Laurence G. Miller

Arsenic(III) fuels anoxygenic photosynthesis in hot spring biofilms from Mono Lake, California

Phylogenetic analysis indicates that microbial arsenic metabolism is ancient and probably extends back to the primordial Earth. In microbial biofilms growing on the rock surfaces of anoxic brine pools fed by hot springs containing arsenite and sulfide at high concentrations, we discovered light-dependent oxidation of arsenite [As(III)] to arsenate [As(V)] occurring under anoxic conditions. The communities were composed primarily of Ectothiorhodospira-like purple bacteria or Oscillatoria-like cyanobacteria. A pure culture of a photosynthetic bacterium grew as a photoautotroph when As(III) was used as the sole photosynthetic electron donor. The strain contained genes encoding a putative As(V) reductase but no detectable homologs of the As(III) oxidase genes of aerobic chemolithotrophs, suggesting a reverse functionality for the reductase. Production of As(V) by anoxygenic photosynthesis probably opened niches for primordial Earths first As(V)-respiring prokaryotes.

Sources and flux of natural gases from Mono Lake, California

Abstract The ability to identify a formation mechanism for natural gas in a particular environment requires consideration of several geochemical factors when there are multiple sources present. Four primary sources of methane have been identified in Mono Lake. Two of these sources were associated with numerous natural gas seeps which occur at various locations in the lake and extend beyond its present boundary; the two other gas sources result from current microbiological processes. In the natural gas seeps, we observed flow rates as high as 160 moles CH4 day−1, and estimate total lakewide annual seep flux to be 2.1 × 106 moles CH4. Geochemical parameters (δ13CH4,δDCH4,CH4/[C2H6+ C3H8]) andδ14CH4measurements revealed that most of the seeps originate from a paleo-biogenic (δ13CH4 = about −70%.). natural gas deposit of Pleistocene age which underlies the current and former lakebed. Gas seeps in the vicinity of hot springs had, in combination with the biogenic gas, a prominent thermogenic gas component resulting from hydrothermal alteration of buried organic matter. Current microbiological processes responsible for sources of natural gas in the lake included pelagic meth- anogenesis and decomposition of terrestrial grasses in the littoral zone. Methanogenesis in the pelagic sediments resulted in methane saturation (2–3 mM at 50 cm; δ13CH4 = about −85%.). Interstitial sulfate decreased from 133 mM at the surface to 35 mM by 110 cm depth, indicating that sulfate-reduction and methanogenesis operated concurrently. Methane diffused out of the sediments resulting in concentrations of about 50 μM in the anoxic bottom waters. Methane oxidation in the oxic/anoxic boundry lowered the concentration by >98%, but values in surface waters (0.1–1.3μM) were supersaturated with respect to the atmosphere. The δ13CH4 (range = −21.8 to −71.8%.) of this unoxidized residual methane was enriched in 13C relative to methane in the bottom water and sediments. Average outward flux of this methane was 2.77 × 107 moles yr−1. A fourth, but minor source of methane (δ13CH4 = −55.2%.) was associated with the decomposition of terrestrial grasses taking place in the lakes recently expanded littoral zone.

Bacterial dissimilatory reduction of arsenate and sulfate in meromictic Mono Lake, California

