Schrödinger's microbes: Tools for distinguishing the living from the dead in microbial ecosystems.

While often obvious for macroscopic organisms, determining whether a microbe is dead or alive is fraught with complications. Fields such as microbial ecology, environmental health, and medical microbiology each determine how best to assess which members of the microbial community are alive, according to their respective scientific and/or regulatory needs. Many of these fields have gone from studying communities on a bulk level to the fine-scale resolution of microbial populations within consortia. For example, advances in nucleic acid sequencing technologies and downstream bioinformatic analyses have allowed for high-resolution insight into microbial community composition and metabolic potential, yet we know very little about whether such community DNA sequences represent viable microorganisms. In this review, we describe a number of techniques, from microscopy- to molecular-based, that have been used to test for viability (live/dead determination) and/or activity in various contexts, including newer techniques that are compatible with or complementary to downstream nucleic acid sequencing. We describe the compatibility of these viability assessments with high-throughput quantification techniques, including flow cytometry and quantitative PCR (qPCR). Although bacterial viability-linked community characterizations are now feasible in many environments and thus are the focus of this critical review, further methods development is needed for complex environmental samples and to more fully capture the diversity of microbes (e.g., eukaryotic microbes and viruses) and metabolic states (e.g., spores) of microbes in natural environments.

Potential for Sulfate Reduction in Mangrove Forest Soils: Comparison between Two Dominant Species of the Americas.

Avicennia and Rhizophora are globally occurring mangrove genera with different traits that place them in different parts of the intertidal zone. It is generally accepted that the oxidizing capacity of Avicennia roots is larger than that of Rhizophora roots, which initiates more reduced conditions in the soil below the latter genus. We hypothesize that the more reduced conditions beneath Rhizophora stands lead to more active sulfate-reducing microbial communities compared to Avicennia stands. To test this hypothesis, we measured sulfate reduction traits in soil samples collected from neighboring Avicennia germinans and Rhizophora mangle stands at three different locations in southern Florida. The traits measured were sulfate reduction rates (SRR) in flow-through reactors containing undisturbed soil layers in the absence and presence of easily degradable carbon compounds, copy numbers of the dsrB gene, which is specific for sulfate-reducing microorganisms, and numbers of sulfate-reducing cells that are able to grow in liquid medium on a mixture of acetate, propionate and lactate as electron donors. At the tidal locations Port of the Islands and South Hutchinson Islands, steady state SRR, dsrB gene copy numbers and numbers of culturable cells were higher at the A. germinans than at the R. mangle stands, although not significantly for the numbers at Port of the Islands. At the non-tidal location North Hutchinson Island, results are mixed with respect to these sulfate reduction traits. At all locations, the fraction of culturable cells were significantly higher at the R. mangle than at the A. germinans stands. The dynamics of the initial SRR implied a more in situ active sulfate-reducing community at the intertidal R. mangle stands. It was concluded that in agreement with our hypothesis R. mangle stands accommodate a more active sulfate-reducing community than A. germinans stands, but only at the tidal locations. The differences between R. mangle and A. germinans stands were absent at the non-tidal, impounded location.

Nitrate ammonification in mangrove soils: a hidden source of nitrite?

Nitrate reduction is considered to be a minor microbial pathway in the oxidation of mangrove-derived organic matter due to a limited supply of nitrate in mangrove soils. At a limited availability of this electron acceptor compared to the supply of degradable carbon, nitrate ammonification is thought to be the preferential pathway of nitrate reduction. Mangrove forest mutually differ in their productivity, which may lead to different available carbon to nitrate ratios in their soil. Hence, nitrate ammonification is expected to be of more importance in high- compared to low-productive forests. The hypothesis was tested in flow-through reactors that contain undisturbed mangrove soils from high-productive Avicennia germinans and Rhizophora mangle forests in Florida and low-productive Avicennia marina forests in Saudi Arabia. Nitrate was undetectable in the soils from both regions. It was assumed that a legacy of nitrate ammonification would be reflected by a higher ammonium production from these soils upon the addition of nitrate. Unexpectedly, the soils from the low-productive forests in Saudi Arabia produced considerably more ammonium than the soils from the high-productive forests in Florida. Hence, other environmental factors than productivity must govern the selection of nitrate ammonification or denitrification. A rather intriguing observation was the 1:1 production of nitrite and ammonium during the consumption of nitrate, more or less independent from sampling region, location, sampling depth, mangrove species and from the absence or presence of additional degradable carbon. This 1:1 ratio points to a coupled production of ammonium and nitrite by one group of nitrate-reducing microorganisms. Such a production of nitrite will be hidden by the presence of active nitrite-reducing microorganisms under the nitrate-limited conditions of most mangrove forest soils.

