Subsurface Microorganisms Research Articles

Monitoring the Metabolic Status of Geobacter Species in Contaminated Groundwater by Quantifying Key Metabolic Proteins with Geobacter-Specific Antibodies

Simple and inexpensive methods for assessing the metabolic status and bioremediation activities of subsurface microorganisms are required before bioremediation practitioners will adopt molecular diagnosis of the bioremediation community as a routine practice for guiding the development of bioremediation strategies. Quantifying gene transcripts can diagnose important aspects of microbial physiology during bioremediation but is technically challenging and does not account for the impact of translational modifications on protein abundance. An alternative strategy is to directly quantify the abundance of key proteins that might be diagnostic of physiological state. To evaluate this strategy, an antibody-based quantification approach was developed to investigate subsurface Geobacter communities. The abundance of citrate synthase corresponded with rates of metabolism of Geobacter bemidjiensis in chemostat cultures. During in situ bioremediation of uranium-contaminated groundwater the quantity of Geobacter citrate synthase increased with the addition of acetate to the groundwater and decreased when acetate amendments stopped. The abundance of the nitrogen-fixation protein, NifD, increased as ammonium became less available in the groundwater and then declined when ammonium concentrations increased. In a petroleum-contaminated aquifer, the abundance of BamB, an enzyme subunit involved in the anaerobic degradation of mono-aromatic compounds by Geobacter species, increased in zones in which Geobacter were expected to play an important role in aromatic hydrocarbon degradation. These results suggest that antibody-based detection of key metabolic proteins, which should be readily adaptable to standardized kits, may be a feasible method for diagnosing the metabolic state of microbial communities responsible for bioremediation, aiding in the rational design of bioremediation strategies.

A c‐type cytochrome and a transcriptional regulator responsible for enhanced extracellular electron transfer in Geobacter sulfurreducens revealed by adaptive evolution

The stimulation of subsurface microbial metabolism often associated with engineered bioremediation of groundwater contaminants presents subsurface microorganisms, which are adapted for slow growth and metabolism in the subsurface, with new selective pressures. In order to better understand how Geobacter species might adapt to selective pressure for faster metal reduction in the subsurface, Geobacter sulfurreducens was put under selective pressure for rapid Fe(III) oxide reduction. The genomes of two resultant strains with rates of Fe(III) oxide reduction that were 10-fold higher than those of the parent strain were resequenced. Both strains contain either a single base-pair change or a 1 nucleotide insertion in a GEMM riboswitch upstream of GSU1761, a gene coding for the periplasmic c-type cytochrome designated PgcA. GSU1771, a gene coding for a SARP regulator, was also mutated in both strains. Introduction of either of the GEMM riboswitch mutations upstream of pgcA in the wild-type increased the abundance of pgcA transcripts, consistent with increased expression of pgcA in the adapted strains. One of the mutations doubled the rate of Fe(III) oxide reduction. Interruption of GSU1771 doubled the Fe(III) oxide reduction rate. This was associated with an increased in expression of pilA, the gene encoding the structural protein for the pili thought to function as microbial nanowires. The combination of the GSU1771 interruption with either of the pgcA mutations resulted in a strain that reduced Fe(III) as fast as the comparable adapted strain. These results suggest that the accumulation of a small number of beneficial mutations under selective pressure, similar to that potentially present during bioremediation, can greatly enhance the capacity for Fe(III) oxide reduction in G. sulfurreducens. Furthermore, the results emphasize the importance of the c-type cytochrome PgcA and pili in Fe(III) oxide reduction and demonstrate how adaptive evolution studies can aid in the elucidation of complex mechanisms, such as extracellular electron transfer.