Abstract The stratified (meromictic) water column of alkaline and hypersaline Mono Lake, California, contains high concentrations of dissolved inorganic arsenic (∼200 μmol/L). Arsenic speciation changes from arsenate [As (V)] to arsenite [As (III)] with the transition from oxic surface waters (mixolimnion) to anoxic bottom waters (monimolimnion). A radioassay was devised to measure the reduction of 73 As (V) to 73 As (III) and tested using cell suspensions of the As (V)-respiring Bacillus selenitireducens , which completely reduced the 73 As (V). In field experiments, no significant activity was noted in the aerobic mixolimnion waters, but reduction of 73 As (V) to 73 As (III) was observed in all the monimolimnion samples. Rate constants ranged from 0.02 to 0.3/day, with the highest values in the samples from the deepest depths (24 and 28 m). The highest activities occurred between 18 and 21 m, where As (V) was abundant (rate, ∼5.9 μmol/L per day). In contrast, sulfate reduction occurred at depths below 21 m, with the highest rates attained at 28 m (rate, ∼2.3 μmol/L per day). These results indicate that As (V) ranks second in importance, after sulfate, as an electron acceptor for anaerobic bacterial respiration in the water column. Annual arsenate respiration may mineralize as much as 14.2% of the pelagic photosynthetic carbon fixed during meromixis. When combined with sulfate-reduction data, anaerobic respiration in the water column can mineralize 32–55% of this primary production. As lakes of this type approach salt saturation, As (V) can become the most important electron acceptor for the biogeochemical cycling of carbon.

Dissimilatory Arsenate and Sulfate Reduction in Sediments of Two Hypersaline, Arsenic-Rich Soda Lakes: Mono and Searles Lakes, California

ABSTRACT A radioisotope method was devised to study bacterial respiratory reduction of arsenate in sediments. The following two arsenic-rich soda lakes in California were chosen for comparison on the basis of their different salinities: Mono Lake (∼90 g/liter) and Searles Lake (∼340 g/liter). Profiles of arsenate reduction and sulfate reduction were constructed for both lakes. Reduction of [73As]arsenate occurred at all depth intervals in the cores from Mono Lake (rate constant [k] = 0.103 to 0.04 h−1) and Searles Lake (k = 0.012 to 0.002 h−1), and the highest activities occurred in the top sections of each core. In contrast, [35S]sulfate reduction was measurable in Mono Lake (k = 7.6 ×104 to 3.2 × 10−6 h−1) but not in Searles Lake. Sediment DNA was extracted, PCR amplified, and separated by denaturing gradient gel electrophoresis (DGGE) to obtain phylogenetic markers (i.e., 16S rRNA genes) and a partial functional gene for dissimilatory arsenate reduction (arrA). The amplified arrA gene product showed a similar trend in both lakes; the signal was strongest in surface sediments and decreased to undetectable levels deeper in the sediments. More arrA gene signal was observed in Mono Lake and was detectable at a greater depth, despite the higher arsenate reduction activity observed in Searles Lake. A partial sequence (about 900 bp) was obtained for a clone (SLAS-3) that matched the dominant DGGE band found in deeper parts of the Searles Lake sample (below 3 cm), and this clone was found to be closely related to SLAS-1, a novel extremophilic arsenate respirer previously cultivated from Searles Lake.

Degradation of methyl bromide in anaerobic sediments.

Methyl bromide (MeBr) was anaerobically degraded in saltmarsh sediments after reaction with sulfide. The product of this nucleophilic substitution reaction was methanethiol, which underwent further chemical and bacterial reactions to form dimethyl sulfide. These two gases appeared transiently during sediment incubations because they were metabolized by methanogenic and sulfate-reducing bacteria. A second, less significant reaction of MeBr was the exchange with chloride, forming methyl chloride, which was also susceptible to attack by sulfide. Incubation of 14 C-labeled methyl iodide as an analogue of MeBr resulted in the formation of 14 C 4 and 14 CO 2 and also indicated that sulfate-reducing bacteria as well as methanogens metabolized the methylated sulfur intermediates

Large carbon isotope fractionation associated with oxidation of methyl halides by methylotrophic bacteria

The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as ≈70‰) shifts in δ13C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70‰) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C1-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.

