Xylano-pectino-cellulolytic enzymes are valuable enzymes of the industrial sector. In our earlier study, we have reported a novel and cost effective methodology for the qualitative screening of cellulase-free xylano-pectinolytic microorganisms by replacing the commercial, highly expensive substrates with agricultural residues, but the microorganisms with xylanolytic, pectinolytic, cellulolytic, xylano-pectinolytic, xylano-cellulolytic, pectino-cellulolytic, and xylano-pectino-cellulolytic potential were obtained. The probability of getting the desired combination was low, so efforts were made to further improve this cost effective methodology for obtaining the high yield of the microbes capable of producing desired combination of enzymes. By inclusion of multiple enrichment steps in sequence, using only practically low cost substrates and without any nutrient media till primary screening stage, this improved novel protocol for screening gave only the desired microorganisms with xylano-pectino-cellulolytic activity. Using this rapid, efficient, cost effective, and improved methodology, microbes with required combination of enzymes can be obtained and the probability of getting the desired microorganisms is cent percent. This is the first report presenting the methodology for the isolation of xylano-pectino-cellulolytic positive microorganisms at low cost and consuming less time.
Lignocellulose, the most abundant natural biopolymer on earth, is an important source for the production of various industrially useful materials. Enzymatic and chemical methods can be used for the degradation of these materials. Chemical methods are performed at high temperatures and alkaline conditions, which produce toxic by-products as compared to the enzymatic methods. Chemical degradation of these materials is toxic to the environment which can be replaced by enzymatic processes or can be merged with enzymatic methods so as to reduce the concentration of toxic chemicals being used conventionally in chemical processes. Various industrial processes involving the use of microbial enzymes are less polluting, highly efficient, and energy saving and also result in lower disposal problems [
Xylano-pectino-cellulolytic enzymes are the industrially important enzymes which specifically degrade the xylan, pectin, and cellulose into sugars and are being used for the extraction of vegetable oil; processing of animal feed, food, and beverages; recycling of waste paper; textile industry; and biofuel production [
Soil is the good source of microorganisms due to the nutrient rich environments, where there is a high proliferation of microorganisms. Soil samples contaminated with the effluents of various paper and textile industries were collected. These were mixed in equal proportions to make the composite samples. Enrichment step was carried out using agricultural wastes and no other nutrient medium was used. Before use, dried green citrus peel and wheat bran were washed separately and dried at 45°C and citrus peel was grinded. One gram of the composite soil sample was suspended in 100 mL Erlenmeyer flask containing 20 mL sterile deionized water, pH 10.0, and kept on shaker set at 50°C, 200 rpm for 1 h in order to uniformly mix the soil sample and centrifuged after 1 h so as to get the clear suspension. Ten percent of the above suspension was added to 20 mL sterile water (pH 10.0) supplemented with only 2% wheat bran and was incubated at 50°C, 200 rpm for 24 h. Wheat bran was used to stimulate the growth of xylanase producing microorganisms. After 24 h of incubation, 10% inoculum from wheat bran enriched culture was used to inoculate another 100 mL Erlenmeyer flask containing 20 mL sterile water (pH 10.0) supplemented with only 2% citrus peel for the enrichment of pectinase producing microorganisms and was incubated at 50°C, 200 rpm for 24 h. After incubation, 10% inoculum from wheat bran-citrus peel enriched culture was used to inoculate another 100 mL Erlenmeyer flask containing 20 mL sterile water (pH 10.0) supplemented with only 2% waste paper and was incubated at 50°C, 200 rpm for 24 h. Waste paper was used to stimulate the growth of cellulase producing microorganisms. After enrichment of xylano-pectino-cellulolytic microorganisms one by one in wheat bran, citrus peel, and waste paper, final enrichment step was carried out using all these substrates (1% each), in 20 mL sterile water (pH 10.0), and the incubation was done under the same conditions as mentioned above. This final multistep enriched culture sample was used for further study.
For the isolation of xylano-pectino-cellulolytic microorganisms, primary screening was carried out on agar plates containing wheat bran, citrus peel, and waste paper individually and no other nutrient medium was used. Different dilutions of the enriched culture sample were plated onto agar plates containing 2% wheat bran (pH 10.0) for the screening of xylanolytic microorganisms and the plates were incubated at 50°C for 24 h. In order to evaluate the efficiency of this new methodology, different isolates having large colony size were selected from the above plates and growth was also checked onto agar plates containing 2% citrus peel and 2% waste paper individually using the same conditions as above for the screening of pectinolytic and cellulolytic microorganisms, respectively.
