Vertical Distribution of Denitrification Potential,Denitrifying Bacteria,and Benzoate Utilization in Intertidal Microbial Mat Communities

Aspects of denitrification and benzoate degradation were studied in two estuarine microbial mat communities on the California coast by measuring the depth distributions of potential denitrification rates, genetic potential for denitrification, nitrate concentration, benzoate mineralization rates, total bacterial abundance, and abundance of a denitrifying strain (TBD-8b) isolated from one of the sites. Potential denitrification was detected in microbial mat cores from both Elkhorn Slough and Tomales Bay. Maximum denitrification rates were more than two orders of magnitude higher at Elkhorn Slough (3.14 mmol N m⁻² d⁻¹) than at Tomales Bay (0.02 mmol N m⁻² d⁻¹), and at both sites, the maximum rates occurred in the 0–2 mm depth interval. Ambient pore [NO₃+NO₂] was substantially higher at Elkhorn Slough than at Tomales Bay. Incorporation and mineralization of benzoate was maximal near the mat surface at Elkhorn Slough. The areal rate of benzoate utilization was 1045 nmol C m⁻² d⁻¹, which represented utilization of 70% of the added substrate in 24 h. Total bacterial and TBD-8b abundances were greatest near the surface at both Tomales Bay and Elkhorn Slough, and TBD-8b represented less than 0.2% of the total. Genetic potential for denitrification, quantified by hybridization with a nitrite reductase gene fragment, was present below the mat surface at average levels representing presence of the gene in approximately 10% of the total cells.     

Spatial variability in photosynthetic and heterotrophic activity drives localized δ13Corg fluctuations and carbonate precipitation in hypersaline microbial mats

Modern laminated photosynthetic microbial mats are ideal environments to study how microbial activity creates and modifies carbon and sulfur isotopic signatures prior to lithification. Laminated microbial mats from a hypersaline lagoon (Guerrero Negro, Baja California, Mexico) maintained in a flume in a greenhouse at NASA Ames Research Center were sampled for δ¹³C of organic material and carbonate to assess the impact of carbon fixation (e.g., photosynthesis) and decomposition (e.g., bacterial respiration) on δ¹³C signatures. In the photic zone, the δ¹³C_org signature records a complex relationship between the activities of cyanobacteria under variable conditions of CO₂ limitation with a significant contribution from green sulfur bacteria using the reductive TCA cycle for carbon fixation. Carbonate is present in some layers of the mat, associated with high concentrations of bacteriochlorophyll e (characteristic of green sulfur bacteria) and exhibits δ¹³C signatures similar to DIC in the overlying water column (?2.0‰), with small but variable decreases consistent with localized heterotrophic activity from sulfate‐reducing bacteria (SRB). Model results indicate respiration rates in the upper 12 mm of the mat alter in situ pH and concentrations to create both phototrophic CO₂ limitation and carbonate supersaturation, leading to local precipitation of carbonate minerals. The measured activity of SRB with depth suggests they variably contribute to decomposition in the mat dependent on organic substrate concentrations. Millimeter‐scale variability in the δ¹³C_org signature beneath the photic zone in the mat is a result of shifting dominance between cyanobacteria and green sulfur bacteria with the aggregate signature overprinted by heterotrophic reworking by SRB and methanogens. These observations highlight the impact of sedimentary microbial processes on δ¹³C_org signatures; these processes need to be considered when attempting to relate observed isotopic signatures in ancient sedimentary strata to conditions in the overlying water column at the time of deposition and associated inferences about carbon cycling.     

Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengers

We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate—one where methanogenesis was allowed and another in which it was completely inhibited with 2‐bromoethane sulfonate. We observed a three‐way syntrophy among ethanol fermenters, acetate‐oxidizing anode‐respiring bacteria (ARB), and a H₂ scavenger. When methanogenesis was allowed, H₂‐oxidizing methanogens were the H₂ scavengers, but when methanogenesis was inhibited, homo‐acetogens became a channel for electron flow from H₂ to current through acetate. We established the presence of homo‐acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo‐acetogens. Both methods documented the presence of the homo‐acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol‐fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H₂‐scavenging syntrophic partners that were either H₂‐oxidizing methanogens or homo‐acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron‐flow distribution and H₂‐scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69–78. © 2009 Wiley Periodicals, Inc.     

Analysis of the Microbial Community in an Acidic Hollow-Fiber Membrane Biofilm Reactor (Hf-MBfR) Used for the Biological Conversion of Carbon Dioxide to Methane

Hydrogenotrophic methanogens can use gaseous substrates, such as H₂ and CO₂, in CH₄ production. H₂ gas is used to reduce CO₂. We have successfully operated a hollow-fiber membrane biofilm reactor (Hf-MBfR) for stable and continuous CH₄ production from CO₂ and H₂. CO₂ and H₂ were diffused into the culture medium through the membrane without bubble formation in the Hf-MBfR, which was operated at pH 4.5–5.5 over 70 days. Focusing on the presence of hydrogenotrophic methanogens, we analyzed the structure of the microbial community in the reactor. Denaturing gradient gel electrophoresis (DGGE) was conducted with bacterial and archaeal 16S rDNA primers. Real-time qPCR was used to track changes in the community composition of methanogens over the course of operation. Finally, the microbial community and its diversity at the time of maximum CH₄ production were analyzed by pyrosequencing methods. Genus Methanobacterium, related to hydrogenotrophic methanogens, dominated the microbial community, but acetate consumption by bacteria, such as unclassified Clostridium sp., restricted the development of acetoclastic methanogens in the acidic CH₄ production process. The results show that acidic operation of a CH₄ production reactor without any pH adjustment inhibited acetogenic growth and enriched the hydrogenotrophic methanogens, decreasing the growth of acetoclastic methanogens.     

Injection of hydrogen gas stimulates acid mine drainage treatment in laboratory‐scale hydroponic root mats

The environmentally benign disposal of acid mine drainage (AMD) is still a technical challenge. In the present study, artificial AMD was treated in a laboratory‐scale floating hydroponic root mat of soft rush, Juncus effusus. This ecotechnological system was operated with hydrogen injection and water recirculation but without an external carbon supply. It achieved a mean increase of ΔpH = 3.3 up to pH ≈ 8.2, high sulfate removal of up to 87%, and efficient removal of iron (100%), aluminum (99.8%), manganese (97.4%), and zinc (99.6%). Sulfide was not detected in the outflow. Treatment performance correlated with the amount of hydrogen loading. Daily oscillations of the redox potential up to amplitudes of ΔE_h ≈ 450 mV in a mean range of E_h ≈ ?150 to +300 mV indicated a correlation of plant physiology and removal processes. Apparently, sulfate and metal removal were the result of chemolithotrophic microbial sulfate reduction supported by the externally provided H₂ and chemoorganotrophic sulfate reduction driven by rhizodeposits. Bicarbonate generated in the microbial transformation of such plant‐derived organic carbon contributed to pH neutralization. The effluent's pH increase was governed further by recirculation of the treated AMD. The flow regime and the injection of hydrogen at the ground of the root mat caused concentration gradients where the most efficient removal occurred in the deepest zone of the root mat. Further investigations should target long‐term stability, plant growth dynamics, load variations, balances of carbon and sulfur, the removal of H₂S and metal precipitates from the system as well as efficient hydrogen supply.     

