Suzanna L. Bräuer

Isolation of a novel acidiphilic methanogen from an acidic peat bog

Acidic peatlands are among the largest natural sources of atmospheric methane and harbour a large diversity of methanogenic Archaea. Despite the ubiquity of methanogens in these peatlands, indigenous methanogens capable of growth at acidic pH values have resisted culture and isolation; these recalcitrant methanogens include members of an uncultured family-level clade in the Methanomicrobiales prevalent in many acidic peat bogs in the Northern Hemisphere. However, we recently succeeded in obtaining a mixed enrichment culture of a member of this clade. Here we describe its isolation and initial characterization. We demonstrate that the optimum pH for methanogenesis by this organism is lower than that of any previously described methanogen.

Methanoregula boonei gen. nov., sp. nov., an acidiphilic methanogen isolated from an acidic peat bog

A novel acidiphilic, hydrogenotrophic methanogen, designated strain 6A8(T), was isolated from an acidic (pH 4.0-4.5) and ombrotrophic (rain-fed) bog located near Ithaca, NY, USA. Cultures were dimorphic, containing thin rods (0.2-0.3 μm in diameter and 0.8-3.0 μm long) and irregular cocci (0.2-0.8 μm in diameter). The culture utilized H(2)/CO(2) to produce methane but did not utilize formate, acetate, methanol, ethanol, 2-propanol, butanol or trimethylamine. Optimal growth conditions were near pH 5.1 and 35 °C. The culture grew in basal medium containing as little as 0.43 mM Na(+) and growth was inhibited completely by 50 mM NaCl. To our knowledge, strain 6A8(T) is one of the most acidiphilic (lowest pH optimum) and salt-sensitive methanogens in pure culture. Acetate, coenzyme M, vitamins and yeast extract were required for growth. It is proposed that a new genus and species be established for this organism, Methanoregula boonei gen. nov., sp. nov. The type strain of Methanoregula boonei is 6A8(T) (=DSM 21154(T) =JCM 14090(T)).

Methanogenesis in McLean Bog, an Acidic Peat Bog in Upstate New York: Stimulation by H2/CO2 in the Presence of Rifampicin, or by Low Concentrations of Acetate

Acidic peat bog soils produce CH4 and although molecular biological studies have demonstrated the presence of diverse methano-genic populations in them, few studies have sustained methanogenesis by adding the CH4 precursors H2/CO2 or acetate, and few indigenous methanogens have been cultured. McLean Bog is a small (ca. 70 m across), acidic (pH 3.4–4.3) Sphagnum -dominated bog in upstate New York. Although addition of H2/CO2 or 10 mM acetate stimulated methanogenesis in soils from a nearby circumneutral-pH fen, neither of these substrates led to sustained methanogenesis in McLean Bog soil slurries. After a brief period of stimulation by H2/CO2, methanogenesis in McLean Bog soil declined, which could be attributed to buildup of large amounts of acetic acid produced from the H2/CO2 by acetogens. Addition of the antibiotic rifampicin inhibited acetogenesis (carried out by Bacteria) and allowed methanogenesis (carried out by Archaea) to continue. Using rifampicin, we were able to study effects of temperature, pH, and salts on methanogenesis from H2/CO2 in McLean Bog soil samples. The enriched H2/CO2-utilizing methanogens showed an optimum for activity near pH 5, and a temperature optimum near 35°C. Methanogenesis was not stimulated by addition of 10 mM acetate, but it was stimulated by 1 mM acetate, and multiple additions were consumed at increasing rates and nearly stoichiometrically converted to CH4. In conclusion, we have found that both hydrogentrophic and aceticlastic methanogens are present in McLean Bog soils, and that methanogenic activity can be stimulated using H2/CO2 in the presence of rifampicin, or using low concentrations of acetate.

