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Open Access
Identification and characterization of enzyme(s) causing browning of button mushroom, Agaricus bisporus (Lange) Sing.
(Punjab Agricultural University, Ludhiana, 2020) Ravneet Kaur; Sodhi, H.S.
Agaricus bisporus is the most acceptable mushroom in the world but being perishable it loses sheen as fresh produce. Present study has dealt with the enzymes related to browning of button mushroom, namely, tyrosinase (E.C 1.14.18.1), laccase (EC 1.10.3.2), peroxidase (EC 1.11.1.7), phenylalanine ammonia lyase (E.C. 4.3.1.5), lipoxygenase (EC 1.13.11.12) and catalase (EC 1.11.1.6). These were estimated in submerged state and the compost/casing (solid substrate). The enzyme activity of A. bisporus mycelium in broth was found to increase with time and maximum activity was that of peroxidase (7.41U/mg) and laccase (6.05U/mg). Mycelial run in compost indicated maximum specific activity of laccase enzyme (12.3 U/mg) while the activity of the other enzymes was significantly lower. During the pin head stage, there was an increase in the activity of tyrosinase, laccase, peroxidase, PAL, catalase, lipoxygenase enzymes as 17.67, 13.98, 14.67, 4.5, 2.33 and 17.75U/mg respectively. After harvesting during the first flush, tyrosinase activity was reduced up to 12.03 U/mg and remained at par during the second and third flush harvesting. A similar pattern was observed for laccase and PAL. Peroxidase activity showed a decline up to second flush and remained stable thereafter. Catalase activity reduced from the pin head stage to first flush and no significant change in the lipoxygenase activity was observed during reproductive phase of mushroom production. During the postharvest period, the activity of melanin synthesizing enzymes that is tyrosinase, laccase and peroxidase was found to increase with time during storage and maximum increase was that for tyrosinase leading to increase in browning index (BI) value. The activity of the lipoxygenase enzyme increased which led to increased electrolytic leakage and hence lipid peroxidation. During the storage period, there was a decrease in the activity of PAL while the activity of catalase was found to increase. Mushroom washing treatments (KMS, Citric acid and Salicylic acid) and fumigation (cinnamon oil and clove oil) along with packing treatments (polypropylene bags, carton trays with cling film cover and plastic trays with cling film cover) were studied up to 16d at 4 day intervals. The washing treatments using 0.2% KMS, 1% citric acid and 200µmole/L salicylic acid showed a significant decline in the browning associated enzymes and BI in all the packagings during 16d storage. In general mushrooms treated with 200µmole/L salicylic acid and packed in PT showed the best results with minimum BI value (23.09) and tyrosinase activity (27.72 U/mg) at 16th day of storage. Cinnamon oil and clove oil fumigation indicated a gradual decline in activity of melanogenic enzymes and BI with increasing oil concentration up to 40 ppm. Tyrosinase enzyme was purified and found active over pH range 6.4 to 7.2 showing 90% and 88% of the maximum activity at pH 7. The enzyme was stable at a temperature range 25-40°C with maximum activity at 35°C. The kinetic studies of enzyme showed that in case of catechol as substrate, the Km was found to be 0.71 mM with Vmax 2518 µmole/min/ml. In case of L-Dopa from Lineweaver-Burk plot, the Km value was found to be 0.87 mM with Vmax 1714 µmole/ml/min. The SDS-PAGE electrophoresis of the enzyme gave a single prominent band at 43 kDa. FTIR spectra of purified enzyme indicated a secondary structure that reflected the amide I and amide II bands. This study indicated that mushroom browning is a complex process triggered after the harvesting involving the enzymatic reactions. The study of the browning associated enzymes at the different stages of the cultivation and during the storage with different postharvest treatments showed that browning can be decelerated by the postharvest treatments which mainly act by restricting the tyrosinase and related enzymes.
