Methane Production Characteristics of an Anaerobic Co-Digestion of Pig Manure and Fermented Liquid Feed.

Methane production characteristics of anaerobic co-digestion of pig manure (PM) and fermented liquid feed (FLF) were investigated in a continuous digester under mesophilic conditions. The experiment followed three phases. PM alone was digested in phase I. In phases II and III, PM and FLF were mixed in a ratio of 95:5 and 90:10 (% v/v), respectively. The specific methane yields (SMYs) during phases I, II, and III were 238, 278, and 326.8 mLCH4·gVS-1-added, respectively. It was due to the effect of balancing the feedstock carbon-to-nitrogen ratio by adding FLF. This improvement can also be attributed to the readily biodegradable compounds in the FLF. The higher SMY obtained in this study showed a positive synergistic effect in the anaerobic co-digestion of PM and FLF. The results also indicate that adding the FLF positively affected and maintained a constant pH level, avoiding volatile fatty acid accumulation and ammonia inhibition in the anaerobic digestion (AD). Thus, this study provides valuable information regarding the usage of unused or wasted FLF as a co-substrate for the practical AD of PM. The production of fermented liquid additives such as FLF to improve the methane production from the AD of PM is a potential novel alternative to food waste recycling in Japan, besides compost and animal feeding.

Complete Genome Sequence of Gelria sp. Strain Kuro-4, a Thermophilic Anaerobe Isolated from a Thermophilic Anaerobic Digestion Reactor Treating Poly(L-Lactic Acid).

Strain Kuro-4 was isolated as a novel member of the genus Gelria from a thermophilic anaerobic digestion reactor treating poly(l-lactic acid). Here, we report a 2,880,462-bp complete circular genome sequence of Kuro-4, with a G+C content of 61.9%. The chromosome harbors 2,831 protein-coding genes and 62 RNA-coding genes.

Trade-Off Relation between Fungicide Sensitivity and Melanin Biosynthesis in Plant Pathogenic Fungi.

Circumventing the emergence of fungicide-resistant strains is a crucial issue for robust disease management in agriculture. The agricultural fungicide ferimzone has been used for the control of rice diseases including rice blast. The emergence of ferimzone-resistant strains in rice fields has not been reported. Here, we identified the copper transport CoICT1 gene as the ferimzone sensitivity gene in Colletotrichum orbiculare and the rice blast fungus Magnaporthe oryzae. Genetic and cytological analyses showed that functional defects in the copper transport pathways, consisting of CoIct1 and P-type ATPase CoCcc2, led to the low sensitivity to ferimzone and the pathogenicity defect due to attenuated melanization in the appressorium. Importantly, the presence of CuSO4 induced high sensitivity to ferimzone even in the coict1 mutant. Our study shows that there is a trade-off relation between the sensitivity to ferimzone and fungal pathogenicity.

Manganese Treatment Alleviates Zinc Deficiency Symptoms in Arabidopsis Seedlings.

Plant phenotypes caused by mineral deficiencies differ depending on growth conditions. We recently reported that the growth of Arabidopsis thaliana was severely inhibited on MGRL-based zinc (Zn)-deficient medium but not on Murashige-Skoog-based Zn-deficient medium. Here, we explored the underlying reason for the phenotypic differences in Arabidopsis grown on the different media. The root growth and chlorophyll contents reduced by Zn deficiency were rescued by the addition of extra manganese (Mn) during short-term growth (10 or 14 d). However, this treatment did not affect the growth recovery after long-term growth (38 d). To investigate the reason for plant recovery from Zn deficiency, we performed the RNA-seq analysis of the roots grown on the Zn-basal medium and the Zn-depleted medium with/without additional Mn. Principal component analysis of the RNA-seq data showed that the gene expression patterns of plants on the Zn-basal medium were similar to those on the Zn-depleted medium with Mn, whereas those on the Zn-depleted medium without Mn were different from the others. The expression of several transcription factors and reactive oxygen species (ROS)-related genes was upregulated in only plants on the Zn-depleted medium without Mn. Consistent with the gene expression data, ROS accumulation in the roots grown on this medium was higher than those grown in other conditions. These results suggest that plants accumulate ROS and reduce their biomass under undesirable growth conditions, such as Zn depletion. Taken together, this study shows that the addition of extra Mn to the Zn-depleted medium induces transcriptional changes in ROS-related genes, thereby alleviating short-term growth inhibition due to Zn deficiency.

