IDENTIFICATION OF COMMERCIAL-SEED BACTERIA FOR PAINT

LIQUID WASTE TREATMENT

IDENTIFIKASI KEBERAGAMAN BAKTERI PADA COMMERCIAL-

SEED PENGOLAH LIMBAH CAIR CAT
Bening Mayanti1 and Herto Dwi Ariesyady2
Environmental Engineering Study Program
Faculty of Civil and Environmental Engineering ITB, Jl Ganesha 10 Bandung 40132
1
bening.mayanti@gmail.com and 2herto@ftsl.itb.ac.id

Abstract: Biological treatment is a kind of treatment which can breakdown organic matter effectively and
efficiently. The basic principle to treat the waste which has the high level of organic matter is utilization of
microorganism activities to breakdown the chemical compounds and turn them into a simple one, in the other
word, microorganism play a role of a biological process. To treat the waste, the bacteria can be isolated from
the commercial seed contain a package of bacteria or from the waste itself. Studying the diversity of bacteria
inside the commercial seed used to treat paint liquid waste through isolation process and gram reaction, then
conventional method with biochemical tests is the purpose of this research. Identification of commercial seed is
important because Approximately 10,000 different dyes and pigments are used industrially.Genus for all of the
bacteria from commercial seed is Bacillus. In this research, three species from commercial seed were identified
successfully. They were Bacillus licheneformis, Bacillus subtilis, and Bacillus cereus. The presence of three
kinds bacteria indicates that the biological process used a consortium bacteria.Every bacteria has different
growth pattern in Nutrient Broth media. It was shown by the difference of growth curve which has been
indicated by generation time(g),interval for binary fision, and growth rate constant of (k)both pure cultures and
mixed culture.. The value of generation time and growth rate constant for Bacillus licheniformis were 25.20
minutes and 1.52 hour-1; Bacillus subtilis 33.43 minutes and 1.15 hour-1 ; Bacillus cereus 30.95 minutes and
1.28 hour-1 ; mixed culture 42.48 minutes and 0.90 hour-1.

Key words: Bacillus, commercial seed, generation time, growth rate constant

Abstrak : Pengolahan secara biologis merupakan pengolahan yang efektif dan efisien dalam mendegradasi
materi organik dengan prinsip memanfaatkan aktivitas mikroorganisme untuk menguraikan/memecahkan
senyawa kimia yang terkandung dalam air buangan menjadi bentuk yang lebih sederhana, dengan kata lain.
mikroorganisme memegang peranan penting dalam proses biologis. Bakteri yang digunakan dapat berupa
commercial seed maupun bakteri yang secara alami tumbuh pada suatu limbah . Untuk itulah, tujuan dari
penelitian ini adalah untuk mempelajari keragaman bakteri yang terdapat dalam commercial seed pengolah
limbah cair cat dengan menggunakan metode isolasi dan pewarnaan Gram serta metoda konvensional
menggunakan serangkaian uij biokimia. Identifikasi bakteri pendegradasi cat perlu dilakukan karena sekitar
10.000 zat warna dan pigmen yang berbeda digunakan untuk keperluan industri. Isolat yang didapat terdiri
atas bakteri gram positif Genus Bacillus. Pada penelitian ini berhasil diidentifikasi spesies dari bakteri yang
terdapat pada commercial seed tersebut, yaitu Bacillus licheniformis, Bacillus subtilis, dan Bacillus cereus.
Keberadaan bakteri-bakteri tersebut menunjukkan proses pengolahan yang menggunakan bakteri konsorsium
yang setiap bakterinya memiliki pola pertumbuhan yang berbeda dalam media Nutirent Broth. Hal tersebut
ditunjukkan oleh perbedaan kurva pertumbuhan yang ditandai waktu generasi(g), yaitu waktu yang diperlukan
untuk memperbanyak diri sebanyak dua kali lipat, yang berbeda dan dengan konstanta laju pertumbuhan (k)
yang nilainya berbeda pula baik untuk kultur murni maupun kultur campuran. Waktu generasi dan konstanta
laju pertumbuhan untuk Bacillus licheniformis adalah 25,20 menit dan 1,52 jam-1; Bacillus subtilis sebesar
33,43 menit dan 1,15 jam-1 ; Bacillus cereus sebesar 30,95 menit dan 1,28 jam-1 ; dan mixed culture sebesar
42,48 menit dan 0,90 jam-1.

