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Enzymatic activity of bacteria isolated from different compost.

Introduction

Composting a natural biological process is the controlled decay of organic matter in a warm moist environment by action of bacteria, fungi and other organisms [1]. Compost is the result of composting which is, by definition, microbial decomposition of organic waste i.e. manures, straw, green waste etc. Microbiological characteristics must be the most effective parameter in considering compost. Composting is a self-heating, aerobic, solid-phase biological accelerated natural process of biodegradation and mineralization of organic matter. The process relies on the right combination of carbon (wood, straw) and nitrogen (pig, poultry manure), the right moisture content and ability to blend and aerate the materials. Composts in disease suppression, adsorption and transport of heavy metals and potential to adsorb or transform hazardous organic pollutants and persistent biological molecules have been studied. The organic waste materials mainly of animal and plant origin are potential sources of organic matter and plant nutrient [2] and the benefits derived from the utilization of these organic materials ranges from improvement of soil fertility to a reliable means of waste disposal. Cow dung manure is a nitrogen rich material and is of economic importance as fertilizer, feed supplement or as energy sources. Composting of green wastes such as vegetable waste has potential to contribute to enhance the preservation of the environment and to increase soil fertility.

Vermicompost is the product of composting utilizing various species of worms, usually earthworms, and white worms to create a heterogeneous mixture of decomposing vegetable or food waste, bedding materials, and vermicast.Vermicompost also contain plenty of 'beneficial soil microbes' which helps in 'soil regeneration' & 'fertility improvement' & protect them from degradation while also promoting growth in plants.[3]. Vermicompost contains some antibiotics and actinomycetes which help in increasing the 'power of resistance' among the crop plants against pest and diseases. So pesticide spray was significantly reduced where earthworms and vermicompost were used in agriculture [4]. In land application of products from organic wastes, such as composts is gaining importance as integrated and biological means as agriculture are becoming increasingly popular. Composts are an important source of nutrients usable by microorganisms. Results on compost amendments generally enhance the development of the microflora and increased the global activity of the soils [5].As compost is one of the diverse environments, so the objective of the work is to study the microbiological diversity and enzymatic activity of the bacteria found in the compost.

Materials & Methods Sample Collection

The three different compost samples were collected. The Vermi compost was collected from the Adivasimela & cow-dung compost was collected from the rural area and the agricultural compost was collected from Agriculture Department of O.U.A.T Bhubaneswar. These samples were brought to the P.G Department Of Microbiology for further processing.

Isolation and culture of microorganisms Isolation

Isolation of bacteria was done by 10fold serial dilution method.. The diluted sample was taken from the tubes and the plated containing Nutrient Agar sterilized in 121[degrees] C and 15lb pressure for 20 mins. To the plates 1 to 2 drops of diluted sample was added and it was uniformly spread over the media with the help of L-shaped loop and rotor. The plates were then incubated for 24hrs at 37[degrees]C inside an incubator. The number of colonies formed were observed & counted with the help of a colony counter. Then the colony forming units (CFU) was calculated.

Maintenance of Pure Culture

The colonies which were streaked out in the plates were maintained in the sterilized slants for further use. The total process was undertaken in LA under aseptic condition. After pure cultures were obtained they were maintained in the refrigerator and different parameters of the isolates were studied.

Growth, Morphology and Staining Of the Organism (Bacteria)

Morphological characters of the bacteria were studied following the methods [6].The isolates were streaked on NA plates, incubated for 24 hours at 30[degrees]C. The shape, size, color, margin and opacity were recorded from isolated colonies.

Isolation of Actinomycetes

The petriplates were sterilized and Actinomycetes Isolation Agar (AIA) was prepared. To it glycerol was added and then the media was sterilized. After sterilization antibiotic Ambystrin & antifungal Rustinin was added to inhibit the growth of bacteria & fungi respectively. Then serial dilution of the compost sample was done. The media was cooled & poured into the petriplates & allowed to solidify. About 0.1ml of the sample was added to each plate & spreaded uniformly with a L-shaped glass spreader. The plates were then incubated at 28o c for 4 to 5 days and their growth was observed. The numbers of colonies were counted by means of a colony counter.

Physiological and biochemical character

Physiological and biochemical characters of the organisms were checked following the standard methods for identification of the isolates [7] Different biochemical tests like Oxidase test, catalase test, urease test, indole production test, methyl red, voges-proskauer (acetone production) test, nitrate reduction test, citrate utilization test, carbohydrate metabolism were studied.

