An agroecological structure model of compost-soil-plant interactions for sustainable organic farming.

Compost is used worldwide as a soil conditioner for crops, but its functions have still been explored. Here, the omics profiles of carrots were investigated, as a root vegetable plant model, in a field amended with compost fermented with thermophilic Bacillaceae for growth and quality indices. Exposure to compost significantly increased the productivity, antioxidant activity, color, and taste of the carrot root and altered the soil bacterial composition with the levels of characteristic metabolites of the leaf, root, and soil. Based on the data, structural equation modeling (SEM) estimated that amino acids, antioxidant activity, flavonoids and/or carotenoids in plants were optimally linked by exposure to compost. The SEM of the soil estimated that the genus Paenibacillus and nitrogen compounds were optimally involved during exposure. These estimates did not show a contradiction between the whole genomic analysis of compost-derived Paenibacillus isolates and the bioactivity data, inferring the presence of a complex cascade of plant growth-promoting effects and modulation of the nitrogen cycle by the compost itself. These observations have provided information on the qualitative indicators of compost in complex soil-plant interactions and offer a new perspective for chemically independent sustainable agriculture through the efficient use of natural nitrogen.

Computational estimation of sediment symbiotic bacterial structures of seagrasses overgrowing downstream of onshore aquaculture.

Coastal seagrass meadows are essential in blue carbon and aquatic ecosystem services. However, this ecosystem has suffered severe eutrophication and destruction due to the expansion of aquaculture. Therefore, methods for the flourishing of seagrass are still being explored. Here, data from 49 public coastal surveys on the distribution of seagrass and seaweed around the onshore aquaculture facilities are revalidated, and an exceptional area where the seagrass Zostera marina thrives was found near the shore downstream of the onshore aquaculture facility. To evaluate the characteristics of the sediment for growing seagrass, physicochemical properties and bacterial ecological evaluations of the sediment were conducted. Evaluation of chemical properties in seagrass sediments confirmed a significant increase in total carbon and a decrease in zinc content. Association analysis and linear discriminant analysis refined bacterial candidates specified in seagrass overgrown- and nonovergrown-sediment. Energy landscape analysis indicated that the symbiotic bacterial groups of seagrass sediment were strongly affected by the distance close to the seagrass-growing aquaculture facility despite their bacterial population appearing to fluctuate seasonally. The bacterial population there showed an apparent decrease in the pathogen candidates belonging to the order Flavobacteriales. Moreover, structure equation modeling and a linear non-Gaussian acyclic model based on the machine learning data estimated an optimal sediment symbiotic bacterial group candidate for seagrass growth as follows: the Lachnospiraceae and Ruminococcaceae families as gut-inhabitant bacteria, Rhodobacteraceae as photosynthetic bacteria, and Desulfobulbaceae as cable bacteria modulating oxygen or nitrate reduction and oxidation of sulfide. These observations confer a novel perspective on the sediment symbiotic bacterial structures critical for blue carbon and low-pathogenic marine ecosystems in aquaculture.

A potential network structure of symbiotic bacteria involved in carbon and nitrogen metabolism of wood-utilizing insect larvae.

