David B. Hedrick

Physiological and taxonomic description of the novel autotrophic, metal oxidizing bacterium, Pseudogulbenkiania sp. strain 2002

A lithoautotrophic, Fe(II) oxidizing, nitrate-reducing bacterium, strain 2002 (ATCC BAA-1479; =DSM 18807), was isolated as part of a study on nitrate-dependent Fe(II) oxidation in freshwater lake sediments. Here we provide an in-depth phenotypic and phylogenetic description of the isolate. Strain 2002 is a gram-negative, non-spore forming, motile, rod-shaped bacterium which tested positive for oxidase, catalase, and urease. Analysis of the complete 16S rRNA gene sequence placed strain 2002 in a clade within the family Neisseriaceae in the order Nessieriales of the Betaproteobacteria 99.3% similar to Pseudogulbenkiania subflava. Similar to P. sublfava, predominant whole cell fatty acids were identified as 16:17c, 42.4%, and 16:0, 34.1%. Whole cell difference spectra of the Fe(II) reduced minus nitrate oxidized cyctochrome content revealed a possible role of c-type cytochromes in nitrate-dependent Fe(II) oxidation. Strain 2002 was unable to oxidize aqueous or solid-phase Mn(II) with nitrate as the electron acceptor. In addition to lithotrophic growth with Fe(II), strain 2002 could alternatively grow heterotrophically with long-chain fatty acids, simple organic acids, carbohydrates, yeast extract, or casamino acids. Nitrate, nitrite, nitrous oxide, and oxygen also served as terminal electron acceptors with acetate as the electron donor.

Measuring soil microbial community diversity using polar lipid fatty acid and denaturing gradient gel electrophoresis data.

The possibility of calculating useful microbial community diversity indices from environmental polar lipid fatty acid and 16S rDNA PCR-DGGE data was investigated. First, the behavior of the species richness, Shannons, and Simpsons diversity indices were determined on polar lipid fatty acid profiles of 115 pure cultures, communities constructed from those profiles with different numbers of species, and constructed communities with different distributions of species. Differences in the species richness of these artificial communities was detected by all three diversity indices, but they were insensitive to the evenness of the distribution of species. Second, data from a field experiment with substrate addition to soil was used to compare the methods developed for lipid- and DNA-based diversity indices. Very good agreement was found between indices calculated from environmental polar lipid fatty acid profiles and denaturing gradient gel electrophoresis profiles from matched samples (Pearsons correlation coefficient r=0.95-0.96). A method for data pre-treatment for diversity calculations is described.

Phospholipid furan fatty acids and ubiquinone-8 : Lipid biomarkers that may protect Dehalococcoides strains from free radicals

ABSTRACT Dehalococcoides species have a highly restricted lifestyle and are only known to derive energy from reductive dehalogenation reactions. The lipid fraction of two Dehalococcoides isolates, strains BAV1 and FL2, and a tetrachloroethene-to-ethene-dechlorinating Dehalococcoides-containing consortium were analyzed for neutral lipids and phospholipid fatty acids. Unusual phospholipid modifications, including the replacement of unsaturated fatty acids with furan fatty acids, were detected in both Dehalococcoides isolates and the mixed culture. The following three furan fatty acids are reported as present in bacterial phospholipids for the first time: 9-(5-pentyl-2-furyl)-nonanoate (Fu18:2ω6), 9-(5-butyl-2-furyl)-nonanoate (Fu17:2ω5), and 8-(5-pentyl-2-furyl)-octanoate (Fu17:2ω6). The neutral lipids of the Dehalococcoides cultures contained unusually large amounts of benzoquinones (i.e., ubiquinones [UQ]), which is unusual for anaerobes. In particular, the UQ-8 content of Dehalococcoides was 5- to 20-fold greater than that generated in aerobically grown Escherichia coli cultures relative to the phospholipid fatty acid content. Naphthoquinone isoprenologues (MK), which are often found in anaerobically grown bacteria and archaea, were also detected. Dehalococcoides shows a difference in isoprenologue pattern between UQ-8 and MK-5 that is atypical of other bacteria capable of producing both quinone types. The difference in UQ-8 and MK-5 isoprenologue patterns strongly suggests a special function for UQ in Dehalococcoides, and Dehalococcoides may utilize structural modifications in its lipid armamentarium to protect against free radicals that are generated in the process of reductive dechlorination.

