Phd thesis on xylanase

Phd thesis on xylanase

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Microbial communities in hot springs at high elevations have been extensively studied worldwide. In the present study, phd thesis on xylanase, a total of thermophilic bacteria were isolated from 12 samples collected from Manikaran and Yumthang hot springs of Indian Himalayas. The bacterial isolates were studied for phylogenetic profiling, growth properties at varying conditions and potential sources of extracellular thermostable hydrolytic enzymes such as protease, amylase, xylanase phd thesis on xylanase cellulase, phd thesis on xylanase.

Based on production of extracellular hydrolases, 51 isolates from Manikaran 28 and Yumthang thermal springs 23 were selected and identified using 16S rRNA gene sequencing which included 37 distinct species of 14 different genera namely Anoxybacillus, Bacillus, Brevibacillus, Brevundimonas, Burkholderia, Geobacillus, Paenibacillus, Planococcus, Pseudomonas, Rhodanobacter, Thermoactinomyces, Thermobacillus, Thermonema and Thiobacillus.

Out of 51 hydrolase producing bacteria, 24 isolates showed stability at wide range of temperature and pH treatments. In present investigation, three thermotolerant bacteria namely, Thermobacillus sp NBM6, Paenibacillus ehimensis NBM24 and Paenibacillus popilliae NBM68 were found to produced cellulase-free xylanase.

These potential extracellular thermostable hydrolytic enzymes producing thermophilic bacteria have a great commercial prospect in various industrial, medical and agriculture applications. Microbes were present in every conceivable ecological niche, includes from the tropics to the poles and hot springs to underwater hydrothermal vents.

Particularly, prokaryotic communities under thermophilic habitats have been undergone the physiological adaptations to high temperature and chemical stress. Recently, these communities have attained the focus of applied research not only in terms of biotechnological prospects but also to understand the use of primitive analogues of biomolecules existed during early Earth environments Raddadi et al. The microbial diversity of cold and snow caped lakes such as cold deserts of Himalaya India Sahay et al.

Apart from cold habitats, Himalayas have unique hot springs at high elevations, such as Soldhar and Ringigad hot springs, Uttaranchal Himalaya Kumar et al.

Thermal springs are hot spots of biodiversity of microbes which can be utilized as source of novel genes, molecules and hydrolytic enzymes for agricultural, medical and industrial processes Saxena et al.

Novel thermophilic microbes have isolated and characterized from thermal extreme environments of world such as Thermotoga elfii Ravot et al. Manikaran and Yumthang thermal springs are the hottest in the country, with a temperature range of 89—95 °C, which can be potential sources of novel genes, molecules and microorganisms. Despite intensive studies on terrestrial thermal springs, very little is known about bacterial diversity of thermal springs at high elevation, phd thesis on xylanase.

Hence, a comprehensive approach is needed to analyze the bacterial diversity of such niche, in terms of their phylogenetic profiling and its potential bioresources for extracellular hydrolytic enzymes.

Thermostable enzymes, isolated from thermophiles, have found potential commercial and industrial significance due to their inherent stability under harsh industrial processes Verma et al. Natural thermal environments or extreme niches are providing support to the primitive form of life; such habitats became special inherent resources of bioactive molecules for modern biotechnologists. Therefore, every country has putting research efforts towards exploration and conservation of the bio-resources available in their natural ecosystems.

Potential microbes from hot springs offer a major advantage of preserving those strains for future studies and exploring them in due course for potential biotechnological applications in medical, industrial and agriculture processes.

The present investigation deals with culturable thermophilic bacteria and their identification using 16S rRNA gene sequencing. Following culturable technique, isolation, characterization, phylogenetic profiling and the hydrolytic enzymes production of thermo-adapted culturable microbes was performed for samples collected from hot springs of Indian Himalayas.

Water and sediment samples were collected from Manikaran and Yumthang hot springs, Indian Himalayas Table 1. The temperatures and pH of samples were recorded at sampling sites. The temperature and pH of Manikaran hot springs ranges from 89 to 95 °C and 7. Six different samples from each hot springs collected and transferred into sterilized thermostatic bottles.

All the samples were kept in sterilized thermostatic flask and were transported to the laboratory in a minimum time. The population of culturable thermophilic bacteria in the 12 different water and sediments were enumerated through enrichment using the standard serial dilution plating technique.

For enrichment, 10 mL of each water sample was inoculated directly into 50 mL of nutrient broth, where as in case of sediment samples, 5 g of sediment was mixed with 10 mL of corresponding filter sterilized water and inoculated into 50 mL of nutrient broth. All the phd thesis on xylanase were incubated at 60 °C for 2 h. Colonies that appeared were purified by repeated streaking to obtain isolated colonies using respective medium plates.