Effective biological utilization of wood biomass is necessary worldwide. Since several insect larvae can use wood biomass as a nutrient source, studies on their digestive microbial structures are expected to reveal a novel rule underlying wood biomass processing. Here, structural inferences for inhabitant bacteria involved in carbon and nitrogen metabolism for beetle larvae, an insect model, were performed to explore the potential rules. Bacterial analysis of larval feces showed enrichment of the phyla Chroloflexi, Gemmatimonadetes, and Planctomycetes, and the genera Bradyrhizobium, Chonella, Corallococcus, Gemmata, Hyphomicrobium, Lutibacterium, Paenibacillus, and Rhodoplanes, as bacteria potential involved in plant growth promotion, nitrogen cycle modulation, and/or environmental protection. The fecal abundances of these bacteria were not necessarily positively correlated with their abundances in the habitat, indicating that they were selectively enriched in the feces of the larvae. Correlation and association analyses predicted that common fecal bacteria might affect carbon and nitrogen metabolism. Based on these hypotheses, structural equation modeling (SEM) statistically estimated that inhabitant bacterial groups involved in carbon and nitrogen metabolism were composed of the phylum Gemmatimonadetes and Planctomycetes, and the genera Bradyrhizobium, Corallococcus, Gemmata, and Paenibacillus, which were among the fecal-enriched bacteria. Nevertheless, the selected common bacteria, i.e., the phyla Acidobacteria, Armatimonadetes, and Bacteroidetes and the genera Candidatus Solibacter, Devosia, Fimbriimonas, Gemmatimonas Opitutus, Sphingobium, and Methanobacterium, were necessary to obtain good fit indices in the SEM. In addition, the composition of the bacterial groups differed depending upon metabolic targets, carbon and nitrogen, and their stable isotopes, δ13C and δ15N, respectively. Thus, the statistically derived causal structural models highlighted that the larval fecal-enriched bacteria and common symbiotic bacteria might selectively play a role in wood biomass carbon and nitrogen metabolism. This information could confer a new perspective that helps us use wood biomass more efficiently and might stimulate innovation in environmental industries in the future.

In Vitro and In Vivo Antibacterial Activities of a Novel Quinolone Compound, OPS-2071, against Clostridioides difficile.

OPS-2071 is a novel quinolone antibacterial agent characterized by low oral absorption that reduces the risk of adverse events typical of fluoroquinolone class antibiotics. The in vitro and in vivo antibacterial activities of OPS-2071 against Clostridioides difficile were evaluated in comparison to vancomycin and fidaxomicin. OPS-2071 exhibited potent antibacterial activity against 54 clinically isolated C. difficile strains with a MIC of 0.125 µg/ml (MIC50) and 0.5 µg/ml (MIC90), making it more active than vancomycin on a concentration basis (MIC50, 2 µg/ml; MIC90, 4 µg/ml) and comparable to fidaxomicin (MIC50, 0.063 µg/ml; MIC90, 8 µg/ml). OPS-2071 showed equally potent antibacterial activity against both hypervirulent and nonhypervirulent strains, while a significant difference in susceptibility to fidaxomicin was observed. Spontaneous resistance to OPS-2071 and vancomycin was not observed; however, resistance to fidaxomicin was observed at 4× MIC. The mutant prevention concentration of OPS-2071 was 16-fold lower than those of fidaxomicin and vancomycin, and the postantibiotic effect of OPS-2071 was longer than those of fidaxomicin and vancomycin. Also, OPS-2071 showed low systemic exposure, with OPS-2071 having 2.9% oral bioavailability at 1 mg/kg in rats. Furthermore, OPS-2071 showed significant in vivo efficacy at 0.0313 mg/kg/day (50% effective doses), 39.0-fold and 52.1-fold lower than those of vancomycin and fidaxomicin, respectively, in a hamster model of C. difficile infection. OPS-2071 has the potential to become a new therapeutic option for treating C. difficile infection.

Development of a systematic feedback isolation approach for targeted strains from mixed culture systems.

