Cellulolytic Bacteria Screened from Qinling (China) for Biomass Degradation and Cellulases First Cloned from Bacillus methylotrophicus

Abstract Background Cellulosic biomass degradation still needed more studies while bioenergy is becoming mainly energy in future and more evaluate bacteria isolation laid a foundation of further study. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. The aim of this work was to find potential cellulolytic bacteria in depredating multiform carbon source cellulose substrate. Results In this study, 55 potential cellulolytic bacteria screened out and were identified. Based on the results of the investigation of cellulase activities and reducing sugar content via different carbon substrate effect, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further taken to hydrolyze wheat straw, corn stover and switchgrass, suggesting that B. methylotrophicus 1EJ7 was the most preponderant bacterium, obtaining highest sugar content (95mg/100mL) in switchgrass, wheat straw and corn stover. Scanning electron microscopy (SEM) and X-ray diffraction results of wheat straw surface and crystallinity indicated the hydrolyzation. By ascertaining the target sequence of cellulase for the cloning and expression in an economical and convenient manner, the genes of β-glucosidase (243 aa) and endoglucanase (499 aa) of B. methylotrophicus 1EJ7. Recombinant β-glucosidase from GH16 family and enzyme activity was 1670.15±18.94 U/mL. Endoglucanase consist of GH5 family catalytic domain and a carbohydrate-binding module belongs to CBM3 family and enzyme activity was 0.130±0.002 U/mL. Conclusions Screened, identified the cellulolytic bacteria from rotten wood of Qinling Mountains and explored their ability in degrading different carbon source cellulose substrate, including purified and natural carbon sources. Bacillus were the predominant species among the isolated strains, and Bacillus methylotrophicus 1EJ7 performant well on cellulose degradation. In the meantime, the β-glucosidase and endoglucanase were successfully cloned and expressed from Bacillus methylotrophicus for the first time. The strain and the recombinant enzyme have potential application in industrial production.

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Screening of Cellulolytic Bacteria from Rotten Wood of Qinling (China) for Biomass Degradation and Cloning of Cellulases from Bacillus methylotrophicus

10.21203/rs.2.14767/v3 ◽

2019 ◽

Author(s):

Lingling Ma ◽

Yingying Lu ◽

Hong Yan ◽

Xin Wang ◽

Yanglei Yi ◽

...

Keyword(s):

Bacillus Subtilis ◽

Wheat Straw ◽

Cellulose Degradation ◽

Good Effect ◽

Qinling Mountains ◽

Cellulolytic Bacteria ◽

Biomass Degradation ◽

Bacillus Methylotrophicus ◽

Rotten Wood ◽

Degradation Effect

Abstract Background Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. Results In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15±18.94 U/mL and 0.130±0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Conclusions Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

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Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus

BMC Biotechnology ◽

10.1186/s12896-019-0593-8 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 3

Author(s):

Lingling Ma ◽

Yingying Lu ◽

Hong Yan ◽

Xin Wang ◽

Yanglei Yi ◽

...

Keyword(s):

Bacillus Subtilis ◽

Wheat Straw ◽

Cellulose Degradation ◽

Good Effect ◽

Qinling Mountains ◽

Cellulolytic Bacteria ◽

Biomass Degradation ◽

Bacillus Methylotrophicus ◽

Rotten Wood ◽

Degradation Effect

Abstract Background Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. Results In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15 ± 18.94 U/mL and 0.130 ± 0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Conclusions Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and B. methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from B. methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

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Screening of Cellulolytic Bacteria from Rotten Wood of Qinling (China) for Biomass Degradation and Cloning of Cellulases from Bacillus methylotrophicus

10.21203/rs.2.14767/v2 ◽

2019 ◽

Author(s):

Lingling Ma ◽

Yingying Lu ◽

Hong Yan ◽

Xin Wang ◽

Yanglei Yi ◽

...

Keyword(s):

