Selective phylogenetic analysis targeting 16S rRNA genes of hyperthermophilic archaea in the deep-subsurface hot biosphere

International drilling projects for the study of microbial communities in the deep-subsurface hot biosphere have been expanded. Core samples obtained by deep drilling are commonly contaminated with mesophilic microorganisms in the drilling fluid, making it difficult to examine the microbial community by 16S rRNA gene clone library analysis. To eliminate mesophilic organism contamination, we previously developed a new method (selective phylogenetic analysis [SePA]) based on the strong correlation between the guanine-plus-cytosine (G+C) contents of the 16S rRNA genes and the optimal growth temperatures of prokaryotes, and we verified the method's effectiveness (H. Kimura, M. Sugihara, K. Kato, and S. Hanada, Appl. Environ. Microbiol. 72:21-27, 2006). In the present study we ascertained SePA's ability to eliminate contamination by archaeal rRNA genes, using deep-sea hydrothermal fluid (117 degrees C) and surface seawater (29.9 degrees C) as substitutes for deep-subsurface geothermal samples and drilling fluid, respectively. Archaeal 16S rRNA gene fragments, PCR amplified from the surface seawater, were denatured at 82 degrees C and completely digested with exonuclease I (Exo I), while gene fragments from the deep-sea hydrothermal fluid remained intact after denaturation at 84 degrees C because of their high G+C contents. An examination using mixtures of DNAs from the two environmental samples showed that denaturation at 84 degrees C and digestion with Exo I completely eliminated archaeal 16S rRNA genes from the surface seawater. Our method was quite useful for culture-independent community analysis of hyperthermophilic archaea in core samples recovered from deep-subsurface geothermal environments.     

Selective Phylogenetic Analysis Targeted at 16S rRNA Genes of Thermophiles and Hyperthermophiles in Deep-Subsurface Geothermal Environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76°C) and river water (14°C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82°C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84°C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84°C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

Selective phylogenetic analysis targeted at 16S rRNA genes of thermophiles and hyperthermophiles in deep-subsurface geothermal environments

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.     

Microbial Community Stratification Controlled by the Subseafloor Fluid Flow and Geothermal Gradient at the Iheya North Hydrothermal Field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331)

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.     

Analysis of Dissimilatory Sulfite Reductase and 16S rRNA Gene Fragments from Deep-Sea Hydrothermal Sites of the Suiyo Seamount, Izu-Bonin Arc, Western Pacific

This study describes the occurrence of unique dissimilatory sulfite reductase (DSR) genes at a depth of 1,380 m from the deep-sea hydrothermal vent field at the Suiyo Seamount, Izu-Bonin Arc, Western Pacific, Japan. The DSR genes were obtained from microbes that grew in a catheter-type in situ growth chamber deployed for 3 days on a vent and from the effluent water of drilled holes at 5°C and natural vent fluids at 7°C. DSR clones SUIYOdsr-A and SUIYOdsr-B were not closely related to cultivated species or environmental clones. Moreover, samples of microbial communities were examined by PCR-denaturing gradient gel electrophoresis (DGGE) analysis of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragments obtained from the vent catheter after a 3-day incubation revealed the occurrence of bacterial DGGE bands affiliated with the Aquificae and γ- and -Proteobacteria as well as the occurrence of archaeal phylotypes affiliated with the Thermococcales and of a unique archaeon sequence that clustered with “Nanoarchaeota.” The DGGE bands obtained from drilled holes and natural vent fluids from 7 to 300°C were affiliated with the δ-Proteobacteria, genus Thiomicrospira, and Pelodictyon. The dominant DGGE bands retrieved from the effluent water of casing pipes at 3 and 4°C were closely related to phylotypes obtained from the Arctic Ocean. Our results suggest the presence of microorganisms corresponding to a unique DSR lineage not detected previously from other geothermal environments.     

