DYNAMICS GROWTH OF THE PREDOMINANT

MICROORGANISMS IN BIOWASTE DEGRADATION USING

ANAEROBIC BATCH REACTOR

DINAMIKA PERTUMBUHAN MIKROORGANISME YANG

BERPERAN PADA DEGRADASI BIOWASTE DALAM REAKTOR
ANAEROB TERCURAH

Asri Suciati1 and Barti Setiani Muntalif2

Study Program of Environmental Engineering
Faculty of Civil and Environmental Engineering, Institut Teknologi Bandung
Ganesha Street no. 10 Bandung 40132
1
cicilia_andrea@yahoo.com dan 2barti_setiani@yahoo.com 
 

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

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INTRODUCTION

Organic waste or biowaste dominant derived from market, or commercial activities. In

Bandung city, the type of organic waste can reach 70% of the total waste generated. The
increasing waste along with increasing number of people, the system of waste management in
Bandung through landfilling not be sufficient to accommodate the existing waste, will
ultimately provide a problem for the environment. Since that, an alternative solution was
developed through the efforts degradation organic material using MBT method (Mechanical
Biological Treatment) with the ability to utilize organic waste that can be decomposed
biologycally by microorganisms. MBT method is able to stabilize the organic waste so it can
be removed safely to landfill or re-processed into fertilizer materials.
Specifically, the biowaste degradation using MBT Method has 2 processes which are
sort and mechanical separation to eliminate non-biodegradable material into biodegradable
material. This biodegradable materials will be processed through the biological compost,
anaerobic digester, or a combination of both. The combination of mechanical and biological
process makes possibility to materials recovery and biowaste stabilization.
Anaerob biological processes are selected with consideration of its use in accordance
with the high concentration waste, that convert energy in the form of biogas (metan) and
sludge generated will be much less (Gallert & Winter, 1999 in Haryanti 2009). Anaerob
decomposition of organic material works due to the activties of microorganisms in
metanogen environment where the degradation of organic material happened in the absence
of inorganic electron acceptors (oxygen, nitrate, sulphate, sulfur, Fe3+, Mn4+). Respiration
and fermentation processes that occur in anaerobic condition utilize proton or bicarbonate as
electron acceptors (Stams, 1994).
Anaerobic fermentation is divided into 4 stages of the decomposition process which
are hydrolysis, acidogenesis (fermentation), acetogenesis and methanogenesis (Raskin et al,
1997). Each stage will involve a different group of bacteria that will work synergically and
form a bacteria consortium. Consortia bacteria itself can be classified into nonmetanogen
bacteria and metanogen bacteria. Bacteria non metanogen divided into groups of bacteria
hydrolytic, fermentative, and acetogenik (Madigan et al, 2003).
The hydrolytic bacteria roles in breakdown of the complex compound into simple
molecules such as glucose, amino acids, fatty acid and glycerin at the initial stage. This initial
stage is longing rapid and almost simultaneously with the fermentation process. In the second
stage, the bacterial fermentation/acidogen bacteria will produce acids organic, alcohol, CO2,
H2 from simple molecules resulted by hydrolysis stage in an anerobic condition. The next
stage is the formation of acetate in addition format, CO2, H2 from the short-chain fatty acid
that involves a group of organisms that are usually called sintrofi bacteria or producing
proton asetogen bacteria. Final phase of anaerobic degradation is metan formation.
Metanogen bacteria is classified to Archaebacteria groups. Based on the metanogenesis
process, it is divided into Hydrogenotrophic methanogens and Acetotrophic methanogens or
acetoclastic bacteria.
Isolation is done with the observed differences in morphology (shape, color) and the
reaction of hemolysis from each colony on the blood agar, further purification is done using
nutrient agar. Identification is performed to find the genus and species of isolated
microorganisms through the observed criteria which are morphology, and physiology.
Morphology criteria consists of form, size, elevation, and the surface of the colony, spore
shape and position. Observation of gram staining also needed to help the identification
process. The physiology criteria is the condition that is needed for the growth of
microorganisms that aimed at knowing the metabolism of microorganisms through the
biochemical test to depict the reaction enzimatik by each microorganisms kind. This criteria

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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

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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.

