Melanie R. Mormile

Anaerobic Biodegradation of Gasoline Oxygenates: Extrapolation of Information to Multiple Sites and Redox Conditions

A series of alcohol, ketone, ester, and ether oxygenates were tested for their susceptibility to anaerobic decay in samples from four chronically contaminated sedimentary environments. The effect of various electron acceptors on oxygenate biodegradation was also evaluated with a single inoculum source. In addition, two acetogenic bacteria were tested for their ability to metabolize selected oxygenate compounds. The susceptibility of the test oxygenates to anaerobic decay could be related to their chemical structure. That is, compounds other than the ethers that possessed primary or secondary substituted carbon atoms were readily degraded under all conditions tested while compounds that had tertiary substituted carbon atoms resisted biodegradation. The ether oxygenates were generally not degraded when incubated with various inocula, regardless of the electron acceptor status. The exceptions included methyl butyl ether, which was depleted in both sulfate-reducing and methanogenic incubations, and the partial transformation of methyl tert-butyl ether to tert-butanol after a 152-day acclimation period in a single replicate from a river sediment chronically contaminated with fuel. Heat-inactivated control incubations suggested that the latter transformation was biologically catalyzed. 47 refs., 1 fig., 3 tabs.

Nitrate Reduction with Halomonas campisalis: Kinetics of Denitrification at pH 9 and 12.5% NaCl

Regeneration of ion exchange resins with NaCl produces brine containing high concentrations of nitrate that can be difficult to remove using standard biological, physical, or chemical technologies. In this study. Halomonas campisalis (ATCC #700597) (Mormile et al., 1999) was shown to completely reduce nitrate at 125 g/L NaCl and pH 9. This organism was also used in experiments to determine nitrate-reduction rates and biomass yields. Kinetic parameters were measured separately with glycerol, lactate. acetate, ethanol, and methanol. The specific nitrate-reduction rate coefficient was highest in cultures amended with acetate, while lactate and glycerol (a natural osmoticum in hypersaline environments) had lower reduction rates. No evidence of nitrate reduction was observed when ethanol or methanol was provided as an electron donor. Kinetic modeling provided values for nitrate and nitrite-reduction rate coefficients and for biomass yields. Measured rates and yields were similar to reported parameters obtained from non-halophilic nitrate-reducing cultures under low salt concentrations. Therefore, for highly saline solutions, the use of halophiles to selectively remove nitrate from these brines may represent a viable treatment option.

Inhibition of microbial metabolism in anaerobic lagoons by selected sulfonamides, tetracyclines, lincomycin, and tylosin tartrate

Antibiotics are used to maintain healthy livestock and to promote weight gain in concentrated animal feed operations. Antibiotics rarely are metabolized completely by livestock and, thus, are often present in livestock waste and in waste-treatment lagoons. The introduction of antibiotics into anaerobic lagoons commonly used for swine waste treatment has the potential for negative impacts on lagoon performance, which relies on a consortium of microbes ranging from fermentative microorganisms to methanogens. To address this concern, the effects of eight common veterinary antibiotics on anaerobic activity were studied. Anaerobic microcosms, prepared from freshly collected lagoon slurries, were amended with individual antibiotics at 10 mg/L for the initial screening study and at 1, 5, and 25 mg/L for the dose-response study. Monitored metabolic indicators included hydrogen, methane, and volatile fatty acid concentrations as well as chemical oxygen demand. The selected antibiotics significantly inhibited methane production relative to unamended controls, thus indicating that antibiotics at concentrations commonly found in swine lagoons can negatively impact anaerobic metabolism. Additionally, historical antibiotic usage seems to be a potential factor in affecting methane production. Specifically, less inhibition of methane production was noted in samples taken from the lagoon with a history of multiple-antibiotic use.

Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

ABSTRACT The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

Complete Genome Sequence of the Haloalkaliphilic, Hydrogen-Producing Bacterium Halanaerobium hydrogeniformans

Halanaerobium hydrogenoformans is an alkaliphilic bacterium capable of biohydrogen production at pH 11 and 7% (wt/vol) salt. We present the 2.6-Mb genome sequence to provide insights into its physiology and potential for bioenergy applications.

Molecular analysis of the microbial communities of Mars analog lakes in Western Australia.

Unique, shallow interdune lakes and groundwaters with extremely low pH and high salinity exist in Australia, along with nearby lakes that possess higher pH values. These acidic hypersaline environments are possibly the best modern terrestrial analogues for past martian environments. However, no previous microbiological analyses of these lakes have been conducted. During the Australian winter of 2005, water samples were taken from several hypersaline lakes located in southern Western Australia that possessed acidic to slightly alkaline pH. These samples were subjected to molecular analysis to identify bacterial communities. DNA extraction and polymerase chain reaction (PCR) amplification of the 16S rRNA gene sequences, by using universal bacterial primers, were also performed on the samples. Extracted DNA was amplified with 1070 forward and 1392 GC-clamped reverse primers and analyzed by using denaturant gradient gel electrophoresis (DGGE). In addition, libraries were developed from DNA retrieved from four lakes, including a marginal marine neutral lake, an inland neutral lake, and two inland acid lakes, and selected clones with distinct operational taxonomic units were sequenced. The DGGE profiles and clone sequence data indicate that there are distinct, abundant, and diverse microbial populations in these Australian hypersaline environments, especially the acidic ones. These results are significant for two reasons: (1) they provide the first microbiological survey of natural acid saline lakes and (2) they hint at the possibility that there could have been a diverse microbial population in acidic hypersaline environments on Mars.

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.

Activity-Based Metagenomic Screening and Biochemical Characterization of Bovine Ruminal Protozoan Glycoside Hydrolases

ABSTRACT The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

A Streamlined Strategy for Biohydrogen Production with Halanaerobium hydrogeniformans, an Alkaliphilic Bacterium

Biofuels are anticipated to enable a shift from fossil fuels for renewable transportation and manufacturing fuels, with biohydrogen considered attractive since it could offer the largest reduction of global carbon budgets. Currently, lignocellulosic biohydrogen production remains inefficient with pretreatments that are heavily fossil fuel-dependent. However, bacteria using alkali-treated biomass could streamline biofuel production while reducing costs and fossil fuel needs. An alkaliphilic bacterium, Halanaerobium hydrogeniformans, is described that is capable of biohydrogen production at levels rivaling neutrophilic strains, but at pH 11 and hypersaline conditions. H. hydrogeniformans ferments a variety of 5- and 6-carbon sugars derived from hemicellulose and cellulose including cellobiose, and forms the end products hydrogen, acetate, and formate. Further, it can also produce biohydrogen from switchgrass and straw pretreated at temperatures far lower than any previously reported and in solutions compatible with growth. Hence, this bacterium can potentially increase the efficiency and efficacy of biohydrogen production from renewable biomass resources.

Biotransformation of Toxic Organic and Inorganic Contaminants by Halophilic Bacteria

In many arid regions, lakes often lose significant amounts of water through evaporation, leading to alkaline and saline aquatic systems. The water in terminal lakes (i.e. lakes with no outlet) may contain from 0.3–30% NaCl and higher (Williams 1998). Saline lakes are globally distributed and account for approximately half of the total volume of all inland surface water in the world (Williams 1998). The alkaline-saline lakes are a special class of saline lakes, with a pH from 9 to almost 12 in addition to high salinity (Duckworth et al. 1996). Lake Magadi (Kenya), Mono Lake (California, USA) and Soap Lake (Washington, USA) are examples of such lakes.