Functional microbial community response to nutrient pulses by artificial groundwater recharge practice in surface soils and subsoils

Subsurface microorganisms are essential constituents of the soil purification processes associated with groundwater quality. In particular, soil enzyme activity determines the biodegradation of organic compounds passing through the soil profile. Transects from surface soil to a depth of 3.5 m were investigated for microbial and chemical soil characteristics at two groundwater recharge sites and one control site. The functional diversity of the microbial community was analyzed via the activity of eight enzymes. Acid phosphomonoesterase was dominant across sites and depths, followed by L-leucine aminopeptidase and beta-glucosidase. Structural [e.g. phospholipid fatty acid (PLFA) pattern] and functional microbial diversities were linked to each other at the nonwatered site, whereas amendment with nutrients (DOC, NO(3)(-)) by flooding uncoupled this relationship. Microbial biomass did not differ between sites, whereas microbial respiration was the highest at the watered sites. Hence, excess nutrients available due to artificial groundwater recharge could not compensate for the limitation by others (e.g. phosphorus as assigned by acid phosphomonoesterase activity). Instead, at a similar microbial biomass, waste respiration via overflow metabolism occurred. In summary, ample supply of carbon by flooding led to a separation of decomposition and microbial growth, which may play an important role in regulating purification processes during groundwater recharge.

Microbial Physiology in Deep Biosphere of the Earth

To date, together with rapidly developed culture-independent molecular techniques, knowledge in biomass, phylogenetic diversity and biogeography of microbial components has been extensively characterized in various terrestrial and oceanic habitats of deep biosphere. However, the general sketch of phenotypic and physiological diversity of subsurface microorganisms has remained poorly understood as most of the indigenous subsurface microbial components still escape from the laboratory cultivations. In this article, the physiological properties of the microorganisms that have been cultivated from various subsurface environments are overviewed.

Coupling a genome-scale metabolic model with a reactive transport model to describe in situ uranium bioremediation.

SummaryThe increasing availability of the genome sequences of microorganisms involved in important bioremediation processes makes it feasible to consider developing genome‐scale models that can aid in predicting the likely outcome of potential subsurface bioremediation strategies. Previous studies of the in situ bioremediation of uranium‐contaminated groundwater have demonstrated that Geobacter species are often the dominant members of the groundwater community during active bioremediation and the primary organisms catalysing U(VI) reduction. Therefore, a genome‐scale, constraint‐based model of the metabolism of Geobacter sulfurreducens was coupled with the reactive transport model HYDROGEOCHEM in an attempt to model in situ uranium bioremediation. In order to simplify the modelling, the influence of only three growth factors was considered: acetate, the electron donor added to stimulate U(VI) reduction; Fe(III), the electron acceptor primarily supporting growth of Geobacter; and ammonium, a key nutrient. The constraint‐based model predicted that growth yields of Geobacter varied significantly based on the availability of these three growth factors and that there are minimum thresholds of acetate and Fe(III) below which growth and activity are not possible. This contrasts with typical, empirical microbial models that assume fixed growth yields and the possibility for complete metabolism of the substrates. The coupled genome‐scale and reactive transport model predicted acetate concentrations and U(VI) reduction rates in a field trial of in situ uranium bioremediation that were comparable to the predictions of a calibrated conventional model, but without the need for empirical calibration, other than specifying the initial biomass of Geobacter. These results suggest that coupling genome‐scale metabolic models with reactive transport models may be a good approach to developing models that can be truly predictive, without empirical calibration, for evaluating the probable response of subsurface microorganisms to possible bioremediation approaches prior to implementation.

Quantifying expression of Geobacter spp. oxidative stress genes in pure culture and during in situ uranium bioremediation

