Current Research in Microbiology and Biotechnology Vol. 2, No. 4 (2014): 416-421 Research Article Open Access

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Isolation and Characterization of Cellulose Degrading Bacteria from Biogas Slurry and their RAPD profiling

Sumit Kumar Dubey1, Rakesh Kumar Meena2, Shweta Sao1, Jitendra Patel1, Sanket Thakur2 and Prashant Shukla2* 1 Dr. C.V. Raman University, Kota, Bilaspur, Chhattisgarh, India. 2 Aditya Biotech Lab and Research Pvt. Ltd. Raipur, Chhattisgarh, India * Corresponding author: Prashant Shukla; email: prashant19782000@gmail.com

ABSTRACT Cellulose occurs exclusively in plants and is the most abundant organic substance in plant kingdom. Biogas from agriculture waste is considered a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process. The present work concentrates on the isolation of cellulose-degrading bacteria from biogas slurry and assessment of their cellulolytic activity and RAPD profiling. Two efficient strains of cellulolytic microorganisms namely Sc and Sw were isolated from biogas slurry showed the remarkable ability to degrade Carboxy-methyl Cellulose in the media. Optimum temperature was 350C, pH 7.0 and Salt concentration 1% for both strain of bacteria (namely Sc and Sw). They also showed their abilities to degrade amylase. Both the strain showed 20% similarities to each other. These bacterial isolates can be used as inoculants for enhancing the degradation process of cellulose present in agricultural waste.

Keywords: Cellulose, Biogas slurry, Morphological, Biochemical, RAPD profiling

INTRODUCTION Cellulose occurs exclusively in plants and is the most abundant organic substance in plant kingdom. Cellulose is a linear, un-branched homo-polysaccharide of D-glucose units. Cellulose is the most versatile organic compound on earth and received much attention as a substrate for the production of biofuels and single cell proteins through enzymatic degradation with the help of microbial cellulases [1]. Many microorganisms have been reported with cellulosic activities including many bacterial and fungal strains by which we can utilize wheat straw resource effectively. Researchers have strong interests in cellulases because of their applications in industries of starch processing, grain alcohol fermentation, malting and brewing, extraction of fruit and vegetable juices, pulp and paper industry, and textile industry as well as environmental benefits. Certain ruminants and herbivorous animals e.g. cattle, goats, sheep, buffalo, deer and camels etc, contain fungal and bacterial group of microorganisms including Trichonympha, Clostridium, Actinomycetes, Bacteroides succinogenes, Butyrivibrio fibrisolvens, Ruminococcus albus, and Methanobrevibacter ruminantium [2] in the

gut which produce enzyme namely cellulose that can cleave β - glycosidic bonds. Hydrolysis of cellulose yields a disaccharide cellobiose, followed by beta-D-glucose. Microorganisms having the ability to degrade cellulosic compounds having great importance toward different biological and ecological improvement and has provided a broad platform for research in determining the physico-biochemical properties of these microorganisms as well as their use in different biotechnological processes [3]. Biogas technology is now prominent energy source toward general house hold requirement after LPG in village area, which is effectively promoted by Indian government body. Efforts are going on to make biogas plants more and more economical. As a part of these efforts, Researchers are also interested to understand microorganisms involved in the biogas production process and their interactions [4, 5, and 6]. Biogas from agriculture waste is considered a valuable source of renewable energy. However, recalcitrant plant cell structures represent a barrier in the fermentative biodegradation process. Therefore, more efficient

Received: 11 June 2014 Accepted: 25 June 2014 Online: 16 July 2014

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degradation of cellulose and hemicellulose to monomeric sugars are required to optimize the fermentation efficiency and to increase methane yields [7] Microorganisms involved in cellulose degradation was reported namely Bacillus sp. [8and 9], Pseudomonas sp. [10] Clostridium sp. [1] and Cellulomonas sp. [11]) as these are known generally as cellulolytic bacteria. The present work concentrates on the isolation of cellulose-degrading bacteria from biogas slurry and assessment of their cellulolytic activity and RAPD profiling. MATERIALS AND METHODS Materials Sample (Biogas slurry) was collected directly from biogas plant situated Aditya Biotech Agricon Research and Development Centre Nandanvan Road, Chandanidih, Raipur (C.G.) PIN - 492001. Pre -sterilized test tube were used for collection purpose and these were brought to laboratory (ABLR, Raipur) for isolation of cellulose degrading bacteria. Methods Isolation of cellulolytic bacteria Cellulolytic bacterial strains were isolated from soil by using serial dilutions. Bacterial culture was inoculated in CMC (Carboxy-methyl cellulose) medium [1] supplemented with 1% CMC (Hi Media) and incubated at 370C for 24hours. After incubation for 24 hours, CMC agar plates were flooded with 0.1- 0.2 % congo red and allowed to stand for 15 min at room temperature. 1M NaCl was thoroughly used for counterstaining the plates. Clear zones were appeared around growing bacterial colonies indicating cellulose hydrolysis. The bacterial colonies having the largest clear zone were selected for pure culture isolation and identification. Morphological and Biochemical Examination of Isolates Morphological Examination The isolates was examined for morphological characteristics like, colony size, shape, color, margin, elevation, opacity, consistency, type of cell wall and motility. Biochemical Examination The isolates were examined for IMViC test, starch and cellulose hydrolysis, gelatin liquefaction, urease, H2S, catalase activity and sugar (sucrose, lactose and mannitol) fermentation test. Effect of Incubation time Different incubation times (24, 48, 72, 96 hours) were employed to study optimum incubation period for bacterial growth. Estimation of temperature, pH and salt tolerance Estimation of Temperature Tubes of Nutrient Agar Media (NAM) broth were inoculated with bacterial culture and kept for incubation at different temperature (25, 30, 35, 40, 45 and 500C) to study growth at different temperature.