RESULTS AND DISCUSSION

ƒ Dynamics of Total Ammount of Bacterial Colony in The Reactor
Enumerations are done using the method of Total Plate Count (TPC) through the
various serial dilutions with the provisions of the colony only on the amount of dilution with
30-300 colonies. From the enumeration data description that shown in Figure 1, we get the
dynamics of the total ammount of predominant bacteria that involved in the biowaste
degradation processs in Anaerob Batch. The total ammount of bacterial colonies on the 0th
day (initial running reactor) is the highest number of colonies counted which is 2.9x109
cfu/ml. This happens due to the optimal condition of the reactor (environmental conditions in
the early running optimally set), the additional seeding that has reached acclimatization
before as the enrichment source of degradation bacterial consortium, and the stage of
anaerobe process that can change the environment conditions has not happened and affect the
growth of bacteria. While in the enumeration on the next week the total ammount of bacterial
colonies fluctuates but did not reach the total amount as in the 0th day. The decreasing in
total ammount of bacterial colony could occur due to environmental changes on the reactor
because the stage of anaerobe process has occurred so that the bacterial colonies that are not
able to adapt to the environment will have higher number of death than the number of
presence and growth of bacterial colonies that are able to live in the environment. The
increase of total ammount of bacterial colony occurred because a number of bacterial
colonies have experienced the adaptation phase to the environmental conditions of the
reactor. It could also occur because of the changes on the environment conditions that support
the growth of the bacterial colony.

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 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 …

Figure 1 Recapitulation of Total Ammount of Bacterial Colony

ƒ Observation Morphology with Blood Agar

Enumeration were performed using the blood agar media on samples taken weekly
from Anaerobic Batch reactor and then the observations are made morphologically based on
the size, color and hemolytic reaction from each colony of bacteria that grow. From the
observation results we obtained 31 kinds of different colonies that were observed and there
were 7 types of colonies that appear (at least they appeared after 5 weeks) and can be
assumed as dominant colony that take role in the liquid biowaste processing, which are A
bacteria, C bacteria, E bacteria, F bacteria, N bacteria, and O bacteria. Each of the dominant
bacteria then would be isolated using the nutrient agar.

ƒ Isolation and Microscope Identification

The next isolation used nutrient agar as the medium to get pure culture (Levett, 1991).
Isolation was done on the enumeration sample results of the 0th, 7th, 28th and 69th days.
Results of this further isolation produced the J, E1, E2, A, F1 bacteria from the 0-day
isolation; the bacteria F2 and K from the 7th day isolation; the F3 and G bacteria from the
28th day isolation; and the C, D and F4 bacteria from the 69th day isolation. The sample
insulation points were selected by considering the fluctuation of the parameter values that
occur on the secondary data that is VSS, and pH.
From the isolation results are shown in Table 3, on four enumeration point we found
12 single colonies with some morphology differences, the type of gram reaction , and shape
of the colony based on Gram strain. The isolation result consists of 10 types of rods-shaped
and 2 types of coccus with the results of Gram strain showing that all of the types have
positive gram reaction. On the 7th day of isolation we found 2 types of bacteria that has the
same morphology characteristics, gram reaction type and colony shape as the 2 types of
bacteria on the 0th day of isolation but they were no longer found in the isolation on the
following day. This result shows that both types of bacteria has the adaptation ability at the
initial condition of the reactor where the pH value decreases because of the acid forming.

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 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

irreguler Irregul Irreguler

ireguler
with irregul irregul er, and at the Circular irregul irreg Circ
Shape irregular with serrate irreguler
serrate er er slipper bottom with core er uler ular
margins
margins y medium
Margin entir entir
Morph lobate lobate lobate serrate curled lobate lobate lobate entire entire
s e e
ology
on Hemoly gam
gamma beta beta alfa gamma gamma gamma gamma gamma beta beta
Blood sis ma
Agar Moder modera mod Mod
Size Big Big Big Big Big moderate small small
ate te erate erate
White White White
Colony’ White and White with and and whit whit
White white white white white
s colour thick and thick dark transpara transpara e e
core nt nt
modera Yelow-
Size small moderate big big big big small big big Big
te small
trans
transpara transluce transpa transpara translu opaq
Optic translucent opaque opaque opaque opaque lucen
nt nt rant nt cent ue
t
Morph Irreguler
Small - irreg irreg
ology Shape circular with serrate irreguler circular irreguler circular circular circular rhizoid
circular uler uler
on margin
Nutrien Elevati
t Agar flat flat flat flat flat flat flat flat flat flat flat Flat
on
permuk Smo Smo
Smooth smooth smooth smooth smooth smooth smooth smooth smooth smooth
aan oth oth
Margin undulat lobat lobat
entire entire undulate lobate entire entire entire entire entire
s e e e
Thic Thic
Rod, thin Thick- Mikro-
Thick- Thin-rod Mikro- Mikro- k-rod k-rod
Shape oval, Rod-shape rod and Rod, coccus rod,
rod and short coccus rod, oval and and
small short short oval
short short
Positiv positiv positiv positiv posit Posit
gram positive positive positive positive postive postive
Gram e e e e ive ive
Strain Single Strep
strepto/chai Strepto/ single but Strepto single but
Colony single diplo diplo single but diplo to/ch
n chain groups /chain groups
groups ain
Spore absenc absence
presenc presenc presenc prese prese
presenc presence presence presence absence e  presence
e e e nce nce
e
Spore
ovoi cente
potitio center ovoid ovoid center center center center
d r
n

