biodegradation of organic waste using bacillus subtilis...

International Journal of Comprehensive Leading Research In Science ( ISSN: 2455-4693 )

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Vol. 1 Issue 1, pp: (1-12), December 2015, Available at: www.ijclris.com

Page No : 1 www.ijclris.com

Biodegradation of Organic Waste Using Bacillus subtilis and

Aspergillus niger

Senbaham D1, Amutha R1*, Sri Ramani M1, Asma Begam S2

1Department of Microbiology

Vivekanandha College of Art and Sciences for women, Elayampalayam

2Department of Biotechnology

Vivekanandha College of Engineering for Women, Elayampalayam

* Corresponding Author Email.id: [email protected]

Organic waste materials are being used in huge amounts around the world. It is used in

improving soil health and its helps to the crop yields. But nowadays using many chemical fertilizers it

spoil the soil which leads to the organic wastes does not degraded in soil results in causing of some

environmental pollution. Pearl millet is the world's fourth most important tropical food cereal.

While early growing and well adapted to droughty, sandy acid soils of low fertility, pearl millet is

highly responsive to fertilizer and moisture on well drained soils. Corn is also widely grown in

tropical, temperate and some cold zones. In the current study, the degradation process was studied

by the enzymes –Amylase from Bacillus spp. and protease from Aspergillus spp. The isolated

microorganisms were enzymes produced in the production media. These enzymes from both wild

strains were partially purified by ammonium sulphate precipitation method and quantitatively

esti ated Lo r ’s ethod. These strai s ere atta hed o the orga i aste a d the

degradation was confirmed by the weight loss. It can also be confirmed by tensile strength and

decrease in viscosity, in some cases, molecular weight distribution, and fragility. By observing the

results, the enzyme weight was gradually decreased in the production media. Corn waste was

degraded faster by the protease enzyme when compared to the amylase enzyme and pearl millet

was degraded faster by the amylase enzyme when compared to the protease enzyme. Hence it may

be concluded that using Bacillus spp. and Aspergillus niger, we can successfully degrade organic

wastes within short duration.

Key words: Organic waste, amylase, protease, biodegradation, purification.

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1. INTRODUCTION

Due to the development of civilization, vast amount of organic waste is being produced. Its

return to the environment is highly desirable, as it is characterized by significant amount of macro-

and micronutrients. However, some of the organic waste cannot be used directly, and needs

improvement in its physical and chemical properties (Slawomir wierzba et al., 2005). Environmental

contamination has the potential to be a major threat to the survival of living organisms. The misuse

of chemical fertilizers and pesticides can contribute to the deterioration of the environment (Kaosol

2009).

The organic waste biodegradation was evaluated with bacterial inoculate containing selected

proteolytic, lipolytic and cellulolytic bacterial strains.Wastes are generated by activities in all

economic sectors and also by goods consumption (Mbonu, et al., 2007).These materials influence

crop yield and affect chlorophyll coloration, due to the amount of nutrients absorbed by the plant

from the soil. In addition to supplying plant nutrients, they improve the physical properties of the

soil (Adeleye et al., 2010) and enhance the soil microbial activities which enhance nutrient supply.

Based on the catalytic site on the substrate, proteases are mainly classified in to

endoproteases and exoproteases (Rao et al., 1998). Endoproteases preferably act at the inner

region of the polypeptide chain. By contrast, exoproteases preferentially act at the end of the

polypeptide chain. Feathers hydrolysed by mechanical or chemical treatment can be converted to

feedstuffs, fertilizers, glues and foils or used for the production of amino acids and peptides.

Enzymes are substances present in the cells of living organisms in minute amounts and are

capable of speeding up chemical reactions (associated with life processes), without themselves

being altered after the reaction. They accelerate the velocity of there action without necessarily

initiating it (Oyeleke and Oduwole, 2009). Microbial enzymes are preferred to those from both plant

and animal sources because they are cheaper to produce, and their enzyme contents are more

predictable, controllable and reliable (Burhan et al., 2003).