Different isolates selected in primary screening were spotted onto the agar plates containing only 0.5% peptone and 1.0% wheat bran (pH 9.0) and were incubated at 50°C for 24 h. After 24 h of incubation, xylanase producing microorganisms were selected by flooding the plates with 0.5% (w/v) Congo red for 15 min followed by repeated washing with 1 M NaCl for analyzing the zones of substrate hydrolysis [
Final screening was done by quantitative estimation of enzymes activity after producing them under submerged fermentation.
To 20 mL sterile water containing 0.5% peptone, we added 2% wheat bran, 2% citrus peel, and 2% wheat straw, pH 8.0, for simultaneous production of xylanase, pectinase, and cellulase enzymes, respectively. In this study, yeast extract, KNO3, KH2PO4, MgSO4, or any other additive has also not been added in the medium, as described in our earlier study [
Birchwood xylan 1%, pectin 0.5%, and carboxymethyl cellulose 1% were used for estimation of xylanase, pectinase, and cellulase activity, respectively. The enzymes activity was determined by measuring the amount of reducing sugars liberated after enzyme substrate reaction using 3,5-dinitrosalicylic acid reagent [
Due to the high cost of commercial substrates for screening purposes, the alternative cost effective agricultural wastes were used in our earlier study for the isolation of xylano-pectinolytic microorganisms [
In qualitative analysis, only 0.5% peptone was added to agar plates containing wheat bran, citrus peel, and waste paper individually. Formation of large clear zone around the colonies indicated that the strains were enzymes producer with good substrate hydrolyzing ability. Zone of substrate hydrolysis obtained after Congo red staining on peptone-wheat bran agar plates indicated the xylanolytic nature of the microorganisms. In order to check the efficiency of this methodology, zones were also analysed on peptone agar plate containing commercial xylan. The clearance zones of substrate hydrolysis shown by isolate on peptone agar plate containing wheat bran/xylan (Figures
Zone of substrate hydrolysis shown on (a) wheat bran; (b) xylan; (c) citrus peel; (d) pectin; (e) waste paper; (f) CMC by xylano-pectino-cellulolytic bacterial isolate AVS13 and zone of substrate hydrolysis produced by a known xylanase producer
Table
Zones of substrate hydrolysis and activity analysis of xylano-pectino-cellulolytic enzymes.
Isolates | Qualitative analysis | Quantitative analysis | |||||||
