ISOLATION AND CHARACTERIZATION OF CELLULOLYTIC BACTERIA FROM DECOMPOSING RICE STRAW

Three cellulolytic bacterial strains were isolated fmm decomposing rice straw. They were able to utilize cellulose, rice straw powder and carboxymethylcellulose as substrates. Two of these strains, Listeria sp. and Enterobacter sp., were abundant during initial stages of decomposition whereas the other strain, Pseudomonas sp., became dominant towards the late stages of the process. Comparison of their endo-1,4~ l u c a n a s e ( ~ I 1 u l a s e ) activity by indirect methods indicated a higher level of enzymatic activity in the Pseudomonas sp. than in the other two strains. Release of glucose by saccharification of cellulose and carboxymethylcellulose was also higher with Pseudomonas sp. compared with Listeria sp.


INTRODUCTION
Most agricultural residues of crop plants, particularly cereals, are rich in lignocellulosic materials.'s2Cellulose, a long-chain polysaccharide made of P(1,4)-linked glucose units, is the principal constituent of lignocelluloses.Association of cellulose with lignin, another complex polymeric molecule composed of phenylpropanoid units, forms the lignocelluloses.Hemicellulose is the other major component of lignocelluloses.I t is a heterogeneous group of long-chain polysaccharides of which basic units are arabinose, xylose, mannose or galactose.Degradation of lignocellulosic material is a slow process and only a relatively narrow taxonomic range of bacteria and fungi are able to degrade such material.The ability of microorganisms to degrade cellulosic material is of considerable interest both in terms of microbial ecology and biotechnology.Degradation of cellulosic material requires the cooperative action of a family of cellulolytic enzymes that have been classified into three major groups: endoglucanases (EC3.2.1.4),exoglucanases (EC 3.2.1.91)and P-glucosidases (EC 3.2.l.21)'.3Cellulolytic properties ofthese enzymes have been studied mostly in fungi4 The bacterial cellulase system has only recently become a focus of investigation.Due to high cellulolytic activity of some bacteria and their short generation time they are a more promising group of organisms in degrading lignocellulosic waste^.^In this paper we report isolation and characterization of three cellulolytic bacterial strains involved in the degradation of rice straw, a widely available agricultural residue in Sri Lanka.
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Isolation of bacteria from rice straw
Rice straw samples representing different stages of the process of decomposition, starting from one week after harvesting, were used for this study.Each sample (5g) was washed with 15 ml ofsterile 0.07 M potassium phosphate buffer, 7.2, with vigorous shaking and the washing was filtered through two Whatman 3 MM filter papers.From each filtrate a dilution series (10-I -10"') was prepared using sterile potassium phosphate buffer, pH 7.2, and O.lml samples were spread on Lagar (log tryptone, 5 g yeast extract, 10 g NaCl, l g glucose and 15g agar per literY plates and incubated a t 37OC.

Identification of cellulolytic bacteria
Different types of bacteria from L-agar plates were inoculated onto the cellulose agar (2.5 g of precipitated cellulose, 0.5g peptone, 0.2 g dipotassium-hydrogen phosphate, 0.2g magnesium sulphate, 0.4 g potassium carbonate, 0.02 g calcium chloride, ferrous sulphate and sodium chloride, and 15g Oxoid ion agar per liter) and incubated at 37OC.Only the bacterial strains capable of degrading cellulose grow well on this medium.None of these strains grew on the agar medium without cellulose.Thus, several cellulolytic bacterial strains were isolated and on the basis of morphological and biochemical characteristics they were identified to the genus level.

Confirmation of cellulolytic activity .
Bacterial strains identified as cellulase-positive were further tested for their cellulolytic activity by growing in media containing rice straw powder or, carboxymethylcellulose (CMC) (BDH, high viscosity), a semisynthetic cellulosic substrate.