Mn(II)-Oxidizing Bacteria Are Abundant And Environmentally Relevant Members Of Ferromanganese Deposits In Caves Of The Upper Tennessee River Basin

The upper Tennessee River Basin contains the highest density of our nations caves; yet, little is known regarding speleogenesis or Fe and Mn biomineralization in these predominantly epigenic systems. Mn:Fe ratios of Mn and Fe oxide-rich biofilms, coatings, and mineral crusts that were abundant in several different caves ranged from ca. 0.1 to 1.0 as measured using ICP-OES. At sites where the Mn:Fe ratio approached 1.0 this represented an order of magnitude increase above the bulk bedrock ratio, suggesting that biomineralization processes play an important role in the formation of these cave ferromanganese deposits. Estimates of total bacterial SSU rRNA genes in ferromanganese biofilms, coatings, and crusts measured approximately 7×107–9×109 cells/g wet weight sample. A SSU-rRNA based molecular survey of biofilm material revealed that 21% of the 34 recovered dominant (non-singleton) OTUs were closely related to known metal-oxidizing bacteria or clones isolated from oxidized metal deposits. Several different isolates that promote the oxidation of Mn(II) compounds were obtained in this study, some from high dilutions (10–8–10–10) of deposit material. In contrast to studies of caves in other regions, SSU rRNA sequences of Mn-oxidizing bacterial isolates in this study most closely matched those of Pseudomonas, Leptothrix, Flavobacterium, and Janthinobacterium. Combined data from geochemical analyses, molecular surveys, and culture-based experiments suggest that a unique consortia of Mn(II)-oxidizing bacteria are abundant and promoting biomineralization processes within the caves of the upper Tennessee River Basin.

Methanolinea Mesophila Sp. Nov., A Hydrogenotrophic Methanogen Isolated From Rice Field Soil, And Proposal Of The Archaeal Family Methanoregulaceae Fam. Nov. Within The Order Methanomicrobiales

A novel mesophilic, hydrogenotrophic methanogen, designated strain TNR(T), was isolated from an anaerobic, propionate-degradation enrichment culture that was originally established from a rice field soil sample from Taiwan. Cells were non-motile rods, 2.0-6.5 µm long by 0.3 µm wide. Filamentous (up to about 100 µm) and coccoid (about 1 µm in diameter) cells were also observed in cultures in the late exponential phase of growth. Strain TNR(T) grew at 20-40 °C (optimally at 37 °C), at pH 6.5-7.4 (optimally at pH 7.0) and in the presence of 0-25 g NaCl l(-1) (optimally at 0 g NaCl l(-1)). The strain utilized H(2)/CO(2) and formate for growth and produced methane. The G+C content of the genomic DNA was 56.4 mol%. Based on sequences of both the 16S rRNA gene and the methanogen-specific marker gene mcrA, strain TNR(T) was related most closely to Methanolinea tarda NOBI-1(T); levels of sequence similarities were 94.8 and 86.4 %, respectively. The 16S rRNA gene sequence similarity indicates that strain TNR(T) and M. tarda NOBI-1(T) represent different species within the same genus. This is supported by shared phenotypic properties, including substrate usage and cell morphology, and differences in growth temperature. Based on these genetic and phenotypic properties, strain TNR(T) is considered to represent a novel species of the genus Methanolinea, for which the name Methanolinea mesophila sp. nov. is proposed; the type strain is TNR(T) ( = NBRC 105659(T) = DSM 23604(T)). In addition, we also suggest family status for the E1/E2 group within the order Methanomicrobiales, for which the name Methanoregulaceae fam. nov. is proposed; the type genus of family is Methanoregula.

