237 Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 CODEN: JPCBBG September - November 2018 ; 6(3):237-249 Online available at https://www.jpcbs.info
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Research Article
Role of Microbial Extracellular Enzymes in the Biodegradation of Wastes Monisha Khanna Kapur1*, Payal Das1, Prateek Kumar1, Munendra Kumar1, Renu Solanki2 1Acharya 2Deen
Narendra Dev College, University of Delhi, Govindpuri, Kalkaji, New Delhi 110019, India Dayal Upadhyaya College, University of Delhi, Sector 3, Dwarka, Opp NSIT, New Delhi,110078,
India *CORRESPONDING AUTHOR Dr. Monisha Khanna Kapur, Principal Investigator, Microbial Technology Lab, Acharya Narendra Dev College, University of Delhi , Govindpuri, Kalkaji, New Delhi 110 019 Email:
[email protected],
[email protected]
ARTICLE INFORMATION Received July 07, 2018 Revised September 02, 2018 Accepted September 12, 2018 Published No vembe r 30, 2018
ABSTRACT Several soil bacteria produce extracellular enzymes having potential to degrade wastes into useful raw materials that can be directly recycled in industries. Actinomycetes are well-known producers of extracellular enzymes. In our study, during primary screening, isolate 196 was found to be efficient in degrading sugarcane bagasse and corn stover and showed high cellulase activity. Similarly isolate 197 showed high xylanase activity, degrading rice straw and wheat bran; isolate 136 showed high chitinase activity and degraded crustacean shells and isolate 244 showed maximum phosphatase activity, degrading fruit pulp. Taxonomic characterization using 16S rDNA study revealed that these bacteria isolated from soil belong to genus Streptomyces. Based on the results of primary screening, 196 and 197 were selected for secondary analysis. In secondary screening isolate 196 showed cellulase activity of 2.86 IU/ml/min at pH 6.80-7.0 and 2.92 IU/ml/min activity at 300C. Isolate 197 showed xylanase activity at the rate of 3.86 IU/ml/min at pH 7.2 and 3.72 IU/ml/min at temperature range of 280C-300C. Statistical analysis of fermentation conditions using one way ANOVA and Post Hoc test analyses signified a notable pH and temperature effect on enzyme activity of isolates 196 and 197. KEYWORDS: Extracellular enzymes; Actinomycetes; environmental wastes; fermentation conditions; statistical analysis.
INTRODUCTION Actinomycetes are group of gram positive filamentous soil bacteria [1, 2] well known as producers of various types of extracellular enzymes. These enzymes are produced within the cell but are secreted and function outside the cell [3]. Extracellular enzymes are of great
importance because they have large scale applications in various industries such as paper, textile, detergent, cosmetics, biosensors, pharmaceuticals, agricultural, bioremediation and biorefineries [5,6]. They can easily degrade wastes into useful raw materials that can be directly recycled in industries.
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Kapur et al Traditional chemical and physical treatments on biodegradable waste materials are not efficient methods for degradation because these processes are costly, take longer time and involve use of chemicals having hazardous properties. Biodegradation using microbial extracellular enzymes is an efficient and economical alternative to the traditional methods [7]. Microorganisms can convert hazardous waste materials into non-hazardous form by enzymatic processes [8]. Microbes like bacteria, fungi and yeast involved in biodegradation process use organic waste material as the source of energy resulting in the complete biodegradation of wastes. In the present study, biodegradable waste samples were collected from agro-industrial, fishery industries and household sites [9]. Samples were pretreated for converting them into powdered substrates. Actinomycete cultures from diverse habitats and producing extracellular enzymes were subjected to primary screening. Isolates showing high production of enzymes were selected and subjected to
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taxonomic characterization by polyphasic approach. 16S rRNA genes of the strains were amplified and sequenced. Sequence analysis was done using “Sequencing analysis 5.1.1” software and “EzTaxon” server, and it was found that isolates 196, 197, 136 and 244 belong to the genus Streptomyces. After analysis of sequence, evolutionary trees based on 16S rDNA were designed with the help of “Phylip-3.69” software package. These isolates were then subjected to secondary screening to determine range of enzyme activity at different pH and temperature. Statistical analyses of fermentation conditions including temperature and pH was done using Post Hoc tests (Turkey HSD) and one way ANOVA by IBM SPSS Statistics 19 software. MATERIALS & METHODS Collection of waste samples from selected sites and their conversion into substrates Collection of waste samples Waste samples were collected from diverse ecological habitats and were transferred to the laboratory in zip-lock bags (Table 1).