Kata kunci : Bacillus, commercial seed,waktu generasi, konstanta laju pertumbuhan

WW6-1
INTRODUCTION

Waste is a by product which is generated from production process and generated from
waste treatment process both industrial scale and domestic scale. Chemically, waste is
classified into two kind, they are organic waste and inorganic waste. The presence of waste
can give the negative effect to environment and health if the concentration stays on the high
level and handled improperly. One kind of so many kinds wastes are liquid waste containing
color agent which is released to environmental without any treatment. Even the dye
concentration may be less than 1 ppm, lower than many other chemicals found in wastewater,
the color is predominant and visible. Approximately 10,000 different dyes and pigments are
used industrially (Sukumar et al., 2007).
Large amount of chemically different dyes are used for various industrial applications
and a significant propotion of these dyes enter the environment in waste water .These dyes
are designed to be resistent to the light, water, and oxidizing agents and are therefore difficult
to be degraded once released into aquatic system.Conventional wastewater treatment systems
are often inefficient and existing physical and chemical technologies are expensive, time
consuming and often methodologically emanding. Therefore, it may be economical to
develop alternative means of dye decolorization, such as bioremediation due to its reputation
as on environmentally friendly and publicy acceptable treatment technology. The treatment
systems having mixed microbial populations are more effective due to concerted metabolic
activities of microbial community. As the catabolic activities of microorganisms in mixed
consortium complement each other, the utilization of microbial consortium offers
considerable advantages over the use of pure cultures in the degradation synthetic dyes. The
individual strains may attack the dye molecule at different positions or may use
decomposition products produced by another strain for further decomposition (Jadhav et al.,
2008).
Biodegradation process can be approached by two main ways, they are seeding and
environmental modification. Seeding is a inoculating process to liquid waste installation.
Inoculating microorganism can be originated from outer polluted location (non-indigenous)
or can be obtained from polluted location (indigenous). On the other side, the purposes of
environmental modification are to optimize metabolism activities of microorganism through
nutrition addition especially nitrogen and phosphor, to increase the number of microorganism
and humidity, that could be accelerated by co-substrate addition as supporting system for
microorganism growth.
In decolorization process of effluents the use of bacteria constitutes an alternative
mode of treatment in aerobic conditions. Some specialized strains of aerobic bacteria have
developed the ability to use azo dyes as sole source of carbon and nitrogen, others only
reduce the azo group by special oxygen-tolerant azo reductases (Sharma et al., 2009).
Generally, seeding is done by using a commercial seed which can be unsuitable to the
characteristics of waste that need to be treated. Beside that, commercial seed has an
expensive cost and can cause competition wiht natural microbe that present in the system.
Indonesia is a potentially-tropical-country to get local isolate from the contaminated area
which has an ability to breakdown the contamination well. Microbe that isolated from
contaminated area contaminated by paint has a potency to breakdown panit liqud waste.
Studies carried out in the past have used undefined microbial consortium or pure
cultures for dye decolorization. In the present study, a defined consortium of two organisms
Galactomyces geotrichum MTCC 1360 and newly isolated Bacillus sp (Jadhav et al., 2008).
Isolation process was done and found a bacterial strain that highly decolorized the
toxic azo dye methyl red (MR) from soil samples by an enrichment culture under aerobic

WW6-2
conditions. This bacterial strain was identified as Bacillus sp. B29 belonging to B. cereus
group (Ooi, et al., 2007)
Over the past decade, biological treatment has been investigated. Microbial
decolorization is an environment-friendly and cost competitive alternative to chemical
decomposition process. Several kinds of microorganism such as fungi (Penicillium
decumbens, Aspergillus sp., Aspergillus niger, Flavadon flavus), white rot fungi
(Phanerochaete sp., Phanerochaete chrysosporium, Trametes versicolor, Coriolus sp.,), yeast
(Citeromyces sp.) and bacteria (Bacillus sp., Pseudomonas sp., acetogenic bacteria) have
been reported regarding their abilities to remove color. There are many reports showing that
white rot fungi are very effective to remove color in the wastewater. But for the application in
large scale treatment, it has been impeded of owing to lack of an appropriate reactor system
capable of coping with relatively slow fungal degradation, loss of extracellular enzymes and
mediator with discharged water (Jiranuntipon et al., 2008).
Pure bacterial or fungal cultures have been studied in order to develop bioprocess for
melanoidins decolorization in molasses wastewater. However, the performance of fungal
decolorization was limited by long growth cycle and moderate decolorization rate. In
contrast, the bacterial decolorization is usually faster, but it may require a mixed community
to decolorize through combined metabolic mode of individual culture. The mixed culture of
Bacillus spp. exhibited a two- to fourfold increase in decolorization over that showed by any
individual Bacillus isolate. Hence, the bacterial consortium seems to be more competent for
treatment due to maintenance of microorganism and co-metabolism to enhance the efficiency
of decolorization (Jiranuntipon et al., 2008).
Special for treating paint-liquid-waste, stressing is done to the ability of bacteria to
utilize chemical compound inside the paint as the carbon source. Because of the composition
inside the paint is not dominant, pigment application is along with thinner as the solvent.
Then, the components which have to be tested are soluble pigment in thinner, acrylic resin
and melamine.