Microbiological reduction of Sb(V) in anoxic freshwater sediments

Microbiological reduction of millimolar concentrations of Sb(V) to Sb(III) was observed in anoxic sediments from two freshwater settings: (1) a Sb- and As-contaminated mine site (Stibnite Mine) in central Idaho and 2) an uncontaminated suburban lake (Searsville Lake) in the San Francisco Bay Area. Rates of Sb(V) reduction in anoxic sediment microcosms and enrichment cultures were enhanced by amendment with lactate or acetate as electron donors but not by H2, and no reduction occurred in sterilized controls. Addition of 2-(14)C-acetate to Stibnite Mine microcosms resulted in the production of (14)CO2 coupled to Sb(V) reduction, suggesting that this process proceeds by a dissimilatory respiratory pathway in those sediments. Antimony(V) reduction in Searsville Lake sediments was not coupled to acetate mineralization and may be associated with Sb-resistance. The microcosms and enrichment cultures also reduced sulfate, and the precipitation of insoluble Sb(III)-sulfide complexes was a major sink for reduced Sb. The reduction of Sb(V) by Stibnite Mine sediments was inhibited by As(V), suggesting that As(V) is a preferred electron acceptor for the indigenous community. These findings indicate a novel pathway for anaerobic microbiological respiration and suggest that communities capable of reducing high concentrations of Sb(V) commonly occur naturally in the environment.

The geochemistry of methane in Lake Fryxell, an amictic, permanently ice-covered, antarctic lake

The abundance and distribution of dissolved CH4 were determined from 1987–1990 in Lake Fryxell, Antarctica, an amictic, permanently ice-covered lake in which solute movement is controlled by diffusion. CH4 concentrations were < 1 υM in the upper oxic waters, but increased below the oxycline to 936 μM at 18 m. Sediment CH4 was 1100 μmol (1 sed)−1 in the 0–5 cm zone. Upward flux from the sediment was the source of the CH4, NH4+, and DOC in the water column; CH4 was 27% of the DOC+CH4 carbon at 18 m. Incubations with surficial sediments indicated that H14CO3− reduction was 0.4 μmol (1 sed)−1 day−1 or 4× the rate of acetate fermentation to CH4. There was no measurable CH4 production in the water column. However, depth profiles of CH4, NH4, and DIC normalized to bottom water concentrations demonstrated that a significant CH4 sink was evident in the anoxic, sulfate-containing zone of the water column (10–18 m). The δ13CH4 in this zone decreased from −72 % at 18 m to −76% at 12 m, indicating that the consumption mechanism did not result in an isotopic enrichment of 13CH4. In contrast, δ13CH4 increased to −55 % at 9 m due to aerobic oxidation, though this was a minor aspect of the CH4 cycle. The water column CH4 profile was modeled by coupling diffusive flux with a first order consumption term; the best-fit rate constant for anaerobic CH4 consumption was 0.012 yr−1. On a total carbon basis, CH4 consumption in the anoxic water column exerted a major effect on the flux of carbonaceous material from the underlying sediments and serves to exemplify the importance of CH4 to carbon cycling in Lake Fryxell.

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 compounds warming effect (on a mass basis) relative to the same mass of CO2. 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.

Electricity generation by anaerobic bacteria and anoxic sediments from hypersaline soda lakes

Anaerobic bacteria and anoxic sediments from soda lakes produced electricity in microbial fuel cells (MFCs). No electricity was generated in the absence of bacterial metabolism. Arsenate respiring bacteria isolated from moderately hypersaline Mono Lake (Bacillus selenitireducens), and salt-saturated Searles Lake, CA (strain SLAS-1) oxidized lactate using arsenate as the electron acceptor. However, these cultures grew equally well without added arsenate using the MFC anode as their electron acceptor, and in the process oxidized lactate more efficiently. The decrease in electricity generation by consumption of added alternative electron acceptors (i.e. arsenate) which competed with the anode for available electrons proved to be a useful indicator of microbial activity and hence life in the fuel cells. Shaken sediment slurries from these two lakes also generated electricity, with or without added lactate. Hydrogen added to sediment slurries was consumed but did not stimulate electricity production. Finally, electricity was generated in statically incubated “intact” sediment cores from these lakes. More power was produced in sediment from Mono Lake than from Searles Lake, however microbial fuel cells could detect low levels of metabolism operating under moderate and extreme conditions of salt stress.