Takeshi Yamada

Acidipila rosea gen. nov., sp. nov., an acidophilic chemoorganotrophic bacterium belonging to the phylum Acidobacteria

Two strains of aerobic acidophilic chemoorganotrophic bacteria designed strains AP8(T) and AP9 were isolated from acid mine drainage and acidic soil, respectively. These isolates were gram-negative, nonmotile cocci and coccobacilli measuring 0.5-0.8 μm in diameter. Cells were capsulated. Colonies on solid media were pink colored. The pH range for growth was 3.0-6.0 (optimum pH 4.5). Sugars, gluconate, and some amino acids were good carbon and energy sources for growth. The main components of cellular fatty acids were C(15:0) iso and C(16:1) ω7c. Menaquinone-8 was the major quinone. The G+C content of genomic DNA was 59.5%. Both strains had identical sequences of 16S rRNA genes that were most closely related to that of the type strain of Acidobacterium capsulatum (96% similarity). There were major differences between the isolates and A. capsulatum in cell morphology, carbon nutrition, and fatty acid profiles. Based on these phylogenetic and phenotypic data, we propose the name Acidipila rosea gen. nov., sp. nov. to accommodate the novel isolates. The type strain is AP8(T) (NBRC 107607(T), KCTC 23427(T)).

Isolation and Functional Gene Analyses of Aromatic-Hydrocarbon-Degrading Bacteria from a Polychlorinated-Dioxin-Dechlorinating Process

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 105 copies of the AhDO large subunit genes g−1 wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from “Dehalococcoides” and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

Bacteria of the candidate phylum TM7 are prevalent in acidophilic nitrifying sequencing-batch reactors.

Laboratory-scale acidophilic nitrifying sequencing-batch reactors (ANSBRs) were constructed by seeding with sewage-activated sludge and cultivating with ammonium-containing acidic mineral medium (pH 4.0) with or without a trace amount of yeast extract. In every batch cycle, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate. Attempts to detect nitrifying functional genes in the fully acclimated ANSBRs by PCR with previously designed primers mostly gave negative results. 16S rRNA gene-targeted PCR and a subsequent denaturating gradient gel electrophoresis analysis revealed that a marked change occurred in the bacterial community during the overall period of operation, in which members of the candidate phylum TM7 and the class Gammaproteobacteria became predominant at the fully acclimated stage. This result was fully supported by a 16S rRNA gene clone library analysis, as the major phylogenetic groups of clones detected (>5% of the total) were TM7 (33%), Gammaproteobacteria (37%), Actinobacteria (10%), and Alphaproteobacteria (8%). Fluorescence in situ hybridization with specific probes also demonstrated the prevalence of TM7 bacteria and Gammaproteobacteria. These results suggest that previously unknown nitrifying microorganisms may play a major role in ANSBRs; however, the ecophysiological significance of the TM7 bacteria predominating in this process remains unclear.

Enhancement of Electricity Production by Graphene Oxide in Soil Microbial Fuel Cells and Plant Microbial Fuel Cells

The effects of graphene oxide (GO) on electricity generation in soil microbial fuel cells (SMFCs) and plant microbial fuel cell (PMFCs) were investigated. GO at concentrations ranging from 0 to 1.9u2009g⋅kg−1 was added to soil and reduced for 10u2009days under anaerobic incubation. All SMFCs (GO-SMFCs) utilizing the soils incubated with GO produced electricity at a greater rate and in higher quantities than the SMFCs which did not contain GO. In fed-batch operations, the overall average electricity generation in GO-SMFCs containing 1.0u2009g⋅kg−1 of GO was 40u2009±u200919u2009mW⋅m−2, which was significantly higher than the value of 6.6u2009±u20098.9u2009mW⋅m−2 generated from GO-free SMFCs (pu2009<u20090.05). The increase in catalytic current at the oxidative potential was observed by cyclic voltammetry (CV) for GO-SMFC, with the CV curve suggesting the enhancement of electron transfer from oxidation of organic substances in the soil by the reduced form of GO. The GO-containing PMFC also displayed a greater generation of electricity compared to the PMFC with no added GO, with GO-PMFC producing 49u2009mW⋅m−2 of electricity after 27u2009days of operation. Collectively, this study demonstrates that GO added to soil can be microbially reduced in soil, and facilitates electron transfer to the anode in both SMFCs and PMFCs.