METHODOLOGY

Place of Research
This research was conducted in two laboratories; Industrial Hygiene and Toxicology
and Solid Waste and Hazardous Waste Laboratory, Environmental Engineering Study
Program.

Research Material
Commercial seed was the main material in this research. The other materials are
Nutrient Agar (Oxoid) to isolate pure culture, 250 ml flasks containing Nutrient Broth
(Oxoid) to enrich the cultures, SBS (Salt Base Solution) as the growth media for bacteria,
materials for biochemical tests to figure out the bacteria’s species, as well as the paint
component such as thinner and pigmen, melamine, and acrylic resins.

Research Instrument
Some instruments were used to support this research. The instruments were four of
250 ml flasks containing Nutrient Broth to enrich the bacteria cultures (pure culture and
mixed culture). The other instruments are pipette, micro pipette, analytical scale, reaction
tubes, petri, baker glass, pH meter, spectrophotometer, incubator, and orbital shaker.

WW6-3
Bacteria Isolation
The purpose of bacteria isolation was to obtain the pure culture, which only contains
one type of bacterium.
Purification process was done through some steps, these were:
• Dilution to commercial seed using sterilized aquadest.
• The different colony was separated through pour plate method.
• Purification was performed using the inoculating needle, then it was streaked to
Nutrient Agar inside the sterilized petri, so that one pure colony can be gained.
• One type of colony was then transferred to sideways Nutrient Agar inside reaction
tube .

Bacteria Identification
Isolation process resulted bacteria which needed to be identified to figure out the
species of these bacteria. Identification was done by a number of chemical tests to find out
the bacteria’s characteristics, so the species can be recognized. Some of the tests were
gelatine hydrolylis, reaction to different carbohydrate, nitrate reduction, etc. based on
Manual for Identification of Medical Bacteriology (Cowan, 1974)

Growth Test
Growth test was done to figure out the growth characteristic of every bacteria through
growth curve. It was made by measuring bacteria growth by means of the optical density in
NB media using spectrophotometer (Spectronic 20 Genesys) from each culture which is
shaked and incubated.

RESULTS AND DISCUSSION

Analyzing of Commercial Seed Bacteria

Direct isolation was conducted with commercial seed as a source .Purification was
done in a purpose to obtain pure culture through pour plate method. The dilution process was
done from 10-1 until-8 duplicate, with the result of counting in Table 1.
Table 1 Total Colony from Pour Plate Method

No. Dilution Level Total Colony (I) Total Colony (II)

1 10-1 Too Numerous To Count (TNTC) TNTC

2 10-2 TNTC TNTC

3 10-3 TNTC TNTC

4 10-4 TNTC TNTC

5 10-5 213 158

6 10-6 49 63

7 10-7 9 11

8 10-8 - -

WW6-4
 Four of dilution process gave a TNTC (Too Numerous To Count) result. It happened
to dilution level from10-1 until 10-4. TNTC was occured to the total colony exceed 300
colonies. This kind of result, cannot be used to analyze the total cells per mililiter. The total
colony more than 300 colonies is not a valid result because microorganisms are too crowded
and can cause attachment between one and another colony. Beside that, this kind of growth
can cause accumulation colonies in one place, thus the real number of colonies cannot be
counted well. On the other side, if the number of colonies less than 30, it indicates that the
bacteria population is too low and cannot represent the real number of bacteria per mililiter.
This kind of growth happened to dilution level from 10-7 until 10-8 . The representative result
was obtained from10-5 until 10-6 dilution level.
There were six pure cultures bacteria which were isolated for identification purpose,
but the results of biochemical tests showed that there were similarity between them. Finally,
there were only three bacteria named BM1, BM2, dan BM3.
Observation to the morphologycal shape was done after the pure culture is available.
Table 2 shows the morphologycal observation to commercial seed bateria.

