DRB SUMMARY

CHAPTER 1 AND 2
• Bioprocess  a process that utilizes microbial cells / plants / animals or its cell components
(enzymes etc.) to run such a process.
In the utilization of microorganisms need to know the cell biochemistry
• Physiology is the study of chemical changes occurring within the cell associated with its
development, growth and life cycle.
• Microbiology is one of the areas of science with other sciences, potentially to be applied in
commercial / industrial scale to produce products (goods and / or services).
• Bioprocess Engineering: application of engineering principles to design, build and analyze
bioprocess for efficient, economical process with high productivity
• Advantages of Bioprocess:
1. The bioprocess reaction conditions are lighter
2. Can be used to make the product difficult or not economical (chemical reaction)
3. The enzymatic reaction is more effective and the reaction rate is faster
4. Generally require raw materials in the form of materials that can be updated
5. Development of recombinant DNA technology provides great opportunities in obtaining superior
microbes
• Lack of Bioprocess:
1. Produced products in the form of complex mixture (difficult to separate)
2. Liquid fermentation liquid and the resulting product is relatively small and unstable (expensive)
3. Bioprocess is prone to contamination (with open / open cultivation)
4. Variability (diversity) of large bioprocess, because:
Microbial cells tend to mutate
Enzymes are sentif and unstable (can be damaged / degraded quality)
• Bioprocess Engineering has an important role in the development and commercialization /
industrialization of bioprocess products. To obtain a bioprocess product that has the best results
(efficiency level and high productivity), optimal results for commercial, and economical need to do
some research / development in bioprocess engineering.

Cultivation on BioProcess
• Microbial cell structure
• Cell theory: all living things are made up of cells
• Cells: the smallest living unit of the body MH  the place of life function
• Cell Type: based on the organization of its cell structure
- Prokaryotes (without cell nuclear membrane)
- Eukaryote (having cell nucleus membrane)
• Prokaryotic (single cell microorganisms (bacteria & cyanobacteria))
Characteristic features :
- does not have a true cell nucleus
- does not have a cell membrane that separates the nucleus from the cytoplasm
- the division of the nucleus by means of cleavage (asexual simple)
- some have flagella
- small and simpler cells than eukaryotic
- cell growth and metabolism faster than eukaryotic
• Eukaryotic (algae, fungi, protozoa, plants and animals)
Characteristic features :

- has a true cell nucleus and internal membrane system

- complex cell nucleus and cell-membrane membrane
- More complicated nuclear divisions (mitosis)
- eukaryotic cells require more varied internal organelles to carry out metabolism, generate
energy and transmit nutrients to all parts of their cells

• ORGANISM MANAGEMENT  System 5 World (Kingdom) (1969 by Robert Whitaker):