Aurantimonas manganoxydans, sp. nov. and Aurantimonas litoralis, sp. nov.: Mn(II) Oxidizing Representatives of a Globally Distributed Clade of alpha-Proteobacteria from the Order Rhizobiales

Several closely related Mn(II)-oxidizing alpha-Proteobacteria were isolated from very different marine environments: strain SI85-9A1 from the oxic/anoxic interface of a stratified Canadian fjord, strain HTCC 2156 from the surface waters off the Oregon coast, and strain AE01 from the dorsal surface of a hydrothermal vent tubeworm. 16S rRNA analysis reveals that these isolates are part of a tight phylogenetic cluster with previously characterized members of the genus Aurantimonas. Other organisms within this clade have been isolated from disparate environments such as surface waters of the Arctic and Mediterranean seas, a deep-sea hydrothermal plume, and a Caribbean coral. Further analysis of all these strains revealed that many of them are capable of oxidizing dissolved Mn(II) and producing particulate Mn(III/IV) oxides. Strains SI85-9A1 and HTCC 2156 were characterized further. Despite sharing nearly identical 16S rRNA gene sequences with the previously described Aurantimonas coralicida, whole genome DNA-DNA hybridization indicated that their overall genomic similarity is low. Polyphasic phenotype characterization further supported distinguishing characteristics among these bacteria. Thus SI85-9A1 and HTCC 2156 are described as two new species within the family ‘Aurantimonadaceae’: Aurantimonas manganoxydans sp. nov. and Aurantimonas litoralis sp. nov. This clade of bacteria is widely distributed around the globe and may be important contributors to Mn cycling in many environments. Our results highlight the difficulty in utilizing 16S rRNA-based approaches to investigate the microbial ecology of Mn(II) oxidation.

Limitations and benefits of ARISA intra-genomic diversity fingerprinting.

Monitoring diversity changes and contamination in mixed cultures and simple microcosms is challenged by fast community structure dynamics, and the need for means allowing fast, cost-efficient and accurate identification of microorganisms at high phylogenetic resolution. The method we explored is a variant of Automated rRNA Intergenic Spacer Analysis based on Intra-Genomic Diversity Fingerprinting (ARISA-IGDF), and identifies phylotypes with multiple 16S-23S rRNA gene Intergenic Transcribed Spacers. We verified the effect of PCR conditions (annealing temperature, duration of final extension, number of cycles, group-specific primers and formamide) on ARISA-IGD fingerprints of 44 strains of Shewanella. We present a digitization algorithm and data analysis procedures needed to determine confidence in strain identification. Though using stringent PCR conditions and group-specific primers allow reasonably accurate identification of strains with three ARISA-IGD amplicons within the 82-1000 bp size range, ARISA-IGDF is best for phylotypes with >or=4 unambiguously different amplicons. This method allows monitoring the occurrence of culturable microbes and can be implemented in applications requiring high phylogenetic resolution, reproducibility, low cost and high throughput such as identifying contamination and monitoring the evolution of diversity in mixed cultures and low diversity microcosms and periodic screening of small microbial culture libraries.

Ubiquitous Dissolved Inorganic Carbon Assimilation by Marine Bacteria in the Pacific Northwest Coastal Ocean as Determined by Stable Isotope Probing

In order to identify bacteria that assimilate dissolved inorganic carbon (DIC) in the northeast Pacific Ocean, stable isotope probing (SIP) experiments were conducted on water collected from 3 different sites off the Oregon and Washington coasts in May 2010, and one site off the Oregon Coast in September 2008 and March 2009. Samples were incubated in the dark with 2 mM 13C-NaHCO3, doubling the average concentration of DIC typically found in the ocean. Our results revealed a surprising diversity of marine bacteria actively assimilating DIC in the dark within the Pacific Northwest coastal waters, indicating that DIC fixation is relevant for the metabolism of different marine bacterial lineages, including putatively heterotrophic taxa. Furthermore, dark DIC-assimilating assemblages were widespread among diverse bacterial classes. Alphaproteobacteria, Gammaproteobacteria, and Bacteroidetes dominated the active DIC-assimilating communities across the samples. Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia were also implicated in DIC assimilation. Alteromonadales and Oceanospirillales contributed significantly to the DIC-assimilating Gammaproteobacteria within May 2010 clone libraries. 16S rRNA gene sequences related to the sulfur-oxidizing symbionts Arctic96BD-19 were observed in all active DIC assimilating clone libraries. Among the Alphaproteobacteria, clones related to the ubiquitous SAR11 clade were found actively assimilating DIC in all samples. Although not a dominant contributor to our active clone libraries, Betaproteobacteria, when identified, were predominantly comprised of Burkholderia. DIC-assimilating bacteria among Deltaproteobacteria included members of the SAR324 cluster. Our research suggests that DIC assimilation is ubiquitous among many bacterial groups in the coastal waters of the Pacific Northwest marine environment and may represent a significant metabolic process.