Potential Activity, Size, and Structure of Sulfate-Reducing Microbial Communities in an Exposed, Grazed and a Sheltered, Non-Grazed Mangrove Stand at the Red Sea Coast.

After oxygen, sulfate is the most important oxidant for the oxidation of organic matter in mangrove forest soils. As sulfate reducers are poor competitors for common electron donors, their relative success depends mostly on the surplus of carbon that is left by aerobic organisms due to oxygen depletion. We therefore hypothesized that sulfate-cycling in mangrove soils is influenced by the size of net primary production, and hence negatively affected by mangrove degradation and exploitation, as well as by carbon-exporting waves. To test this, we compared quantitative and qualitative traits of sulfate-reducing communities in two Saudi-Arabian mangrove stands near Jeddah, where co-occurring differences in camel-grazing pressure and tidal exposure led to a markedly different stand height and hence primary production. Potential sulfate reduction rates measured in anoxic flow-through reactors in the absence and presence of additional carbon sources were significantly higher in the samples from the non-grazed site. Near the surface (0-2 cm depth), numbers of dsrB gene copies and culturable cells also tended to be higher in the non-grazed sites, while these differences were not detected in the sub-surface (4-6 cm depth). It was concluded that sulfate-reducing microbes at the surface were indeed repressed at the low-productive site as could be expected from our hypothesis. At both sites, sulfate reduction rates as well as numbers of the dsrB gene copies and viable cells increased with depth suggesting repression of sulfate reduction near the surface in both irrespective of production level. Additionally, sequence analysis of DNA bands obtained from DGGE gels based on the dsrB gene, showed a clear difference in dominance of sulfate-reducing genera belonging to the Deltaproteobacteria and the Firmicutes between sampling sites and depths.

(Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., isolated from an underground gas storage.

A mesophilic bacterium, strain An4, was isolated from an underground gas storage reservoir with methanol as substrate and perchlorate as electron acceptor. Cells were Gram-negative, spore-forming, straight to curved rods, 0.5-0.8 microm in diameter, and 2-8 microm in length, growing as single cells or in pairs. The cells grew optimally at 37 degrees C, and the pH optimum was around 7. Strain An4 converted various alcohols, organic acids, fructose, acetoin, and H(2)/CO(2) to acetate, usually as the only product. Succinate was decarboxylated to propionate. The isolate was able to respire with (per)chlorate, nitrate, and CO(2). The G+C content of the DNA was 42.6 mol%. Based on the 16S rRNA gene sequence analysis, strain An4 was most closely related to Sporomusa ovata (98% similarity). The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell-free extracts.

Isolation and characterization of a new CO-utilizing strain, Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans, isolated from a geothermal spring in Turkey.

A novel anaerobic, thermophilic, Gram-positive, spore-forming, and sugar-fermenting bacterium (strain TLO) was isolated from a geothermal spring in Ayas, Turkey. The cells were straight to curved rods, 0.4-0.6 microm in diameter and 3.5-10 microm in length. Spores were terminal and round. The temperature range for growth was 40-80 degrees C, with an optimum at 70 degrees C. The pH optimum was between 6.3 and 6.8. Strain TLO has the capability to ferment a wide variety of mono-, di-, and polysaccharides and proteinaceous substrates, producing mainly lactate, next to acetate, ethanol, alanine, H(2), and CO(2). Remarkably, the bacterium was able to grow in an atmosphere of up to 25% of CO as sole electron donor. CO oxidation was coupled to H(2) and CO(2) formation. The G + C content of the genomic DNA was 35.1 mol%. Based on 16S rRNA gene sequence analysis and the DNA-DNA hybridization data, this bacterium is most closely related to Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacter siderophilus (99% similarity for both). However, strain TLO differs from Thermoanaerobacter thermohydrosulfuricus in important aspects, such as CO-utilization and lipid composition. These differences led us to propose that strain TLO represents a subspecies of Thermoanaerobacter thermohydrosulfuricus, and we therefore name it Thermoanaerobacter thermohydrosulfuricus subsp. carboxydovorans.