1. Kingdom Prokaryote (Monera): bacteria
- Known as the oldest cell
- It has no cell nucleus and membranes in its organelles
Other Kingdoms are: The eekota who has a cell nucleus and
membrane, consisting of:
2. Kingdom Protista: mostly unicellular, has no network structure and most have flagella.
Example: protozoa, single celled algae
3. Kingdom Fungi: can be unicellular (yeast) or multicellular (mold). Many are "saprotrophs".
4. Kingdom Plantae: multicellular, photosynthetic  multicellular algae, plants
5. Kingdom Animalia: Multicellular, heterotrophic  animal
• MICROBAL CHARACTERISTICS  Useful for regulating microbial metabolism and microbial
identification / classification)
1. Cultural Characteristics
- growth media (liquid / solid form, media components etc) - temperature, pH (acid, neutral,
alkaline)
- availability of oxygen (aerobic / anaerobic / facultative)
2. Morphological Characteristics
- Visual  observation with the naked eye
- Shape (length, width, diameter)  microscopic
• Morphology (Forms) Bacteria (certain bacterial characteristics):
a. cocci (spherical / spherical)  Streptococcus
b. bacilli (rod-like / stem)  E. coli
c. spirilla (spiral)  Spira
• Reproduction of bacteria generally by binary division
• Bacterial use:
- As a decomposer
- Fixing nitrogen in soil (nitrobacter)
- Use by humans (on an inudstri / household scale)
• Fungi (Eukaryotic Microorganisms)
- Unicellular and multicellular (hifa)
- Saprophyte, parasite and mutualistic (heterotrophic organisms, lack chlorophyll)
- Temperature distribution - 20 s.d 30oC
- Prefers acidic pH  prevents bacterial contamination
- Most aerobic or facultative (few)
• Yeast
- kemoorganotrof, using organic compounds as sources of energy.
- Unicellular, several multicellular species in the presence of pseudo hyphae / mycelium
- Cell shape: round, oval (like lemon / pear) or cylindrical
- Size is very varied, bigger than bacteria
- Asexual reproduction: "budding" (budding), binary splitting / sporulation (askospora)
- The main carbon source of hexose sugar (glucose, fructose), disaccharide (sucrose, maltose). Some
species may use pentose sugars (ribose), alcohols and organic acids  can be for identification
- Usually requires source of N in the form of hydrolyzate protein (pepton, yeast extract etc)
- Aerobic obligate, facultative anaerobes (S. cerevisiae  no O2 respiration  biomass & when no
fermentation 22 ethanol)
- Grows well at neutral pH or slightly acid
• Kapang
- More resistant to dryness than yeast or bacteria
- Mesophilic & prefer acidic conditions
- Generally can only live in the presence of oxygen (obligate aerob)  need aeration; thick
overlap is limited to cultivation of solid substrates (fermentation of tempe, soy sauce, citric
acid etc)
- How to reproduce: cell division, asexual or more (sexual) and sexual (fewer & lesser)
- Structured or non-septate hypha structures  note agitation on bioreactors, preferably air
mixing (non-mechanical)
- Multicellular, growth at the tip of hyphae  forming mycelium  cell separation by
membrane filtration
• How to Take Nutrient by Microbes (Uptake of Nutrients)
1. Phagocytosis - Protozoa
2. Permeability Absorption - mostly m.o
Passive Transport
Simple diffusion
Facilitated diffusion
Active Transport
Translocation Group
• Passive diffusion
- The process of migrating molecules through membranes from high concentration areas to
lower concentrations.
- The diffusion rate is proportional to the driving force of the concentration difference across
the membrane
- Some compounds such as O2, CO2 & glycerol can pass through the plasma membrane by
passive diffusion
• Facilitated diffusion
- Using protein carrier  the rate of diffusion through selective permeable membranes can be
increased by the use of "carrier protein" (sometimes called permease), embedded in the
membrane
- The rate of facilitated diffusion is increased by the concentration gradient
• Active transport
- Active transport is the process of transporting solute molecules to higher concentration
areas or
- Moving transport rates use metabolic and opposite energy inputs with concentration gradients
- Some compounds such as glucose, amino acids, lactose, galactose, Na +, K +

• Translocation group
An example of a translocation group  phosphoenolpyruvate: sugar phosphotransferase system
(PTS), which transports various sugars into prokaryotic cells and simultaneously phosphorylates
using phosphoenolpyruvate (PEP) as a phosphate donor
PEP + sugar (outside) pyruvate + sugar-P (inside)

DRB SUMMARY
CHAPTER 3
METABOLISM (Energy Requirement & Source for Microorganism Cells)