Table 2 Morphologycal Characteristics of Commercial Seed Bacteria.

Bacteria
No. Characteristics
BM1 BM2 BM3

1 Shape Rounded, reef edge irregular Rounded, reef edge

2 Edge undulate branched,undulate lobate

3 Elevation raised raised raised

4 Size small large large

5 Texture soft soft soft

6 Appearance dull dull dull

7 Pigment nonpigment nonpigment nonpigment

8 Optical opaque translucent translucent

Observation process to the morphologycal characteristics or usually called

morphologycal screening is useful to help the observer to put one or more bacteria which
indicate the same spescies into a group, especially when a lot of bacteria are need to be
identified through conventional method. It can simplified biochemical tests, because the
bacteria that has similarity can be eliminated to make identification process easier. However,
morphologycal screening process need a special ability, so that the eliminated bacteria is the
same bacteria.
After morphologycal screening process, preliminary tests were done to determine the
genus of bacteria which is showed on Table 3.

Table 3 Result of the Preface Test from Commercial Seed Bacteria

No. Prosedure BM1 BM2 BM3

1 Gram reaction + + +

2 Shape Rod Rod Rod

WW6-5
 No. Prosedure BM1 BM2 BM3

3 Motility + + +

4 Spore reaction + + +

5 Catalese test + + +

6 Growth on glucose broth + + +

Notes: (+) positive reaction

(-) negative reaction

After the Gram reaction of every bacteria was obtained, the preface tests were done to
determine the genus of bacteria. These tests based on Manual for the Identification of
Medical Bacteria (Cowan, 1974) and the bacteria belong to Bacillus genus.
Bacillus is rod shaped bacteria, came under positive gram bacteria in young culture,
motil (nonmotil reaction can be happened), produce endospore which usually heat resistant,
aerob (some species are anaerob facultative), positive catalase reaction,vary on oxidizing.
Every species gives a different reaction in sugar usage, half can attack and the rest cannot
(Cowan, 1974).
Bacillus are gram positive bacteria, however this endospore poducer will turn into
Gram negative bacteria when they enter stationary phase of their growth . Most of Bacillus
are mesophille bacteria which has the optimum temperature of 30-45°C, although there are
some of Bacillus that belongs to thermophille group with optimum temperature of 65°C
(Todar, 2009).
The next steps were identification process to figure out the species of these bacteria
through biochemical tests showed in Table 4.

Table 4 Result of the Biochemical Test from Commercial Seed Bacteria

Bacteria
No. Tests
BM1 BM2 BM3

1 Gram reaction (J) + +

2 Motility + + +

3 Morphologycal group 1 1 1

4 Spore shape ovale ovale ovale

5 Spore position center center center

6 Swollen bacillary body - - -

7 Growth on 45°C + + +

8 Growth on 65°C - - -

9 Growth on pH 5,7 + + +

10 Growth on 7% NaCl + + +

11 Citrate utilization + + +

WW6-6
 Bacteria
No. Tests
BM1 BM2 BM3

12 Anaerobic growth on glucose broth + - +

13 Carbohydrate from:

glucose + + +

arabinose - + -

mannitol + + -

xylose + + -

14 VP test + - +

15 Starch hydrolysis + + +

16 Nitrate reduction + - +

17 Indole - - -

18 Gelatine hydrolysis + + +

19 Casein hydrolysis + + +

20 Urease test - - weak

Notes: (J) positive on young culture, inconstant to old culture

(+) positive reaction
(-) negative reaction
(1) spore shaped ovale or cylinder; center, terminal, or sub terminal; bacillary body only slightly
swollen or not at all.

A group of biochemical tests were used to determine the species of Bacillus bacteria.
Biochemical tests are conventional method used phenomenological system, that is the result
of the tests is compared with the literature table (Cowan, 1974).
Biochemical tests produce result of three kinds bacteria. The bacteria inside the
commercial seed are shown in Table 5.

