Optimization of cellulose Production by bacteria isolated from saw dust

Cellulase enzymes are of enormous value in various industries as well as in treatment of wastes particularly lignocellulosic wastes. This high degree of applications of cellulase necessitates inexhaustible search for more sources of these enzymes. The aim of this study was to optimize the production of cellulase by bacteria from saw dust. Bacteria were isolated from sawdust samples, characterized phenotypically and screened for cellulase production. The production of the enzyme was optimized using different carbon and nitrogen sources, temperature, pH and lignocellulosic wastes. Five cellulase-producing bacteria were isolated and identified as A7 and A7 ( Bacillus spp.) B3 B5 and 2 2, 2B5 ( Pseudomonas spp.). Two percent (2%) of carboxymethyl cellulose and 1 % 3 yeast extract gave the highest cellulase production for all the isolates except A7 2 in the case of yeast extract while pH 6.0 was preferred for maximum enzyme production for all except 2B5 which preferred pH 7. The highest sugar was 3 produced by isolate 2B5 (1.38±0.54 mg/ml) in the presence of sawdust among 3 the lignocellulosic wastes. This study showed that the cellulase produced by these isolates can be used to generate sugar for industrial uses from lignocellulosic wastes and plant origin.

Introduction lignocellulosic materials need to be converted to Lignocellulosic materials are one of the simple sugars before fermentation, through a abundant natural complex organic carbons in process called hydrolysis (Taherzadeh and form of plant biomass, which is highly renewable Karimi, 2007). Hydrolysis of cellulosic materials natural resource in the world (Zhu et. al., 2006). is usually by cellulase enzyme complex. The This biomass holds remarkable potential for biodegradation of waste materials occurs by the conversion into commodity products presenting concerted action of various microorganisms double advantages of sustainable resource which produce a series of enzymes that supply and environmental quality (Damisa et. al., contribute to the bioconversion process (Pérez 2008).
The accumulation of lignocellulose et. al., 2002). Cellulose degradation comes from wastes causes environmental problems, while the study of the mesophilic fungi and anaerobic the non-use of these materials constitutes a loss thermophilic cellulolytic bacteria. The bacterial of potentially valuable sources (Mishra and cellulases have very high activities against of enzyme solution. crystalline celluloses like cotton or avicel and are also more thermostable in comparison to fungal Effect of environmental factors on enzyme cellulases (Rani et. al., 2013). Hence, this study production aimed at isolating bacteria with cellulase Various parameters were studied to producing potential and to investigate ability of determine the optimum conditions for enzyme the isolates in degrading lignocellulosic wastes. Sawdust samples were collected from yeast extract and urea); Oke gada sawmill in Ede North Local pH ranges(4, 5, 6 and Government, Osun state, Nigeria. The samples 7.0) and Carbon sources (galactose, lactose, were taken from different depths at 5,15,25,35 sorbose, sorbitol, glucose and sucrose). and 45 cm using soil auger, put into different The effects of lignocellulosic substrates sterile polythene bags and transported to (sawdust, Groundnut shell and Corn cob) on o laboratory. The samples were stored at 4 C for cellulase production were also studied. The further analysis.
substrates were alkali-treated by autoclaving the washed and dried lignocellulosic substrates at o Isolation of Bacteria 121 C for 30mins with 0.25 M NaOH (20 ml/g One gram of sample (sawdust) was substrate). The substrates recovered by filtration added to 10.0 ml sterile distilled water in two through muslin cloth were thoroughly washed different test tubes under aseptic condition. The with deionised water and neutralized with 0.25 M samples were serially diluted in ten folds. From HCl. The substrates were finally washed with -4 -6 -8 10 , 10 and 10 , 1.0 ml of each dilution was many changes of deionized water and dried at 65 0 inoculated onto carboxymethylcellulose agar C to constant weight (Singh et. al., 1988). One (2.0 g tryptone, 0.2 g yeast extract, 0.1 g K HPO gram of each dried substrates was measured as 2 4 and 2.0 g agar in 200 ml of distilled water) plates carbon source with 1 g of tryptone, 0.05 g of using pour plate method. The plates were K HPO and 0.1 g yeast extract in 100 ml. 10 ml 2 4 incubated for 24 hours at 35°C. Pure cultures of each was dispensed in and o the isolates were obtained by series of substerilized at 121 C for 15 mins. Each bottle was culturing on CMC agar plates.

Characterizati
Pure cultures of the isolates were identified on the basis of colony and cell morphology, Gram staining and biochemical (Immanuel et. al., 2006). characterization (including Catalase, Oxidase, Triple sugar iron, MRVP, Motility, Spore, H S, 2

Results
Urase, Gelatinase, NO reduction and Sugar 3 Screening for cellulase production: In fermentation tests) as described by Chessbough, table 1, the highest enzyme production on the (2000) with reference to Bergey's Manual of plates was observed with isolate 2B53 (1.15 cm) Systematic Bacteriology (Sneath, 1986). and the least qualitative value of enzyme was obtained with B53 (0.2 cm).

Cellulase Assay
Cellulase productions by the isolates were determined using dinitrosalicylic acid (DNSA) reagent method .

