Relationship Between Rumen Microbial Composition and Fibrolytic Isozyme Activity During the Biodegradation of Rice Straw Powder Using Rumen Fluid.

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 ana-lysis 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.

Shifts in xylanases and the microbial community associated with xylan biodegradation during treatment with rumen fluid.

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.

Characteristics of various fibrolytic isozyme activities in the rumen microbial communities of Japanese Black and Holstein Friesian cattle under different conditions.

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.

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

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.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using 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 of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased 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.

Response of the denitrifier community and its relationship with multiple N2O emission peaks after mature compost addition into dairy manure compost with forced aeration.

Animal manure is a source of the greenhouse gas nitrous oxide (N2O), therefore understanding the mechanisms underlying its production is essential for developing mitigating strategies and sustainable livestock production system. In this study, microbial communities potentially involved in multiple emission peaks during initial stage of laboratory-scale dairy manure composting with forced aeration system were investigated. Mature compost was used for the bulking agent. Change of overall bacterial community and nitrification-denitrification gene abundance were monitored by using 16S rRNA gene amoA, nirS, nirK or nosZ genes, respectively. Three N2O emission peaks were observed when the temperature reached at 45, 60 and 72 °C, at the same timing of oxygen consumption peaks. The maximum N2O emission peak was 3.86 mg h-1 kg-1 TS when the temperature reached at 60 °C. The shift of bacterial community among these experimental periods was significant, orders Flavobacteriales, Burkholderiales and Xanthomonadales increased, while orders belong to Bacillales, Lactobacillales, Clostridiales and Bacteroidales decreased. In addition, abundance of two denitrification genes (nirS and nosZ) significantly increased during this period. Clone library analysis of these genes showed that significantly increased sequences belonged to Pseudomonas-like clusters for both genes, indicates that denitrifiers possesses these genes are involved for these N2O emission peaks caused by mature compost addition.

Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of [Pasteurella] pneumotropica.

[Pasteurella] pneumotropica is a ubiquitous bacterium frequently isolated from laboratory rodents. Although this bacterium causes various diseases in immunosuppressed animals, little is known about major virulence factors and their roles in pathogenicity. To identify virulence factors, we sequenced the genome of [P.] pneumotropica biotype Heyl strain ATCC 12555, and compared the resulting non-contiguous draft genome sequence with the genome of biotype Jawetz strain ATCC 35149. Among a large number of genes encoding virulence-associated factors in both strains, four genes encoding for YadA-like proteins, which are known virulence factors that function in host cell adherence and invasion in many pathogens. In this study, we assessed YadA distribution and biological activity as an example of one of virulence-associated factor shared, with biotype Jawetz and Heyl. More than half of mouse isolates were found to have at least one of these genes; whereas, the majority of rat isolates did not. Autoagglutination activity, and ability to bind to mouse collagen type IV and mouse fibroblast cells, was significantly higher in YadA-positive than YadA-negative strains. To conclude, we identified a large number of candidate genes predicted to influence [P.] pneumotropica pathogenesis.

Complete Genome Sequence of the Unclassified Iron-Oxidizing, Chemolithoautotrophic Burkholderiales Bacterium GJ-E10, Isolated from an Acidic River.

Burkholderiales bacterium GJ-E10, isolated from the Tamagawa River in Akita Prefecture, Japan, is an unclassified, iron-oxidizing chemolithoautotrophic bacterium. Its single circular genome, consisting of 3,276,549 bp, was sequenced by using three types of next-generation sequencers and the sequences were then confirmed by PCR-based Sanger sequencing.

Draft Genome Sequence of the Rodent Opportunistic Pathogen Pasteurella pneumotropica ATCC 35149T.

Pasteurella pneumotropica is an opportunistic pathogen in rodents that is commonly isolated from upper respiratory tracts in laboratory rodents. Here, we report the draft genome sequence of the P. pneumotropica type strain ATCC 35149, which was first isolated and characterized as biotype Jawetz.

Seawater inundation from the 2011 Tohoku tsunami continues to strongly affect soil bacterial communities 1 year later.

The effects of inundation caused by the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields were evaluated. Bacterial communities were compared across three different types of soil, unflooded field (UF) soil, soil flooded for 2 weeks (short term (ST)), and soil flooded for 2 months (long term (LT)), using polymerase chain reaction-pyrosequencing of 16S rRNA genes. Acidobacteria were dominant in UF, with a relative abundance of approximately 35 %, and Proteobacteria dominated flooded soils (30-67 %). Hierarchical cluster analysis indicated that the community structure of soil bacteria in flooded soils (ST and LT) clearly differed from that in UF. Differences between LT and ST fields were rarely observed in terms of chemical properties and microbial community structure at the phylum level. However, sulfur-oxidizing bacteria (SOB) and nitrite-oxidizing bacteria (NOB) in LT tended to occur at high and low abundances, respectively. Halothiobacillus, a halotolerant SOB, was detected in all LT fields. Unexpectedly, a zeta-Proteobacteria, which had previously only been detected in marine environments, was detected in LT fields only. Our results demonstrate that the effects of the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields may have lasted at least 1 year. Furthermore, SOB, NOB, and zeta-Proteobacteria may serve as indicators of the effects of seawater inundation on microorganisms.