Open Access
Characterization of stress tolerant microalgae strains for biogas production
(Punjab Agricultural University, Ludhiana, 2019) Sharma, Nishu; Phutela, Urmila Gupta
The present research work was aimed at characterization of stress tolerant microalgae strains for biogas production. A total of six strains, five stress tolerant microalgae strains viz. BGLR4, BGLR7, BGLR10, BGLR18, BGLRS and one standard strain Spirulina sp. NCIM 5143 were used in the present study. These strains were screened for maximum biomass production, chlorophyll content, protein, carbohydrate and lipids on four different media viz. Blue green-11(BG-11), Bold’s basal medium (BBM), Algal culture medium (ACM) and Zarrouk’s medium (ZM) and on different concentrations of unsterilized dairy wastewater (20%, 40%, 60%, 80% and 100%). Among all the six strains, BGLR18 and BGLRS were found to produce maximum biomass BBM and ACM respectively while Spirulina sp. NCIM 5143 produced maximum biomass on ZM. BGLR18 and BGLRS showed maximum growth on 60% concentration of dairy wastewater whereas Spirulina sp. NCIM 5143 on 100% concentration of dairy wastewater. Maximum percent reduction in various physico-chemical parameters of dairy wastewater was shown by BGLR18 and BGLRS at 60% dairy wastewater while Spirulina sp. NCIM 5143 at 100%. Further, studies were carried out with strains BGLR18 and BGLRS and compared with standard strain Spirulina sp. NCIM 5143. Cultural conditions of BGLR18, BGLRS and Spirulina sp. NCIM 5143 were optimized using response surface methodology (RSM) based on central composite design. The optimized and most desirable cultural conditions of pH, temperature, light intensity, growth period and inoculum concentration for BGRL18 were 10.02, 20.00°C, 7599.41 Lux, 21.75 days and 1.00% ; for BGLRS were 11.17, 26.03 °C, 4000.02 Lux, 39.37 days and 10.00% and for Spirulina sp. NCIM 5143 were 10.35, 24.38 °C, 8000.00 Lux, 25.13 days and 1.00% respectively. On molecular identification, BGLR18 showed 96% resemblance to Chlorosarcinopsis eremi and BGLRS showed 99% resemblance with Scenedesmus sp. MKB. These three strains showed wide variation in their total nitrogen, total protein,total phenols, antioxidant activity, DPPH radical scavenging activity. ICP-AES analysis showed that heavy metals were negligible in all the three strains while essential elements like Ca, Fe, P and Mg were present in higher concentrations than Spirulina sp. NCIM 5143. GC-MS analysis of methanolic extract showed that many bioactive compounds were present in all the strains. These strains were evaluated for their biogas production potential alone and in co-digestion with paddy straw. The highest biogas was produced by BGLRS (Scenedesmus sp. MKB) (28.80 Litres) with highest biogas production potential (P) of 30.85 mLg-1 VS showing maximum biogas production rate (Rm) of 0.58 mLg-1d -1 with a lag phase (λ) of 1.30 days. While in co-digestion experiment, the highest biogas production was recorded by BGLR18 (Chlorosarcinopsis eremi) (168.46 Litres) with maximum biogas production potential (P) of 214.30 mLg-1 VS showing maximum biogas production rate (Rm) of 8.76 mLg-1 d -1 with a lag phase (λ) of 12.10 days. Hence, the stress tolerant microalgae strains can be co-digested with paddy straw for enhancing biogas production.
Open Access
Development of bacterial consortium to improve green manure degradation and nutrient availability in kharif maize
(Punjab Agricultural University, Ludhiana, 2019) Jaspreet Kaur; Gosal, S.K.