Amylases and glucoamylase are produced by various microorganisms, including bacteria;

fungi and yeast, but a single strain can produce both these enzymes as well. These enzymes have

found applications in processed-food industry, fermentation technology, textile and paper

industries, etc. The SSF has been employed to produce amylases. It has been reported by Krishna



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a d Cha drasekara that a a a fruit stalk aste a e used for the produ tio of α-

amylase employing Bacillus subtilis (CBTK 106) by solid state fermentation (SSF).

Composting is a preferred and environmentally sound method where by organic waste is

reduced to organic fertilizer and soil conditioners through biological processes (Alexander et al.,

1999). The high organic carbon content and biological activity of compost make it effective for

applications such as erosion control and revegetation (Anastasi et al., 2005). Several industrial

bioprocesses have been developed using as much as possible the enzymatic potential of these

microorganisms to convert useless cellulose materials into useful products.

Agro-industrial residues are generally considered the best substrates for the solid state

fermentation processes, and use of solid state fermentation for the production of enzymes is no

exception to that. A number of such substrates have been employed for the cultivation of

microorganisms to produce host of enzymes (Mitra et al., 1994).

2. MATERIALS AND METHODS:

2.1 Sample collection

Two different samples (soil/humus) were collected from ten different areas of Kodaikanal &

Salem. Each sample (100 g) was collected in separate sterile screw capped tube, stored at 4°C for

further use.

2.2 Isolation of Bacteria

One gram of soil was serially diluted in 9 ml of sterile distilled water (10-2

to 10-6

). From each

dilution tube, 1 ml was transferred into culture media nutrient agar plate and incubated at 37°C for

24 h. The colonies were observed (Zaved et al., 2008).

2.3 Morphological and Biochemical Characteristics:

Bacteria were identified morphologically by Gram staining, Motility and biochemically by

Indole production test, Methyl red, Vogues-Proskauer test, Citrate utilization test, Triple sugar iron

test, Nitrate reduction test, Catalase, Oxidase, Gelatin liquefaction, Urease, H2S production,

Hydrolysis of casein and starch hydrolysis test. The fungal isolates were morphologically identified

by using Lacto Phenol Cotton Blue wet mount technique.

2.4 Screening method

2.4.1 Screening for protease Activity of Aspergillus Niger



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The amylolitic activity of A. Niger was determined using the starch agar plate method. This

was done by inoculating the identified organism into Sabourad Dextrose Agar medium which was

supplemented with 1g of starch. The agar plates were then incubated at 30℃ for 5 days. After the

i u atio period, Lugol’s iodi e solutio as added to the culture plate to identify the zones

around the cultures. The diameter formed after the addition of iodine solution was measured to

represent the amylolytic activity (Oyeleke et al., 2011).

2.4.2. Screening for Amylolytic Activity of Bacillus subtilis

The amylolitic activity of the test isolates was determined by using the starch agar plate method, by

inoculating the identified Bacillus species into Nutrient Agar medium which was supplemented with

1g of starch. The agar plates were then incubated at 37℃ for 24hrs. After the incubation period,

Lugol’s iodi e solutio as added to the ulture plate to ide tif the zo es arou d the ultures. The

diameter formed after the addition of iodine solution was measured to represent the amylolytic

activity (Oyeleke et al., 2011).

2.5. Preparation of Medium Used for Amylase Production by Bacillus subtilis

The medium was prepared by weighing the following medium composition in g\L. Bacteriological

Peptone-6g, MgSo4.7H2O-0.5g, Kcl-0.5g, Substrate-1.0g. The above medium composition were

dissolved in 1000ml of distilled water after which 100ml of the medium was measured into a conical

flask (250ml capacity each) heated on hot plate to homogenize and then sterilized in an autoclave at

121℃ for 15 minutes after which they were removed and allowed to cool before the organism was

inoculated.