Microstructure and molecular microbiology of rapidly formed hydrogen‐ and methane‐producing granules in a phase‐separated anaerobic environment

Hydrogen and methane were simultaneously produced in a two‐phase reactor, operated to separate the reactions of hydrogen and methanogen production. Each reactor was inoculated with a seed enriched with different microbial consortia. The first phase was operated with a hydraulic retention time of 7 days and at an organic loading rate of 7.7 g VS L^?1 d^?1 that produced a stable pH of 5.5. This suppressed the growth of methanogens and as a result, the off gas contained up to 27% hydrogen. The second phase was operated with a hydraulic retention time of 12 days and at an organic loading rate of 3.6 g VS L^?1 d^?1. This permitted the growth of hydrogenotrophs and methanogens to produce methane at a concentration of 60%. Examination of the microbial population of the two reactors both microscopically and using PCR, showed an effective separation of hydrogen‐ and methane‐producing microbial communities. The study revealed that the suppression of hydrogentrophs and methanogens can be achieved by adopting rapid method that leads the growth of hydrogen‐ and methane‐producing granules in phase‐separated anaerobic environment.     

Control of biogenic H(2)S production with nitrite and molybdate

The effects of the metabolic inhibitors, sodium nitrite and ammonium molybdate, on production of H₂S by a pure culture of the sulfate-reducing bacterium (SRB) Desulfovibrio sp. strain Lac6 and a consortium of SRB, enriched from produced water of a Canadian oil field, were investigated. Addition of 0.1 mM nitrite or 0.024 mM molybdate at the start of growth prevented the production of H₂S by strain Lac6. With exponentially growing cultures, higher levels of inhibitors, 0.25 mM nitrite or 0.095 mM molybdate, were required to suppress the production of H₂S. Simultaneous addition of nitrite and molybdate had a synergistic effect: at time 0, 0.05 mM nitrite and 0.01 mM molybdate, whereas during the exponential phase, 0.1 mM nitrite and 0.047 mM molybdate were sufficient to stop H₂S production. With an exponentially growing consortium of SRB, enriched from produced water of the Coleville oil field, much higher levels of inhibitors, 4 mM nitrite or 0.47 mM molybdate, were needed to stop the production of H₂S. The addition of these inhibitors had no effect on the composition of the microbial community, as shown by reverse sample genome probing. The results indicate that the efficiency of inhibitors in containment of SRB depends on the composition and metabolic state of the microbial community. Journal of Industrial Microbiology & Biotechnology (2001) 26, 350–355. Received 02 August 2000/ Accepted in revised form 17 April 2001     

Diversity of dimethylsulfide-degrading methanogens and sulfate-reducing bacteria in anoxic sediments along the Medway Estuary,UK

Methane is a powerful greenhouse gas but the microbial diversity mediating methylotrophic methanogenesis is not well-characterized. One overlooked route to methane is via the degradation of dimethylsulfide (DMS), an abundant organosulfur compound in the environment. Methanogens and sulfate-reducing bacteria (SRB) can degrade DMS in anoxic sediments depending on sulfate availability. However, we know little about the underlying microbial community and how sulfate availability affects DMS degradation in anoxic sediments. We studied DMS-dependent methane production along the salinity gradient of the Medway Estuary (UK) and characterized, for the first time, the DMS-degrading methanogens and SRB using cultivation-independent tools. DMS metabolism resulted in high methane yield (39%–42% of the theoretical methane yield) in anoxic sediments regardless of their sulfate content. Methanomethylovorans, Methanolobus and Methanococcoides were dominant methanogens in freshwater, brackish and marine incubations respectively, suggesting niche-partitioning of the methanogens likely driven by DMS amendment and sulfate concentrations. Adding DMS also led to significant changes in SRB composition and abundance in the sediments. Increases in the abundance of Sulfurimonas and SRB suggest cryptic sulfur cycling coupled to DMS degradation. Our study highlights a potentially important pathway to methane production in sediments with contrasting sulfate content and sheds light on the diversity of DMS degraders.     

