Geology topics
Ronald S. Oremland
Publications and source records attributed to Ronald S. Oremland.
Coupled arsenotrophy in a hot spring photosynthetic biofilm at Mono Lake, California
Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated (∼ 85% of 16S rRNA gene clones) by sequences from the photosynthetic Ectothiorhodospira genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with 73 As(V) demonstrated that reduction to 73 As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [ 14 C]acetate oxidized the radiolabel to 14 CO 2 in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [ 14 C]bicarbonate into cell material was stimulated by either H 2 or HS − . Functional genes for the arsenate respiratory reductase ( arrA ) and arsenic resistance ( arsB ) were detected in sequenced amplicons of extracted DNA, with about half of the arrA sequences closely related (∼98% translated amino acid identity) to those from the family Ectothiorhodospiraceae . Surprisingly, no authentic PCR products for arsenite oxidase ( aoxB ) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms. Oxyanions of the group 15 element arsenic, arsenate [As(V)] and arsenite [As(III)], have been known for millennia to be potent poisons. Despite its well-established toxicity to life, the phenomenon of arsenic resistance was discovered whereby some microorganisms maintain an otherwise “normal” existence in the presence of high concentrations of As(V) or As(III) (17, 29, 31). More recently it has become recognized that certain representatives from the bacterial and archaeal domains can actually exploit the electrochemical potential of the As(V)/As(III) redox couple (+130 mV) to gain energy for growth. This can be achieved either by employing As(III) as an autotrophic electron donor or by using As(V) as a respiratory electron acceptor (18, 21, 34). The latter phenomenon, although most commonly associated with chemoheterotrophy, can also employ inorganic substances like sulfide or H 2 . Indeed, As(V)-respiring anaerobes displaying a capacity for chemoautotrophy with these electron donors have been isolated and described (5, 7, 16). We recently reported that photoautotrophy is supported by As(III) in anoxic biofilms located in hot springs on Paoha Island in Mono Lake, CA (15). This process represented a novel means of As(III) oxidation achieved via anoxygenic photosynthesis occurring in certain photosynthetic bacteria (i.e., Ectothiorhodospira ) and possibly within some cyanobacteria as well (e.g., “ Oscillatoria ”). Whether or not a microbial habitat is overtly oxic or anoxic, or temporally shifts between these two states over a diel cycle, critical energy linkages between aerobes and anaerobes have long been known for the biogeochemical cycles of key elements, such as sulfur, iron, and nitrogen. Most prominently studied is the case of nitrogen, whereby an ecological coupling exists between the processes of nitrification and denitrification (9, 10, 28). The former process provides energy to aerobic nitrifiers, while the latter process consumes the nitrate produced by this reaction, thereby meeting the energy needs of the denitrifiers. For arsenic, the detection of both As(III) oxidation and As(V) reduction in oxic and anoxic incubations of freshly collected periphyton suggested that an analogous coupled process may also occur for this element (12). Similarly, several uncontaminated soils in Japan displayed a capacity for either As(V) reduction or As(III) oxidation upon arsenic oxyanion amendment and whether they were incubated under oxic or anoxic conditions (39). A defined coculture consisting of an aerobic As(III) oxidizer (strain OL1) and an anaerobic As(V) respirer (strain Y5) was shown to function in this fashion under manipulated laboratory conditions of oxygen tension (26). We pursued the phenomenon of coupled arsenic metabolism further by using materials collected from the hot spring biofilms in Mono Lake, but we focused on examination of the cycling of arsenic under anoxic conditions. In this paper we report results obtained by manipulated incubations of red-pigmented biofilms found in the hot springs of Paoha Island. Preliminary community characterizations of these biofilms show that they are dominated by B acteria from the genus Ectothiorhodospira but also harbor an assemblage of Archaea related to the Halobacteriacaea . Incubation results have demonstrated the presence of the following arsenic metabolic activities: respiratory As(V) reduction, photosynthetic anaerobic As(III) oxidation, and aerobic As(III) oxidation, along with the ecophysiological conditions under which they occur. Surprisingly, we were unable to obtain authentic PCR products for arsenite oxidase genes ( aoxB ), despite observing aerobic As(III) oxidation activity. These biofilms serve as a model system for how anaerobic cycling of arsenic can be sustained with oxidation of As(III) by anoxygenic photosynthesis coupled to regeneration of this electron donor via dissimilatory As(V) reduction. The significance that such a light-driven anaerobic ecosystem may have played in the Archean Earth is discussed.
