Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey–predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.     

Development of a biomarker for Geobacter activity and strain composition; Proteogenomic analysis of the citrate synthase protein during bioremediation of U(VI)

Monitoring the activity of target microorganisms during stimulated bioremediation is a key problem for the development of effective remediation strategies. At the US Department of Energy's Integrated Field Research Challenge (IFRC) site in Rifle, CO, the stimulation of Geobacter growth and activity via subsurface acetate addition leads to precipitation of U(VI) from groundwater as U(IV). Citrate synthase (gltA) is a key enzyme in Geobacter central metabolism that controls flux into the TCA cycle. Here, we utilize shotgun proteomic methods to demonstrate that the measurement of gltA peptides can be used to track Geobacter activity and strain evolution during in situ biostimulation. Abundances of conserved gltA peptides tracked Fe(III) reduction and changes in U(VI) concentrations during biostimulation, whereas changing patterns of unique peptide abundances between samples suggested sample‐specific strain shifts within the Geobacter population. Abundances of unique peptides indicated potential differences at the strain level between Fe(III)‐reducing populations stimulated during in situ biostimulation experiments conducted a year apart at the Rifle IFRC. These results offer a novel technique for the rapid screening of large numbers of proteomic samples for Geobacter species and will aid monitoring of subsurface bioremediation efforts that rely on metal reduction for desired outcomes.     

Stimulating the In Situ Activity of Geobacter Species To Remove Uranium from the Groundwater of a Uranium-Contaminated Aquifer

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 μM in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.     

The design of long-term effective uranium bioremediation strategy using a community metabolic model

Acetate amendment at uranium contaminated sites in Rifle, CO. leads to an initial bloom of Geobacter accompanied by the removal of U(VI) from the groundwater, followed by an increase of sulfate‐reducing bacteria (SRBs) which are poor reducers of U(VI). One of the challenges associated with bioremediation is the decay in Geobacter abundance, which has been attributed to the depletion of bio‐accessible Fe(III), motivating the investigation of simultaneous amendments of acetate and Fe(III) as an alternative bioremediation strategy. In order to understand the community metabolism of Geobacter and SRBs during artificial substrate amendment, we have created a genome‐scale dynamic community model of Geobacter and SRBs using the previously described Dynamic Multi‐species Metabolic Modeling framework. Optimization techniques are used to determine the optimal acetate and Fe(III) addition profile. Field‐scale simulation of acetate addition accurately predicted the in situ data. The simulations suggest that batch amendment of Fe(III) along with continuous acetate addition is insufficient to promote long‐term bioremediation, while continuous amendment of Fe(III) along with continuous acetate addition is sufficient to promote long‐term bioremediation. By computationally minimizing the acetate and Fe(III) addition rates as well as the difference between the predicted and target uranium concentration, we showed that it is possible to maintain the uranium concentration below the environmental safety standard while minimizing the cost of chemical additions. These simulations show that simultaneous addition of acetate and Fe(III) has the potential to be an effective uranium bioremediation strategy. They also show that computational modeling of microbial community is an important tool to design effective strategies for practical applications in environmental biotechnology. Biotechnol. Bioeng. 2012; 109: 2475–2483. © 2012 Wiley Periodicals, Inc.     

Enrichment of dissimilatory Fe(III)-reducing bacteria from groundwater of the Siklós BTEX-contaminated site (Hungary)

Dissimilatory iron-reducing bacteria are commonly found in microbial communities of aromatic hydrocarbon-contaminated subsurface environments where they often play key role in the degradation of the contaminants. The Siklós benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated area is one of the best characterized petroleum hydrocarbon-contaminated sites of Hungary. Continuous monitoring of the microbial community in the center of the contaminant plume indicated the presence of an emerging Geobacter population and a Rhodoferax phylotype highly associated with aromatic hydrocarbon-contaminated subsurface environments. The aim of the present study was to make an initial effort to enrich Rhodoferax-related and other dissimilatory iron-reducing bacteria from this environment. Accordingly, four slightly different freshwater media were used to enrich Fe(III) reducers, differing only in the form of nitrogen source (organic, inorganic nitrogen or gaseous headspace nitrogen). Although enrichment of the desired Rhodoferax phylotype was not succeeded, Geobacter-related bacteria were readily enriched. Moreover, the different nitrogen sources caused the enrichment of different Geobacter species. Investigation of the diversity of benzylsuccinate synthase gene both in the enrichments and in the initial groundwater sample indicated that the Geobacter population in the center of the contaminant plume may not play a significant role in the anaerobic degradation of toluene.     