Atypical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermoacetica strain.

A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bioreactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the acetogenic bacterium Moorella thermoacetica DSM 521(T) (98.3% sequence similarity). DNA-DNA hybridization showed 75.2 +/- 4.7% similarity to M. thermoacetica DSM 521(T), suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sustained in coculture with a hydrogen-consuming methanogen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulfide formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H(2)/CO(2), unlike M. thermoacetica (DSM 521(T)). The lack of growth with H(2)/CO(2) likely is due to the absence of cytochrome b in strain AMP.

Desulfatirhabdium butyrativorans gen. nov., sp. nov., a butyrate-oxidizing, sulfate-reducing bacterium isolated from an anaerobic bioreactor.

A novel sulfate-reducing bacterium, strain HB1(T), was isolated from an upflow anaerobic sludge blanket (UASB) reactor treating paper-mill wastewater operated at 37 degrees C. Cells of strain HB1(T) were oval to rod-shaped, 1-1.3 microm wide and 2.6-3.5 microm long and Gram-negative. The optimum temperature for growth was 28-30 degrees C. In the presence of sulfate, the isolate was able to grow on H(2)/acetate, formate, ethanol, propionate, fumarate, succinate, butyrate, crotonate, catechol, benzoate, 4-hydroxybenzoate, palmitate and stearate. The isolate only grew on H(2) when acetate was added as a carbon source; when grown on formate, acetate was not required. Growth was also possible on pyruvate and crotonate without an electron acceptor. The isolate showed very poor growth on acetate. Thiosulfate and sulfate were used as electron acceptors. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain HB1(T) represents a novel lineage within the Deltaproteobacteria; sequence similarities between strain HB1(T) and members of other related genera were less than 91%. Strain HB1(T) was also distinguished from members of related genera based on differences in several phenotypic characteristics. It is a member of the family Desulfobacteraceae. The major cellular fatty acids of strain HB1(T) were C(16:0), iso-C(15:0), anteiso-C(15:0) and C(14:0). beta-Hydroxy fatty acids were also present in the range of C(14:0) to C(18:0), of which C(16:0) was the most abundant. The G+C content of the DNA was 55.1 mol%. Based on physiological, biochemical and chemotaxonomic traits together with results of comparative 16S rRNA gene sequence analysis, strain HB1(T) is considered to represent a novel species in a new genus, for which the name Desulfatirhabdium butyrativorans gen. nov., sp. nov. is proposed. The type strain of Desulfatirhabdium butyrativorans is HB1(T) (=DSM 18734(T) =JCM 14470(T)).

(Per)chlorate reduction by the thermophilic bacterium Moorella perchloratireducens sp. nov., isolated from underground gas storage.

A thermophilic bacterium, strain An10, was isolated from underground gas storage with methanol as a substrate and perchlorate as an electron acceptor. Cells were gram-positive straight rods, 0.4 to 0.6 mum in diameter and 2 to 8 mum in length, growing as single cells or in pairs. Spores were terminal with a bulged sporangium. The temperature range for growth was 40 to 70 degrees C, with an optimum at 55 to 60 degrees C. The pH optimum was around 7. The salinity range for growth was between 0 and 40 g NaCl liter(-1) with an optimum at 10 g liter(-1). Strain An10 was able to grow on CO, methanol, pyruvate, glucose, fructose, cellobiose, mannose, xylose, and pectin. The isolate was able to respire with (per)chlorate, nitrate, thiosulfate, neutralized Fe(III) complexes, and anthraquinone-2,6-disulfonate. The G+C content of the DNA was 57.6 mol%. On the basis of 16S rRNA analysis, strain An10 was most closely related to Moorella thermoacetica and Moorella thermoautotrophica. The bacterium reduced perchlorate and chlorate completely to chloride. Key enzymes, perchlorate reductase and chlorite dismutase, were detected in cell extracts. Strain An10 is the first thermophilic and gram-positive bacterium with the ability to use (per)chlorate as a terminal electron acceptor.