The present investigation was conducted to isolate cellulose degrading bacteria with plant growth promoting traits to enhance green manure (Crotolaria juncea) decomposition as well as nutrient mineralization in maize crop. A total of 75 different bacteria were isolated from soil samples collected from various locations. Only 20 isolates were selected on their ability to degrade cellulose. Cultural and morphological characterization showed that most of these bacterial isolates were gram positive, non-motile and non-endospore forming cocci. Biochemical characterization of the isolates revealed that most of these isolates were positive for oxidase, catalase, methyl red, starch and citrate utilization test. Only two bacterial isolates (CD4 and CD6) were urease positive, one (CD11) was indole positive and three (CD1, CD11 and CD15) were found to HCN positive. Functional characterization of these isolates showed that the isolates CD2 and CD9 had highest cellulase (27.543 mg/ml) and β-glucosidase activity (15.46 μg pNP released/ml), respectively. Highest IAA production (42.25 μg/ml), phosphorous solubilization index (1.80) and quantitative P- solubilization (25.39 μg/ml) were recorded for CD9 bacterial isolate. All the isolates were found to have nitrogen fixing ability. The maximum amount of ammonia (6.659 μM/ml) was excreted by isolate CD18 on N-free Jensen‟s medium. On the basis of functional characteristics, three best isolates (CD2, CD9 and CD14) were evaluated for green manure decomposition and nutrient mineralization studies, under pot conditions under different soil (sterilized and unsterilized) and moisture regimes (aerobic and flooded). The application of isolates as consortium was found best for increasing green manure decomposition, nutrient mineralization and soil biological activities relative to uninoculated control treatments. These effects of consortium inoculation were found more pronounced in aerobic conditions as compared to flooded conditions. A field experiment was conducted for two consecutive years (2016 and 2017). A significant decomposition of green manure was observed with application of CDBC biofertilizer within the period of seven days in field condition. A total of eight treatments having different combinations of biofertilizer (CDBC), green manure and inorganic nitrogen fertilizers (75% and 100%) were used to study their effect on soil microbial dynamics, soil chemical properties and yield of maize crops (var. PMH1 and var. PMH4). Microbial population (total bacteria, fungi, P-solubilizer, amylolytic and cellulolytic bacteria) and enzymatic activities (dehydrogenase, alkaline phosphatase, urease, cellulase, amylase and invertase) in maize rhizosphere significantly increased in treatments having integrated application of CDBC, green manure and 100% whereas, soil actinomycetes and diazotrophic population were found highest in treatments with sole application of inorganic nitrogen fertilizer. Higher soil microbial populations and enzymatic activities were observed during second year of field study relative to first year. Soil chemical properties viz., electrical conductivity, organic carbon, available nitrogen, available phosphorous and potassium content were found highest under integrated application of CDBC with green manure and recommended dose of nitrogen fertilizer whereas soil pH varied non-significantly among the treatments. Maximum plant height, yield attributes and yield were observed in same treatment. The results indicated that application of CDBC biofertilizer not only decreased the fallow period of green manure decomposition but also enhanced soil health, fertility and yield of maize crop. So, application of CDBC along with green manure and inorganic nitrogen fertilizer can serve as sustainable agricultural practice for maize cultivation.
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Comparison of shelf life and bioefficacy of liquid and charcoal based biofertilizer in chickpea
(Punjab Agricultural University, Ludhiana, 2019) Anjali; Sharma, Poonam
The present investigation was carried out to compare the shelf life and bioefficacy of liquid and charcoal based biofertilizers of LGR33+RB1 amended with different additives viz. 0.1% CMC, 2% PVP and 10mM trehalose stored at 4⁰ and 28⁰C upto 300 days in chickpea. Viable cell count as well as plant growth promoting (PGP) traits viz. IAA production, P solubilization and siderophore production were obtained at an interval of 30 days and results revealed that all liquid based biofertilizer treatments with additives recorded significantly high viable population, IAA production and P solubilisation during the entire study period (300 days) at 4⁰ and 28⁰C as compared to charcoal based biofertilizer treatments. However, among different additives amendment with 0.1% CMC showed maximum viable population and PGP traits in both liquid as well as charcoal based biofertilizers. In vitro seed germination experiment was conducted to evaluate the effect of liquid and charcoal based biofertilizer amended with different additives on growth parameters in chickpea. Liquid biofertilizer treatment LGR33+RB1+0.1% CMC recorded significantly high plumule length (12.5cm), radical length (5.8cm), fresh weight and dry weight of seedling (0.72 and 0.68 g, respectively) along with improved seed survival rate of inoculants on chickpea seeds upto 48 hrs (8.27log cfu/ml). In vivo studies all liquid as well as charcoal based biofertilizer treatments with additives improved growth parameters in chickpea as compared to recommended consortium treatment without additives. Liquid biofertilizer treatment LGR33+RB1+0.1% CMC recorded maximum seed emergence, plant height, chlorophyll content, number of nodules, dry weight of nodules as well as leghaemoglobin content, soil enzyme activity, total N and P content of shoot and soil along with improvement in grain yield over uninoculated control.