Tidal movements of female leopard sharks (<Emphasis Type="Italic">Triakis semifasciata</Emphasis>) in Elkhorn Slough,California

The leopard shark (Triakis semifasciata) is one of the most common species of elasmobranch in California, and uses the shallow bays and estuaries of California extensively throughout its life history. To examine the role that tides and time of day play on the distribution and movements of leopard sharks in an estuarine environment, a total of 22 female leopard sharks (78–140 cm TL) were tagged with acoustic transmitters in Elkhorn Slough, California, USA. Eight sharks were manually tracked for 20–71.5 h, and 13 sharks were monitored for 4–280 days using an array of acoustic receivers. Overall, the distribution and movements of sharks were strongly influenced by the tides and to a lesser extent by period of day, although general patterns of movement differed depending on what region of Elkhorn Slough the sharks were using. In the main channel of Elkhorn Slough, sharks generally moved with the tide, maximizing the area over which they could forage on a more dispersed prey field. Conversely, leopard sharks within the Elkhorn Slough National Estuarine Research Reserve regularly swam against strong currents to remain in proximity to the intertidal mudflats. This high degree of fidelity to a specific region was probably due to an abundance of important prey in the area. These results indicate that movements, and thus the foraging ecology, of leopard sharks show a high degree of plasticity and are influenced by tidal stage, tidal current, availability of suitable habitat, and availability and distribution of important prey items.     

Trophic links between fermenters and methanogens in a moderately acidic fen soil

Trophic links between fermentation and methanogenesis of soil derived from a methane‐emitting, moderately acidic temperate fen (pH 4.5) were investigated. Initial CO₂:CH₄ production ratios in anoxic microcosms indicated that methanogenesis was concomitant to other terminal anaerobic processes. Methane production in anoxic microcosms at in situ pH was stimulated by supplemental H₂–CO₂, formate or methanol; supplemental acetate did not stimulate methanogenesis. Supplemental H₂–CO₂, formate or methanol also stimulated the formation of acetate, indicating that the fen harbours moderately acid‐tolerant acetogens. Supplemental monosaccharides (glucose, N‐acetylglucosamine and xylose) stimulated the production of CO₂, H₂, acetate and other fermentation products when methanogenesis was inhibited with 2‐bromoethane sulfonate 20 mM. Glucose stimulated methanogenesis in the absence of BES. Upper soil depths yielded higher anaerobic activities and also higher numbers of cells. Detected archaeal 16S rRNA genes were indicative of H₂–CO₂‐ and formate‐consuming methanogens (Methanomicrobiaceae), obligate acetoclastic methanogens (Methanosaetaceae) and crenarchaeotes (groups I.1a, I.1c and I.3). Molecular analyses of partial sequences of 16S rRNA genes revealed the presence of Acidobacteria, Nitrospirales, Clamydiales, Clostridiales, Alpha‐, Gamma‐, Deltaproteobacteria and Cyanobacteria. These collective results suggest that this moderately acidic fen harbours phylogenetically diverse, moderately acid tolerant fermenters (both facultative aerobes and obligate anaerobes) that are trophically linked to methanogenesis.     

The genome of the Gram‐positive metal‐ and sulfate‐reducing bacterium Desulfotomaculum reducens strain MI‐1

Spore‐forming, Gram‐positive sulfate‐reducing bacteria (SRB) represent a group of SRB that dominates the deep subsurface as well as niches in which resistance to oxygen and dessication is an advantage. Desulfotomaculum reducens strain MI‐1 is one of the few cultured representatives of that group with a complete genome sequence available. The metabolic versatility of this organism is reflected in the presence of genes encoding for the oxidation of various electron donors, including three‐ and four‐carbon fatty acids and alcohols. Synteny in genes involved in sulfate reduction across all four sequenced Gram‐positive SRB suggests a distinct sulfate‐reduction mechanism for this group of bacteria. Based on the genomic information obtained for sulfate reduction in D. reducens, the transfer of electrons to the sulfite and APS reductases is proposed to take place via the quinone pool and heterodisulfide reductases respectively. In addition, both H₂‐evolving and H₂‐consuming cytoplasmic hydrogenases were identified in the genome, pointing to potential cytoplasmic H₂ cycling in the bacterium. The mechanism of metal reduction remains unknown.     