Table 1: Collection sites for waste materials Waste material used as substrate
Collection site
Waste degraded by enzyme
Sugarcane bagasse
Govindpuri Local Juice Corner
Cellulase
Corn Stover
Okhla PhaseI, New Delhi
Wheat Bran
Local atta chakki, Govindpuri, New Delhi
Rice straw
Matloda, Hisar, Haryana
Crustacean shells
Ghazipur, New Delhi, Fish Market
Chitinase
Fruit pulp
Jawahar Colony, Faridabad Local Juice
Phosphatase
Pre-treatment of waste samples to be used as substrate A multitude of different pre-treatment methods have been suggested in literature. These are placed under (a.) physical (grinding, irradiation, milling), (b.) chemical methods (alkali/dilute acid treatment, effects of organic solvents, oxidizing acids), (c.) physicochemical (steam pretreatment/auto-hydrolysis, wet oxidation, hydrothermolysis) (d.) biological (combined use of
Xylanase
lignin degrading enzymes like peroxidases and laccases) [10-12]. Screening of isolates at primary level for checking production of Xylanase, Cellulase, Chitinase, Phosphatase Reviving of actinomycete cultures Actinomycete cultures representing different ecological habitats and known to be producing extracellular enzymes were selected (Table 2).
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Kapur et al The cultures were revived and restreaked on Yeast extract – Malt extract (YM agar) [13, 14]
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for getting purified culture plates.
Table 2: List of actinomycete isolates selected for Screening S.No.
Properties
Habitats
1. 2. 3.
Number assigned to isolates/Colonies
Agricultural Soil- Dhanaura, UP, India Agricultural Soil- Yamuna River, Delhi, India Dumping Site- Sarai Kale Khan, Delhi, India Purana Quila- Delhi, India
Cellulose degrading isolates
4. 5.
Diversity Park- Sarai Kale Khan, Delhi, India Dumping Site- Sarai Kale Khan, Delhi, India
6. 1.
4.
Agricultural Soil- Dhanaura, UP, India Agricultural Soil- Yamuna River, Delhi, India Sarai kale Khan, Dumping Site, Delhi, India Sugar Plant- Dhanaura, UP, India
5.
Sugar Plant- Dhanaura, UP, India
1.
Agricultural Soil- Dhanaura, UP, India Agricultural Soil- Yamuna River, Delhi, India Sarai Kale Khan, Dumping Site Delhi, India Sugar Plant- Dhanaura, UP, India
2. 3.
2. 3.
Xylan degrading isolates
Chitin degrading isolates
4. 5.
Chemical Plant- Faridabad, HR, India Chemical Plant- Faridabad HR, India
6. 1.
196 191 106 186 Isolate 194, Positive Control (Previous study) 43 88 197 61 Isolate 169, Positive Control (Previous study) 4 222 88 66 136 Isolate 130, Positive Control (Previous study)
Agricultural Soil- Nainital, UK, India
116
Agricultural Soil- Kashipur, UK, India
161
3.
Yamuna Bank- Delhi, India
79
4.
Great Himalayan National ParkTeerthan Valley, HP, India
244
5.
Agricultural soil- Kashipur, UK, India
Isolate 165, Positive Control (Previous study)
2.
Phosphate degrading isolates
84
Primary Screening Media were prepared and supplemented with specific pre-treated waste samples or with
commercial substrates. 1. Basal agar medium (pH 6.8-7.0) for cellulase (composition (gL-1) - (NH4)2SO4
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Kapur et al
2.
3.
4.
