Life Sciences and Medicine Research, Volume 2011: LSMR-25

Isolation of Cellulytic Bacterial Strains from Soil for Effective and Efficient Bioconversion of Solid Waste
D Barman, ZA Saud, MR Habib, MF Islam, K Hossain, T Yeasmin* Department of Biochemistry and Molecular Biology, University of Rajshahi, Rajshahi 6205, Bangladesh.
*Correspondence to: Tanzima Yeasmin, yeasmin_bio@yahoo.com
Accepted: March 22, 2011; Published: July 30, 2011
Abstract The present investigation was conducted to find out the effective cellulytic bacteria for biodegradation of solid kitchen and agricultural wastes as organic manure or compost. Bacterial strains of Moraxella sp., Cellulomonas sp. and Planococcus sp. were isolated from soil and cultured on nutrient agar media. Changes of temperature and pH, CO2 release, crude fiber loss, protein, sugar and fat content as well as the activity of endoglucanase and cellobiase of waste were noted for selecting the most effective strain. In comparison to Cellulomonas sp. and Planococcus sp., inoculation of Moraxella sp. enhanced the degradation of kitchen and agricultural waste, shown by the increased CO2 release (54.3 and 37.62 mg), crude fiber loss (46.86% and 45.11%), total sugar reduction (72.52% and 74.27%), fat reduction (65.20% and 61.22%), endoglucanase -1 -1 -1 -1 (0.097 mg.hr .ml ) and cellobiase (0.82 mg.hr .ml ) activities. Inoculation of Cellulomonas sp. strain (53.89% and 77.96%) showed high protein reduction in comparison with inoculation of Moraxella sp. strain (20.04% and 63.42%) for kitchen and agricultural wastes. The overall findings of this investigation demonstrate the effectiveness of Moraxella sp. as a useful strain for bioconversion of solid organic waste. Keywords: Cellulytic bacteria; biodegradation; kitchen waste; agricultural waste.

1. Introduction Bacteria use wastes for their own metabolism and finally they produce some simple and useful compounds which are important for soil health, plant growing and over all to keep well balance of natural ecosystem. Bacteria along with saprobic fungi are an important contributor to optimal agricultural and kitchen wastes bioconversion [1]. Bangladesh is a densely populated country. Every day a huge quantity of solid waste is produced from its urban area and country site [2]. The municipal solid waste in the urban centers of Bangladesh is generated mainly from domestic, commercial and industrial sources [3, 4]. It contains mostly organic wastes which can be decomposed by composting process [5]. Composting is the microbial decomposition of biodegradable organic solid waste into an end product which is stable and soil like material called compost. Since kitchen and agricultural wastes are less harmful to the environment than industrial waste, composting of wastes other than industrial draws considerable attention. It would significantly reduce the amount of waste and the end product can be used as compost or biofertilizer which can be handled, stored, transported and applied to the field without adversely affecting the environment [6]. Therefore, presently the composting is emerging to be a popular waste management alternative both in developed and developing countries including in Bangladesh [5, 7, 8]. The implementation of the composting technology has a great potential for mitigating several problems related to ecological imbalance as well as water, soil and air pollutions. Various composters are currently commercially available or several types of in-vessel composting systems have been developed for installation in food service establishments to manage food waste as a recyclable resource. It is difficult to maintain steady degradation due to the instability of the microflora within the composter due to the raw material, pH, temperature and other environmental conditions [9-13]. Cellulolytic enzymes play an important role in natural biodegradation processes in which plant lignocellulosic materials are efficiently degraded by cellulolytic fungi, bacteria, actinomycetes and protozoa. In industry, these enzymes have found novel applications in the production of fermentable sugars and ethanol [14-16], organic acids [17], detergents and other chemicals [18]. The present study was carried out to isolate effective soil bacterial strains and those were applied to decompose kitchen and agricultural wastes in vitro condition and

