Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Abstract : Anaerobic decomposition of organic material done with the help of microorganisms and occurs in the
methanogen environment where organic matter degraded without presence of inorganic electron acceptor
(oxygen, nitrate, sulphate, sulphur, Fe3+, Mn4+). Respiration and fermentation proccess which occurs on
anaerobic condition uses proton or bicarbonate as electron acceptor. Optimization of organic material
anaerobic degradation is the result of a variety of bacteria that work in a consortium. Integrated analysis of the
overall dynamics of the growth and diversity of predominant bacteria in the degradation process required, as
the process will not run if there is only one of the bacteria species, it requires a consortium of more than one
species. The objective of this research is to observe the dynamics of population and microbia diversity which
take a role in the liquid phase of biowaste degradation stages using the anaerobic batch reactor. 6L-sized
reactor operated for 70 days and samples taken for counting total bacteria colonies per 1 week. Morphology
observation and the gram-reaction test on the continued isolated sample at day-0. day-7, day-28, and day-69
resulted in 12 isolate with pure gram-positive reaction that consists of 10 rod-shaped cells and 2 coccus cells
The identification using API 20A resulted bacteria of Clostridium beijerinckii/butyricum, Clostridium
botulinum/sporogenes, Actinomyces meyeri/odontolyticus, Streptococcus intermedius, Strepcoccus constellatus,
and Clostridium innocuum, where the group of Clostridium sp become the dominant bacterial group in the work
of the anaerobic batch reactor. Growth dynamics of microorganisms is influenced by environmental factors
such as pH and in line with the VSS parameters.
Key words: anaerobic digester, dynamics growth, isolation, Gram staining, API 20A
Abstrak : Penguraian materi organik secara anaerob dilakukan dengan bantuan mikroorganisme dan terjadi
pada lingkungan metanogen dimana materi organik didegradasi tanpa kehadiran elektron akseptor anorganik
(oksigen, nitrat, sulfat, sulfur, Fe3+, Mn4+). Proses fermentasi dan respirasi yang terjadi pada kondisi anaerob
memanfaatkan proton atau bikarbonat sebagai elektron akseptor. Optimalisasi degradasi materi organik secara
anaerob merupakan hasil kerja berbagai bakteri yang bekerja secara konsorsium. Analisis yang terintegrasi
secara menyeluruh terhadap dinamika pertumbuhan dan keanekaragaman bakteri yang berperan dalam proses
degradasi diperlukan karena proses tidak akan berjalan jika hanya terdapat salah satu bakteri saja, konsorsium
memerlukan lebih dari satu spesies. Tujuan dari penelitian ini yaitu untuk mengamati dinamika populasi dan
keanekaragaman mikrobia yang berperan dalam tahapan degradasi biowaste fasa cair dengan menggunakan
anaerobic batch reactor. Reaktor berukuran 6L dioperasikan selama 70 hari dan diambil sampel untuk
pengecekkan total koloni bakteri setiap 1 minggu. Hasil pengamatan morfologi dan pengujian reaksi gram pada
isolasi lanjutan sampel hari ke-0, hari ke-7, hari ke-28 dan hari ke-69 menghasilkan 12 isolat murni dengan
reaksi gram positif yang terdiri dari 10 sel batang dan 2 sel kokus. Identifikasi dengan API 20A menghasilkan
bakteri Clostridium beijerinckii/butyricum, Clostridium botulinum/sporogenes, Actinomyces
meyeri/odontolyticus, Streptococcus intermedius, Strepcoccus constellatus, dan Clostridium innocuum.
Kelompok genus Clostridium sp menjadi kelompok bakteri dominan dalam kerja reaktor batch anaerob.
Dinamika pertumbuhan mikroorganisme dipengaruhi faktor lingkungan berupa pH dan sejalan dengan
parameter VSS.
Kata kunci: reaktor batch anaerob, dinamika pertumbuhan,isolasi, pewarnaan Gram, API 20A
SW8-1
INTRODUCTION
SW8-2
is carried out with the API (Analytical Profile Index) 20A method for the kind of the anaerob
group's bacteria.