Studies on some cultural factors and
stimulate highest cellulose production apart from enzyme production A72.

production by the isolates Effects of different Carbon sources on enzyme production
The results obtained showed that different isolates have their most preferred carbon source for enzyme production. For isolate A72 and 2B53 the highest cellulase production was observed in the presence of sucrose (0.22 and 0.2 mg/ml) while it was glucose for A74 (0.32 mg/ml), galactose for B32 and B53 (0.24 mg/ml). The least cellulase production was A74 (0.02 mg/ml) and in the medium containing lactose.

Effects of different Nitrogen sources on the
Effects of different lignocellulosic wastes on the enzyme production by the isolates enzyme production by the isolates The effects of different nitrogen sources Ability of different lignocellulosic wastes on enzyme production of bacterial isolates (A72, to elicit the production of cellulase by the isolates A74, B32, B53 and 2B53) was examined in the is shown in Table 2. For isolates Bacillus medium and the results are shown in Figure  spp.(A72 and A74), the maximum cellulase 2.Only yeast extract produce considerably high production was at 24 hr incubation in sawdust amount of enzyme among the nitrogen sources and corn cob containing media but 48 hr in for Pseudomonas spp., 3.31, 1.12 and 2.93 ground nut shell. For Pseudomonas sp. (B32), mg/ml for B32, B53 and 2B53 respectively. maximum enzyme (0.46 mg/ml) production was observed at 24 hr incubation in sawdust while it Effects of different temperatures on the enzyme was at 48 hr of incubation for Pseudomonas spp.
( B53 and 2B53) that maximum cellulase production by the isolates (0.14±0.01 and 1.38±0.54 mg/ml) for B53 and In figure 3, A74 showed the highest 0 2B53 respectively) production was noticed. For cellulase production at the temperature of 45 C corn cob, the highest values of cellulase (0.26 mg/l). Highest cellulase (0.8 mg/ml) was 0 production was observed at the 24 hr of obtained at 40 C by isolate 2B53. The enzyme incubation for isolates B32 and B53 while there production declined as the temperature was fluctuations in the amount of the enzyme increased, A74, B32 and B53 produced the o produced by isolate 2B53 . minimal cellulase activity (0.02 mg/ml) at 60 C.
The mean values of cellulase produced at different hours and among the isolates, using Effects of different pH on and enzyme production various lignocellulosic wastes are shown in Table  by the isolates 2. The statistical significant variations were In Figure 4 pH 7 was found to be observed among the quantities of enzyme favourable for isolate 2B53 for maximal enzyme produced. production whereas, others preferred pH 6.0 to

Effects of different concentrations of CMC on the cellulase
Isolate B32 was found to have the highest cellulase production at concentration of 2.0 w/v of CMC. The minimum cellulase production for 2B53 is at concentration of 0.5 w/v when compared to other isolates at the same concentration. Isolate A74 and 2B53 showed substantial increase in cellulase production from 0.5 w/v and 1.0 w/v CMC concentration, while for isolates A72, B32 and B53 the same trend of substantial increase in production was observed from 0.5-2.0 w/v concentrations (Figure 1).

Discussion
48 h in sawdust medium while the enzyme In this study, extracellular cellulase was production (0.01±0.01 mg/ml) was recorded produced by bacterial isolates from sawdust. It for A74 at 72 hr in the same sawdust medium. was discovered that there was a progressive The results obtained in this study are increase in cellulase production by all the consistent with that earlier reported by Ojumu isolates from 0.5-2.0 % concentration of CMC. et. al. (2003) and Ja'afaru and Fagade, (2010) The use of CMC in cellulase production is in that sawdust stimulated cellulase production agreement with results of Narashima et.al. than other lignocellulosic wastes. (2006) who reported high level of cellulase The difference in the production of production using cellulose for the growth of cellulolytic enzyme on variety of lignocellulosic materials by different organisms might be due Aspergillus and Bacillus spp.
to various factors like differences in cellulose Sources of nitrogen in the medium of content in the lignocellulose obtained from growth for an organism are factors in different plant sources, variation in structure propagation of such isolate. Yeast extract was and cellulolytic abilities of the organisms at best utilized by most of the isolates. The same different degree as well as culture conditions observation was reported by Okeke and Obi, (Ja`afaru and Fagade, 2007;Chinedu et. al., (1993) and Naruma and Jirapa, (2007 incubation. This observation might be due to Highest cellulase production was high concentration of rapidly metabolised observed between pH 6.0 and 7.0 for all the carbon source after 24 hr or 48 hr (Chinedu et. isolates. This is in agreement with that Bakri et. al., 2008) Annamalai et. al. (2013. The authors reported References pH 6.0 to 7.5 for Bacillus sp and Pseudomonas Annamalaia N, Rajeswarib M. V., Elayaraja S sp enzyme production. a n d B a l a s u b ra m a n i a n T. ( 2 0 1 3 ) . Different isolates have their most Thermostable, haloalkaline cellulase from preferred carbon source for enzyme Bacillus halodurans CAS 1 by conversion of production. The least cellulase production was A74 in the medium containing lactose while l i g n o c e l l u l o s i c w a s t e s . D O I : the highest cellulase production was also from 10.1016/j.carbpol.94(1) :409 -15. A74 found in the presence of glucose. This report is accordance with Sonla et. al. (2013).