Structural characterization of diabolic acid-based tetraester, tetraether and mixed ether/ester, membrane-spanning lipids of bacteria from the order Thermotogales.

The distribution of core lipids in the membranes of nine different species of the order Thermotogales, one of the early and deep branching lineages in the Bacteria, were examined by HPLC/MS and demonstrated to consist of membrane-spanning diglycerol lipids comprised of diabolic acid-derived alkyl moieties. In the Thermotoga species the core membrane lipids are characterized by the presence of both ester and ether bonds, whereas in the phylogenetically more distinct Thermosipho and Fervidobacterium spp. only ester bonds occur. A tentative biosynthetic route for the biosynthesis of these membrane-spanning lipids is proposed. Since species of the order Thermotogales are assumed to have occurred early during the evolution of life on Earth, as suggested by its position in the phylogenetic tree of life, these data suggest that the ability to produce both ether and ester glycerol membrane lipids developed relatively early during microbial evolution.

Methanol utilizing Desulfotomaculum species utilizes hydrogen in a methanol-fed sulfate-reducing bioreactor.

A sulfate-reducing bacterium, strain WW1, was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as sole energy source in the presence of sulfate. Growth of strain WW1 on methanol or acetate was inhibited at a sulfide concentration of 200 mg l(-1), while on H2/CO2, no apparent inhibition occurred up to a concentration of 500 mg l(-1). When strain WW1 was co-cultured under the same conditions with the methanol-utilizing, non-sulfate-reducing bacteria, Thermotoga lettingae and Moorella mulderi, both originating from the same bioreactor, growth and sulfide formation were observed up to 430 mg l(-1). These results indicated that in the co-cultures, a major part of the electron flow was directed from methanol via H2/CO2 to the reduction of sulfate to sulfide. Besides methanol, acetate, and hydrogen, strain WW1 was also able to use formate, malate, fumarate, propionate, succinate, butyrate, ethanol, propanol, butanol, isobutanol, with concomitant reduction of sulfate to sulfide. In the absence of sulfate, strain WW1 grew only on pyruvate and lactate. On the basis of 16S rRNA analysis, strain WW1 was most closely related to Desulfotomaculum thermocisternum and Desulfotomaculum australicum. However, physiological properties of strain WW1 differed in some aspects from those of the two related bacteria.

Reductive dechlorination of beta-hexachlorocyclohexane (beta-HCH) by a Dehalobacter species in coculture with a Sedimentibacter sp.

An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the beta-HCH isomer to benzene and chlorobenzene in a ratio of 0.5-2 depending on the amount of beta-HCH degraded. The culture grows with H(2) as electron donor and beta-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate beta-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of beta-HCH by a defined anaerobic bacterial culture.

Methanol utilization by a novel thermophilic homoacetogenic bacterium, Moorella mulderi sp. nov., isolated from a bioreactor.

A thermophilic, anaerobic, spore-forming bacterium (strain TMS) was isolated from a thermophilic bioreactor operated at 65 degrees C with methanol as the energy source. Cells were gram-positive straight rods, 0.4-0.6 microm x 2-8 microm, growing as single cells or in pairs. The temperature range for growth was 40-70 degrees C with an optimum at 65 degrees C. Growth was observed from pH 5.5 to 8.5, and the optimum pH was around 7. The salinity range for growth was 0-45 g NaCl l(-1 )with an optimum at 10 g l(-1). The isolate was able to grow on methanol, H(2)-CO(2 )(80/20%, v/v), formate, lactate, pyruvate, glucose, fructose, cellobiose and pectin. The bacterium reduced thiosulfate to sulfide. The G+C content of the DNA was 53 mol%. Comparison of 16S rRNA genes revealed that strain TMS is related to Moorella glycerini (96%, sequence similarity), Moorella thermoacetica (92%) and Moorella thermoautotrophica (92%). On the basis of physiological and phylogenetic differences, strain TMS is proposed as a new species within the genus Moorella, Moorella mulderi sp. nov. (=DSM 14980, =ATCC BAA-608).