In various research studies, the stages that occur in the processing anaerob
(hydrolysis, fermentation, asetogenesis, metanogenesis) has been well described, but the
relationship between the composition of groups of microbes, diversity, and dynamics, with
the performance of reactor has not been explained well. Research studies conducted more on
the relationship dynamics of the target population in a specific (eg metanogen groups archae,
bacteria sintropi) on the performance of reactor (Raskin et al., 1994; Griffin et al., 1998;
Angenent et al., 2002; Godon et al., 1997; Sekiguchi et al., 1998 dalam Raskin et al.,2007).
The objective of this research is to observe the dynamics growth of the predominant bacteria
and diversity microbial that role in the degradation stages of biowaste using the liquid phase
in anaerobic batch reactor. Optimization of organic material anaerobic degradation is the
result of a variety of bacteria that work in a consortium(Burke, 2001). Therefore, from this
research is expected to study the diversity bacteria in the anaerob processing and give
information concerning the dynamics of the population and succession of the bacterial group
that happened. Identification of ecological microbial that found from the isolated daily
sample spot can be used to observe the activity of each group in reactor performance.
METHODOLOGY
This research was conducted at the Laboratory of Water Quality, Environmental
Microbiology Laboratory and the Laboratory of Industrial Hygiene and Toxicology Study
Program Environmental Engineering ITB.
Reactor Processing Biowaste
Reactor system that is used in the batch system is Anaerobic Batch Reactor sized 6 L
and operated for 70 days.
Sample Taken
Sampling method used in this research is is grab sample. Sampling was done once
every week as much as 20 ml. Sample from the anaerob batch reactor was only done in the
form of washing water variations such as tap water where sampling point was located in the
middle of the reactor to get the optimal assumption of homogenity point.
Media
Blood agar (Merck, Germany) is used as a selective medium with the composition in
grams per liter: beef extract (10 g), peptone (10 g), NaCl (5.0 g), agar (15 gr), aquadest (1
liter) and sterile horse blood (5-10%). Nutrient agar (Merck, Germany) is used as the
insulation medium and purification culture with the composition in grams per liter: beef
extract (1 g), yeast extract (2 g), peptone (5 g), NaCl (5 g), agar (15 gr), and aquadest (1
liter). Each media sterilized using autoclave.
Enumeration and Insulation Different microorganisms
The cultivation of microorganism is done by inoculate microorganism in to blood
agar. Inoculation technique used the pour plate technique, previously done with the dilution
that results obtained colonies grown a pure culture. Enumeration calculation of the total
colonies growth is observed after incubation in anaerobic condition for 48 hours (temperature
370C) which formed 30-300 colonies. Enumeration of each weekly sample will be observed
by morphology criteria and types of hemolysis produced from each colony. If there is a
number of colonies which are assumed as dominant colony, it will be cultivated with
continue isolation . Isolation done using the nutrient agar and purification carried out to
SW8-3
obtain a single colony with a streak plate method: the quadrant streak (Method B).
Morphology criteria of each single colony also observed in order. Isolation the anaerob
bacteria should be done using equipment that formed anaerobic conditions such as anaerobic
glove box, anaerobic chamber, anaerobic jar (SD, Herbert, 1978).
Examination with the microscope
Examination was conducted to observe the characteristics of the cell walls of bacteria
and the shape of the cell using a light microscope (Swift Instrument International SA
No.7864820) with 1000x enlargement and using the immerse oil. The observations were
done using the Gram strain method.
Identification with API 20A Method
The API 20A method consists of 21 kinds of biochemical test that is used to identify
species of anaerob bacteria. Biochemical test consists of testing indol; urease; fermentation of
glucose, mannitol, sacrose, lacktose, maltose, salicin, xylose, arabinose, gliserol, cellobiose,
mannose, melezitose, raffinose, sorbitol, rhamnose, trehalose; gelatin hydrolysis; esculin
hydrolysis (ß-glucosidase ); catalase. The principle of Analytical Profile Index method is to
inoculate identified pure bacteria suspense into 21 types of microtube or well containing the
respective substrate or medium for the biochemical test. Conducted during the next 48 hours
incubation at 370C in the anaerobic jar. During the incubation process, the bacteria will make
the metabolism and cause a color changing in both media spontaneously or with the addition
of a reagent first. Reaction on the results of each test will get the next combination of
numbers using the API 20A software for identified the species of bacteria.