2.6. Preparation of Medium used for Protease Production by Aspergillus niger

The medium was prepared by weighing the following composition in (g /L): peptone, 5; yeast

extract 3; malt extract 2, substrate 2 and pH 8. The above medium composition were dissolved in

1000ml of distilled water after which 100ml of the medium was measured into a conical flask (250ml

capacity each) heated on hot plate to homogenize and then sterilized in an autoclave at 121℃ for

15 minutes after which they were removed and allowed to cool before inoculating.

2.7. Extraction of Amylase Enzyme from Bacillus subtilis



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After incubation, the production medium was centrifuged at 6000rpm for 30 min to separate

the cells. The supernatant was collected as it contained the crude enzyme and stored at 4°C till

further use (Suganthi R., et al., 2011).

2.8. Extraction of protease enzyme from Aspergillus niger

After the incubation period, extraction of the crude enzyme was done by centrifugation of

the fermented media at 2000rpm (revolution per minute) for 5 minute. Supernatant collected were

then filtered off using What a ’s u er filter paper. The filtrate o tai ed the rude a lase

enzyme (Suganthi et al., 2011).

2.9. Lowry’s Method:

Different dilutions of BSA solutions are prepared by mixing stock BSA solution (1 mg/ ml) and

ater i the test tu e. The fi al olu e i ea h of the test tu es is l. The BSA ra ge is . to

mg/ ml. From these different dilutions, pipette out 0.2 ml protein solution to different test tubes

and add 2 ml of alkaline copper sulphate reagent (analytical reagent). Mix the solutions well. This

solution is incubated at room temperature for 10 mins. Then add 0.2 ml of reagent Folin Ciocalteau

solution (reagent solutions) to each tube and incubate for 30 min. Zero the colorimeter with blank

and take the optical density (measure the absorbance) at 660 nm. Plot the absorbance against

protein concentration to get a standard calibration curve. Check the absorbance of unknown sample

and determine the concentration of the unknown sample using the standard curve plotted above

(Oyeleke et al., 2011).

3. RESULTS AND OBSERVATION

3.1Sample collection

Soil samples were collected from natural habitat from Berijam forest, Kodaikanal, Tamil

Nadu. (Fig :1)



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Fig: 1 Sample collection

3.2. Isolation of Bacillus spp

Bacillus spp was isolated from the sample collected from the regions around the vegetable

market Salem. It was serially diluted and cultured on the nutrient agar plates, white colour rounded

colonies were observed (Fig 2).

Fig. 2 Isolation of Bacillus spp

3.3. Isolation of Aspergillus spp

Aspergillus spp was isolated from the sample collected from the regions around the Berijam

forest, Kodaika al. It as seriall diluted a d ultured o the Sa oroud’s De trose agar edia. The

Aspergillus spp was identified by the Lacto phenol cotton blue staining (Fig: 3).



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Fig: 3 Isolation of Aspergillus spp

3.4. Screening of Amylase & Protease producers

Amylase producing Bacillus spp & protease producing Aspergillus spp was confirmed by over

layering of 1% iodine on the Starch agar plate and a clear zone was observed within few minutes

(Fig. 4).

Fig. 4 Screening of Amylase & Protease producers

3.5. Amylase & protease production media

Amylase was produced by Bacillus substilis in production media and protease was produced

by Aspergillus niger. Both the enzymes were confirmed through starch hydrolysis for the production

medium (Fig: 5).

Figure: 5

Amylase Protease



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3.6. Extraction of Amylase Enzyme from Bacillus subtilis

Amylase was extracted from production media by centrifuging the broth culture at 6000 rpm for

10 min. The collected supernatant was used for partial purification of the enzyme.

3.7. Extraction of Protease Enzyme from Aspergillus niger

Extraction of protease from production media was done by centrifuging at 2000 rpm for 10

min. The collected supernatant was used for partial purification of the enzyme.

3.8. Partial purification of Enzyme samples

All the enzyme samples isolated from wild strains were partially purified by ammonium

sulphate precipitation method. The precipitate was collected by centrifuging the solution (Fig. 6).