Thermotoga lettingae sp. nov., a novel thermophilic, methanol-degrading bacterium isolated from a thermophilic anaerobic reactor.

A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate. The G+C content of the DNA of strain TMOT was 39.2 mol%. The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively. Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii. Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation. In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine. In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2. However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors. Yeast extract was required for growth. Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan. Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation. Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%). The type strain is TMOT (= DSM 14385T = ATCC BAA-301T). On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.

Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum subsp. nov., a thermophilic, syntrophic, propionate-oxidizing, spore-forming bacterium.

From granular sludge from a laboratory-scale upflow anaerobic sludge bed reactor operated at 55 degrees C with a mixture of volatile fatty acids as feed, a novel anaerobic, moderately thermophilic, syntrophic, spore-forming bacterium, strain TPO, was enriched on propionate in co-culture with Methanobacterium thermoautotrophicum Z245. The axenic culture was obtained by using pyruvate as the sole source of carbon and energy. The cells were straight rods with pointed ends and became lens-shaped when sporulation started. The cells were slightly motile. The optimum growth temperature was 55 degrees C and growth was possible between 45 and 62 degrees C. The pH range for growth of strain TPO was 6-8, with an optimum at pH 7-7.5. Propionate was converted to acetate, CO2 and CH4 by a co-culture of strain TPO with Methanobacterium thermoautotrophicum Z245. In pure culture, strain TPO could grow fermentatively on benzoate, fumarate, H2/CO2, pyruvate and lactate. Sulphate could serve as inorganic electron acceptor when strain TPO was grown on propionate, lactate, pyruvate and H2/CO2. The G+C content was 53.7 mol%. Comparison of 16S rDNA sequences revealed that strain TPO is related to Desulfotomaculum thermobenzoicum (98%) and Desulfotomaculum thermoacetoxidans (98%). DNA-DNA hybridization revealed 88.2% reassociation between strain TPO and D. thermobenzoicum and 83.8% between strain TPO and D. thermoacetoxidans. However, both organisms differ physiologically from strain TPO and are not capable of syntrophic propionate oxidation. It is proposed that strain TPO should be classified as new subspecies of D. thermobenzoicum as D. thermobenzoicum subsp. thermosyntrophicum.

Gelria glutamica gen. nov., sp. nov., a thermophilic, obligately syntrophic, glutamate-degrading anaerobe.

A novel anaerobic, gram-positive, thermophilic, spore-forming, obligately syntrophic, glutamate-degrading bacterium, strain TGO(T), was isolated from a propionate-oxidizing methanogenic enrichment culture. The axenic culture was obtained by growing the bacterium on pyruvate. Cells were rod-shaped and non-motile. The optimal temperature for growth was 50-55 degrees C and growth occurred between 37 and 60 degrees C. The pH range for growth was 5.5-8 with optimum growth at pH 7. In pure culture, strain TGO(T) could grow on pyruvate, lactate, glycerol and several sugars. In co-culture with the hydrogenotrophic methanogen Methanobacterium thermautotrophicum strain Z-245, strain TGO(T) could grow on glutamate, proline and Casamino acids. Glutamate was converted to H2, CO2, propionate and traces of succinate. Strain TGO(T) was not able to utilize sulphate, sulphite, thiosulphate, nitrate or fumarate as electron acceptors. The G+C content was 33.8 mol%. Sequence analysis of the 16S rDNA revealed that strain TGO(T) belongs to the thermophilic, endospore-forming anaerobes, though no close relations were found. Its closest relations were Moorella glycerini (92%) and Moorella thermoacetica (90%). Strain TGOT had an unusually long 16S rDNA of more than 1700 bp. The additional base pairs were found as long loops in the V1, V7 and V9 regions of the 16S rDNA. However, the loops were not found in the 16S rRNA. The name Gelria glutamica gen. nov., sp. nov. is proposed for strain TGO(T).