Syntrophic interactions among anode respiring bacteria (ARB) and Non‐ARB in a biofilm anode: electron balances

We demonstrate that the coulombic efficiency (CE) of a microbial electrolytic cell (MEC) fueled with a fermentable substrate, ethanol, depended on the interactions among anode respiring bacteria (ARB) and other groups of micro‐organisms, particularly fermenters and methanogens. When we allowed methanogenesis, we obtained a CE of 60%, and 26% of the electrons were lost as methane. The only methanogenic genus detected by quantitative real‐time PCR was the hydrogenotrophic genus, Methanobacteriales, which presumably consumed all the hydrogen produced during ethanol fermentation (～30% of total electrons). We did not detect acetoclastic methanogenic genera, indicating that acetate‐oxidizing ARB out‐competed acetoclastic methanogens. Current production and methane formation increased in parallel, suggesting a syntrophic interaction between methanogens and acetate‐consuming ARB. When we inhibited methanogenesis with 50 mM 2‐bromoethane sulfonic acid (BES), the CE increased to 84%, and methane was not produced. With no methanogenesis, the electrons from hydrogen were converted to electrical current, either directly by the ARB or channeled to acetate through homo‐acetogenesis. This illustrates the key role of competition among the various H₂ scavengers and that, when the hydrogen‐consuming methanogens were present, they out‐competed the other groups. These findings also demonstrate the importance of a three‐way syntrophic relationship among fermenters, acetate‐consuming ARB, and a H₂ consumer during the utilization of a fermentable substrate. To obtain high coulombic efficiencies with fermentable substrates in a mixed population, methanogens must be suppressed to promote new interactions at the anode that ultimately channel the electrons from hydrogen to current. Biotechnol. Bioeng. 2009;103: 513–523. © 2009 Wiley Periodicals, Inc.     

Effects of Amendment with Ferrihydrite and Gypsum on the Structure and Activity of Methanogenic Populations in Rice Field Soil

Methane emission from paddy fields may be reduced by the addition of electron acceptors to stimulate microbial populations competitive to methanogens. We have studied the effects of ferrihydrite and gypsum (CaSO₄·2H₂O) amendment on methanogenesis and population dynamics of methanogens after flooding of Italian rice field soil slurries. Changes in methanogen community structure were followed by archaeal small subunit (SSU) ribosomal DNA (rDNA)- and rRNA-based terminal restriction fragment length polymorphism analysis and by quantitative SSU rRNA hybridization probing. Under ferrihydrite amendment, acetate was consumed efficiently (<60 μM) and a rapid but incomplete inhibition of methanogenesis occurred after 3 days. In contrast to unamended controls, the dynamics of Methanosarcina populations were largely suppressed as indicated by rDNA and rRNA analysis. However, the low acetate availability was still sufficient for activation of Methanosaeta spp., as indicated by a strong increase of SSU rRNA but not of relative rDNA frequencies. Unexpectedly, rRNA amounts of the novel rice cluster I (RC-I) methanogens increased significantly, while methanogenesis was low, which may be indicative of transient energy conservation coupled to Fe(III) reduction by these methanogens. Under gypsum addition, hydrogen was rapidly consumed to low levels (~0.4 Pa), indicating the presence of a competitive population of hydrogenotrophic sulfate-reducing bacteria (SRB). This was paralleled by a suppressed activity of the hydrogenotrophic RC-I methanogens as indicated by the lowest SSU rRNA quantities detected in all experiments. Full inhibition of methanogenesis only became apparent when acetate was depleted to nonpermissive thresholds (<5 μM) after 10 days. Apparently, a competitive, acetotrophic population of SRB was not present initially, and hence, acetotrophic methanosarcinal populations were less suppressed than under ferrihydrite amendment. In conclusion, although methane production was inhibited effectively under both mitigation regimens, different methanogenic populations were either suppressed or stimulated, which demonstrates that functionally similar disturbances of an ecosystem may result in distinct responses of the populations involved.     