Functional environmental proteomics: elucidating the role of a c-type cytochrome abundant during uranium bioremediation

Studies with pure cultures of dissimilatory metal-reducing microorganisms have demonstrated that outer-surface c-type cytochromes are important electron transfer agents for the reduction of metals, but previous environmental proteomic studies have typically not recovered cytochrome sequences from subsurface environments in which metal reduction is important. Gel-separation, heme-staining and mass spectrometry of proteins in groundwater from in situ uranium bioremediation experiments identified a putative c-type cytochrome, designated Geobacter subsurface c-type cytochrome A (GscA), encoded within the genome of strain M18, a Geobacter isolate previously recovered from the site. Homologs of GscA were identified in the genomes of other Geobacter isolates in the phylogenetic cluster known as subsurface clade 1, which predominates in a diversity of Fe(III)-reducing subsurface environments. Most of the gscA sequences recovered from groundwater genomic DNA clustered in a tight phylogenetic group closely related to strain M18. GscA was most abundant in groundwater samples in which Geobacter sp. predominated. Expression of gscA in a strain of Geobacter sulfurreducens that lacked the gene for the c-type cytochrome OmcS, thought to facilitate electron transfer from conductive pili to Fe(III) oxide, restored the capacity for Fe(III) oxide reduction. Atomic force microscopy provided evidence that GscA was associated with the pili. These results demonstrate that a c-type cytochrome with an apparent function similar to that of OmcS is abundant when Geobacter sp. are abundant in the subsurface, providing insight into the mechanisms for the growth of subsurface Geobacter sp. on Fe(III) oxide and suggesting an approach for functional analysis of other Geobacter proteins found in the subsurface.     

Stimulating the in situ activity of Geobacter species to remove uranium from the groundwater of a uranium-contaminated aquifer

The potential for removing uranium from contaminated groundwater by stimulating the in situ activity of dissimilatory metal-reducing microorganisms was evaluated in a uranium-contaminated aquifer located in Rifle, Colo. Acetate (1 to 3 mM) was injected into the subsurface over a 3-month period via an injection gallery composed of 20 injection wells, which was installed upgradient from a series of 15 monitoring wells. U(VI) concentrations decreased in as little as 9 days after acetate injection was initiated, and within 50 days uranium had declined below the prescribed treatment level of 0.18 micro M in some of the monitoring wells. Analysis of 16S ribosomal DNA (rDNA) sequences and phospholipid fatty acid profiles demonstrated that the initial loss of uranium from the groundwater was associated with an enrichment of Geobacter species in the treatment zone. Fe(II) in the groundwater also increased during this period, suggesting that U(VI) reduction was coincident with Fe(III) reduction. As the acetate injection continued over 50 days there was a loss of sulfate from the groundwater and an accumulation of sulfide and the composition of the microbial community changed. Organisms with 16S rDNA sequences most closely related to those of sulfate reducers became predominant, and Geobacter species became a minor component of the community. This apparent switch from Fe(III) reduction to sulfate reduction as the terminal electron accepting process for the oxidation of the injected acetate was associated with an increase in uranium concentration in the groundwater. These results demonstrate that in situ bioremediation of uranium-contaminated groundwater is feasible but suggest that the strategy should be optimized to better maintain long-term activity of Geobacter species.     

Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth

Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L_2,3 absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)–Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.     

Enrichment of Members of the Family Geobacteraceae Associated with Stimulation of Dissimilatory Metal Reduction in Uranium-Contaminated Aquifer Sediments

Stimulating microbial reduction of soluble U(VI) to insoluble U(IV) shows promise as a strategy for immobilizing uranium in uranium-contaminated subsurface environments. In order to learn more about which microorganisms might be involved in U(VI) reduction in situ, the changes in the microbial community when U(VI) reduction was stimulated with the addition of acetate were monitored in sediments from three different uranium-contaminated sites in the floodplain of the San Juan River in Shiprock, N.Mex. In all three sediments U(VI) reduction was accompanied by concurrent Fe(III) reduction and a dramatic enrichment of microorganisms in the family Geobacteraceae, which are known U(VI)- and Fe(III)-reducing microorganisms. At the point when U(VI) reduction and Fe(III) reduction were nearing completion, Geobacteraceae accounted for ca. 40% of the 16S ribosomal DNA (rDNA) sequences recovered from the sediments with bacterial PCR primers, whereas Geobacteraceae accounted for fewer than 5% of the 16S rDNA sequences in control sediments that were not amended with acetate and in which U(VI) and Fe(III) reduction were not stimulated. Between 55 and 65% of these Geobacteraceae sequences were most similar to sequences from Desulfuromonas species, with the remainder being most closely related to Geobacter species. Quantitative analysis of Geobacteraceae sequences with most-probable-number PCR and TaqMan analyses indicated that the number of Geobacteraceae sequences increased from 2 to 4 orders of magnitude over the course of U(VI) and Fe(III) reduction in the acetate-amended sediments from the three sites. No increase in Geobacteraceae sequences was observed in control sediments. In contrast to the predominance of Geobacteraceae sequences, no sequences related to other known Fe(III)-reducing microorganisms were detected in sediments. These results compare favorably with an increasing number of studies which have demonstrated that Geobacteraceae are important components of the microbial community in a diversity of subsurface environments in which Fe(III) reduction is an important process. The combination of these results with the finding that U(VI) reduction takes place during Fe(III) reduction and prior to sulfate reduction suggests that Geobacteraceae will be responsible for much of the Fe(III) and U(VI) reduction during uranium bioremediation in these sediments.     

