Motoo Utsumi

Biogeography and biodiversity in sulfide structures of active and inactive vents at deep-sea hydrothermal fields of the Southern Mariana Trough.

ABSTRACT The abundance, diversity, activity, and composition of microbial communities in sulfide structures both of active and inactive vents were investigated by culture-independent methods. These sulfide structures were collected at four hydrothermal fields, both on- and off-axis of the back-arc spreading center of the Southern Mariana Trough. The microbial abundance and activity in the samples were determined by analyzing total organic content, enzymatic activity, and copy number of the 16S rRNA gene. To assess the diversity and composition of the microbial communities, 16S rRNA gene clone libraries including bacterial and archaeal phylotypes were constructed from the sulfide structures. Despite the differences in the geological settings among the sampling points, phylotypes related to the Epsilonproteobacteria and cultured hyperthermophilic archaea were abundant in the libraries from the samples of active vents. In contrast, the relative abundance of these phylotypes was extremely low in the libraries from the samples of inactive vents. These results suggest that the composition of microbial communities within sulfide structures dramatically changes depending on the degree of hydrothermal activity, which was supported by statistical analyses. Comparative analyses suggest that the abundance, activity and diversity of microbial communities within sulfide structures of inactive vents are likely to be comparable to or higher than those in active vent structures, even though the microbial community composition is different between these two types of vents. The microbial community compositions in the sulfide structures of inactive vents were similar to those in seafloor basaltic rocks rather than those in marine sediments or the sulfide structures of active vents, suggesting that the microbial community compositions on the seafloor may be constrained by the available energy sources. Our findings provide helpful information for understanding the biogeography, biodiversity and microbial ecosystems in marine environments.

Abundance of Zetaproteobacteria within crustal fluids in back-arc hydrothermal fields of the Southern Mariana Trough

To extend knowledge of subseafloor microbial communities within the oceanic crust, the abundance, diversity and composition of microbial communities in crustal fluids at back-arc hydrothermal fields of the Southern Mariana Trough (SMT) were investigated using culture-independent molecular techniques based on 16S rRNA gene sequences. Seafloor drilling was carried out at two hydrothermal fields, on- and off-ridge of the back-arc spreading centre of the SMT. 16S rRNA gene clone libraries for bacterial and archaeal communities were constructed from the fluid samples collected from the boreholes. Phylotypes related to Thiomicrospira in the Gammaproteobacteria (putative sulfide-oxidizers) and Mariprofundus in the Zetaproteobacteria (putative iron-oxidizers) were recovered from the fluid samples. A number of unique archaeal phylotypes were also recovered. Fluorescence in situ hybridization (FISH) analysis indicated the presence of active bacterial and archaeal populations in the fluids. The Zetaproteobacteria accounted for up to 32% of the total prokaryotic cell number as shown by FISH analysis using a specific probe designed in this study. Our results lead to the hypothesis that the Zetaproteobacteria play a role in iron oxidation within the oceanic crust.

Methane production from rice straw pretreated by a mixture of acetic-propionic acid.

Rice straw was treated with a mixed solution of acetic acid and propionic acid to enhance its biodegradability. The effect of acid concentration, pretreatment time, and the ratio of solid to liquid on the delignification performance of rice straw were investigated. It was found that the optimal conditions for hydrolysis were 0.75 mol/L acid concentration, 2h pretreatment time and 1:20 solid to liquid ratio. Batch methane fermentation of untreated rice straw, pretreated rice straw, and the hydrolysates (the liquid fraction) of pretreatment were conducted at 35 degrees C for 30 days, and the results indicated that methane production of rice straw can be enhanced by dilute organic acid pretreatment. Moreover, most of the acid in hydrolysates can also be converted into methane gas.

Characteristics of a Microcystin-Degrading Bacterium under Alkaline Environmental Conditions

The pH of the water associated with toxic blooms of cyanobacteria is typically in the alkaline range; however, previously only microcystin-degrading bacteria growing in neutral pH conditions have been isolated. Therefore, we sought to isolate and characterize an alkali-tolerant microcystin-degrading bacterium from a water bloom using microcystin-LR. Analysis of the 16S rRNA gene sequence revealed that the isolated bacterium belonged to the genus Sphingopyxis, and the strain was named C-1. Sphingopyxis sp. C-1 can grow; at pH 11.0; however, the optimum pH for growth was pH 7.0. The microcystin degradation activity of the bacterium was the greatest between pH 6.52 and pH 8.45 but was also detected at pH 10.0. The mlrA homolog encoding the microcystin-degrading enzyme in the C-1 strain was conserved. We concluded that alkali-tolerant microcystin-degrading bacterium played a key role in triggering the rapid degradation of microcystin, leading to the disappearance of toxic water blooms in aquatic environments.

Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1

The mlr gene cluster consisting of mlrA, mlrB, mlrC, and mlrD is involved in the degradation of the cyanobacterial toxin microcystin. However, it is unclear which degradation intermediates are metabolized by MlrB and MlrC. To address these questions, we constructed recombinant Escherichia coli to overproduce MlrB and MlrC from Sphingopyxis sp. C-1, and determined which intermediates were degraded in cell-free extracts. The cell-free extract containing MlrB degraded linearized microcystin-LR, giving rise to a tetrapeptide. The cell-free extract of MlrC degraded linearized microcystin-LR and also degraded the tetrapeptide to the amino acid Adda. These results indicate that linearized microcystin-LR is degraded by both MlrB and MlrC, and tetrapeptide is degraded by specifically by MlrC in Sphingopyxis sp. C-1.