Nitrate removal performance and diversity of active denitrifying bacteria in denitrification reactors using poly(L-lactic acid) with enhanced chemical hydrolyzability.

Poly(L-lactic acid) (PLLA) can be used as an external electron donor in denitrification reactors to treat drinking water, aquaculture water, and industrial wastewater with an imbalanced carbon/nitrogen ratio. However, for PLLA to function in these applications, its chemical hydrolyzability requires improvement. Although the adjustment of the crystallinity (Xc) is effective in improving the hydrolyzability of PLLA, the condition for the Xc of PLLA, in which a sufficient amount of lactic acid is released for denitrification, must be clarified. Therefore, this study investigated the effective Xc range and optimal PLLA content as an electron donor for continuous nitrate removal in denitrification reactors. This study also explored the abundance, succession, and diversity of active denitrifying bacteria in denitrification reactors. The nitrate removal activity of activated sludge using the highly crystalline PLLA (Xc = 39.4%) was 1.8 mg NO3- -N g MLSS-1 h-1, which is 2.4 times higher than that using the nearly amorphous PLLA (Xc = 0.9%). During the 57 days of operation, the denitrification reactor with 3% (w/v) highly crystalline PLLA continued to completely remove nitrate, with a maximum nitrate removal activity of 22.8 mg NO3- -N g MLSS-1 h-1. The 16S rRNA amplicon sequencing and clone library analyses are using transcripts of two nitrite reductase genes, encoding cytochrome cd1 nitrite reductase, and copper-containing nitrite reductase revealed that bacteria belonging to the families Comamonadaceae, Rhodocyclaceae, and Alcaligenaceae were active denitrifying bacteria in the denitrification reactor using PLLA.

Prokaryotic Community Structures in a Thermophilic Anaerobic Digestion Reactor Converting Poly(l-Lactic Acid) for a Long Period Revealed by 16S rRNA Gene Amplicon Sequencing.

Little information on poly(l-lactic acid) (PLLA) treatment-associated microbiota in thermophilic anaerobic digestion reactors is available. Here, we provide 16S rRNA gene sequence data on microbiota in a thermophilic anaerobic digestion reactor converting PLLA to methane for 336 days. Data comprising 99,566 total high-quality reads were tabulated at the taxonomic class level.

Draft Genome Sequence of Thermodesulfovibrio sp. Strain Kuro-1, a Thermophilic, Lactate-Degrading Anaerobe Isolated from a Thermophilic Anaerobic Digestion Reactor.

Thermodesulfovibrio sp. strain Kuro-1, newly isolated from a thermophilic anaerobic digestion reactor, is a thermophilic anaerobe that can utilize l-lactic acid in fermentation, sulfate respiration, and cocultivation with hydrogenotrophic methanogens. Here, we report its draft genome sequence, consisting of a 1.93-Mb sequence with a G+C content of 34.0%.

Improvement of methanogenic activity of anaerobic digestion using poly(l-lactic acid) with enhanced chemical hydrolyzability based on physicochemical parameters.

Because packing bags and disposable items of poly (l-lactic acid) (PLLA) waste are discharged together with other organic waste including garbage, anaerobic co-digestion of PLLA and other organic waste is required. However, because of low hydrolyzability of PLLA products, the chemical hydrolyzability must be improved for PLLA treatment during anaerobic digestion. This study aimed to assess weight-average molecular weight (Mw) and crystallinity (Xc), to determine the chemical hydrolyzability of PLLA, for PLLA treatment during anaerobic digestion. Moreover, the possibility of anaerobic co-digestion of the PLLA after improvement of chemical hydrolyzability and other organic waste was also discussed. Detectable methanogenic activity of the mesophilic and thermophilic anaerobic sludges of PLLA occurred in the Mw range of 6,800 to 16,500, and 6,800 and 38,000, respectively. The methanogenic activity of mesophilic and thermophilic anaerobic sludge was higher with PLLA with a high crystallinity (Xc = 39.9-46.1%) than with nearly amorphous PLLA (Xc = 0.3-3.5%). The maximum methanogenic activity of anaerobic sludge using PLLA with an Xc of approximately 40-45% and with a Mw of 10,300 and 16,500 for mesophilic and thermophilic anaerobic sludge were 0.013 gCOD·gVS-1·d-1 and 0.13 gCOD·gVS-1·d-1, respectively. A survey on the possibility of anaerobic co-digestion of PLLA after improvement in chemical hydrolyzability based on Mw and Xc and organic wastes revealed that thermophilic conditions at 55 °C are more advantageous than mesophilic conditions at 37 °C.