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Bioefficacy of endophytic actinomycetes for plant growth promotion and management of fungal diseases in vegetables
(Punjab Agricultural University, Ludhiana, 2019) Khushboo; Khanna, Veena
Endophytic actinomycetes are associated with internal tissues of plants without any disease symptoms and are promising source of novel secondary metabolites. The present study was conducted with a view to screen endophytic actinomycetes from four vegetable crops (Solanum lycopersicum, Capsicum annum, solanum tuberosum and Cucumis melo) and evaluate their potential to produce plant growth promoting and biocontrol agents. A total of 240 isolates were isolated from roots, stems and leaves tissues of vegetables and presumptively identified as identified as Streptomyces sp. (n=163), Micromonospora sp. (n=25), Microbispora sp. (n=11), Actinopolyspora sp. (n=4), Sacchropolyspora sp. (n=8), Nocardia sp. (n=22) and Psuedonocardia sp. (n=7). The functional potentialities of the isolates recorded were phosphate solubilization, siderophore, IAA, gibberallic acid and ammonia production. Streptomyces fulvissimus (TR60), a Solanum lycopersicum isolate exhibited maximum phosphate solubilization activity (32.42 mg/100ml) while Streptomyces flavoviridis (PS53), a Solanum tuberosum isolate showed high yield of IAA (132.74 μg/ml). Streptomyces diastaticus subsp. Ardesiacus (CR36), a Capsicum annum isolate exhibited maximum siderophore production: catechols 147.3 μg/ml and hydroxamate 129.4 μg/ml. Eighty isolates out of 240 displayed [S.lycopersicum (n=21), C.annum (n=15), S.tuberosum (n=20) and C. melo (n=24)] activity against different plant pathogenic fungi (Fusarium oxysporum, R. solani, Pythium sp. and Sclerotium rolfsii) In present study, field trial conducted with these potential isolates based on results of in vitro plant growth promoting traits and also offered protection against fungal diseases under controlled glass house condition besides stimulating plant growth.
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Studies on lignocellulolytic enzymes from spent mushroom straw for bioethanol production
(Punjab Agricultural University, Ludhiana, 2019) Rajni Devi; Kapoor, Shammi
Spent mushroom substrates (SMS), a lignocellulosic residue of recommended mushroom cultures Agaricus bisporus, Pleurotus florida, Calocybe indica and Volvariella volvacea were evaluated as a fermentable substrate for ethanol production. Activity of Lignocellulolytic enzymes (oxidative and hydrolytic) produced by these four fungal species in SMS at different stages of growth (spawn run, primordial, 1st flush, 2nd flush and end of crop) in nonaxenic and on different interval of days (7th, 14th, 21st and 28th) under axenic condition were evaluated. Considerable amount of oxidative (Lignin peroxidase, Manganese peroxidase, versatile peroxidase and laccase) and hydrolytic (carboxymethylcellulase, xylanase and cellobiohydrolase) enzymes were found in SMS’s. Maximum oxidative enzymes activity was found in SMS of A. bisporus and P. florida whereas, higher hydrolytic enzyme activity was observed in SMS of V. volvacea and C. indica under both axenic and non-axenic conditions. Among oxidative enzymes, maximum specific activity of manganese peroxidase (25.61 U/mg of protein) was found in SMS of A. bisporus at primordial stage and in P. florida (28.89 U/mg of protein) at end of crop on 28th day of incubation. Versatile peroxidase was observed in SMS of all three fungal species except A. bisporus and maximum specific activity value 14.35 U/mg of protein (SMS of V. volvacea) was found at primordial stage under non-axenic condition while 23.33 U/mg of protein (SMS of P. florida) was recorded on 28th day under axenic condition. Lignin peroxidase with maximum value of 8.24 U/mg of protein was found only in SMS of C. indica. Under non axenic conditions, among hydrolytic enzymes, xylanase (21.02 U/mg of protein) was found maximum at end of crop in SMS of V. volvacea while maximum CMCase (32.82 U/mg of protein) was found in SMS of P. florida under axenic condition. These enzymes were partially purified and concentrated by lyophilisation and used for further biological treatment of spent mushroom substrates. Relative proportions of lignocellulosic components (lignin, cellulose, and hemicellulose) were also analyzed. Maximum percent reduction of cellulose and hemicellulose compared to lignin was observed in SMS’s of all three fungal species except A. bisporus in which lignin (43.02%) showed maximum percent reduction. Physico-chemical modifications in SMS were observed by SEM, FTIR and XRD respectively. Biologically pretreated SMS’s were processed for bioethanol production by using two different yeasts i.e. Saccharomyces cerevisiae and Pachysolen tannophilus, and it was also compared with the bioethanol produced from the SMS treated with alkali and partially concentrated enzymes. Maximum ethanol of 13.90 g/l (V. volvacea) and 10.48 g/l (C. indica) were recorded after pretreatment with lyophilized enzymes. According to these results; the spent mushroom substrate of all four fungal species can be a cheap convenient substrate for sustainable production of bioethanol with maximum lignocellulolytic enzymes recovery.
