to reuse, recycle and reduced due to the threat of global warming, the more this inventions significance will be appreciated and bring into practice in most of the developing countries of the world. PIBiT holds the promise of being a multipurpose analytical process that is inexpensive, user-friendly, and environment-friendly and exhibits almost no energy consumption and zero emissions. PIBiT was featured in the prestigious September 17 issue of the Sustainable Practices 2004: Innovations, Technologies, and Products through Mr. David Schaller. Sustainable Practices 2004 is compiled and provided by the EPA (Environmental Protection Agency of the United States.) Region Eights Sustainable Practices and State Partnerships Program. EPAs Region Eight serves Colorado, Montana, North Dakota, South Dakota, Utah and Wyoming. Surely I could have made the invention complex and complicated by introducing a light-measuring device instead of just using plain scotopic or dark vision to observe the light signals of the luminous microbes. But it defeats the purpose of making it readily available to the vast majority of the poor people of the world. Digital Photography is a much simpler and far less expensive alternative to the use and procurement of a luminometer although

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its light measuring ability may not be as sensitive. I subscribe to the school of thought known as KISS, which I first heard from an MIT professor giving a lecture in the BIOTECHNICA 1988 in Hannover, Germany. He said that at MIT, they keep everything including research output KISS which means Keep It Simple Stupid. Hopefully, the day will come when the World Health Organization (WHO) and UNDP and also UNESCO will have adopted this simple invention in many of the developing countries of the world as a simple and inexpensive means to guard the safety of their drinking water and for the protection of their environment. I am sharing a simple note that I sent to the people, I believed that I am indebted to most particularly to the 2001 YIA judges, the people at FEER and HP. It goes

My winning the Gold in the Asia-Pacific Young Inventors Award Is very much like my culture of luminous microbes One microbe alone cannot shine on its own Much less attain a state of brilliant luminosity But by being in the company of other microbes Can one truly shine and together as a whole attain amazing luminosity It is with joyous pride and profound gratitude that I myself have likewise attained this state of brilliant luminosity

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Only because, I have been in the company of the other 2001 brilliant inventors.

Myself with Mr. Chau Tak Hay, secretary of commerce & industry of the government of Hong Kong Semi Autonomous Region (HKSAR) receiving the gold trophy of the 2001 Young Inventors Award

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The winners of the YIA through the years

Year Wang Qijie Gold China Randall Law Silver Singapore Bronze Liang Xiaojun, Sun Yi, &Zhang Xuming China Nanyang Technological University Ryuji Inai [2003] Japan Sangjin Han South Korea Joanna Tan Hwa Lay Singapore Ngee Ann Polytechnic Growth Market Anthony Samir [2002] Australia Robert Fearn Australia Michael Zheng Zhongming Singapore National University of Singapore The Fungus Among Us Edward Quinto [2001] Philippines T.B. Bini Singapore Yu Chen-Chi R.O.C Taiwan National Cheng Kung University What a Catch Mulyoto Pangestu Monash University [2000] BIOLOGY Australia Tse Kowk-Kuen HongKong City University of Hong Kong SOLAR ENERGY China Chong Wai Yin, Kelvin Singapore Ngee Ann Polytechnic GENETICS

[2004] Nanyang Technological University

National University of Singapore

National University of Singapore Material Advantage

Seoul National University Fuel-Cell Thrift

University of Melbourne Life-saving Precision

University of New South Wales Music to My Ears

University of Santo Tomas A Luminous Vision

National University of Singapore Nerves of Steel

www.ntu.edu.sg/home/EXJLiang/YIA2004.htm

Go west, life is peaceful there; Go west, where the skies are blue To finally complete the prize of having won the Gold in the Asia Pacific 2001 Young Inventors Award, I had the rare privileged of being flown on an all-expense paid trip to Palo Alto, California through the courtesy of HewlettPackard Invent. The trip entailed visiting Stanford University and the

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Hewlett-Packard Nanotech Laboratories and most of all to meet the winners of the 2001 Collegiate Inventors Contest considered Americas most brilliant young minds. I was supposed to fly to California with the two other winners of the 2001 Young Inventors Award. But Bini Thumbarathy and Chen-chi both cannot fly to the U.S. Bini was on her eight month of pregnancy and Chen-chi had academic commitments and so I have to go to the U.S. alone. The visit to the U.S. was something that I could not miss for the world. The chance to visit the U.S. came to me once again and this time around, it was offered to me as a gift for having won the Young Inventors Awards (YIA)! Vividly, I recalled my sad experience in 1993 when my earnest desire to go to the U.S. was squashed by the U.S. consul in Bonn by denying me a visa. I left the embassy so broken hearted that day because I do not know when the opportunity to visit the U.S. will ever knock again. Astonishingly in 2002, it did knocked again and even opened up the door for me! It was so different in 2002 than in 1993, so confident and proud am I of going to the U.S. embassy in Manila for I know fully well that I will get my coveted visa this time. The processing of my visa in the U.S. embassy was a breeze. With just a question from the consul on what it is that I have invented that won me the award and presenting my certificate of employment from UST and the official letter of invitation from HP I finally got my precious six-month visa the

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following day. I was so pleased with how I was treated by the woman consul that I left her a copy of the FEER magazine containing a write-up of my award winning entry. The saying that the best things in life are free meant so much to me than ever before. So far, all of my foreign trips had been free of charge. Recently, in 2007, I requested again for a U.S. visa to enable me to attend a conference in Texas. Lo and behold, I was so pleased and thankful that I was given a ten-year visa. I woke up very early at around 2:00 am in the morning of 2001 Easter Sunday in my rented place near UST. Immediately, I organized myself and soon I was at the international airport at 4:00 am. From Manila, I first flew to Hong Kong to get a connecting flight that will take me all the way to the City by the Bay - San Francisco. The journey to California was quite long. I felt so excited, somewhat daunted because it is going to be my first trip to the United States. Surely I have made many long trips to Germany before, always flying westward across the vast landmasses of Asia and Europe. Now, I am flying eastwards across the vast expanse of the Pacific Ocean. Flying via United Airlines, we cruised at an altitude of more than 30,000 ft. Seated beside the window, I did not see the ocean below just a thick blanket of white-grayish clouds extending all the way yonder.

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Sojourn in the famous Silicon Valley

America here I finally come! I reached San Francisco in the early morning of Easter Sunday after about fourteen hours of flight. It was a very memorable 2002 Easter Sunday for me because I finally set foot on the land of milk and honey. The land that many Filipinos will do everything possible to enter, settle down and work. It was indeed a relief that I am standing once again on firm American ground. I can hear in the back of my mind the Mamas and the Papas singing California Dreamin, and I almost put some flowers in my hair. The long trip made me feel so plane-sick that I felt sleepy and groggy upon exiting the plane. Mustering enough energy, I began my long trek in the imposing corridors of the magnificent airport moving towards the area of the U.S. immigration officials. The cold early sunny Easter morning was truly a welcome relief. The mere thought of being in the U.S. revitalized me and gave me enough push to move on. It really felt so great to be finally in the U.S. of A. The first thing I noticed was the presence of so many Filipinos holding various types of job in the airport. The immigration official that gave me a six-month stay and the woman in charge of the money exchange looked like Filipinos. Often one can hear Filipino being spoken in some quarters of the

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Airport and in the streets of San Francisco. I guess that really makes San Francisco a home away from home for many Filipinos. Unfortunately, I missed the person who was supposed to fetch me at the airport and who will bring me to the Sheraton Hotel in Palo Alto. It made me a bit nervous because I dont have enough money to take a taxi ride to the Sheraton. So, I bravely went out of the Airport holding on to my luggage to explore the public transportation system of San Francisco. What I did was to take the free shuttle ride to Caltrain from where I hope to travel to Palo Alto. Since it was a Sunday, the scheduled Caltrain trips were far apart in between. I decided to return to the Airport and soon enough I was so happy to discover a bus route that finally took me to the Shopping Mall in Palo Alto located close to the Sheraton hotel. It was a delightfully languid day. The weather had a welcome charm, it was sunny and cool, and the sky was blue as cheery American eyes. Alluring two-storey houses lined the suburban streets; showy and wellmaintained green lawns and a modern vibrant city were my first memorable glimpses of San Francisco. Palo Alto is a captivating place with the widest of flowing free ways and seemingly boundless parks and vast locales for the multi-national business companies. Ah yes! It is the envy of many Californias ostentatious display of limitless open spaces. Stanford University

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and Silicon Valley are the famous landmarks of this region nestled several kilometers south of San Francisco. I reached the Sheraton Hotel at noontime, some of my relatives visited me at mid noon, and at 6 pm I was already in bed deeply asleep due to the long exhausting journey that originated from the other side of the globe. The following refreshing morning, in the Lobby of the Sheraton Hotel, still suffering from an intense jet lag, I had the pleasure of being introduced to the five winners of the 2001 U.S. Collegiate Inventors Contest. Steve Anderson, Manager of HP Brand Sponsorship, a tall black American who is an avid fan of the Giants and who likes Filipino dishes like noodles (pancit) and spring rolls (lumpia), was our HP host. We toured the HP Main Office in Palo Alto, the original offices of Dave Packard and Bill Hewlett where a literal open door policy was implemented to all the employees of their corporation. This policy surely made HP a very competitive corporation that is able to continuously re-invent itself to meet the challenges and the needs of the world.

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Meeting the winners of the 2001 U.S. Collegiate Inventors Contest

Due to my jet lag, I almost fell from my seat while trying so hard to keep myself awake listening to HPs CTO Mr. Stephen Squires message. He is the chief technical officer of HP who gave a short inspiring round-table discussion on HP Invent and on innovation. During the light lunch tendered by the HP administration in the scenic HP garden, I finally got the chance to really know in person the winners of the U.S. Collegiate Inventors Contest. Two of the winners, Michael Oddy and Daniel Fletcher recently earned their Ph.D. in Engineering from the prestigious Stanford University. Would you believe that the research endowment fund of Stanford University alone stood at 7.6 Billion US Dollars, which was roughly the size of the Philippine government national budget in 2001! Sascha Welz is also an Engineering Ph.D. holder from the University of Illinois at Chicago. Sascha comes from the Saar region in Germany. The Saarland for me is a region full of wonderful memories and in whose university I finished a one-year postgraduate certificate course in Biogeography and Environmental Assessment way back in 1992. Small world isnt it! Dana Perkins, the only rose in the group, recently earned her Ph.D. in Pharmacology and Experimental Therapy from the University of Maryland. Incidentally, there

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was a big celebration that Monday evening among us because the University of Maryland made history by winning for the first time in the NCAA against Indiana University. Dana proudly said that she was born in the Dracula-famed Transylvania region of Romania and ultimately migrated to the US. Dhaval Doshi was an Indian national who like the others earned his Ph.D. in engineering recently from the University of New Mexico. The sixth winner who was not able to make it to Palo Alto was Xiangfeng Duan from Harvard University.

What is it that they invented? Xiangfeng won by developing nanowire building blocks. Michael who comes from Arizona invented a novel device for the rapid stirring of micro and nano liter (extremely small) solutions for molecular diagnostics with potential for aiding genetic and drug discovery research. Daniel a true-blooded Californian invented a microsurgical tool that provides more precise tissue dissection and drug injection capability than is currently possible with existing procedures. Sascha invented a costeffective technique to produce dynamic seals coating for vehicle engines to improve life and prevent failure. Dana successfully utilized a Herpes virus as transfer agent to combat degenerative brain diseases like Alzheimer in laboratory animals. She said that clinical trails would follow soon. Dhaval

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developed a technique using UV light to alter a thin films pore size, optical characteristics and other properties to improve microelectronics. Inventors are not nerds, which is usually how Hollywood portrays them. Just like any other young people, inventors know how to have a good time. They enjoy going out in the company of friends to dine in good restaurants, drink beer, laugh at good jokes, and watch basket ball games. Traits that unite them are being open-minded and inquisitive, analytical in thinking and a strong dedication to science. An inventor sees things not with his eyes but with a mind that dares to confront the conventional and challenge mediocrity. They are gifted with sparks of imagination that can make huge leaps and bounds in technological development and scientific investigations. One simply

needs to look back at numerous events in history when the sons and daughters of science initiated and led revolutions in ideas and ways of life. They started a cascading reaction of events that created milestones and breakthroughs in many areas of human endeavors leading to the twentiethcenturys conception of gene-manipulation based Biotechnology and the computer-based Information Technology. In todays world of modern living, every aspect of the level of progress and the high quality of life that we now enjoy so much can be traced to the ideas of great minds. The discovery of antibiotics, structure of proteins, functions of nucleic acids and other

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biomolecules, and the invention of the printing press, gyroscope, plastics, jet-propulsion, robotics, photography, DNA recombinant technology,

polymerase chain reaction (PCR), computerization, LCD, LED, and nanotechnolgy has brought all aspects of human living well beyond the space age.

The Hewlett Packard experience

We also visited the HP Archives, the HP Cooltown Experience which was really so cool, the Computer Museum in Mountain View and the Explotatorium in the Bay Area. The vicinity of the Exploratorium with its tall huge roman columns is memorable for I have seen it several times in the movies. The Pacific Bell Park home of the San Francisco Giants is very impressive and modern. We took our meals in some of the famous restaurants in the Bay Area and we visited the famed HP Laboratories where research in Nanotechnology, the current stuff of science fiction, was conducted. Imagine developing a chip the size of your thumb that can hold several terabytes of stored information as well as creating machines the size of molecules. We also visited the famous Garage, birthplace of the famed Silicon Valley, where HP had its origin. Dave and Bill started their business

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of inventing electronic gadgets like oscilloscopes and calculators and transformed it into the multi Billion-dollar computer and information technology company known today with pride as HP. It is also amazing to know that Dave Packard worked as an engineer for General Electric (GE) during the great depression only to resign and to put up a company that now rivals General Electric itself. Even though other companies like Dell have over taken HP in terms of personal computer sale, it still is one of the best brands to consider when buying computers, computer accessories, and computer services. In fact, my second more modern laptop is an HP Compaq Pressario.

The afternoon of my last day in San Francisco was truly hectic. I got to see some of my relatives, which brought me to Daly City where they lived. A visit to San Francisco will not be complete without the trip to the Golden Gate Bridge. Uncle Jesse and I made a rapid buying spree for gifts to be brought back home. My relatives, some of which I saw for the first time, were all so generous to give me whatever dollar bills they had and I got to be sixty-four dollars richer than when I entered the U.S. carrying a mere 100 dollar bill. When I finally got back home in Manila in the wee hours of the next Sunday morning after almost a week of a whirlwind visit in San

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Francisco, I switched on my FM stereo to help me unwind and reflect on my memorable sojourn in San Francisco. The very first song that I heard,

unbelievably, was the song I left my Heart in San Francisco by Tony Bennett! Absolutely true, I swear.

It is my wish to visit San Francisco again someday. San Francisco is a welcoming, wonderful, and energetic city with so much to offer to the unwary visitor for him to stay on for good. It is a metropolis for the worlds dreamers and visionaries where they create the future of technology. Silicon Valley has pioneered many technological innovations whose impact on modern living continues to affect the way we live and view the world today. The experience of having won the Gold in the 2001 Asia Pacific Young Inventors Award is really the chance of a lifetime that opened up new doors of opportunity for me. I wish all our budding scientists and inventors across this vast archipelago all the best in joining the various science contests that are held annually in the Philippines. I am hopeful indeed that just like Germany and the U.S. we can finally move our country forward into the globalized world of Biotechnology and Information Technology through innovative ideas.

