Holly C. Pinkart

Aerobic biodegradation of methyl tert-butyl ether by aquifer bacteria from leaking underground storage tank sites.

ABSTRACT The potential for aerobic methyl tert-butyl ether (MTBE) degradation was investigated with microcosms containing aquifer sediment and groundwater from four MTBE-contaminated sites characterized by oxygen-limited in situ conditions. MTBE depletion was observed for sediments from two sites (e.g., 4.5 mg/liter degraded in 15 days after a 4-day lag period), whereas no consumption of MTBE was observed for sediments from the other sites after 75 days. For sediments in which MTBE was consumed, 43 to 54% of added [U-14C]MTBE was mineralized to14CO2. Molecular phylogenetic analyses of these sediments indicated the enrichment of species closely related to a known MTBE-degrading bacterium, strain PM1. At only one site, the presence of water-soluble gasoline components significantly inhibited MTBE degradation and led to a more pronounced accumulation of the metabolite tert-butyl alcohol. Overall, these results suggest that the effects of oxygen and water-soluble gasoline components on in situ MTBE degradation will vary from site to site and that phylogenetic analysis may be a promising predictor of MTBE biodegradation potential.

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.

Sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake with an unprecedented high sulfide content.

ABSTRACT Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 107 cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.

Diversity of extremophilic purple phototrophic bacteria in Soap Lake, a Central Washington (USA) Soda Lake

Culture-based and culture-independent methods were used to explore the diversity of phototrophic purple bacteria in Soap Lake, a small meromictic soda lake in the western USA. Among soda lakes, Soap Lake is unusual because it consists of distinct upper and lower water bodies of vastly different salinities, and its deep waters contain up to 175 mM sulfide. From Soap Lake water new alkaliphilic purple sulfur bacteria of the families Chromatiaceae and Ectothiorhodospiraceae were cultured, and one purple non-sulfur bacterium was isolated. Comparative sequence analysis of pufM, a gene that encodes a key photosynthetic reaction centre protein universally found in purple bacteria, was used to measure the diversity of purple bacteria in Soap Lake. Denaturing gradient gel electrophoresis and subsequent phylogenetic analyses of pufMs amplified from Soap Lake water revealed that a significant diversity of purple bacteria inhabit this soda lake. Although close relatives of several of the pufM phylotypes obtained from cultured species could also be detected in Soap Lake water, several other more divergent pufM phylotypes were also detected. It is possible that Soap Lake purple bacteria are major contributors of organic matter into the ecosystem of this lake, especially in its extensive anoxic and sulfidic deep waters.

Low-substrate regulated microaerophilic behavior as a stress response of aquatic and soil bacteria

Low-substrate regulated microaerophilic behavior (LSRMB) was observed in 10–54% of the bacteria isolated from several fresh-water lakes or ponds, subsurface soils, activated sludge, and Antarctic dry valley soils. Five Pseudomonas and two Bacillus type species showed LSRMB. A subsurface Pseudomonas jessenii strain was used as a model to show the metabolic interaction between substrate and oxygen concentrations, cell band movement, and the appearance of unique stress lipids and proteins. When the oxygen in the P. jessenii culture medium was increased from 11% to 100% saturation under atmospheric condition, the concentration of 17:0 cyclopropane fatty acid, a stress indicator, increased five-fold, and four unique proteins were also detected. This stress response occurred only in low-substrate media. It is our hypothesis that LSRMB is a common but under-appreciated trait of many aquatic and soil bacteria.

Perchlorate and Nitrate Remediation Efficiency and Microbial Diversity in a Containerized Wetland Bioreactor

We have developed a method to remove perchlorate (14–27 μg/L) and nitrate (48 mg/L) from contaminated groundwater using a wetland bioreactor. The bioreactor has operated continuously in a remote field location for more than 2 yr with a stable ecosystem of indigenous organisms. This study assesses the bioreactor for long-term perchlorate and nitrate remediation by evaluating influent and effluent groundwater for oxidation-reduction conditions and nitrate and perchlorate concentrations. Total community DNA was extracted and purified from 10-g sediment samples retrieved from vertical coring of the bioreactor during winter. Analysis by denaturing gradient gel electrophoresis of short, 16S rDNA, polymerase-chain-reaction products was used to identify dominant microorganisms. Bacteria genera identified were closely affiliated with bacteria widely distributed in soils, mud layers, and fresh water. Of the 17 dominant bands sequenced, most were gram negative and capable of aerobic or anaerobic respiration with nitrate as the terminal electron acceptor (Pseudomonas, Acinetobacter, Halomonas, and Nitrospira). Several identified genera (Rhizobium, Acinetobactor, and Xanthomonas) are capable of fixing atmospheric nitrogen into a combined form (ammonia) usable by host plants. Isolates were identified from the Proteobacteria class, known for the ability to reduce perchlorate. Initial bacterial assessments of sediments confirm the prevalence of facultative anaerobic bacteria capable of reducing perchlorate and nitrate insitu.