Visualization of CMC (P-D-Glucan) hydrolysis
One of the enzymes required to convert cellulose to glucose is endo-1,4-0g l u c a n a ~e , ~ which can be detected by the hydrolysis of CMC.Enzymic activity of the isolated bacterial strains was visualized as described8 with a slight modification.Inoculum from each fresh bacterial culture was patched on CMC agar which contained CMC (1 mglml) instead of cellulose in the cellulose agar medium, and incubated at 37OC for 8h.Plates were then flooded with an aqueous solution of congo red for 15min.After pouring off the congo red solution, plates were flooded with 1M NaCl for 15 min.After that zones of hydrolysis could be seen as clear areas.

Release of glucose by saccharification'of cellulose and CMC
Cellulose broth containing 0.5% cellulose (wlv) and all the other ingredients except agar found'in the cellulose agar medium was used to determine the' s a c c h d c a t i o n of cellulose.CMC broth was p r e p d using 0.5% CMC (whr) instead of cellulose..Samples of badrial culturea (0.5 ml) grown in them broths to a same cell density were used to i n d a t e 501x11 of h s h broth in 250 ml flasks.These cultures were incubated at 37°C with continuous ehaldng, for24 h, and then centPifuged at 5000 g for 10 m i . .Glucose concentration ofthe supernatants were determined as de~cribed.~A 0.1 ml sample of the supernatant was boiled

RESULTS
L-agar plates prepared with diluted straw-washings were used initially to determine different types of bacteria present at various stages of decomposition (sampling period was from one week after harvesting to three months).In all samples there were three prominent types of bacteria that could be distinguished by their colony characteristics.Only these strains grew on cellulose agar demonstrating their cellulolytic activity.Morphological and biochemical characterisitics showed that these strains belong to the genera Pseudomonas, Listeria and Enterobacter (Table 1).These three strains were present in similar numbers in the straw samples which were less than one month old.However, in the samples taken from straw Release of glucose by the saccharification of cellulose and CMC was examined only with the Pseudomonas sp. and Listeria sp.Psez~domonas sp.released 2.0 and 1.8mmoLA glucose when cellulose and CMC were used respectively.Concentrations of glucose produced by Listeria sp. were 1.8mmol.lwith cellulose and 1.5mmoLA with CMC.

DISCUSSION
Cellulolytic bacterial strains isolated from decomposing rice straw were able to utilize both natural and semi-synthetic cellulosic substrates.Variation of the frequencj of occurrence of these three genera with time is evidence of existence of a cellulolytic bacterial succession on this substrate.The role of Listeria sp. and Enterobacter sp.seems to be more important during the early stages of decomposition.Increasing frequency of the Pseudomonas sp. after about one month suggests that the action of the enzymes produced by this strain is required during the later stages As detectcd from the congo red method, this Pseudomonas sp. has a high level of end0-1,4-~~lucanase activity.This is one of the enzymes required for the conversion of cellalose to glucose.Saccharification of wheat straw yields a number of products including reducing sugars xylose and glucose.1°Since glucose or its oligomers have not been found in cereal straw hemicellulose fractions," the production of glucose confirms hydrolysis of the cellulose component of straw.Release of high amounts of glucose from both cellulose and CMC by Pseudomonas sp.compared to the amounts of released glucose with Listeria sp.codrms its high level of production of extracellular cellulases.Pseudomonas sp. with cellulase adivity has been isolated previou~ly.'~Pseudomonas sp.isolated in the present study appears to have high cellulase activity in comparison with the bacteria isolated earlierI2 and may be suitable for genetic manipulation to achieve a higher efficiency of degradation of rice straw.

with 5
ml o-toluidine for 10 min, and O D , , w a ~ determined.Corresponding glucose concentration was read from a standard calibration curve of glucose concentration (0 -15 mmou).

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Positive result or growth, b -Negative reault or nb growth, c -Not tested.

Table 1 :
Characteristics used to identify the bacteirial strainsCharacteristicsListeria sp.Enterobacter sp.Pseudomonas sp.