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to search their potentiality as a bioconversion agent and compare their biodegradation capability on kitchen and agricultural wastes. 2. Methods 2.1. Source of sample Kitchen waste was collected in polythene bag from the kitchen dustbin of Bangabandhu Sheikh Muzibur Rahaman Hall of Rajshahi University and debris (left by farmers) was collected as agricultural waste from crop fields near the University campus. Kitchen waste is the mixture of husk and waste part of vegetables (such as potato, pumpkin, papaya, green plantain, snake-gourd, spinach, ladys finger, green amaranth, brinjal, etc.) and the composition of agricultural waste is plant stems, crop shells, wood shavings, saw dust, etc. 2.2. Isolation of bacteria Serial dilution technique [19] was used for the isolation of bacteria from soil. For isolation of effective cellulytic bacteria, samples (soil) were collected from garbage of Rajshahi University campus where waste materials from relevant area of university campus, were collected. The samples were taken from 10-15 cm depth with sterile spoons and collected in sterile polythene bags which were transported to the laboratory and stored at 4C until used. 2.3. Identification of isolated bacteria The isolated bacteria with strong cellulytic property were purified according to the procedure described by Dal Peiulyt [20]. In this method, single colony was inoculated into an autoclaved test tube containing filter paper and distilled water and incubated at 37C for 15 days. Cellulytic bacterial strain degraded filter paper in compare to control tube (without bacteria). Finally, the isolated and purified cellulytic bacterial strains, designated as B1, B2 and B3, were identified by means of morphological examination, cultural studies and biochemical characterization according to the methods of Buchanon and Gibbons [21]. 2.4. Preparation of reagent bottle For biodegradation of kitchen and agricultural wastes, seven sets of bottles were taken and each set contained six bottles. 1st, 2nd, 3rd, 4th and 5th sets were used for pH measurement, estimation of crude fiber loss, carbon dioxide release test, protein, fat and carbohydrate content. The 6th and 7th sets were used for cellulolytic activity determination. All the experiments had been done using control containing non-sterilized waste samples and control treatments were also performed with no inoculation. Such controls were used here to examine how parameters in waste were changed by native microflora of waste and by isolated strain only. The pre-weighted bottle of 250 mL capacity was half filled with 30 g of each sterilized sample (except control) separately. The samples were saturated by adding 100 mL sterilized distilled water (Biomass to water 6 proportion = 30:100). The bottles were inoculated with 2 mL bacterial suspension (2 x 10 bacteria per mL) under laminar air flow cabinet and then kept at room temperature for 28 days keeping close the bottle with cotton plug and a sterile thermometer to maintain proper aseptic condition. 2.5. Biodegradation parameter study Temperature and pH change of treated kitchen and agricultural wastes were observed after 28 days. Crude fiber content was measured using the procedure of AOAC method [22]. CO2 release during biodegradation was measured as described by Abubakar et al. [23]. Water soluble protein amount was determined by the method of Lowry [24] using bovine serum albumin (BSA) as standard. Lipid content of sample at different levels was determined by the method of Bligh and Dyer [25]. The sugar content of decomposed sample was determined colorimetrically by the anthrone method [26]. 2.6. Enzyme extraction and assays After 28 days, enzyme assays were performed by the method described by Jain and Singhai [27] at two different pH containing buffer to examine enzyme activity in different pH. Enzymes were extracted using

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Life Sciences and Medicine Research, Volume 2011: LSMR-25

different acetate buffer i) 0.25 M acetate buffer of pH 5.2 and ii) 0.05 M acetate buffer of pH 5.8. For the extraction of enzymes, 60 mL of buffer was added to the bottle having decomposed substrate and shaken well at 150 rpm in a rotating shaker for two hours. The buffer from the bottle was then decant and centrifuged at 3000 rpm for 30 minutes to obtain clear supernatant. The supernatant was then used as crude enzyme samples to assay the activity of endoglucanase and cellobiase. 2.7. Endoglucanase activity Endoglucanase activity was determined following the method described by Fergus [8]. The reaction mixture containing 9 mL of 5.5% carboxymethyl cellulose (CMC) solution in 0.25 M acetate buffer (pH 5.2) and 1 mL of enzyme sample were incubated at 45oC for 1 hour. An aliquot of 1 mL of this reaction mixture was taken and added in a separate tube containing dinitrosalicylic acid (DNS) reagent [9]. The tubes were boiled in a boiling water bath for 10 minutes and the color that developed was read at 550 nm. Then, enzyme activity was calculated as the amount of glucose released from substrate (CMC) per hour per ml (mg.hr-1.mL-1). Similar procedure was carried out for 0.05 M acetate buffer (pH 5.8) to determine the endoglucanase activity. 2.8. Cellobiase activity Cellobiase activity was determined according to the method described by Eriksson [10]. For the study, the reaction mixture was prepared as 1.5 mL of 0.25 M acetate buffer (pH 5.2), 2.5 mL 5 mM cellobiose solution in acetate buffer and 1 mL of enzyme sample were mixed simultaneously and then incubated at 45C for 1 hr. An aliquot of 1 mL of this reaction mixture was mixed with 3 mL of dinitrosalicylic acid (DNS) reagent. After boiling the reaction mixture for 10 minutes, the absorbance of the color developed was measured at 550 nm. The enzyme activity was expressed as the amount of glucose released per hour per ml enzyme sample (mg.hr-1.mL1 ) from the substrate. Similarly, cellobiase activity was determined by using 0.05 M acetate buffer (pH 5.8). 2.9. Statistical analysis All values were expressed as mean S.E.M (Standard Error of Mean). Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Dunnettst test using SPSS statistical software of 10 version. P<0.05 were considered to be statistically significant when compared with control

