Relationship between the vertical distribution of fine roots and residual soil nitrogen along a gradient of hardwood mixture in a conifer plantation.

In forest ecosystems, understanding the relationship between the vertical distribution of fine roots and residual soil nitrogen is essential for clarifying the diversity-productivity-water purification relationship. Vertical distributions of fine-root biomass (FRB) and concentrations of nitrate-nitrogen (NO3 -N) in soil water were investigated in a conifer plantation with three thinning intensities (Control, Weak and Intensive), in which hardwood abundance and diversity were low, moderate and high, respectively. Intensive thinning led to the lowest NO3 -N concentration in soil water at all depths (0-100 cm) and highest FRB at shallow depths (0-50 cm). The NO3 -N concentration at a given depth was negatively correlated with total FRB from the surface to the depth at which NO3 -N concentration was measured, especially at shallow depths, indicating that more abundant fine roots led to lower levels of downward NO3 -N leaching. FRB contributed positively to nitrogen content of hardwood leaves. These findings demonstrate that a hardwood mixture in conifer plantations resulted in sufficient uptake of NO3 -N from soil by well developed fine-root systems, and translocation to canopy foliage. This study suggests that productivity and water purification can be achieved through a hardwood mixture in conifer plantations.

Functional Group Distribution of the Carrier Surface Influences Adhesion of Methanothermobacter thermautotrophicus.

Various support carriers are used for high-density retention of methanogenic archaea in anaerobic wastewater treatment systems. Although the physicochemical properties of carrier materials and microorganisms influence the adhesion of methanogenic archaea, details about the underlying mechanism remain poorly characterized. We applied seven types of chemical surface modifications to carbon felts to clarify the adhesion properties of Methanothermobacter thermautotrophicus, a representative thermophilic hydrogenotrophic methanogen. The relationship between carrier surface properties and methanogen adhesion was evaluated. M. thermautotrophicus adhesion was significantly increased up to 2.6 times in comparison with control on carbon felts treated with NaOH, HCl, H2SO4, or Na2HPO4. Treated carbon felts showed a lower water contact angle, but no correlation between the carrier surface contact angle and methanogen adhesion was observed. On the other hand, at the surface of the carrier that showed improved adhesion of methanogens, the ratio of -COOH : -OH was 1 : 0.65. Such a ratio was not observed with treated carriers for which methanogen adhesion was not improved. Therefore, in the adhesion of M. thermautotrophicus, the functional group abundance was important as well as physical surface properties such as the hydrophobicity. Hydrogenotrophic methanogens are involved in active methanation during the startup of anaerobic digestion. Additionally, these methanogenic archaea function as methanogenic cathode catalysts. Therefore, anaerobic digestion performance will greatly improve by controlling the adhesion of hydrogenotrophic methanogens such as M. thermautotrophicus.

Prevalence and first genotyping of Giardia duodenalis in beef calves in Vietnam.

Little information is available on the epidemiology of Giardia duodenalis in beef cattle from Vietnam. This study was performed to determine the prevalence and genotypes/assemblages of G. duodenalis in native beef calves younger than 6 months in the region. A total of 412 calf fecal samples, randomly selected from 99 small-scale farms located in DacLac and KhanhHoa provinces, central Vietnam, were screened for the presence of G. duodenalis cysts using the zinc-sulfate flotation method followed by iodine staining. The overall prevalence on the sample and herd levels were 13.8% (57/412) and 42.4% (42/99), respectively. Molecular analysis in the ß-giardin and triosephosphate isomerase genes demonstrated the presence of only G. duodenalis assemblage E in the animals. Since assemblage E has been rarely reported in humans, the zoonotic risk in beef calves in the region appears to be minimal.

Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila.

Heat shock protein 70 (Hsp70) is a member of a family of conserved chaperone proteins whose function is well investigated in many model organisms. Here we focus on an Hsp70 called Ssa5 in the ciliate protozoan Tetrahymena thermophila, and reveal that its translation is heat inducible as for general Hsps. Moreover, the protein is abundantly expressed in the cytoplasm during sexual reproduction (conjugation) as well as in response to heat-stress. Knocking out of SSA5 (ΔSSA5) does not affect the survival of the cell under heat-stress, likely due to other Hsp70 paralogs compensating for the defect. During conjugation, ΔSSA5 leads to a fertilization defect in which the two pronuclei are in close proximity but never fuse. The unfertilized pronuclei differentiate, resulting in a heterokaryon with developed haploid germline and somatic nuclei. In addition, degeneration of the parental somatic nucleus is not affected. These results suggest a specific involvement of Ssa5 in pronuclear fusion and fertilization.