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Bioluminescence projects shining brightly in science fair contests

After going through years of conducting demonstrations on microbial bioluminescence in numerous lectures, seminar-workshops, training courses and in annual conventions held in UST and in other universities, microbial bioluminescence has finally aroused the curiosity of the countrys education sector. High school students came to UST motivated by their desire to apply bioluminescence as a tool to study problems in biology for their science fair projects.

Shining colonies of Vibrio fischeri in the dark

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In 1998, Dean Emeritus Carmen G. Kanapi of USTs College of Science introduced me to a group of four high school students from the Assumption in Makati. One of the girls was the niece of Dean Kanapi. The Assumption girls were pretty and zesty, so confident of themselves and so enthusiastically dedicated to their science project. These girls were definitely far from being the picture of spoiled rich girls that most ordinary people have of the Assumptionistas. They worked hard for three weeks in the universitys old research center located in the charity hospital until the early hours of evening and were so ecstatic to see the beauty of bioluminescence. They were holding brightly glowing flasks of bioluminescence high up in the air and swaying as if they we were in a rock music concert. Suffice it to say, they won first place in their high schools science fair contest.

In 1999, a lone female high school student from the UST Pay High School reached the national level contest of the Intel Science and Engineering Fair with her study on the use of bioluminescence for the ecotoxicological monitoring of rivers in Bulacan. Many rivers in Bulacan are contaminated with heavy metals due to the heavy presence of the tanning industry. The Tanning industry throws out wastewater containing high concentration of chromium. Her entry won first prize for the UST Pay High

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School in the Intel Science and Engineering Fair Contest for the National Capital Region (NCR). The NCR is roughly all of the Metropolitan Manila area. She went on to compete in the Intel National Level Contest and won second place. But only the winner of the first prize goes on to the US to represent the Philippines in the International Intel Science and Engineering Fair. It has been said that when it rains, it pours. In 2004, after five years, high school students composed of an individual from Davaos Assumption High School and a group of three students from the Quezon City Science High School had once again worked on a project dealing with the marine luminous microbes.

Winning in the Intel science and engineering fairs

The first group of students that I took in who did their project in USTs new Thomas Aquinas Research Complex (TARC) was from the Quezon City Science High School. One of my favorite strains of luminous microbes was Vibrio fischeri USTCMS 1063, a brightly shining luminous microbe that I isolated from the fresh juices of squids. Vibrio fischeri is one of the safest of microbes that one can work with for a science project according to the

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Chicago Science Fair Board. When the students from the Quezon City Science High School composed of a boy and two girls named Jayson Reggie Obos, Melanie C. Melchor and Trina G. Napasindayao came to me one afternoon they wanted to work on disinfectants. It was indeed timely to work on disinfectants because SARS had just then ravaged Canada and Hong Kong. Using disinfectants should prevent the spread of infectious diseases like SARS and flu. At first, I turned down their request of supervising them simply because I find the project on disinfectants too common something that lacks novelty. Nevertheless, Jasyon simply will not give up and their collective persuasion and perseverance finally paid off. I finally took them in and they were granted permission to do their project in the Pure and Applied Microbiology Lab. However, instead of working with disinfectants, I gave them a different science project something that involves bioluminescence. A year before, I had worked with the use of microbial bioluminescence to measure the dissolved oxygen content or the degree of pollution of water and wastewater samples. In a screw-capped tube, a suspension of luminous microbes is mixed with the water sample to be tested. Once mixed by shaking and swirling, the liquid inside the tube shines uniformly with bluish-green hue in the dark. This novel method should enable bioluminescence to measure the degree of pollution in water

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samples. My findings have shown that the longer the luminance of the water sample inside the tube persist, the more dissolved oxygen there is in the water and therefore the less polluted it is. Polluted water contains small amounts of dissolved oxygen thereby sustaining the period of luminance, which needs oxygen, at a much shorter period. In non-polluted water where the amount of dissolved oxygen is high, the period of luminance of the liquid inside the tube should persist longer. I gave this project to the QueSci students, a nickname that they fondly call their school. The QueSci students were bright, articulate, and assertive enabling them to explain their work so extemporaneously. Surely, they have what it takes to win. They had full confidence and they were able to organize their thoughts rather quickly and speak it out with coherence and clarity. Rarely have I seen such qualities in students not even with most of my college students. Thus, this group from the Quezon City Science High School was given Vibrio fischeri USTCMS 1063 for their science project. It dealt with the simple and rapid measurement of dissolved oxygen (DO) in wastewater samples based on the length of time it takes for the luminance of the liquid inside the tube to black out. They collected water and wastewater samples from different areas in Manila and evaluated their degree of pollution using the method, which I called Tube Luminance Extinction. Using a calculator and their

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knowledge of basic statistics, they processed their data into a beautiful equation showing inverse relationship between the length of time of extinction of the luminance inside the tube and the degree of pollution in the water samples.

What a Grand Slam this project brought them. In a year they won several prizes in various science contests held in the Philippines. Their science project entry earned the QueSci students, the following national and international awards:

Best Science Project sponsored by the College of Engineering of UP Diliman for the countrys science high schools Best Science Project in the First National Science Fair Contest (2004) sponsored by the Department of Education (DECS) in Sta. Cruz, Laguna First Place in the Physical/Applied Sciences category at the Intel 2004 Regional Level Contest First Place in the Intel 2004 National Level Contest held at the Bahay ng Alumni, University of the Philippines at Diliman Intel 2004 Best Science Project Intel 2004 Excellence in Environmental Science Award

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 Cash Price from the US based Science News Magazine Fourth Place in the 2004 Grand Awards of the Intel International Science and Engineering Fair held in Portland, Oregon; USA.

Another group of three girls from La Consolacion High School in Manila used Vibrio fischeri USTCMS 1063 to determine the presence of residual chlorine in various tap water samples collected all over Metro Manila. Residual chlorine in tap water is an effective shield against the spread of water-borne diseases like typhoid fever, cholera and dysentery. Indeed using bioluminescence, they found out that residual chlorine is no longer present in almost all water samples collected in various places in Metro Manila. Water samples from Tondo had practically no detectable residual chlorine, which explains several outbreaks of water-borne diseases in that area. Several people died from these outbreaks caused by illegal water connections and leaking pipes. The La Consolacion students won first place in their schools contest and won as well in the National Capital Region (NCR) Division of the Intel Science and Engineering Fair Contest. Unfortunately, they failed to reach the Intel National Level Competition. I really thought that this project owing to its novelty and public health significance should have won in the national level. Probably the girls were

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simply not able to impress the judges with the impact of their research project.

A third group of four students from the Paraaque Science High School completed a project on the use of Vibrio fischeri for the rapid, simple and inexpensive way of screening our indigenous medicinal plants for the presence of antimicrobial compounds. The luminous microbes have been found from previous studies to be very sensitive to the action of antimicrobial agents in plant extracts. Indeed a less than one-percent garlic extract in water was found to be most potent in exhibiting antimicrobial activity when compared with several plants that have been tested. Onion, ginger, Malunggay and Lagundi were just some of the plants they found to possess significant antimicrobial activities based on the length of time it would take for the plant extract to extinguish the luminance of the microbes. The shorter the period of time for the luminance of the plant extract treated luminous microbes to fade out, the potent the antimicrobial activity of the plant extract is. They won prizes in the Intel regional science fair contest held in Alabang as well as in the first nationwide Science Fair Contest held in Sta. Cruz, Laguna sponsored by the Philippines Department of Education. Lastly, a girl from the Assumption High School in Davao made used of the marine

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luminous microbes to determine the water pollution level of environmental water samples. She won first place in the Intel regional level and went on to represent her region at the Intel national level competition.

One evening in 2005, while I was all alone in Microbiology Laboratory, the students from QueSci dropped by for a surprise visit to thank me for all the assistance. As a token of their appreciation, they left me their ISEF (International Science and Engineering Fair) pins from the contest that they just attended in Portland, Oregon. They relayed to me with so much excitement and happiness the wonderful experience of having been given the opportunity to represent the country in the Intel International Science and Engineering Fair (ISEF). It was remarkable for them to be in the U.S. at a very young age. What they had achieved was the kind of fairy tale contest adventure that millions of Filipino high school students can only dream of achieving. To have been given such honors and the privilege of representing their country in an international competition in America is for them the chance of a lifetime. Meeting thousands of other young participants in the contest from countries all over the world must have enriched and changed their outlook and ambition in life. Two of them went on to De La Salle University to pursue a degree in engineering and one went on to the

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Ateneo de Manila University to pursue a career in mathematics. They are really the future scientists and engineers who will someday take their control post and chart the course of science and engineering in this country and surely its direction into socio-economic prosperity. When they left me that evening, it was for me deja vecu! All of a sudden, I was transported back to the year 2002 my annus mirabilis and relived once more in my thoughts the thrill of victory, of what it felt like to win the Gold in the Young Inventors Awards. The fond memories of receiving my award at the top of Hong Kongs tallest building The Central Plaza and the trip to Silicon Valley in California to visit the HPs headquarter and the Nanotech Laboratories came back to find me wistfully longing. The year 2004 was truly for these QueSci students what 2002 was for me. It was a year that they and their school will always look back to with so much nostalgia and pride; a year that they will always reminisce with so much happiness in their lifetime. Through it all, my research work on the marine luminous microbes have been a great blessing to me and to many of the younger generation of Filipino scientists. Every now and then, I still received queries on bioluminescence from students coming from the Philippine Science High School, the Manila Science High School and from the other science high schools. I still get a feeling of amazement and gratitude when I am alone with my luminous microbes in

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the dark. They can surely heighten ones awareness of what it is like to be alive. Seeing and feeling the cool bright bluish-green light that they generate is also their way of telling me how happy they are to be alive as well. A speck of life has indeed made contact with another more advanced life form through the gift of light and the complexity of vision. Through it all, I am confident and secured in the notion that whatever the future holds, my Microbes of Light will continue to shine a path that will guide me to new and rewarding endeavors. Bioluminescence, the light of life, coming from whatever organism, be it a protist, a copepod, a firefly or even a fish, will continue to inspire our countrys young brilliant minds to be analytical and innovative; bold and daring and to explore the great and the small! Indeed, Vibrio fischeri is teaching us lessons in green that can be used to safeguard our, the environments and the planets health. Very recently, I supervised a group of three boys from Espaas Ramon Magsaysay High School on their own original special project. They worked on the effect of different wavelengths in the drying of guava leaves on its antibacterial activity. I am glad that they won in the NCR and went on to the National Level. Recently, a group of students from the Manila Science High School visited me and told me the good news that their science project won several awards in the 6th Regional Congress: Search for SEAMEO Young Scientists

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(SSYS) Awards held last 3 6 March 2008 at the SEAMEO Recsam, Penang Malaysia. Emmanuel D. Delocado, Justine Timothy P. Cruz, Jose Noel Gamba, Edilberto Barcelona and Mr. Jonathan P. Derez their adviser won 2nd place in the Science Category 1 for Best Project Exhibit and 3rd Place for Science Category 3 for Best Presentation for their project entitled Bioluminescent Bacteria (V.fischeri and V. phosphoreum) To Direct A Killing Mechanism on Leukemia Cells. The group of Emmanuel Delocado was indeed so generous to give me a cake, a key chain from Malaysia and the souvenir program of the 6th SEAMEO Regional Congress.

The key chain from Malaysia given to me by Emmanuel Delocado

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When Emmanuel Delocado inquired on Bioluminescence a year earlier he already had in mind a clear idea as to what he wants to do. He wanted to use the energetic blue-green light to kill cancer cells. Indeed, I was skeptical of the projects success. I provided him the luminous microbes and he used it the way he intended to. The rest was history. He came back to me with several awards to be proud of. His group also won 2nd place in the Intel Science and Engineering Fair at the National Level held at UP Diliman. They went on to pursue biology at UP Diliman and Ateneo de Manila University.

The contest where the group of Emmanuel Delocado from the Manila Science High School won with their project on the use of Vibrio fischeri

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Participants pins for the Intel Science and Engineering Fair 2004 held in Portland, Oregon given to me by the students of QueSci

A photograph taken in the dark of luminous agar plates of Vibrio fischeri USTCMS 1063 as single letter/number proclaiming the Quadricentennial celebration of UST in the year 2011. Picture was taken during the day of the launching of the University of Santo Tomas Collection of Microbial Strains (USTCMS) in the Thomas Aquinas Research Complex.

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From all the years I had serving as supervising scientists to students coming from various high schools , I can reckon that 9 groups in one way or another were granted the privilege to work in the Microbiology Laboratory of the Research Center for the Natural Sciences (RCNS). Seven made it to the regional level of the Intel Science and Engineering Fair competition. Five made it to the national level held for many years now at the Bahay ng Alumni in UP Diliman and one made it to the US and won a grand award.

Below is a picture of the famous HP Garage bearing a historical marker with the inscription Birthplace of Silicon Valley and meeting the winners of the prestigious US Collegiate Inventors Contest. From left D. Doshi (Univ. New Mexico), D. Fletcher (Stanford), S. Welz (Univ. Chicago), D. Perkins (Univ. Maryland), myself and M. Oddy (Stanford)

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Chapter II: Of Light

A light shines in the darkness and the darkness overcame it not New Testament

The nature and habitats of the marine luminous microbes Bioluminescence is an awe-inspiring attribute manifested by a wide array of terrestrial and marine organisms like fishes, squids, insects, crustaceans, fungi, and microorganisms. It is more prevalent among the marine organisms were it is employed to either attract mates and potential preys or distract enemies and their predators. The species of the marine luminous microbes are ubiquitously found in seawater as well as in association with many of its inhabitants: plants and animals, of this vast realm known as the hydrosphere. Indeed fishes, squids, crustaceans, shellfishes, as well as seaweeds have been known to harbor luminous microbes. The marine luminous species are classified into three microbial groupings or genera: vibrio, photobacterium and alteromonas. However, another microbial group Xenorhabdus, a genus whose member species is Xenorhabdus luminescens, is not marine but terrestrial in nature. Terrestrial means that the member species are found on land and infects a specific type of nematode (worm). Strains that I have isolated and preserved in the USTCMS are identified as species of Vibrio harveyi, Vibrio fischeri and photobacterium leiognathi. Since these microbes are of marine origin, their
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growth medium whether in solid or in liquid form must contain salt at a concentration of three percent by weight of salt per volume of water. This three percent concentration of salt required by the marine luminous microbes simulates the salt content of seawater their main habitat. Without the required amount of salt in their liquid world, these marine microbes will die instantly. Plain water exerts a phenomenon known in biology as hypoosmosis to the cells of these halophilic or salt-loving microbes. Hypoosmosis simply means that there are more dissolved substances in the liquid inside the cells than in the liquid outside the cells. This imbalance in concentration of dissolved substance with more inside and less outside the cell will cause water outside the cells to exert a net uni-directionally movement into the cell causing it to swell gradually and subsequently to burst and die. However, when salt is dissolve at a final concentration of three percent in the liquid medium outside the cells where they are suspended they neither swell nor shrink in size. This biological phenomenon is known as isoosmosis indicating an equality in the amount of dissolved substances found inside and outside the cell. Since the concentration is equal, the amount of water entering the cell is equal to the amount of water leaving the cell thereby achieving a state of structural equilibrium and viable integrity for the cells. The net movement of water molecules into and out of

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the cell is zero in isoosmosis. Cells in isoosmotic liquid medium live and grow normally. The strict dependence on salt and the non-disease causing nature of these luminous microbes, particularly Vibrio fischeri and Photobacterium phosphoreum and Photobacterium leiognathi on humans make them suitable and safe test microbes for many biological experiments that can be developed into sound and original science projects. Washing these microbes from the tap is enough to kill them thereby precluding their ability to spread and multiply in the sewer system. Employing sound microbiological pure culture techniques, it will be easy to isolate these marine luminous microbes from squids and all salt-water fishes. The brownish fecal contents of the intestines of fishes that can be squeezed out easily by the fingers, the juice that readily drips out of the bodies of squids and the sticky and slippery bodily surfaces of saltwater fishes are good and rich sources of the luminous Vibrio and Photobacterium species.