• Metabolism:
All chemical activity occurring in the cell, both decomposing (catabolism) and forming
(anabolic) functions  principally oxidation-reduction reactions
• Function of Metabolism
- Break down complex compounds (nutrients) into substrate / precursor (initial compound)
- Capturing energy released from nutrient breakdown (catabolism)
- Molecular synthesis required (anabolism)
• Relationships of catabolism and anabolism
- Catabolism: oxidative; exergonic (releasing energy)
- Anabolism: Reductive, endergonic (requires energy)
• Energy is used by microbial cells for activity
- BIOCHEMICAL PROCESS OF ENERGY (CATABOLISM)
1. Energy Requirement & Sources for Microbes
2. Chemical Energy & Energy Transfer
3. ATP synthesis by microbes
4. The Path of Nutrient Catabolism
- BIOCHEMICAL PROCESS YG NEED ENERGY (ANABOLISM / BIOSINTETIC)  studied in Metabolism II
1. Energy Usage for Biosynthetic Processes
2. Biosynthesis of Nitrogen-containing Compounds:
Biosynthesis of Amino Acids & Proteins
Nucleotide Biochemical & Nucleic Acid
3. Carbohydrate Biosynthesis
4. Lipid Biosynthesis (Long Chain Fatty Acid and Phospholipide)
5. Energy Usage for Processes Other Than Biosynthesis:
Nutrient Transport into Cell & Motility
• Chemical Energy: energy contained in chemical bonds
Bacteria get this energy with the chemical compound catabolism energy is stored temporarily
generally on ATP
• ATP (adenosine triphosphate) stores and transfers energy by receiving / releasing inorganic
phosphate (Pi)
  disintesis dari ADP (adenosine diphosphate)
 (Kandungan energi 1 mol ATP = 7.3 kkal/mol)
energi
ADP + Pi ATP + air
energi
ATP + air ADP + fosfat
• High energy bonds are formed through oxidation reactions
• ATP synthesis by microbes
1. Substrate Level Phosphorylation (energy formation (ATP) generally in fermentation) - The
inorganic phosphate addition process in the oxidizing compound in the metabolic pathway
- There are 2 stages in Glycolysis and 1 stage in the Krebs Cycle (TCA) that produces ATP
through Substrate Level Phosphorylation
2. Oxidative phosphorylation (Kemiosmosis) (occurs in the process of respiration)
- The last electron acceptor on RTE is O2 and H2O will be formed by generating energy
(ATP).
- Involves a series of electron displacements involving electron-carrying compounds:
reduced nicotine adenine dinucleotide (NADH) / FADH2 and cytochome used for the
synthesis of ATP from ADP
3. Photophosphorylation
- Occurs in organisms that have chlorophyll
- Solar light is used to generate protonmotive force that drives ATP synthesis  ATP
production using energy from sunlight (not from breaking organic substrates)  occurs in
photosynthesis
• RTE is the final stage of cellular respiration in which energy in NADH & FADH2 is
released, then transferred and stored in ATP
• RTE takes place due to the formation of H + ion gradients between matrix and space
between mitochondrial membranes  drives ATP synthesis through Oxidative
Phosphorylation (Kemiosmosis)
• Location of the RTE:
Eukaryotes: - mitochondria
Prokaryotes: - cytoplasmic membranes
• Catabolism of Complex Nutrients
- M.o can use various types of compounds of energy sources  complex molecules (proteins,
fats, polysaccharides) hydrolysed enzymatically into simple compounds
- The simple molecule can then be converted into another compound that can enter the
trajectory of cell dissimilation:
1. Proteins  peptides  amino acids  form asampiruvat  into the Krebs Cycle
2. Fat  glycerol  to Glycolysis; fatty acids  Acetyl CoA  to the Krebs Cycle
3. Polysaccharides  Monosaccharide / glucose to Glycolysis
• How microbes get energy

- Fermentation:
oxidation-reduction reactions in biological systems that produce energy  as electron donors and
acceptors are used organic compounds (need not be O2)  inefficient (carbohydrate compounds in
the form of glucose).
Involves glycolysis but does not involve RTE. Produces 2ATP through substrate level phosphorylation.
- Aerobic respiration
The cell has an oxidase enzyme, so it can use O2 as the last electron acceptor. Energy produced 20x
from fermentation.
Involves metabolic pathways:
1. Glycolysis
2. The conversion of pyruvate to acetyl Co-A
(Pyruvic acid (from glycolysis) is oxidized to Acetyl CoA)  as the initial substrate of Krebs Silk by
ingesting 1 NADH
3. The Krebs Cycle
4. Electron Transport Chain & Oxidative Phosphorylation
- Anaerobic respiration
Respiration without the use of oxygen from the outside, but using inorganic compounds (CO2,
sulfate, or nitrate) present in the substrate as the last electron acceptor  Less energy produced than
aerobic respiration.
 Anaerobic Respiration involves:
Glycolysis, Cyclic Acid Cycle and RTE
 The total energy yield per oxidized glucose molecule is smaller or equal to 36 ATP (less than
Aerobic Respiration, but more than fermentation)
- Photosynthesis
Uses solar energy to increase the energy of electrons generated by oxidation of water into oxygen
Reaction: n CO2 + H2O (CH2O) n
When light is absorbed, the electrons are released from the chlorophyll and will enter the Electron

Rantai Transpor Elektron (RTE)  energi (ATP)

DRB SUMMARY
CHAPTER 4
METABOLISM II (CATABOLISM & ANABOLISM)