Middle-thermophilic sulfur-oxidizing bacteria Thiomonas sp. RAN5 strain for hydrogen sulfide removal.

Hydrogen sulfide (H2S) is one of the most toxic and offensively odorous gases and is generated in anaerobic bioreactors. A middle-thermophilic sulfur-oxidizing bacterium (SOB), Thiomonas sp. strain RAN5, was isolated and applied for H2S removal from both artificial and anaerobically digested gas. When a bioreactor containing medium inoculated with RAN5 was aerated continuously with artificial gas (containing 100 ppm H2S) at 45 degrees C for 156 hr, the H2S concentration in the vented gas was reduced by 99%. This was not affected by the presence of other microbes in the bioreactor The H2S removal efficiency of the RAN5 bioreactor for anaerobically digested gas was greater than 99% at influent H2S concentrations ranging from 2 to 1800 ppm; the efficiency decreased to 90% at influent H2S concentrations greater than 2000 ppm. Thiomonas sp. strain RAN5 cannot survive at room temperature, and thus its leakage from a wastewater treatment plant would not damage sewage systems. These data suggest that Thiomonas sp. strain RAN5 may be a useful microorganism for H2S removal.

Growth of ammonia-oxidizing archaea and bacteria in cattle manure compost under various temperatures and ammonia concentrations.

A recent study showed that ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) coexist in the process of cattle manure composting. To investigate their physiological characteristics, liquid cultures seeded with fermenting cattle manure compost were incubated at various temperatures (37°C, 46°C, or 60°C) and ammonium concentrations (0.5, 1, 4, or 10 mM NH (4) (+) -N). The growth rates of the AOB and AOA were monitored using real-time polymerase chain reaction analysis targeting the bacterial and archaeal ammonia monooxygenase subunit A genes. AOB grew at 37°C and 4 or 10 mM NH (4) (+) -N, whereas AOA grew at 46°C and 10 mM NH (4) (+) -N. Incubation with allylthiourea indicated that the AOB and AOA grew by oxidizing ammonia. Denaturing gradient gel electrophoresis and subsequent sequencing analyses revealed that a bacterium related to Nitrosomonas halophila and an archaeon related to Candidatus Nitrososphaera gargensis were the predominant AOB and AOA, respectively, in the seed compost and in cultures after incubation. This is the first report to demonstrate that the predominant AOA in cattle manure compost can grow and can probably oxidize ammonia under moderately thermophilic conditions.

Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction.

Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.

Archaeal community dynamics and detection of ammonia-oxidizing archaea during composting of cattle manure using culture-independent DNA analysis.

The composting process is carried out under aerobic conditions involving bacteria, archaea, and fungi. Little is known about the diversity of archaeal community in compost, although they may play an important role in methane production and ammonia oxidation. In the present study, archaeal community dynamics during cattle manure composting were analyzed using a clone library of the archaeal 16S rRNA gene. The results indicated that methane-producing archaea (methanogen) and ammonia-oxidizing archaea (AOA) may be the dominant microbes throughout the composting. The community consisted primarily of Methanocorpusculum-like and Methanosarcina-like sequences until day 2, while the number of Candidatus Nitrososphaera-like sequences increased from day 6 to day 30. Methanosarcina thermophila-like sequences were dominant from day 2, suggesting that M. thermophila-like species can adapt to increasing temperature or nutrient loss. A denaturant gradient gel electrophoresis analysis of the archaeal amoA genes revealed that the dominant amoA gene sequence with 99% homology to that of Candidatus Nitrososphaera gargensis was identical to those obtained from a different composting facility. These data suggested that AOA may play a role in ammonia oxidation in several composting practices. Our results provide fundamental information regarding archaeal community dynamics that will help in understanding the collective microbial community in compost.

Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure.

