Identification of bacteria involved in the decomposition of lignocellulosic biomass treated with cow rumen fluid by metagenomic analysis

https://doi.org/10.1016/j.jbiosc.2020.03.010Get rights and content

We had developed a new pretreatment system using cow rumen fluid to improve the methane production from lignocellulosic substrates. However, the pretreatment conditions differ from the in-situ rumen environment, therefore different microbes may be involved in plant cell wall decomposition. In the current study, shotgun metagenomic analysis using MiSeq platform was performed to elucidate the bacteria which produce cellulase and hemicellulase in this pretreatment system. The rumen fluid which contained waste paper pieces (0.1% w/v) were incubated at 37°C during 120 h. The fluid samples were collected from the reactor at each time-point and analyzed for chemical properties. Rumen microbial DNA was extracted from 0-h and 60-h samples and subjected to shotgun-metagenomic analysis. After pretreatment, approximately half of cellulose and hemicellulose contents of the waste paper were decomposed and some volatile fatty acids were accumulated. Clostridia (e.g., Ruminococcus and Clostridium) were the predominant bacteria before and after 60-h pretreatment, and their relative abundance was increased during pretreatment. However, Prevotella and Fibrobacter, one of the most dominant bacteria in-situ rumen fluid, were observed less than 3% before incubation and they were decreased after pretreatment. Genes encoding cellulase and hemicellulase were mainly found in Ruminococcus, Clostridium, and Caldicellulosiruptor. Calicellulosiruptor, which had not been previously identified as the predominant genus in lignocellulose decomposition in in-situ rumen conditions, might be considered as the main fibrolytic bacterium in this system. Thus, this study demonstrated that the composition of fibrolytic bacteria in this system was greatly different from those in the in-situ rumen.

Section snippets

Pretreatment of wastepaper using rumen fluid

The treatment condition was accordance with the method described by Baba et al. (12). Rumen contents were collected orally from grass-fed cow and filtered with a mesh strainer (1 mm × 1 mm) to remove coarse solids. The waste paper (0.3 g, 35 mm long × 25 mm wide) were soaked in 300 ml of rumen fluid containing 0.3 g of l-cysteine as a reducing agent for anaerobic rumen bacteria growth. Headspace gas in the bottle was then replaced with nitrogen gas. They were incubated at 37°C on a rotary

Pretreatment of lignocellulosic biomass using rumen fluid

Approximately half of the cellulose, hemicellulose, and lignin in the wastepaper reaction were decomposed after 60 h of pretreatment with rumen fluid (Table 1). The decomposition of substrates increased dCOD and two of the three VFAs, namely acetic acid and butyric acid, leading to a lowered pH (Fig. 1). VFA concentrations and dCOD increased continuously during pretreatment up to 120 h. Our previous study showed that half of the paper decomposed during 24 h to pretreatment. However, the results

Discussion

In this study, we conducted a metagenomic analysis to reveal the microbes involved in lignocellulose decomposition in the pretreatment system using cow rumen fluid. The system was not pH controlling, since it was not supplied with saliva, and the VFAs from lignocellulose were accumulated. However, the chemical properties of this system, including pH and VFAs content, were similar with to that in in-situ rumen condition (24). Plant cell wall degradation process in-situ rumen environment was

Acknowledgments

We thank Mr. Kazuya Sato and Ms. Rie Yamamoto of Field Science Center, Tohoku University, for their technical support during the collection of cow rumen contents. We would like to thank Editage for English language editing. This work was supported by the Next-generation Energies for Tohoku Recovery (NET) project.

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    Present address: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184–8588, Japan.

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    Present address: Japan Collection of Microorganisms, RIKEN BioResource Research Center, Tsukuba, Ibaraki 305-0074, Japan.

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    Present address: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Suematsu, Nonoichi, Ishikawa 921–8836, Japan.

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    Present address: Faculty of Agro-Food Science, Niigata Agro-Food University, Tainai, Niigata 959–2702, Japan.

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