The composition, localization and function of low-temperature-adapted microbial communities involved in methanogenic degradations of cellulose and chitin from Qinghai-Tibetan Plateau wetland soils

Energy Science & Engineering

Yin

Wang

et al. 2019

The main objective of this study was to establish the metabolic pathway of a biogas fermentation system active at low temperature, and to describe the bacterial and archaeal methanogenic species responsible. A biogas fermentation inoculum adapted to be active at 9°C was used in a 10‐L batch‐type temperature‐controlled fermentor, using pig manure as the raw material. Fermentation at 9°C was followed for 120 days. A combination of nonbiological and biological factors was analyzed. Species were identified from OTU analysis based on 16S rDNA amplicon sequencing, and correlation analysis was used to study the effect of nonbiological factors, bacterial communities, and archaea communities. Biogas production was most effective between days 50 and 90, where gas production was above 0.70 L/d and the CH4 yield was above 0.36 L/g‐volatile solid. The relative abundance of the first dominant bacterial OTU with 99% identity to Clostridium cellulovorans varied between 16.17% and 27.30%. These bacteria typically degrade cellulose and hemicellulose. The second dominant bacterial OTU (relative abundance 15.23%‐27.15%) was 99% identical to Terrisporobacter petrolearius, a typical fermentative species. The most abundant (4.15%‐37.14%) archaeal OTU was 98% identical to Methanocorpusculum sinense, which is a typical hydrogenotrophic methanogen. A low‐temperature biogas metabolic pathway was constructed, based on abundant bacterial and methanogen OTUs. The most suitable metabolic pathway describes that hydrogen is produced by one part of the community and then used to reduce the produced carbon dioxide into CH4; this seems to be the major pathway by which biogas is produced under low‐temperature conditions.

Section: Discussionmentioning

confidence: 88%

Construction of biogas metabolic pathway in a low‐temperature biogas fermentation system

Energy Science & Engineering

Yin

Wang

et al. 2019

“…2); therefore, P1724 should belong to an anaerobe as well. So far, only few studies focused on chitin degradation in cold-area wetlands under anoxic conditions although anaerobic fermentation of chitin could be one of the important processes of nitrogen cycling in such environments [22,26,27]. Using the next-generation metagenomic method, we found this novel chitinases P1724 and avoided culturing the cold-favoring chitinolytic anaerobes, such as Paludicola psychrotolerans, which were normally hard to handle and very slow-growing [28].…”

Section: Discussionmentioning

confidence: 97%

“…After subcultured repeatedly for two years, the microbiome was collected by centrifugation and used for next-generation metagenomic sequencing. For more detailed information on sample processing, please refer to our previous publication [22].…”

Section: Microbial Consortium Enrichmentmentioning

confidence: 99%

“…Here we reported the first metagenome-sourced chitinase that contained two GH18 catalytic domains. In our previous study, we found the microbial communities from the Qinghai-Tibetan Plateau wetland soils could effectively convert chitin to methane at low temperatures [22]. We continued to anaerobically enrich the microbial community from Haiyan wetland soil with chitin flakes as a sole carbon resource, and then next-generation metagenomic sequencing was done on the community.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cloning, Purification, and Characterization of a Novel Metagenome-Sourced Microbial Chitinase with Dual Catalytic Domains

Dai

Liu

et al. 2020

Preprint

Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases. Results: A metagenomic approach was used to discover the chitinases from a wetland on the Qinghai-Tibetan plateau. Gene P1724 was found and predicted to have two GH18 catalytic domains. Gene sequence containing P1724 , only its N-terminal GH18 domain ( P1724nGH18 ) or only C-terminal GH18 domain ( P1724cGH18 ) were cloned and expressed in Escherichia coli BL21 (DE3). These purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C. The activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ . All three chitinases showed both chitotiosidase and endochitinase activities, and produced predominantly N, N΄-Diacetylchitobiose from colloid chitin. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity in temperature and pH stabilities, NaCl tolerance and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18’s, which indicated that two GH18 domains of P1724 works synergistically in degrading chitin. Conclusion: Compared to the most of microbial chitinases containing only one catalytic domain, chitinases P1724 with two GH18 catalytic domains was discovered firstly by the metagenomic approach. P1724 is a novel chitinase with unique amino acid sequences and hydrolytic mode, and could be used in cold environments or industries.

“…Here we reported the first metagenome-sourced chitinase that contained two GH18 catalytic domains. In our previous study, we found the microbial communities from the Qinghai-Tibetan Plateau wetland soils could effectively convert chitin to methane at low temperatures [25]. We continued to anaerobically enrich the microbial community from Haiyan wetland soil with chitin flakes as a sole carbon resource, and then next-generation metagenomic sequencing was done on the community.…”

Section: Introductionmentioning

confidence: 99%

The heterologous expression and characterization of a novel chitinase with dual catalytic domains discovered from a wetland soil metagenome

Dai

Liu

et al. 2020

Preprint

Background: Microbial chitinases have attracted a lot of attention because of their great potential in many applications. Metagenome-based approach obtains the target genes from the environment directly without culturing the microbes and is becoming a powerful tool to discover the novel chitinases.Results: Gene P1724 was found in a chitin-enriched microbial community from a wetland on the Qinghai-Tibetan plateau using the metagenomic approach. The translated protein sequence of P1724 was predicted to have two GH18 catalytic domains and showed very low similarities with any known and hypothetical chitinases. The gene sequence of P1724 , its N-terminal GH18 domain ( P1724nGH18 ), or C-terminal GH18 domain ( P1724cGH18 ) was cloned and expressed in Escherichia coli BL21 (DE3). Using colloid chitin as substrate, these purified recombinant chitinases showed maximum hydrolytic activities at 40 °C, pH 5.0-6.0 and 0-0.5 M NaCl, and were cold adaptive since they were still active at 4 °C; the activities of three chitinases were decreased with the presence of Cu 2+ and EDTA, but increased with Ba 2+ and Ca 3+ ; all three chitinases showed both chitotiosidase and endochitinase activities, and could hydrolyze chitosan as well. Other than these common characteristics, P1724 and P1724nGH18 shared more similarity in temperature and pH stabilities, NaCl tolerance and substrate affinity, suggesting the N-terminal GH18 domain contributed more than the C-terminal GH18 did in biochemical characteristics of P1724. k cat / K m value (catalytic efficiency) of P1724 was significantly higher than the sum values of P1724nGH18 and P1724cGH18, which indicated that two GH18 domains of P1724 worked cooperatively in degrading chitin.Conclusion: Compared to most microbial chitinases that contain only one catalytic domain, P1724 contains two and was firstly discovered by the metagenomic approach. P1724, its N-terminal and C-terminal catalytic domains were heterologously expressed and characterized. These three recombinant chitinases are phylogenetically distant to any chitinases studied so far, have unique hydrolytic mode, high catalytic efficiency and so new promising candidates for chitinases in applications. In addition, this study broadens the knowledge of unknown chitinases in nature and shows a natural strategy for enzyme engineering by adding other catalytic domain(s).