The sulfur cycle in a permanently meromictic haloalkaline lake

Soap Lake is a haloalkaline lake located in central Washington. This lake is a remnant of the Missoula flood events that created the landscape of western Montana, the southeastern portion of Washington state, and much of Oregon. It is 15,000 - 20,000 years old, and has maintained a stable meromixis for the last 10,000 years. This carbonate lake is characterized by a brackish mixolimnion, and a monimolimnion with a salinity of ~14%. The pH of both layers of the lake is approximately 10. Both layers also have a high concentration of dissolved sulfate, with the mineral mirabilite (Na2SO4•10H2O) found in the monimolimnion sediments. Sulfide concentrations in the monimolimnion exceed 100 mM. As part of the mission of the NSF Soap Lake Microbial Observatory, microorganisms involved in the sulfur cycle in this lake were studied in terms of their diversity and function. High rates of sulfate reduction were measured in both layers of the lake, with new species of sulfate-reducing bacteria seen in both areas. A particularly novel psychrophilic sulfur oxidizer was isolated from the monimolimnion. This organism has the ability to induce the formation of mirabilite, which was assumed to be an abiotically deposited evaporite mineral. This is the first evidence for a biogenic origin of this mineral. This leads to the possibility that related sulfate minerals, such as those reported on the Mars surface, may have a biogenic origin.

Diversity, evolution, and horizontal gene transfer (HGT) in soda lakes

Soap Lake is a hypersaline, alkaline lake in Central Washington State (USA). For the past five years the lake has been the site of an NSF Microbial Observatory project devoted to identifying critical geochemical and microbial characteristics of the monimolimnion sediment and water column, and has demonstrated rich multispecies communities occupy all areas of the lake. Soap Lake and similar soda lakes are subject to repeated transient periods of extreme evaporation characterized by significant repetitive alterations in salinity, pH, and total water volume, yet maintain high genetic and metabolic diversity. It has been argued that this repetitive cycle for salinity, alkalinity, and sulfur concentration has been a major driver for prokaryote evolution and diversity. The rapidity of wet-dry cycling places special demands on genome evolution, requirements that are beyond the relatively conservative eukaryotic evolutionary strategy of serial alteration of existing gene sequences in a relatively stable genome. Although HGT is most likely responsible for adding a significant amount of noise to the genetic record, analysis of HGT activity can also provide us with a much-needed probe for exploration of prokaryotic genome evolution and the origin of diversity. Packaging of genetic information within the protective protein capsid of a bacteriophage would seem preferable to exposing naked DNA to the highly alkaline conditions in the lake. In this study, we present preliminary data demonstrating the presence of a diverse group of phage integrases in Soap Lake. Integrase is the viral enzyme responsible for the insertion of phage DNA into the bacterial hosts chromosome. The presence of the integrase sequence in bacterial chromosomes is evidence of lysogeny, and the diversity of integrase sequences reported here suggests a wide variety of temperate phage exist in this system, and are especially active in transition zones.

Co-evolution of cyanophage and cyanobacteria in Antarctic lakes: adaptive responses to high UV flux and global warming

Rapid adaptation to acute environmental change demands co-evolution of indigenous viral populations and their hosts. Horizontal gene transfer (HGT) is a highly efficient adaptive mechanism, but a difficult phenomena to dectect. The mosaic nature of bacteriophage genomes resulting from HGT has generally been explored using phylogenetic analysis of coding regions. Focusing on the proteome certainly provides one window into the origin and evolution of genome information storage. However, the original fitness function for a nucleotide polymer would arise from a more primal survival imperative predating the appearance of a coding function. Multivariate analysis of a genome information storage metric (lossless compression), nucleotide distributions, and distributions of the three major physiochemical characteristics of the polymer (triple:double bonding [G+C], purine:pyrimidine [G+A], and keto:amine [G+T] fractions) produces a metric to detect and characterize mosaicism in both coding and non-coding regions of a genome. We discuss possibilities and limitations of using these techniques to investigate HGT and the origins and evolution of genome complexity. Analysis of available virus (n= 2374) and bacteriophage genomes (n=417) indicates these probes can perform whole-genome taxonomy tasks or sliding window searches for evidence of HGT in a single genome. HGT responses may serve as a canary or bell-weather for global environmental change. We discuss one area of application of considerable interest to our institute: the response of cyanophage and their cyanobacteria hosts to variations in ultraviolet solar flux in geographically isolated Antarctic lakes.

Cost efficient prediction of Cabernet Sauvignon wine quality

The quality of wines can be assessed both from chemical/biological tests and sensory tests (which rely mainly on human experts). Determining which is the subset of tests to be used is a difficult problem. Each test has its own contribution for predicting the quality of wines and, in addition, its own cost. We use our own database, consisting of 32 wine characteristics applied to 180 wine samples. In addition we use wine quality labels assigned by a wine expert. To the extent of our knowledge, this is the first study of this kind on wines from Washington State, and also the first wine study in general to include cost minimization of the measurements as a goal. Our approach is based on two stages. First, we identify reasonably good classifiers (from a given set of classifiers). Next, we search for the optimal subset of features to maximize the performance of the best classifier and also minimize the overall cost of the measurements. As a result, through our method we can answer queries like “the best performing subset of tests for a given threshold cost”.