Peter D. Franzmann

Methanogenium frigidum sp. nov., a psychrophilic, H2-Using methanogen from Ace Lake, Antarctica

Methanogenium frigidum sp. nov. was isolated from the perennially cold, anoxic hypolimnion of Ace Lake in the Vesfold Hills of Antarctica. The cells were psychrophilic, exhibiting most rapid growth at 15 degrees C and no growth at temperatures above 18 to 20 degrees C. The cells were irregular, nonmotile coccoids (diameter, 1.2 to 2.5 microns) that occurred singly and grew by CO2 reduction by using H2 as a reductant. Formate could replace H2, but growth was slower. Acetate, methanol, and trimethylamine were not catabolized. Cells grew with acetate as the only organic compounds in the culture medium, but growth was much faster in medium also supplemented with peptones and yeast extract. The cells were slightly halophilic; good growth occurred in medium supplemented with 350 to 600 mM Na+, but no growth occurred with 100 or 850 mM Na+. The pH range for growth was 6.5 to 7.9; no growth occurred at pH 6.0 or 8.5. Growth was slow (maximum specific growth rate, 0.24 day-1; doubling time, 2.9 days). This is the first report of a psychrophilic methanogen growing by CO2 reduction.

Halomonadaceae fam. nov., a New Family of the Class Proteobacteria to Accommodate the Genera Halomonas and Deleya

Summary The phylogenetic position of Halomonas subglaciescola ACAM 12 and Deleya aesta NCMB 1980 was elucidated by 16S rRNA cataloguing. Together with Halomonas elongata ATCC 33173 and the misclassified organism Flavobacterium halmophilum NCMB 1971, whose DNA G+C content was corrected to 63 mol%, they constitute an individual subline of descent within the gamma subclass of the Proteobacteria . The transfer of Flavobacterium halmophilum into Halomonas as Halomonas halmophila comb. nov. is proposed. The branching point of the Halomonas-Deleya line of descent from other sublines in terms of similarity coefficients (S AB ) is as low as 0.45. This justifies the description of a new family, for which the name Halomonadaceae is proposed.

The role of iron-hydroxy precipitates in the passivation of chalcopyrite during bioleaching

Abstract The bioleaching of chalcopyrite in an acidic sulphate nutrient medium was investigated using Sulfobacillus thermosulfidooxidans, a moderately thermophilic iron- and sulphur oxidising bacterium. Copper release to solution was initially rapid but this slowed significantly after about SO hours. The decrease in chalcopyrite dissolution rate coincided with significant precipitation of jarosite on the mineral surface. Cultures of the moderately thermophilic acidophilic bacteria Acidimicrobium ferrooxidans, Sulfobacillus acidophilus and Sulfobacillus thermosulfidooxidans were grown in anaerobic media containing chalcopyrite passivated by jarosite. The moderate thermophiles used the ferric ion in the jarositic surface precipitate as a terminal electron acceptor in place of oxygen in the anoxic environment. Despite extensive bioreduction of the iron-hydroxy precipitates, it was found that the jarosite was not completely removed and that subsequent biooxidation of the treated concentrate achieved no significant increases in copper release compared with concentrate that had not been subjected to prior biooxidation or bioreduction.

Halobacterium lacusprofundi sp. nov., a Halophilic Bacterium Isolated from Deep Lake, Antarctica

Summary Halobacterium lacusprofundi sp. nov. is described based on the characteristics of two strains (ACAM 32, ACAM 34) of red halophilic bacteria isolated from Deep Lake, a hypersaline, Antarctic lake. Bacteria which could grow on media prepared with undiluted Deep Lake water have not been isolated previously from Deep Lake. Cells were pleomorphic rods which lysed when suspended in distilled water. Unlike other halobacteria, the strains grew at 4°C, albeit very slowly. Fastest generation times (11 h) occurred between 31–37°C. The strains were not proteolytic, did not produce acids from sugars, but utilized a wide range of carbon sources including sugars, alcohols, and organic acids for growth. The strains grew in media with Mg ++ concentrations ranging from 0.005 to 1.0 mol/l and with NaCl concentrations ranging from 1.5 mol/l to saturation. Cells contained isoprenoid neutral lipids typical of halophilic archaebacteria. The major constituents were diphytanyl glyceryl ether, squalene and dihydrophytol. Approximately 30% of the total ether lipids consisted of at least four glyceryl ethers containing one or more double bonds in the phytenyl group. Also present were squalane, dihydrosqualene, tetrahydrosqualene, dihydrophytol and phytol. The G+C content of the major component DNA was 65 to 66 mol%. Satellite band DNA was present with G+C contents ranging from 54 to 57 mol%. Analysis of the 16S rRNA catalogue showed strain ACAM 34 was most closely related to Halobacterium saccharovorum ATCC 29252 (S AB value 0.74) but only distantly related to Haloferax volcanii ATCC 29605 (S AB value 0.43), Haloarcula vallismortis ATCC 29715 (S AB value 0.36), Halococcus morrhuae ATCC 17082 (S AB value 0.39) or Natronococcus occultus NCMB 2192 (S AB value 0.47). The type strain is UQM 3107 (ACAM 34).