Draft Genome Sequence of Moorella sp. Strain Hama-1, a Novel Acetogenic Bacterium Isolated from a Thermophilic Digestion Reactor.

Moorella sp. strain Hama-1 was isolated from a thermophilic anaerobic digestion reactor treating poly(l-lactic acid). The strain is a thermophilic acetogen capable of lactate oxidation under anaerobic conditions. Here, we report the draft genome sequence of strain Hama-1, comprising 3.27 Mb in 48 contigs, with a G+C content of 56.6%.

Supercritical fluid extraction of bacterial and archaeal lipid biomarkers from anaerobically digested sludge.

Supercritical fluid extraction (SFE) was used in the analysis of bacterial respiratory quinone (RQ), bacterial phospholipid fatty acid (PLFA), and archaeal phospholipid ether lipid (PLEL) from anaerobically digested sludge. Bacterial RQ were determined using ultra performance liquid chromatography (UPLC). Determination of bacterial PLFA and archaeal PLEL was simultaneously performed using gas chromatography-mass spectrometry (GC-MS). The effects of pressure, temperature, and modifier concentration on the total amounts of RQ, PLFA, and PLEL were investigated by 23 experiments with five settings chosen for each variable. The optimal extraction conditions that were obtained through a multiple-response optimization included a pressure of 23.6 MPa, temperature of 77.6 °C, and 10.6% (v/v) of methanol as the modifier. Thirty nine components of microbial lipid biomarkers were identified in the anaerobically digested sludge. Overall, the SFE method proved to be more effective, rapid, and quantitative for simultaneously extracting bacterial and archaeal lipid biomarkers, compared to conventional organic solvent extraction. This work shows the potential application of SFE as a routine method for the comprehensive analysis of microbial community structures in environmental assessments using the lipid biomarkers profile.

Supercritical fluid extraction and ultra performance liquid chromatography of respiratory quinones for microbial community analysis in environmental and biological samples.

Microbial community structure plays a significant role in environmental assessment and animal health management. The development of a superior analytical strategy for the characterization of microbial community structure is an ongoing challenge. In this study, we developed an effective supercritical fluid extraction (SFE) and ultra performance liquid chromatography (UPLC) method for the analysis of bacterial respiratory quinones (RQ) in environmental and biological samples. RQ profile analysis is one of the most widely used culture-independent tools for characterizing microbial community structure. A UPLC equipped with a photo diode array (PDA) detector was successfully applied to the simultaneous determination of ubiquinones (UQ) and menaquinones (MK) without tedious pretreatment. Supercritical carbon dioxide (scCO(2)) extraction with the solid-phase cartridge trap proved to be a more effective and rapid method for extracting respiratory quinones, compared to a conventional organic solvent extraction method. This methodology leads to a successful analytical procedure that involves a significant reduction in the complexity and sample preparation time. Application of the optimized methodology to characterize microbial communities based on the RQ profile was demonstrated for a variety of environmental samples (activated sludge, digested sludge, and compost) and biological samples (swine and Japanese quail feces).

Epigenetic modifications of the 35S promoter in cultured gentian cells.