Estimation of pH Tubes of NAM broth were made with different pH (4, 5, 6, 7, 8 and 9) inoculated with bacterial culture and kept at 370C for 24 hours for growth. Estimation of salt tolerance capability Tubes of NAM broth were prepared with different concentration of NaCl (1, 2, 3, 4, 5 and 6%) and were inoculated with bacterial culture and kept at 370C for 24 hours for growth. Effect of carbon sources To identify the suitable carbon source for the Cellulase production, the carbon source (CMC) of the production medium was replaced with various carbon sources like Sucrose, Lactose and Maltose. The assay was carried out after 72 hours of incubation. Growth curve Growth curve of bacteria was determined by inoculating bacteria in different test tube containing NAM broth and obtained OD of the sample after 24 hours interval (e.g. 24, 48, 72 and 96) using Spectrophotometer (ELICO/BL-200). Isolation of genomic DNA and RAPD profiling Isolation of genomic DNA was done by SDS (10% Sodium dodecyl sulphate method). Quantification of extracted sample Genomic DNA was quantified by using 0.8 % agarose gel and Nano Drop Spectrophotometer. PCR Amplification RAPD PCR was performed on bacterial DNA samples using different primers (from suing universal primer) according to the method described by Williams et al. 1990. The thermal cycle profile was as follow: 4 min initial denaturation at 95ºC, 44 cycles of 1 min at 92ºC, 1 min at 30ºC, 1 min at 72ºC, followed by a final extension at 72ºC for 10 min. PCR product were analyzed in Gel Electrophoresis (Tarson M1-01) 2% agarose gel stained with ethidium bromide . Gels were photographed by Gel Documentation system (Bio Rad- Gel doc EZ imager). The phylogeny tree of bacterial isolates was made according to statistical program analysis (SYSpc- ver. 2.0). RESULTS AND DISCUSSION The seven bacteria were screened for cellulase production using CMC medium. Among all these two bacterial strains Sc and Sw strains were further identified and their morphological and biochemical tests were performed. Bacteria were screened on CMC medium [10] where 0.1% congo red was used whereas [12] 1% congo red was used. Pure culture was isolated from screened bacterial colony for further study. Two strains of bacteria namely Sc and Sw were selected for further study. Microscopic examination of Sc isolate was showed gram negative and short rod shaped bacteria in the

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same manner colonies Sw isolate was reported gram negative and rod shaped bacteria (Table 4). Sc was negative for indole and phosphatase and positive for MR, VP, citrate, catalase, amylase and urease but Sw was negative for indole production, MR test, urease and phosphatase and positive for VP, citrate, catalase and amylase (Table 5). [13] reported that colonies of ASN2 (isolate ASN2 was identified as Cellulomonas sp.) was negative for indole production, Voges Proskauer test and citrate utilization and positive for catalase and nitrate reduction. Similarly with Sc could ferment sucrose and maltose whereas Sw could ferment lactose, Sucrose and maltose (Table 5 and Fig.5,6,7,8,9,10,11,12,13,14,15 and 16). [1] revealed that closest relative of strain CTL-6 was Clostridium thermocellum utilize cellulose, cellobiose, glucose, and sorbitol as the sole carbon source. No growth of strain CTL-6 was observed with glycerol, mannose, sucrose, and raffinose. Result indicated that optimum temperature was 350C, pH 7.0 and Salt concentration 1%, and salt tolerance till 6% recorded for both strain of bacteria namely Sc and Sw (Table 6) whereas Prasad et al. (2013) investigated that optimal pH was 7.0, temperature 260C. [14] and [12] both found temperature 35o C and pH 7·5 were optimal for growth and on the basis of that growth curve has been drawn (Table 7 and Fig 1, 2, 3 and 4). Biochemical test performed was checked by (ABIS) online bacterial identification tool in which result indicated (according to biochemical test results) for Sc was Bacillus subtilis (possibility of B. atrophaeus) ~ 73% (acc: 28%), Bacillus licheniformis ~ 70% (acc: 28%), Bacillus cereus ~ 70% (acc: 28%), and for Sw was Bacillus licheniformis ~ 78% (acc: 30%), Paenibacillus cineris ~ 71% (acc: 30%), Paenibacillus cookii ~ 71% (acc: 30%). [15] Irfan et al investigated five cellulose degrading bacteria were Sphingomonas sp., Pseudomonas sp.M1, Achromobacter sp., Pseudomonas sp. M2, and Stenotrophomonas sp. and in 2012 Irfan et al., observed Cellulomonas sp. ASN2 and [16] Ahmad et al., (2013) observed among the 15 isolates one belonged to Pseudomonas spp., one to Aeromonas spp., one to Pasteurella spp., two belonged to Staphylococcus spp. and then to Bacillus genus.

