Dataset Information

Biomethane production from sugar beet pulp under cocultivation with Clostridium cellulovorans and methanogens.

ABSTRACT: This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH₄). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was identified and classified based on several computer programs. As a result, Methanosarcina mazei (1.34% of total counts) and the other methanogens were found in the MFMP. Interestingly, the simultaneous utilization of hydrogen (H₂) and carbon dioxide (CO₂) for methanogenesis was observed in the coculture with Consortium of C. cellulovorans with the MFMP (CCeM) including M. mazei. Furthermore, the CCeM degraded 87.3% of SBP without any pretreatment and produced 34.0 L of CH₄ per 1 kg of dry weight of SBP. Thus, a gas metabolic shift in the fermentation pattern of C. cellulovorans was observed in the CCeM coculture. These results indicated that degradation of agricultural wastes was able to be carried out simultaneously with CH₄ production by C. cellulovorans and the MFMP.

SUBMITTER: Tomita H

PROVIDER: S-EPMC6379495 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Biomethane production from sugar beet pulp under cocultivation with Clostridium cellulovorans and methanogens.

Tomita Hisao H Okazaki Fumiyoshi F Tamaru Yutaka Y

AMB Express 20190218 1

This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH<sub>4</sub>). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was ident ...[more]

PMID: 30778890

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Project description:BACKGROUND:Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. RESULTS:Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. CONCLUSIONS:This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.

Project description:Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing adhE1 and ctfA-ctfB-adc genes from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward n-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.IMPORTANCE Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer of n-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for n-butanol production from lignocellulose by CBP.

Project description:In nature, filamentous fungi are exposed to diverse nutritional sources and changes in substrate availability. Conversely, in submerged cultures, mycelia are continuously exposed to the existing substrates, which are depleted over time. Submerged cultures are the preferred choice for experimental setups in laboratory and industry and are often used for understanding the physiology of fungi. However, to what extent the cultivation method affects fungal physiology, with respect to utilization of natural substrates, has not been addressed in detail. Here, we compared the transcriptomic responses of Aspergillus niger grown in submerged culture and solid culture, both containing sugar beet pulp (SBP) as a carbon source. The results showed that expression of CAZy (Carbohydrate Active enZyme)-encoding and sugar catabolic genes in liquid SBP was time dependent. Moreover, additional components of SBP delayed the A. niger response to the degradation of pectin present in SBP. In addition, we demonstrated that liquid cultures induced wider transcriptome variability than solid cultures. Although there was a correlation regarding sugar metabolic gene expression patterns between liquid and solid cultures, it decreased in the case of CAZyme-encoding genes. In conclusion, the transcriptomic response of A. niger to SBP is influenced by the culturing method, limiting the value of liquid cultures for understanding the behavior of fungi in natural habitats. IMPORTANCE Understanding the interaction between filamentous fungi and their natural and biotechnological environments has been of great interest for the scientific community. Submerged cultures are preferred over solid cultures at a laboratory scale to study the natural response of fungi to different stimuli found in nature (e.g., carbon/nitrogen sources, pH). However, whether and to what extent submerged cultures introduce variation in the physiology of fungi during growth on plant biomass have not been studied in detail. In this study, we compared the transcriptomic responses of Aspergillus niger to growth on liquid and solid cultures containing sugar beet pulp (a by-product of the sugar industry) as a carbon source. We demonstrate that the transcriptomic response of A. niger was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment could not fully explain the behavior of the fungus in a solid environment. This could partially explain the differences often observed between the phenotypes on plates compared to liquid cultures.

Dataset Information

Biomethane production from sugar beet pulp under cocultivation with Clostridium cellulovorans and methanogens.

Publications

Biomethane production from sugar beet pulp under cocultivation with Clostridium cellulovorans and methanogens.

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