Our previous studies found strict gene silencing associated with CaMV-35S promoter-specific de novo methylation in transgenic gentian plants. To dissect the de novo methylation machinery, especially in association with histone modification, 35S-driven sGFP-expressing and -silenced gentian cultured cell lines that originated from a single transformation event were produced and used for epigenetic analyses. A sGFP-expressing primarily induced cell suspension culture (PS) was hypomethylated in the 35S promoter region, although a low level of de novo methylation at the 35S enhancer region (-148 to -85) was detected. In contrast, a sGFP-silenced re-induced cell suspension culture (RS), which originated from leaf tissues of a transgenic plant, was hypermethylated in the 35S promoter region. Chromatin immunoprecipitation analysis showed that in RS, histone H3 of the silenced 35S promoter region was deacetylated and also dimethylated on lysine 9. Interestingly, in the silenced 35S promoter 3' region, dimethylation of histone H3 lysine 4 was also observed. When hypomethylation and histone H3 acetylation of the 35S region occurred in PS, de novo methylation at the 35S enhancer region had already taken place. The de novo methylation status was also resistant to 5-aza-2'-deoxycytidine treatment. These results suggest that de novo methylation of the enhancer region is a primitive process of 35S silencing that triggers histone H3 deacetylation.

Supercritical fluid extraction of microbial phospholipid fatty acids from activated sludge.

Supercritical carbon dioxide (scCO2) extraction was applied for the determination of microbial phospholipid fatty acids (PLFA) in activated sludge. Quantification was performed by using gas chromatography-mass spectrometry (GC-MS). The highest extraction yields of PLFA, at a concentration of 7.28 nmol/mg-dry activated sludge, was obtained at a temperature of 80°C, pressure of 25 MPa and 10% (v/v) methanol for a 15-min extraction time. ScCO2 extraction results obtained in these conditions were comparable with those obtained by liquid organic solvent extraction (LSE) based on diversity and equalibility indices. The repeatability test showed that the relative standard deviation values were less than 13%. The experimental results show that the scCO2 extraction saves time and uses much less organic solvent. In addition, scCO2 extraction is a promising and alternative method for the analysis of microbial community structure in environmental assessment using the PLFA profile.

Strict de novo methylation of the 35S enhancer sequence in gentian.

A novel transgene silencing phenomenon was found in the ornamental plant, gentian (Gentiana triflora x G. scabra), in which the introduced Cauliflower mosaic virus (CaMV) 35S promoter region was strictly methylated, irrespective of the transgene copy number and integrated loci. Transgenic tobacco having the same vector did not show the silencing behavior. Not only unmodified, but also modified 35S promoters containing a 35S enhancer sequence were found to be highly methylated in the single copy transgenic gentian lines. The 35S core promoter (-90)-introduced transgenic lines showed a small degree of methylation, implying that the 35S enhancer sequence was involved in the methylation machinery. The rigorous silencing phenomenon enabled us to analyze methylation in a number of the transgenic lines in parallel, which led to the discovery of a consensus target region for de novo methylation, which comprised an asymmetric cytosine (CpHpH; H is A, C or T) sequence. Consequently, distinct footprints of de novo methylation were detected in each (modified) 35S promoter sequence, and the enhancer region (-148 to -85) was identified as a crucial target for de novo methylation. Electrophoretic mobility shift assay (EMSA) showed that complexes formed in gentian nuclear extract with the -149 to -124 and -107 to -83 region probes were distinct from those of tobacco nuclear extracts, suggesting that the complexes might contribute to de novo methylation. Our results provide insights into the phenomenon of sequence- and species- specific gene silencing in higher plants.

Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge.

Supercritical CO2 (scCO2) extraction, with methanol as modifier, was applied to the determination of ubiquinones and menaquinones in activated sludge. Four ubiquinones and 12 menaquinones species were identified based on retention time and UV spectrum in 0.1g dried activated sludge. The optimum extraction conditions were at a pressure of 25 MPa, a temperature of 55 degrees C, and 10% (v/v) methanol for 15 min. At this condition, the concentrations of extracted ubiquinones and menaquinones were found to be 0.181 and 0.326 micromol/g-dry-cell, respectively. The results were comparable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Furthermore, the method was evaluated in term of repeatability, which resulted in an RSD of < or =10%. The experimental results have demonstrated the technique to be simple, fast, and with less consumption of organic solvents. This work shows the potential application of supercritical CO2 extraction to microbial community analysis using quinone profile.