As part of an effort to diagnose the physiological status of Geobacter species during in situ bioremediation of uranium-contaminated groundwater, transcript levels for two genes potentially associated with oxidative stress, cydA and sodA, were quantified throughout a bioremediation field study in Rifle, CO, USA. Despite the accumulation of Fe(II) in the groundwater, which is inconsistent with the presence of dissolved oxygen, both genes were highly expressed during the bioremediation process. Therefore, the response to oxidative stress was further evaluated with Geobacter uraniireducens, an isolate from the Rifle site. When G. uraniireducens cultured with fumarate as the electron acceptor was exposed to 5% oxygen for 8 h, there was a significant increase in cydA and sodA transcripts as well as other genes associated with oxygen respiration or oxidative stress. Oxygen-exposed cells had lower transcript abundance for genes associated with anaerobic respiration, metabolism and motility. Short-term oxygen exposure had little impact on cydA transcript levels, as more than 1 h was required for increases to levels comparable to the subsurface. Abundance of cydA and sodA transcripts for the isolate G. sulfurreducens were always higher in cells cultured with Fe(III) compared with fumarate as an electron acceptor, even when fumarate-grown cells were exposed to oxygen, and Fe(III)-grown cells were grown anaerobically. These results suggest that the apparently high Geobacter cydA and sodA expression during bioremediation cannot necessarily be attributed to oxidative stress and demonstrate that diagnosis of the metabolic status of subsurface microorganisms through transcript analysis should be coupled with appropriate geochemical analyses.

Flooding forested groundwater recharge areas modifies microbial communities from top soil to groundwater table

Subsurface microorganisms are crucial for contaminant degradation and maintenance of groundwater quality. This study investigates the microbial biomass and community composition [by phospholipid fatty acids (PLFAs)], as well as physical and chemical soil characteristics at woodland flooding sites of an artificial groundwater recharge system used for drinking water production. Vertical soil profiles to c. 4 m at two watered and one nonwatered site were analyzed. The microbial biomass was equal in watered and nonwatered sites, and considerable fractions (25-42%) were located in 40-340 cm depth. The microbial community structure differed significantly between watered and nonwatered sites, predominantly below 100 cm depth. Proportions of the bacterial PLFAs 16:1omega5, 16:1omega7, cy17:0 and 18:1omega9t, and the long-chained PLFAs 22:1omega9 and 24:1omega9 were more prominent at the watered sites, whereas branched, saturated PLFAs (iso/anteiso) dominated at the nonwatered site. PLFA community indices indicated stress response (trans/cis ratio), higher nutrient availability (unsaturation index) and changes in membrane fluidity (iso/anteiso ratio) due to flooding. In conclusion, water recharge processes led to nutrient input and altered environmental conditions, which resulted in a highly active and adapted microbial community residing in the vadose zone that effectively degraded organic compounds.

In situ bacterial colonization of compacted bentonite under deep geological high-level radioactive waste repository conditions

Subsurface microorganisms are expected to invade, colonize, and influence the safety performance of deep geological spent nuclear fuel (SNF) repositories. An understanding of the interactions of subsurface dwelling microbial communities with the storage is thus essential. For this to be achieved, experiments must be conducted under in situ conditions. We investigated the presence of groundwater microorganisms in repository bentonite saturated with groundwater recovered from tests conducted at the Aspö underground Hard Rock Laboratory in Sweden. A 16S ribosomal RNA and dissimilatory bisulfite reductase gene distribution between the bentonite and groundwater samples suggested that the sulfate-reducing bacteria widespread in the aquifers were not common in the clay. Aerophilic bacteria could be cultured from samples run at <or=55 degrees C but not at >or=67 degrees C. Generally, the largely gram-negative groundwater microorganisms were poorly represented in the bentonite while the gram-positive bacteria commonly found in the clay predominated. Thus, bentonite compacted to a density of approximately 2 g cm(-3) together with elevated temperatures might discourage the mass introduction of the predominantly mesophilic granitic aquifer bacteria into future SNF repositories in the long run.

Session 33. Subsurface Microorganisms in Terrestrial and Extraterrestrial Environments

Session 33. Subsurface Microorganisms in Terrestrial and Extraterrestrial Environments

Feast and famine — microbial life in the deep-sea bed

The seabed is a diverse environment that ranges from the desert-like deep seafloor to the rich oases that are present at seeps, vents, and food falls such as whales, wood or kelp. As well as the sedimentation of organic material from above, geological processes transport chemical energy--hydrogen, methane, hydrogen sulphide and iron--to the seafloor from the subsurface below, which provides a significant proportion of the deep-sea energy. At the sites on the seafloor where chemical energy is delivered, rich and diverse microbial communities thrive. However, most subsurface microorganisms live in conditions of extreme energy limitation, with mean generation times of up to thousands of years. Even in the most remote subsurface habitats, temperature rather than energy seems to set the ultimate limit for life, and in the deep biosphere, where energy is most depleted, life might even be based on the cleavage of water by natural radioisotopes. Here, we review microbial biodiversity and function in these intriguing environments.