How does Vibrio fischeri look like?

Vibrio fischeri and their kinds, the marine microbes, were most likely one of the very first living organisms to debut on earth more than three and half billion years ago. To get a very good picture of this enormously

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incomprehensible time scale, I used the one-year analogy that I first learned from Carl Sagan in his TV show: Cosmos. Let us compress the billions of years that the microbes have been on earth in a span of one year and set the time of the microbes emergence on earth at 12 oclock AM of January first New Years day. In this geological time-scale, the emergence of humans is registered at 3 oclock PM in the afternoon of December 31, on the last day of the year. Microbes have been on earth from January to December while humans have been on earth only in the last 9 hours of the year. In short, based on this one-year scale, the microbes have been on earth much longer than we humans have been on earth. Vibrio fischeri, like any other microbe, leads a very simple life lacking all the complexities associated with the structure and functions of higher plants and animals. Because of their lifes simplicity, microbes are robust survivors. They have lived through several waves of major extinctions and global destruction that struck planet earth in its long history of evolutionary development. These cataclysmic events wiped out ecosystems and communities of living organisms forever in the past. The Vibrios are roughly short and bent rod-shaped single celled organism with an outer envelope composed of fat-carbohydrate-protein mixture. Vibrio fischeri has three to seven whip-like structures conspicuously protruding out from one end of its body. These whip-like structures are

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called Flagella which they use for moving rapidly like a torpedo in their watery world. The rigid flagella rotate and they get propelled. Because of these flagella Vibrio fischeri is one of the fastest swimming microbe known. Inside their plump rod bag-shaped bodies are complex interacting biological structures and many biomolecules that sustain life and subsequently produces bright light. The whole cell converts large amount of biochemical energy into radiant energy, causing it to light up like an incandescent bulb whenever they acquire the critical population needed to express bioluminescence. Bioluminescent cells consume a large percentage of its ATP (Adenosine Triphosphate), an energy chemical currency, into radiant energy. Hereditary units called genes are found inside their cells carrying the genetic information coding for the very essence of their own biological identity and the capability to reproduce and propagate their kind. This biomolecule functioning as carrier and repository of genetic information is called Deoxyribonucleic Acid or simply DNA. DNA is a very long fibrous polymer made up of billions of building blocks called nucleotide. Genes or coding portions of DNA are attached together in tandems separated by segments of non-coding regions of DNA in highly coiled and tangled fibers called chromosome. In addition to chromosomes found inside the cells, there are other numerous specialized biomolecules called enzymes.

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Enzymes perform specific functions like making DNA and proteins essential for the maintenance of life. Enzymes are the protein engineers and builders of the cell; they speed up the building up and breaking down of biological structures. All the chemical reactions that take place inside the cell are referred to as metabolism. Some chemical reactions are involved in the breakdown of chemical compounds like simple sugars and fats to provide energy. For instance, glucose is broken down in the presence of oxygen primarily via a gradual combustion process to produce carbon dioxide, water, and energy. The oxygen requiring process of the breakdown of chemicals like sugars inside the cell is accompanied by the release of a tremendous amount of energy. This breaking down of chemical substances into simpler substances that usually provide energy is called catabolism specifically respiration. On the other hand, some reactions that lead to the building up of large substances like cellular proteins, nucleic acids, carbohydrates, and fats comprising the very structure of the cell itself require energy. This building up process that requires energy is called anabolism. Catabolic reactions provide energy that fuels anabolic reactions to proceed to completion. A tough external covering called the cell wall encloses microbial cells. It protects and maintains the shape and integrity of the cell. Located beneath this tough outer covering is a more fragile cell film called

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the membrane. It defines and delineates the cell into the intra from the extracellular region and is responsible for transporting inwardly the dissolved nutrients like sugars and peptides that it meets in their liquid vicinity. Microbial cells consume food for growth and reproduction and excrete out waste products like organic acids and carbon dioxide. Organic acids and carbon dioxide are the products of their metabolism.

Each cell shown below by a simple drawing reproduces by dividing into two daughter cells after a time interval through a process called binary fission. Each of the two daughter cells ultimately become parent cells themselves dividing further again to give rise to four daughter cells and then to eight and so on. The length of time required for a Vibrio fischeri cell to reproduce itself, known as the generation time, is roughly twenty minutes long. Before one knows it, a single cell will have given rise to more than a million progeny cells after one day or twenty-four hours. It is vital that every cell of Vibrio fischeri need to be placed in a three-percent saline to live well and happily. This is due to the marine home of these microbes. If they are suspended in water without substantial amount of table salt like tap water or distilled water, they will die instantly. Without salt, water will swiftly enter

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their cellular body causing it to swell like an inflating balloon and ultimately bursting.

A Single Cell of Vibrio fischeri can have 3 to 7 prominent whip-like appendages called flagella used for locomotion

A photograph of a 1,000 times microscopic picture of the cells of Vibrio fischeri USTCMS 1063 shown as small blue oval-shaped structures occurring singly and some in pairs

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Where is Vibrio fischeri found? The marine luminous microbes particularly Vibrio fischeri are universally found in seawater where they exist in planktonic and free-living forms. Planktonic means that the individual cells of the luminous microbes move with the flow of the sea current and free-living means being able to live on their own initiatives independent of the intervention of other organism. Vibrio fischeri from its Binomial Latin name describes vibrio, a bent, short, and rod-shaped microbe found on the external surfaces and in the intestines of saltwater fishes, squids, and crustaceans. Thus, the second name fischeri referring to the saltwater fishes. Even Hollywoods Nemo should harbor luminous microbes on its body and in its tummy. The marine luminous microbes can establish various types of biological relationships with many marine animals like mutualism, commensal, and parasitism.

Where my Vibrio fischeri USTCMS 1063 was isolated. Inky black liquids from squids is a rich source of marine luminous microbes

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Some luminous microbes have evolved to become the component of the light organs of fishes and squids exhibiting shared benefits between fish and microbe called mutualism. I remember a news article several years ago in the Philippines reporting a group of fishermen from Negros Island seeing large bright illuminations deep in the sea at night. Many of the fishermen believed then that these undersea rapidly moving illuminations are UFOs or more appropriately USOs (Unidentified Swimming Object). Only to be explained rationally by marine biologists from the prestigious Silliman University that these undersea illuminations seen at night are large schools of lantern fishes. This fish possesses light organs scattered all over its body and swimming together in large numbers called schools can give them collective brightness in the sea. At night a school can shine brightly together thereby producing large moving undersea illuminations.

American

microbiologists

discovered

an

astounding

symbiotic

relationship between the luminous microbe Vibrio fischeri and the small bobtail squid found in the Hawaiian seas. The luminous microbes get food and shelter from the squid. On the other hand, the small and cute squid is rendered invisible by its luminous microbial symbiont thus preventing it from ending up in the belly of some huge hungry fishes. In the sea, the Bobtail
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squid uses the symbiotic luminous microbes to illuminate its body when the moon above is also bright. This ingeniously evasive action by the Bobtail squid erases or cancels its own shadow completely and like Harry Potter with his magical invisible cloak disappears from the sight of voracious predators that may be swimming beneath it. Isnt that neat; a squid that can make itself invisible to hungry eyes! However, in the abysmal pitch-black depths of the oceans of the world where the sperm whale battles the giant squid the opposite effect occurs. National Geographic tells us that at this depth where no light from the sun much less the moon ever reaches, the location of the giant squid is rendered visible to the sperm whale by the light that the squid emits through the associated marine luminous microbes.

Photo of the small and cute Hawaiian Bobtail Squid (From Internet:www.ou.edu.)

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The luminous microbes are in significant quantity found as commensal in the gills and on the skin of marine fishes where they become an integral part of the existing surface layer of numerous microbial communities. Commensalism is a relationship between two organisms in which one derives advantages like food and shelter without harming or benefiting the other. The marine luminous microbes are abundantly found in the intestinal content of salt-water fishes comprising as much as 90% of the aerobic microbial population. The microbes exist as commensals for the saltwater fishes and other marine animals. This is exactly the main source of my isolated luminous microbes when I first discovered them after I got back to the Philippines from Germany. The juices, surface slime, and gut content of saltwater fishes and squids are rich sources of the commensal marine luminous microbes.

Some species of luminous microbes particularly Vibrio harveyi can be parasitic on crustaceans. Parasitism is a relationship between two organisms in which one gets the upper hand and benefits from it to the detriment of the other. Vibrio harveyi, a cousin of Vibrio fischeri elaborates chitinase a protein product that helps in attacking crustaceans like shrimps and prawns. This serious disease inflicted on crustaceans is called luminous

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vibriosis. Chitinase dissolves the hard and glistening chitinous outer covering of crustaceans called exoskeleton allowing the microbe to gain entry inside their body and start the process of infection. Luminous vibriosis is a big problem for the prawn industry.

Marine animals like fishes, squids, and prawns are goldmine of the marine luminous microbes of which the Philippines and other coastal regions are abundantly blessed

Lastly, these microbes are also saprophytic meaning they clean up the marine environment. Like many other microbes, they are involved in the natural decomposition of organic matter and speed up the process of decay thereby maintaining the cleanliness of the marine environment.

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The Milky Sea Seafarers of ancient times have reported rare phenomenon in which large areas of the sea light up or glow brightly at night. Cruising through this shining sea has been likened to being in the midst of a sea of white much like milk that extends all the way to the horizons. The glow is actually blue but may appear white to human eyes. Based on its whitish apparition, this rare occurrence in the sea is known in scientific literature as the Milky Sea. Marine luminous bacteria undergo a population explosion, a bloom, made possible by still hitherto unknown physico-chemical conditions. When their population becomes very dense, the individual cells shine by virtue of quorum sensing and collectively the entire seawater as well. From a few hundred cells per liter of non-milky seawater, the cell count can go up into the trillions for the milky seawater. It must be a very exhilarating experience to be inside a Milky Sea which even mentioned by Jules Verne in his famous book: 20,000 Leagues Under the Sea. Recently, Steven Miller of the U.S. Naval Research Laboratory was able to confirm 1995 Satellite images taken by a merchant ship. U.S. Defense Meteorological Satellite Program was that of a large milky area. The Milky Sea, located off the coast of Somalia, covered an area of 15,400 square kilometer a little bigger than Samar Island. It brightly lit up this part of

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the Arabian Sea for three consecutive nights in 25, 26 and 27 January of 1995. From my experience with luminous microbes, they will have to adhere together on some solid surface. Immobilization causes them to shine much longer than if they are suspended in seawater. But in all the times that I have observed these luminous microbes in the dark, there was no instance when I saw them shining white. All the luminous observations I made with them in darkness even under scotopic vision they shined with a stable blue-green light.

Cultivation of the luminous microbes on a solid culture medium

The use of a solid medium for the cultivation of microbes goes all the way back to the pioneering effort of a famous German microbiologist and Nobel prize winner named Robert Heinrich Koch in 1882. His techniques and the lab glass wares he used are still very much utilized in microbiology today. Kochs significant contributions to microbiology and to modern medicine have been recognized so well in Germany and in other countries that streets, hospitals, and research institutions were named after him. Microbes like humans need nutrients like soluble proteins and carbohydrates to sustain growth and development. Growth for humans is associated with

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an increase in size. Among microbes, it is manifested by an increase in numbers or in population size over a period of time. Soups or broths are prepared by boiling chunks of beef or pork or chicken in water resulting in a liquid that is rich in soluble proteins, vitamins, and minerals. If soups or broths are left unattended for long exposed to the air at room temperature they will spoil rapidly. Spoilage indicates that microbes have gotten into the soup from air-borne dusts or from the non-sterile bowl containing the soup and have grown tremendously into the millions of microbial cells per drop of the soup. The microbes have eaten up the nutrients in the soup and subsequently secreted out waste substances into their surroundings. These waste substances accumulate rapidly because of the booming population of microbes causing the development of undesirable changes in the properties of the soup. The soup after a day or two becomes turbid, smells foul, and tastes bad, which are all signs of microbial contamination. In microbiology, a solid medium is called Nutrient Agar and a liquid medium is called Nutrient Broth which are commonly used for the cultivation of microbes. Nutrient agar is composed of a broth and a solidifying agent, usually agar. Agar is a complex carbohydrate extracted from the red marine seaweed that possesses gelling and stabilizing property. It is employed in desserts to prepare colorful and delicious solid matrices containing embedded pieces of

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fruits. Nutrient agar is prepared as a hot yellowish liquid of broth that hardens once it cools down. If marine luminous microbes are to be cultivated, there is also a need to add sodium chloride at an amount of three percent in the nutrient agar before they are allowed to solidify. Microbes grow inside the nutrient agar or on the surface as visible round objects called colonies. Traditionally soups or infusions of cow or pigs meat, heart and brain mixed with agar are the ones prepared as solid culture media used for the cultivation of microorganisms. At present, microbes food has become highly sophisticated and now comes in powdered dehydrated form of many kinds for special purposes. The light yellowish powder of the commercially prepared microbes food, looking much like infant formula, must first be weighed, added to a specified volume of distilled water, heated to dissolve and sterilized at high temperature. Sterilization kills

microorganisms that may have already been present in the nutrient medium or may have entered it during the preparation method. After the sterilization process, while still warm and in the liquid state, nutrient agar is poured into a Petri dish. A Petri dish is an enclosing pair of glass dishes. The bottom shallow glass dish is enclosed loosely by a cover glass dish. Once poured inside the bottom glass dish, it is closed with the cover glass dish and the culture medium inside is allowed to solidify as it cools down to room

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temperature. The entire procedure in the preparation of culture media is much like preparing fruit flavored gelatin. A fixed weight of the powdered gelatin product contained in a foil sachet is added to a defined volume of water; mixed by stirring; heated to boiling to dissolve the contents; poured while still warm and liquid in specially shaped plastic containers called moulds and allowed to solidify upon cooling. Next step in the cultivation of microbes requires the use of a metal wire called an inoculating loop or simply a wire loop. It is a nichrome or platinum wire about a third of a foot long in which one end is closed into a loop the size of the tip of a pen. From the opposite open end of the wire loop, a metal rod is attached as a handle. This special device is used for transferring microbes from their sources to any solid or liquid nutrient medium. The source can be a culture medium already harboring trillions of actively thriving microbes or it can be a soil suspension, saliva, mucus secretions, or dirty water that is known to contain microbes by the millions. For the marine luminous microbes, their sources are juices, the surface slime, and the gut content of salt-water fishes.

A Wire Loop, with handle at its left end and a loop at its right end

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While holding the handle of the wire loop much like holding a pen, the other circular end called the loop is touched or dipped in materials called sources deemed to contain large numbers of microbes. The loop now containing millions of clinging microbial cells can be introduced inside a nutrient agar plate. Removing the upper glass cover of the petri dish exposes the solid nutrient agar inside the bottom glass dish. The loop by now contains millions of clinging living microbial cells. It is then touch gently on the surface of the exposed nutrient agar and at once streaked repeatedly on the face or surface of the nutrient agar moving from side to side of the glass dish until the entire surface has been streaked. After the streaking procedure, the cover dish is used to close the petri dish. Covering the petri dish ensures that unwanted microbial contaminant from the immediate surroundings like air dusts does not get inside the petri dish where the nutrient medium is found. Using the method above, called pure culture technique, assures that only the microbes coming from the wire loop will be the ones that will grow inside the petri dish.

