Biochemical characterization and biolog based identifi cation of effi cient jute retting bacterial isolates from retting water

Jute is considered as one of the cheapest sources of natural fiber after cotton in terms of its production and its uses (Ahmed and Nijam, 2008). Among all natural industrial fibers nearly 15% of total outputs are supplied from jute which indicates its economic importance (Haque et al., 2001a). Retting, the most important post-harvest operation carried out by various types of retting microorganism, ultimately improves the quality of jute fiber and its economic value thereafter (Islam and Rahaman, 2013). It may be defined as the process of separation and extraction of fiber from non-fibrous tissues and woody part of the stem through dissolution and decomposition of pectins, gums and other mucilaginous substances (Dasgupta et al., 1977; Majumdar and Dey, 1977). This is because a jute retting microbe is supposed to be highly efficient which is having high Pectinolytic and Xylanase (EC 3.2.1.8) (Collins et al., 2005) activity with no or less Cellulase (EC 3.2.1.4) (Sarrouh et al., 2012) activity. During retting, degradation of pectin carried out by Pectinase mainly Polygalacturonase (PG) and pectin lyase (PLN) (EC 4.2.2.10) (Duvetter et al., 2009) activity of retting microbes while partial degradation of Xylan (major component of hemicellulose) done by Xylanase to make the fiber soft in nature (Ahmed and Akhter, 2001; Das et al., 2012a). Application of mixed enzyme (Cellulase, Xylanase and Pectinase) on jute shown significant positive changes on physical characteristics of jute fibers in terms of whiteness index, brightness index (Vigneswaran and Jayapriya, 2009). Until now there were several attempts that has been taken to test the pectinolytic activity of microorganisms like fungi (Haque et al.,2001b), bacteria (Das et al., 2012a) for retting and pilling of jute (Banik and Ghosh, 2008) and identified through technique like 16S rDNA analysis (Das et al., 2012a; Das et al., 2011; Munshi and Chattoo, 2008) or through Bergey’s manual (Banik and Ghosh, 2008; Ali, 1958; Jalaluddin, 1965) and assessment of bacterial community done through biolog ecoplates under different stages of jute retting at different location (Das et al., 2013). Until now there is no report available on the identification of retting microorganisms through using BIOLOG Microplate system from retting water. Biochemical characterization and biolog based identifi cation of effi cient jute retting bacterial isolates from retting water


INTRODUCTION
Jute is considered as one of the cheapest sources of natural fiber after cotton in terms of its production and its uses (Ahmed and Nijam, 2008).Among all natural industrial fibers nearly 15% of total outputs are supplied from jute which indicates its economic importance (Haque et al., 2001a).Retting, the most important post-harvest operation carried out by various types of retting microorganism, ultimately improves the quality of jute fiber and its economic value thereafter (Islam and Rahaman, 2013).It may be defined as the process of separation and extraction of fiber from non-fibrous tissues and woody part of the stem through dissolution and decomposition of pectins, gums and other mucilaginous substances (Dasgupta et al., 1977;Majumdar and Dey, 1977).This is because a jute retting microbe is supposed to be highly efficient which is having high Pectinolytic and Xylanase (EC 3.2.1.8)(Collins et al., 2005) activity with no or less Cellulase (EC 3.2.1.4)(Sarrouh et al., 2012) activity.During retting, degradation of pectin carried out by Pectinase mainly Polygalacturonase (PG) and pectin lyase (PLN) (EC 4.2.2.10) (Duvetter et al., 2009) activity of retting microbes while partial degradation of Xylan (major component of hemicellulose) done by Xylanase to make the fiber soft in nature (Ahmed and Akhter, 2001;Das et al., 2012a).Application of mixed enzyme (Cellulase, Xylanase and Pectinase) on jute shown significant positive changes on physical characteristics of jute fibers in terms of whiteness index, brightness index (Vigneswaran and Jayapriya, 2009).Until now there were several attempts that has been taken to test the pectinolytic activity of microorganisms like fungi (Haque et al.,2001b), bacteria (Das et al., 2012a) for retting and pilling of jute (Banik and Ghosh, 2008) and identified through technique like 16S rDNA analysis (Das et al., 2012a;Das et al., 2011;Munshi and Chattoo, 2008) or through Bergey's manual (Banik and Ghosh, 2008;Ali, 1958;Jalaluddin, 1965) and assessment of bacterial community done through biolog ecoplates under different stages of jute retting at different location (Das et al., 2013).Until now there is no report available on the identification of retting microorganisms through using BIOLOG Microplate system from retting water.
Beside enzymatic activity, water is a crucial factor for retting of jute.At water stress condition improper retting leads to low-quality jute which found recently in major jute growing areas of Bangladesh (Islam and Rahaman, 2013).Under this situation, the identification of efficient retting microorganisms can provide a suitable way for the development of new retting technique, especially under water scarcity condition.The present study was undertaken to characterize and identify the efficient retting microbes present in the jute retting water on the basis of their enzymatic activity, i.e., Pectinolytic, Xylanse, and Cellulase activity.