Elucidation of functions of bacteria in a mixed culture system (MCS) such as composting, activated sludge system is difficult, since the system is complicating with many unisolated bacteria. Here, we developed a systematic feedback isolation strategy for the isolation and rapid screening of multiple targeted strains from MCS. Six major strains (Corynebacterium sphenisci, Bacillus thermocloacae, Bacillus thermoamylovorans, Bacillus smithii, Bacillus humi, and Bacillus coagulans), which are detected by denaturing gradient gel electrophoresis (DGGE) analysis in our previous study on MCS for l-lactic acid production, were targeted for isolation. Based on information of suitable cultivation conditions (e.g., media, pH, temperature) from the literature, feedback isolation was performed to form 136 colonies. The following direct colony matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was optimised as the second screening to narrow down 20 candidate colonies from similar spectra patterns with six closest type strains. This step could distinguish bacteria at the species level with distance similarity scores ≥0.55 corresponding to 16S rRNA gene sequence similarity ≥98.2%, suggesting that this is an effective technique to minimize isolates close to targeted type strains. Analysis of 16S rRNA gene sequences indicated that two targeted strains and one strain related to the target had successfully been isolated, showing high similarities (99.5-100%) with the sequences from the DGGE bands, and that the other candidates were affiliated with three strains that were closely related to the target species. This study proposes a new method for systematic feedback isolation that may be useful for isolating targeted strains from MCS for further investigation.

Antimalarial Phenanthroindolizine Alkaloids from Ficus septica.

During the screening of antimalarial substances, MeOH extract from the twigs of Ficus septica was shown to have potent antimalarial activity. Bioassay-guided fractionation of a methanol extract of the twigs of F. septica led to the isolation of a new seco-phenanthroindolizine alkaloid and three known phenanthroindolizine alkaloids. Their structures were elucidated on the basis of NMR analysis. All isolated compounds were tested against Plasmodium falciparum. Compounds 2-4 displayed antimalarial activity against the 3D7 strain of P. falciparum with IC50 values 0.028-0.42 µM, whereas a new compound 1 exhibited a moderate antimalarial activity.

Thermophile-fermented compost extract as a possible feed additive to enhance fecundity in the laying hen and pig: Modulation of gut metabolism.

Recently, we reported that the oral administration of an extract of compost fermented with marine animal resources and thermophilic Bacillus species should confer health benefits in fish, pigs and rodents. Herein, the relations between fecundity and gut metabolites in laying hens and pigs on farms after oral exposure to compost were investigated. On the hen farms, the egg production of hens continuously administered the extract was maintained at significantly higher levels compared with the hens not administered the extract. On the swine farms, after the compost treatment, the shipping dates of fattening pigs were shortened, with an improvement in the death rate of the pigs. When the levels of fecal organic acids, such as short-chain fatty acids, lactate, and ammonium, as indicators of gut metabolism and energy sources for peripheral tissues, were examined, the levels of the acetate, propionate, and butyrate in the feces of the hens and pigs in the compost-treated group were not always different from those in the untreated control group. However, the levels of lactate were consistently low in the feces of both animals after the compost treatment. The fecal ammonium concentrations in old hens (age 597-672 days) and 2-month-old piglets from the compost-fed mother sows were low when compared with the untreated groups. The concentrations of free organic acids and their related compounds in the animal products (eggs and pig loins) were nearly equal to those in the untreated control products. Thus, the oral administration of the thermophile-fermented compost should improve the fecundity of hens and pigs by modifying their gut metabolism.

Thermophile-fermented compost as a fish feed additive modulates lipid peroxidation and free amino acid contents in the muscle of the carp, Cyprinus carpio.

Recently, a compost fermented with marine animals with thermophilic Bacillaceae in a clean and exclusive process at high temperature was reported as a possible feed additive to improve the healthy balance in sea fish and mammals (i.e., pigs and rodents). Here, the effects of the oral administration of the compost on the muscle and internal organs of carp (Cyprinus carpio) as a freshwater fish model were investigated. The fatty acid composition was different in the muscle of the carp fed with or without the compost extract, but there was little difference in the hepatopancreas. The accumulation of triacylglycerols, cholesterol, lipid peroxide and hydroxyl lipids decreased in the muscle after the oral administration of the compost extract in the carps over 12 weeks, but the accumulation did not always decrease in the hepatopancreas. In contrast, free-radical-scavenging activities and the concentrations of free amino acids in the muscle did not always increase and was dependent on the dose of the compost at 12 weeks. The scavenging activities and part of free amino acid levels in the muscle of the carp were improved at 24 weeks after a high dose of compost exposure, and then the survival rates of the carp were maintained. Thus, the oral administration of thermophile-fermented compost can prevent peroxidation and increase the content of free amino acids in the muscle of the freshwater fish, depending on the dose and term of the administration, and may be associated with the viability of the fish.