Polar lipid fatty acids, LPS-hydroxy fatty acids, and respiratory quinones of three Geobacter strains, and variation with electron acceptor

The polar lipid fatty acids, lipopolysaccharide hydroxy-fatty acids, and respiratory quinones of Geobacter metallireducens str. GS-15, Geobacter sulfurreducens str. PCA, and Geobacter bemidjiensis str. Bem are reported. Also, the lipids of G. metallireducens were compared when grown with Fe3+ or nitrate as electron acceptors and G. sulfurreducens with Fe3+ or fumarate. In all experiments, the most abundant polar lipid fatty acids were 14:0, i15:0, 16:1ω7c, 16:1ω5c, and 16:0; lipopolysaccharide hydroxy-fatty acids were dominated by 3oh16:0, 3oh14:0, 9oh16:0, and 10oh16:0; and menaquinone-8 was the most abundant respiratory quinone. Some variation in lipid profiles with strain were observed, but not with electron acceptor.

Combined lipid/DNA extraction method for environmental samples

Abstract Previously, separate methods have been developed for the extraction and purification of lipids and DNA from soils and sediments. This paper describes a new method for the isolation of both lipids and DNA from the same environmental sample. This combined method is based on the Bligh and Dyer lipid extraction technique. Upon phase separation, lipids partition into the organic phase and DNA partitions into the aqueous phase. DNA extraction and recovery from the solid phase is necessary under certain conditions. Preliminary experiments performed with 32P-labeled DNA in the absence of soil showed that greater than 98% of the total DNA was present in the aqueous phase after the modified Bligh and Dyer extraction. Analysis of the DNA by polyacrylamide gel electrophoresis and autoradiography demonstrated that no degradation of DNA occurred during the lipid extraction procedure. Lipid extraction of lyophilized cells showed that DNA was released from Pseudomonas putida and Bacillus subtilis cells corresponding to 26±5 and 14±4% of the theoretical DNA yield, respectively. The combined lipid/DNA extraction method was applied to both lyophilized cells and wet cells added to soil. Analysis by DNA:DNA hybridization showed that approx. 40–50% of the DNA from cells added to soil was recovered after lipid extraction relative to samples treated only with conventional DNA extraction. Estimation of cell number per gram soil based on either lipid or DNA analysis showed good agreement with actual numbers added based on plate counts of the inocula. DNA extracts from samples which had been lipid-extracted also had lower amounts of humic material. Although some DNA was not recovered after lipid extraction, that which was recovered was of sufficiently high quality for hybridization analysis. This method shows utility for the co-recovery of both lipids and DNA from a single sample; this is particularly useful when a small sample size is all that is available or procurable.

Utility of radiotracer activity measurements for subsurface microbiology studies

Radiotracer activity measurements were conducted on subsurface sediments collected from the Savannah River Plant, Aiken, SC. Sediments were aseptically extruded from stainless steel core liners into a nitrogen flushed glove bag. Subsurface materials were immediately inoculated into aerobic and/or anaerobic tubes for time course experiments. Mineralization experiments utilized 14C-2-acetate and 14C-UL-glucose, while radiotracer uptake experiments included 14C-1-acetate incorporation into lipids and 3H-methyl-thymidine incorporation into microbial DNA. Microbiological activity of subsurface sediments varied 5 orders of magnitude between dense compacted clays and water-bearing sands. Aquifers tens of meters beneath the earths surface exhibited activities greater than some of the near-surface soils. Radiotracer techniques proved to be sensitive, reproducible and applicable to field implementation. Agreement was observed between water abundance, sand content and microbial activities.