SW8-4
Log bcteri ammount
3,0E+09
3,0E+08
(cfu/ml)
3,0E+07
3,0E+06
3,0E+05
0 10 20 30 40 50 60 70 80
Hari Ke‐ total koloni …
SW8-5
Table 1 Isolation of Results Sample Day-0, to-7, to-28, to-69
Name J E1 E2 A F1 F2 K F3 G C D F4
SW8-6
Figure 2 (J)Small Rod-Cocci, Figure 3 (E1)small Rod, Figure 4 (E2) Rods,
Gram positive thin, Gram postive Gram postive
Figure 5 (A) Small-Rods, Gram Postive Gambar 6 (F1) Rod-Cocci, Gram Positive
Figure7 (F2) Short-Rods, Gram Postive Gambar 8 (K) Coccus, gram positive
SW8-7
Figure 9 (F3) Mikroccous, Gram positive Gambar 10 (G) Micro-rods, gram positive
SW8-8
Table 2 Biochemical test result with API 20A
Fermentation Hidrolysis Fermentation
Symb Ind catala spor gra co
urease
ol ol se e m cc
Glu Man Lak Sac Mal Sal Xyl Ara Gel Esc Gli Cel Mne Mlz Raf Sor Rha Tre
J ‐ ‐ + ‐ ‐ + + + ‐ ‐ ‐ + ‐ ‐ + ‐ ‐ ‐ ‐ + ‐ + + ‐
E1 ‐ ‐ + ‐ ‐ ‐ ‐ ‐ ‐ ‐ + + ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ + + ‐
E2 ‐ ‐ + + ‐ + + + ‐ + ‐ + ‐ + + ‐ ‐ ‐ + + ‐ + + ‐
A ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ + + ‐ ‐ ‐ ‐ ‐ ‐ ‐ + ‐ + + ‐
F1 ‐ ‐ + ‐ ‐ + + ‐ ‐ ‐ ‐ + ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ + ‐
F2 ‐ ‐ + ‐ ‐ + + ‐ ‐ ‐ + + ‐ ‐ ‐ ‐ ‐ ‐ ‐ + ‐ + + ‐
K ‐ ‐ + ‐ + + + + ‐ ‐ ‐ + ‐ + + ‐ ‐ ‐ ‐ + ‐ ‐ + +
F3 ‐ ‐ + ‐ ‐ ‐ + + ‐ ‐ ‐ ‐ ‐ ‐ + ‐ ‐ ‐ ‐ + ‐ ‐ + +
G ‐ ‐ + + ‐ + ‐ + ‐ ‐ ‐ + ‐ + + ‐ ‐ ‐ ‐ ‐ ‐ + + ‐
C ‐ ‐ + + ‐ + + + ‐ + ‐ + + + + ‐ ‐ ‐ ‐ + ‐ + + ‐
D ‐ ‐ + ‐ ‐ + + + ‐ ‐ ‐ + ‐ ‐ ‐ ‐ ‐ ‐ ‐ + ‐ + + ‐
F4 ‐ ‐ + ‐ ‐ + + + ‐ ‐ ‐ + ‐ ‐ + ‐ ‐ ‐ ‐ + ‐ + + ‐
E1 Clostridium botulinum/sporogenes
F1 Actinomyces meyeri/odontolyticus
F2 Clostridium botulinum/sporogenes
7th day
K Streptococcus intermedius
F3 Strepcoccus constellatus
28th day
G Clostridium innocuum
C Clostridium beijerinckii/butyricum
69th day D Clostridium sp
F4 Clostridium sp
SW8-9
6000
VSS (mg/l) and Bacteril
3,0E+09
Total Colony (cfu/ml)
5000
4000 3,0E+08 VSS
3000
3,0E+07
2000
1000 3,0E+06
total
0 3,0E+05 koloni
0 10 20 30 40 50 60 70 80 Bakteri
Day
Figure 2 Relationships between Total Ammount of Bacterial Colony with VSS Parameters
The relationship between the total ammount of bacterial colony and the environment
physical conditions such as pH is shown in Figure 3. The graph shows a decrease in pH after
the 0-day that describes the forming of fermentation /acidogenesis stages that causes a low
pH environment. The changes of the environment conditions will affect the growth and early
life of the bacteria, so the bacteria that are not able to adapt to such conditions will die in the
environment that does not support the metabolism process of bacteria. Along with the
increase of pH to the neutral stage, where most of the metanogen bacteria grow on optimal
conditions (Supriatin, 2008), the total amount of the bacterial colony also re-increase.