Distribution of anaerobic methane-oxidizing and sulfate-reducing communities in the G11 Nyegga pockmark,Norwegian Sea

Pockmarks are seabed geological structures sustaining methane seepage in cold seeps. Based on RNA-derived sequences the active fraction of the archaeal community was analysed in sediments associated with the G11 pockmark, in the Nyegga region of the Norwegian Sea. The anaerobic methanotrophic Archaea (ANME) and sulfate-reducing bacteria (SRB) communities were studied as well. The vertical distribution of the archaeal community assessed by PCR-DGGE highlighted the presence of ANME-2 in surface sediments, and ANME-1 in deeper sediments. Enrichments of methanogens showed the presence of hydrogenotrophic methanogens of the Methanogenium genus in surface sediment layers as well. The active fraction of the archaeal community was uniquely composed of ANME-2 in the shallow sulfate-rich sediments. Functional methyl coenzyme M reductase gene libraries showed that sequences affiliated with the ANME-1 and ANME-3 groups appeared in the deeper sediments but ANME-2 dominated both surface and deeper layers. Finally, dissimilatory sulfite reductase gene libraries revealed a high SRB diversity (i.e. Desulfobacteraceae, Desulfobulbaceae, Syntrophobacteraceae and Firmicutes) in the shallow sulfate-rich sediments. The SRB diversity was much lower in the deeper section. Overall, these results show that the microbial community in sediments associated with a pockmark harbour classical cold seep ANME and SRB communities.     

Possible nonanthropogenic origin of two methanogenic isolates from oil‐producing wells in the san miguelito field,ventura county,California

Two novel strains of rod‐shaped methanogens were isolated from oil‐producing wells of high temperature and moderate salinity in the San Miguelito field. The strains grow at 65°C in media containing 6% NaCl, form large aggregates of cell materials in liquid culture, and produce methane from H₂ and CO₂ only. The geochemistry and microbiology of the oil reservoir and surrounding areas support the view that not all methanogens isolated from oil wells are results of colonization during or after human exploration, and that methanogens can exist in situ under thermal, haline conditions.     

Wo lebten die ersten Zellen – und wovon?

How, and where, did the first cells on Earth grow? The last universal common ancestor of all cells (Luca) was long considered as the common ancestor of bacteria, archaea and eukaryotes. New trees of life have a host for the origin of mitochondria (of eukaryotes) branching within the archaea, making Luca the common ancestor of bacteria and archaea. New comparative genomic investigations have reconstructed Luca's microbial ecology. The 355 protein families that trace back to Luca by phylogenetic criteria describe Luca as anaerobic, CO₂ ‐ and N₂ ‐fixing, H₂ ‐dependent and thermophilic. Luca's biochemistry was replete with FeS clusters and radical reaction mechanisms, its cofactors reveal an essential role for transition metals in its metabolism. Luca lived in an anaerobic geochemical active environment rich in H₂ , CO₂ and iron. This lifestyle is similar to modern acetogens (bacteria) and methanogens (archaea), the physiologically most ancient microbes.     

Formate production and utilization by methanogens and by sewage sludge consortia — interference with the concept of interspecies formate transfer

Pure cultures of H₂/CO₂- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H₂/CO₂ at H₂ partial pressure in the gas phase above 200 kPa. At decreasing H₂ partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (–180 to –200 mV), formate-utilizing methanogens produced high amounts of formate from H₂/CO₂. No formate was excreted by the species, which could only utilize H₂/CO₂ for methanogenesis. In contrast, H₂ formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H₂/CO₂ and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H₂, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H₂ transfer. Correspondence to: J. Winter