Disturbance, starvation, and overfeeding stresses detected by microbial lipid biomarkers in high-solids high-yield methanogenic reactors

SummaryMicrobial biomass and community structure of methanogenic anaerobic biomass reactors can be quantitatively monitored by signature, lipid analysis. The eubacterial and eukaryotic polar lipid fatty acids and the methanogen polar lipid ethers are reliable measures of their respective biomasses. The pattern of polar lipid fatty acids yields information on the community structure and metabolic state of the eubacteria and eukaryotes. These biomarker methods were applied over a 2-day feeding cycle of a highly productive batch-fed high-solids anaerobic biomass reactor. It was sampled before feeding, 6 h after feeding (disturbed)., at maximum gas production (healthy, 24 h), and after feedstock utilization (starved, 48h). Relative to the healthy condition, the disturbance of feeding significantly decreased eubacterial biomass and the proportion of unsaturated fatty acids, and increased branched fatty acids and the eubacterial stress biomarker,trans/cis 16: 1ω7. The starved condition was not significantly different from the healthy in biomass or proportions of fatty acids, but did show a significant increase in the proportion of the eubacterial stress biomarkertrans/cis 18: 1ω7. This reactor was compared to a second of the same design which had been overfed and showed significantly less productivity. The overfed reactor had a significantly lower methanogenic biomass,iso-branched fatty acids, and higher eubacterial stress markers Cy17:0 andtrans/cis 18: 1ω7 than the highly productive reactor.

Uranium reduction and microbial community development in response to stimulation with different electron donors

Stimulating microbial reduction of soluble U(VI) to less soluble U(IV) shows promise as an in situ bioremediation strategy for uranium contaminated groundwater, but the optimal electron donors for promoting this process have yet to be identified. The purpose of this study was to better understand how the addition of various electron donors to uranium-contaminated subsurface sediments affected U(VI) reduction and the composition of the microbial community. The simple electron donors, acetate or lactate, or the more complex donors, hydrogen-release compound (HRC) or vegetable oil, were added to the sediments incubated in flow-through columns. The composition of the microbial communities was evaluated with quantitative PCR probing specific 16S rRNA genes and functional genes, phospholipid fatty acid analysis, and clone libraries. All the electron donors promoted U(VI) removal, even though the composition of the microbial communities was different with each donor. In general, the overall biomass, rather than the specific bacterial species, was the factor most related to U(VI) removal. Vegetable oil and HRC were more effective in stimulating U(VI) removal than acetate. These results suggest that the addition of more complex organic electron donors could be an excellent option for in situ bioremediation of uranium-contaminated groundwater.

Microbial biomass and community structure of a phase-separated methanogenic reactor determined by lipid analysis

SummaryAn anaerobic phase-separation biomass reactor was established on cellulose with the hydrolysis and fermentation steps occurring in the first stage, and acetogenesis and methanogenesis in the second stage. Based upon lipid biomarker analysis, eubacterial and eukaryotic cells accounted for approximately 6% of the volatile solids of the first stage and 17% of the second, while methanogens were approximately 1% of the volatile solids in the first stage and 9% of the second. Clustering the polar lipid fatty acids into groups based upon their distributions between the two stages of the reactor clarified the differences in community structure caused by phase-separated operation. Although inoculated from the same source, the two stages maintained very different microbial communities. Signature fatty acids known as indicators of unbalanced growth in eubacteria were significantly higher in the first stage of the reactor.

The effects of oxygen and chloroform on microbial activities in a high-solids, high-productivity anaerobic biomass reactor

The effects of oxygen and chloroform addition on microbial metabolic activities were measured on a unique thermophilic high-solids high-productivity methanogenic reactor using a combination of radiotracer, lipid analysis, and microcosm techniques. Methane production and the incorporation of 14C-2-acetate into methane were decreased by both oxygen and chloroform treatment. Oxygen increased and chloroform decreased total CO2 production and CO2 production from labeled acetate. Introduction of oxygen decreased methanogenesis by facultative anaerobes competing for reducing equivalents. Chloroform was directly toxic to methanogens at the low application level, and inhibited all metabolic activities at the high application level. Increased diether lipid synthesis by methanogens was observed with oxygen addition, and methanogen lipid synthesis continued at a low level in the absence of detectable methanogenesis with chloroform addition. These responses may be due to previously unknown adaptations to toxic compounds by methanogens.