Acetate oxidation by syntrophic association between Geobacter sulfurreducens and a hydrogen-utilizing exoelectrogen

Anodic microbial communities in acetate-fed microbial fuel cells (MFCs) were analyzed using stable-isotope probing of 16S rRNA genes followed by denaturing gradient gel electrophoresis. The results revealed that Geobacter sulfurreducens and Hydrogenophaga sp. predominated in the anodic biofilm. Although the predominance of Geobacter sp. as acetoclastic exoelectrogens in acetate-fed MFC systems has been often reported, the ecophysiological role of Hydrogenophaga sp. is unknown. Therefore, we isolated and characterized a bacterium closely related to Hydrogenophaga sp. (designated strain AR20). The newly isolated strain AR20 could use molecular hydrogen (H₂), but not acetate, with carbon electrode as the electron acceptor, indicating that the strain AR20 was a hydrogenotrophic exoelectrogen. This evidence raises a hypothesis that acetate was oxidized by G. sulfurreducens in syntrophic cooperation with the strain AR20 as a hydrogen-consuming partner in the acetate-fed MFC. To prove this hypothesis, G. sulfurreducens strain PCA was cocultivated with the strain AR20 in the acetate-fed MFC without any dissolved electron acceptors. In the coculture MFC of G. sulfurreducens and strain AR20, current generation and acetate degradation were the highest, and the growth of strain AR20 was observed. No current generation, acetate degradation and cell growth occurred in the strain AR20 pure culture MFC. These results show for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with the isolated Hydrogenophaga sp. strain AR20, with electrode as the electron acceptor.     

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.     

Proteogenomic Monitoring of Geobacter Physiology during Stimulated Uranium Bioremediation

Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.Enzymatic reduction of U(VI) to insoluble U(IV) by dissimilatory Fe(III)-reducing bacteria (DIRB) can limit subsurface U(VI) migration (2, 19, 20). This observation provided the basis for an important new biotechnology that involves remediation of contaminated groundwater by organic amendment-based stimulation of the activities of DIRB. Despite the obvious high potential value, there are significant technological challenges that must be overcome before this approach can be deployed as a functional biotechnology. In situ U(VI) reduction rates are directly coupled with microbial physiology and community composition, both of which change as bioremediation progresses (22, 24, 28). Thus, a key need is the ability to monitor changes in microbial community membership and function as they occur, so that optimal management strategies can be implemented to achieve the desired geochemical outcomes. This challenge is significant because metal reduction occurs within pore spaces deep in the aquifer and the process is associated with vast numbers of microbial cells distributed in the subsurface.Changes in the complements of DIRB proteins should reflect shifts in microbial physiology and could be used to monitor in situ bioremediation technologies if microbial proteomes could be tracked during organic amendment. Proteomic methods have previously been used to detect physiological responses of microorganisms growing in pure culture (8, 16, 17) and in genomically characterized natural microbial communities (7, 18, 26, 34). Strain-resolved proteomic approaches (18) have the potential to also track changes in microbial community composition. Previously, the use of proteomic analysis techniques to monitor subsurface bioremediation has been precluded by the lack of metagenomic data. Here, we used seven isolate Geobacter genomes to generate a reference database against which peptide data measured using two-dimensional (2D) liquid chromatography (LC)-based high-resolution tandem mass spectrometry (MS-MS) were compared for protein identification. Although differences between environmental and isolate peptide sequences may preclude peptide identification, peptides common to multiple Geobacter types and isolates enable protein identification, and peptides unique to one isolate constrain environmental genotypes. Simultaneous analysis of community structure and function in natural microbial communities that relies upon proteomics-derived genomic insights is referred to as proteogenomics (Fig. ​(Fig.1).1). In the current study, a proteogenomic approach was applied to monitor the progress of an in situ U bioremediation project carried out at the Department of Energy (DOE) Integrated Field Research Challenge site in Rifle, CO. Despite strain complexity in the recovered samples, proteomic data provided insights into the community structure and physiology of planktonic Geobacter isolates in aquifer solutions as groundwater U(VI) concentrations decreased.Open in a separate windowFIG. 1.Strain-resolved proteogenomic techniques allow identification of unique peptides and constrain the genotypes present in environmental samples. (1) Proteins were predicted from the seven isolate Geobacter genomes. (2) Proteins were aligned. In the example shown, they share a conserved region (similar colors in all seven regions) and have a variable region (indicated by the variety of colors for each protein). (3) Tryptic digest patterns are used to predict peptide sequences. Proteins were extracted from the experimental sample (4) and digested into peptides by using trypsin (5). These peptides were separated using LC (6) before high-resolution mass spectrometry on a fraction of peptides from an elution peak (7) to determine the mass of the parent ions. (8) The peptides are fragmented and mass spectrometry measurements made on fragment ions. (9) Mass spectral data are matched to peptide sequences predicted in step 3 to allow peptide identification. Peptide sequences differing by only one amino acid can be identified, as shown in the spectral diagram. (10) Detected peptides are mapped onto aligned protein sequences to identify peptides shared or unique to each of the isolate species. Spectral counts can be mapped together with peptides. Where coexisting unique peptides are detected, ratios of unique spectral counts can be used to infer relative strain abundance. As shown, the identification of a unique peptide indicates that this protein is most closely related to that of G. bemidjiensis.     