3. Results and Discussion 3.1. Identification of B1, B2 and B3 By comparing the results of morphological, physiological and biochemical characteristics with literature [2830], the isolate B1, B2, and B3 were identified as Moraxella sp., Cellulomonas sp. and Planococcus sp., respectively. 3.2. Temperature, pH change and CO2 release After 28 days of inoculation of bacterial strains (B1, B2, and B3) into the kitchen and agricultural wastes, the temperature of B1, B2 and B3 inoculated kitchen waste were found as 35oC, 32oC and 34oC, respectively and control temperature was 30oC. But there were no remarkable changes found in agricultural waste compared with control. The final pH of degraded sample of agricultural waste were 8.86, 8.90, and 8.95 for B1, B2 and B3 strain respectively after 28 days of inoculation and pH was measured as 8.26 in control. In case of kitchen waste, the final pH was 8.22, 8.82 and 8.15 for B1, B2 and B3 strain respectively and pH of control was 7.53. Before inoculation of bacterial strain, the pH was 6.8 and 6.48 of agricultural and kitchen wastes respectively. In this study, results regarding pH change indicated that pH of decomposed sample was changed from acidic to basic value. The pH of decomposed sample is increased gradually during composting by thermo-tolerant bacteria due to the degradation of nitrogen containing materials to soluble organic nitrogen, the formation of + ammonium ion (NH4 ) and the release of hydroxide ion by hydrolysis [23]. The above reasons may be the possible cause for increasing of pH in decomposed sample used in this study.

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Table 1: CO2 released of decomposed sample of kitchen and agricultural wastes.

Total CO2 release (mg) Strain Kitchen waste Control 28.38 1.23 B1 54.30 1.39* B2 38.94 2.12 B3 38.50 1.60 *Significantly different from control (Kitchen waste); P<0.05 Agricultural waste 14.83 0.89 37.62 2.98 32.14 3.17 24.52 1.95

The biodegradation potential of B1, B2 and B3 strains were estimated through carbon dioxide release. The maximum amount of carbon dioxide release (54.3 mg) (P<0.05) was found for B1 and followed the order B1>B2>B3 (Table 1). The high release of CO2 by B1 strain indicated the potential efficacy of this strain as cellulose degradative bacteria. Abubacker et al. [23] also found such type of results during the investigation on some fast cellulose degrading fungi. 3.3. Crude fiber loss In respect to B2 and B3 strains, the highest crude fiber loss of kitchen (46.86%) and agriculture (45.11%) waste was observed for B1 (P<0.05) strain after 28 days of inoculation. In case of control treatment, reductions were 23.76% and 28.21% for kitchen and agricultural wastes, respectively (Table 2). High loss of crude fiber by B1 strain in both kitchen and agricultural wastes also revealed its strong biodegradative potentiality like Phanerochaete chrysosporium [11] which showed high crude fiber reduction in case of wheat. Organic waste containing polymer of aromatic compounds like lignin became resistant and relatively difficult for cellulolytic organisms to decompose [31]. So in this study, composition of kitchen and agricultural wastes may be the possible reason for high crude fiber loss by isolated strain. 3.4. Water soluble protein, total sugar and fat contents of kitchen and agricultural wastes The percentage (%) reduction in water soluble protein, total sugar and fat content of kitchen and agricultural wastes by B1, B2 and B3 strains, is presented in table 2. In case of water soluble protein, the highest and significant (P<0.05) reduction was observed for inoculation of B2 with kitchen (53.89%) and agricultural (77.96%) wastes after 28 days. After 28 days of inoculation with B1 strain, the highest amount of total sugar (72.52% and 74.27%) and fat (65.20% and 61.22%) was significantly (P<0.05) reduced in kitchen and agricultural wastes in comparison to that of B2 and B3 strain. Our findings for carbohydrate reduction were quite similar with the study of Slawomir et al. [32]. They showed that the amount of carbohydrate in wastes was decreased to decomposed matter which accounted for 48.8% reduction. The amount was even more decreased after 3 months and reached the value 56.3%. In this study, all polymeric substances like water soluble protein, fat etc. may be cleaved by the enzymes produced by degrading microflora with respect to control. Table 2: Percentage (%) reduction of crude fiber, water soluble protein, total sugar and fat after 28 days of inoculation with B1, B2 and B3.
Strain Crude fiber loss (%) 23.76 46.86 35.64 26.40 Kitchen waste Protein Sugar reduction reduction (%) (%) 8.16 38.37 20.04 72.52 53.89 70.49 43.39 50.82 Agricultural waste Protein Sugar reduction reduction (%) (%) 42.73 36.45 63.42 74.27 77.96 62.70 66.78 44.29