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Diameter (mm) of zone of substrate |
Diameter (mm) of zone of substrate |
Enzyme activity |
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Wheat bran | Citrus peel | Waste paper | Xylan | Pectin | Cellulose | Xylanase | Pectinase | Cellulase | |
AVS 1 | 27 ± 1 | 20 ± 1 | 5 ± 1 | 28 ± 1 | 21 ± 1 | 5 ± 1 | 167 ± 28 | 134 ± 17 | 3.5 ± 0.5 |
AVS 2 | 22 ± 1 | 20 ± 2 | 13 ± 1 | 22 ± 1 | 20 ± 1 | 14 ± 3 | 83 ± 16 | 117 ± 13 | 22 ± 1.5 |
AVS 3 | 31 ± 1 | 14 ± 1 | 5 ± 1 | 32 ± 1 | 15 ± 1 | 5 ± 1 | 233 ± 35 | 50 ± 8 | 3.2 ± 0.6 |
AVS 4 | 20 ± 2 | 23 ± 2 | 18 ± 1 | 20 ± 1 | 24 ± 2 | 18 ± 1 | 69 ± 8 | 184 ± 26 | 30 ± 3.5 |
AVS 5 | 25 ± 1 | 19 ± 1 | 25 ± 1 | 26 ± 1 | 19 ± 1 | 26 ± 1 | 150 ± 18 | 101 ± 18 | 42 ± 4 |
AVS 6 | 24 ± 1 | 14 ± 1 | 29 ± 2 | 24 ± 1 | 14 ± 1 | 30 ± 3 | 117 ± 14 | 50 ± 8 | 54 ± 5.2 |
AVS 7 | 32 ± 1 | 13 ± 1 | 26 ± 1 | 33 ± 1 | 13 ± 1 | 26 ± 1 | 267 ± 30 | 25 ± 5 | 41 ± 5 |
AVS 8 | 38 ± 1 | 11 ± 1 | 6 ± 1 | 39 ± 2 | 11 ± 1 | 6 ± 1 | 852 ± 62 | 20 ± 3.5 | 5 ± 0.8 |
AVS 9 | 28 ± 1 | 14 ± 1 | 10 ± 1 | 28 ± 1 | 13 ± 1 | 10 ± 1 | 184 ± 31 | 35 ± 4 | 17 ± 1 |
AVS 10 | 18 ± 2 | 16 ± 1 | 7 ± 1 | 17 ± 1 | 16 ± 1 | 7 ± 1 | 35 ± 7 | 67 ± 9 | 8.5 ± 0.8 |
AVS 11 | 17 ± 1 | 29 ± 3 | 12 ± 1 | 17 ± 1 | 30 ± 3 | 12 ± 1 | 25 ± 3.5 | 434 ± 41 | 20 ± 1.5 |
AVS 12 | 14 ± 1 | 11 ± 1 | 8 ± 1 | 14 ± 1 | 10 ± 1 | 8 ± 1 | 9 ± 1.5 | 17 ± 2.5 | 11 ± 1 |
AVS 13 |
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AVS 14 | 19 ± 2 | 23 ± 1 | 22 ± 1 | 19 ± 1 | 24 ± 1 | 24 ± 1 | 57 ± 11 | 167 ± 18 | 40 ± 4 |
AVS 15 | 32 ± 1 | 18 ± 1 | 11 ± 1 | 34 ± 1 | 18 ± 1 | 10 ± 1 | 284 ± 17 | 84 ± 9 | 14 ± 1.2 |
AVS 16 | 32 ± 1 | 13 ± 1 | 7 ± 1 | 33 ± 2 | 13 ± 1 | 7 ± 1 | 267 ± 29 | 34 ± 3.5 | 8 ± 1 |
AVS 17 | 32 ± 1 | 20 ± 2 | 22 ± 1 | 33 ± 1 | 21 ± 2 | 22 ± 3 | 251 ± 14 | 117 ± 14 | 30 ± 3 |
AVS 18 | 20 ± 3 | 8 ± 1 | 26 ± 1 | 20 ± 1 | 9 ± 1 | 27 ± 1 | 69 ± 10 | 9 ± 1 | 45 ± 4.2 |
AVS 19 | 24 ± 1 | 20 ± 1 | 5 ± 1 | 24 ± 1 | 21 ± 1 | 6 ± 1 | 117 ± 16 | 104 ± 15 | 3.5 ± 0.5 |
AVS 20 | 30 ± 1 | 8 ± 1 | 18 ± 1 | 31 ± 1 | 9 ± 1 | 18 ± 1 | 217 ± 25 | 14 ± 1.5 | 25 ± 2.5 |
AVS 21 | 30 ± 1 | 13 ± 1 | 12 ± 1 | 30 ± 1 | 14 ± 1 | 12 ± 1 | 201 ± 17 | 50 ± 6 | 20 ± 1.5 |
AVS 22 | 34 ± 1 | 16 ± 1 | 17 ± 1 | 35 ± 1 | 16 ± 1 | 17 ± 1 | 384 ± 33 | 58 ± 10 | 27 ± 2 |
AVS 23 | 34 ± 2 | 13 ± 1 | 26 ± 1 | 35 ± 3 | 13 ± 1 | 27 ± 1 | 401 ± 28 | 34 ± 3.5 | 35 ± 3.5 |
AVS 24 | 22 ± 1 | 10 ± 1 | 14 ± 1 | 23 ± 1 | 11 ± 1 | 14 ± 1 | 84 ± 0.9 | 13 ± 1.5 | 24 ± 2.5 |