Looking carefully on the nutrient agar inside the petri dish, no noticeable change on the surface of the nutrient agar after the streaking procedure can be seen. But there are actually several thousands of microbial cells

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scattered on the surface of the nutrient agar that are simply too small to be seen. But given the time for these single cells scattered on the nutrient agars surface to grow and multiply to two cells and the two to four cells and then to eight cells and so on. A single isolated cell, after a day or two, would have given rise to millions of daughter cells all piled up together into a visible mass called a colony. For the luminous microbes, each of the small round colonies will be glowing bluish-green in the dark due to bioluminescence. A period of one to two days is just right length of time for these colonies to render themselves visible to the unaided human eye. All the millions of cells found in a single colony are clones, replicas, or mirror images of the very first single progenitor cell that gave rise to all of them by continuous and rapid binary fissions. A cell undergoing binary fission enlarges and then split into two cells. We really cannot view each cell of the millions of cells making up a colony or a population called culture as truly distinct like each human on this planet is unique. The individual identity of a single microbial cell simply does not exist since the cells making up a population are all identical to one another. It therefore makes the entire population act as one and renders it as a single organismic entity. Most microbes are prolific; they can grow very fast in a short period. Vibrio natriegens, a close non-luminous cousin of Vibrio fischeri has a generation time or doubling time of only ten

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minutes. Compare that to the humans generation time or doubling time measured in years. A microbes generation time is more than a million times faster than humans generation time. This short generation time allows them to mutate or change themselves faster allowing them greater flexibility in adapting to any alterations in their environmental conditions. And adapting they did! Not only have microbes persisted for billions of years; they even survived the cataclysmic events in earths history that have caused waves of large-scale extinction of plant and animal life. Today, mans activities have caused the extinction of many animals like the Dodo and the Tasmanian Tiger but has not yet caused the extinction of any living microbe. The world would probably be a better place if the extinction of microbes like Mycobacterium tuberculosis or Porphyromonas gingivalis can ever be achieved.

Photograph of numerous brightly luminous colonies of Vibrio fischeri growing on nutrient agar inside a Petri dish against the slit of a door

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Cultivation of the luminous microbes in a liquid culture medium A liquid culture medium is a soup or broth that retains its fluidity even at room temperature simply because no solidifying agent is added to it. The wire loop having millions of clinging microbes coming from a source is simply dip into the liquid culture medium contained in a glass vessel like a test tube or a flask. Once dipped, the cells from the loop are dispersed and suspended in the fresh liquid nutrient medium where they can grow and multiply. Right after dipping the loop in the broth you will not see any observable change in the liquid. But again given the time, the initially introduced cells in the culture medium grow and multiply into the trillions. After a day, the liquid will turn cloudy and turbid indicating the presence of millions of microbes per drop of the liquid. Once again, if the cells are of the marine luminous microbes, salt will have to be added to the medium. Growing to large a population will ultimately cause the medium containing the marine luminous microbes to instantly shine bluish-green in the dark after several hours.

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Microbes that can talk with one another

Individual cells of a population of Vibrio fischeri are able to communicate with one another by using a chemical signal. Microbiologists have isolated and identified the chemical signal that luminous microbes used for cell to cell communication as homoserine lactone. This phenomenon is called Quorum Sensing. Individual cells are able to know or sense how big their population is; based on the amount of the homoserine lactone that is found within their immediate vicinity. When the cells of luminous microbes are introduced into a fresh nutrient medium, the individual cells that are found in small number dont light up just yet. The cells are all dim and are spaced f ar apart from one another. But as the cells reproduce and the population rapidly increases in number, a point is reached wherein all the cells light up at the same time causing the population as a whole to shine brightly. It is much like a star that explodes radiantly into a supernova outshining the entire galaxy where it is located. It has been estimated by microbiologist that this critical mass of cells that must be attained to make the entire population shine brightly is about 50,000,000 cells per drop of the liquid containing them. That is indeed an astronomical number!

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For us humans we are able to know how many we are in a place by talking to one another and doing a roll call. For the luminous microbes they are able to know their population size or sense their quorum by talking to one another through their unique chemical signal. The concentration of the signal homoserine lactone in the liquid medium accumulates and goes up when the population increases in size as well. The larger the population, the more cells are present all producing and secreting the chemical signal into their surrounding environment. The space between cells becomes smaller as they increase in number and the concentration of homoserine lactone goes up as well since more cells are making it and secreting it out of their cellular bodies. The rising concentration of the chemical signal in the liquid medium will soon bathe each of the millions of cells making up the population, ultimately bringing about the switching on of special group of genes in their chromosomes called lux found inside each cell. Lux is an appropriate term since in Latin it means Light. The high concentration of Homoserine Lactone in the medium will cause it to enter each cell in the population at the same time and switch on the lux genes of individual cells causing them to instantly shine brightly. It is much like switching on the lights of the large and tall Christmas tree in the Big Apple, all the bulbs lighting up

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at the same time once it gets plugged in. The lux genes found in each luminous microbial cell govern the expression of the bioluminescence trait.

The identification of the marine luminous microbes

As part of my research work in UST, I am constantly isolating more strains of marine luminous microbes. The luminous microbial holdings of the USTCMS will continue to increase and indeed boasts of being the biggest in the Philippines. Classification and identification of the marine luminous microbes and other microbes require isolating them in pure form and subjecting them through a series of scientific investigative process resulting in the assignment of new binomial names to each of them. This investigative process studies the characteristics or attributes of the microbial isolates in terms of their microscopic appearance, appearance as colonies, kinds of nutrient on which they can grow, physico-chemical conditions affecting their growth, innate abilities to transform specific chemicals, information found in their DNA and their habitats. Results for unknown microbial isolates are compared with that of a known or a previously identified microbial species as well as information contained in an authoritative book called the Bergeys Manual of Determinative Bacteriology. High degree of similarities in the

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studied characteristics obtained for an unknown isolate with a known microbial strain indicates that they are of the same species. However, dissimilar characteristics between unknown and known microbial strains are indicative that they are not the same and therefore belong to different species. If an unknown isolate have characteristics that does not jibe with a known strain that has been reported in scientific journals and technical books then the unknown strain must be a new or a novel species. Finding a novel species is actually like having discovered a new form of life much like discovering a new plant or animal. In biology, the discoverer has the privilege and rights to give the novel species its new name, a binomial name composed of a genus and species by which it is made known to all. Relying on the very rich diversity of marine living organisms found in our country, it is indeed a matter of time before novel indigenous species of marine luminous microbes will have been discovered. If I were to fine one, I will surely name it Vibrio manilanensis or Vibrio philippinensis in honor of our country! Application of a holistic approach on identification, like polyphasic characterizations coupled with the information that can be derive from the 16S rDNA sequencing should facilitate the attainment of this noble goal. Known luminous species in the genus vibrio are Vibrio fischeri, Vibrio

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harveyi,Vibrio logei and Vibrio vulnificus. From the genus photobacterium, Photobacterium phosphoreum and Photobacterium leiognathi are luminous.

The chemistry behind bioluminescence

Microbial bioluminescence rendered visible as shining colonies in agar plates or as glowing liquid broth culture is truly the most awe-inspiring phenomenon that microbiology can unravel before human eyes. Imagine living organisms generating their own cold bright bluish-green light in the dark. All life processes are possible and sustained by getting energized electrons in molecules found in foods called carbohydrates. These energized electrons from carbohydrates are brought inside the cell and once inside, the energy that they possess is removed causing them to be deenergized. The energy that removed from the electrons is needed to sustain and maintain the life of all cells. Living organisms will die if not provided with these energized electrons into their cells, which come only from the foods consumed. Carbohydrates are describe as the main source of energy for living organisms precisely because they are the major source of energized electrons. These substances are also the most abundant type of food found in the biosphere. However, on a weight-to-weight basis, fats and oils contain

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far more energized electrons than carbohydrates. In all living systems, the energy removed from the energized or activated electrons is use to produce a universally known energy molecule known simply as ATP. ATP is a versatile molecule that can be used to either withdraw energy from when needed or store energy into when there is an abundance of it. It is like money that can be withdrawn from banks when needed and can be deposited in banks if there is a surplus.

The movement of the energized electrons in the process of light production (bioluminescence) is actually an alternative pathway or a detour (Shunt) of the electron transport chain or respiratory chain. This lightgenerating metabolic process is the responsibility of the enzyme Luciferase, an enzyme present as well in other bioluminescent organisms like firefly. Luciferase accepts activated electrons from a special molecule coming from their food. The energized electrons instead of being passed on to the cytochromes the usual terminal route of the traffic of the energized electrons in respiration, are instead rerouted to the enzyme Luciferase, which orchestrates the bioluminescence reactions. Luciferase carrying the highly energized electrons passes them on to oxygen, where the energized electrons are stripped of their energy thus ending up as low energy electrons

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bound tightly in water. The energy difference between the initial energized electrons and the final de-energized electrons is emitted as the cold bluishgreen light that is visible as bioluminescence, the gift of light.

Humans also get energy in practically the same way as described previously for the luminous microbes. Electrons coming from high calorie foods like sugars, bread and French fries are in the activated or energized state. Once these foods are eaten the activated electrons are transported inside our cells by special molecules where they are then stripped of the energy they carry. The energy removed from them is utilized to power life processes like thinking, walking, talking, and breathing. The once energized electrons, now devoid of energy, go down to a lower energy level and taken up by oxygen that ends up as water produced in cells. To prevent the gain of weight through the consumption of large quantities of fats and

carbohydrates, one can burn off these excess calories through rigorous workouts performed called aerobic exercises. Exertion is a form of burning off the excess calories or chemical energy through heavy aerobic or oxygenrequiring workouts. Breathing gets deeper because of the need for more oxygen from air by our respiring cells. Heat is produce in our body at the peak of these strenuous exercises resulting in the production of massive

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amount of perspiration. This oxygen-requiring metabolic process of burningoff excess calories is called Respiration. Water comes out of our skin dripping profusely and evaporates rapidly into the surrounding air taking with it the excess heat that the body has produced. This cooling effect is important for human survival for without perspiration and water evaporation; heat is trap in our body causing body temperature to rise rapidly to dangerous levels causing irreparable damage to tissues and organs. Respiration is similar to bioluminescence; both processes need oxygen to extract the energy from electrons present in high energy or calorie molecules. However, in respiration, the energy obtained from the electrons is released as heat while in bioluminescence, the energy is released in the form of visible light. In physics, light propagates itself through space in the form of waves. The length of a wave from crest to crest is its wavelength and wavelength determines the color of the light that humans can see. The light produced by luminous microbes has a wavelength of approximately 490 nm making the radiation appear bluish-green to our sight. The abbreviation nm stands for nanometer, which is a very small unit of measurement. A nanometer is a unit of measurement for length; a ruler for instance contains 305,000,000 nanometers. A light with a wavelength of 410 nm will appear

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violet and 700 nm appears red in color. The overall reaction for the bioluminescence of the marine microbes is shown as follows:

Reduced Flavin Mononucleotide (RFM)

+ Oxygen + Long Chain Aldehyde (LCA)

ENERGY (LIGHT)
Oxidized Flavin Mononucleotide (OFM)

+ water

+ Long Chain Fatty Acid (LCFA)

The bioluminescence reaction is not complicated at all; it can just so easily be likened to a game of basketball. The terms above, in the first row, are the players in a basketball game and the ball is the electron. The energized electron or the ball initially comes from LCA, a high-energy biomolecule. LCA then passed on the ball to RFM. RFM accepts the ball and passes it on further to oxygen. Oxygen accepts the ball and holds on to it permanently. In the process, oxygen is immediately converted to water located in the second row. The electron in waters possession is in the deenergized form. The energy removed from the initially energized electron is emitted as the visible cold bluish-green light of bioluminescence. LCA is converted to LCFA as it gives up the electron to RFM. And RFM is converted to OFM as it loses the electron when it passes on the ball to oxygen. Oxygen is the final electron acceptor and it gets transformed
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immediately into water. The electron now deprived of its energy ends lockup in water. The LCFA can be broken down into several two-carbon long chemical compounds by a special pathway known in biochemistry as betaoxidation. The two-carbon long compounds can be fed into a terminal cyclic oxidative pathway called the Krebs Cycle in biochemistry where it is converted to carbon dioxide and water and the production of more energy in the form of ATP.

Focus your attention on oxygen and water (bold terms), it can be seen that bioluminescence, the generation of light, depends on the availability of oxygen. If the amount of oxygen in the reaction is restricted compared to the other the upper row chemical reactants, then the bioluminescence reaction can be made to measure the amount of available oxygen in a liquid medium where the luminous microbes are found. More oxygen translates into longer sustained periods of bioluminescence and less oxygen means shorter periods of bioluminescence. This is the principle of the award-winning project of the QueSci students. Vibrio fischeri and the other microbes made their debut on earth billions of years ago, three and a half to be exact, in a world that is totally different from what it is now. They were the very first living organism that appeared

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on earth at the dawn of time; only after around a billion years after earth was formed as a planet in its fiery cauldron of birth. The microbes were very much ahead of the emergence of the dinosaurs, the fishes, the plants, the trilobites and all the other groups of living organisms existing today. The world in which they originally found themselves is a world totally devoid of oxygen where processes so seemingly natural as burning and combustion cannot and do not happen at all. Fire simply does not exist. The absence of oxygen in this primeval world was created by the massive geological upheaval that imprisoned gaseous oxygen. Oxygen was bound forcefully in gaseous compounds in the atmosphere, in liquid water with hydrogen and in the solid form of hard rocks and minerals that form the earths crust. An atmosphere without gaseous molecular oxygen thus tenaciously acquired the initial stronghold and blanketed the world with such endurance that it lasted for something like 80% of earths history. Using Carl Sagans analogy of compressing in a one-year period, the history of planet earth, then that would mean that the atmosphere was devoid of oxygen (anaerobic) from January to October and attained its present aerobic condition only in November to December. What caused the atmosphere to change abruptly on a global scale? Something momentous must have occurred that altered the course of planetary evolution forever. Some of Vibrio fischeris relative

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microbes were able to develop and acquire a green, light-reactive pigment. The green pigment enabled them to become the first producers; capturing energy from sunlight in an instant and utilizing it to convert a gas carbon dioxide and water into carbohydrates, food for all. That is the main reaction. However, a side reaction was able to split water into hydrogen and oxygen. This made the big difference in atmospheric evolution. In short, this special group of microbes was able to invent an essential biological process called photosynthesis. Photosynthesis enabled the chlorophyllous

microorganisms to virtually conquer the planet and vanquished the other microbes simply by religiously producing oxygen. Microbes that remained strict anaerobes cannot tolerate oxygen and were killed by its accumulation in the atmosphere. The photosynthetic microbes are known as the Bluegreen Algae, Blue-green bacteria or Cyanobacteria and from them descended more complex type of algae and ultimately all modern plants existing today. A whole new kingdom was born, the realm of the green whose members possess the light-sensitive pigment - chlorophyll. The chlorophyllous organisms performed photosynthesis with fidelity for billions of years of their existence on this planet changing the world completely and eventually setting the stage for the emergence of another Kingdom - the animals. Oxygen accumulated in the atmosphere converting it from being

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anaerobic to aerobic. Ozone, one of the chemical manifestations of oxygen, was formed high up in the sky shielding the earth from harmful extraterrestrial radiation. This important shield paved the way for the eventual colonization of the land by living organisms. Life, which originated in the sea finally ventured into land and it started an entirely new way of living on the continents. Life previously marine has now become terrestrial. Fire too finally showed up in the world; a sign that the processes of burning and combustion are now possible. For the realm of the tiny, the microbes, this is a huge change to contend with. Some of them adapted by becoming aerobic organisms and those that cannot tolerate oxygen, the strictly anaerobic microbes were almost wiped out from the face of the planet surviving today only in habitats totally devoid of oxygen. These anoxic environments are found deep in the mud and sediments, in the great depths of the ocean floors and would you believe inside the animals intestinal tract. Vibrio fischeri must have been a survivor; it developed the ability to grow in the presence of oxygen. In microbiological term, it is Facultative meaning it can grow in the presence or absence of oxygen. In a world that has gone aerobic Vibrio fischeri was able to produce something wonderful, the gift of light. Indeed some scientists believe that the process of bioluminescence is Vibrio fischeris way of removing oxygen from the atmosphere. If it were true,

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it is a huge exercise in futility but a beautiful one at that! Bioluminescence is like burning or combustion in producing light and radiance but unlike burning or combustion almost no heat is produced. Bioluminescence can be likened to a special fire giving off light but does not generate heat, a cold fire!