Fluctuations of the number of bacterial colonies occur because of the growth and death of the
bacteria due to environmental factor such as the pH.
Ttotal amount of bactery
9 3E+09
8
300000000
(cfu/ml)
7
6 pH
30000000
5
4 3000000
3 Total koloni
2 300000 bakteri
(cfu/ml)
0 20 40 60 80
pH
Figure 3 Relationships of Total Ammount of Bacterial Colony with pH parameter
CONCLUSION
In the processing of biowaste using Anaerob Batch Reactor, some fluctuations of the
total number of bacterial colony occurred. The initial morphology observation gave the
results of 31 bacterial species with 7 types of bacteria that were assumed dominant. The
Bacteria that were resulted from the further insulation were the Gram-positive bacteria
consist of 10 rod-shaped cells, 2 cell-shaped kokus. Identification with API 20 A acquired
bacterial Clostridium beijerinckii / butyricum, Clostridium botulinum / sporogenes,
Actinomyces meyeri / odontolyticus, Streptococcus intermedius, Strepcoccus constellatus, and
Clostridium innocuum. The group of genus Clostridium sp become the dominant groups of
bacteria that appear and identified at biowaste processing in Anaerob batch reactor. Total
dynamics of the colony is influenced by environmental parameters such as pH, and it is
proportional with the VSS parameters.
SW8-10
ACKNOWLEDGEMENT
This research is part of the joint Bandung Institute of Technology, Indonesia;
Lembaga Ilmu Pengetahuan Indonesia (LIPI) Bandung, Indonesia with Karlshure University,
Germany.
References
Batra, Navneet dan Bali, Vandana, dkk, 2009, Metagenomics: Concept, methodology,
ecological inference and recent advance, Department of Biotechnology, Panjab University,
Chandigarh, India, Biotechnologi Journal
Bitton, Gabriel, 1994, Wastewater Microbiology, Departement of Nevironmental
Engineering Sciences, University of Florida, Wiley-Liss Press: Newyork
Gallert, C & Winter, J. 1999. Bacterial Metabolism in Wastewater Treatment Systems.
Environmental Processes I. 11A:17-48
Hwang, Seokhwan, dan Kim, Jaai, dkk, 2005, Use of Real-Time PCR for Group-
Specific Quantification of Aceticlastic Methanogens in Anaerobic Processes: Population
Dynamics and Community Structures, School of Environmental Science and Engineering,
Pohang University of Science andTechnology, Pohang, Kyungbuk
Levett.1991. ‘Anaerobic microbiology. A practical approach’. Oxford University
Press. New York
Raskin, Lutgarde, Mackie, I.Roderick, McMahon, D.Katherine, Griffin, E.Matt, 1997.
Methanogenic Population Dynamics during Start-Up of Anaerobic Digesters Treating
Municipal Solid Waste and BiosolidsMatt. Environmental Engineering and Science, Civil
Engineering Laboratory, University of Illinois at Urbana–Champaign.
Raskin, Lutgarde dan Tumbleson, E.Mike, 2007, Microbial diversity and dynamics in
multi- and single-compartment anaerobic bioreactors processing sulfate-rich waste streams,
Department of Civil and Environmental Engineering,University of Illinois at Urbana-
Champaign, Urbana, IL 61801, USA. Environmental Microbiology Journal Salle. 1980.
‘Fundamental Principles of bacteriology’. McGraw-Hill Book Company. Tokyo
Stams, Alfons J.M. 1994. Metabolic Interactions between Anaerobic Bacteria in
Methanogenic Environments. Antonie van Leeuwenhoek.66:271-294.
Supriatin, Yati. 2008. Kajian Produksi Biogas Skala Laboratorium dengan Inokulum
Konsorsium Alami Metanogen dalam Substrat Bungkil Jarak Pagar (Jatropha curcas L).
Tesis Bioteknologi ITB.
SW8-11