16S rRNA Phylogenetic Investigation of the Candidate Division “Korarchaeota”

The environmental distribution and phylogeny of “Korarchaeota,” a proposed ancient archaeal division, was investigated by using the 16S rRNA gene framework. Korarchaeota-specific primers were designed based on previously published sequences and used to screen a variety of environments. Korarchaeota 16S rRNA genes were amplified exclusively from high temperature Yellowstone National Park hot springs and a 9°N East Pacific Rise deep-sea hydrothermal vent. Phylogenetic analyses of these and all available sequences suggest that Korarchaeota exhibit a high level of endemicity.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Anaerobic oxidation of methane at different temperature regimes in Guaymas Basin hydrothermal sediments

Anaerobic oxidation of methane (AOM) was investigated in hydrothermal sediments of Guaymas Basin based on δ¹³C signatures of CH₄, dissolved inorganic carbon and porewater concentration profiles of CH₄ and sulfate. Cool, warm and hot in-situ temperature regimes (15–20 °C, 30–35 °C and 70–95 °C) were selected from hydrothermal locations in Guaymas Basin to compare AOM geochemistry and 16S ribosomal RNA (rRNA), mcrA and dsrAB genes of the microbial communities. 16S rRNA gene clone libraries from the cool and hot AOM cores yielded similar archaeal types such as Miscellaneous Crenarchaeotal Group, Thermoproteales and anaerobic methane-oxidizing archaea (ANME)-1; some of the ANME-1 archaea formed a separate 16S rRNA lineage that at present seems to be limited to Guaymas Basin. Congruent results were obtained by mcrA gene analysis. The warm AOM core, chemically distinct by lower porewater sulfide concentrations, hosted a different archaeal community dominated by the two deep subsurface archaeal lineages Marine Benthic Group D and Marine Benthic Group B, and by members of the Methanosarcinales including ANME-2 archaea. This distinct composition of the methane-cycling archaeal community in the warm AOM core was confirmed by mcrA gene analysis. Functional genes of sulfate-reducing bacteria and archaea, dsrAB, showed more overlap between all cores, regardless of the core temperature. 16S rRNA gene clone libraries with Euryarchaeota-specific primers detected members of the Archaeoglobus clade in the cool and hot cores. A V6-tag high-throughput sequencing survey generally supported the clone library results while providing high-resolution detail on archaeal and bacterial community structure. These results indicate that AOM and the responsible archaeal communities persist over a wide temperature range.     

Culture-Dependent and -Independent Characterization of Microbial Communities Associated with a Shallow Submarine Hydrothermal System Occurring within a Coral Reef off Taketomi Island, Japan

Microbial communities in a shallow submarine hydrothermal system near Taketomi Island, Japan, were investigated using cultivation-based and molecular techniques. The main hydrothermal activity occurred in a craterlike basin (depth, ~23 m) on the coral reef seafloor. The vent fluid (maximum temperature, >52°C) contained 175 μM H₂S and gas bubbles mainly composed of CH₄ (69%) and N₂ (29%). A liquid serial dilution cultivation technique targeting a variety of metabolism types quantified each population in the vent fluid and in a white microbial mat located near the vent. The most abundant microorganisms cultivated from both the fluid and the mat were autotrophic sulfur oxidizers, including mesophilic Thiomicrospira spp. and thermophilic Sulfurivirga caldicuralii. Methane oxidizers were the second most abundant organisms in the fluid; one novel type I methanotroph exhibited optimum growth at 37°C, and another novel type I methanotroph exhibited optimum growth at 45°C. The number of hydrogen oxidizers cultivated only from the mat was less than the number of sulfur and methane oxidizers, although a novel mesophilic hydrogen-oxidizing member of the Epsilonproteobacteria was isolated. Various mesophilic to hyperthermophilic heterotrophs, including sulfate-reducing Desulfovibrio spp., iron-reducing Deferribacter sp., and sulfur-reducing Thermococcus spp., were also cultivated. Culture-independent 16S rRNA gene clone analysis of the vent fluid and mat revealed highly diverse archaeal communities. In the bacterial community, S. caldicuralii was identified as the predominant phylotype in the fluid (clonal frequency, 25%). Both bacterial clone libraries indicated that there were bacterial communities involved in sulfur, hydrogen, and methane oxidation and sulfate reduction. Our results indicate that there are unique microbial communities that are sustained by active chemosynthetic primary production rather than by photosynthetic production in a shallow hydrothermal system where sunlight is abundant.     

Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.     

Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.     

Phylogenetic Analysis of Nonthermophilic Members of the Kingdom Crenarchaeota and Their Diversity and Abundance in Soils

Within the last several years, molecular techniques have uncovered numerous 16S rRNA gene (rDNA) sequences which represent a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species. rDNA sequences of members of this novel crenarchaeotal group have been recovered from low- to moderate-temperature environments (−1.5 to 32°C), in contrast to the high-temperature environments (temperature, >80°C) required for growth of the currently recognized crenarchaeotal species. We determined the diversity and abundance of the nonthermophilic members of the Crenarchaeota in soil samples taken from cultivated and uncultivated fields located at the Kellogg Biological Station’s Long-Term Ecological Research site (Hickory Corners, Mich.). Clones were generated from 16S rDNA that was amplified by using broad-specificity archaeal PCR primers. Twelve crenarchaeotal sequences were identified, and the phylogenetic relationships between these sequences and previously described crenarchaeotal 16S rDNA sequences were determined. Phylogenetic analyses included nonthermophilic crenarchaeotal sequences found in public databases and revealed that the nonthermophilic Crenarchaeota group is composed of at least four distinct phylogenetic clusters. A 16S rRNA-targeted oligonucleotide probe specific for all known nonthermophilic crenarchaeotal sequences was designed and used to determine their abundance in soil samples. The nonthermophilic Crenarchaeota accounted for as much as 1.42% ± 0.42% of the 16S rRNA in the soils analyzed.     

Occurrence of Shewanella algae in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival

The marine bacterium Shewanella algae, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of S. algae decreased rapidly from 10⁷ CFU/ml to 10¹ CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 10⁵ to 10⁷ CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that S. algae would survive cold exposure better if in the biofilm state; however, culturable counts from S. algae biofilms decreased as rapidly as did counts of planktonic cells.     

Humboldt's spa: microbial diversity is controlled by temperature in geothermal environments

Over 200 years ago Alexander von Humboldt (1808) observed that plant and animal diversity peaks at tropical latitudes and decreases toward the poles, a trend he attributed to more favorable temperatures in the tropics. Studies to date suggest that this temperature–diversity gradient is weak or nonexistent for Bacteria and Archaea. To test the impacts of temperature as well as pH on bacterial and archaeal diversity, we performed pyrotag sequencing of 16S rRNA genes retrieved from 165 soil, sediment and biomat samples of 36 geothermal areas in Canada and New Zealand, covering a temperature range of 7.5–99 °C and a pH range of 1.8–9.0. This represents the widest ranges of temperature and pH yet examined in a single microbial diversity study. Species richness and diversity indices were strongly correlated to temperature, with R² values up to 0.62 for neutral–alkaline springs. The distributions were unimodal, with peak diversity at 24 °C and decreasing diversity at higher and lower temperature extremes. There was also a significant pH effect on diversity; however, in contrast to previous studies of soil microbial diversity, pH explained less of the variability (13–20%) than temperature in the geothermal samples. No correlation was observed between diversity values and latitude from the equator, and we therefore infer a direct temperature effect in our data set. These results demonstrate that temperature exerts a strong control on microbial diversity when considered over most of the temperature range within which life is possible.     

Molecular Relationship between Two Groups of the Genus Leptospirillum and the Finding that Leptospirillum ferriphilum sp. nov. Dominates South African Commercial Biooxidation Tanks That Operate at 40°C

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.     

Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30°C) and higher (35 to 38°C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.     