Bacillus Subtilis ◽

Wheat Straw ◽

Cellulose Degradation ◽

Good Effect ◽

Qinling Mountains ◽

Cellulolytic Bacteria ◽

Biomass Degradation ◽

Bacillus Methylotrophicus ◽

Rotten Wood ◽

Degradation Effect

Abstract Background Cellulosic biomass degradation still needs to be paid more attentions as bioenergy is the most likely to replace fossil energy in the future, and more evaluable cellulolytic bacteria isolation will lay a foundation for this filed. Qinling Mountains have unique biodiversity, acting as promising source of cellulose-degrading bacteria exhibiting noteworthy properties. Therefore, the aim of this work was to find potential cellulolytic bacteria and verify the possibility of the cloning of cellulases from the selected powerful bacteria. Results In present study, 55 potential cellulolytic bacteria were screened and identified from the rotten wood of Qinling Mountains. Based on the investigation of cellulase activities and degradation effect on different cellulose substrates, Bacillus methylotrophicus 1EJ7, Bacillus subtilis 1AJ3 and Bacillus subtilis 3BJ4 were further applied to hydrolyze wheat straw, corn stover and switchgrass, and the results suggested that B. methylotrophicus 1EJ7 was the most preponderant bacterium, and which also indicated that Bacillus was the main cellulolytic bacteria in rotten wood. Furthermore, scanning electron microscopy (SEM) and X-ray diffraction analysis of micromorphology and crystallinity of wheat straw also verified the significant hydrolyzation. With ascertaining the target sequence of cellulase β-glucosidase (243 aa) and endoglucanase (499 aa) were successfully heterogeneously cloned and expressed from B. methylotrophicus 1EJ7, and which performed a good effect on cellulose degradation with enzyme activity of 1670.15±18.94 U/mL and 0.130±0.002 U/mL, respectively. In addition, based on analysis of amino acid sequence, it found that β-glucosidase were belonged to GH16 family, and endoglucanase was composed of GH5 family catalytic domain and a carbohydrate-binding module of CBM3 family. Conclusions Based on the screening, identification and cellulose degradation effect evaluation of cellulolytic bacteria from rotten wood of Qinling Mountains, it found that Bacillus were the predominant species among the isolated strains, and Bacillus methylotrophicus 1EJ7 performed best on cellulose degradation. Meanwhile, the β-glucosidase and endoglucanase were successfully cloned and expressed from Bacillus methylotrophicus for the first time, which provided new materials of both strain and the recombinant enzymes for the study of cellulose degradation and its application in industry.

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Produksi Enzim Selulase Kasar dari Isolat Bakteri B2S8 menggunakan Substrat Brangkasan Jagung dengan Perlakuan Konsentrasi Inokulum dan Komposisi Media yang berbeda

JURNAL REKAYASA DAN MANAJEMEN AGROINDUSTRI ◽

10.24843/jrma.2020.v08.i02.p11 ◽

2020 ◽

Vol 8 (2) ◽

pp. 267

Author(s):

Nursatria Purba ◽

Ida Bagus Wayan Gunam ◽

I Made Mahaputra Wijaya

Keyword(s):

Enzyme Activity ◽

Protein Content ◽

Corn Stover ◽

Bacterial Isolate ◽

Specific Activity ◽

Block Design ◽

Cellulolytic Bacteria ◽

Endoglucanase Activity ◽

Cellulase Enzyme ◽

The Media

The purpose of this study was determined the media and concentration of cellulolytic bacterial isolates to produce high cellulase enzyme activity. Production of crude cellulase enzyme in media and concentration of different bacterial isolate used a factorial Randomized Block Design (RBD) which consist of two factors. The first factor was the media production of different cellulase enzyme consisting of 3 levels, namely media 1, 2 and 3. The second factor was the concentration of bacterial isolate consisting of 5 levels namely 1, 2, 3, 4 and 5%. This study used a B2S8 cellulolytic bacterial isolate that has the highest value of cellulase enzyme activity and the highest degradation rate of cellulose in previous studied and determined the ability of exoglucanase enzyme activity, endoglucanase enzyme and dissolved protein content produced from cellulolytic bacterial isolate. This study used Carboxymethyl Cellulose (CMC) for enzyme activity test and 1% corn stover as a substrate on the media to produce crude cellulase enzyme. The result showed that the highest cellulase enzyme activity in the third media and 5% cellulolytic bacterial inoculum concentration resulted in endoglucanase activity of 0.0332 IU/mL, exoglucanases enzyme activity of 0.0060 IU/mL, dissolved protein content in the amount of 0.5670 mg/mL, the specific endoglucanase activity of 0.0807 IU/mg and the specific activity of exoglucanase of 0.0123 IU/mg. Keywords: Cellulolytic bacteria, Cellulase enzymes, Enzyme activity, Corn stover

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Effects of Bacillus subtilis on growth, digestive enzyme activity, and non-specific immunity in hybrid sturgeon (Acipenser baeri ♂×Acipenser schrenkii ♀) juveniles

Journal of Fishery Sciences of China ◽

10.3724/sp.j.1118.2011.01315 ◽

2013 ◽

Vol 18 (6) ◽

pp. 1315-1320 ◽

Cited By ~ 2

Author(s):

Xiaoyong LIU ◽

Ying ZHANG ◽

Qian QI ◽

Mingjun ZHAO ◽

Likai MAI ◽

...

