Login in here. The Journal of General and Applied Microbiology. Journal home Advance online publication Journal issue About the journal. Full Papers. Isolation of cellulase-producing Microbulbifer sp. Keywords: bacteria , cellulase , low temperature , marine fish. View Article Download Cite References Statistics Share Abstract Cellulase is one of the most economically important enzyme, which aids in catalyzing cellulolysis, the decomposition of cellulose and other related polysaccharides.
Keywords: Cellulase bacterial isolates sugarcane waste soil enzyme catalyzing cellulolysis P. How to Cite Hassan, A. Characterization of a thermophilic cellulase from Geobacillus sp. HTA, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass. PLoS One. A novel strain of Trichoderma viride shows complete lignocellulolytic activities. Shonukan, Purification and characterization of cellulase from wild type and two improved mutants of Pseudomonas flourescens.
African Journal of Biotechnology. Okeke BC, Lu J. Characterization of a defined cellulolytic and xylanolytic bacteria consortium for bioprocessing of cellulose and hemicelluloses. Approaches to Dispersing Medical Biofilms". American Journal of BioScience. The researchers conclude that Paenibacillus sp's strain shows high potential for the optimal development of cellulase in a 1. Besides, some cellulose-decomposing bacteria showing CMCase activity within the 0.
Deka et al. In addition, setting up an optimized condition, Da Vinha et al. Hp strain, Sheng et al. Global Cellulase's Market Report for reveals that Asia Pacific is the largest purchaser of cellulases and added that cellulase application would hit USD million by , which will increase by 5. The production of cellulase enzymes in the future most exciting advanced field of research by biotechnology with several factors, including cost, genetic engineering, a particular activity, and purification steps that are feasible in research interests.
The degradation of cellulosic materials has been identified in numerous investigations. Still, few studies have examined which microorganisms, especially bacteria, could be used as the best source of cellulase enzyme production for industrial purposes. For this reason, there is a growing demand for the isolation of bacterial aerobic strains, which reasonably produce higher cellulase activity. Our study has conducted by isolating aerobic bacteria from a local soil sample that could hydrolyze cellulosic material.
Besides, further identified those bacterial isolates by the growth of selective media. Current work was also done to optimize media parameters for improving the output of cellulose employing identified bacterial isolates.
In conclusion, the bacterial isolates CBM21 produces 1. The flow diagram of the study plan was shown in supplementary Figure 1. Traditional serial dilution technique has been used to isolate cellulolytic bacteria through agar plate about 10 -1 to 10 -7 dilution factor.
From the test tubes labeled 10 -6 and 10 -7 , 0. Different discrete colonies were found on an agar plate and were selected further. CMC agar plates had been soaked with iodine solution for approximately 5 minutes and then allowed to stand at room temperature. Several colonies were shown a clear circular zone on the agar plate.
The respective broth culture which were shown clear zone were streak on CMC agar media containing 1. The plates were further flooded with iodine and allowed to stand at room temperature for around five minutes. The CMC hydrolysis clear zone and colony diameter ratio were measured and reported. For cellulase development in a submerged environment, the bacterial colonies with the largest ratio were selected.
Bacterial isolates were pre-sumptuously characterized by morphology, as well as some biochemical analysis.
Pseudomonas sp. Cetrimide media composition pancreatic digest of gelatin 2. The selective agent of cetrimide inhibits most bacteria as it functions as a detergent. For several years, therefore, 0. Potential isolates for enzyme efficiency have been examined. The maximum cellulase production isolates were considered for the following study. The maximum hydrolysis zone of these isolates is grown in 20 ml inoculum production medium [composition: Carboxymethylcellulose CMC 0.
It was used as an inoculum medium subsequently the source of production medium The media were inoculated with 2. At the end of centrifugation, the clear supernatant was collected to serve as a crude enzyme source and used to determine enzymatic activity The DNSA method was used to determine how much reducing sugar was liberated during the hydrolysis.
Under the following standard assay conditions, the enzyme unit EU was calculated as the amount of cellulase required to release 1 mole of reducing sugar per ml per minute The cellulase activities of each of the bacterial strains were assayed in triplicate. The reaction mixes included a buffer of 1ml 0. Following incubation, 1.
The absorbance was measured at nm. Glucose release was measured with the glucose calibration curve 26 , and enzymatic activity was determined by the following equation Cellulase activity of the culture supernatant of bacteria was grown at different temperatures, pH, carbon sources, and incubation periods.
The optimal substrate and carbon source concentration are taken, and the broth pH is balanced at 6. Cultures are inoculated and cultivated at specific temperatures. The initial pH 7 of the production medium was adjusted and then inoculated with selected bacterial isolates that had been grown overnight.