Circular metagenome-assembled genome of Candidatus Cloacimonadota recovered from anaerobic digestion sludge.

This study reports a circular metagenome-assembled genome (cMAG) of Candidatus Cloacimonadota recovered from a mesophilic full-scale food waste treatment plant. The cMAG spans 2,298,113 bp, with 980× coverage and 1 contig.

Circular metagenome-assembled genome of Candidatus Patescibacteria recovered from anaerobic digestion sludge.

A single-contig, circular metagenome-assembled genome (cMAG) of Candidatus (Ca.) Patescibacteria was reconstructed from a mesophilic full-scale food waste treatment plant in Japan. The genome is of small size and lacks fundamental biosynthetic pathways. Taxonomic analysis using the Genome Taxonomy Database revealed that this cMAG belonged to the genus JAEZRQ01 (Ca. Parcubacteria).

Prevalence and molecular characterization of Cryptosporidium in ostriches (Struthio camelus) on a farm in central Vietnam.

This study was performed to determine the prevalence and molecular characterization of Cryptosporidium in ostriches on a farm in Khanh Hoa province, central Vietnam. A total of 464 ostrich fecal samples were examined Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method, and 110 (overall prevalence 23.7%) were identified as positive by microscopy. Prevalence of Cryptosporidium in animals of <45 days, 45-60 days, 61-90 days, 91 days-12 months and >12 months was 23.5% (16/68), 33.3% (22/66), 35.2% (68/193), 0 and 5.8% (4/69), respectively (p<0.05). The majority of positive samples scored as the 3+ level of intensity of infection were from 61 to 90 days ostriches. Molecular analysis in the 18S ribosomal RNA, 70 kDa heat shock protein and actin genes demonstrated the presence of only Cryptosporidium avian genotype II in ostriches in central Vietnam.

Molecular characterization of Cryptosporidium in pigs in central Vietnam.

Little information is available on the epidemiology of Cryptosporidium in pigs in central Vietnam. The aims of this study were to investigate the prevalence and to characterize the genotype distribution of Cryptosporidium isolates in pigs in this region. A total of 193 pig fecal samples were screened for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method, and 28 (overall prevalence 14.5 %) were identified as positive by microscopic observation. Positive samples were further analyzed by polymerase chain reaction amplification and sequencing. Genetic identification based on the 18S ribosomal RNA and 70 kDa heat shock protein genes revealed that pigs in Vietnam are infected with two species/genotypes (Cryptosporidium suis and Cryptosporidium pig genotype II). This study is the first molecular characterization of Cryptosporidium in pigs in Vietnam. The presence of these host-adapted species/genotypes suggests that pigs may not pose a significant public health risk in this area. More extensive studies are necessary to ascertain the zoonotic potential of Cryptosporidium in porcine hosts in Vietnam.

Species-specific Primer and Probe Sets for Detection of Syntrophic Long-chain Fatty Acid-degrading Bacteria in Anaerobic Digestion Using Quantitative PCR.

Lipid-rich wastes are energy-dense substrates for anaerobic digestion. However, long-chain fatty acids (LCFAs), key intermediates in lipid degradation, inhibit methanogenic activity. In this study, TaqMan-based qPCR assays targeting the 16S rRNA gene of the cardinal LCFA-degrading bacterial species Syntrophomonas palmitatica and S. zehnderi were developed and validated. A trial experiment showed the advantage of species-specific quantification versus genus-specific quantification in assessing bacterial capacity for lipidic waste degradation. These qPCR assays will serve as monitoring tools for estimating the LCFA-degrading capacity of anaerobic digester communities and developing an effective strategy to enrich LCFA-degrading bacteria.

Semi-wet methanogen cathode composed of oak white charcoal for developing sustainable microbial fuel cells.

The present study aimed to evaluate a semi-wet biocathode composed of oak white charcoal and agarose gel as an alternative to the standard carbon felt biocathodes used in microbial fuel cells (MFCs). The MFC containing the oak white charcoal cathode (Oak-MFC) recorded a higher current value than that of the MFC containing a carbon felt cathode (CF-MFC). The Oak-MFC produced approximately 4.0-fold more electrons in the external circuit and 1.7-fold more methane (CH4) than the CF-MFC. A real-time PCR targeting mcrA showed that the number of methanogens adhering to the oak white charcoal cathode was approximately 15-fold that adhering to the carbon felt cathode. These results suggest that the methanogens attached to the cathode of both MFCs received electrons and CH4 was produced from carbon dioxide (CO2). Furthermore, Oak-MFC performed better than CF-MFC, thereby suggesting that oak white charcoal bound by agarose gel can be used as an alternative methanogen cathode. The propionic acid degradation rate of Oak-MFC was faster than that of CF-MFC suggesting that the cathodic reaction may affect the anodic reaction. The use of oak-derived electrode as a methanogen cathode also could contribute to sustainable forest management and promote regular thinning of oak trees. Further, its use will enable carbon fixation and efficient energy conversion from CO2 to CH4, thus contributing to sustainable energy use.