Enzymatic activity

The activities of various extra cellular enzymes produced by the isolates were studied by the following tests:

Starch hydrolysis test: Capacity of the organism to hydrolyze starch into simple substances like dextrin, glucose, maltose, etc. by the amylase enzymes were detected by spot inoculating the bacterial cultures on NA plates containing 1% soluble starch. After incubation for 24 hours at 30oC, the plates were flooded with iodine solution for 5 minutes, excess solutions were decanted off and starch hydrolysis was noted from a clear zone formed around the colonies. Clear area around the colonies indicated partial hydrolysis of starch.

Lipase test: Lipase production by the organisms was tested using different lipid sources

On plates indicated at 30 degree for 3 days. Tributyrin (or Vegetable oil) hydrolysis test: The Tributyrin (or vegetable oil) is broken down by the lipase produced by the organisms to water soluble butyric acid and forms a clear around the lipase positive colonies.

Protein hydrolysis test: Proteolytic activity of bacteria was detected from the following tests on NA plates containing the protein sources.

Gelatin hydrolysis test: Gelatin liquefaction was detected by spot inoculating the bacteria on NA plates containing 1% gelatin followed by the incubation at 30 degree for 24-48 hours. The plates were flooded with acidic mercuric chloride solution (15%), waited for 5-10 minutes, the excess solution was decanted off and appearance of a clear zone around the colonies was indicative of hydrolysis of gelatin by the enzyme gelatinase.

Casein hydrolyzing test: Casein hydrolyzing activity of the bacteria was recorded from liquefaction of casein by the bacteria on NA plates containing 1% casein, spot inoculated and incubated at 30 degree for 24-48 hours. The plate were flooded with acidic HgCl2 (15%), excess solution was decanted off and clear zone formation was observed.

Pectin hydrolysis test: The ability of the microorganisms to hydrolyze pectin in to pectic acid was assessed by the test. NA plates containing 1% pectin was spotted with the bacteria, incubated at 30 degree for 72 hours. The plates were flooded with 1% which precipitates pectin and a clear zone around the colony will be formed

Chitin hydrolysis test: Chitin hydrolysis ability of the microorganisms to glucosamine was assessed by the test. Chitin at 1% level was added to NA medium; organisms were spotted and incubated at 30 degree for 72 hours. A clear zone formation was observed after the incubation period. hexadecyltrimethyl ammonium bromide i.e. cetyl trimethyl ammonium bromide (CTAB)

Result and Discussion

Out of three different compost samples, the maximum number of bacteria and actinomycetes were obtained in Vermicompost (12x[10.sup.10] and 5x[10.sup.5]) followed by cow dung compost and agricultural compost.

Out of 11 isolates, five were Gram's positive rods, five were Gram's positive Cocci, and only one was Gram's negative Cocci which were isolated from different composts.C1,C2,C3 from cow dung compost,V1,V2 from vermicompost and A1, A2, A3,A4,A5,A6,A7 from agricultural compost

Two actinomycetes were isolated and identified as Nocardia sp. and Streptomyces sp. on the basis of morphology and colony characteristics

The biochemical characterization of 11 isolates were studied in terms of MR-VP test, citrate tests, Mannitol and motility tests, C1,C3,V1,A1, A2, A3,A5 were found methyl red positive. Only one isolates A2 was positive for VP-test.C1,C3,A2, A3, A5 showed positive for Simmon's Citrate tests.A2, A3, A4, A5,A6, A7 were showed Mannitol positive and C1, C3, V1, A2, A4, A5, A6, A7 were found to be motility positive.

Out of 11 isolates, 27.2% were found to be amylase positive, 36.3% were lipase positive, 27.2% were gelatinase positive, caseinase all were negative, 9.0% were pectinase positive and all were chitinase negative. So maximum number of lipase producing microorganisms are present in compost samples.

Three intracellular enzymes viz, oxidase, catalase and urease were studied in all isolates and nitrate reduction tests was studied only for Bacillus species. Except three, other two Bacilli (A3 & A5) showed nitrate reduction positive.

Twelve sugars were taken and tested for sugar utilization test by Bacillus species.

Maximum number of bacteria was obtained in vermicompost sample in comparison to cow dung and agricultural compost samples. This result is similar with the result of Aira, et.al [8] who were also obtained maximum number of bacteria from vermicompost sample. The two actinomycetes organisms were isolated and identified as Streptomyces and Nocardia sp. on the basis of morphology and colony characteristics.