Effects of temperature and pH on growth of all the representative isolates were investigated. All the bacterial isolates were grown for 24 h at 40 °C, after that µL of bacterial broth were inoculated into micro well plates having thermus liquid medium and incubated in temperature controlled Automated Microbiology Growth Analysis System Oy Growth Curve Ab Ltd, Finland.

The optical density at nm was measured at regular intervals. All the isolates were analyzed in triplicates and growth curves were derived using the analysis software provided with the instrument.

CFU have been recorded for each selected isolated after 24 h by inoculating µL of broth on thermus medium. All the bacterial isolates were screened for the production of extracellular thermostable hydrolases such as protease, amylase, xylanase and cellulase. The primary screening of all isolates for enzymes production was carried out as described earlier Sahay et al. The all isolates were inoculated in the basal medium phd thesis on xylanase Yeast extract, 1 g; KH 2 PO 41 g; MgSO 4 ·7H 2 O; 0.

Purified bacterial strains were grown in mL flasks containing 50 mL of nutrient broth and 5 µL of culture was spot inoculated on basal medium plates and incubated at 45, 55 and 65 °C for 3—5 days. Plates were observed for the formation of clear zone for protease.

The clear zone around the colony was observed in flooded with 0. The cultures were tested for amylolytic activity using basal medium supplemented with 0. After incubation for 5 days, in each plate 0, phd thesis on xylanase.

The formation of zone around cultures indicated the amylase activity. The stability of each enzyme was estimated at different temperature and pH. Selected strains were inoculated into mL flasks containing mL basal medium having corresponding polymer as substrate and incubated at its respective optimum growth temperature and pH for 3—5 days.

An aliquot of the crude enzyme cell free extract was pre-incubated at different temperatures 45, 55, 65, phd thesis on xylanase, 75 and 85 °C for 30 min at pH 7. The stability of enzyme activity SE at variable temperatures and pH was calculated as per the formula described in our earlier studies Sahay et al.

All the enzyme assays were conducted in triplicates and data was subjected to analysis of variance ANOVA using software SPSS ver. Genomic DNA of selected 51 strains was extracted by the method described earlier by Sahay et al. The amplification was phd thesis on xylanase out in a μL volume and amplification conditions were used as described earlier Sahay et al. The PCR amplified 16S rDNA were purified with a Quiaquick purification kit Qiagen.

PCR products of partial 16S rRNA gene were sequenced with fluorescent terminators Big Dye, Applied Biosystems and run in × phd thesis on xylanase Applied Biosystems ABI prism automated DNA sequencer at SCI Genome Chennai, India. The 16S rRNA gene sequences were aligned to those of closely related bacterial species available at GenBank database using BLASTn program.

The phylogenetic tree was constructed on the aligned datasets using the neighbour-joining method NJ implemented in the program MEGA 4. Bootstrap analysis was performed on random samples taken from the multiple phd thesis on xylanase. The partial 16S rRNA gene sequences of 51 strains were submitted to NCBI GenBank and Accession Numbers assigned were HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ phd thesis on xylanase Manikaran hot springs and HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ, HQ from Yumthang hot phd thesis on xylanase. All the 51 strains were deposited at National Bureau of Agriculturally Important Microorganisms NBAIM culture collection facility, phd thesis on xylanase, Mau, Uttar Pradesh, India.

The physico-chemical analysis showed specific variations among sediment and water samples collected from Manikaran and Yumthang hot springs. The samples from both springs were acidic in nature; however there was a wide variation in the temperatures of the two springs. In Manikaran, the maximum temperature was as high as 99 °C; while in Yumthang hot spring maximum temperature was 63 °C.

Total six culture media were employed to retrieve the culturable bacteria from both hot springs. A total bacteria were isolated from Manikaran and Yumthang hot springs. The variation was observed in bacterial species obtained from two thermal springs of distinct geographic locations. Similarly, in recent reports on natural extreme environments the culture-based approaches provided thermophiles, with highly significant for biotechnological applications Suman et al.

All isolates were screened for tolerance to range of temperatures and isolated were grouped into two category as thermophilic 60—90 °C and thermotolerant 37—60 °C. Maximum isolates were grown at 45 °C; while only 19 isolates NBM 19, NBM 48, NBM37, NBM31, NBM71, NBM75, NBM24, NBM38, NBM40, NBM49, NBY8, NBY23, NBY4, NBY33, NBY36, NBY16, NBY37, NBY28, and NBY57 showed growth at phd thesis on xylanase °C.