The bluish-green wavelength of bioluminescence carries more energy than green, yellow, orange, and red wavelengths. It can be concluded that since the conversion of chemical energy to light energy in bioluminescence is highly conserved, so much so that very little of the transformed energy is wasted as heat thereby highly energetic blue-green light is generated. Since the blue-green light can travel farthest under the sea and can therefore reach as many eyes as possible compared with other visible colors, then evolution thus favored a bluish-green color for bioluminescence as a means of signaling for the marine denizens. Many scientists had surmised that the marine luminous microbes played a significant role in the evolution of the vertebrate eyes and therefore of vision. Since most of the lower animal life forms are from the sea, a molecule that can respond sensitively to bioluminescence must have first evolved. This light-sensitive molecule was later on incorporated in special light sensitive organs called Eye. Eyes in higher life forms can then generate visions. Sea creatures, great, and small,

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in the unbelievable vastness and depths of the worlds oceans used bioluminescence as the major means of communication. It is the main reason why the pictures or films of the marine world appear in shades of blue allowing blue-green light to be absorbed less and to travel far distances.

Hand grasping a flask containing billions of happily shining cell suspension of Vibrio fischeri

References:
Jones, B.W. and M.K. Nishiguchi. Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda), Marine Biology 2004

Kaplan, H.B. and E P Greenberg (1985) Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J Bacteriol. 163(3): 12101214.

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Nealson, K. and Hastings, J.W. (1991) The luminous bacteria. In: The Prokaryotes 2nd Edition (A. Balows, H.G. Trper, M. Dworkin, W. Harder and K.H. Schleifer, eds.) pp. 625-639, Volume I, Part 2, Ch. 25. (Springer-Verlag, NY) The emergence of complex life. www.globalchange.umich.edu/globalchange1/current/lectures/complex_life/complex_life. html

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Chapter III: Of Vision

His eyes were like fireballs, fearfully blazing Cats

Catty, the praying tabby Cat

The Eye Vision is a complex reaction of life processes triggered by light that enables us to perceive and interpret the world around us in full living colors. Science tells us that the stimulus that ultimately creates vision comes to us in the form of light particles called photons. A photon is a particle of light that carries a fixed package of energy. Countless numbers of photon come

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from outside and enters the eye through the cornea containing a special lens for focusing them. They then continue traveling by traversing the main internal transparent gel-like portion of the eye known as the vitreous body. The photons finally end their journey when they hit the back portion of the eye known as the optic part of the retina. The retina contains hundreds of millions of photoreceptive cells, the rods and cones, which interact sensitively with light. Photons hit a special biomolecule called rhodopsin found inside the eyes photoreceptor cells and transfers its fixed package of energy to it resulting in a cascade of chemical reactions that generates a nerve impulse. The nerve impulse, much like an electric current, travels along the optic nerve bringing it ultimately to the brain. The optic nerve is like a copper wire connecting the eye and the brain. Eventually, the swiftly traveling nerve impulse reaches the brain creating a sensation in the mind known as vision. During daytime or in well-lit conditions the cones are activated and function in receiving signals resulting in bright or photopic vision. However, in the dark, the cones lose their functions to very dim light and the rods take over. Activating the rods results in another kind of vision, a vision called scotopic or dark vision. Dark vision is not well known since one does not normally find himself in a dark environment completely devoid of light. The use of artificial lights of various kinds: incandescent and

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fluorescent,

powered by electrical energy has

brightened up the

surroundings even during the night that caused photopic vision to still be employed by the eyes. This might eventually cause scotopic or dark vision to be relegated into permanent disuse and once a biological structure or function is rendered useless, evolution will eventually remove it completely. Many times in the past when people discover new, isolated ecosystems found deep underground where not a single photon of light can ever enter penetrate, creatures previously with eyes that get trapped in this dark world loses their sight and vision. Their useless eyes degenerate into nonfunctional or vestigial organs eventually making them completely blind. Evolution directs this course of development since there is no use eyes in a world without light.

The luminous marine microbes must have played a leading role in the emergence and evolution of the first eye since they have been producing light in the realm of the earths vast hydrosphere even before the first animal appeared on earth. Since all life in general and animal life in particular first emerged in the sea, the first eye must have been biologically developed to respond to the bluish-green color of microbial bioluminescence. In its most primitive form or first step in the evolution of vision, a molecule must be

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developed inside a special cell of an organism that can alter its shape when exposed to the light of bioluminescence. This special cell must have been incorporated as part of a system of cells or an organ, which ultimately acquired the form of an eye. Thus Vibrio fischeri must have been a crucial participant in the evolution of eyes and vision. Why do humans possess scotopic or dark vision that responds most sensitively to the bluish-green color of bioluminescence when we are terrestrial dwellers not marine? Remember that bioluminescence is the major means of communication by the denizens of the marine world. The answer was simply there all along; it wonderfully verifies the very essence, the very foundation of biology itself evolution. Scotopic vision in humans is the most likely remnant of the vision that our great great fore creature had used in the darker marine world five hundred million years ago. This first period in the first and longest of three major biogeologic time scales known as the Paleozoic Era or ancient life is called the Cambrian period. It is the time when the first fish appeared and existed as the dominant and most intelligent creature of that time. This fishy forefather left the sea and became a land dweller roughly four hundred million years ago, scotopic vision must have been relegated to secondary status by evolution with another type of vision called photopic vision. Photopic vision is suitable in a terrestrial world,

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out of the sea, where it is much brighter compared to the dimmer marine world where scotopic vision is preferred. Scotopic vision is what I used in observing the dimmer manifestations of microbial bioluminescence specially the light coming from PIBiT. Scotopic vision requires absolute darkness and so there is a need to stay long in a dark room. Upon entering a dark room one does not immediately see some of the dimmer manifestations of microbial bioluminescence because it takes time for the eye to switch from photopic to scotopic vision. A period of around fifteen to twenty minutes of immersing oneself in absolute darkness called dark adaptation.

The standard microbial bioluminescence toxicity test

The cold bluish-green light generated by the various species of marine luminous microbes like Vibrio fischeri, Vibrio harveyi, Photobacterium phosphoreum and Photobacterium leiognathi, is a visually observable and measurable biological property known as bioluminescence. Since

bioluminescence is the product of a stable and complex metabolic activity, it lends itself well as a highly accurate and sensitive indicator of the well being and viability of the luminous microbes. If the luminous microbes are happy

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and are in a state of maximum well being; they can tell us by shining brightly over time. But if they are sad and their state of well being is low their light dims rapidly and even fades out to the point of complete inhibition. Any physical agent like heat and toxic chemicals that can damage the integrity of the cell will cause partial or complete inhibition of their light output. The rapid dimming of the luminous cells exposed to inhibitory compounds usually takes place almost instantly from a few seconds to several minutes to an hour or more depending on the degree of toxicity and the concentration of the chemical agent.

The conventional method of the microbial bioluminescence toxicity test in the evaluation of the toxic nature of a water sample if it is safe or unsafe, is to add the right amount of luminous microbes in the water sample itself. Before adding the luminous microbes in the water sample to be tested, it must first be made three-percent saline by adding the right amount of table salt. This is required since the luminous microbes are of marine origin; without the required amount of salt, the luminous microbes will die instantly. The water sample to be tested is placed inside a small glass test tube, which glows bluish-green upon the addition of luminous microbes. Another tube contains the negative control. The negative control simply contains clean

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and pure water. Luminous microbes are likewise added in the negative control. The two tubes, one bearing the water sample and one bearing the negative control, are both inserted into the luminometer side by side. A luminometer is an instrument that measures quantitatively the luminance of the two tubes. There are two possible outcomes from this toxicity test: the luminance of the water sample fades rapidly as compared with the luminance of the negative control after 30 minutes to an hour. An alternative result is when the luminance of the water sample does not fade and remains just as bright as the luminance of the negative control after 30 minutes to an hour. If there is a big difference between the luminance of the water sample and the negative control with the water sample much dimmer than the negative control, then the water sample is dirty and unsafe to drink it contains something toxic. If there is no difference between the luminance of the water sample and the negative control with the water sample persisting just as bright as the negative control then the water sample is as clean and pure as the negative control. The water sample does not contain any toxic agent. The presence of toxic substances in the water sample harms the luminous microbes causing them to decrease and at times even shut down completely their light production. Any unknown compound or substance,

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whose toxicity is suspected, can likewise easily be verified and evaluated using this method.

Outcome of a Clean and Pure Water Sample Contained in Tubes

After half to one hour

Water Sample

Negative Control

Water Sample

Negative Control

Outcome of a Dirty and Unsafe Water Sample Contained in Tubes

After half to one hour

Water Sample

Negative Control

Water Sample

Negative Control

N.B. Yellow means sustained luminance; gray means decreased luminance

The luminance of the negative control should persist brightly over a period of time since it contains pure water with no substance to harm the luminous microbes. It is the luminance of the water sample that will either persist or decrease. That is the simplicity of the bioluminescence toxicity test

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making it very useful and practical. The only stumbling block for its widespread use in the developing countries is the very expensive luminometer. A luminometer costs approximately one and a half million pesos depending on the brand.

The Bioluminescence oxygen demand (BiOD) for water pollution

Water pollution is the undesirable change in the quality of water due to human or non-human activities. Polluted water can exert irreparable damage the ecosystem. The quality of water is measured based on the following parameters: pH, dissolved oxygen, temperature, and dissolved chemical substances. The pH of non-polluted water usually ranges from 6 to 9. Dissolved oxygen (DO) an important parameter that measures the amount of oxygen in mom-polluted water is usually 6 ppm. DO support aquatic animal life like arthropods and fishes. Should DO fall much lower than 6 ppm, animal life dies due to insufficient oxygen. An aquarium needs continuous aeration to increase DO and thereby providing ample amount of oxygen for the resident fishes. If the aeration is stopped, then not enough oxygen can be provided to the resident fishes and soon they die. Temperature is also a factor in determining water pollution. High

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temperature can cause the amount of dissolved oxygen to decrease. Dissolved chemicals like magnesium, calcium, sulfate, bicarbonate, chloride and others determine water hardness. Water hardness if high can affect the ability of soap to foam. Bacterial bioluminescence is also affected by pH, oxygen and temperature. So theoretically, bioluminescence be used to evaluate the quality of the water sample, its ability to sustain aquatic life, based on the response of the luminous microbes in it. Once again, salt has to be dissolved in the wastewater samples to a 3 percent solution contained in test tube before the addition of the luminous microbes. Brightly shining blue-green colonies can easily be scraped off from the nutrient agar plates in a small volume of 3 percent saline to make a heavy suspension. These brightly shining suspensions are then added to wastewater samples contained in screw capped test tubes. After capping and inverting the tube twice to homogenize the distribution of the luminous cells, the entire liquid shines bluish-green in the dark. However, the bioluminescence after a while breaks apart, fades and blacks out completely except for a thin zone of bioluminescence that remains on the surface of the liquid. Tube

bioluminescence will fade once dissolved oxygen in the liquid (wastewater sample) is depleted. The longer the length of time of the bioluminescence in the tube, the less polluted the water is. The shorter the length of time of the

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bioluminescence in the tube, the more polluted it is. Bioluminescence like respiration is a biological process that needs oxygen. So if there is little of dissolved oxygen in the water, indicative of heavy pollution, the shorter is the length of time to sustain. The tube bioluminescence fades swiftly to the point of blacking out completely. Below is a picture of homogeneous tube

bioluminescence except for the second tube from left that is already in the process of blacking out. Using BiOD, it is hereby proposed that a wastewater sample blacking out, undergoing tube luminescence extinction, in 90 minutes and less is classified as heavily polluted. A tube luminescence extinction of more than 90 to 120 minutes for a water sample tested with BiOD is classified as moderately polluted and a period of tube luminescence extinction of greater than 120 minutes is non-polluted. The tube bioluminescence extinction technology (TuBET) This innovation making used of bioluminescence in test tubes for various assays is given the name Tube Bioluminescence Extinction Technology or in short TuBET. Tubet can be used to determine the freshness of various food samples like milk, pork products, beef products, chicken products, sea foods, and many dried foods. Since freshness connotes cleanliness and something that has not yet undergone degradation or decomposition then bioluminescence conducted in test tubes can be

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employed for its evaluation. Meat and milk products that have undergone significant stages of degradation due to autolysis or microbial action generate volatile amines resulting from protein breakdown. The presence of large amounts of these biogenic amines in meat products due to extensive autolysis and microbial activity put into question the freshness of the food samples and concomitantly its safety. It is primarily the reason why solutions or suspensions of meat products become alkaline when they undergo microbial spoilage. Bacteria break down proteins to produce these offensive smelling volatile amines like putrescine and cadaverine. Meat coming from double dead animals are specially worrisome since undesirable changes in its quality set in immediately at the time of death. Since TuBET is very sensitive to high bacterial load and the biogenic amines, it can readily be used for the evaluation of freshness of the samples. Saline washings of meat and other food samples that yield short periods of extinction using TuBET would indicate a condition of high bacterial load and high biogenic amines, which reflects the unhygienic and dirty state of the tested samples. Parameters and standards of clean and acceptable food quality can be based on sound investigations using TuBET. Furthermore, the use of TuBET should make the analyses of food for freshness faster and cheaper but without sacrificing assay sensitivity.

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From left, tubes 1, 3, and 4 still exhibit homogeneous tube luminescence while tube 2 has undergone extinction (black out). Take note of the bright thin layer of luminescence at the surface is due to an abundant supply of oxygen from the air-space.

Aside from a novel parameter for the classification of water pollution using TuBET, other applications include rapid determination of the bacterial load of and sterilized quality of pasteurized and UHT treated liquid milk, chocolate drinks, and other non-acidic beverages. I never thought before that a brightly shining suspension of the luminous microbes can make opaque drinks like milk and chocolates shine in the dark. Indeed bioluminescence carries much energy that it can make plenty of liquids shine in darkness.
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Tubes containing UHT-treated and pasteurized milk mixed with a suspension of luminous microbes. The 3 tubes from left have undergone bioluminescence extinction and the 4th tube from left is starting to black out.

Preparation of small paper-discs Marine luminous microbes have been found to adhere readily on practically any solid surface of various compositions like glass, plastic, metal, and paper. They have an outer covering that is rich in carbohydrates and fats making it quite sticky. Once the luminous microbes are rendered motionless on a solid material many useful and amazing things can be done with them. Paper among other materials holds the promise of availability and ease of use for immobilizing the luminous microbes. Whatmann filter papers

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have been found to be suitable for this purpose. Whatmann is a special type of paper that is sold in the market used by wet laboratory researchers and analysts. Whatmann filter paper comes in different grades and each grade exhibits specific properties for thickness, porosity, etc required for a scientific investigation. I used Whatmann grade one filter paper, the most commonly employed grade that comes in large white sheets the size of a gift-wrapping paper. I then cut it into even smaller sheets the size of a table napkin, which is subsequently punched out into small discs using an ordinary office paper puncher. The punch out paper discs have a diameter of roughly six millimeter, which were all collected in a petri dish.

