Tatsuhiko Hoshino

Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor

A deep sleep in coal beds Deep below the ocean floor, microorganisms from forest soils continue to thrive. Inagaki et al. analyzed the microbial communities in several drill cores off the coast of Japan, some sampling more than 2 km below the seafloor (see the Perspective by Huber). Although cell counts decreased with depth, deep coal beds harbored active communities of methanogenic bacteria. These communities were more similar to those found in forest soils than in other deep marine sediments. Science, this issue p. 420; see also p. 376 Coal beds more than 2 kilometers below the seafloor host methanogenic bacteria related to those found in forest soils. [Also see Perspective by Huber] Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~104 cells cm−3. Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

ABSTRACT Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

Molecular quantification of environmental DNA using microfluidics and digital PCR

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58 ng μL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng μL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2) copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats.

Direct Detection by In Situ PCR of the amoA Gene in Biofilm Resulting from a Nitrogen Removal Process

ABSTRACT Ammonia oxidation is a rate-limiting step in the biological removal of nitrogen from wastewater. Analysis of microbial communities possessing the amoA gene, which is a small subunit of the gene encoding ammonia monooxygenase, is important for controlling nitrogen removal. In this study, the amoA gene present in Nitrosomonas europaea cells in a pure culture and biofilms in a nitrifying reactor was amplified by in situ PCR. In this procedure, fixed cells were permeabilized with lysozyme and subjected to seminested PCR with a digoxigenin-labeled primer. Then, the amplicon was detected with an alkaline phosphatase-labeled antidigoxigenin antibody and HNPP (2-hydroxy-3-naphthoic acid-2′-phenylanilide phosphate), which was combined with Fast Red TR, and with an Alexa Fluor 488-labeled antidigoxigenin antibody. The amoAgene in the biofilms was detected with an unavoidable nonspecific signal when the former method was used for detection. On the other hand, the amoA gene in the biofilms was detected without a nonspecific signal, and the cells possessing the amoAgene were clearly observed near the surface of the biofilm when Alexa Fluor 488-labeled antidigoxigenin antibody was used for detection. Although functional gene expression was not detected in this study, detection of cells in a biofilm based on their function was demonstrated.

Molecular analysis of microbial population transition associated with the start of denitrification in a wastewater treatment process

Aims:  The objective of this study is to determine the bacteria playing an important role in denitrification by monitoring the molecular dynamics accompanying the start of denitrification.

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

ABSTRACT A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.

Comparative Study of Subseafloor Microbial Community Structures in Deeply Buried Coral Fossils and Sediment Matrices from the Challenger Mound in the Porcupine Seabight

Subseafloor sedimentary environments harbor remarkably diverse microbial communities. However, it remains unknown if the deeply buried fossils in these sediments play ecological roles in deep microbial habitats, or whether the microbial communities inhabiting such fossils differ from those in the surrounding sediment matrix. Here we compare the community structures of subseafloor microbes in cold-water coral carbonates (Madrepora oculata and Lophelia pertusa) and the clay matrix. Samples were obtained from the Challenger Mound in the Porcupine Seabight at Site U1317 Hole A during the Integrated Ocean Drilling Program Expedition 307. DNA was extracted from coral fossils and the surrounding sedimentary matrix at 4, 20, and 105 m below the seafloor. 16S rRNA genes of Bacteria and Archaea were amplified by PCR, and a total of 213,792 16S rRNA gene-tagged sequences were analyzed. At the phylum level, dominant microbial components in both habitats consisted of Proteobacteria, Firmicutes, Nitrospirae, Chloroflexi, and Miscellaneous Crenarchaeota Group (MCG) at all three of the depths examined. However, at the genus and/or species level (similarity threshold 97.0%), the community compositions were found to be very different, with 69–75 and 46–57% of bacterial and archaeal phylotypes not overlapping in coral fossils and the clay matrix, respectively. Species richness analysis revealed that bacterial communities were generally more diverse than archaea, and that the diversity scores of coral fossils were lower than those in sediment matrix. However, the evenness of microbial communities was not significantly different in all the samples examined. No eukaryotic DNA sequences, such as 18S rRNA genes, were obtained from the corals. The findings suggested that, even at the same or similar depths, the sedimentological characteristics of a habitat are important factors affecting microbial diversity and community structure in deep subseafloor sedimentary habitats.

Biological CO2 conversion to acetate in subsurface coal-sand formation using a high-pressure reactor system

Geological CO2 sequestration in unmineable subsurface oil/gas fields and coal formations has been proposed as a means of reducing anthropogenic greenhouse gasses in the atmosphere. However, the feasibility of injecting CO2 into subsurface depends upon a variety of geological and economic conditions, and the ecological consequences are largely unpredictable. In this study, we developed a new flow-through-type reactor system to examine potential geophysical, geochemical and microbiological impacts associated with CO2 injection by simulating in-situ pressure (0–100 MPa) and temperature (0–70°C) conditions. Using the reactor system, anaerobic artificial fluid and CO2 (flow rate: 0.002 and 0.00001 ml/min, respectively) were continuously supplemented into a column comprised of bituminous coal and sand under a pore pressure of 40 MPa (confined pressure: 41 MPa) at 40°C for 56 days. 16S rRNA gene analysis of the bacterial components showed distinct spatial separation of the predominant taxa in the coal and sand over the course of the experiment. Cultivation experiments using sub-sampled fluids revealed that some microbes survived, or were metabolically active, under CO2-rich conditions. However, no methanogens were activated during the experiment, even though hydrogenotrophic and methylotrophic methanogens were obtained from conventional batch-type cultivation at 20°C. During the reactor experiment, the acetate and methanol concentration in the fluids increased while the δ13Cacetate, H2 and CO2 concentrations decreased, indicating the occurrence of homo-acetogenesis. 16S rRNA genes of homo-acetogenic spore-forming bacteria related to the genus Sporomusa were consistently detected from the sandstone after the reactor experiment. Our results suggest that the injection of CO2 into a natural coal-sand formation preferentially stimulates homo-acetogenesis rather than methanogenesis, and that this process is accompanied by biogenic CO2 conversion to acetate.

Monitoring the microbial population dynamics at the start-up stage of wastewater treatment reactor by terminal restriction fragment length polymorphism analysis based on 16S rDNA and rRNA gene sequences

The microbial population dynamics at the start-up stage of a wastewater treatment reactor was investigated using terminal restriction fragment length polymorphism (T-RFLP) analysis based on 16S rDNA and rRNA gene sequences. The results of fragment peaks suggested that the number and activity of nitrifying bacteria increased in association with the start of nitrification, and the relative ratios of 16S rRNA of these bacteria changed prior to those of the 16S rDNA. Furthermore, multidimensional scaling (MDS) analysis revealed that the 16S rRNA exhibited wider dispersion than the 16S rDNA at the start-up stage, indicating that the diversity of 16S rRNA in the microbial communities was strongly affected by environmental changes.