Identification of a novel arsenite oxidase gene, arxA, in the haloalkaliphilic, arsenite-oxidizing bacterium alkalilimnicola ehrlichii strain MLHE-1
Although arsenic is highly toxic to most organisms, certain prokaryotes are known to grow on and respire toxic metalloids of arsenic (i.e., arsenate and arsenite). Two enzymes are known to be required for this arsenic-based metabolism: (i) the arsenate respiratory reductase (ArrA) and (ii) arsenite oxidase (AoxB). Both catalytic enzymes contain molybdopterin cofactors and form distinct phylogenetic clades (ArrA and AoxB) within the dimethyl sulfoxide (DMSO) reductase family of enzymes. Here we report on the genetic identification of a “new” type of arsenite oxidase that fills a phylogenetic gap between the ArrA and AoxB clades of arsenic metabolic enzymes. This “new” arsenite oxidase is referred to as ArxA and was identified in the genome sequence of the Mono Lake isolate Alkalilimnicola ehrlichii MLHE-1, a chemolithoautotroph that can couple arsenite oxidation to nitrate reduction. A genetic system was developed for MLHE-1 and used to show that arxA (gene locus ID mlg _ 0216 ) was required for chemoautotrophic arsenite oxidation. Transcription analysis also showed that mlg _ 0216 was only expressed under anaerobic conditions in the presence of arsenite. The mlg _ 0216 gene is referred to as arxA because of its greater homology to arrA relative to aoxB and previous reports that implicated Mlg_0216 (ArxA) of MLHE-1 in reversible arsenite oxidation and arsenate reduction in vitro . Our results and past observations support the position that ArxA is a distinct clade within the DMSO reductase family of proteins. These results raise further questions about the evolutionary relationships between arsenite oxidases (AoxB) and arsenate respiratory reductases (ArrA). Arsenic is toxic to most organisms and is known to cause cancer in humans. However, bacteria have adapted several biotransformation pathways that function to either couple the reduction or oxidation of arsenicals to energy conservation and growth (1). The enzymologies of these two pathways have several features in common. The arsenate respiratory reductase (ArrAB) and arsenite oxidase (AoxAB) enzymes are usually composed of at least two subunits, a small iron-sulfur cluster-containing subunit (ArrB and AoxA) and a larger molybdopterin-containing catalytic subunit (ArrA and AoxB). Although they catalyze arsenic redox chemistry, ArrA and AoxB form distinct phylogenetic clades within the dimethyl sulfoxide (DMSO) reductase family of molybdenum-containing enzymes (16, 24). Culture-dependent approaches have resulted in the isolation of a variety of diverse bacteria that metabolize arsenic (reviewed in reference 26). Many of these isolates have had their genomes sequenced, which has been insightful for understanding the composition and diversity of arr and aox gene clusters. In the arsenite-oxidizing nitrate reducer Alkalilimnicola ehrlichii strain MLHE-1 (a haloalkaliphile isolated from Mono Lake [CA]) (10, 15), bioinformatic analysis of its genome revealed the absence of genes homologous to the arsenite oxidase genes of the aoxB type. Instead, two genes ( mlg _ 0216 and mlg _ 2426 ) were identified that better resembled the catalytic subunit of the arsenate respiratory reductase (20); however, MLHE-1 has not been shown to respire (or reduce) arsenate (15). Recent work by Richey et al. (20) showed that the Mlg_0216 protein (and not Mlg_2426) was expressed under chemolithoautotrophic (10 mM arsenite and 10 mM nitrate) growth conditions. Moreover, it was shown that Mlg_0216 exhibits both arsenate reductase and arsenite oxidase activities in vitro . These observations raised the question, is the mlg _ 0216 gene required for arsenite oxidation in vivo ? In this report, we addressed this question by developing a genetic system in MLHE-1, generating strains with mutations in mlg _ 0216 and mlg _ 2426 , and physiologically characterizing the resulting strains. Our results implicate mlg _ 0216 in chemolithoautotrophic arsenite oxidation coupled to nitrate respiration.