How to make the paper-discs bioluminous

It is very easy to make these paper-discs bioluminous. A species of Vibrio fischeri USTCMS 1063, is sensitive to toxicants and also shine brightly over long period. This strain with an accession number 1063 was isolated from squids (Loligo sp.) was bought at the Trabaho Market near UST. It was preserved and maintained in the USTCMS (University of Santo Tomas Collection of Microbial Strains) and was used for the innovative paper-disc immobilized bioluminescence technology (PIBIT). Vibrio fischeri

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USTCMS 1063 was streaked in a nutrient agar plates. After the streaking procedure, the plates were incubated at room temperature in a locker for about fifteen hours. After the incubation period, the plates were examined for luxuriant growth as shown by the presence of numerous brightly shining colonies in the dark. Three percent saline was prepared by dissolving three grams of table salt in approximately two cups of distilled water. A little volume of the saline was poured into the bottom dish containing the nutrient agar. Saline was poured on the brightly shining colonies of Vibrio fischeri just enough to flood the surface of the agar. Using a wire loop, the colonies were gently scraped off from the surface of the agar medium. This was done easily by using the usual streaking procedure described previously. The heavily turbid saline suspension was poured into a bigger glass flask. The suspension in the flask was vigorously shaken by hand for about a minute to homogenize it. It was then examined in the dark for its bright bioluminescence. In darkness, the liquid inside the flask should glow brightly bluish-green as though it were radioactive.

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A brightly shining suspension of luminous microbes

The turbidity of the cell suspension was then adjusted according to the level of turbidity of a McFarland Number One Barium Sulfate Turbidity Standard. There is a need to adjust the turbidity of the cell suspension because it can vary widely. Its like being ask how should the degree or intensity of the turbidity of the cell suspension be, should it be less or more with respect to what standard of comparison? Without adjusting the degree of cloudiness or turbidity to a McFarland Number 1 Standard there is no way on earth by which you and I can arrive at the same level of turbidity and also of luminance intensity. The level of turbidity used can surely affect the results of the toxicity test. Microbiologists are well verse in preparing these

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increasing levels of McFarland Standards numbered one to ten. A McFarland Number One is just a barely cloudy cell suspension when visually examined in a test tube but a cell suspension of similar turbidity contains 300,000,000 cells in a milliliter of the cell suspension. The degree of

turbidity of a McFarland Number One can be reproduced in the kitchen by mixing a cup of water with more or less two to three drops of liquid whole milk. By adding more saline in the flask containing the heavily turbid cell suspension it can be diluted to something less turbid, less cloudy and ultimately approximating that of a McFarland Number One Turbidity Standard. After the level of the turbidity of the cell suspension has been adjusted to the turbidity of a McFarland Number One, it can now be used for the very simple procedure of immobilizing the luminous cells. Using a stainless steel curved forceps with serrated tips; one paperdisc was taken out from a collection of it contained in a petri dish. The single paper-disc held at the tip of the forceps is simply dipped into the previously standardized McFarland Number One cell suspension. Simply immersing the paper-disc into the luminous cell suspension is enough to cause large numbers of luminous cells to be adsorbed and entrapped in the paper-disc resulting in their immobilization. I estimated that there must be something like fifteen million luminous cells immobilized in a paper-disc. This large

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numbers of luminous cells packed and immobilized together like sardines in the small confines of the paper-disc is important in the cells continuous state of bright bioluminescence. Recall that these cells require a sense of quorum of being part of a very large population for them to continue shining brightly. Packing millions of luminous cells in the matrices of a small paperdisc tricks them into thinking exactly just that of the cells b eing a part of a big population. Dispersing the luminous cells in a big volume of saline will make them lose their bright luminosity for lack of close contact with one another. This would make them think that their population is small and so the individual cells become dim. The bioluminous paper disc is then directly transferred into and allowed to sink in a screw-capped test tube containing the water sample or chemical solution to be tested for toxicity. The bioluminous paper-discs are introduced individually into four tubes containing water of known purity, which serve as the negative control. A negative control simply means water free of any toxic contaminant; known to be clean and pure. Next step would be to introduce bioluminous paper-discs individually into four test tubes containing a water sample or a chemical solution whose toxicity is to be determined. The water sample can be from bottled water, groundwater, river and even seawater or it can also be a solution of any chemical whose toxicity is suspected. In all

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cases, the fresh water samples have to be made saline by adding table salt to make a three-percent solution. Likewise, chemicals to be tested will have to be dissolved in a three-percent saline. The luminous microbes require the three-percent salinity because of their marine origin. Another essential factor to be checked before introducing the bioluminous paper-disc is the pH of the water sample or chemical solution prepared. The pH is a measure of the acidity or alkalinity of a water sample or any solution. For this toxicity test to be valid, the pH must be in the range of about five to nine. A pH of seven will be perfect for this purpose since it is neutral neither acidic nor alkaline. The luminous microbes love a neutral pH, which is pH seven. Examples of acids are vinegar and tomato sauce. Vinegar has a pH of three and tomato sauce has a pH of four. Examples of alkaline substances are milk of magnesia and baking powder. Milk of magnesia has a pH of 11 and baking powder has a pH of 9. Exposure of the luminous microbes to extreme acidic or alkaline pH; pH values below 5 or above 9 will cause the cells to die resulting in the fading of their luminance.

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Tales of light from the bioluminous paper-discs

The four screw-capped test tubes each containing the bioluminous paper-disc are examined in the dark. All the test tubes will have to be placed in a metal rack, which can hold a maximum of forty tubes. I would hold and lift up the rack above my head and observe the tubes from their butts so to speak in a dark room. In this way, the tubes disappear in the dark and I see only an array of bioluminous paper-discs arranged in rows and columns. The luminance of the four bioluminous paper-discs contained in the negative control tubes as well as those in the four water sample tubes was examined. If the period of luminance of the bioluminous paper-discs in the sample tubes lasted as long as those in the negative control tubes then the water samples tested is as clean as the water in the negative control tubes. The water sample is therefore clean and safe to drink. But if the luminance of the bioluminous paper-discs in the water samples fades rapidly and at times black out in comparison to that of the negative control tubes then there is something toxic in the water samples tested. The water sample is not safe to drink since they contain toxicants that caused the luminance of the paperdiscs inside them to fade out rapidly. These are the two possible outcomes of this simple toxicity test; either the luminance in the sample persists as

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bright as the negative control or the luminance of the sample fades out much faster than that of the negative control. The longer the luminance of the bioluminous paper-discs persists, the safer and the less toxic the sample or the chemical solution is. Degree of toxicity can also be determined as well by dissolving each chemical to be tested at the same concentration in saline and monitoring the length of time each will cause the bioluminous paper-disc to fade out or even to black out completely.

Outcome of a Clean and Pure Water Sample Using PIBiT

Negative Control

After half to one hour

Negative Control

Water Sample

Water Sample

Outcome of a Dirty and Unsafe Water Sample Using PIBiT

Negative Control

After half to one hour

Negative Control

Water Sample

Water Sample

N.B. Yellow means sustained luminance; gray means decreased luminance

The shorter the time period to fade completely the more toxic the chemical is. And the longer the time period for the luminance of the solution

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to fade out completely, the less toxic the chemical. Using this method, chemicals can be arranged individually from least toxic to the most toxic.

Background information behind bioluminosity and immobilization

A paper-disc when dipped into a brightly shining blue liquid absorbs the millions of luminous microbial cells and will retain its bright luminosity even when transferred and immersed in another saline. The paper disc absorbs a small amount of liquid and becomes bioluminous because of the millions of luminous microbial cells rendered bound, intertwined, and trapped in the network of cellulosic fibrils found in the paper disc itself. The body of the luminous microbes contains sticky substances on their bodys outer covering making them adhere to the cellulosic fine and intertwining fibers of paper. This simple method causes the immobilization of millions of luminous microbes in the paper-disc making it brightly bluish-green when viewed in the dark. The readily visible bioluminous paper discs thereby precludes the use of expensive and complex light-measuring instruments called luminometers in testing the toxicity of water samples and unknown solutions. The human eye can distinguish between varying degrees of brightness and are very

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sensitive even to low magnitude light sources when it has undergone dark adaptation. Dark adaptation enlarges the opening of the iris allowing more photons to get into the eye and activates the photoreceptor rod cells of the retina. Dark adaptation usually takes fifteen to thirty minutes of switching our vision from photopic to scotopic, from bright to dark vision. Science has determined that scotopic or dark vision is so sensitive to light that even a single photon that reaches the retina will elicit a vision. A bioluminous paperdisc contains millions of luminous microbes each of which is producing hundreds of photons per second. Collectively the luminous cells in a single bioluminous paper-disc can send photons into our eyes by the millions.

Paper-disc Immobilized Bioluminescence Technology (PIBiT)

The method of using bioluminous paper-discs for the bioluminescence toxicity test using scotopic or dark vision is hereby presented with the proposed term Paper-disc Immobilized Bioluminescence Technology or simply the acronym PIBiT. PIBiT is an innovative, simple and highly economical, non-instrument and non-culture medium based technique with wide ranging biotechnological applications. The use of scotopic vision in PIBiT thereby precludes the use of complex and expensive luminometers.

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Moreover, since saline, a non-growth medium for microbes is used all through out the procedure in the preparation and in the testing of samples, the required microbiological measures to keep out unwanted microbial contaminant called aseptic techniques can be abandoned completely. PIBiT is user-friendly because of its simplicity and environment-friendly due to the significantly reduced consumption of chemicals, manpower, and energy.

A luminous suspension contained in an eppendorf tube roughly half the size of our little finger can produce one hundred bioluminous paper-discs. A hundred bioluminous paper-discs can test twenty-four water samples with four replicates per sample and four replicates as well for the negative control. One brightly shining plate culture can give a luminous suspension that produce hundreds of thousands of bioluminous paper discs! In addition, PIBiT can be made resource sustainable, which should make it very useful and practicable in poor countries where a need to regularly monitor their drinking water for safety and cleanliness is needed.

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Punched out filter paper-discs contained in a petri dish, held by a forcep ready to be immersed in a flask containing suspension of luminous microbes and contained in a screw-capped tube whose water content is being tested for toxicity.

Clean and safe drinking water I was born in the sign of water and its there that I feel my best, the albatross and the whales they are my brothers is a part of a lyric of a beautiful song. The song entitled Cool Change by the Little River Band, appealed to very much for Indeed I was also born in the sign of water and my winning entry in the Young Inventors Awards has been designated in the issue of the Far Eastern Economic Review magazine as Testing the Waters. It really seems that I have such close affinity with everything that has to do with water though I really never learned how to swim! Water is defined by science as the universal solvent. Universal means that compared to other chemical solvents more substances: solid, liquid and even gas; crystalline and powder are soluble in water. Many of these substances are associated with or components of living organisms and are appropriately called biomolecules (Life Molecules). Our cells are bathed in and out with
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water. Seventy to eighty percent of the composition of many living organisms is water; for jellyfishes the water content probably approaches a hundred percent. Water is the best medium in which our life processes can take place. Chemical reactions and cellular processes occurring at the molecular level are happening in a solution of water and salts. Since todays terrestrial organisms have move out of the sea a long, long time ago; there is a need to continuously replenish the water within them from an external source. Animals lose water from urination, perspiration, and respiration. Plants lose theirs by transpiration. That is why animals have mouths and plants have roots; for food assimilation and to replace the water that escapes constantly to the environment. Thus, water becomes an essential resource for the sustenance and maintenance of life. Waters primal significance means that access to clean and safe water must be ensured at all cost. Simply putting it arithmetically, safe and clean water equal healthy and long life for all living organisms. Dirty and polluted water is unhealthy because it poisons living organisms resulting in diseases and therefore short life.

The old adage that water is life has never meant so much to us now than ever before. Cyanide contaminated seawater in Sorsogon, Philippines;

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benzene in a river in China; arsenic in the groundwater in Bangladesh, and mercury, chromium, lead, and cadmium in a lake in Massachusetts are just some of the news that grabs the headlines in the media and internet these days. Contamination of our ecosystem in general and drinking water in particular by toxic chemicals has caused serious health problems like poisoning, cancer and birth defects. Indeed, our drinking waters cleanliness and safeness is threatened as never before by a plethora of chemicals artificially created in large quantities spewed out by a rapidly industrializing world. Metallic and non-metallic pollutants abound within our immediate vicinity that their presence though at minute amounts have already been detected in the water that we drink and in the fresh produce that we eat. Many of these chemicals have the ability to react with and change the chemical structure of DNA, the blue-print of life. Changing DNA can lead to birth defects if what has been affected is the DNA of sperms, egg cells or the developing fetus itself. However, if what is affected is the DNA of one of the billions of cells of our bodies then this can lead to cancer and malignancy. On a grand scale, chemicals can also destroy swathes of ecosystems and put many organisms at the risk of extinction. Chemical toxicants like heavy metals, chlorinated hydrocarbons, aromatic compounds, formalin, cyanides, detergents, phenolics, pesticides, mycotoxins and even some disinfectants

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and antiseptic agents have already found their way into our bodies. Like a book, no one can judge water by its appearance; water can look crystal clear, smell right but deadly! Very small concentrations of various toxicants at the parts per million (ppm) or even parts per billion (ppb) level will hardly affect the physical qualities of water but can cause chronic life threatening diseases when taken over long periods of time. The detection of toxicants in water can be expensive and complicated using the instrumental analysis employed today in modern laboratory. PIBiT is definitely the solution to this seemingly helpless predicament of safeguarding the cleanliness of our water. PIBiT is first and foremost to be used for the simple, rapid and cheap toxicity testing of all forms of water samples like groundwater, surface water as well as bottled mineral and distilled water. The beauty of using PIBiT is that it can sensitively detect a wide variety of toxicants: metallic and nonmetallic. Mercury, lead, cadmium, barium, nickel, cobalt, chromium, arsenic, copper, cyanides, formaldehyde, dyes, detergents, and pesticides are all detectable at their maximum allowable concentration in water as specified by the United States Environmental Protection Agency (EPA) in as short as half to an hour. PIBiT tells us if the water is toxic or not; whether it is safe or unsafe to drink. However, PIBiT cannot tell us the identity of the toxicant or toxicants that are present in the water.