On the basis of staining, maximum number of bacteria was Gram's positive rods, followed by Gram's positive Cocci and only one isolate was Gram's negative Cocci. On the basis of biochemical tests the 11 isolates were identified as C1- Micrococcus luteus, C2-Streptococcus sp. C3-Bacillus fusiformis, v1-Neisseria sp. A1- Micrococcus sp., A2- Bacillus cereus, A3-Bacillus subtilis, A4-Paenibacillus sp., A5-Brevibacillus sp., A6-Stapylococcus sp, A7-Streptococcus mutans from vermicompost, agricultural compost and cow dung compost samples. D.V.Adegunloye, et. al [9] obtained 86% bacteria from cow dung compost samples. These bacteria were identified as Micrococcus luteus, Bacillus pumilus, Bacillus macereans, pseudomonas aeroginnosa, Enterobacter aerogenes, Proteus mirabilis etc.Jeaine.I, Boulter et. al [10]obtained 78% of bacteria from compost sample which were Gram's negative bacteria such as Pseudomonas sp., Serratia sp., klebsilla sp., & Enterobacter sp., and Gram's positive were identified as Bacillus sp.

The bacteria isolated from different compost samples showed lipase positive, amylase positive, gelatinase positive, pectinase positive, catalase positive, and urease positive. This result is similar to the result of Nipun Silwat et. al-*- [11]who obtained different types of microorganisms from vermicompost sample showing amylase, lipase, urease and catalase activity.

Sugar tests were performed for Bacillus spp. And out of five isolates, four isolates were positive for Sacarose, Trehalose, Raffinose, and Eesculine hydrolysis on the basis of PIBwin software, the Bacillus spp. were identified as Bacillus fusiformis, Bacillus cereus, Bacillus subtilis, and Paenibacillus sp. and Brevibacillus sp. Due to the high potential for enzymatic activity, the bacterial isolates can be utilized in different biotechnological industry.

References

[1] Salvator, K., and Sabee, W.E., 1995. Evaluation of Fertilizer Value and Nutrient Release From Corn and Soybean Residues Under Laboratory and Greenhouse. Conditions. Commu. Soil/ Sci., Plant Anal., 26: 469-484.

[2] Adeniran, J.A., Taiwo, L.B., and Sobulo, R.A., 2003. Effects of Organic wastes and Method of composting on compost maturity, Nutrient Composition of Compost and Yields of Two Vegetable Crops. J. of Sustainable Agriculture, Vol. 22: 95-101.

[3] De Brito Alvarez, M.A., Gagne, S., and Antoun, H., 1995. Effect of compost on rhizosphere microflora of the tomato and on the incidence of plant-growth promoting rhizobacteria. J. of Applied and Environmental Microbiology, 61: 194-199.

[4] Singh, R.D., 1993. Harnessing the Earthworms for Sustainable Agriculture. Institute of National Organic Agriculture, Pune, India, pp: and L. Dendooven b 1-16.

[5] Bailey, K.L., Lazarovits, G., 2003. Suppressing soil-borne diseases with residue management and organic amendments. Soil Tillage Res. 72, 169-180.

[6] Norris J.R., Swain H., 1971. Staining bacteria: Materials in Microbiol. 5A, 105-134.

[7] Bryant TN (2004). PIBWin-software for probabilistic identification. Journal Appl. Microbiol. www.som.soton.ac.uk/staff/tnb/pib.htm 97(6): 1326-1327.

[8] Aira, M., Monroy, F., Dominguez, J., 2007. Earthworms strongly modify microbial biomass and activity triggering enzymatic activities during vermicomposting independently of the application rates of pig slurry. Sci. Total Environ. 385, 252-261.

[9] Adegunloye, D.V., Adetuyi, F.C., Akinyosoye, F.A., and Doyeni, M.O., 2007.Microbial Analysis of Compost Using Cow dung as Booster, Pakistan Journal of Nutrition 6 (5): 506-510.

[10] Jeanine I., Boulter, Jack T., Trevors and Greg J., Boland, 2002. Microbial studies of compost: bacterial identification, and their potential for turfgrass pathogen suppression, World Journal of Microbiology & Biotechnology 18: 661-671.

[11] Nipun Silawat, Shweta Chouhan, Pramod Sairkar, Garg, R. K., Neetu Vijay and Mehrotra, N. N., 2009. Estimation of bacterial diversity in soil and vermi compost using sole source carbon utilization (SSCU) profile. African Journal of Microbiology Research Vol. 4 (4), pp. 255-266.

Kalyani Sahoo (1), Dr. Pratima Ray * (2) and Dr. Sukantibala Mohapatra (2)

(1) Academy of Managememt and Information Technology, Utkal University, Bhubaneswar, India (2) Centre for Post Graduate Studies, Orissa University of Agriculture and Technology, Bhbaneswar, India

* Corresponding Author E-mail: pratimary@yahoo.com
Table 1: The bacterial & actinomycetes load of compost sample.

