Yutaka Nakai

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Rumen fibrolytic microorganisms have been used to increase the rate of lignocellulosic biomass biodegradation; however, the microbial and isozymatic characteristics of biodegradation remain unclear. Therefore, the present study investigated the relationship between rumen microorganisms and fibrolytic isozymes associated with lignocellulosic biomass hydrolysis. Rice straw, a widely available agricultural byproduct, was ground and used as a substrate. The biodegradation of rice straw powder was performed anaerobically in rumen fluid for 48 h. The results obtained revealed that 31.6 and 23.3% of cellulose and hemicellulose, respectively, were degraded. The total concentration of volatile fatty acids showed a 1.8-fold increase (from 85.4 to 151.6‍ ‍mM) in 48 h, and 1,230.1‍ ‍mL L–1 of CO2 and 523.5‍ ‍mL L–1 of CH4 were produced. The major isozymes identified by zymograms during the first 12‍ ‍h were 51- and 140-kDa carboxymethyl cellulases (CMCases) and 23- and 57-kDa xylanases. The band densities of 37-, 53-, and 58-kDa CMCases and 38-, 44-, and 130-kDa xylanases increased from 24 to 36 h. A microbial ana­lysis indicated that the relative abundances of Prevotella, Fibrobacter, and Bacteroidales RF16 bacteria, Neocallimastix and Cyllamyces fungi, and Dasytricha and Polyplastron protozoa were related to fibrolytic isozyme activity. The present results provide novel insights into the relationships between fibrolytic isozymes and rumen microorganisms during lignocellulose biodegradation.

Summary: Treatment with rumen fluid improves methane production from non‐degradable lignocellulosic biomass during subsequent methane fermentation; however, the kinetics of xylanases during treatment with rumen fluid remain unclear. This study aimed to identify key xylanases contributing to xylan degradation and their individual activities during xylan treatment with bovine rumen microorganisms. Xylan was treated with bovine rumen fluid at 37°C for 48 h under anaerobic conditions. Total solids were degraded into volatile fatty acids and gases during the first 24 h. Zymography showed that xylanases of 24, 34, 85, 180, and 200 kDa were highly active during the first 24 h. Therefore, these xylanases are considered to be crucial for xylan degradation during treatment with rumen fluid. Metagenomic analysis revealed that the rumen microbial community’s structure and metabolic function temporally shifted during xylan biodegradation. Although statistical analyses did not reveal significantly positive correlations between xylanase activities and known xylanolytic bacterial genera, they positively correlated with protozoal (e.g., Entodinium , Diploplastron , and Eudiplodinium ) and fungal (e.g., Neocallimastix , Orpinomyces , and Olpidium ) genera and unclassified bacteria. Our findings suggest that rumen protozoa, fungi, and unclassified bacteria are associated with key xylanase activities, accelerating xylan biodegradation into volatile fatty acids and gases, during treatment of lignocellulosic biomass with rumen fluid.

Rumen microorganisms produce various fibrolytic enzymes and degrade lignocellulosic materials into nutrient sources for ruminants; therefore, the characterization of fibrolytic enzymes contributing to the polysaccharide degradation in the rumen microbiota is important for efficient animal production. This study characterized the fibrolytic isozyme activities of a rumen microbiota from four groups of housed cattle (1, breeding Japanese Black; 2, feedlot Japanese Black; 3, lactating Holstein Friesian; 4, dry Holstein Friesian). Rumen fluids in all cattle groups showed similar concentrations of total volatile fatty acids and reducing sugars, whereas acetic acid contents and pH were different among them. Predominant genera were commonly detected in all cattle, although the bacterial compositions were different among cattle groups. Zymograms of whole proteins in rumen fluids showed endoglucanase activities at 55 and 57 kDa and xylanase activity at 44 kDa in all cattle. Meanwhile, several fibrolytic isozyme activities differed among cattle groups and individuals. Treponema , Succinivibrio , Anaeroplasma , Succiniclasticum , Ruminococcus , and Butyrivibrio showed positive correlations with fibrolytic isozyme activities. Further, endoglucanase activity at 68 kDa was positively correlated with pH. This study suggests the characteristics of fibrolytic isozyme activities and their correlations with the rumen microbiota.

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances ofSelenomonas,Eudiplodinium, andMetadiniumdecreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances ofPorphyromonas,Didinium, unclassified Bacteroidetes,Clostridialesfamily XI,LachnospiraceaeandSphingobacteriaceaeincreased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.

The 2011 Tohoku tsunami had a serious impact, such as an increase in harmful substances and salinity over a large area. Herein, we evaluated transitions in bacterial communities in agricultural fields in the four years after the 2011 Tohoku tsunami. Bacterial communities were compared across four different types of soil—unflooded field (UF) soil, soil flooded for a short term (ST), soil flooded for the long term (LT), soil flooded long term and cultivated fields (LTC), and marine environmental materials (bay sediment, sea sand and sea water), using a polymerase chain reaction (PCR) and pyrosequencing of 16S ribosomal RNA genes. In the soil bacterial communities that were flooded by the 2011 Tohoku tsunami, these effects were not seen after 2013. Although the difference in bacterial communities between LT and UF became smaller during the four years, the bacterial communities in LT were different from those in UF in several ways, such as a higher tendency frequency of sulfur-oxidizing bacteria (SOB) and the presence of halotolerant SOB. Therefore, it is thought that the Tohoku tsunami affected the microbial communities in the soil for more than four years. Especially genus Halothiobacillus, which is Halotolerant SOB in flooded soils, was detected neither in unflooded soil nor in the marine environment. Therefore, it is thought that inundation by a tsunami produces a unique environment with bacterial communities to form in soil. Further, SOB structure, especially halotolerant, might serve as a good indicators of the impacts of inundation on bacterial communities in agricultural fields over the long term.

Rumen fluid has been applied to lignocellulosic biomass digest for methane production, and various feed supplements have been shown to improve ruminant digestion of lignocellulosic biomass. Therefore, we investigated the effects of sodium dodecyl sulfate (SDS) on the pretreatment of lignocellulosic biomass with rumen fluid and subsequent methane fermentation. SDS was mixed with rumen fluid at concentrations of 0.1, 0.2, 0.4, and 0.8 g/L. After SDS addition, the waste paper was pretreated with rumen fluid at 37 °C for 6 h. SDS addition decreased the number of surviving rumen ciliates after pretreatment. SDS addition increased the dissolved chemical oxygen demand during pretreatment; however, SDS addition did not increase the volatile fatty acid concentration. After pretreatment, batch methane fermentation of pretreated waste paper was performed at 35 °C for 45 days. SDS addition at 0.1 and 0.2 g/L shortened the waste paper digestion time and enhanced methane gas production compared to the control. By contrast, SDS addition at 0.4 and 0.8 g/L remarkably inhibited methane production from waste paper. These findings suggest that low concentrations of SDS can improve the efficiency of lignocellulosic biomass pretreatment with rumen fluid, and can enhance methane production from waste paper.

Sulfur-oxidizing bacterial diversity at the surface of cattle manure was characterized throughout the composting process using a sulfur oxidation gene (soxB) clone library approach. In the mesophilic phase, clones related to the genera Hydrogenophaga and Hydmgenophilus were characteristically detected. In the thermophilic phase, clones related to the genera Ilydrogenophaga and Thiohalobacter were predominant. In the cooling phase, the predominant soxB sequences were related to the genus Pseudaminobacter and a new sulfur-oxidizing bacterium belonging to the class Alphaproteobacteria. The present study showed changes in the community composition of sulfur-oxidizing bacteria at the surface of compost throughout the composting process.