2.64, KH2PO4 2.38, K2HPO4.3H2O 5.65, MgSO4.7H2O, trace salt solution (composition gL-1) 1ml. CuSO4.5H2O 6.43, FeSO4.7H2O 1.1, MnCl2.4H2O 7.9, ZnSO4.7H2O 1.5, agar 15 [13]. The medium was supplemented with sugarcane bagasse, corn stover and cellulose (commercial substrate) respectively. Mineral Salt agar medium (pH 7.2) for xylanase (composition (gL-1) NaNO3 2.0, KCl 0.5, Fe2 (SO4)3 0.01, MgSO4.7H2O 0.5, KH2PO4 0.14, K2HPO4.3H2O 1.2, Yeast extract 0.02, agar 15 [15]. The medium was supplemented with rice straw, wheat bran and birchwood xylan (commercial substrate) respectively. Chitin medium (pH 8.0) for chitinase (composition gL-1) KH2PO4 0.3, ZnSO4.7H2O 0.001, MgSO4.5H2O 0.5, K2HPO4 0.7, FeSO4.7H2O 0.01, agar 20 [16, 17]. The medium was supplemented with crustacean shells and colloidal chitin (commercial substrate) respectively. Pikovskaya’s medium (pH 7.2) for phosphatase (composition gL-1) ZnSO4.7H2O 0.001, K2HPO4 0.7, MgSO4.5H2O 0.5, KH2PO40.5, FeSO4.7H2O 0.01, Bromophenol blue (composition- 0.4g in ethanol, pH 6.5 using NaOH, working concentration= 0.08%, 0.16%, 0.24%, 0.32%, 0.40%), agar 20 [18, 19]. The medium was supplemented with fruit pulp and tricalcium phosphate (commercial
240 substrate) respectively. The strains were subjected to plate assay (primary screening) by spot inoculating them on different media plates. Inoculated plates were incubated for 7-21 days at 280C for secretion of extracellular enzymes. Clear zone surrounding the bacterial colonies indicates production of enzyme. Zones of hydrolysis were calculated by subtracting the size of inoculum from the total diameter of zone. Each experiment was carried out independently with three replicates.
Study of taxonomic status of the strains by Polyphasic approach using Bioinformatics tools: The highest enzyme producers (isolates 196,197,136 and 244) were selected for determining their taxonomic status. Genomic DNA isolation of highest enzyme producers by Phenol-Chloroform method: Genomic DNA was isolated by methods standardized for actinomycetes [13, 20]. Polymerase chain reaction (PCR) of 16s rRNA gene of isolates 196, 197, 136, 244: Genomic DNA of the selected isolates was subjected to 16s rRNA gene amplification using specific primers and under defined conditions (Table 3 and 4). The final product was analyzed on agarose gel [1, 13, 20, 21].
Table 3: 16s rRNA gene specific Primers S.No.
Primer
Sequence of Primer
1. 2. 3. 4.
8F 1492R 27F 1542R
5’-AAGGAGGTGATCCAGCCGCA-3’ 5’-AGAGGTGATCCAGCCGCA-3’ 5’-AGAGTTTGATCCTGGCTCA-3’ 5’-AAGGAGGTGATCCAGCCGCA-3’
Table 4: The PCR conditions used for 16s rRNA gene amplification PCR reaction mix (100µl)
PCR conditions
MQ= 74.2µl gDNA(template)= 3µl 10X PCR Buffer= 10µl
35 cycles Initial denaturation: 5min. at 95°C Denaturation: 30sec. at 95°C
dNTP(s)= 1.5µl Primer 8F „OR‟ 27F= 5µl Primer 1492R „OR‟ 1542R= 5µl
Primer annealing: 30sec. at 55°C Primer extension: 1min. 30sec. at 72°C Final extension: 10min. at 72°C
Taq DNA polymerase= 1.3µl
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Kapur et al Elution and sequencing of amplified PCR product(s) of isolates 196, 197, 136, 244: The amplified PCR fragments were processed using the QIAGEN gel extraction kit and were submitted for sequencing. The sequences obtained were analyzed and assembled using Sequence analysis 5.1.1 software. The resultant assembled sequence was then searched using EZ Taxon and NCBI database and the FASTA format sequences of its homologies (20 in total) were copied and pasted in a notepad file [13,20 ,22]. Phylogenetic tree construction using “Phylip-3.69” for Taxonomical analysis of Isolates 196,197,136,244: The multiple FASTA sequence file was aligned using ClustalX software. The resultant output sequences were then used to construct phylogenetic tree using Phylip 3.69. The tree was viewed using Treeview software [1, 21, 23]. Secondary screening Optimization of fermentation conditions for cellulase and xylanase Based on the results of primary screening, the colonies showing maximum zone of clearance in case of cellulase and xylanase were subjected to submerged fermentation process (SmF). The selected isolates were cultured in 25ml 148G broth (g/L-1)- Beef extract 4g, Glucose 2.2g, Bacto-peptone 5g, Tryptone 3g, NaCl 1.5g, Yeast extract 0.5g (pH 7.5) incubated at 28֯C (5 to 6 days) at 200rpm on incubator shaker [6]. A standard inoculum having average viable count 105 to 107 CFUs/ml was inoculated in respective medium for enzyme production. Culture flasks were kept for incubation at range of temperature, pH values for determination of optimum conditions for enzyme action.