Table 5 Identification Result of Commercial Seed Bacteria

No. Code Bacteria

1 BM1 Bacillus licheniformis

2 BM2 Bacillus subtilis

3 BM3 Bacillus cereus

The final result of identification process showed three different bacteria from Bacillus
genus. They were Bacillus licheniformis, Bacillus cereus, and Bacillus subtilis. In the other
words, the commercial seed contains consortium bacteria which used to breakdown paint
component. Previous research showed that Bacillus subtilis has ability to decolorize and
degrade a few a dyes (Itoh et al., 1993).

WW6-7
 Except the advantages from positive interaction of usage the consortium bacteria by
the completion of metabolic reaction, there should be awareness to the presence of
competition interaction. This competition could develop a condition in which the faster
growing species will outgrow the slower growing one, unless growth rates are identical
(Sterrit and Lester, 1988).
Therefore, the determination of constant growth rate using pure culture and mixed
culture were necessary.

Microorganisms Growth Rate

Microorganism growth rate was conducted by inoculating bacteria, both pure culture
and mixed culture in NB media (beef extract 1 g/L, yeast extract 2 g/L, peptone 5 g/L,
sodium chloride 5 g/L), incubated 37 °C, and the OD during 24 hours was measured in every
hour.
Growth curve from every pure cultures of Bacillus and mixed culture were then
plotted on logarithmic scale (Figure 1).

(a) 1 (c) 1
0 4 8 12 16 20 24 0 4 8 12 16 20 24

Absorbance
0.1
Absorbance

0.1

0.01

0.01
0.001 Time (hour)
Time (hour)

1 1
0 4 8 12 16 20 24 0 4 8 12 16 20 24
(b) (d)
Absorbance
Absorbance

0.1
0.1

0.01
0.01
Time (hour)
Time (hour)

Figure 1 Growth curve of Pure Cultures and Mixed Culture

(a) Bacillus licheniformis Growth Curve
(b) Bacillus subtilis Growth Curve
(c) Bacillus cereus Growth Curve
(d) Mixed Culture Growth Curve

Growth curves showed some phases of bacteria growth. In some bacteria the lag
phase could not be seen, it was caused by the one-hour-measurement, so the changing which
was happened between the one hour interval was unknown.

WW6-8
 The result of extrapolation at exponential phase used to counted the generation time
(g) and growth rate constant can be seen in Figure 2.

1 1
(a) (c) 0 1 2 3 4 5
0 1 2 3 4 5
Absorbance

Absorbance
0.1
0.1
0.01 Slope = 0.7168
Slope = 0.5836

0.001 0.01
Time (hour) Time (hour)

1 1
0 1 2 3 4 5 0 1 2 3 4
(b) (d)
Absorbance

Absorbance
0.1 0.1

Slope = 0.5403 Slope =0.4216

0.01 0.01
Time (hour) Time (hour)

Figure 2 Extrapolation of Exponential Phase from Pure Cultures and Mixed Cultures
(a) Bacillus licheneformis Extrapolation
(b) Bacillus subtilis Extrapolation
(c) Bacillus cereus Extrapolation
(d) Mixed Culture Extrapolation

In the laboratory with optimum condition for Bacillus growth, these species showed
the 25 minutes generation time (Todar, 2009).
Exponential phase will give the value of g (generation time) dan k (growth
rateconstant), they can be calculated by following formula (Madigan et al., 2003):
g = 0,301 / slope
k = 0.693 / g
Thus, the number of generation time and growth rate constant were determined (Table 6).

Table 6 Generation Time and Growth Rate Constant of Pure Cultures and Mixed Culture

Value
No. Bacteria
g (minute) k (hour-1)
1 Bacillus licheneformis 25.20 1.52
2 Bacillus subtilis 33.43 1.15
3 Bacillus cereus 30.95 1.28
4 Mixed culture 42.84 0.90

Every culture gives the different value of generation time both pure culture and mixed
culture. Generation time for pure culture varied from 25.20 minutes until 33.43 minutes , the
results were in accordance with previous results (Todar, 2009). It showed that the optimum
condition for the growth process is fulfilled, whereas the growth rate constant showed the
generation rate of bacteria which grow exponentially. Growth rate constants from every