Investigations into the biodegradation of microcystin-LR mediated by the biofilm in wintertime from a biological treatment facility in a drinking-water treatment plant

The potential of winter biofilm for microcystin-LR (MCLR) biodegradation was comparatively evaluated under various nutrient conditions. Results indicated that MCLR was completely biodegraded by Day 7 without nutrient addition. MCLR-biodegradation was inhibited in the presence of phosphate or glucose addition, with complete MCLR removal observed by Day 10. MCLR was totally biodegraded by Day 7 with dual nutrients comprising glucose and nitrate, suggesting that additional nitrate alleviated the inhibitory effect of glucose alone on the biodegradation. Simultaneously, MCLR-degrading gene (mlrA) abundance were detected to increase with increasing amount of MCLR being degraded under the respective conditions, implying that MCLR-biodegradation depended on the population of indigenous MCLR-degrading bacteria (MCLRDB), which was related to the population of non-degrading bacteria in the biofilm. MCLRDB was found to primarily use MCLR for proliferation rather than other nutrients. This is the first report verifying MCLR as a primary substrate for bacteria under various nutrient conditions.

Comparative study for the effects of variable nutrient conditions on the biodegradation of microcystin-LR and concurrent dynamics in microcystin-degrading gene abundance.

Microcystin-LR (MCLR) degradation capability of biofilm was investigated with and without additional nutrients (nitrate, ammonium, peptone and glucose) at concentrations of 100 and 1000 mg L(-1). The MCLR-degradation was stimulated with nitrate and inhibited with other nutrients, except for that glucose of low concentration had no obvious effect. Both stimulatory and inhibitory effects enhanced with increasing concentration of corresponding nutrient. Quantitative polymerase chain reaction (qPCR) indicated that enhanced inhibition in biodegradation correlated to increased inhibition in functional gene (mlrA) abundance, as nutrient concentration increased. Stimulated biodegradation under low nitrate concentration may result from more rapid increase in mlrA gene abundance. These suggested that MCLR-degradation largely depended upon responsible bacterial population, which was affected by population of other bacteria in biofilm according to 16S rDNA-targeting qPCR. However, inhibited mlrA gene abundance implied that the stimulated biodegradation under high nitrate concentration might be involved in the mechanisms not related to MCLRDB population.

Dynamics of the functional gene copy number and overall bacterial community during microcystin-LR degradation by a biological treatment facility in a drinking water treatment plant.

Information is limited on the potential for microcystins (MCs) degradation by carrier-attached biofilms obtained in winter that were not exposed to detectable levels of MCs in the preceding months. Under controlled laboratory conditions, we confirmed that microcystin-LR (MCLR) was effectively biodegraded within 5.5 days in cultures of the biofilm sampled in winter. Quantitative polymerase chain reaction (qPCR) assays revealed that seasonal variations in the MCLR-degradation potential of the biofilm were closely related to the initial MCLR-degrader population in the biofilm. Indigenous MCLR-degraders in the biofilm could accumulate by exposure to natural MCLR in the water column, accelerating MCLR-degradation. The qPCR assay suggested that MCLR may be a primary substrate for the degraders in the presence of another labile organic carbon associated with the biofilm under the present study conditions. qPCR and PCR-denaturing gradient gel electrophoresis (DGGE) for 16S rDNA demonstrated that the overall bacterial population from the winter biofilm rapidly increased with the MCLR-degrader population and remained stable after day 3.5, while the overall bacterial community structure shifted throughout the entire biodegradation period. This study is important to the in-depth understanding of microbial degradation of MCs and could facilitate the bioremediation of MCs in polluted habitats.

Anaerobic Coculture of Microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C Enhances Generation of n-Alkane-Rich Biofuels after Pyrolysis

ABSTRACT We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH4 production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Characteristics of Microbial Communities in Crustal Fluids in a Deep-Sea Hydrothermal Field of the Suiyo Seamount

To directly access the sub-seafloor microbial communities, seafloor drilling has been done in a deep-sea hydrothermal field of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific. In the present study, crustal fluids were collected from the boreholes, and the bacterial and archaeal communities in the fluids were investigated by culture-independent molecular analysis based on 16S rRNA gene sequences. Bottom seawater, sands, rocks, sulfide mound, and chimneys were also collected around the boreholes and analyzed for comparisons. Comprehensive analysis revealed the characteristics of the microbial community composition in the crustal fluids. Phylotypes closely related to cultured species, e.g., Alteromonas, Halomonas, Marinobacter, were relatively abundant in some crustal fluid samples, whereas the phylotypes related to Pelagibacter and the SUP05-group were relatively abundant in the seawater samples. Phylotypes related to other uncultured environmental clones in Alphaproteobacteria and Gammaproteobacteria were relatively abundant in the sand, rock, sulfide mound, and chimney samples. Furthermore, comparative analysis with previous studies of the Suiyo Seamount crustal fluids indicates the change in the microbial community composition for 3 years. Our results provide novel insights into the characteristics of the microbial communities in crustal fluids beneath a deep-sea hydrothermal field.