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Enzyme activity estimation for cellulase and xylanase Well grown, week old cultures were centrifuged to obtain cell free supernatant which was taken as crude enzyme source. Dinitrosalicyclic (DNS) method was used to estimate enzyme activity for both enzymes in crude extract. 0.5ml of crude cell free extract from the c u l t u re fl a s k s of b o t h p r o c e s s e d w a s t e s and c o m m e r c i a l s u b s t r a t e s were added to 1.5ml volume of 2% cellulose, prepared in 0.05M sodium citrate buffer having pH value of 4.8 and incubated at 40°C for 30 minutes. To each tube, 3ml DNS reagent was added, tubes were then incubated for 5 minutes at 100°C [3, 13, 24, 25]. Absorbance was recorded at 575 nm using spectrophotometer (UV Vis Elico SI-159). Glucose standard curve was made and by extrapolating the absorbance values on the graph, amount of glucose in each sample was determined. One unit of enzyme activity is the amount of enzyme that releases 1μM of glucose/ml/min. Each experiment was carried out independently with three replicates. Analyses of enzymatic activity by univariate and multivariate methods using statistical software Experimental data was statistically evaluated using Post-Hoc and ANOVA analyses at p < 0.05 (significance level) by using software IBM SPSS 19. RESULTS Collection of waste samples from selected sites and their conversion into substrates. In the present study, the waste samples collected from various sites (Figure 1) were processed and converted into powdered substrates by different pre-treatment methods. Utilization of enzymatic potential of bacterial cultures for degradation of waste
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Figure 1: Collection sites of waste samples from varied habitats and their processing into substrates Reviving of actinomycete cultures Actinomycete cultures representing different ecological habitats and known to be producing extracellular enzymes were selected and revived. Primary Screening During primary screening, isolate no. 196 was found to be most efficient in degrading sugarcane
Colony 196 on BM + bagasse plate (congo red staining)
bagasse and corn stover and showed maximum cellulase activity (32mm on bagasse and 24 mm on corn stover substrates) followed by isolate no. 194 (positive control, 26mm and18mmm), isolate no. 106 (12mm), isolate no. 84 (7mm and 5mm), isolate 186 (3mm) and isolate 191 (no zone on bagasse and corn stover substrates) (Figure 2).
Colony194 (Positive control) on BM + bagasse plate (congo red staining)
Colony 186 on BM + bagasse plate (congo red staining)
Figure 2: Colonies with Cellulase enzyme activity showing degradation of processed waste supplemented in growth medium plates in form of zones of clearance (denoted by arrows)
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Kapur et al Similarly isolate 197 showed maximum xylanase activity (21mm on rice straw 15mm and wheat bran substrates) followed by isolate no. 169
Colony 197 on MSA + wheat bran plate (congo red staining)
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(positive control, 13mm and 18mm), isolate no. 61 (10mm and 7mm), isolate no. 85 (6mm and 4mm) and isolate no. 43 (3mm) (Figure 3).
Colony 88 on MSA + wheat bran plate (congo red staining)
Colony 169 (Positive control) on MSA + wheat bran plate
Figure 3: Colonies with Xylanase enzyme activity showing degradation of processed waste supplemented in growth medium plates in form of zones of clearance (denoted by arrows) In case of chitinase, isolate no. 136 showed maximum chitinase activity (30mm on Crustacean shells substrate) followed by isolate no. 130 (positive control, 22mm), isolate no. 222 (12mm), isolate no. 88 (9mm) and isolate no. 04 (5mm) ( (Figure 4). In phosphatase, isolate no.
Colony 136 on CM+ crustacean shells plate
244 showed maximum phosphatase activity (20mm on Fruit pulp substrate) followed by isolate no. 79 (15mm), isolate no. 165 (positive control, 12mm), isolate no. 161 (9mm) and isolate no. 116 (1mm) (Figure 5).