Effect of Incubation Temperature on the Detection of Thermophilic Campylobacter Species from Freshwater Beaches,Nearby Wastewater Effluents,and Bird Fecal Droppings

This large-scale study compared incubation temperatures (37°C versus 42°C) to study the detection of thermophilic Campylobacter species, including Campylobacter jejuni, C. coli, and C. lari, in various surface water samples and bird fecal droppings around Hamilton Harbor, Lake Ontario. The putative culture isolates obtained from incubation temperatures of 37 and 42°C were confirmed by Campylobacter genus- and species-specific triplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA gene internal transcribed spacer (ITS) region. A total of 759 water, wastewater, and bird fecal dropping samples were tested. Positive amplification reactions for the genus Campylobacter were found for 454 (60%) samples incubated at 37°C, compared to 258 (34%) samples incubated at 42°C. C. jejuni (16%) and C. lari (12%) were detected significantly more frequently at the 42°C incubation temperature than at 37°C (8% and 5%, respectively). In contrast, significantly higher rates of C. coli (14%) and other Campylobacter spp. (36%) were detected at the 37°C incubation temperature than at 42°C (8% and 7%, respectively). These results were consistent across surface water, wastewater, and bird fecal dropping samples. At times, Campylobacter spp. were recovered and detected at 37°C (3% for C. jejuni, 10% for C. coli, and 3% for C. lari) when the same samples incubated at 42°C were negative. A significantly higher rate of other Campylobacter spp. was detected only at 37°C (32%) than only at 42°C (3%). These results indicate that incubation temperature can significantly influence the culturability and detection of thermophilic and other fastidious Campylobacter spp. and that a comprehensive characterization of the Campylobacter spp. in surface water, wastewaters, or bird fecal droppings will require incubation at both 37 and 42°C.     

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the division Proteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).     

Expanding the Verrucomicrobial Methanotrophic World: Description of Three Novel Species of Methylacidimicrobium gen. nov.

Methanotrophic Verrucomicrobia have been found in geothermal environments characterized by high temperatures and low pH values. However, it has recently been hypothesized that methanotrophic Verrucomicrobia could be present under a broader range of environmental conditions. Here we describe the isolation and characterization of three new species of mesophilic acidophilic verrucomicrobial methanotrophs from a volcanic soil in Italy. The three new species showed 97% to 98% 16S rRNA gene identity to each other but were related only distantly (89% to 90% on the 16S rRNA level) to the thermophilic genus Methylacidiphilum. We propose the new genus Methylacidimicrobium, including the novel species Methylacidimicrobium fagopyrum, Methylacidimicrobium tartarophylax, and Methylacidimicrobium cyclopophantes. These mesophilic Methylacidimicrobium spp. were more acid tolerant than their thermophilic relatives; the most tolerant species, M. tartarophylax, still grew at pH 0.5. The variation in growth temperature optima (35 to 44°C) and maximum growth rates (µmax; 0.013 to 0.040 h⁻¹) suggested that all species were adapted to a specific niche within the geothermal environment. All three species grew autotrophically using the Calvin cycle. The cells of all species contained glycogen particles and electron-dense particles in their cytoplasm as visualized by electron microscopy. In addition, the cells of one of the species (M. fagopyrum) contained intracytoplasmic membrane stacks. The discovery of these three new species and their growth characteristics expands the known diversity of verrucomicrobial methanotrophs and shows that they are present in many more ecosystems than previously assumed.     

Simultaneous Direct Counting of Total and Specific Microbial Cells in Seawater, Using a Deep-Sea Microbe as Target

To rapidly and accurately enumerate total and specific microbes in aquatic samples, fluorescent in situ hybridization was combined with direct counting via direct immobilization of cells on a polymer-coated Nuclepore filter. The technique, named FISH-DC, achieved almost complete recovery of total cells and reproducibility of Psychrobacter pacificensis cells of deep-sea origin (error, ≤3%) in a mixed culture and in natural seawater. Target cells immobilized on the filter were also successfully enumerated after stringent 3-cycle hybridization and even after a 16-month preservation at −30°C.