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In detail, metals like Lead is detectable by PIBiT in mineral water at its U.S. EPA maximum allowable concentration of 0.015 ppm in just one hour; cadmium at 0.005 ppm also in one hour; barium at 2 ppm in one hour and the transition metals nickel at 0.1 ppm in one and half an hour and copper at 1.3 ppm in one and half an hour. The organic toxicants like the detergent sodium dodecylsulfate detectable at 0.05 ppm in two hours; the stain methylene blue at 0.1 ppm in two hours; the chlorinated hydrocarbon Chloroform at 0.05 ppm in an hour; Phenol at 0.5 ppm in one and half an hour and the preservative formalin at 0.05 ppm in one hour. PIBiTs ability to detect mercury in mineral water is at 10 ppb (parts per billion) in thirty minutes. When the bioluminous paper-discs are exposed to 1 ppb of mercury even om hard water, the luminance fades in an hour. One ppb is mercurys maximum allowable concentration in drinking water as specified by U.S. E.P.A. Moreover, when exposed to 0.1 ppb the luminance of the bioluminous paper-discs fades in two hours and finally when exposed to a mercury concentration of 0.01 ppb or 10 ppt (parts per trillion) the bioluminous paper-discs fades in three hours. One part per trillion is the limit of detection for mercury in mineral water or hard water using PIBiT. While the bioluminous paper-discs in the negative control blacks out completely in six hours. These results show how ultra-sensitive PIBiT can be in detecting

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a wide array of inorganic toxicants like heavy metals. PIBiT rivals the detection ability of a sophisticated instrument called the Atomic Absorption Spectrophotometer (AAS). An AAS can cost three hundred thousand US dollars but PIBiT cost only a mere one dollar to analyze one thousand or more water samples for toxicity. In addition to heavy metals and other organic toxicants, PIBiT can also detect the presence of volatile organic compounds (VOCs) in bottled distilled water. Distillation process units can produce toxic distilled water that can cause the bioluminous paper-discs to black out in as short as fifteen minutes due to the presence of VOCs. But if the distilled water is boiled for ten minutes in an open container, the resulting water can sustain the brightness of the bioluminous paper-discs for as long as five hours. How come there is a big difference in the length of time of luminance of the bioluminous paperdiscs between distilled water and boiled distilled water? Why did boiling reduce the toxicity of the distilled water? The answer is very simple. Boiling expels the highly volatile VOCs from the distilled water. These VOCs are chlorinated hydrocarbons and therefore have the potential to be

carcinogenic. VOCs are produced when the surface water undergoes chlorination in the water treatment plant converting it into tap water. When tap water is distilled, the VOCs being more volatile than water evaporates

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faster and so accumulates largely in the condensing water as it cools down and collected. The distillation of tap water has an undesirable side effect, the VOCs present in the tap at low concentrations get heavily concentrated in the distilled water. Is it possible to remove VOCs from distilled water? The answer is yes. A special steam vent should be present in the distillation apparatus that will result in the removal of VOCs from distilled water. Can the VOCs be removed completely from distilled water during the distillation process most probably not. The distilled water will have to be passed through special filtration columns containing activated carbon that should remove all of the VOCs. A distilled water sample in which the luminance of the boiled is significantly longer than that of the unboiled should be suspected of containing prohibitive concentrations of VOCs. Ozone treatment of water can also produce toxic byproducts in water, which can be detected by PIBiT.

Brands of shining mineral water containing luminous microbes

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Between bottled distilled and mineral water, PIBiT favors the bottled mineral water. This indicates that the bioluminous paper-discs remain bright much longer in mineral or hard water than in distilled water. Mineral water contains dissolved substances like magnesium, calcium, bicarbonate, etc that have beneficial effects on the integrity of the luminous cells. Besides, humans have been drinking mineral water since the dawn of time for almost three million years now and distilled water has only been around for about twenty years. Among the commercially available distilled water in the Philippines, PIBiT stays brighter longer in Brand B than in Brand C and among the mineral water, PIBiT favors Brand A over Brand D and Brand D is better than brand E an ozone treated mineral water. It would be better if we take a second look in the use of ozone to clean up mineral water for drinking purposes because doing so may bring in more harm than good. Many nasty chemicals can be produce in water by an ozone treatment and most of these are carcinogenic. Ozonolysis or the degradation of minute amount of organic compounds in water using ozone is one of the treatments utilized by German industrial plants for their wastewater. The higher incidence of cancer in the population can be traced to prolonged exposure to a plethora of artificial chemicals found in our modern way of life. In summary, a water sample tested for toxicity using PIBiT that resulted in the

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rapid inhibition of luminance in comparison with a negative control in as short as an hour or less should be questionable as to its cleanliness and safety.

PIBiTs applications and its resource sustainability

PIBiT can be an alternative and novel method of antimicrobial assay without making use of the conventional nutrient broth and nutrient agar for the antimicrobial testing. Complex and expensive nutrient agar media used initially for the cultivation of the marine luminous microbes can readily be replaced by local substitutes. I found out that skimmed milk powder, which is available in all local market, could be used as a good substitute for the cultivation of marine luminous microbes. The skimmed milk powder once suspended in water can first be treated with the protein-degrading enzyme papain. Papain can be obtained from the milky latex of the papaya tree when its stem or the immature fruit is cut. The papaya tree is abundantly found in all the countries of the tropics. Casein, milk protein, is a large molecule that needs to be digested by proteolytic enzymes to shorter peptides and even amino acids so that it becomes soluble and more absorbable to microbial cells. Another possible substitute for papain is the

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pancreatic juice or juice from the small intestine of slaughtered pigs or cows. The juice contains the enzyme trypsin that can breakdown the large protein molecule in milk called casein into smaller more soluble portions that can be absorbed easily by the luminous microbes. Agar is also a substance that is readily available in the tropical coastal regions particularly from seaweeds. The glass petri dishes can be re-used.

Luminous microbes growing and glowing brightly on self-made locally available seawater salt, skimmed milk and agar-agar bars

Coarse salt can always be taken easily from the sea by sunlightpowered evaporation and this is the kind of salt that the marine luminous microbes are really accustomed to. Turbidity standardization using a McFarland Standard # 1 contained in screw-capped test tube can be prepared and reused repeatedly. The whatmann paper can possibly be replaced by commercially available rough paper towel or some dried leaves

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that can be punched out into small discs. PIBiT can find applications in the following:

Using this method, fifteen different plants prepared as one percent extracts in saline caused the bioluminous paper-discs in garlic, ginger, onion and bitter gourd to black out much faster than the negative control. Garlic causes the bioluminous paper-discs to black out in as short a period as five minutes. PIBiT can therefore be used to screen plant extracts rapidly for antimicrobial activity. Three plant extracts studied in UST showed potent antimicrobial activity with complete light inhibition being achieved in less than five minutes, three extracts are moderately active with complete light inhibition being achieved in three to four hours and eight showed no antimicrobial activity indicating much longer period of sustained luminance. PIBiT also responds sensitively to common disinfectants containing chlorox and phenolics and health-care products like soaps and tooth pastes with triclosan, rubbing alcohol, contact lens solution, eye drops, mouthwashes etc. making their antimicrobial assays easier, cheaper and simpler. Results showed that chlorox and chlorox-based disinfectants are most potent disinfectants since they can achieve

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inhibition of light output of the bioluminous paper-discs at a much lower dilution levels than the phenolic based disinfectants. Soaps and toothpastes containing triclosan likewise inhibit light output of the bioluminous paper-discs faster and at much lower dilutions than identical commercial products without it. Acute toxicity testing of grains like rice and corn, cereal-based snacks, food colorings and chemical additives, copra, root crops like cassava, vegetables and other foods for the presence of mycotoxins, Benzo(a)pyrenes, pesticides, cyanides, formalin etc. can be easily and cheaply monitored to ensure safe and clean foods for the people. Toxicity testing of fishes and seafoods for the presence of malachite green and other poisonous compounds. Fishermen can probably use PIBiT as natural, cheap, luminous and attractive baits for various saltwater fishes. Fishes seemed to be attracted to the cold bluish-green light of marine bioluminescence. Thousands of luminous paper discs can easily be prepared in the laboratory and released into the sea at night to attract various fishes thereby facilitating their capture. Since the luminous bacteria used have not been genetically modified and indeed are of marine origin, their use would be a very natural process. In addition, the movement

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and luminosity of these bioluminous discs in the sea can easily be monitored for probable oceanographic studies. Epidemiological studies and diagnosis of chemical poisoning of the populace can be achieved by collecting and analyzing urine samples and testing it for heavy metal poisoning. Marine plant extracts can be assayed using PIBiT to screen for antivibriosis compounds. Luminous vibriosis is responsible for the infestations of prawn farms. Extracts from sea plants and seaweeds can be screened and subsequently utilized for antivibriosis activity. This should be more environmentally appropriate than the use of antibiotics and other synthetic antimicrobial agent. It is also highly probable that since luminous bacteria are normal microflora of the intestine of marine fishes, they may play a probiotic role (beneficial) enhancing the well being of these marine animals. PIBiT can also be used to introduce these bacteria in the gut of saltwater fishes raised in captivity by mouth so as to increase their resistance against microbial pathogens. Since bacterial bioluminescence is highly dependent on dissolved oxygen, PIBiT can most likely be utilized to measure dissolved oxygen (DO) in water as well as well as evaluate the BOD at a much cheaper

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and faster rate. The amount of dissolved oxygen in water is used to determine the pollution level in the water. The more oxygen is dissolved in a water sample the less polluted it is. The less oxygen, the more polluted the water is. PIBiT can also be made to monitor cheaply the amount of residual chlorine in drinking water. Chlorine in drinking water protects us from waterborne diseases. PIBiT can also be used to easily determine the amount of salt in perspiration, which can be utilized for the initial diagnosis of cystic fibrosis. PIBiT is also sensitive to ultraviolet radiation and can determine their effectiveness for disinfecting purposes in the operating rooms of hospitals and clean benches of research institutions. TuBET can rapidly determine the Freshness of fishes, seafoods, pork, beef, and chicken. Lastly, PIBiT can be used to check any unknown liquid or solution whose toxicity is suspected. Paints and organic materials suspected to contain heavy metals like lead, mercury, and cadmium can first undergo incineration. The resulting residue is dissolved in saline and tested using PIBiT. Here in UST, I intend to check unknown liquids

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dripping from any of the tubes hanging up in our ceilings or even water coming as condensate from an autoclave. Any unknown solid that is readily soluble in water can likewise be tested for toxicity.

The use of PIBiT can be programmed in such a way that it can be prepared during daytime and observation of the glowing discs can favorably and easily be done during nighttime. This should facilitate the testing process and the evaluation of the results. PIBiT is very natural, environmentally friendly, non-harmful and almost zero energy consuming analytical process. The paper-discs are highly biodegradable material. It should significantly reduced consumption of chemicals and culture media, glasswares, manpower, and energy than is conventionally required in a typical chemical analysis. Significantly, less consumption of commercially available chemicals and other materials would mean significantly less industrial output of the greenhouse gases. These characteristics will surely endear PIBiT to people concerned with environmental protection and delaying global warming problems. PIBiT can address the Philippine Clean Water Act with certainty. PIBiT can safeguard the protection of the La Mesa Watershed through its highly sensitive capability to detect the presence of minute amounts of toxic chemicals that can come from without. Mies once

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said. less is more. PIBiT is just exactly that; its simplicity and minimalistic attributes hold the promise of affordability to the vast number of poor people throughout the world. Its simplicity and minimalistic attributes also hold the promise of being an alternative to existing technologies that even contribute to the strength of climate change. PIBiTs presents itself as an environmentally friendly technique now that the apocalyptic effects of global warming threaten the world.

EQ phone NASA

It was very touching for me to hear from Pres. George Bush, the planned construction of the International Space Station by the National Aeronautics and Space Administration or NASA. What a great achievement that will be for mankind, imagine a permanent abode in the firmaments which will eventually serve as the launching pad for modern space crafts that will carry humans to the outer planets of the solar system and beyond. People and animals will someday dwell in the ISS. Ever since mans great voyages from the Old World to the New, a substantial supply of clean and safe drinking water is needed. Water in the ISS, will be a very precious commodity that requiring it to be rationed and reused. Water present in all

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human secretions like urine, sweats, mouth water vapor and hand washings will have to be recycled by special machines. These special machines installed in the ISS will be fed with the collected bodily fluids of the occupants. These fluids will then converted into ultra-pure drinkable water! Between the urine and the drinkable water are series of continuous purification operations and processes. More often than not, contaminants can gain entry into the water as it undergoes various purification treatments. If I may recommend, NASA can make use of PIBiT to check on a regular basis the quality of their recycled drinking water. PIBiTs ultra-sensitivity to minute amounts of toxicants in water can safeguard the health and well being of the space crews for many years. Bringing the marine luminous microbes in space will be the least of all problems. They can be stored in small plastic cryotubes and preserved easily in small canisters of liquid nitrogen or in small ultra-low temperature freezers. The luminous microbes kept under suspended animation can be revived easily into viable cells and subsequently utilized in PIBiT. Microbial bioluminescence can also be

employed to directly measure the life support system of other planets that we humans might consider to reside on. Physiological conditions like temperature, pH, oxygen availability and ionic concentration needed to sustain life are also compatible with those of Vibrio fischeri. For indeed,

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bioluminescence is oxygen and oxygen is bioluminescence. Mans inherent exploratory nature will bring us to extra-terrestrial worlds someday. Surely, in those days bioluminescence and PIBiT will play an important role in ensuring safe and clean drinking water to the space travelers. In addition, since Vibrio fischeri and Photobacterium leiognathi are honest-to-goodness denizens of the earths marine hydrosphere, then we can used them to monitor bodies of water in other planets like the oceans of Europa or the icecold geysers of Enceladus for their similarity to the physic-chemical properties of earths seawater.

Future plans for PIBiT

Scientists share the results of their findings by publishing it in technical journal devoted to a particular discipline. The other alternative is to submit research studies or findings in the form of an abstract for oral and poster presentation in a special annual gathering of colleagues and fellow scientists. In both cases, peers in the scientific community review the soundness of the hypotheses, experimental results and validity of the study. After reviewing the submitted abstract based on established protocols, a group of scientists working together as a committee makes a decision as to

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its acceptance or rejection for presentation. This special gathering is called a congress, convention, or symposium and is organized by a small highly specialized group or a large international organization of scientists. It is with much excitement that I dream of someday presenting my findings in a scientific organization held in the US or Europe.

I was so thrilled last February 2006 when my submitted abstract on PIBiTs ultra-sensitive ability to detect mercury in hard water got accepted for a poster presentation in the prestigious 106th annual convention of the American Society for Microbiology (ASM). The ASM will be held in Orlando, Florida in May of 2006. My abstract was peer reviewed by ASM and was accepted for presentation. It is indeed an honor and a privilege for a research work to be recognized by an international body of scientists like the prestigious American Society for Microbiology. Although excited, as I was to participate in the ASM, my planned participation fell through for lack of support from the USTs College of Science administration. Presumably, for the administration, financial assistance for travel is only awarded for an oral and not for a poster presentation. It was truly disappointing for I have looked forward with much enthusiasm to visit Florida and to participate in the convention. Hopefully, in 2008 in Boston, I can be allowed to participate in

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the prestigious annual convention of the American Society for Microbiology. Before ASM, my abstract on PIBiT was also accepted for a poster presentation in the gathering of the International Union of Microbiological Societies (IUMS) late in 2005. IUMS was held at the Moscone Center in San Francisco. Again for lack of funds, I was not able to attend and participate. In both IUMS and ASM, I am pleased that my abstract on PIBiT underwent rigorous screening and was considered worthy of presentation. In fact, ASM screens submitted abstracts thoroughly based on high standards and accepted protocols before an acceptance is granted to the author. In June 2005, another aspect of PIBiT, submitted as an abstract, was accepted for poster presentation in the 24th International Congress of Chemotherapy. The International Society of Chemotherapy, based in London, sponsors the Congress for Infection and Cancer. However, hope indeed springs eternal. One day while searching the internet, I came across two other special gatherings in the US that still receives submitted papers for presentation that deals on my research area. One of these is a smaller and more specialized meeting of the International Society of Bioluminescence and

Chemiluminescence (ISBC). The 14th annual convention of the ISBC will take place in San Diego, California in October of 2006. This convention is probably the one suited for me since it specializes in bioluminescence and

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chemiluminescence and that is what PIBiT is all about. The other gathering, much bigger, is the annual convention of the American Association for the Advancement of Science AAAS. It will be held in San Francisco in February of 2007. Its theme on sustainable practices to protect the environment and provide safe drinking water makes PIBiT suitable for a poster presentation. Besides, PIBiT presented in the AAAS can be made known to a wider and bigger community of scientists coming from various disciplines. The World Water Forum held biannually in different countries the last one was in Mexico will also be one of the best venues, where I can present PIBiT. The World Water Forum deals with how water is utilized, managed, protected, distributed and studied in third world countries. Lastly, I am still searching for annual conventions and symposia in the U.S. concerned with water, the environment or microbiology. PIBiT will surely be useful in these conventions for its ability to detect minute amounts of contaminants in water that pose health risk to the populace. Very recently, my years of work on PIBiT gained recognition from my colleagues in the Philippine Society for Microbiology (PSM) when I was awarded the Best Poster Paper Award for 2007. It is indeed an honor to be included in the roster of the PSMs Best Poster Paper Awardees with the likes of Prof. Sanchez, Raymundo, Dogma, Matias,

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Natividad, Barraquio, Kobayashi, Rivera, Rivero, Monsalud, Tapay, de Ungria, Dalmacio and many others.