Colony 61 on CM+ crustacean shells plate
Colony 136 on CM+ crustacean shells plate
Figure 4: Colonies with Chitinase enzyme activity showing degradation of processed waste supplemented in growth medium plates in form of zones of clearance (denoted by arrows)
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Colony 161 on PKV+ fruit pulp plate (bromophenol blue staining)
Colony 79 on PKV+ fruit pulp plate (bromophenol blue staining)
244
Colony 244 on PKV+ fruit pulp plate (bromophenol blue staining)
Figure 5: Colonies with phosphatase enzyme activity showing degradation of processed waste supplemented in growth medium plates in form of zones of clearance (denoted by arrows). Study of taxonomic status of strains by Polyphasic approach using Bioinformatics tools Genomic DNA isolation of highest enzyme producers by Phenol-Chloroform method Taxonomic characterization of highest enzyme producers (isolates 196,197,136 & 244) was done by polyphasic approach. Genomic DNA profile on the gel showed a sharp single band for all the strains. Polymerase chain reaction (PCR) of 16s rRNA gene of isolates 196, 197, 136, 244 Genomic DNA of the selected isolates were subjected to 16s rRNA gene amplification using specific primers. The final product was analyzed on 0.8% agarose gel. Using Lambda DNA EcoRI/HindIII double digested ladder as Phylogenetic tree construction using “Phylip-3.69” for taxonomical analysis of isolates 196,197,136,244 After sequencing, the resultant assembled sequences were compared with the sequences of close Streptomyces species deposited in database. The results showed that all the strains belong to the genus Streptomyces. Actinomycete isolates 196, 197, 136, 244 were represented as distinct clades in their respective rooted evolutionary tress designed by neighbor-joining method. Isolate 196: 16s rRNA gene sequence of 1409 nucleotides was generated for isolate 196. Phylogenetic studies revealed that isolate 196 showed 100% similarity with Streptomyces
reference it was found that the size of eluted products of isolates 96, 197, 136, 244 was ≈1500bp which is equal to the size of 16s rRNA gene. Elution and sequencing of amplified PCR product(s) of isolates 196, 197, 136, 244 The amplified product were then eluted and size of the band obtained in each case was found to be≈1500bp. The eluted product samples were submitted for sequencing. The sequences obtained were analyzed and assembled using Sequence analysis 5.1.1 software. The resultant assembled sequence was then searched using EZ Taxon and the FASTA sequences of it homologs (20 in total) were copied and pasted in a notepad file which was used for construction of phylogenetic trees. albolongus followed by 99.93%, 99.50%, 99.43% with Streptomyces cavourensis, Streptomyces puniceus, Streptomyces bacillaris respectively. Isolate 197: 16s rRNA gene sequence of 1428 nucleotides was generated for isolate 197. Phylogenetic studies revealed that isolate 197 has highest 16s rRNA similarity of 100% with Streptomyces griseorubens followed by 99.79%, 99.79%, 99.79% with Streptomyces althioticus. Isolate 136: 16s rRNA gene sequence of 1431 nucleotides was generated for isolate 136. Phylogenetic studies revealed that isolate 136 has highest 16s rRNA similarity of 100% with Streptomyces parvulus followed by 99.37%, 99.30%, 99.30% with Streptomyces malachitospinus, Streptomyces olivaceus,
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Kapur et al Streptomyces pactum. Isolate 244: 16s rRNA gene sequence of 1441 nucleotides was generated for isolate 244. Phylogenetic studies revealed the isolate 244 has highest 16s rRNA similarity of 100% with Streptomyces parvulus followed by 99.37%, 99.30%, 99.30% with Streptomycesmalachitospinus, Streptomyces olivaceus, Streptomyces pactum. Secondary screening Based on the results of primary screening,
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isolates 196 (for cellulase) and 197 (for xylanase) were selected for quantitative analyses using fermentation parameters of pH and temperature so as to identify the conditions at which enzymes show highest levels of activity. In case of isolate 196, maximum cellulase activity was 2.86 IU/ml/min at pH 6.8-7.0 and 2.92 IU/ml/min activity at temperature 300C (Table 5, Figure 6 and 7). Isolate 197 showed maximum xylanase activity, 3.86IU/ml/min at pH 7.2 and 3.72 IU/ml/min activity at temperature range of 280C-300C (Table 6, Figure 8 & 9).
Table 5: Optimization of fermentation conditions for Isolate 196 (Cellulase producer) Cultures
pH
Enzyme activity (IU/ml/min)
Temp. (°C)
Enzyme activity (IU/ml/min)
Isolate 196 (Agricultural soil, Yamuna river, Delhi)
6.4
0.67
26
0.90
6.6 6.8
0.89 2.86
28 30
0.92 2.92
7.0
2.80
32
0.65
7.2 7.4
0.63 0.056
34 36
0.12 0.09
SubstrateSugarcane baggase
Table 6: Optimization of fermentation conditions for isolate 197 (Xylanase producer) CULTURES Isolate 197 (Dumping Site, Sarai kale Khan, Delhi) (Substrate-Rice straw)
pH
Enzyme activity (IU/ml/min)
Temp.