Application of modified supercritical carbon dioxide extraction to microbial quinone analysis.

Supercritical carbon dioxide (scCO2) was applied to extract microbial quinones from activated sludge. Identification and analysis was then performed using high-performance liquid chromatography (HPLC) equipped with ultraviolet-visible (UV-Vis) detector and photodiode array detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scCO2 were similar to that of the conventional method (organic solvent extraction).

Enhancement of CLAIM (clinical accounting information) for a localized Chinese version.

CLinical Accounting InforMation (CLAIM) is a standard for the exchange of data between patient accounting systems and electronic medical record (EMR) systems. It uses eXtensible Markup Language (XML) as a meta-language and was developed in Japan. CLAIM is subordinate to the Medical Markup Language (MML) standard, which allows the exchange of medical data between different medical institutions. It has inherited the basic structure of MML 2.x and the current version, version 2.1, contains two modules and nine data definition tables. In China, no data exchange standard yet exists that links EMR systems to accounting systems. Taking advantage of CLAIM's flexibility, we created a localized Chinese version based on CLAIM 2.1. Since Chinese receipt systems differ from those of Japan, some information such as prescription formats, etc. are also different from those in Japan. Two CLAIM modules were re-engineered and six data definition tables were either added or redefined. The Chinese version of CLAIM takes local needs into account, and consequently it is now possible to transfer data between the patient accounting systems and EMR systems of Chinese medical institutions effectively.

CLAIM (CLinical Accounting InforMation)--an XML-based data exchange standard for connecting electronic medical record systems to patient accounting systems.

With the evolving and diverse electronic medical record (EMR) systems, there appears to be an ever greater need to link EMR systems and patient accounting systems with a standardized data exchange format. To this end, the CLinical Accounting InforMation (CLAIM) data exchange standard was developed. CLAIM is subordinate to the Medical Markup Language (MML) standard, which allows the exchange of medical data among different medical institutions. CLAIM uses eXtensible Markup Language (XML) as a meta-language. The current version, 2.1, inherited the basic structure of MML 2.x and contains two modules including information related to registration, appointment, procedure and charging. CLAIM 2.1 was implemented successfully in Japan in 2001. Consequently, it was confirmed that CLAIM could be used as an effective data exchange format between EMR systems and patient accounting systems.

Metal elution from Ni- and Fe-based alloy reactors under hydrothermal conditions.

Elution of metals from Ni- and Fe-based alloy (i.e. Inconel 625 and SUS 316) under hydrothermal conditions was investigated. Results showed that metals could be eluted even in a short contact time. At subcritical conditions, a significant amount of Cr was extracted from SUS 316, while only traces of Ni, Fe, Mo, and Mn were eluted. In contrast, Ni was removed in significant amounts compared to Cr when Inconel 625 was tested. Several factors including temperature and contact time were found to affect elution behavior. The presence of air in the fluid even promoted elution under subcritical conditions.

Change of monochloroacetic acid to biodegradable organic acids by hydrothermal reaction.

The feasibility of biodegradability improvement induced from the structural conversion of refractory pollutants by hydrothermal reaction was investigated. Monochloroacetic acid (MCAA) was selected as a preliminary material represented for linear hydrocarbon structured refractory pollutants. Under the tested conditions, MCAA was partially destructed and then converted to biodegradable reaction products by hydrolysis, dehydration and thermal decomposition. The identified products were glycolic acid, citric acid and formic acid. Total organic carbon (TOC) reduction during the structural conversion did not exceed 24%, except the results at the reaction conditions of 350 degrees C and 17 MPa. However, Produced biodegradable organic acids were reduced by thermal decomposition with increasing reaction temperature and time. At the reaction temperature of 250 and 300 degrees C, biodegradability (BOD/COD(Cr)) was reached at 0.51 in 6.9 min and 0.52 in 7.4 min despite the presence of dissociated chlorine ions. The detachment of recalcitrant chlorine ion from MCAA and the production of biodegradable organic acids by hydrothermal reaction were directly related to the biodegradability improvement of reaction products.