Keyword(s):

Enzyme Activity ◽

Bacillus Subtilis ◽

Digestive Enzyme ◽

Digestive Enzyme Activity ◽

Acipenser Baeri ◽

Specific Immunity ◽

Hybrid Sturgeon

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Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Molecules ◽

10.3390/molecules26092527 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2527

Author(s):

Zahra Azzouz ◽

Azzeddine Bettache ◽

Nawel Boucherba ◽

Alicia Prieto ◽

Maria Jesus Martinez ◽

...

Keyword(s):

Aspergillus Niger ◽

Wheat Straw ◽

Plant Biomass ◽

Residual Activity ◽

Exchange Chromatography ◽

Glycosyl Hydrolases ◽

Biomass Degradation ◽

Xylan Hydrolysis ◽

Rsm Optimization ◽

Single Carbon Source

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL−1 and Vmax = 5.647 U·mg−1). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

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Torrefaction of Woody and Agricultural Biomass: Influence of the Presence of Water Vapor in the Gaseous Atmosphere

Processes ◽

10.3390/pr9010030 ◽

2020 ◽

Vol 9 (1) ◽

pp. 30

Author(s):

María González Martínez ◽

Estéban Hélias ◽

Gilles Ratel ◽

Sébastien Thiéry ◽

Thierry Melkior

Keyword(s):

Water Vapor ◽

Water Content ◽

Wheat Straw ◽

Beech Wood ◽

High Water ◽

High Water Content ◽

Biomass Degradation ◽

Moist Atmosphere ◽

Thermochemical Transformation ◽

Degradation Reactions

Biomass preheating in torrefaction at an industrial scale is possible through a direct contact with the hot gases released. However, their high water-content implies introducing moisture (around 20% v/v) in the torrefaction atmosphere, which may impact biomass thermochemical transformation. In this work, this situation was investigated for wheat straw, beech wood and pine forest residue in torrefaction in two complementary experimental devices. Firstly, experiments in chemical regime carried out in a thermogravimetric analyzer (TGA) showed that biomass degradation started from lower temperatures and was faster under a moist atmosphere (20% v/v water content) for all biomass samples. This suggests that moisture might promote biomass components’ degradation reactions from lower temperatures than those observed under a dry atmosphere. Furthermore, biomass inorganic composition might play a role in the extent of biomass degradation in torrefaction in the presence of moisture. Secondly, torrefaction experiments on a lab-scale device made possible to assess the influence of temperature and residence time under dry and 100% moist atmosphere. In this case, the difference in solid mass loss between dry and moist torrefaction was only significant for wheat straw. Globally, an effect of water vapor on biomass transformation through torrefaction was observed (maximum 10%db), which appeared to be dependent on the biomass type and composition.

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Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO

Green Chemistry ◽

10.1039/c4gc02179g ◽

2015 ◽

Vol 17 (5) ◽

pp. 2896-2903 ◽

Cited By ~ 53

Author(s):

Ursula Fabiola Rodríguez-Zúñiga ◽

David Cannella ◽

Roberto de Campos Giordano ◽

Raquel de Lima Camargo Giordano ◽

Henning Jørgensen ◽

...

Keyword(s):

Wheat Straw ◽

Corn Stover ◽

Sugarcane Bagasse ◽

Cellulosic Ethanol ◽

Cellulose Oxidation ◽

Available Resources

Sugarcane bagasse, corn stover, and wheat straw are among the most available resources for the production of cellulosic ethanol.

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Effect of temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37

Journal of Science Natural Science ◽

10.18173/2354-1059.2021-0009 ◽

2021 ◽

Vol 66 (1) ◽

pp. 72-79

Author(s):

Thuoc Doan Van ◽

Hung Nguyen Phuc

Keyword(s):

Enzyme Activity ◽

Bacillus Subtilis ◽

Animal Feed ◽

Culture Conditions ◽

Effect Of Temperature ◽

Physical Parameters ◽

Original Activity ◽

Optimum Ph ◽

Production Activity ◽

Ph Range

The effect of physical parameters such as temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37 was investigated. The results indicated that the optimum culture conditions for enzyme activity were pH 7.0 and 35 oC. The optimum pH and temperature for enzyme activity were 6.0 and 70 oC. The crude enzyme was found to be stable in the pH range of 5.0 to 7.0. The enzyme was stable for 1 h at a temperature from 30 to 80 oC; nearly 100% of enzyme activity remained at temperatures of 30 - 40 oC, and about 34% of original activity remained at a temperature of 80 oC. These features demonstrated that α-amylase from B. subtilis V37 can be applied in many areas such as the food, fermentation, and animal feed industries.

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