The filtrate of cell-free culture is collected after 24 hours of incubation and used as a source of crude enzymes. Several carbon substitutes, including fructose, lactose, and sugar, have been used as an initial source of carbon in growth media to compensate for glucose. The incubation period has optimized for the cellulase production and the pH of the production media broth was adjusted to 7.
The fermentation product was withdrawn at different time intervals, e. After centrifugation, for cellulase testing, the supernatant was used to approximate a standard glucose curve. In this analysis, ten samples were collected from 5 different locations, yielding 40 isolates from each sample using the dilution 10 -6 and 10 -7 plates. From these, out of isolates were removed due to non-cellulolytic. The 40 isolates were then screened for cellulase activity on CMC agar using iodine staining, whereas congo red staining did not yield an effective result.
According to Gohel et al. Gomashe et al. As a consequence, native microbes can be a source of cellulase that can be investigated for use in a variety of applications. The CMCase activity is shown in Figure 1 ; of both the four isolates, the highest ratio of the apparent zone diameter to the colony diameter on the CMC agar plate was determined compared to other isolates Figure 2.
Indole test These tests were performed by inoculating a loop-full of culture in tryptophan broth and then incubated at 37 0 C. After incubation for 24 hours kovacs reagent was added to the tubes. If the cherry red colour ring is formed indicates positive for indole production. After incubation period half of the culture was transferred to a clean tube and other tube of culture was used to conduct VP test.
Catalase test To 3 ml of hydrogen peroxide, add a loop full of culture using inoculating loop on a clean glass slide, evolution of effervescence indicates positive test Experiments in microbiology, plant pathology and biotechnology by K R Aneja After incubation, the plates were treated with iodine to conform the hydrolysis of starch.
Mannitol test This test determines ability of organism to utilize mannitol as source of carbon. A slant of mannitol agar in clean and sterile test tube is streaked with a loop full of culture and incubated for 24hrs.
If the color changes from pink to yellow, it refers positive to mannitol test. Urease test These tests were performed by inoculating a loop full of culture in urease agar media and then incubated at C for 24 hours.
If the media turns into pink colour shows positive test for urease. Optimisation of Cellulose Effect of ph To determine the optimum pH for cellulase production, Media containing 0. Whole flask samples were withdrawn and cellulase activity in the supernatant was assayed. Effect of temperature To determine the optimum temperature for cellulase production media containing 0.
Whole-flask samples were used for the cellulase assay. The reaction mixture contains 1ml of 0. Acetate Buffer pH 5. The absorbance was measured at nm. UV visible spectrophotometer, Model: Hitachi-U One unit of cellulase activity is defined as the quantity of cellulase required to liberate 1. Results And Discussion:- For isolation of cellulose degrading bacteria, termites were collected from woody habitat, as it contains cellulose degrading bacteria in its gut and digestive tract.
The colony morphology was studied in detail and the results are presented in Table. After incubation Cong red test was performed for screening of the cellulolytic organisms. Total of thirteen bacterial isolates found to be positive on screening media cellulose Cong red agar by producing clear zone around their colonies Figure.
The optimization of the isolated bacteria which as the potentiality for the production of cellulose was carried out and results are shown in Table. In recent years, more attention has been directed towards screening of novel microbial strain that has broad spectrum enzyme activity. The high cost of cellulase production and low enzyme activities limit their industrial use such as bio-fuel production, bio fertilizers, and textile industry.
So effort are to be taken to economize and increase the yield of cellulase production by media optimization Ma. J et al. Gupta et al. So we collected faecal matter of livestock animals such as Sheep and Goat from rural area Haliyal and Malamaddi, Dharwad as our site for sample collection. We selected this source for our study because these animals feed on wide variety of plant biomass which is rich in cellulose diversity able to degrade different types of cellulose as they contain most of cellulase producing microorganisms able to degrade cellulose and we collected termite belonging to family kalotermitidae in woody habitat from Botanical Garden Karnataka University Dharwad.
We selected these samples because termite has symbiotic microorganisms in Gut and digestive tract which helps in digesting the cellulose containing food. Collected samples were serially diluted and plated on minimal salt media CMC agar media , Shankar et al. Out of 9 isolates 3 isolates isolate1 S3, isolate2 G3, isolate3 T4 showed maximum zone, further 3 cultures used for biochemical characterization.
Same 3 isolates isolate S3, isolate G3, isolate T4 showed maximum enzyme activity, based on standard calibration curve by DNS method. Overall isolate S3 showed maximum enzyme activity. Hence we can conclude that the isolate can be used for production and isolation of cellulose which can be used for industrial processes. Table Colony morphology of isolated organisms.
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