Carbon isotopic fractionation associated with methylotrophic methanogenesis

Methanogenesis from “noncompetitive” substrates such as trimethylamine (TMA) and dimethylsulfide (DMS) may be quantitatively important in global methane budgets. This is because choline, glycine betaine, trimethylamine-N-oxide and related compounds, the precursors of TMA, are produced abundantly and ubiquitously by prokaryotic and eukaryotic organisms, particularly those from marine environments. “Noncompetitive” substrates may be a particularly important source for the methane which occurs in the surface mixed layer of the ocean at or above saturation levels. In this study, we measured isotopic fractionation factors for methane and polyisoprenoid lipids formed by methanogens utilizing trimethylamine as their principal carbon source. Methanosarcina barkeri showed isotope effects (e) of 50.2‰ for the conversion of TMA to methane and 20.2‰ for TMA-biomass. Moreover, phytanyl chains of M. barkeri polar lipids were depleted by as much as 18‰ compared to biomass as was the co-occurring hydrocarbon PME. For the Antarctic methanogen Methanococcoides burtonii we measured even greater e values of 71‰ (TMA to CH4), 49.6‰ (TMA to biomass) and 79.9‰ (TMA to phytanyl ether). It should be stressed that these large fractionations represent the maximum or near maximum values possible when the substrate concentrations are non-limiting. The isotopic compositions of methane and methanogen lipid formed by these organisms in natural environments will depend on how completely the substrates are consumed and on how this carbon is partitioned between assimilation and dissimilation processes. The e values for methylotrophic methane formation measured here are significantly higher than those reported for aceticlastic methanogenesis (approx 21‰) and in the same range as those reported for reduction of carbon dioxide (32 to 79‰). The highly 13C-depleted signature of polyisoprenoid moieties compared to biomass of cultured methanogens suggests that there is significant isotopic fractionation inherent in the lipid biosynthetic pathways of Archaea.

Simple organic electron donors support diverse sulfate-reducing communities in fluidized-bed reactors treating acidic metal- and sulfate-containing wastewater

Bacterial diversity of lactate- and ethanol-utilizing sulfate-reducing fluidized-bed reactor (FBR) communities was investigated with culture-independent methods. The FBRs were fed for 500 days with synthetic mineral processing wastewater containing sulfate, zinc and iron with hydraulic retention time of 16-24 h. Sodium lactate or ethanol was used as electron donor for microbial sulfate reduction. For microbial characterization, 16S rRNA gene clone libraries and denaturing gradient gel electrophoresis (DGGE) fingerprinting were employed. The FBR communities were diverse and contained many previously undescribed bacteria. The clone library indicated significant differences between bacterial communities of the two reactors. Most notable was the large number of Proteobacterium sequences retrieved from the ethanol-fed reactor, whereas in the lactate-fed reactor, sequences clustering with Nitrospira phylum were most abundant. Ethanol-utilizing FBR culture was more diverse than the lactate-utilizing one. Some sequences from each reactor were closely related to known sulfate reducers, such as Desulfobacca acetoxidans, Desulforhabdus amnigenus, and species of Desulfovibrio. DGGE profiling showed some changes in the bacterial communities over 393 days of continuous FBR operation. This study showed that it is possible to maintain diverse sulfate-reducing consortia using simple electron donors, lactate or ethanol in an open engineered ecosystem.

Performance and ethanol oxidation kinetics of a sulfate-reducing fluidized-bed reactor treating acidic metal-containing wastewater.

The treatment of simulated acidic wastewater (pH 2.5–5)containing sulfate (1.0–2.2 g l-1), zinc (15–340 mg l -1) and iron (57 mg l -1) was studied in a sulfate-reducing fluidized-bed reactor (FBR) at 35 °C.The original lactate feed for enrichment and maintenance of the FBRculture was replaced stepwise with ethanol over 50 days. The robustnessof the process was studied by increasing stepwise the Zn, sulfate andethanol feed concentrations and decreasing the feed pH. The following precipitation rates were obtained: 360 mg l -1 d -1 for Zn and 86 mg l -1 d -1 for Fe, with over 99.8% Zn and Fe removal, with a hydraulic retention time of 16 h. Under these conditions, 77–95% of the electrons were accepted by sulfate reduction. The alkalinity produced from ethanol oxidation increased the wastewater pH from 2.5 to 7.5–8.5. Michaelis–Menten constants (Km) determined in batch FBR experiments, were 4.3–7.1 mg l -1 and 2.7–3.5 mg l -1 for ethanol and acetateoxidation, respectively. The maximum oxidation velocities (Vmax)were 0.19–0.22 mg gVS -1 min -1 and0.033–0.035 mg gVS -1 min -1, for ethanol and acetate, respectively. In summary, the FBR process produced a good quality effluent as indicated by its low organic content and Zn and Fe concentrations below0.1 mg l -1.