Acidiphilium iwatense sp. nov., isolated from an acid mine drainage treatment plant, and emendation of the genus Acidiphilium

Several strains of aerobic, acidophilic, chemo-organotrophic bacteria belonging to the genus Acidiphilium were isolated from an acid mine drainage (AMD) (pH 2.2) treatment plant. 16S rRNA gene sequence comparisons showed that most of the novel isolates formed a phylogenetically coherent group (designated Group Ia) distinguishable from any of the previously established species of the genus Acidiphilium at <98% similarity. This was supported by genomic DNA-DNA hybridization assays. The Group Ia isolates were characterized phenotypically by an oval cell morphology, non-motility, growth in the range pH 2.0-5.5 (optimum pH 3.5), lack of photosynthetic pigment and the presence of C19:0 cyclo ω8c as the main component of the cellular fatty acids and ubiquinone-10 as the major quinone. On the basis of these data, the name Acidiphilium iwatense sp. nov. is proposed to accommodate the Group Ia isolates, and the description of the genus Acidiphilium is emended. The type strain of Acidiphilium iwatense sp. nov. is MS8(T) (u200a=NBRC 107608(T)=KCTC 23505(T)).

Real-time optical monitoring of microbial growth using optimal combination of light-emitting diodes

Abstract. We developed a real-time optical monitoring system consisting of a monochrome complementary metal-oxide semiconductor (CMOS) camera and two light-emitting diodes (LEDs) with a constant temperature incubator for the rapid detection of microbial growth on solid media. As a target organism, we used Alicyclobacillus acidocaldarius, which is an acidophilic thermophilic endospore-forming bacterium able to survive in pasteurization processes and grow in acidic drink products such as apple juice. This bacterium was cultured on agar medium with a redox dye applied to improve detection sensitivity. On the basis of spectroscopic properties of the colony, medium, and LEDs, an optimal combination of two LED illuminations was selected to maximize the contrast between the colony and medium areas. We measured A. acidocaldarius and Escherichia coli at two different dilution levels using these two LEDs. From the results of time-course changes in the number of detected pixels in the detection images, a similar growth rate was estimated amongst the same species of microbes, regardless of the dilution level. This system has the ability to detect a colony of approximately 26 μm in diameter in a detection image, and it can be interpreted that the size corresponds to less than 20 μm diameter in visual inspection.

Ammonia-oxidizing activity and microbial community structure in acid tea (Camellia sinensis) orchard soil

The purpose of this study was to determine the ammonia-oxidizing activity and the phylogentic composition of microorganisms involved in acid tea (Camellia sinensis) orchard soil. All soil samples were collected from three sites located in Tahara and Toyohashi, Aichi Prefecture, Japan. The potential nitrification rate (PNR) was measured by the chlorate inhibition method. The soil pH of tea orchards studied ranged from 2.78 to 4.84, differing significantly from sample to sample, whereas that of meadow and unplanted fields ranged from 5.78 to 6.35. The PNR ranged from 0.050 to 0.193 μg NO2−-Ng−1 h−1 and were positively correlated with the soil pH (r2 = 0.382, p<0.001). Bulk DNA was extracted from a tea orchard soil (pH 4.8; PNR, 0.078 μg NO2−-Ng−1 h−1) and subjected to PCR-aided clone library analyses targeting archaeal and bacterial amoA genes. The detected archaeal clones separated from the cluster of the Soil clones and tightly clustered with the clones originating from other acidic soil environments including the Chinese tea orchard soil. These results suggest that the specific archaeal populations dominate as the ammonia oxidizers in acid tea-orchard soils and possibly other acid soils, independent of geographic locations, which results from the adaptation to specific ecological niches.

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 (Xcu202f=u202f39.9-46.1%) than with nearly amorphous PLLA (Xcu202f=u202f0.3-3.5%). 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 55u202f°C are more advantageous than mesophilic conditions at 37u202f°C.