6.6 6.8 7.0
0.72 0.92 1.12
240C 260C 280C
Enzyme activity (IU/ml/min) 0.68 0.78 3.72
7.2
3.86
300C
3.70
0.96
320C
1.12
0.091
340C
0.61
7.4 7.6
Analyses of enzymatic activity by univariate and multivariate methods using statistical software One way ANOVA was used for statistical analyses of fermentation conditions including temperature and pH, which showed a notable temperature and pH effect on enzyme activity of isolates 196 and 197. For isolate 196 it was F (5, 12) = 111288.172, p = .000 for pH and F (5, 12) = 111288.172, p = .000 for temperature.
For isolate 197 it was F (5, 12) = 66581.475, p = .000 for pH and F (5, 12) = 137621.778, p = .000 for temperature. Results were re-checked using Post Hoc analysis where a statistically significant variation in activity was observed at various values of temperature and pH. In conclusion, with increasing temperature and pH, enzyme activity increases initially, then attains a maximum level and finally decreases gradually
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Kapur et al hence producing a bell shaped curve. For isolate 196, maximum cellulase activity was at pH range 6.80-7.0, temperature 300C (Figure
pH Figure 10 (a): Bell shaped curve of activity at different pH shown by isolate 196 DISCUSSION Actinomycetes are well known producers of extracellular enzymes [26, 27, 28, 29]. In the current study actinomycetes were isolated from diverse habitats including Agricultural SoilYamuna River, Delhi; waste dumping site (Sarai Kale Khan) Delhi; chemical plant (NTPC)Faridabad and Great Himalayan National Park- Teerthan Valley and were screened for their ability to produce extracellular enzymes. Isolation of actinomycete strains from varied habitats have been reported by researchers earlier too for screening of extracellular enzyme producers [30-33]. They have isolated bacterial strains from diverse habitats and screened the cultures for presence of cellulase, xylanase, chitinase and phosphatase activity respectively. During primary screening, isolate no. 196 was found to be most efficient in degrading sugarcane bagasse and corn stover and showed maximum cellulase activity (32mm on bagasse and 24mm on corn stover substrates) as compared to other colonies and positive control. Similarly isolate 197 showed maximum xylanase activity (21mm on rice straw and 15mm on wheat bran substrate), isolate 136 showed maximum chitinase activity (30mm on crustacean shells substrate) and isolate 244 showed maximum phosphatase activity (20mm on fruit pulp substrate).
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10(a, b)) and for isolate 197, maximum xylanase activity was at pH 7.2, temperature range 280C -300C (Figure 11 (a, b)).
Temperature Figure 10 (b): Bell shaped curve of activity at different temperature shown by isolate Nayaka and Vidyasagar [34], screened isolates from different habitats and found them to yield zones of clearance for degradation of sugarcane bagasse in the range of 19-52mm. Porsuk et al., [35] obtained zones of clearance of 12-25mm for rice straw degrading bacterial isolates. Similar experiments with wheat bran, crustacean shell and fruit pulp degrading bacteria have been reported in literature [33, 36-38]. Taxonomic characterization of selected isolates was done by polyphasic approach. 16SrRNA gene sequence analyses showed that isolates 196, 197, 136 and 244 belong to the genus Streptomyces. Sequence similarity between isolate 196 with Streptomyces albolongus was 100%, isolate 197 with Streptomyces griseorubens was 100%, isolate 244 with Streptomyces parvulus was 100% and isolate 136 with Streptomyces parvulus was 100%. Yassein et al., [39] performed taxonomic study of the selected cellulase producing Streptomyces isolate C188 by 16S rRNA gene analysis. The results of 16S rRNA genes showed high similarity (98 to 100%) with Streptomyces. Ninawe et al., [40] analyzed 16S rRNA gene sequences of three xylanase producing strains and found that the strains belong to the genus Strepomyces. Similar work has been reported which includes the use of 16SrRNA gene sequence for taxonomic characterization of actinomycete isolates and
