Brian G. Rahm

Absolute quantification of Dehalococcoides proteins: enzyme bioindicators of chlorinated ethene dehalorespiration

The quantification of trace proteins in complex environmental samples and mixed microbial communities would be a valuable monitoring tool in countless applications, including the bioremediation of groundwater contaminated with chlorinated solvents. Measuring the concentrations of specific proteins provides unique information about the activity and physiological state of organisms in a sample. We developed sensitive (< 5 fmol), selective bioindicator assays for the absolute quantification of select proteins used by Dehalococcoides spp. when reducing carbon atoms in the common pollutants trichloroethene (TCE) and tetrachloroethene (PCE). From complex whole-sample digests of two different dechlorinating mixed communities, we monitored the chromatographic peaks of selected tryptic peptides chosen to represent 19 specific Dehalococcoides proteins. This was accomplished using multiple-reaction monitoring (MRM) assays using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS), which provided the selectivity, sensitivity and reproducibility required to quantify Dehalococcoides proteins in complex samples. We observed reproducible peak areas (average CV = 0.14 over 4 days, n = 3) and linear responses in standard curves (n = 5, R(2) > 0.98) using synthetic peptide standards spiked into a background matrix of sediment peptides. We detected and quantified TCE reductive dehalogenase (TceA) at 7.6 +/- 1.7 x 10(3) proteins cell(-1) in the KB1 bioaugmentation culture, previously thought to be lacking TceA. Fragmentation data from MS/MS shotgun proteomics experiments were helpful in developing the MRM targets. Similar shotgun proteomics data are emerging in labs around the world for many environmentally relevant microbial proteins, and these data are a valuable resource for the future development of MRM assays. We expect targeted peptide quantification in environmental samples to be a useful tool in environmental monitoring.

Greenhouse Gas Emissions from Septic Systems in New York State.

Onsite septic systems use microbial processes to eliminate organic wastes and nutrients such as nitrogen; these processes can contribute to air pollution through the release of greenhouse gases (GHGs). Current USEPA estimates for septic system GHG emissions are based on one study conducted in north-central California and are limited to methane; therefore, the contribution of these systems to the overall GHG emission budget is unclear. This study quantified and compared septic system GHG emissions from the soil over leach fields and the roof vent, which are the most likely locations for gas emissions during normal septic system operation. At each of eight septic systems, we measured fluxes of CH, CO, and NO using a static chamber method. The roof vent released the majority of septic system gas emissions. In addition, the leach field was a significant source of NO fluxes. Comparisons between leach field and vent emissions suggest that biological processes in the leach field soil may influence the type and quantity of gas released. Overall, our results suggest that (i) revisions are needed in USEPA guidance (e.g., septic systems are not currently listed as a source of NO emissions) and (ii) similar studies representing a wider range of climatic and geographic settings are needed. The total vent, sand filter, and leach field GHG emissions were 0.17, 0.045, and 0.050 t CO-equivalents capita yr, respectively. In total, this represents about 1.5% of the annual carbon footprint of an individual living in the United States.

Methane and nitrous oxide cycling microbial communities in soils above septic leach fields: Abundances with depth and correlations with net surface emissions

Onsite septic systems use soil microbial communities to treat wastewater, in the process creating potent greenhouse gases (GHGs): methane (CH4) and nitrous oxide (N2O). Subsurface soil dispersal systems of septic tank overflow, known as leach fields, are an important part of wastewater treatment and have the potential to contribute significantly to GHG cycling. This study aimed to characterize soil microbial communities associated with leach field systems and quantify the abundance and distribution of microbial populations involved in CH4 and N2O cycling. Functional genes were used to target populations producing and consuming GHGs, specifically methyl coenzyme M reductase (mcrA) and particulate methane monooxygenase (pmoA) for CH4 and nitric oxide reductase (cnorB) and nitrous oxide reductase (nosZ) for N2O. All biomarker genes were found in all soil samples regardless of treatment (leach field, sand filter, or control) or depth (surface or subsurface). In general, biomarker genes were more abundant in surface soils than subsurface soils suggesting the majority of GHG cycling is occurring in near-surface soils. Ratios of production to consumption gene abundances showed a positive relationship with CH4 emissions (mcrA:pmoA, p < 0.001) but not with N2O emission (cnorB:nosZ, p > 0.05). Of the three measured soil parameters (volumetric water content (VWC), temperature, and conductivity), only VWC was significantly correlated to a biomarker gene, mcrA (p = 0.0398) but not pmoA or either of the N2O cycling genes (p > 0.05 for cnorB and nosZ). 16S rRNA amplicon library sequencing results revealed soil VWC, CH4 flux and N2O flux together explained 64% of the microbial community diversity between samples. Sequencing of mcrA and pmoA amplicon libraries revealed treatment had little effect on diversity of CH4 cycling organisms. Overall, these results suggest GHG cycling occurs in all soils regardless of whether or not they are associated with a leach field system.

Gas emission data from eight New York State septic systems

This data was collected as part of a study for the New York State Water Resources Institute and the New York State Department of Environmental Conservation Hudson River Estuary Program, with support from the New York State Environmental Protection Fund, under New York State Department of Environmental Conservation service contract #C006135. A.M. Truhlar was supported for the duration of the study by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144153.