• Isolation on the 0th Day

From isolation on the 0th day, there were 5 colonies, named J, E1, E2, A, F1, found after
48 hours incubation in the anaerobic condition. The characteristics of each colony were
known after the Gram strain. The results of the Gram strain can be seen in Figure 2, small
bacteria and scattered; Figure 3, short bacteria and chain formed colonies; Figure 4, medium
size bacteria and have eksospore. Figure 5, smallest size of most bacteria, and bacteria in
Figure 6, have the edge taper and form colonies.

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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

• Isolation on the 7th Day

On the 7th day of isolation there were 2 colonies, named F2 and K, found after 48 hours
incubation in the anaerobic condition. The characteristics of each colony were known after
the Gram strain. The results of the Gram strain can be seen in Figure 7, rod -shaped bacteria
and chain form colonies, and Figure 8, bacteria with scattered coccus.

Figure7 (F2) Short-Rods, Gram Postive Gambar 8 (K) Coccus, gram positive

• Isolation on the 28th Day

On the 28th day of isolation there were 2 colonies, named F3 and G, found after 48 hours
incubation in the anaerobic condition. The characteristics of each colony were known after
the Gram strain. The results of the Gram strain can be seen in Figure 9, bacteria in
micrcoccus shape and form colonies, and Figure 10, small rod-shaped bacteria and scattered.

SW8-7 
 
Figure 9 (F3) Mikroccous, Gram positive Gambar 10 (G) Micro-rods, gram positive

• Isolation on the 69th Day

On the 28th day of isolation there were 3 colonies, named C, D and F4, found after 48
hours incubation in the anaerobic condition. The characteristics of each colony were known
after the Gram strain. The results of the Gram strain can be seen in Figure 11, rod-shaped
and the smallest size of three bacteria were found; Figure 12, rod-shaped and taper edge;
and Figure 1, rod-shaped bacteria and chain form colonies.

Figure 11(C) Micro-Rods, Figure 12 (D) Rods, Figure 13 (F4) Short-rods,

short, gram positive gram positive gram positive

ƒ Identification of bacteria with Analytical Profile Index 20A

Identification is done with API 20A is used for specific types of anaerob bacteria in
general. Identification indicated for the genus to the species of each bacterial isolation results.
As shown in Table 2 is biochemical test results, show that metabolic activity of bacteria with
the test medium. Individual reactions to the medium will be the object of analysis in the
determination of the genus and species of bacteria using the API 20 A software.
Identification result by using the software are shown in Table 3. The Identification
results obtained the type of bacteria, namely Clostridium beijerinckii/butyricum, Clostridium
botulinum/sporogenes, Actinomyces meyeri/odontolyticus, Streptococcus intermedius,
Strepcoccus constellatus, and Clostridium innocuum. Groups of bacteria Clostridium sp
found and identified on the entire isolation sample point. Although the groups of bacteria
Actinomyces were appear in isolation day-0 and groups of bacteria Streptococcus sp were
appear in isolation day-7 and day-28, but in overall, the condition describe that genus
Clostridium sp is the dominant group of bacteria and have a role in biowaste processing in a
Anaerob Batch Reactor.

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 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  ‐  ‐  +  ‐  ‐  +  +  +  ‐  ‐  ‐  +  ‐  ‐  +  ‐  ‐  ‐  ‐  +  ‐  +  +  ‐ 

Table 3 API 20A Identification Result

Isolasi Symbol Genus/Spesies
J Clostridium beijerinckii/butyricum

E1 Clostridium botulinum/sporogenes

0-day E2 Clostridium beijerinckii/butyricum

A 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

ƒ The Relationships Between Dynamic Total Ammount of Bacterial Colony and

Secondary Parameters
Relationship between total ammount of bacterial colony during the time of processing
and the value of volatile Suspended Solid (VSS) is shown in Figure 14. The graph shows that
there are proportional fluctuation trend between the total ammount of bacterial colony and the
value of VSS. The common VSS parameters were used to find out how much biomass
contained in the suspension. Therefore, the decrease of the total number of colonies will
affect the value of the measured VSS.

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 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.

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ACKNOWLEDGEMENT
This research is part of the joint Bandung Institute of Technology, Indonesia;
Lembaga Ilmu Pengetahuan Indonesia (LIPI) Bandung, Indonesia with Karlshure University,
Germany.

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