Similarly, in case of thermophilic Bacilli obtained from hot spring showed tolerance to high temperature 70—90 °C Lentini et al. In growth kinetic studies, the multiplication of isolates belongs to thermophilic group was observed after 12—24 h of inoculation, where as the remaining isolates from thermotolerant groups, cells growth and multiplication was observed after 20—24 h of inoculation, phd thesis on xylanase. Our results indicated that most of the isolates from both springs showed wide pH ranges i.

However, the isolates such as NBM24, NBM6, phd thesis on xylanase, NBY38, NBY4, NBY5, phd thesis on xylanase, NBY33, NBY36, NBY16, NBY26, NBY37, and NBY55, 11 isolates have showed a narrow pH range of 5—8.

Comparative studied has been done for each isolate for pH and temperature tolerances, it was observed that thermotolerant isolates with growth temperature range 45—65 °C showed the pH range between 3 and 9 Table 3. Extreme environments of high temperatures can be a source for novel species of microbes, as they can tolerate extremes of temperature stress. In recent years, several studies have been conducted to look for the diversity of microbes in extreme high temperatures environments, such as geothermal areas of Deception island, in the South Shetland Archipelago Llarch et al.

All the were examined for their ability to produce extracellular hydrolytic enzymes at different temperatures and it was found that protease, amylase, cellulase and xylanase were exhibited by 30, phd thesis on xylanase, 30, 23 and 18 strains respectively Table 3.

Among51 bacterial isolates phd thesis on xylanase one or more enzymes of protease, amylase, cellulase and xylanase Fig. Among 51 bacterial strains only, 4, 6, 5, and 3 produced protease, amylase, cellulase and xylanase respectively. Among selected isolates, only 5 9. The culturable bacterial isolates of both hot springs showed thermostable enzyme activities with wide range of pH variation Table 3.

Our present results were in support with the earlier study that demonstrated thermostable cellulase production at an optimum temperature of 50 °C by Brevibacillus thermoruber Derekova et al. Thermophilic microorganisms with the ability to produce proteases and amylases have also been reported from geothermal sites or thermal springs Kumar et al.

In the present investigation, strains capable of producing thermostable proteases and amylases were identified. The thermophilic bacteria produced thermo-active enzymes, which is of great interest for both fundamental research and industrial applications. Different thermo-active enzymes production by microbes, in this study may find applications in various industries viz.

food, detergents, pharmaceutical, biofuels, etc. a Diversity and distribution of 51 bacterial isolates for four different hydrolytic enzymes production at high temperature; b the Venn diagram illustrates the number of extracellular hydrolytic enzymes producing bacteria. Xylanases are the endoactive enzymes which are generally produced in the medium containing xylan and also containing xylanase hydrolysate as the carbon source and attack the xylan chain in a random manner, causing a decrease in degree of polymerization of the substrates and liberating shorter oligomers, xylobiose and xylose.

Cellulase free xylanase are of paramount significance in some of industries Adhyaru et al. Treatment with xylanase at elevated temperature disrupts the cell wall structure, facilitates lignin removal in the various stages of bleaching of paper, Therefore xylanase must lack cellulolytic activity. In present investigation, three thermotolerant bacteria namely Thermobacillus sp NBM6, Paenibacillus ehimensis NBM24 and Paenibacillus popilliae NBM68 were found to produced cellulase-free xylanase Fig.

There are phd thesis on xylanase report on cellulase-free xylanase production by thermophilic bacteria e, phd thesis on xylanase. Paenibacillus sp. N1 Pathania et al. Recently, interest in xylanase has markedly increased due its wide variety of biotechnological applications such as pre-bleaching of pulp, improving the digestibility of animal feed stocks, modification of cereal-based stuffs, bioconversion of lignocellulosic material and agro-wastes to fermentable products, clarification of fruit juices and degumming of plant fibers.

The phylogenetic trees were constructed to determine the affiliations for 28 and 23 bacteria isolated from thermal springs of Indian Himalayas Fig. Overall all identified bacteria belong to 37 distinct species of 14 different genera namely Anoxybacillus, Bacillus, Brevibacillus, Brevundimonas, phd thesis on xylanase, Burkholderia, Geobacillus, Paenibacillus, Planococcus, Pseudomonas, Rhodanobacter, Thermoactinomyces, Thermobacillus, Thermonema and Thiobacillus.

Phylogenetic analysis of thermo-active hydrolytic phd thesis on xylanase producing bacteria phd thesis on xylanase from a Manikaran hot springs; b Yumthang hot springs.

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