Recognitions through the years on my work with Bioluminescence

Lately, while surfing the web I came across a site (http://tiee.ecoed.net) the Ecology Education Publications dated April 2005 maintained by the Ecological Society of America and supported by the National Science Foundation. Listed in its website was my very first ISI (Institute for Scientific Information) publication in 2001 on bioluminescence that appeared in the journal of Biological Education. What an honor indeed it was to receive such recognition from a prestigious body of teachers and scientists in the U.S.

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TIEE TEACHING ISSUES AND EXPERIMENTS IN ECOLOGY

a peer reviewed publication of ecological educational materials by the Ecological Society of America CURRENT VOLUME ALL VOLUMES ABOUT TIEE SUBMIT WORK SEARCH

From the www.tiee.ecoed.net

PIBiT was also submitted for poster presentation and accepted in the 15th International Congress of Photobiology (ICP), which will be held on June 2009 in Dsseldorf, Germany. Another prestigious congress where it was screened and accepted for poster and group discussions presentation was in the 2009 FEMS (Federation of European Microbiological Societies) that will be held in July of 2009 in Gothenburg, Sweden. We dream of a much better place, a distant and perfect world of our deepest yearning. A world we try so hard to realize in this present life through our own little ways of achieving it. A world free of pollution, wars, diseases, defects, intolerance, sadness, senseless killings, and where no one is poor and deprived of dignity and self-worth. This generation of ours may never see that distant world come into fruition. It definitely will not come
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in my lifetime. But to dream and strive for it, little by little, each day of our lives is like going through a dark tunnel and seeing a light, the distant world, from afar getting ever so closer.

All of us take pleasure, guidance and strength from a song that comes along in our life that is full of meaning and purpose; a song that embodies our dreams and aspirations. For me this song is from Disneys animated movie Hercules entitled Go the Distance. For from where I am now, the US could be that far off place in space and time where I dream of presenting PIBiT to an association of the learned men and women of science. To hear with much anticipation what they think of PIBiT and how it can be used, applied and even improved for the betterment of life. Here are the inspiring lyrics to that wonderful song:

I have often dreamed, of a far off place Where a heros welcome, would be waiting for me Where the crowds will cheer, when they see my face And a voice keeps saying, this is where Im meant to be Ill be there someday, if I can go the distance I will find my way, if I can be strong

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I know every mile, will be worth my while When I go the distance, Ill be right where I belong Down an unknown road, to embrace my fate Though that road may wander, it will lead me to you And a thousand years, would be worth the wait It might take a lifetime, but somehow Ill see it through And I wont look back, cause I can go the distance And Ill stay on track, no, I wont accept defeat Its an uphill slope, but I wont lose hope Till I go the distance, and my journey is complete But to look beyond the glory is the hardest part For a heros strength is measured by his heart Like a shooting star, I will go the distance I will search this world, I will face its harms I dont care how far, cause I can go the distance Till I find my heros welcome, waiting in your arms I will search this world, I will face its harms Till I find my heros welcome, waiting in your arms

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The healing effects of the good blue light

Plate cultures of bioluminescent microbes can generate bright and stable blue light for the entire nighttime of 12 hours or more. Several shining plates can be enclosed in special glass receptacles and placed inside hotel rooms or any dark exhibition lobby or also in science discovery or exploratory hall. These bioluminescent lamps placed above or near beds can function as novel fixtures striking a chord with the occupants of our grand fight against global warming and a new green way of living. This is particularly effective since microbial bioluminescence creates bright light without consuming electricity, generating heat or producing green house gases. In addition, the good blue light of 490 nm wavelength has been found to repair serious cellular damages due to our exposure to sunlight that can carry ultraviolet radiation. Ultraviolet radiation causes numerous lesions in our DNA, the repository of our genetic information, which if left unrepaired can lead to cancer or programmed cell death. During daytime, we are exposed to the damaging part of sunlight, its energetic ultraviolet portion. So why not expose ourselves to bioluminescence coming from special lamps at nighttime, when we sleep, this should help our cells repair and heal themselves. Indeed, recent scientific studies showed that it is blue light,

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which effectively and efficiently repair the damage in our DNA thus allowing sick cells to heal themselves. Healing using blue light allow them to recover their original health and vitality. In fact, many scientists believe that one of the reasons for the existence of bioluminescence, the production of cold light, is specifically to activate enzymes that repair DNA damaged by harmful chemicals and physical agents called mutagens. I can see the day when bioluminescence will become a part of our everyday life. They can indeed become aesthetic displays in our homes, in hotel rooms, in lobbies and also science exploration centers like aquaria, plants and flowers.

Stacked luminous plate cultures brightly shining that allows one to read prints in utter darkness

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It is rare, even if it ever happens, for Vibrio fischeri to unfurl its brightest luminous wonders in nature, as it would do so in the laboratory. For instance, in seawater Vibrio fischeris population density is so low probably occurring in only ten cells in a cup of seawater. This extremely low number precludes their ability to attain the magic number or the quorum of 10,000,000 cells/mL to initiate a brilliant bioluminescence. Nevertheless, packing them close together in large numbers to attain the magic number needed to switch on their lux genes to initiate bioluminescence has been achieved in the light organs of fishes and squids. But still the kind of

sustained maximum brilliance several folds brighter than the light organs of fishes and squids and lasting long for several hours has only been achieved by Vibrio fischeri in the artificial world of the laboratory. Modern microbiology has provided the right sustenance and the optimum conditions to Vibrio fischeri for it to shine in full splendor. Perhaps Vibrio fischeri has been waiting for billions years of its existence on this planet; pre-empting the emergence of the first sponge, the first crustacean, the first plant, the first fish, the first amphibian, the first reptile, the first bird, the first mammal and most of all the first intelligent being. A thinking species finally found the means to unlock the vast bioluminescent potentials of the marine luminous

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microbes. Vibrio fischeri, maker of light, originating from the earliest periods of earths history must have been instrumental in the development of vision in primitive animals that gradually evolved into the most complex organ of higher life forms - the eye. Luminous microbes were most likely the first bioluminescent organisms producing blue-green radiant energy that interacted specifically with light-sensitive chemical pigment known as carotenoids. The interaction converts light energy into electrical energy,

which travels to the brain of animals where the unique sense of vision is created. At the peak of creation is man endowed with an intelligent mind for whom dominion over all creatures, grand and humble, is given and with whom luminous microbes alone in the dark has made a close encounter of the third kind. The contact can give man a deep sense of appreciation and an inspiration of awe for this microbes wonderful gift of light. The photons that Vibrio fischeri creates, leave its source from the cell, travel through the long inter-organismic space to reach human eyes, trigger visual reactions and bring about a new vision in man. A vision that can inspire us to learn and value the greatness of this humble speck of life and indeed all other living organisms, great and small, with which we share this planet with. It is indeed a new vision of oneness with all living organisms for molecular biology has proven that we all share a common lineage, a single ancestry.

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Microbes and plants are our cousins and animals are our brothers and sisters. A pet is defined as a plant or animal kept for amusement and entertainment or something loved and cherished. The luminous microbe to me is more than just a pet. It is the cause for a much greener world and a better life.

A photo of the first Christmas tree in history illuminated by bioluminescence

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A photo of the Worlds First Bioluminescence Illuminated Christmas Tree serving as a screensaver for computer desktops, notebooks and tablets

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BIOLUMINESCENCE:
Cold Light
Light of Life

Living Light
Milky Sea

Light of the Abyss The good blue light of a Marine Luminous Microbe:

Vibrio fischeri isolated from

a local squid shining in water

without heat and using no

electricity

STOP GLOBAL WARMING! A Merry Bright Bioluminescent Christmas for 2008

Edward Quinto, UST, Manila, The Philippines

Wishing all the grand and humble denizens of our Mother Earth

Having fun with the bioluminescence A bioluminescent Santa Claus

They say that the grand symphonies of Beethoven have to await the evolution of a twentieth century full modern orchestra to unravel their ultimate beauty and grandeur. The same can be said of Vibrio fischeri, a twentieth century modern microbiology has finally unlock the full potentials of the wonders of this microbe and the beauty of its celestial gift:

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bioluminescence. Observing the awe-inspiring beauty of its blue-green light has made the study of the intricacies of life processes and the means to protect it more meaningful, more practical, and less daunting. This smartest and heavenliest of microbes must have stolen light from the gods like the mythical Prometheus and gave it to us to see a new world in a different bluer light. Health as many say is wealth, for truly what is life if it is not lived and enjoyed in the best of health. Protecting health is what this microbe does; it gives off its own Light of Life to teach us new ways on how to keep our water and our environment clean. A clean water and environment should bring us all a healthy and beautiful life. Indeed the microbes of light are giving us lessons in green. Lessons that can make us learn how to protect effectively the health of the environment, the whole planet, and all of us. Suddenly, a world tarnished by pollution due to rapid industrialization and a booming population is showing signs of rehabilitation. I believe that we can still avert the predicted dawning of the apocalyptic effects of Global Warming due to our wanton and reckless neglect of the environment. As an amateur astronomer and a microbiologist, my eyes have seen the light from the farthest the Andromeda Galaxy to the deepest bioluminescence; the light from outer space to the light of inner space; the light from the hottest stars to the coldest bioluminescence; the light of the nonliving to the living light!

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Let us see the world in a different light, the light of bioluminescence. What a beautiful world it will be! It will surely be a greener world, a world where the water is safe to drink and the environment is clean and free of harmful pollutants. After having shared my personal and fulfilling experience with the Microbes of Light with you, I implore and enjoin all to begin the exploration of new ways of living through bioluminescence, a clean form of life energy. To bring to light the publics awareness on the usefulness of

bioluminescence, the good blue light, let us strive to request UNESCO to declare 26 January as World Bioluminescence Day. This day was not chosen because its my birthday or its Australia Day or its the Day of the Republic of India but on this day the brightest recorded apparition of a Milky Sea that appeared in the Arabian Sea was recorded by a U.S. satellite. Therefore, with utmost urgency I plead to all our Biophiles, the lovers of life of this world to help me launch this bioluminescence revolution, a new way of green living for all of us. It will surely be a new way of healthy and aesthetic life for all of us, a beautiful life illuminated by the good blue light shining a pathway to the future, to a clean and safe new world.

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A beautiful and clean view of the West Philippine Sea from the top of the world

References: Aruldoss, J.A., Viraraghavan, T., (1998) Toxicity Testing of Refinery Wastewater Using Microtox. Bull. Environ. Contam. Toxicol. 60 456 - 463

Bulich, A.A., Tung, K.K. and G. Scheibner (1990) The Luminescent Bacteria Toxicity Test. Its Potential as an In Vitro Alternative. J. Biolumin. Chemilumin. 5(2) 71 77.

Peter, S., Siersdorfer, C., Kaltwasser, H. and M. Geiger (1995) Toxicity Estimation of Treated Coke Plant Wastewater Using the Luminescent Bacteria Assay and the Algal Growth Inhibition Test. Environ. Tox. And Water Quality. 10: 179 184.

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Quinto, E. A Simple Water Toxicity Test Using Photobacterium leiognathi. Journal of Biological Education. 35(2) pp. 89 92

Schnapf, J., "How Photoreceptors Respond to Light", Scientific American, April 1987 Tchounwou, P.B. and L. Reed (1999) Assessment of Lead Toxicity to the marine bacterium, Vibrio fischeri, and to a Heterogeneous Population of Microorganisms Derived from the Pearl River in Jackson, Mississippi, USA. Reviews on Environmental Health 14(2) 51 61. Yates, I.E. and J.K. Porter (1982) Bacterial Bioluminescence as a Bioassay for Mycotoxins. Appl. Environ. Microbiol., 44(5):1072-1075.

Water on the Space Station. http://science.nasa.gov/headlines/y2000/ast02nov_1.htm

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About the Author The author is a tenured faculty member teaching microbiology and molecular biology at the Department of Biological Sciences, College of Science; University of Santo Tomas. He completed his B.S. Chemistry in 1982 and M.S. in Microbiology in 1998 in UST and earned a postgraduate certificate course in Biogeography and Environmental Assessment (Aufbaustudium) from the University of Saarland, Germany in 1993. He is a registered chemist and an elected regular member of the National Research Council of the Philippines (NRCP) in the division of Biological Sciences. Recently, he was elected Diplomate of the esteemed Philippine Academy of Microbiology (PAM). A contributor of the section in microbiology of the book: Guidebook to Plant Screening: Chemical and Biological that won the National Academy of Science and Technology (NAST) Outstanding Book Award in 2006. His national awards include winning three times (1997, 1999 and 2000) in the professional category of the Best Poster Competition sponsored by the Philippine Council for Health Research and Development (PCHRD) of the Department of Science and Technology (DOST). Recently, he won the celebrated 2007 Best Poster Paper Award, which was followed by the 2008 Best Oral Paper Award of the Philippine Society for Microbiology (PSM). UST awarded him three consecutive Silver Series Awards (2000, 2002 and 2004), the International Publication Award, Dangal ng UST Santo Domingo Award for Best in Innovation, and the Parangal ng Science Award (2006 and 2007). His international awards include the Gold in the 2001 Young Inventors Awards sponsored by the prestigious Far Eastern Economic Review (FEER) and Hewlett-Packard (HP) Invent and in 2005, the Tom Bergan Memorial Award on the occasion of the 24th International Congress of Chemotherapy in Manila given by the 212

International Society of Chemotherapy (ISC - London). He is listed in the 2006 - 2007 Marquis Whos Who in Science and Engineering. In 2003, he founded and launched the University of Santo Tomas Collection of Microbial Strains (USTCMS) whose maiden catalogue and brochure were printed through a generous grant provided him by the Philippine Council for Advanced Science and Technology Research Development (PCASTRD). His collected strains of marine luminous bacteria served as the initial microbial holdings that evolved into the present USTCMS. He is an active full member of the American Society for Microbiology (ASM) and an active life member of the Philippine Society for Microbiology (PSM), the Biology Teachers Association of the Philippines (BIOTA) and the Philippine Association of German Academic Exchange Scholars (PAGAES). He is a recipient of scholarship grants from the German Academic Exchange Service (DAAD) in 1992-1993 and in 2000; the Carl Duisberg Gesellschaft (CDG) in 1990 and the Gesellschaft fr Biotechnologische Forschung (GBF) in 1988. He has been deeply involved in microbiology and chemistry education in the Philippines with 23 years of experience through his numerous publications in local and international peer-reviewed journals, seminar-workshops, training courses, lectures, patents, paper and poster presentations, and the writing of laboratory manuals. His research interests are in the fields of microbial bioluminescence, microbial taxonomy, bioactive microbial natural products, and lactic acid fermentations. For years now, he has supervised many high school students in their science projects that went on to win national and international awards especially in the Intel Science and Engineering Fair (ISEF). His hobby includes amateur astronomy, traveling, reading, and writing.

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