An acidulocomposting system for the treatment of cattle manure with little emission of ammonia gas was developed, and the structure of its microbial community was investigated by denaturing gradient gel electrophoresis (DGGE) and clone library construction. An acidulocomposting apparatus (BC20, 20 L) was operated for 79 days to treat 2 kg (wet wt) of garbage per 1 or 2 days. On day 80 of operation, the substrate was changed from garbage to cattle manure (1 kg of beef cattle manure was added to the apparatus every 2 or 3 days), and the system continued operating from days 80 to 158. The compost in the vessel was under acidic conditions at pH 5.2-5.8, and ammonia emission was below the detectable level (<5 ppm) throughout the period of cattle manure feeding. Total nitrogen and total carbon in the compost were 26-29 and 439-466 mg/g of dry weight, respectively, which are higher than those in general cattle manure compost. The main acids accumulated during operation were lactic and acetic. Sequencing analysis targeting the 16S rRNA gene revealed the stable dominance of the bacterial phylum Firmicutes, with a high proportion of the isolates belonging to the genus Bacillus. Using a culturing method with MRS agar, we isolated lactic acid bacteria (LAB) related to Pediococcus acidilactici, Weissella paramesenteroides, and Lactobacillus salivarius, indicating the existence of LAB in the system. These results indicate that acidulocomposting treatment of cattle manure is not accompanied by ammonia emission and that Bacillus and LAB may be the key components in the system.

Bacterial populations in epilithic biofilms along two oligotrophic rivers in the Tohoku region in Japan.

Bacterial populations in epilithic biofilms collected from two distinct oligotrophic rivers of Japan were studied using denaturing gradient gel electrophoresis (DGGE). PCR-DGGE of the 16S rRNA gene and subsequent sequencing analysis suggested that in freshwater biofilms, members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group were the most dominant, followed by those of alpha, beta, gamma, and delta-Proteobacteria; Leptospiraceae; and unidentified bacteria. Members of the CFB group, alpha-Proteobacteria, and cyanobacteria/plastid DNA were also detected from the biofilms collected from the estuary site, but the species in these samples differed from those detected in biofilms in the freshwater areas of the rivers. A comparison between the determined sequences revealed that similar bacterial species existed in biofilms at different sites of a river, and identical species existed in biofilms of distinct rivers. The results suggested that bacterial species in biofilms found in the estuary were different from those found in the freshwater areas of the rivers; however, the common bacterial species were distributed in biofilms collected from not only different sites along the same river but also sites in distinct oligotrophic rivers.

Reduction in excess sludge production in a dairy wastewater treatment plant via nozzle-cavitation treatment: case study of an on-farm wastewater treatment plant.

Nozzle-cavitation treatment was used to reduce excess sludge production in a dairy wastewater treatment plant. During the 450-d pilot-scale membrane bioreactor (MBR) operation, when 300 l of the sludge mixed liquor (1/10 of the MBR volume) was disintegrated per day by the nozzle-cavitation treatment with the addition of sodium hydrate (final concentration: 0.01% W/W) and returned to the MBR, the amount of excess sludge produced was reduced by 80% compared with that when sludge was not disintegrated. On the basis of the efficiency of CODCr removal and the ammonia oxidation reaction, it was concluded that the nozzle-cavitation treatment did not have a negative impact on the performance of the MBR. The estimation of the inorganic material balance showed that when the mass of the excess sludge was decreased, the inorganic content of the activated sludge increased and some part of the inorganic material was simultaneously solubilized in the effluent.

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination.

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.

Molecular analysis of ammonia-oxidizing bacteria community in intermittent aeration sequencing batch reactors used for animal wastewater treatment.

Bacterial communities and betaproteobacterial ammonia-oxidizing bacteria (AOB) communities were evaluated seasonally in an intermittent-aeration sequencing batch process (SBR, plant A) and in 12 other livestock wastewater treatment plants (WWTP): eight SBRs and four conventional activated-sludge systems. Microbial communities were analysed by reverse transcription polymerase chain reaction followed by denaturing-gradient gel electrophoresis (DGGE) and the construction of clone libraries for 16S rRNA and ammonia monooxygenase (amoA) genes. In plant A, the dominant bacteria were as-yet-uncultured bacteria of Bacteroidetes and Proteobacteria, and the DGGE profiles showed that the bacterial communities were stable during a given treatment cycle, but changed seasonally. In betaproteobacterial AOB communities, two AOB phylotypes (members of the Nitrosomonas ureae-oligotropha-marina cluster) were dominant during the seasons in plant A. Although the dominant AOB phylotypes differed among the 13 WWTPs, dominance by one or two AOB phylotypes was commonly observed in all plants. Sequencing of the DGGE bands indicated that amoA sequences belonging to the Nitrosomonas europaea-eutropha cluster were dominant in 11 plants, where the ammonia-nitrogen concentration was high in the raw wastewater, whereas those belonging to the Nitrosomonas ureae-oligotropha-marina cluster were dominant in two plants where the concentration was relatively low. Even though we detected many minor amoA sequences by means of five clone libraries for the A to D plants, no libraries comprised both amoA sequences belonging to the two clusters, indicating that the dominant AOBs were defined by cluster level in each plant.