Open Access
Collection, identification and domestication of wild edible Agaricales from Punjab
(Punjab Agricultural University, Ludhiana, 2019) Amanpreet Kaur; Sodhi, H.S.
Wild edible Agaricales were surveyed and collected during the month of July-September in the years 2015-2018 from five agro-climatic zones of Punjab. Total 127 mushrooms were collected; 34 mushrooms were collected from sub-mountain undulating zone, 19 wild mushrooms were collected from undulating plain zone, 36 mushrooms were collected from central plain zone, 17 from western plain zone, 21 from western zone. The mushrooms collected belong to genera Agaricus, Lepiota/ Macrolepiota, Pleurotus, Poadaxis, Clitocybe. Tissue culture of 92 wild mushrooms were obtained and total cellulases, cellobiase, endo-β-1,4-glucanase, endoxylanase and laccasse were estimated from pure cultures. Total cellulases ranged between 0.01 to 4.36 U/ml, cellobiase between 0.01 to 8.34 U/ml, endo-β-1,4-glucanase between 0.009 to 6.97 U/ml, endoxylanase between 0.28 to 16.18U/ml and laccasse was 0.02-3.74 U/ml. Based on enzyme producing capability, 18 cultures were identified to study linear growth (mm) on complete yeast agar medium (CYM) and potato dextrose agar (PDA) media, different pH range (5-8), temperature range (25°C-28°C). CYM was proved to be the best medium for AMN 05, AMN 71, AMN 132 and PDA was found good for AMN 14 and AMN 60. The pH 7 was proved as best pH for most of the cultures. AMN 14, AMN 39, AMN 52, AMN 54 and AMN 170 showed maximum growth at 30°C. AMN 39 and AMN 202 were successfully domesticated on wheat straw as substrate. These two cultures were subjected to molecular characterization through 18S rRNA sequencing. AMN 39 showed 100% homology with Pleurotus sapidus and AMN 202 showed the same with Pleurotus floridanus. Yield of Pleurotus sapidus (AMN 39) and Pleurotus floridanus (AMN 202) were compared with Pleurotus sajor-caju and Pleurotus florida and found that yield of Pleurotus sapidus (40 kg/q of dry substrate) and Pleurotus floridanus (47 kg/q of dry substrate) was lower than Pleurotus sajor-caju (52 kg/q of dry substrate) and Pleurotus florida (60 kg/q of dry substrate). It was observed through scanning electron microscope that hyphae of Pleurotus florida was seen to be more tightly packed as compared to Pleurotus sapidus (AMN 39) and Pleurotus floridanus (AMN 202). SDS PAGE was performed to know the heterogeneity among the mushroom proteins. AMN 39 had protein with molecular weight of 16.16 KDa and AMN 202 shown to have two proteins with molecular weight of 17.47 KDa and 42.8 KDa.
Open Access
Characterization of psychrotolerant rhizobacteria for Zn biofortification in lentil (Lens culinaris L. Medikus)
(Punjab Agricultural University, Ludhiana, 2018) Jaskiran Kaur; Khanna, Veena
Seventy psychrotolerant rhizobacterial isolates were evaluated for their PGP traits at 10°, 20° and 30°C. Highest IAA production was recorded with isolates PRh-9, PRh-14, PRh- 15, PRh-60, PRh-61 and that was confirmed by HPLC (8.98, 4.91, 5.66, 2.73 and 1.62 μg/ml respectively). Isolate PRh-14 recorded maximum Zn-solubilization potential of 354, 304.8 and 284.5 ppm at 10°, 20° and 30°C. It also recorded the maximum P-solubilization efficiency, gibberellins, EPS production and biofilm formation at 10°, 20° and 30°C. Eleven isolates amplified acdS gene which confirmed the presence of ACCD activity. Maximum naringin and quercetin production was shown by isolate PRh-61 and PRh-9, further confirmed by TLC studies. Highest antagonistic activity was recorded by PRh-9 against Rhizoctonia bataticola and Fusarium oxysporum (33.3% and 48.1% respectively) and was also established by SEM analysis. Potent siderophore, salicyclic acid and HCN producers were PRh-9, PRh- 61 and PRh-60. The SEM analysis of root surface revealed that coinoculation with Rhiobium and PRh-14 helped in maintaining the root integrity owing to biofilm formation on the root surface. Under field conditions coinoculation of Rhizobium with PRh-15, PRh-14, PRh-9, PRh-61, PRh-60 and PRh-30 further increased by grain yield by 1.7, 4.4, 3.7, 2.5, 2.09, 1.5% respectively, over Rhizobium alone. A significant increase in the grain and straw zinc content was recorded with the treatment R+PRh-14 (105.9 and 141.0 ppm respectively). The DGGE analysis of soil DNA depicted a composite banding pattern that reflected a high microbial diversity. The promising rhizobacterial isolates were identified as PRh-9 (Pseudomonas flourescens), PRh-14 (Aeromonas hydrophila), PRh-60 (Lysinibacillus fusiformis) and PRh-61 (Pseudomonas korensis) based on 16S rDNA sequencing.