AVS 25 | 31 ± 1 | 12 ± 1 | 8 ± 1 | 32 ± 1 | 12 ± 1 | 8 ± 1 | 234 ± 20 | 20 ± 2 | 12 ± 1.2 |
AVS 26 | 30 ± 2 | 15 ± 1 | 10 ± 1 | 31 ± 1 | 16 ± 1 | 10 ± 1 | 217 ± 18 | 42 ± 5 | 14 ± 1.5 |
AVS 27 | 19 ± 1 | 13 ± 1 | 5 ± 1 | 20 ± 1 | 13 ± 1 | 6 ± 1 | 58 ± 6 | 27 ± 3 | 3.2 ± 0.8 |
AVS 28 | 34 ± 1 | 26 ± 3 | 27 ± 1 | 35 ± 2 | 28 ± 2 | 28 ± 1 | 317 ± 32 | 267 ± 31 | 44 ± 4.5 |
AVS 29 | 31 ± 1 | 24 ± 1 | 33 ± 2 | 32 ± 1 | 25 ± 2 | 34 ± 1 | 234 ± 21 | 217 ± 26 | 92 ± 13 |
AVS 30 | 30 ± 1 | 9 ± 1 | 29 ± 3 | 30 ± 1 | 8 ± 1 | 30 ± 2 | 201 ± 13 | 9 ± 0.8 | 67 ± 8.5 |
AVS 31 | 32 ± 1 | 24 ± 2 | 30 ± 1 | 33 ± 2 | 25 ± 1 | 30 ± 1 | 267 ± 27 | 200 ± 17 | 54 ± 6 |
AVS 32 | 25 ± 1 | 18 ± 1 | 7 ± 1 | 25 ± 1 | 18 ± 1 | 8 ± 1 | 134 ± 12 | 87 ± 8 | 12 ± 1 |
AVS 33 | 32 ± 2 | 15 ± 1 | 23 ± 1 | 32 ± 1 | 15 ± 1 | 24 ± 1 | 251 ± 15 | 39 ± 4 | 35 ± 3.2 |
AVS 34 | 35 ± 2 | 25 ± 1 | 5 ± 1 | 37 ± 1 | 25 ± 1 | 5 ± 1 | 585 ± 61 | 184 ± 17 | 3.5 ± 0.8 |
AVS 35 | 30 ± 1 | 24 ± 2 | 27 ± 1 | 31 ± 1 | 24 ± 3 | 28 ± 1 | 217 ± 30 | 201 ± 25 | 44 ± 4 |
AVS 36 | 17 ± 2 | 8 ± 1 | 13 ± 1 | 18 ± 1 | 8 ± 1 | 14 ± 1 | 27 ± 3.2 | 12 ± 1 | 21 ± 2 |
AVS 37 | 32 ± 1 | 18 ± 1 | 22 ± 2 | 32 ± 1 | 19 ± 1 | 23 ± 1 | 234 ± 23 | 100 ± 17 | 34 ± 3.5 |
AVS 38 | 20 ± 1 | 16 ± 1 | 15 ± 1 | 20 ± 1 | 16 ± 1 | 16 ± 1 | 67 ± 14 | 54 ± 8 | 25 ± 2 |
AVS 39 | 32 ± 1 | 14 ± 1 | 8 ± 1 | 32 ± 2 | 15 ± 1 | 8 ± 1 | 251 ± 25 | 35 ± 3.5 | 12 ± 1.2 |
AVS 40 | 32 ± 1 | 15 ± 2 | 22 ± 1 | 32 ± 1 | 15 ± 2 | 24 ± 1 | 234 ± 20 | 51 ± 5 | 36 ± 4 |
AVS 41 | 34 ± 1 | 8 ± 1 | 29 ± 2 | 35 ± 1 | 8 ± 1 | 30 ± 3 | 351 ± 33 | 8 ± 0.75 | 54 ± 5 |
AVS 42 | 36 ± 3 | 14 ± 1 | 6 ± 1 | 38 ± 2 | 14 ± 1 | 6 ± 1 | 702 ± 42 | 40 ± 3.5 | 7 ± 1.5 |
AVS 43 | 20 ± 1 | 24 ± 1 | 6 ± 1 | 20 ± 1 | 25 ± 1 | 6 ± 1 | 67 ± 16 | 201 ± 23 | 5 ± 1 |
AVS 44 | 25 ± 2 | 28 ± 2 | 8 ± 1 | 25 ± 1 | 30 ± 2 | 8 ± 1 | 134 ± 21 | 434 ± 37 | 10 ± 1.5 |
AVS 45 | 19 ± 1 | 26 ± 1 | 7 ± 1 | 19 ± 1 | 27 ± 3 | 7 ± 1 | 50 ± 7 | 267 ± 28 | 9 ± 1.2 |
AVS 46 | 32 ± 2 | 26 ± 1 | 33 ± 2 | 33 ± 1 | 27 ± 1 | 34 ± 2 | 267 ± 24 | 117 ± 16 | 82 ± 15 |
AVS 47 | 35 ± 2 | 14 ± 1 | 8 ± 1 | 36 ± 1 | 14 ± 1 | 8 ± 1 | 518 ± 38 | 34 ± 3.5 | 10 ± 1.5 |
AVS 48 | 32 ± 1 | 15 ± 1 | 13 ± 1 | 32 ± 1 | 16 ± 1 | 13 ± 1 | 251 ± 23 | 57 ± 5 | 21 ± 2 |
AVS 49 | 25 ± 1 | 14 ± 1 | 8 ± 1 | 25 ± 1 | 15 ± 1 | 8 ± 1 | 134 ± 16 | 50 ± 4 | 8.5 ± 1.2 |
AVS 50 | 30 ± 1 | 8 ± 1 | 22 ± 1 | 31 ± 1 | 8 ± 1 | 23 ± 2 | 217 ± 20 | 14 ± 1.5 | 37 ± 3.2 |
Morphological, physiological, biochemical tests and 16S rDNA sequencing of the isolated microorganism AVS 13.