Thermodynamic and kinetic characterization of two methyl-accepting chemotaxis heme sensors from Geobacter sulfurreducens reveals the structural origin of their functional difference

The periplasmic sensor domains GSU582 and GSU935 are part of methyl-accepting chemotaxis proteins of the bacterium Geobacter sulfurreducens containing one c-type heme and a PAS-like fold. Their spectroscopic properties were shown previously to share similar spectral features. In both sensors, the heme group is in the high-spin form in the oxidized state and low-spin after reduction and binding of a methionine residue. Therefore, it was proposed that this redox-linked ligand switch might be related to the signal transduction mechanism. We now report the thermodynamic and kinetic characterization of the sensors GSU582 and GSU935 by visible spectroscopy and stopped-flow techniques, at several pH and ionic strength values. Despite their similar spectroscopic features, the midpoint reduction potentials and the rate constants for reduction by dithionite are considerably different in the two sensors. The reduction potentials of both sensors are negative and well framed within the typical anoxic subsurface environments in which Geobacter species predominate. The midpoint reduction potentials of sensor GSU935 are lower than those of GSU582 at all pH and ionic strength values and the same was observed for the reduction rate constants. The origin of the different functional properties of these closely related sensors is rationalized in the terms of the structures. The results suggest that the sensors are designed to function in different working potential ranges, allowing the bacteria to trigger an adequate cellular response in different anoxic subsurface environments. These findings provide an explanation for the co-existence of two similar methyl-accepting chemotaxis proteins in G. sulfurreducens.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling

Geobacter sulfurreducens is a well-studied representative of the Geobacteraceae, which play a critical role in organic matter oxidation coupled to Fe(III) reduction, bioremediation of groundwater contaminated with organics or metals, and electricity production from waste organic matter. In order to investigate G. sulfurreducens central metabolism and electron transport, a metabolic model which integrated genome-based predictions with available genetic and physiological data was developed via the constraint-based modeling approach. Evaluation of the rates of proton production and consumption in the extracellular and cytoplasmic compartments revealed that energy conservation with extracellular electron acceptors, such as Fe(III), was limited relative to that associated with intracellular acceptors. This limitation was attributed to lack of cytoplasmic proton consumption during reduction of extracellular electron acceptors. Model-based analysis of the metabolic cost of producing an extracellular electron shuttle to promote electron transfer to insoluble Fe(III) oxides demonstrated why Geobacter species, which do not produce shuttles, have an energetic advantage over shuttle-producing Fe(III) reducers in subsurface environments. In silico analysis also revealed that the metabolic network of G. sulfurreducens could synthesize amino acids more efficiently than that of Escherichia coli due to the presence of a pyruvate-ferredoxin oxidoreductase, which catalyzes synthesis of pyruvate from acetate and carbon dioxide in a single step. In silico phenotypic analysis of deletion mutants demonstrated the capability of the model to explore the flexibility of G. sulfurreducens central metabolism and correctly predict mutant phenotypes. These results demonstrate that iterative modeling coupled with experimentation can accelerate the understanding of the physiology of poorly studied but environmentally relevant organisms and may help optimize their practical applications.