Among them, Bacillus spp. has shown a remarkable ability of cellulose hydrolysis in terms of Congo red assay by giving a zone ratio of 3.4. [14] Meher and Ranade (1993) had found various anaerobic hydrolytic and methanogenic bacteria active in cattle dung biogas plants including Syntrophobacter wolinii a propionate degrading bacterium in co-culture with hydrogen utilizing methanogen viz., Methanobacterium formicicum. Genetic diversity analysis Genetic diversity analysis was performed by Rbac (Universal primer for bacteria) RAPD based markers. Total 18 markers used for the genetic diversity analysis. The range of alleles amplified was 6 to 10 for all the primers. Total 108 alleles amplified. The dendogram generated by using NTSYS-pc ver. 2.02 software UPGMA matrices. Both the bacteria showed 20% similarities to each other, in other words, they are 85% dissimilar to each other (Fig17, 18, 19, 20and 21). Table 4. Colony Characterization

S No

Morphological Characterization

Sc Sw

1 Size 0.2-0.3 0.1-0.2 2 Shape Short Rod Rod 3 Color Creamish White 4 Size 0.2-0.3 0.1-0.2 5 Elevation Raised Raised 6 Margin Round Round 7 Opacity Opaque Opaque 8 Gram’s Nature -ve -ve 9 Motility Motile Motile

Table 5. Biochemical Characterization

S No

Biochemical Characterization

Sc Sw

1 Indole (-) (-) 2 MR (+) (-) 3 VP (+) (+) 4 Citrate (+) (+) 5 Catalase (+) (+) 6 Gelatin hydrolysis test (-) (-) 7 Amylase (+) (+) 8 Cellulase (+) (+) 9 Urease (+) (-) 10 Phosphatease (-) (-) 11 Lactose Fermentation (-) (+) 12 Sucrose Fermentation (+) (+) 13 Maltose Fermentation (+) (+)

Table 6. Estimation of Temperature, pH and salt tolerance

S No

Optimization Tem Sc Sw pH Sc Sw NaCl Sc Sw

1 25 0.9947 1.2423 4 0.1738 0.1327 1 0.9764 0.9683 2 30 1.2679 1.4586 5 0.5971 0.5871 2 0.6714 0.8718 3 35 1.5915 1.5718 6 0.9619 1.1432 3 0.5308 0.6931 4 40 1.3535 1.0828 7 0.9947 1.2423 4 0.5537 0.4749 5 45 0.1985 0.0678 8 0.8657 1.0361 5 0.4028 0.2035 6 50 0.0888 0.0181 9 0.4838 0.7836 6 0.3724 0.1063

Table 7. Growth curve

Bacterial Strain

Time (in hours) 24 48 72 96

Sc 1.5387 1.5548 1.6835 1.6285 Sw 1.5635 1.5957 1.6071 1.5357

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Figure 1. Effect of temperature on bacterial strains

Figure 2. Effect of pH on bacterial strains

Figure 3. Effect of salt concentration on bacterial strains

Figure 4. Growth curve of bacterial strains

Figure 5. Cellulose degradation (Sc)

Figure 6. Cellulose degradation (Sw)

Figure 7. Indole test

Figure 8. Vogues Proskeur test

Figure 9. Methyl red test

Figure 10. Amylase test

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Figure 11. Citrate test

Figure 12. Amylase test

Figure 13. Gelatinase enzyme test

Figure 14. Fermentation (Sucrose)

Figure 15. Fermentation (Lactose)

Figure 16. Fermentation (Maltose)

Figure 17. Rbac06

Figure 18. Rbac07

Figure 19. Rbac08

Figure 20. Rbac09

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Figure 21. Dendrogram generated by using NTSYS-pc ver. 2.02 software UPGMA matrices. REFERENCES

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