Bacillus hisashii sp. nov., isolated from the caeca of gnotobiotic mice fed with thermophile-fermented compost.

A taxonomic study was performed on 15 bacterial isolates from the caeca of gnotobiotic mice that had been fed with thermophile-fermented compost. The 15 isolates were thermophilic, Gram-stain-positive, facultatively anaerobic, endospore-forming bacteria, and were most closely related to Bacillus thermoamylovorans CNCM I-1378T. The 16S rRNA gene sequence of strain N-11T, selected as representative of this new group, showed a similarity of 99.4 % with Bacillus thermoamylovorans CNCM I-1378T, 94.7 % with Bacillus thermolactis R-6488T, and 94.4 % with Bacillus kokeshiiformis MO-04T. The isolates were then classified into two distinct groups based on a (GTG)5-fingerprint analysis. Two isolates, N-11T and N-21, were the representatives of these two groups, respectively.` The N-11T and N-21 isolates showed 66-71 % DNA-DNA relatedness with one other, but had less than 37 % DNA-DNA relatedness with B. thermoamylovorans LMG 18084T. The other 13 isolates showed DNA-DNA relatedness values above 74 % with the N-11T isolate. All 15 isolates grew at 25-60 °C (optimum 50 °C), pH 6-8 (optimum pH 7) and were capable of growing on a medium containing 6 % (w/v) NaCl (optimum 0.5 %). The 15 isolates could be distinguished from B. thermoamylovorans LMG 18084T because they showed Tween 80 hydrolysis activity and did not produce acid from melibiose. The major fatty acids were anteiso-C15 : 0, C16 : 0, iso-C15 : 0, iso-C14 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and several unidentified phospholipids. The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The menaquinone was MK-7. The DNA G+C content was 37.9 mol%. Based on the phenotypic properties, the 15 strains represent a novel species of the genus Bacillus, for which the name Bacillus hisashii sp. nov. is proposed. The type strain is N-11T ( = NRBC 110226T = LMG 28201T).

Direct starch fermentation to L-lactic acid by a newly isolated thermophilic strain, Bacillus sp. MC-07.

A newly isolated Bacillus sp. MC-07 showed 99.2 % 16S rRNA gene sequence similarity with the Bacillus thermoamylovorans LMG 18084(T). It demonstrated optimum and maximum growth temperatures of 50 and 62 °C, respectively. The ability of MC-07 to produce optically pure L-lactic acid via direct fermentation of starch without enzymatic hydrolysis was investigated at different pH values (6.0-8.0) by intermittent adjustments every 12 h. During batch fermentation in mineral salt medium containing 0.001 % yeast extract at pH 7.0, 20 g/L of soluble starch was utilized to produce 16.6 g/L L-lactic acid at 50 °C within 24 h of fermentation, with 100 % optical purity, 92.1 % lactic acid selectivity, and an L-lactic acid yield of 0.977 g/g. Direct starch fermentation at pHs 6.0, 6.5, 7.5, and 8.0 resulted in considerably lower concentrations of lactic acid than did at pH 7.0. Compared with B. thermoamylovorans LMG 18084(T), the ability of strain MC-07 to produce L-lactic acid was superior.

Thermotolerant Bacillus kokeshiiformis sp. nov. isolated from marine animal resources compost.