Isolation and Selection
Retting water samples were collected from different retting tank.The enumeration of retting microbes was done following serial dilution technique and pour plate method (Parmer and Sclunidt, 1965).The retting water was serially diluted up to 10 −6 times.Each dilution was plated in Pectin agar media (Pectin -1%, Yeast extract -1%, Sodium chloride -0.5%, and Agar -2%) and was incubated for three days at 34°C.The pure cultures of 40 morphologically distinct isolates on Pectin agar plate were developed for further analysis.

Enzymatic Study of Isolated Microorganism
For the qualitative study of 40 isolates with respect to formation of halozone (Plate 1a-c), spot inoculations of the isolates were done on Pectin agar plate, Xylan agar plate following the modified method of Kaur et al. (2011) and in case of carboxymethyl cellulase (CMC) agar plate it was according to modified method of Baharuddin et al. (2010).Then the plates were incubated for 72 h in Orbital Incubator Shaker (Model CIS-24 BL, Make REMI).After that media surface of Pectin and Xylan, agar plate was kept flooded with 2% CTAB (Cetyl Trimethyl Ammonium Bromide) for 30 min.The flooding was done with 0.03% Congo red in case of CMC agar plate.Then the plate surfaces were washed using distilled water to get the halozone on media surface.Potency index (zone diameter/colony diameter) of each strain measured with three replication (Phutela et al., 2005).
All 40 strains then were used for quantitative analysis of Pectinolytic activity in terms of PG and PLN at 48 and 72 h of incubation following modified enzymatic assay of Phutela et al. (2005) and Nedja et al. (2001) respectively.But out of 40 only 8 strains showing higher potency index for Xylanase and lower potency index for cellulase were selected for the enzymatic assay of Xylanase and Cellulase following the method of Monisha et al. (2009) and Miller et al. (1960) respectively at 48 and 72 h after inoculation in Xylan broth and CMC broth.
Statistical analyses were performed using SPSS v16.0 for Windows (SPSS Inc., USA) with 5% probability level of statistical significance.

Identifi cation of Isolates
After analyzing data, microbial strains having significantly high Pectinolytic and Xylanase activity with low Cellulase activity were identified up to species level by using Biolog (Model: BioLoG Microstation ™ ) system based on the metabolic fingerprinting pattern of isolates using the software ML_51_01_ml3.

Qualitative Analysis
Among 40 strains, most strains exhibited high Pectin and Xylan degrading capacity defined by high potency index and maximum of them were shown high potency index in CMC agar plate.All data of qualitative analysis were not given (Table 1).All microbial strains were formed halozone in Pectin agar plate indicating all had considerable jute retting capacity.For this reason,  In CMC agar plate halozone formed surrounding area of the colonies (Plate 1b) represented that the isolates (S15, S17, S18, S20) having the cellulose degrading capacity, so they were not considered for retting.Xylanse activity of microbial strain is act as a bleaching agent which increase the brightness of fiber.For this reason, out of 40 strains only 8 strains were selected for quantitative enzymatic analysis of Cellulase and Xylanse activity on the basis of low (<1.5) and high potency index (>1.5) in CMC and Xylan agar plate respectively (Table 1).

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A quantitative study of Pectinolytic enzyme involved estimation of PG and PLN activity at 48 and 72 h interval.It was found that most of the microbial strains have high PG activity at 72 than 48 h (Figures 1 and 2).At 48 h, PG activity ranges from 2.08 ± 0.07 to 6.8 ± 0.42 μmol/ml/min while at 72 h it ranges from 2.30 ± 0.023 to 7.71 ± 0.1 μmol/ml/min.Highest and lowest PG activity found in S37 and S24 respectively at 48 h.At 72 h, PG activity found highest in S20 and lowest in S1 (Table 2).During PLN estimation result was opposite (Figures 3 and 4).Most of the microbial strains shown high PLN activity at 48 h than 72 h.PLN activity of strains at 48 h ranged from 11.70 ± 0.12 to 184.83 ± 0.2 U/ml and at 72 h it ranged from 20.63 ± 1.18 to 161.20 ± 1.38 U/ml of culture filtrate (Table 2).In 48 h, PLN activity found highest in S3 while it was lowest in S18, S19, S21, S22, S23, S24 and S27.From this view, we can found that S24 was worst performer among 40 strains on the basis of Pectinase activity.The result clearly indicates that during the early stage of retting water complex Pectinolytic compound breaks down to simple form as PG acid through several enzymes like PLN which later breaks down by PG.Another thing which may conclude from this view (Table 2) that in jute retting microorganisms PG activity and PLN activity both are independent of each other because we have not get any microbial strains having both of high PLN and high PG activity at 48 and 72 h.Similar results obtained by Das et al. (2012a).where microbial strains having higher PG activity failed to show higher PLN activity.