Halomonas subglaciescola, a new species of halotolerant bacteria isolated from Antarctica

Halomonas subglaciescola sp. nov. is proposed, based on the characteristics of 29 strains of halotolerant, nonpigmented bacteria isolated from an Antarctic, hypersaline, meromictic lake. These strains and three reference strains of halotolerant bacteria were tested for 92 attributes. The data were analyzed by numerical taxonomic procedures. The new isolates did not cluster with the three reference strains, which included the type strain of Halomonas elongata. However, some of the isolates did share the following attributes which are characteristic of members of the genus Halomonas: a guanine-plus-cytosine content of 60.9 ± 1.0 to 62.9 ± 0.7 mol%, halotolerancse, largely oxidative mode of metabolism, motility, and peritrichous flagellation. The following are distinguishing features of the new species: cytochrome oxidase positive, no growth at 37°C, and glucose and other sugars are not utilized for growth. The type strain is strain ACAM 12 (= UQM 2926). The species has two biovars; biovar I contains motile strains and is represented by the type strain, and biovar II contains nonmotile strains and is represented by strain ACAM 21 (= UQM 2927).

Microbial community structure and biomass estimates of a methanogenic Antarctic Lake ecosystem as determined by phospholipid analyses

Phospholipid analyses were performed on water column particulate and sediment samples from Ace Lake, a meromictic lake in the Vestfold Hills, Antarctica, to estimate the viable microbial biomass and community structure in the lake. In the water column, methanogenic bacterial phospholipids were present below 17 m in depth at concentrations which converted to a biomass of between 1 and 7×108 cells/liter. Methanogenic biomass in the sediment ranged from 17.7×109 cells/g dry weight of sediment at the surface to 0.1×109 cells/g dry weight at 2 m in depth. This relatively high methanogenic biomass implies that current microbial degradation of organic carbon in Ace Lake sediments may occur at extremely slow rates. Total microbial biomass increased from 4.4×108 cells/ liter at 2 m in depth to 19.4×108 cells/liter at 23 m, near the bottom of the water column. Total nonarchaebacterial biomass decreased from 4.2 ×109 cells/g dry weight in the surface sediment (1/4 the biomass of methanogens) to 0.06×108 cells/g dry weight at 2 m in depth in the sediment. Phospholipid fatty acid profiles showed that microeukaryotes were the major microbial group present in the oxylimnion of the lake, while bacteria dominated the lower, anoxic zone. Sulfate-reducing bacteria (SRB) comprised 25% of the microbial population at 23 m in depth in the water column particulates and were present in the surface sediment but to a lesser extent. Biomass estimates and community structure of the Ace Lake eco-system are discussed in relation to previously measured metabolic rates for this and other antarctic and temperate ecosystems. This is the first instance, to our knowledge, in which the viable biomass of methanogenic and SRB have been estimated for an antarctic microbial community.

The effects of nitrogen and water on mineralisation of hydrocarbons in diesel-contaminated terrestrial Antarctic soils

Bioremediation of petroleum-contaminated soil in the Antarctic will be logistically and technically difficult and will cost more than similar treatment in temperate regions or the Arctic because of the remote location and unfavourable environmental conditions. To optimise nutrient amendments for the remediation of a long-term hydrocarbon-contaminated site at Old Casey Station in Antarctica, we investigated the effects of nitrogen (and phosphorus) amendments on microbial mineralisation using radiometric microcosm experiments and gas chromatography. Hydrocarbon mineralisation at nine different inorganic nitrogen concentrations (ranging from 85 to over 27,000 mg N kg-soil-H2O−1) was monitored over 95-day incubation at 10 °C. Total 14C-octadecane mineralisation increased with increasing nutrient concentration peaking in the range 1000–1600 mg N kg-soil-H2O−1. The microcosms with the lowest and highest concentrations of N had extended lag phases of over 12.5 days prior to significant mineralisation. Gas chromatographic analysis of the aliphatic components of Special Antarctic Blend (SAB) diesel in the contaminated soil showed good agreement with the 14C-octadecane mineralisation outcomes. Ratios of n-C17/pristane and n-C18/phytane indicated that low nutrient concentrations rather than water were the main limiting factor for biodegradation of hydrocarbons in the soil collected from Old Casey Station when incubated at 10 °C. However, because the soils from this site are characterised by low water holding capacities, it would be difficult to maintain optimal nutrient concentrations during full-scale treatment, and thus the use of a controlled release nutrient is being considered as a nutrient source in the bioremediation of SAB-contaminated Antarctic soils.