Control B1 B2 B3

Fat reduction (%) 16.76 65.20 62.65 64.50

Crude fiber loss (%) 28.21 45.11 33.75 33.43

Fat reduction (%) 12.92 61.22 44.44 34.46

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Table 3: Activity of endoglucanase and cellobiase in extracts obtained from B1, B2 and B3 inoculated kitchen and agricultural wastes.
Enzyme sample Control Type of waste Kitchen waste Extraction buffer Endoglucanase -1 -1 (mg.hr .mL ) 0.031 0.001 0.032 0.003 0.035 0.004 0.032 0.002 0.094 0.001* 0.097 0.002* 0.089 0.005* 0.088 0.003* 0.066 0.002 0.070 0.003 0.062 0.006 0.065 0.002 0.056 0.005 0.052 0.002 0.051 0.005 0.050 0.004 Cellobiase -1 -1 (mg.hr .mL ) 0.050 0.003 0.048 0.002 0.042 0.003 0.047 0.005 t 0.820 0.004 t 0.780 0.006 t 0.807 0.003 t 0.802 0.007 0.520 0.001 0.501 0.004 0.470 0.005 0.495 0.003 0.390 0.003 0.320 0.007 0.352 0.005 0.370 0.002

0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) Agricultural waste 0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) Kitchen waste 0.25M acetate buffer (pH 5.2) B1 0.05M acetate buffer (pH 5.8) Agricultural waste 0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) Kitchen waste 0.25M acetate buffer (pH 5.2) B2 0.05M acetate buffer (pH 5.8) Agricultural waste 0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) B3 Kitchen waste 0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) Agricultural waste 0.25M acetate buffer (pH 5.2) 0.05M acetate buffer (pH 5.8) *Significantly different from control (Kitchen waste); P<0.05 t Significantly different from control (Agricultural waste); P<0.05

3.5. Activity of endoglucanase and cellobiase Biodegradation of crop residues by microorganism is dependent on the plant and microbial species. Residues reduction varied markedly with plant species. However, there is a relatively good relation between residues biodegradation and microbial lignocellulytic enzyme activities [27]. In this study, comparatively higher level of endoglucanase activity was observed in samples obtained from B1 inoculated kitchen (0.094 and 0.097 mg.hr1 .mL-1 at pH 5.2 and 5.8) and agricultural (0.089 and 0.088 mg.hr-1.mL-1 at pH 5.2 and 5.8) wastes in respect to B2 and B3 strains (Table 3). High level of cellobiase activity was also found for samples obtained from B1 inoculated kitchen (0.820 and 0.780 mg.hr-1.mL-1 at pH 5.2 and 5.8) and agricultural (0.807 and 0.802 mg.hr-1.mL-1 at pH 5.2 and 5.8) wastes. Enzyme samples obtained from B2 inoculated kitchen and agricultural waste showed moderate endoglucanase and cellobiase activities in comparison to that of results obtained from B1 and B3. Actually, the bacterial strain B1 (i.e., Moraxella sp.) grew fast and produced more hemicellulytic and cellulytic enzymes for kitchen and agricultural wastes. We have found lower endoglucanase activity than cellobiase activity in both wastes. This present result agrees with the findings of Sinegani et al. [11] who studied on agricultural residues and found lower endoglucanase activity of wheat bran and jute than cellobiase. 4. Conclusion Considering the results, it may be concluded that the strain B1 will be the potential strain for degradation which will be used effectively to prepare compost within a short period to protect our natural environment and to get rid of solid wastes problem in Bangladesh. Competing Interests The authors declare that they have no competing interests. Authors Contributions DB: Experimental work, manuscript preparation; ZAS: Conception of research work, manuscript preparation; RH: Technical support of research work, manuscript preparation; FI: Technical support of research work; KH & TY: Conception of research work, manuscript preparation.

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