Seasonal Changes In Methanogenesis And Methanogenic Community In Three Peatlands, New York State

Fluctuating environmental conditions can promote diversity and control dominance in community composition. In addition to seasonal temperature and moisture changes, seasonal supply of metabolic substrates selects populations temporally. Here we demonstrate cascading effects in the supply of metabolic substrates on methanogenesis and community composition of anaerobic methanogenic archaea in three contrasting peatlands in upstate New York. Fresh samples of peat soils, collected about every 3 months for 20 months and incubated at 22 ± 2°C regardless of the in situ temperature, exhibited potential rates of methane (CH4) production of 0.02–0.2 mmol L−1 day−1 [380–3800 nmol g−1 (dry) day−1). The addition of acetate stimulated rates of CH4 production in a fen peatland soil, whereas addition of hydrogen (H2), and simultaneous inhibition of H2-consuming acetogenic bacteria with rifampicin, stimulated CH4 production in two acidic bog soils, especially, in autumn and winter. The methanogenic community structure was characterized using T-RFLP analyses of SSU rRNA genes. The E2 group of methanogens (Methanoregulaceae) dominated in the two acidic bog peatlands with relatively greater abundance in winter. In the fen peatland, the E1 group (Methanoregulaceae) and members of the Methanosaetaceae were co-dominant, with E1 having a high relative abundance in spring. Change in relative abundance profiles among methanogenic groups in response to added metabolic substrates was as predicted. The acetate-amendment increased abundance of Methanosarcinaceae, and H2-amendment enhanced abundance of E2 group in all peat soils studied, respectively. Additionally, addition of acetate increased abundance of Methanosaetaceae only in the bog soils. Variation in the supply of metabolic substrates helps explain the moderate diversity of methanogens in peatlands.

Culturable Rhodobacter and Shewanella species are abundant in estuarine turbidity maxima of the Columbia River

Measurements of dissolved, ascorbate-reducible and total Mn by ICP-OES revealed significantly higher concentrations during estuarine turbidity maxima (ETM) events, compared with non-events in the Columbia River. Most probable number (MPN) counts of Mn-oxidizing or Mn-reducing heterotrophs were not statistically different from that of other heterotrophs (10³ -10⁴ cells ml⁻¹) when grown in defined media, but counts of Mn oxidizers were significantly lower in nutrient-rich medium (13 cells ml⁻¹). MPN counts of Mn oxidizers were also significantly lower on Mn(III)-pyrophosphate and glycerol (21 cells ml⁻¹). Large numbers of Rhodobacter spp. were cultured from dilutions of 10⁻² to 10⁻⁵, and many of these were capable of Mn(III) oxidation. Up to c. 30% of the colonies tested LBB positive, and all 77 of the successfully sequenced LBB positive colonies (of varying morphology) yielded sequences related to Rhodobacter spp. qPCR indicated that a cluster of Rhodobacter isolates and closely related strains (95-99% identity) represented approximately 1-3% of the total Bacteria, consistent with clone library results. Copy numbers of SSU rRNA genes for either Rhodobacter spp. or Bacteria were four to eightfold greater during ETM events compared with non-events. Strains of a Shewanella sp. were retrieved from the highest dilutions (10⁻⁵) of Mn reducers, and were also capable of Mn oxidation. The SSU rRNA gene sequences from these strains shared a high identity score (98%) with sequences obtained in clone libraries. Our results support previous findings that ETMs are zones with high microbial activity. Results indicated that Shewanella and Rhodobacter species were present in environmentally relevant concentrations, and further demonstrated that a large proportion of culturable bacteria, including Shewanella and Rhodobacter spp., were capable of Mn cycling in vitro.