地下圏微生物活動に対する難透水層の役割

Microorganisms have a variety of functions that relates to the remediation of subsurface contaminants. Therefore, they are important in subsurface environments. Studies on subsurface microbial ecology with the aim of remediating subsurface contamination have mainly focused on groundwater or aquifer materials because the aquitard (composed of clay or silt) has been considered as a place with little or no microbial activity. However, recent studies on microbiology in aquitard materials have revealed new aspects of this environment. The aquitard can be a source of nutrients, such as organic matter, to the adjacent aquifer, and stimulate microbial activity in the aquifer. Microbial activity is considered to be limited by the pore size distribution of the aquitard, and relatively higher microbial activity has been detected in a younger and shallower aquitard with larger pore size. In this article, I introduce these relationships between subsurface microorganisms and the aquitard.

Detection of Methane in the Atmosphere of Mars

We report a detection of methane in the martian atmosphere by the Planetary Fourier Spectrometer onboard the Mars Express spacecraft. The global average methane mixing ratio is found to be 10 +/- 5 parts per billion by volume (ppbv). However, the mixing ratio varies between 0 and 30 ppbv over the planet. The source of methane could be either biogenic or nonbiogenic, including past or present subsurface microorganisms, hydrothermal activity, or cometary impacts.

Microbiology of formation waters from the deep repository of liquid radioactive wastes Severnyi

The presence, diversity, and geochemical activity of microorganisms in the Severnyi repository of liquid radioactive wastes were studied. Cultivable anaerobic denitrifiers, fermenters, sulfate-reducers, and methanogens were found in water samples from a depth of 162–405 m below sea level. Subsurface microorganisms produced methane from [2- 14C]acetate and [ 14C]CO 2, formed hydrogen sulfide from Na 2 35SO 4, and reduced nitrate to dinitrogen in medium with acetate. The cell numbers of all studied groups of microorganisms and rates of anaerobic processes were higher in the zone of dispersion of radioactive wastes. Microbial communities present in the repository were able to utilise a wide range of organic and inorganic compounds and components of waste (acetate, nitrate, and sulfate) both aerobically and anaerobically. Bacterial production of gases may result in a local increase of the pressure in the repository and consequent discharge of wastes onto the surface. Microorganisms can indirectly decrease the mobility of radionuclides due to consumption of oxygen and production of sulfide, which favours deposition of metals. These results show the necessity of long-term microbiological and radiochemical monitoring of the repository.

Mineral stimulation of subsurface microorganisms: release of limiting nutrients from silicates

Microorganisms play an important role in the weathering of silicate minerals in many subsurface environments, but an unanswered question is whether the mineral plays an important role in the microbial ecology. Silicate minerals often contain nutrients necessary for microbial growth, but whether the microbial community benefits from their release during weathering is unclear. In this study, we used field and laboratory approaches to investigate microbial interactions with minerals and glasses containing beneficial nutrients and metals. Field experiments from a petroleum-contaminated aquifer, where silicate weathering is substantially accelerated in the contaminated zone, revealed that phosphorus (P) and iron (Fe)-bearing silicate glasses were preferentially colonized and weathered, while glasses without these elements were typically barren of colonizing microorganisms, corroborating previous studies using feldspars. In laboratory studies, we investigated microbial weathering of silicates and the release of nutrients using a model ligand-promoted pathway. A metal-chelating organic ligand 3,4 dihydroxybenzoic acid (3,4 DHBA) was used as a source of chelated ferric iron, and a carbon source, to investigate mineral weathering rate and microbial metabolism. In the investigated aquifer, we hypothesize that microbes produce organic ligands to chelate metals, particularly Fe, for metabolic processes and also form stable complexes with Al and occasionally with Si. Further, the concentration of these ligands is apparently sufficient near an attached microorganism to destroy the silicate framework while releasing the nutrient of interest. In microcosms containing silicates and glasses with trace phosphate mineral inclusions, microbial biomass increased, indicating that the microbial community can use silicate-bound phosphate inclusions. The addition of a native microbial consortium to microcosms containing silicates or glasses with iron oxide inclusions correlated to accelerated weathering and release of Si into solution as well as the accelerated degradation of the model substrate 3,4 DHBA. We propose that silicate-bound P and Fe inclusions are bioavailable, and microorganisms may use organic ligands to dissolve the silicate matrix and access these otherwise limiting nutrients.