A novel Gram-staining-positive, endospore-forming, rod-shaped, facultatively anaerobic, thermotolerant bacterium, designated strain MO-04(T), was isolated from a marine animal resources (MAR) compost. The 16S rRNA gene sequence of strain MO-04(T) showed 99.4% similarity with Bacillus thermolactis R-6488(T), 94.1% similarity with Bacillus thermoamylovorans CNCM I-1378(T), 93.3% similarity with Bacillus humi LMG 22167(T), 93.2% similarity with Bacillus niacini IFO 15566(T) and the similarities with other species were less than 93%. DNA-DNA relatedness between strain MO-04(T) and B. thermolactis DSM 23332(T) was 45%. The DNA G+C content of strain MO-04(T) was 33.4 mol%, comparatively lower than that of B. thermolactis R-6488(T) (35.0 mol%). Strain MO-04(T) grew at 35-61 °C (optimum 50 °C), pH 4.5-9.0 (optimum pH 7.2) and tolerated up to 8.0% (w/v) NaCl (optimum 2%). The MO-04(T) cell wall peptidoglycan type was meso-2,6-diaminopimelic acid, and the major fatty acids were C(16 : 1), C(14 : 1), C(17 : 0) and C(17 : 1). The major polar lipids were represented by diphosphatidylglycerol and phosphatidylglycerol and two unidentified phospholipids. The analysed polyphasic data presented here clearly indicate that the isolate MO-04(T) is considered to represent a novel species within the genus Bacillus for which the name Bacillus kokeshiiformis sp. nov. is proposed. The type strain of B. kokeshiiformis is MO-04(T) ( = JCM 19325(T) = KCTC 33163(T)).

A novel production process for optically pure L-lactic acid from kitchen refuse using a bacterial consortium at high temperatures.

We investigated L-lactic acid production in static batch fermentation of kitchen refuse using a bacterial consortium from marine-animal-resource (MAR) composts at temperatures ranging from 30 to 65 °C. At relatively low temperatures butyric acid accumulated, whereas at higher temperatures L-lactic acid was produced. In particular, fermentation at 50 °C produced 34.5 g L(-1) L-lactic acid with 90% lactic acid selectivity and 100% optical purity. Denaturing gradient gel electrophoresis indicated that dominant bacteria present in the original MAR composts diminished rapidly and Bacillus coagulans strains became the dominant contributors to L-lactic acid production at 45, 50 and 55 °C. This is the first report of the achievement of 100% optical purity of L-lactic acid using a bacterial consortium.

Thermophile-fermented compost as a possible scavenging feed additive to prevent peroxidation.

The extract of compost from fermented marine animals and thermophiles, including Bacillaceae, confers health benefits as a feed additive for fish and pigs. However, little research has explored how such compost extracts affect the physiological functions of the animals. In this study, the physiological effects of oral administration of the compost extract on the liver and muscle of rats are evaluated. After long-term administration of the compost extract in rats fed with either a normal diet or a high-fat diet over 3 months, accumulation of lipid peroxide and malondialdehyde, a marker of peroxidation, in the livers was reduced. Under such conditions, the unsaturated fatty acid composition in the liver was not significantly different in the rats fed either with or without the compost extract. In contrast, analyzes of 2,2-diphenyl-1-picrylhydrazyl (DPPH) revealed that free-radical-scavenging activity was increased in the livers of rats fed with the compost extract, although the extract itself had little of this activity. Glutathione, an antioxidant, was slightly increased following compost exposure. In addition, the levels of glutamate and glutamine, sources of glutathione, were slightly raised. Such a tendency was also observed in the muscle. Thus, thermophile-fermented compost can be a fermented feed additive to prevent peroxidation in the liver and muscle, and the effects of this additive may, in part, be associated with the retention of antioxidants and free amino acids within the organs.

Denitrification in soil amended with thermophile-fermented compost suppresses nitrate accumulation in plants.