• Metabolic flux
 Intermediate transfer rate along the metabolic pathway.
 The rate varies depending on the activity of the enzyme, so the identification of key enzymes that
control the rate of carbon flow etc for the synthesis of various products is very important in. increase
productivity
• How to Control Metabolism:
1. Setting Nutrient Taking
- Beginning with the up-take arrangement of nutrients  transport across the cytoplasmic membrane
(specific transport mechanism), except oxygen and other little C compounds, after sufficient nutrient
levels, the nutrient-taking process stops.
- The rate of taking (consumption) of carbon (glucose) sources can be a limiting process for cell
growth
- The compound molecule enters the cell through the cytoplasmic membrane by:
 1). The Mechanism is Not Dependent on Energy
a). Passive Diffusion
b). Diffusion facilitated
2). Depending on Energy Mechanism
a). Active Transport
b). Translocation Group
2. Compartmentalization of the Metabolic Trajectory
- The simplest form of metabolic control is the use of the compartment (organelle) within the cell, so
that the accumulation of certain metabolites separate  organelles are used to control the reaction
inside the cell
- Example:
1) The Krebs cycle that occurs in mitochondrial eukaryotes separate from glycolysis (in the
cytoplasm)
2) The location of eukaryotic fatty acid biosynthesis (in the cytoplasm) is separate from its
degradation sites (peroxisome organelles)
- Bacteria (prokaryotes) do not have compartmentalization, so use another method for controlling
their metabolism e.g RTE (electron transport chain) on the cytoplasmic membrane
- Eukaryotes use organelles to comparte metabolic pathways, so metabolic pathways occur at
specific locations (mitochondria, Golgi body, lysosone, nucleus, fine RE, rough RE, etc.)
3. Control of Enzyme Synthesis
- Enzymes in cells are constitutively present (present at all times) and others exist when necessary
(induced) or when no longer needed to be lost (repression)
- Induction and repression are governed by genes that encode protein synthesis:
DNA transcription  mRNA  translation  protein synthesis
- Catabolite repression
The relative decrease in the rate of synthesis of a specific enzyme, since there are more assimilated
carbon sources (glucose) = Glucose Effects, other than carbon sources (nitrogen source, phosphate
and sulfur), the usefulness of various sources for this catabolite process is that cells can use the most
substrate both (efficiently and effectively) in the metabolite process.
4. Modification of Enzyme Activity
- The activity of the synthesized enzyme can be arranged in various ways:
1) "Post-Transcriptional" Modification (chemical modification) The synthesized enzyme is modified
to be active or inactive by converting by enzyme phosphorylation of the enzyme
2) Effect of Effector (Inhibition of Feedback)
ABCCDE
- High concentrations of end products may cause enzyme synthesis repression on metabolic
pathways  rapid response to high end product accumulation which may cause disturbed
metabolism
• CATABOLISM
In microbes known 4 lanes breaking glucose into pyruvic acid
1. Embden-Meyerhof Parnas (EMP) or Glycolysis (independent of oxygen) pathway
 eukaryotes (fungi) & prokaryotes (mostly bacteria)
2. Pathway of Hexosamonophosphate (HMF) = Pentosa Phosphate = Phosphogluconate
 bacteria, various organisms
3. Entner-Doudoroff Line (ED)
  some bacteria, for example: Zymomonas mobilis producing ethanol; lactic acid bacteria
(homolactate)
4. Path Phosphoketolase (FK)
 lactic acid bacteria heterolactate
• ANABOLISM
• Biosynthesis of Amino Acids & Proteins
1) Asino Amino Biosistesis: Principle  transamination reaction, ie binding of ammonia (NH3) groups
to carboxylic groups of α-ketoglutarate compounds (from the Krebs Cycle). The binding of the amine
group is 2 ways: direct and indirect.
2) Biosynthesis of proteins: Amino acids should be energized before joining
into protein. Amino acids + ATP  amino acids-AMP + pyrophosphates (2P)
• Fatty Acid Biosynthesis
- Fat biosynthesis starts from the biosynthesis of fatty acids
- Precursors: Acetyl Co-A
- Reaction to fatty acid biosynthesis:
1) Transfer of an acetyl group and a malonil group from CoA to ACP: acetyl / malonil-CoA  acetyl-
ACP & malonil-ACP
2) Acetyl-ACP + malonil-ACP condensation forms acetoacetyl-ACP by releasing CO2.
3) Reduction and dehydration reaction reaction  produces butyryl-ACP  butyric acid
• Carbohydrate Biosynthesis
- Glycolysis & Pentococcal Phosphate Path (HMF) is used for the interconversion of sugar
compounds
- Monosaccharides in order to join into polysaccharides must be energized (from ATP).
• Nucleotide biosynthesis