Intrinsic remediation of trichloroethene driven by tetraalkoxysilanes as co‐contaminants: Results of microcosm and field studies

AbstractBiological reduction of trichloroethene (TCE), driven by the transformation products of tetraalkoxysilanes, was investigated in seasonal field monitorings and anaerobic groundwater microcosms. Under anaerobic conditions, tetraalkoxysilanes such as tetrabutoxysilane (TBOS) and tetrakis(2‐ethylbutoxy)silane (TKEBS) are abiotically hydrolyzed to their corresponding alcohols, 1‐butanol and 2‐ethylbutanol, respectively, and silicic acid. These alcohols are then fermented by subsurface microorganisms to their corresponding acids and hydrogen, which likely serves as the ultimate electron donor facilitating reductive dechlorination of TCE. At Site 300, Lawrence Livermore National Laboratory (LLNL), California, tetraalkoxysilanes are present along with TCE as subsurface contaminants. Intrinsic transformation of TCE to cis‐dichloroethene (c‐DCE) was observed at this site, and this was promoted by the transformation products of these tetraalkoxysilanes. Efficient transformation of high concentrations of TCE (150–300 mg/l aqueous concentration) was observed in anaerobic microcosms constructed with groundwater from this site. The lack of transformation of c‐DCE to vinyl chloride (VC) and ethene in the microcosms is consistent with studies of the microbial community in the site groundwater reported by Lowe et al. (2002). © 2003 Wiley Periodicals, Inc.

Search for life in deep biospheres

The life in deep biospheres bridges conventional biology and future exobiology. This review focuses the microbiological studies from the selected deep biospheres, i.e., deep-sea hydrothermal vents, sub-hydrothermal vents, terrestrial subsurface and a sub-glacier lake. The dark biospheres facilitate the emergence of organisms and communities dependent on chemolithoautotrophy, which are overwhelmed by photoautotrophy (photosynthesis) in the surface biospheres. The life at deep-sea hydrothermal vents owes much to chemolithoautotrophy based on the oxidation of sulfide emitted from the vents. It is likely that similarly active bodies such as the Jovian satellite Europa may have hydrothermal vents and associated biological communities. Anoxic or anaerobic condition is characteristic of deep subsurface biospheres. Subsurface microorganisms exploit available oxidants, or terminal electron acceptors (TEA), for anaerobic respiration. Sulfate, nitrate, iron (III) and CO2 are the representative TEAs in the deep subsurface. Below the 3000-4000 m-thick glacier on Antarctica, there have been >70 lakes with liquid water located. One of such sub-glacial lakes, Lake Vostok, is about to be drill-penetrated for microbiological studies. These deep biosphere "platforms" provide new knowledge about the diversity and potential of the Earth's life. The expertise obtained from the deep biosphere expeditions will facilitate the capability of exobiologial exploration.