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Kapur et al found that the strains belong to the genus Streptomyces [1, 21-23, 33]. Based on the results of primary screening, isolates 196 (for cellulase) and 197 (for xylanase) were selected for quantitative analyses using fermentation parameters of pH and temperature so as to identify the conditions at which enzymes show highest levels of activity. In case of isolate 196, maximum cellulase activity was 2.86 IU/ml/min at pH 6.8 and 2.92IU/ml/min activity at 300C. Isolate 197 showed maximum xylanase activity, 3.86IU/ml/min at pH 7.2 and 3.72IU/ml/min activity at 280C. Golinska and Dahm, [41] estimated cellulase activity in different Streptomyces sp. at different pH and temperature and found enzyme activity in the range of 8-13IU/ml using sugarcane bagasse as substrate. Rawashdeh et al., [42] estimated maximum xylanase activity of 5.12IU/ml/min at pH 6.5 and 5.52IU/ml/min at 0
temperature 60 C using rice straw as substrate. Statistical study of fermentation conditions including temperature and pH was done using Post-Hoc test (Turkey HSD) and ANOVA (one way) test signified a notable pH and temperature effect on enzyme activity of isolates 196 and 197. Researchers used the same approach for statistical analysis of fermentation conditions and observed similar results [22, 43-45]. CONCLUSION Actinomycete isolates from diverse habitats were studied for their potential of waste degradation during primary screening. It was observed that isolates 196, 197, 136 and 244 are high producers of cellulase, xylanase, chitinase and phosphatase respectively and were found efficient in degradation of sugarcane bagasse and corn stover; rice straw and wheat bran; crustacean shells; fruit pulp.Secondary screening was performed for 196 (cellulase producer) and 197 (xylanase producers) and with the help of statistical analyses it was concluded that there is a notable effect of pH and temperature on enzyme activity. On increasing pH and temperature, enzyme activity increases initially, then attains a maximum level and finally decrease gradually hence producing a bell shaped curve. As a conclusion of our study, actinomycete isolates were found efficient in degrading various agro- industrial, fishery and household wastes. Future studies involve
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lyophilization of the bacterial cultures, using their liquid formulations for degradation of wastes kept either in compost bags under lab conditions or alternatively degradation of wastes directly in the field. ACKNOWLEDGEMENT The authors acknowledge University of Delhi for granting the financial assistance for research work under the Innovation Project scheme 20152016. Acharya Narendra Dev College (ANDC), University of Delhi is gratefully acknowledged for providing infrastructural facilities. CONFLICT OF INTEREST The authors declare no conflict of interest REFERENCES 1. Ventura M, Canchaya C, Tauch A, Chandra G, Fitzgerald GF, Chater KF, Sinderen DV. Genomics of Actinobacteria: Tracing the Evolutionary History of an Ancient Phylum. Microbiol Mol Biol Rev 2007; 71: 495-548. 2. Janaki T. Enzymes from actinomycetesReview. Int J Chemtech Res 2017; 10(2): 176-182. 3. Das P, Solanki R, Khanna M. Characterization of Extracellular Enzymes from Soil Actinomycetes: A Molecular Approach. Int J Biotech Trends Technol 2015; 5(1): 36-46. 4. Anbu P, Gopinath SCB, Chaulagain BP, Lakshmipriya T. Microbial Enzymes and Their Applications in Industries and Medicine. Biomed Res Int 2017; 1-3. 5. Sharma M. Actinomycetes: Source, Identification, and Their Applications. Int J Curr Microbiol App Sci 2014; 3(2): 801832. 6. Hassard F, Biddle J, Harnett R, Stephenson T. Microbial extracellular enzyme activity affects performance in a full-scale modified activated sludge process. Sci Total Environ 2018; 625: 1527-1534. 7. Joutey NT, Bahafid W, Sayel H, Ghachtouli NE. Biodegradation: Involved Microorganisms and Genetically Engineered Microorganisms. Intech Open Science, Open Minds 2013; p 11. 8. Karigar CS, Rao SS. Role of Microbial Enzymes in the Bioremediation of Pollutants: A Review. Enzyme Res 2011; 111.
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Cite this article as: Monisha Khanna Kapur, Payal Das, Prateek Kumar, Munendra Kumar, Renu Solanki. Role of Microbial Extracellular Enzymes in the Biodegradation of Wastes. J Pharm Chem Biol Sci 2018; 6(3):237-249
J Pharm Chem Biol Sci, September - November 2018 ; 6(3):237-249