Fig. 6 Partial purification of Enzyme samples

3.9. Estimation of Enzyme samples

Partially purified enzyme was estimated by Lower ’s ethod i hi h o i e seru al u i

is used as standard and the total yield of protease and amylase enzyme production media was

estimated and it found to be μg, μg respectively (Table 1).

Table 1 Protocol of Lowery’s Method and OD values at 660 nm

Blank S1 S2 S3 S4 S5 T1

(amylase)

T2

(protease)

Standard protein(ml) - 0.2 0.4 0.6 0.8 1.0 0.5 0.5

Concentration(µg) - 200 400 600 800 1000 - -



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Distilled water (ml) 1 .8 0.6 0.4 0.2 0.5 0.5

Lo r ’s reagent (ml) 5 5 5 5 5 5 5 5

Incubation 10 minute at room temperature

Foli ’s phe ol

reagent(ml)

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Incubation 30 minute at room temperature

OD at 660nm 0.000 0.28 0.49 0.63 0.85 0.89 0.66s 0.94

3.10. Confirmation of Biodegradation of organic waste

The organic waste of about 2 gm were weighed, inoculated in the enzyme production media

and incubated at 30°C. After one week incubation, the organic waste were taken from the media,

weight was checked.

By observing the results, the weight was gradually decreased in the mutant enzyme

production media (Table 2).



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Amount of Degraded Table: 2

Amylase media Protease media

Corn waste Pearl mellet Interval period Corn waste Pearl mellet

215.437mg 219.247mg 1day 218.247mg 218.247mg

213.006mg 215.112mg 5day 214.293mg 216.904mg

209.559mg 211.014mg 9day 211.365mg 213.998mg

205.674mg 206.354mg 13day 207.556mg 211.105mg

DISCUSSION

The soil samples were analyzed with respect to different types of microorganisms. The

isolation and characterization of bacterial and fungal strains from different places (Kodaikannal and

Salem regions) were undertaken. The maximum bacterial density is found in regions of fairly high

moisture content and the optimum level for the activities of aerobic bacteria often is a 50 to 75 % of

the soil moisture holding capacity (Alexander et al., 1961). The production of amylase and protease

enzymes by B. subtilis and A. niger in a production media. A. niger showed highest protease enzyme

activity in a production media, at temperature range from 34-38 ℃. Protease enzyme activity

decreases from 60 ℃, this may be due to the fact that at higher temperature enzymes are de-

natured. Microbial proteases have a number of commercial applications in industries like food,

leather, meat processing and cheese making. A major commercial use is the addition of microbial

proteases to domestic detergents for the digestion of pertinacious stains of fabrics (Sharma et al.,

1980). Aspergillus niger BAN 3E was found to be the best amylase producer and specific activity of

amylase produced by this strain was 52 U/mg in Submerged fermentation. Similar values of enzyme

production, for Aspergillus niger isolates JGI24 and GCB – 34 have been reported. So, this potential

strain was selected for further optimization of culture conditions (Varalakshmi et al., 2009). To meet

the growing demands in the area of industry, it is essential to improve the performance of enzyme

extraction techniques and thus increase the yield without increasing the expenses of production.

Amylase purification has mainly been restricted to a few species of fungi (Abouzeid, 1997).



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CONCLUSION

Comparing the crude and partially purified enzymes, partially purified enzyme has better activity

in the degradation of organic wastes like corn wastes and pearl millet waste. Corn waste is degraded

faster by the protease enzyme when compared to the amylase enzyme. Pearl millet is degraded

faster by the amylase enzyme when compared to the protease enzyme. Hence it can be concluded

that using Bacillus species and Aspergillus niger, we can successfully degrade organic wastes within

short duration.

ACKNOWLEDGEMENT:

The authors are thankful to Management of Vivekanandha Educational Institutions,

Elayampalayam, for providing all the facilities for our research work.

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