Response to comment on "Arsenic(III) Fuels Anoxygenic Photosynthesis in Hot Spring Biofilms from Mono Lake, California"
Schoepp-Cothenet et al . bring a welcome conceptual debate to the question of which came first in the course of planetary biological evolution, arsenite [As(III)] oxidation or dissimilatory arsenate [As(V)] reduction. However, we disagree with their reasoning and stand by our original conclusion.
Bacterial Cycling of Methyl Halides
This chapter focuses on the monohalogenated methanes methyl chloride (MeCl) and methyl bromide (MeBr), their natural and anthropogenic sources, and their degradation by microorganisms, specifically by aerobic bacteria that can use MeBr and MeCl as sole source of carbon and energy. The biogeochemical cycle of methyl halides and the microbiology, biochemistry, genetics, and biotechnological potential of methyl halide-degrading microorganisms are discussed in the chapter. Methyl halides are the dominant halocarbons in the atmosphere. They play an important role in regulating stratospheric ozone concentrations and global warming as well, two factors governing planetary habitability. The monohalomethanes—methyl chloride (MeCl), methyl bromide (MeBr), and methyl iodide (MeI)—are trace gases in the atmosphere with average tropospheric-mixing ratios of 600, 10, and 2 parts per trillion (ppt), respectively. However, methyl halides are radiatively active and hence contribute to global warming by absorbing radiation in the infrared region. This is evident in their elevated global warming potential (GWP), a value calculated to quantify each compound's warming effect (on a mass basis) relative to the same mass of CO 2 . Compounds with high GWP including those like methyl halides with low concentrations may have considerable impact on atmospheric warming when compared with other “greenhouse” gases with low GWP.
Formation of tellurium nanocrystals during anaerobic growth of bacteria that use Te oxyanions as respiratory electron acceptors
Certain toxic elements support the metabolism of diverse prokaryotes by serving as respiratory electron acceptors for growth. Here, we demonstrate that two anaerobes previously shown to be capable of respiring oxyanions of selenium also achieve growth by reduction of either tellurate [Te(VI)] or tellurite [Te(IV)] to elemental tellurium [Te(0)]. This reduction achieves a sizeable stable-Te-isotopic fractionation (isotopic enrichment factor [ε] = −0.4 to −1.0 per ml per atomic mass unit) and results in the formation of unique crystalline Te(0) nanoarchitectures as end products. The Te(0) crystals occur internally within but mainly externally from the cells, and each microorganism forms a distinctly different structure. Those formed by Bacillus selenitireducens initially are nanorods (∼10-nm diameter by 200-nm length), which cluster together, forming larger (∼1,000-nm) rosettes composed of numerous individual shards (∼100-nm width by 1,000-nm length). In contrast, Sulfurospirillum barnesii forms extremely small, irregularly shaped nanospheres (diameter < 50 nm) that coalesce into larger composite aggregates. Energy-dispersive X-ray spectroscopy and selected area electron diffraction indicate that both biominerals are composed entirely of Te and are crystalline, while Raman spectroscopy confirms that they are in the elemental state. These Te biominerals have specific spectral signatures (UV-visible light, Raman) that also provide clues to their internal structures. The use of microorganisms to generate Te nanomaterials may be an alternative for bench-scale syntheses. Additionally, they may also generate products with unique properties unattainable by conventional physical/chemical methods.