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.

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.

Molecular characterization of Cryptosporidium in native beef calves in central Vietnam.

The aims of this study were to investigate the prevalence of Cryptosporidium and to characterize the genotype distribution of Cryptosporidium isolates in native beef calves 2-6 months old in Dac Lac province, central Vietnam. The presence of Cryptosporidium oocysts was determined using the modified Ziehl-Neelsen staining method. The overall prevalence on the sample and herd levels were 18.9% (44/232) and 50% (20/40), respectively. Genotyping based on PCR and sequence analysis of the 18 S rRNA gene revealed occurrence of the two nonzoonotic species Cryptosporidium ryanae and Cryptosporidium bovis, with the former as a dominant species in the animals. The absence of the zoonotic species Cryptosporidium parvum in calves examined suggests that the native beef calves 2-6 months old in the study area are unlikely to contribute to human cryptosporidiosis transmission.

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.

Exploration of microbial communities contributing to effective methane production from scum under anaerobic digestion.

Scum is formed by the adsorption of long-chain fatty acids (LCFAs) onto biomass surface in anaerobic digestion of oily substrates. Since scum is a recalcitrant substrate to be digested, it is disposed via landfilling or incineration, which results in biomass washout and a decrease in methane yield. The microbes contributing to scum degradation are unclear. This study aimed to investigate the cardinal microorganisms in anaerobic scum digestion. We pre-incubated a sludge with scum to enrich scum-degrading microbes. Using this sludge, a 1.3-times higher methane conversion rate (73%) and a faster LCFA degradation compared with control sludge were attained. Then, we analyzed the cardinal scum-degrading microbes in this pre-incubated sludge by changing the initial scum-loading rates. Increased 16S rRNA copy numbers for the syntrophic fatty-acid degrader Syntrophomonas and hydrogenotrophic methanogens were observed in scum high-loaded samples. 16S rRNA amplicon sequencing indicated that Syntrophomonas was the most abundant genus in all the samples. The amino-acid degrader Aminobacterium and hydrolytic genera such as Defluviitoga and Sporanaerobacter became more dominant as the scum-loading rate increased. Moreover, phylogenic analysis on Syntrophomonas revealed that Syntrophomonas palmitatica, which is capable of degrading LCFAs, related species became more dominant as the scum-loading rate increased. These results indicate that a variety of microorganisms that degrade LCFAs, proteins, and sugars are involved in effective scum degradation.

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.

Isolation of bacteriocin-like substances producing bacteria from finished cattle-manure compost and activity evaluation against some food-borne pathogenic and spoilage bacteria.

A finished compost sample was examined for bacteriocin-like substance production against five pathogenic bacteria: Salmonella typhimurium EF 85-9, Escherichia coli O157:H7 ATCC 43888, Enterococcus faecalis JCM 8726, Staphylococcus aureus JCM 2151, and Yersinia enterocolitica JCM 7577. At the preliminary detection of bacterial strains exhibiting antimicrobial activity from the compost sample, thirteen strains could be isolated. Screening of the inhibitory activity was done using agar-well diffusion assay and Microtiter plate growth assay. Six bacterial strains from the compost showed an antimicrobial activity against one or more of the tested indicator strains. Four strains (M1-M4) belonged to Shigella species and the other two strains (M5 and M6) belonged to Salmonella species. The antimicrobial activity was sensitive for alpha-chymotrypsin and papain. The antimicrobial substances from M3, M4 and M6 were heat stable when heated for 15 min at 121 degrees C with 100% relative activity. The bacteriocin-like substance produced by strain M2 was partially characterized. It exhibited an inhibitory activity against the tested food-borne pathogenic and spoilage bacteria, except Enterobacter aerogenes JCM 1235 and Lactobacillus plantarum subsp. plantarum JCM 1149. It was stable at a wide range of pH (3-11). There was no loss of activity for up to 3 weeks when stored at 4 and -20 degrees C or for up to 2 weeks when stored at 28 and -80 degrees C. This is the first report indicating the presence of bacteriocin-like activity in animal manure compost.

Diversity of Sulfur-oxidizing Bacteria at the Surface of Cattle Manure Composting Assessed by an Analysis of the Sulfur Oxidation Gene soxB.

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 Hydrogenophilus were characteristically detected. In the thermophilic phase, clones related to the genera Hydrogenophaga 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.

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.