NO (3) (-) is a major nitrogen source for plant nutrition, and plant cells store NO (3) (-) in their vacuoles. Here, we report that a unique compost made from marine animal resources by thermophiles represses NO (3) (-) accumulation in plants. A decrease in the leaf NO (3) (-) content occurred in parallel with a decrease in the soil NO (3) (-) level, and the degree of the soil NO (3) (-) decrease was proportional to the compost concentration in the soil. The compost-induced reduction of the soil NO (3) (-) level was blocked by incubation with chloramphenicol, indicating that the soil NO (3) (-) was reduced by chloramphenicol-sensitive microbes. The compost-induced denitrification activity was assessed by the acetylene block method. To eliminate denitrification by the soil bacterial habitants, soil was sterilized with γ irradiation and then compost was amended. After the 24-h incubation, the N(2)O level in the compost soil with presence of acetylene was approximately fourfold higher than that in the compost soil with absence of acetylene. These results indicate that the low NO (3) (-) levels that are often found in the leaves of organic vegetables can be explained by compost-mediated denitrification in the soil.

Impact of oral administration of compost extract on gene expression in the rat gastrointestinal tract.

Oral administration of an extract consisting of compost fermented with thermophiles to pigs reduces the incidence of stillbirth and promotes piglet growth. However, the mechanism by which the compost extract modulates the physiological conditions of the animals remains largely unknown. Here, we investigate the effects of compost extract on the physiological responses in the intestine of a mammalian rat model. The level of fecal immunoglobulin A (IgA), which provides protection against pathogens and is secreted from the small intestine, was significantly higher in rats treated with continuous administration of the compost extract than in untreated rats after 2 months, but not after 1 month. However, the fecal IgA level was not significantly different in rats that received the filtered compost extract compared with the untreated rats or the rats that received the compost extract. Gene expression analyses of the small intestine indicated that several immune-related genes were upregulated following compost exposure. Specifically, the expression levels of lymphocyte chemoattractant chemokine CXCL13 and Granzyme B, which is released within cytotoxic T lymphocytes and natural killer cells, increased in the small intestinal tract following compost exposure. Based on these observations, it was postulated that the increased level of fecal IgA following compost exposure was associated with the expression of CXCL13 and Granzyme B in the intestinal tract. Thus, thermophile-fermented compost could contain microbes or substances that activate the rat's gut mucosal immune response.

The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets.

Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n=330-1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p≦0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p<0.0001), while the ratio of weanlings per liveborn piglets was increased by more than 6.5% in the compost extract-fed group. Thus thermophiles and their products in the compost extract might promote growth and reduce stillbirths of piglets during the birth to weaning period.

Stereoselective synthesis of alpha-alkyl-beta-keto imides via asymmetric redox C-C bond formation between alpha-alkyl-alpha-diazocarbonyl compounds and aldehydes.

Asymmetric redox C-C bond formation between alpha-alkyl-alpha-diazocarbonyl compounds and aldehydes was developed as a practical and general method for the construction of alpha-alkyl-beta-keto imides having a chiral nonracemic tertiary stereogenic center.

Microbial analysis of a composted product of marine animal resources and isolation of bacteria antagonistic to a plant pathogen from the compost.

A composting product of marine animal resources has been used as a fertilizer and a soil conditioner in Japan. This compost was produced by a repeated fed-batch fermentation system with three successive aerobic bioreactors. Composting temperature reached about 75 degrees C without heating. The bacterial diversity in this compost was investigated by denaturing gradient gel electrophoresis (DGGE) and sequence determination of the V3 region in the 16S rRNA genes. The sequence analysis showed that a majority of retrieved sequences corresponded to those of Bacillaceae, and we frequently found sequences similar to the 16S rDNA sequences of Bacillus thermocloacae and Bacillus thermoamylovorans. In addition, a bacterium antagonistic to a Fusarium strain was isolated from the compost. The isolate (Bacillus sp. NP-1) produced an antifungal compound, iturin A. These results suggest that this compost serves as a valuable source of plant growth-promoting rhizobacteria including the antifungal bacteria.