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Since the strains having low potency index were selected for this purpose all shown low Carboxymethyl cellulase (CMCase) activity at 48 and 72 h interval.At 48 h, the Cellulase activity ranges from 2.4 ± 0.03 to 3.23 ±.07 μmol/ml/min while at 72 h it was from 1.29 ± 0.12 to 2.56 ± 0.02 μmol/ml/min (Table 3).It was found that all these strains have high Cellulase activity maximum in 48 than 72 h (Figures 5 and 6).At both 48 and 72 h minimum CMCase activity found in S1.Since Pectinase activity was low in S1 in comparison to other strains made it unsuitable for jute retting.The strains S33 and S37 were found to be efficient retting microbes because both Xylanase and Pectinase were found comparatively high (Tables 1 and 2).
Overall potency index for Pentinase and Xylanse activity were high coupled with low potency index for Cellulase activity were recorded for three strains No. 11, 33, and 37. Besides, the quantitative assay showed that all these strains having high Pectinolytic activity as well Xylanse activity Sig value for all significant value 0.00.but lower Cellulase activity (Tables 2 and 3).From this result, it was evident that S11, S33 and S37 found most effective strain in terms of their enzymatic activity.S11  having moderate Pectinase coupled with highest Xylanse and lower CMCase value may also be suitably used for retting.

Identifi cation of Selected Retting Microbes
The Biolog Identification System is a new bacterial identification method that establishes an identification based on the exchange of electrons generated during respiration, leading to a subsequent tetrazolium-based color change.Identification through biolog system, it was found 87.3% correct at the genus level and 75.6% correct at the species level at 24 h (Miller and Rhoden, 1991).These three strains (S11, S33 and S37) were then identified by using biolog, an advanced tool for characterization and identification of microorganisms based on the metabolic fingerprinting pattern of the isolates using the software ML_51_01_ml3.
In the present study, all strains identified as Bacillus spp.The Pectinolytic strains of Bacillus spp.showed cellusolytic activities.These results were in confirmation with the finding of Tamburini et al., (2004) who reported the presence of cellusolytic activities in the Pectinolytic strains that are Bacillus subtilis.Zhang et al., (2000) also found low cellulolytic activities (0.04-0.17U/cm 3 ) of some strains used in degumming of ramie fibres.Few Bacillus strains recorded the Xylanase activity of 11.50 IU/ml and Cellulase activity 1.2-1.5 IU/ml as reported by Subramaniyan and Prema (2002).The presence of Bacillus sp. in retting water was also reported by Jalaluddin (1965), and specifically B. pumilus by Ali (1958).In comparison to earlier reported microbes belonging to Bacillus genus, these strain recorded similar pattern of Xylanase activity with significantly less Cellulase activity which could make essentially more efficient jute retting microbes.

CONCLUSION
Out of 40 Pectinolytic bacterial isolates tested for PG, PLN, Xylanase and Cellulase enzymatic activities, only three isolates were found to have very high Pectinolytic and Xylanase activity with very less Cellulase activity.These identified two microorganisms may provide a suitable means to develop a new retting technique under water scarcity condition to get quality fibre from Jute.If these organisms are to be used as mixed in particular ratio culture, their fibre quality due to retting will be improved, but for this purpose required trial and error method to find out the best ratio to make the mixed culture.

Plate 1 :
(a) Qualitative study of Pectinolytic enzyme using Pectin as substrate (Halozone formation around the colony in Pectin agar plate).(b) Qualitative study of Cellulase enzyme using carboxymethyl cellulase (CMC) as substrate (Halozone formation around the colony in CMC agar plate).(c) Qualitative study of Xylanase enzyme using Xylan (Halozone formation around the colony in Pectin Xylan agar plate)

Figure 7 :
Figure 7: Xylanse activity at 48 h of selected microbial strains Figure 8: Xylanase activity at 72 h of selected microbial strains

Table 3 : PG and PLN activity of all 40 strains at 48 and 72 h
PG: Polygalacturonase, PLN: Pectin lyase Sig value for all significant value 0.00.That means significant at 1% ∞