Arsenic in ground water: A review of current knowledge and relation to the CALFED solution area with recommendations for needed research
Ground water with arsenic concentrations greater than the U.S. Environmental Protection Agency drinking water standard exists throughout much of the CALFED solution area. These high concentrations are of con-cern from the standpoint of both existing water supply and development of conjunctive use projects. Much is known about arsenic mobility in ground water subject to different hydrologic and geochemical conditions. However, some important knowledge gaps exist that limit the ability to design water supply projects that could prevent arsenic mobilization or promote arsenic removal from ground water. A few well studied sys-tems could provide a much better understanding of methods for preventing or eliminating high arsenic problems. Within the context of the examination of a few detailed field studies, some important research needs include: 1.) Determining the significance of metal-bridging aqueous complexes involving inorgan-ic arsenic and natural organic matter, 2.) In the con-text of in situ remediation, determining whether of metal oxides. Little is known about the quantitative significance competition of inorganic arsenic with other inorganic aqueous species in natu-ral systems. Experiments should be conducted with actual aquifer materials, as the effects of aging on arsenic desorption in laboratory studies are quite sig-nificant. 3.) Devise methods to detect and quantify rates of oxidation/reduction reactions of arsenic that are carried out by microorganisms at ambient concen-trations of arsenic and under in situ conditions. The findings from detailed field studies have the potential for greatly reducing the cost of meeting the new drinking-water standard for arsenic. The research would benefit a broad constituency.
Arsenic and selenium in microbial metabolism
Arsenic and selenium are readily metabolized by prokaryotes, participating in a full range of metabolic functions including assimilation, methylation, detoxification, and anaerobic respiration. Arsenic speciation and mobility is affected by microbes through oxidation/reduction reactions as part of resistance and respiratory processes. A robust arsenic cycle has been demonstrated in diverse environments. Respiratory arsenate reductases, arsenic methyltransferases, and new components in arsenic resistance have been recently described. The requirement for selenium stems primarily from its incorporation into selenocysteine and its function in selenoenzymes. Selenium oxyanions can serve as an electron acceptor in anaerobic respiration, forming distinct nanoparticles of elemental selenium that may be enriched in (76)Se. The biogenesis of selenoproteins has been elucidated, and selenium methyltransferases and a respiratory selenate reductase have also been described. This review highlights recent advances in ecology, biochemistry, and molecular biology and provides a prelude to the impact of genomics studies.
Arsenic, microbes and contaminated aquifers
The health of tens of millions of people world-wide is at risk from drinking arsenic-contaminated well water. In most cases this arsenic occurs naturally within the sub-surface aquifers, rather than being derived from identifiable point sources of pollution. The mobilization of arsenic into the aqueous phase is the first crucial step in a process that eventually leads to human arsenicosis. Increasing evidence suggests that this is a microbiological phenomenon.
The microbial arsenic cycle in Mono Lake, California
Significant concentrations of dissolved inorganic arsenic can be found in the waters of a number of lakes located in the western USA and in other water bodies around the world. These lakes are often situated in arid, volcanic terrain. The highest concentrations of arsenic occur in hypersaline, closed basin soda lakes and their remnant brines. Although arsenic is a well-known toxicant to eukaryotes and prokaryotes alike, some prokaryotes have evolved biochemical mechanisms to exploit arsenic oxyanions (i.e., arsenate and arsenite); they can use them either as an electron acceptor for anaerobic respiration (arsenate), or as an electron donor (arsenite) to support chemoautotrophic fixation of CO 2 into cell carbon. Unlike in freshwater or marine ecosystems, these processes may assume quantitative significance with respect to the carbon cycle in arsenic-rich soda lakes. For the past several years our research has focused on the occurrence and biogeochemical manifestations of these processes in Mono Lake, a particularly arsenic-rich environment. Herein we review some of our findings concerning the biogeochemical arsenic cycle in this lake, with the hope that it may broaden the understanding of the influence of microorganisms upon the speciation of arsenic in more common, less “extreme” environments, such as drinking water aquifers.