Tests | Results | Tests | Results |
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Morphological tests: | |||
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Colony Morphology | |||
Configuration | Circular | Margin | Entire |
Elevation | Raised | Surface | Rough |
Texture | Mucoid | Pigment | Off-white |
Opacity | Opaque | Gram’s Reaction | + |
Cell shape | Rod | Spore(s) | + |
Motility | + | ||
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Physiological tests: | |||
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Growth at temperatures | |||
4°C | − | 15°C | − |
25°C | + | 30°C | + |
37°C | + | 42°C | + |
55°C | + | ||
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Growth at pH | |||
pH 5.0 | + | pH 6.0 | + |
pH 7.0 | + | pH 8.0 | + |
pH 9.0 | + | pH 10.0 | + |
pH 11.0 | + | pH 12.0 | + |
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Growth on NaCl (%) | |||
2.0 | + | 4.0 | + |
6.0 | + | 8.0 | + |
10.0 | + | 11.0 | + |
12.0 | + | ||
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Biochemical tests: | |||
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Growth on MacConkey | Non Lactose Fermenting | Indole test | − |
Methyl red test | − | Voges Proskauer test | + |
Citrate utilization | − | Casein hydrolysis | + |
Esculin hydrolysis | + | Gelatin hydrolysis | + |
Starch hydrolysis | + | Nitrate reduction | + |
Ornithine decarboxylase | + | Lysine decarboxylase | + |
Catalase Test | + | Oxidase test | + |
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Acid production from | |||
Adonitol | − | Trehalose | + |
Rhamnose | − | Salicin | + |
Dulcitol | − | Galactose | − |
Melibiose | + | Raffinose | − |
Inulin | + | Sorbitol | + |
Fructose | + | Sucrose | + |
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16S rDNA SEQUENCING | |||
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TGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAAC | |||
ACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATG | |||
GTTGTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACC | |||
CGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACC | |||
TGAGAGGGTGTCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCA | |||
GTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGG | |||
TTTTCGGATCGTAAAGTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACC | |||
TTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTA | |||
GGTGGCAAGCGTTGTCCGGAATATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTG | |||
ATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGA | |||
AGAGGAGAGTGGAATTCCACGTGTAGGGTGAAATGCGTAGAGATGTGGAGGAACACCAGT | |||
GGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACA | |||
GGATTAGATACCCTGGTAGTCCACGCCGTAAAGATGAGTGCTAAGTGTTAGGGGGTTTCCG | |||
CCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGA | |||
AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGAGCATGTGGTTTAATTCGAAGCA | |||
ACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGAGATAGGACGTCCCC | |||
TTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCCGTGTCGTGAGATGTTGGGT | |||
TAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAG | |||
GTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAACATCATGCCCCTTAT | |||
GACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTA | |||
AGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTCGTGAAGCT | |||
GGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACA | |||
CCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTAGG |
+: Positive; −: Negative.
In our earlier study, we got both positive and negative desired enzymes producing microorganisms [
Concurrent production of these industrially important enzymes from a microbial isolate simultaneously in the same production medium will reduce the production cost, maintenance cost, man power, and so forth, to a higher extent in comparison to production of individual enzymes. This novel, improved, and efficient protocol for the screening of microorganisms gave only the desired xylano-pectino-cellulolytic positive microorganisms. Using this methodology, probability of getting the desired microorganisms is high, so this methodology would definitely save time and cost for getting the required microbial isolates. Enzymes producing industries are still looking for microbial isolates having combination of various enzymes, suitable to the needs of the industry. This methodology would also definitely help the industries in the isolation of microbes capable of producing multiple enzymes with high titre at practically low cost.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors gratefully acknowledge the financial support provided by the University Grant Commission (UGC), New Delhi, India. Fellowship awarded to Avtar Singh in the form of MANF by UGC is kindly acknowledged.