• Biosynthesis of Antibiotics
• etc

DRB SUMMARY
CHAPTER 5
ENZYME

• Enzymes:
- The protein molecules produced by living cells
- Biochemical reaction catalyst  specific reaction
- Molecular: a protein composed of amino acids in a regular composition and sequence and
still  form a 3-dimensional structure. "Active Side" (bag surface of the enzyme that interacts
with the substrate)
• Enzyme and Classification Names
- Based on enzyme source  trade name (papain, bromelin, fisin, pancreatin)
- Based on the type of reaction, the type of substrate or form produced  (+) - ase (lipase,
protease)
- "International Union of Biochemistry" E.C. (Enzyme Commision) + 4 numbers
Ex: (alcohol dehydrogenase  E.C.1.1.1.1)
Digit 1: enzyme class: oxidoreductase, transferase, hydrolase, liase, isomerase, and ligase
Digit 2: subclass enzyme
Digit 3: sub-subclass enzyme
Digit 4: Serial serial number on sub-subclass
• How it Works Enzymes:
 accelerate, increase chemical reaction.
 Lokasi pengikatan substrat (lokasi aktif dengan sejumlah ikatan non kovalen)
Ikatan yang terlibat :

 Jenis pengikatan enzim ada 2:

 1) Lock and key: The active side is rigid, so the enzyme can bind to the substrate having the correct
structure and space (S and E)
2) Induced-fit: The active side is not rigid  substrate (glucose) & enzyme amino acid induces shg
formed the exact space structure between E and S
Cofactors: compounds
that can pass through
the membrane analysis.
Cofactors require
enzymes to react in full

Apoenzim: a compound
that does not pass
through the protein part

• Enzyme activity: the rate at which the enzyme converts the substrate into a
product.
•
 Enzyme work: speed up the reaction by decreasing the activation energy (Ea)
When the reaction time is monitored, the rate at a given time (v or vt) is a
tangent gradient (slope) of the curve
d [ P] d[S ]
v  
dt dt

 Aktivitas enzim dinyatakan sebagai unit “U”

- ‘1 unit : sejumlah enzim yang akan mengkonversi 1 mikro mol (μmol) substrat per menit pada
kondisi pH dan suhu tertentu
- 1 kat (1 katal) = jumlah enzim yang mengkatalisis pengubahan 1 gmol substrat per detik
1 kat = 1 gmol per detik = 60 gmol per menit
= 60 x 106 mikromol per menit
 = 6 x 107 U
Konsentrasi enzim  U/ml atau nkat/ml
 Aktivitas enzim spesifik (ukuran kemurnian enzim)
The activity only gives an idea of how many enzymes are contained in the sample. If
desired, the purity of the isolated enzyme.
Aktifitas Spesifik = aktivitas enzim/protein terlarut
 Faktor Yang Berpengaruh Terhadap Aktifitas Enzim
1) The concentration of enzymes
2) Substrate concentration
3) Concentration of product
4) Specific activities (purity)
5) Cofactor
6) Temperature
7) pH
8) Ionic power
9) Solvent
10) Inhibitor (heavy metal
)dan aktivator (ADP adalah aktivator enzim PFK.)
• Activity calculation
- Vmax is the theoretical maximum rate of enzymatic reactions.
- Km is a measure of the affinity of the enzyme against a particular substrate (constant (constant) so
that its value is independent of substrate concentration / enzyme)  substrate concentration [S]
which gives 0.5 x Vmax.
- The greater the Km value, the greater the substrate concentration required to reach ½ Vmax  the
lower the affinity, the greater the substrate concentration required to reach ½ Vmax
• Example problem: Calculate the change in substrate concentration required to increase the
reaction rate from 15% to 75% of the maximum reaction rate, when the reaction rate follows the
Michaelis-Menten Law

 Jawab :

 Perubahan konsentrasi S yang dibutuhkan untuk meningkatkan laju reaksi dari V1→V2 = 3
Km/0.1765 Km
= 16.9972 kali  17 kali
 Inhibisi Enzim
- Inhibitor  mengurangi laju reaksi yang dikatalisis enzim
- Studi inhibisi enzim, mempelajari :
Bagian aktif enzim, mekanisme kerja enzim, gugus fungsi aktif, fisiologi enzim
- Jenis penghambatan :
Kompetitif
Non kompetitif
Unkompetitif
Oleh produk  A E1 B E2 C E3 D


• Source of Enzyme:
1) Plants (papain, bromelin, ficin)
2) Animals (renin)
3) Microbes
• Commercial Enzymes:
1)  amylase: Aspergillus oryzae, Bacillus subtilis
2) Selulase: Aspergillus sp., Tricoderma reesei
3) Glucoamylase: A. niger, Rhizopus sp.
4) Lipase: Aspergillus sp., Rhizopus sp., Candida cylindraceae
5) Protease: Aspergillus niger, A. oryzae, Rhizopus sp., Bacillus sp.