Dissimilatory arsenate reduction with sulfide as the electron donor--Experiments with Mono Lake water and isolation of strain MLMS-1, a chemoautotrophic arsenate-respirer
Anoxic bottom water from Mono Lake, California, can biologically reduce added arsenate without any addition of electron donors. Of the possible in situ inorganic electron donors present, only sulfide was sufficiently abundant to drive this reaction. We tested the ability of sulfide to serve as an electron donor for arsenate reduction in experiments with lake water. Reduction of arsenate to arsenite occurred simultaneously with the removal of sulfide. No loss of sulfide occurred in controls without arsenate or in sterilized samples containing both arsenate and sulfide. The rate of arsenate reduction in lake water was dependent on the amount of available arsenate. We enriched for a bacterium that could achieve growth with sulfide and arsenate in a defined, mineral medium and purified it by serial dilution. The isolate, strain MLMS-1, is a gram-negative, motile curved rod that grows by oxidizing sulfide to sulfate while reducing arsenate to arsenite. Chemoautotrophy was confirmed by the incorporation of H 14 CO 3 − into dark-incubated cells, but preliminary gene probing tests with primers for ribulose-1,5-biphosphate carboxylase/oxygenase did not yield PCR-amplified products. Alignment of 16S rRNA sequences indicated that strain MLMS-1 was in the δ- Proteobacteria , located near sulfate reducers like Desulfobulbus sp. (88 to 90% similarity) but more closely related (97%) to unidentified sequences amplified previously from Mono Lake. However, strain MLMS-1 does not grow with sulfate as its electron acceptor.
Methanogenic activity in plankton samples and fish intestines A mechanism for in situ methanogenesis in oceanic surface waters
When plankton samples were incubated anaerobically with a cysteine-sulfide reducing agent, pronounced methane evolution occurred. This activity was inhibited by air, CHCl 3 , C 2 H 2 , and 2-bromoethanesulfonic acid. Adding [ 14 C]CO 3 2− resulted in accumulation of [ 14 C]CH 4 . Portions of the digestive tracts of three fishes were incubated in methanogenic media, and two of the samples showed the presence of methanogenic bacteria.
Big Soda Lake (Nevada). 1. Pelagic bacterial heterotrophy and biomass
Bacterial activities and abundance were measured seasonally in the water column of meromictic Big Soda Lake which is divided into three chemically distinct zones: aerobic mixolimnion, anaerobic mixolimnion, and anaerobic monimolimnion. Bacterial abundance ranged between 5 and 52 x 10 6 cells ml −1 , with highest biomass at the interfaces between these zones: 2–4 mg C liter −1 in the photosynthetic bacterial layer (oxycline) and 0.8–2.0 mg C liter −1 in the chemocline. Bacterial cell size and morphology also varied with depth: small coccoid cells were dominant in the aerobic mixolimnion, whereas the monimolimnion had a more diverse population that included cocci, rods, and large filaments. Heterotrophic activity was measured by [methyl- 3 H]thymidine incorporation and [ 14 C]glutamate uptake. Highest uptake rates were at or just below the photosynthetic bacterial layer and were attributable to small (<1 µ m) heterotrophs rather than the larger photosynthetic bacteria. These high rates of heterotrophic uptake were apparently linked with fermentation; rates of other mineralization processes (e.g. sulfate reduction, methanogenesis, denitrification) in the anoxic mixolimnion were insignificant. Heterotrophic activity in the highly reduced monimolimnion was generally much lower than elsewhere in the water column. Therefore, although the monimolimnion contained most of the bacterial abundance and biomass (∼60%), most of the cells there were inactive.
Big Soda Lake (Nevada). 2. Pelagic sulfate reduction
The epilimnion of hypersaline, alkaline, meromictic Big Soda Lake contains an average 58 mmol sulfate liter −1 and 0.4 µ mol dissolved iron liter −1 . The monimolimnion, which is permanently anoxic, has a sulfide concentration ranging seasonally from 4 to 7 mmol liter −1 . Depth profiles of sulfate reduction in the monimolimnion, assayed with a 35 S tracer technique and in situ incubations, demonstrated that sulfate reduction occurs within the water column of this extreme environment. The average rate of reduction in the monimolimnion was 3 µ mol sulfate liter −1 d −1 in May compared to 0.9 in October. These values are comparable to rates of sulfate reduction reported for anoxic waters of more moderate environments. Sulfate reduction also occurred in the anoxic zone of the mixolimnion, though at significantly lower rates (0.025–0.090 µ mol liter −1 d −1 at 25 m). Additions of FeS (1.0 mmol liter −1 ) doubled the endogenous rate of sulfate reduction in the monimolimnion, while MnS and kaolinite had no effect. These results suggest that sulfate reduction in Big Soda Lake is iron limited and controlled by seasonal variables other than temperature. Estimates of the organic carbon mineralized by sulfate reduction exceed measured fluxes of particulate organic carbon sinking from the mixolimnion. Thus, additional sources of electron donors (other than those derived from the sinking of pelagic autotrophs) may also fuel monimolimnetic sulfate reduction in the lake.