Bioturbation effects of Chironomus riparius on the benthic N-cycle as measured using microsensors and microbiological assays

Chironomus riparius (Diptera) larvae were added to laboratory microcosms containing defaunated sediments sampled at 2 NO 3 -polluted field sites. Following a 3 wk incubation, the larval influence on the sedimentary nitrogen conversions was studied using microsensors (O 2 , NO 3 - , NH 4 + ) and microbial bulk parameters (microbial biomass, community respiration). At the sediment surface the chironomid larvae fed on particles (deposit-feeding layer), while in the subsurface zone the larvae moved through the sediment and ventilated transient or permanent burrows (ventilation layer). In the deposit-feeding layer of the chironomids, NO 3 - production and NH 4 + consumption were lower and microbial biomass decreased. In the ventilation layer of the chironomids, NO 3 - and NH 4 + conversion maxima were shifted downwards, and both microbial biomass and community respiration were increased. The observed changes in the vertical stratigraphy of the benthic microbial community were ascribed to the depth-specific larval behaviour as: (1) particle ingestion and removal of adhering microorganisms in the deposit-feeding layer; and (2) stimulation of subsurface microorganisms due to an increased supply of O 2 and NO 3 - along with larval ventilation.

Microbial H2 Cycling Does Not Affect δ2H Values of Ground Water.

AbstractStable hydrogen‐isotope values of ground water (δ2H) and dissolved hydrogen concentrations (H2(aq)) were quantified in a petroleum‐hydrocarbon contaminated aquifer to determine whether the production/consumption of H2 by subsurface microorganisms affects ground water δ2H values. The range of δ2H observed in monitoring wells sampled (−27.8 %c to −15.5 %c) was best explained, however, by seasonal differences in recharge temperature as indicated using ground water δ18O values, rather than iso‐topic exchange reactions involving the microbial cycling of H2 during anaerobic petroleum‐hydrocarbon biodegradation. The absence of a measurable hydrogen‐isotope exchange between microbialy cycled H2 and ground water reflects the fact that the amount of H2 available from the anaerobic decomposition of petroleum hydrocarbons is small relative to the amount of hydrogen present in water, even though milligram per liter concentrations of readily biodegradable contaminants are present at the study site. Additionally, isotopic fractionation calculations indicate that in order for H2 cycling processes to affect δ2H values of ground water, relatively high concentrations of H2 (&gt;0.080 M) would have to be maintained, considerably higher than the 0.2 to 26 nM present at this site and characteristic of anaerobic conditions in general. These observations suggest that the conventional approach of using δ2H and δ18O values to determine recharge history is appropriate even for those ground water systems characterized by anaerobic conditions and extensive microbial H2 cycling.

Nucleic-acid characterization of the identity and activity of subsurface microorganisms

Nucleic-acid characterization of the identity and activity of subsurface microorganisms

Modelling nutrient-periphyton dynamics in streams with surface–subsurface exchange

Stream ecosystems include both surface and subsurface components that are connected by the flow of water. Processes occurring in subsurface sediments affect those in the surface, and vice versa. A model was developed to investigate the effects of nutrient transformations occurring in subsurface sediments on the growth of surface-dwelling periphyton. In the model the stream is divided into three zones: free-flowing surface water, a surface storage zone where flow is minimal and where periphyton growth occurs, and a subsurface zone. Parameters were based on information from Sycamore Creek, Arizona, a nitrogen-limited desert stream that has been extensively studied. The behavior of the model was examined both at steady state and as it approached steady state after periphyton biomass was reduced to low values, simulating the effects of a scouring flood. Previous work has shown that subsurface sediments are a source of inorganic nutrients, mainly nitrogen, to surface water and to periphyton communities in Sycamore Creek. Thus it was expected that in this model, periphyton biomass would increase when exchange between surface and subsurface zones was increased, and biomass would also increase when the rate of nutrient transformation in subsurface sediments was increased. Model results confirmed the expectations and highlighted the important role of organic nitrogen in mediating the periphyton-nutrient feedback. Post-flood recovery of periphyton biomass was particularly sensitive to elevated concentrations of inorganic nitrogen in flood water. The model shows that processes in the subsurface zone can have a large effect on surface organisms, and illustrates how surface periphyton communities interact with subsurface microorganisms through the recycling of nutrients.