Open Access
Impact of long term organic and inorganic fertilization on microbial community and screening for cellulose degrading bacteria
(Punjab Agricultural University, Ludhiana, 2018) Bhagat, Poonam
Impact of long term incorporation of rice straw and inorganic nitrogen was studied on microbial communities, soil enzymatic activities and on soil parameters at different time intervals (0, 45, 90 and 120 DAS) for two consecutive years. The different doses of rice straw (0, 5, 7.5 and 10 t/ha) along with different doses of inorganic nitrogen (0, 90, 120 and 150 kg/ha) were incorporated in the wheat field. The first year results revealed maximum total bacterial count (8.31 log 10 cfu/g of soil), fungal count (4.89 log 10 cfu/g of soil), celllulse degrading bacteria count (7.11 log 10 cfu/g of soil) with 7.5 t/ha RS + 120 kg N/ha while actinomycetes count (7.14 log 10 cfu/g of soil), diazotrophic count (6.07 log 10 cfu/g of soil) with 10 t/ha RS alone at 45 DAS. The maximum dehydrogenase activity (23.96 µg TPF/g soil/hr), alkaline phosphatase activity (14.77 µg pNP/g soil/hr) and urease activity (330.0 µg urea/hr/g soil) observed at 45 DAS with 7.5 t/ha RS plus 120 kg N/ha. The microbial population and enzymatic activities found higher in second year over first year. The soil pH was found to be altered from 7.82 to 6.99 and EC from 0.219 to 0.201 dSm-1 with treatment 10 t/ha RS +120 kg N/ha whereas OC from 0.26% to 0.49% with 10 t/ha RS + 150 kg N/ha in two years at 120 DAS. The maximum soil available nutrients viz. nitrogen (136.7 kg/ha), phosphorous (29.94 kg/ha) observed with treatment10 t/ha RS + 150 kg N/ha, potassium (119.78 kg/ha) with treatment 7.5 t/ha RS + 120 kg N/ha at 45 DAS in the first year. The maximum straw and grain nitrogen (0.46%) and (1.37%), phosphorus (0.15%) and (0.38%), potassium (1.72%) and (0.53%) contents respectively in first year found with 10 t/ha RS + 150 kg N/ha. The maximum straw and grain yield (9.65 t/ha) and (6.28 t/ha) respectively observed in first year with 7.5 t/ha RS + 120 kg N/ha. The available nutrients and yield found higher in second year than first year. The cellulose degrading bacteria were isolated and halozone formation confirmed their cellulolytic activity. Seventeen isolates were characterized biochemically and most were positive for oxidase and catalase while negative for H2S test, indole test and MR-VP test and few were positive for citrate utilization, gelatin solubilization and starch hydrolysis. The functional characterization of isolates showed highest cellulolytic activity (1.44µg/ml), IAA production (34.85µg/ml), ammonia excretion (2.048µg/ml) and phosphate solubility (28.56 µg/ml) was exhibited by isolate SKPB3. Isolates were analysed phylogenetically by 16S rDNA sequencing. Isolates clustered in phylogentic trees indicated high similarity and the abundance of particular cellulolytic strains. Identification of representative cultures using parial sequencing of 16S rDNA revealed presence of Acinetobacter sp, Pseudomonas sp, Stenotrophomonas sp, Bacillus sp.

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