Big Soda Lake (Nevada). 3. Pelagic methanogenesis and anaerobic methane oxidation
In situ rates of methanogenesis and methane oxidation were measured in meromictic Big Soda Lake. Methane production was measured by the accumulation of methane in the headspaces of anaerobically sealed water samples; radiotracer was used to follow methane oxidation. Nearly all the methane oxidation occurred in the anoxic zones of the lake. Rates of anaerobic oxidation exceeded production at all depths studied in both the mixolimnion (2–6 vs. 0.1–1 nmol liter −1 d −1 ) and monimolimnion (49–85 vs. 1.6–12 nmol liter −1 d −1 ) of the lake. Thus, a net consumption of methane equivalent to 1.36 mmol m −2 d −1 occurred in the anoxic water column. Anaerobic methane oxidation had a first-order rate constant of 8.1±0.5 × 10 −4 d −1 , and activity was eliminated by filter sterilization. However, in situ methane oxidation was of insufficient magnitude to cause a noticeable decrease of ambient dissolved methane levels over an incubation period of 97 h.
Bioreactors for removing methyl bromide following contained fumigations
Use of methyl bromide (MeBr) as a quarantine, commodity, or structural fumigant is under scrutiny because its release to the atmosphere contributes to the depletion of stratospheric ozone. A closed-system bioreactor consisting of 0.5 L of a growing culture of a previously described bacterium, strain IMB-1, removed MeBr (>110 μmol L -1 ) from recirculating air. Strain IMB-1 grew slowly to high cell densities in the bioreactor using MeBr as its sole carbon and energy source. Bacterial oxidation of MeBr produced CO 2 and hydrobromic acid (HBr), which required continuous neutralization with NaOH for the system to operate effectively. Strain IMB-1 was capable of sustained oxidation of large amounts of MeBr (170 mmol in 46 d). In an open-system bioreactor (10-L fermenter), strain IMB-1 oxidized a continuous supply of MeBr (220 μmol L -1 in air). Growth was continuous, and 0.5 mol of MeBr was removed from the air supply in 14 d. The specific rate of MeBr oxidation was 7 × 10 -16 mol cell -1 h -1 . Bioreactors such as these can therefore be used to remove large quantities of contaminant MeBr, which opens the possibility of biodegradation as a practical means for its disposal.
Microbial degradation of atmospheric halocarbons
Halocarbons are present in the atmosphere at parts-per-trillion (ppt) mixing ratios and are represented by such substances as chlorofluorocarbons (CFCs), hydrochlolofluorocarbons (HCFCs), hydrofluorocarbons (HFCs), and methyl halides like methyl bromide (MeBr) and their further substituted halomethane analogues (e. g., dibromomethane, bromoform). Many Halocarbons have only an anthropogenic origin (e.g., CFCs, HCFCs, HFCs) whereas others are of mixed anthropogenic and natural origin (e.g., MeBr). The molecular composition of the various CFCs, HCFCs, and HFCs can be deduced from their numerical classification by adding 90 to their designated numbers. The first digit (hundreds place) refers to the number of carbon atoms, the second to the number of hydrogens, and the third to the number of fluorines. For example, the molecular structure of CFC-12 is obtained from the resultant number of 102, and refers to a one carbon molecule containing no hydrogens and two fluorines. By inference from its CFC designation two chlorines must also be included. Similarly, the compound HFC-134 has a resultant sum of 224, which implies a two carbon molecule containing two hydrogens and four fluorines (tetrafluoroethane).