WW6-9
bacteria were different, according to metabolic ability in every bacteria. It depends on the
enzyme’s ability in every bacteria which play a role in metabolism process due to every
compound inside the Nutrient Broth media. The value of growth rate constant varied from
0.9 hour-1until 1.52 hour-1. The highest growth rate was occured for Bacillus licheniformis,
whereas the lowest growth rate constant was occured at mixed culture. The growth for mixed
culture slower than the pure ones because of the interaction between one and another
cultures. It indicated that the competition happened in order to get the substrate, made the
bacteria growth became slow.
The research of waste degradation process in laboratory scale usually used the co-
substrate as the first food for the microorganism, and the waste that need to be treated
become a second food for microorganism. That’s why the selection process should be based
on growth curve of every bacteria. Consortium bacteria will be chosen from the second
exponential phase. The pre-requirement is the different time of entering the second
exponential phase. The purpose of this way is to avoid the fight among the bacteria which
want to use the waste as the second food (substrate for cell growth), because the second food
is the main core in the degradation process.

CONCLUSIONS

Bacteria inside the commercial seed had the same genus, Bacillus. They were Bacillus
licheniformis, Bacillus cereus, and Bacillus subtilis. These bacteria was identified with
conventional method through a group of biochemical tests. The precense of these bacteria
inside the commercial seed indicates that paint liquid waste was treated using bacteria
consortium.
Every bacteria has different growth pattern in Nutrient Broth media. It was shown by
the difference of growth curve which has been indicated by generation time (g), interval for
binary fision, and growth rate constant (k) of both pure cultures and mixed culture. The value
of generation time and growth rate constant for Bacillus licheneformis were25.20 minutes
and 1.52 hour-1; Bacillus subtilis 33.43 minutes and 1.15 hour-1 ; Bacillus cereus 29.9
minutes and 1.28 hour-1 ; mixed culture 42.48 minutes and 0.90 hour-1.

ACKNOWLEDGEMENT

This study was funded by PT. Showa Mfg. according to project contract with
Yayasan LAPI ITB, year 2009.

REFERENCES

Cowan S.T. 1974. Manual for Identification of Medical Bacteria. Cambridge University
Press. Cambridge .
Itoh, K.,Yatome,C.,Ogawa,T. 1993. Biodegradation of Anthraquinone Dyes by Bacillus
subtilis. Environmental Contamination and Toxicology. Vol 50. pp 522-527.
Jadhav, S.U., Jadhav,U.U.,Dawkar,V.V., dan Govindwar, S.P. 2008. Biodegradation of
Disperse Dye Brown 3REL by Microbial Consortium of Galactomyces geotrichum
TCC 1360 and Bacillus sp. VUS. Biotechnology and Bioprocess Engineering. Vol
13. pp 232-239.
Jiranuntipon,S., Chareonpornwattana,S., Damronglerd, S., Albasi, C.,Delia, M.L.2008.
Decolorization of synthetic Melanoidins-Containing Wastewater by a Bacterial
Consortium. Industrial Microbiology & Biotechnology. Vol 35. pp 1313-1321.

WW6-10
Madigan, M.T.,Martinko,J.M.,Parker, P. 2003. Biology of Microorganism. 10th Edition.
Prentice Hall.USA.
Ooi, T., Shibata, T., Sato, R., Ohno, H., Kinoshita,S., Thuoc,T.L., Taguchi, S. 2007. An
Azoreductase, Aerobic NADH-dependent Flavoprotein Discovered from Bacillus
sp.:Functional Expression and Enzymatic Characterization. Applied Microbiology
and Biotechnology. Vol 75. pp 377-386
Sharma, P., Singh,L., Dilbaghi,N. 2009.Optimization of Process Variable for Decolorization
of Disperse Yellow 211 by Bacillus subtilis using Box-Behnken Design. Journal of
Hazardous Materials. Vol 164. pp 1024-1029.
Sterrit, R.M., J.N. Lester. 1988. Microbiology for Environmental and Public Health
Engineers. E&FN Spon Ltd. London.
Sukumar, M., Sivasamy,A., Swaminathan, G. 2007. Decolorization of Textile Dye Effluent
by Genetically Improved Bacterial Strains. Applied Biochemistry and
Biotechnology. Vol 136 .pp 53-62.
Todar, Kenneth. 2009. The Genus Bacillus. http://www.textbookofbacteriology.net. Accessed
on June 15th 2009.

WW6-11