Sl.no Organisms Cow-dung
 Compost

1 Bacteria 24 x [10.sup.8]
2 Actinomycetes 32 x [10.sup.4]

Sl.no Vermi Agricultural
 Compost Compost

1 12 x [10.sup.10] 94 x [10.sup.4]
2 5 x [10.sup.5] 6 x [10.sup.4]

Table 2: Colony Characteristics of Bacteria.

Bacteria (Isolates) Colony Morphology Gram's Staining

C1 White, round Cocci +ve
C2 Round, off white Cocci +ve
C3 Wrinkle, off white Rod +ve

V1 Round, off white Cocci -ve
A1 Off white not round Cocci +ve
A2 Round, white Rod +ve
A3 Slightly yellow Rod +ve
A4 Wrinkled, white Rod +ve
A5 Round, slightly orange Rod +ve
A6 White not round Cocci +ve
A7 Round, yellow Cocci +ve

Table 3: Colony Characteristics of Actinomycetes.

Actinomycetes Colony Morphology Gram's Identification
(Isolates) Staining

Organism-1 waxy, shiny; several +ve Nocardia sp.
 millimeters in
 diameter; aerial
 filaments are formed,
 the colony surface
 become dull and fuzzy.

Oganism-2 Powdery colony appears +ve Streptomyces
 convex, concave or
 flat surface; white,
 gray to pinkish
 color Colony.

Table 4: Biochemical Tests of Bacterial Isolates.

Organisms MR-Test VP-Test Simmon Mannitol Motility
(Isolates) citrate Test Test

C1 +ve -ve +ve -ve +ve
C2 -ve -ve -ve -ve -ve
C3 +ve -ve +ve -ve +ve
V1 +ve -ve -ve -ve +ve
A1 +ve -ve -ve -ve -ve
A2 +ve +ve +ve +ve +ve
A3 +ve -ve +ve +ve -ve
A4 -ve -ve -ve +ve +ve
A5 +ve -ve +ve +ve +ve
A6 -ve +ve +ve +ve +ve
A7 -ve -ve -ve +ve +ve

Table 5: Extracellular Enzyme Activity of Bacterial Isolates.

Organisms Starch Lipase Gelatin
(Isolates) Hydrolysis Hydrolysis Hydrolysis

C1 -ve +ve -ve
C2 -ve -ve -ve
C3 -ve -ve -ve
V1 +ve +ve -ve
A1 -ve +ve +ve
A2 +ve -ve +ve
A3 -ve +ve -ve
A4 +ve +ve +ve
A5 -ve -ve -ve
A6 -ve -ve -ve
A7 -ve -ve -ve

Organisms Casein Pectin Chitin
(Isolates) Hydrolysis Hydrolysis Hdrolysis

C1 -ve -ve -ve
C2 -ve -ve -ve
C3 -ve -ve -ve
V1 -ve -ve -ve
A1 -ve -ve -ve
A2 -ve -ve -ve
A3 -ve -ve -ve
A4 -ve +ve -ve
A5 -ve -ve -ve
A6 -ve -ve -ve
A7 -ve -ve -ve

Table 6: Intracellular Enzyme Activity.

Organisms Oxidase Catalase Urease Nitrate
(Isolates) reduction

C1 +ve -ve +ve
C2 +ve -ve -ve
C3 +ve -ve +ve -ve
V1 +ve -ve -ve
A1 +ve -ve +ve
A2 +ve -ve -ve -ve
A3 +ve -ve -ve +ve
A4 +ve -ve +ve -ve
A5 +ve -ve -ve +ve
A6 +ve -ve +ve
A7 -ve -ve -ve

Table 7: Sugar Test for Bacillus Species.

Sugars C3 A2 A3 A4 A5

Esculine Hdrolysis -ve +ve +ve +ve +ve
Arabinose -ve +ve +ve -ve +ve
Xylose -ve -ve -ve -ve -ve
Adonitol -ve -ve -ve -ve -ve
Rhomnose -ve -ve -ve +ve -ve
Cellobiose -ve -ve +ve +ve +ve
Melibinose -ve +ve -ve +ve -ve
Raffinose -ve +ve +ve +ve -ve
Trehalose -ve +ve +ve +ve +ve
Glucose -ve +ve +ve +ve +ve
Lactose -ve +ve -ve +ve -ve
Sacarose -ve +ve +ve +ve +ve
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Author:Sahoo, Kalyani; Ray, Pratima; Mohapatra, Sukantibala
Publication:International Journal of Applied Environmental Sciences
Date:May 1, 2012
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