Mary A. Voytek

METHODS FOR MEASURING DENITRIFICATION: DIVERSE APPROACHES TO A DIFFICULT PROBLEM

Denitrification, the reduction of the nitrogen (N) oxides, nitrate (NO3-) and nitrite (NO2-), to the gases nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2), is important to primary production, water quality, and the chemistry and physics of the atmosphere at ecosystem, landscape, regional, and global scales. Unfortunately, this process is very difficult to measure, and existing methods are problematic for different reasons in different places at different times. In this paper, we review the major approaches that have been taken to measure denitrification in terrestrial and aquatic environments and discuss the strengths, weaknesses, and future prospects for the different methods. Methodological approaches covered include (1) acetylene-based methods, (2) 15N tracers, (3) direct N2 quantification, (4) N2:Ar ratio quantification, (5) mass balance approaches, (6) stoichiometric approaches, (7) methods based on stable isotopes, (8) in situ gradients with atmospheric environmental tracers, and (9) molecular approaches. Our review makes it clear that the prospects for improved quantification of denitrification vary greatly in different environments and at different scales. While current methodology allows for the production of accurate estimates of denitrification at scales relevant to water and air quality and ecosystem fertility questions in some systems (e.g., aquatic sediments, well-defined aquifers), methodology for other systems, especially upland terrestrial areas, still needs development. Comparison of mass balance and stoichiometric approaches that constrain estimates of denitrification at large scales with point measurements (made using multiple methods), in multiple systems, is likely to propel more improvement in denitrification methods over the next few years.

ENVIRONMENTAL CONTROLS ON DENITRIFYING COMMUNITIES AND DENITRIFICATION RATES: INSIGHTS FROM MOLECULAR METHODS

The advent of molecular techniques has improved our understanding of the microbial communities responsible for denitrification and is beginning to address their role in controlling denitrification processes. There is a large diversity of bacteria, archaea, and fungi capable of denitrification, and their community composition is structured by long-term environmental drivers. The range of temperature and moisture conditions, substrate availability, competition, and disturbances have long-lasting legacies on denitrifier community structure. These communities may differ in physiology, environmental tolerances to pH and O2, growth rate, and enzyme kinetics. Although factors such as O2, pH, C availability, and NO3- pools affect instantaneous rates, these drivers act through the biotic community. This review summarizes the results of molecular investigations of denitrifier communities in natural environments and provides a framework for developing future research for addressing connections between denitrifier community structure and function.

Stimulation of Methane Generation from Nonproductive Coal by Addition of Nutrients or a Microbial Consortium

ABSTRACT Biogenic formation of methane from coal is of great interest as an underexploited source of clean energy. The goal of some coal bed producers is to extend coal bed methane productivity and to utilize hydrocarbon wastes such as coal slurry to generate new methane. However, the process and factors controlling the process, and thus ways to stimulate it, are poorly understood. Subbituminous coal from a nonproductive well in south Texas was stimulated to produce methane in microcosms when the native population was supplemented with nutrients (biostimulation) or when nutrients and a consortium of bacteria and methanogens enriched from wetland sediment were added (bioaugmentation). The native population enriched by nutrient addition included Pseudomonas spp., Veillonellaceae, and Methanosarcina barkeri. The bioaugmented microcosm generated methane more rapidly and to a higher concentration than the biostimulated microcosm. Dissolved organics, including long-chain fatty acids, single-ring aromatics, and long-chain alkanes accumulated in the first 39 days of the bioaugmented microcosm and were then degraded, accompanied by generation of methane. The bioaugmented microcosm was dominated by Geobacter sp., and most of the methane generation was associated with growth of Methanosaeta concilii. The ability of the bioaugmentation culture to produce methane from coal intermediates was confirmed in incubations of culture with representative organic compounds. This study indicates that methane production could be stimulated at the nonproductive field site and that low microbial biomass may be limiting in situ methane generation. In addition, the microcosm study suggests that the pathway for generating methane from coal involves complex microbial partnerships.

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (aw) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650–0.605 aw. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 aw). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 aw for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 aw for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life.

Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

ABSTRACT To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

Multiplication of microbes below 0.690 water activity : implications for terrestrial and extraterrestrial life

Since a key requirement of known life forms is available water (water activity; aw ), recent searches for signatures of past life in terrestrial and extraterrestrial environments have targeted places known to have contained significant quantities of biologically available water. However, early life on Earth inhabited high-salt environments, suggesting an ability to withstand low water-activity. The lower limit of water activity that enables cell division appears to be ∼ 0.605 which, until now, was only known to be exhibited by a single eukaryote, the sugar-tolerant, fungal xerophile Xeromyces bisporus. The first forms of life on Earth were, though, prokaryotic. Recent evidence now indicates that some halophilic Archaea and Bacteria have water-activity limits more or less equal to those of X. bisporus. We discuss water activity in relation to the limits of Earths present-day biosphere; the possibility of microbial multiplication by utilizing water from thin, aqueous films or non-liquid sources; whether prokaryotes were the first organisms able to multiply close to the 0.605-aw limit; and whether extraterrestrial aqueous milieux of ≥ 0.605 aw can resemble fertile microbial habitats found on Earth.

Denitrification in nitrate-rich streams: Application of N2:Ar and 15N-tracer methods in intact cores

Rates of benthic denitrification were measured using two techniques, membrane inlet mass spectrometry (MIMS) and isotope ratio mass spectrometry (IRMS), applied to sediment cores from two NO3(-)-rich streams draining agricultural land in the upper Mississippi River Basin. Denitrification was estimated simultaneously from measurements of N2:Ar (MIMS) and 15N[N2] (IRMS) after the addition of low-level 15NO3- tracer (15N:N = 0.03-0.08) in stream water overlying intact sediment cores. Denitrification rates ranged from about 0 to 4400 micromol N x m(-2) x h(-1) in Sugar Creek and from 0 to 1300 micromol N x m(-2) x h(-1) in Iroquois River, the latter of which possesses greater streamflow discharge and a more homogeneous streambed and water column. Within the uncertainties of the two techniques, there is good agreement between the MIMS and IRMS results, which indicates that the production of N2 by the coupled process of nitrification/denitrification was relatively unimportant and surface-water NO3- was the dominant source of NO3- for benthic denitrification in these streams. Variation in stream NO3- concentration (from about 20 micromol/L during low discharge to 1000 micromol/L during high discharge) was a significant control of benthic denitrification rates, judging from the more abundant MIMS data. The interpretation that NO3- concentration directly affects denitrification rate was corroborated by increased rates of denitrification in cores amended with NO3-. Denitrification in Sugar Creek removed < or = 11% per day of the instream NO3- in late spring and removed roughly 15-20% in late summer. The fraction of NO3- removed in Iroquois River was less than that of Sugar Creek. Although benthic denitrification rates were relatively high during periods of high stream flow, when NO3 concentrations were also high, the increase in benthic denitrification could not compensate for the much larger increase in stream NO3- fluxes during high flow. Consequently, fractional NO3- losses were relatively low during high flow.

Deep Drilling into the Chesapeake Bay Impact Structure

Samples from a 1.76-kilometer-deep corehole drilled near the center of the late Eocene Chesapeake Bay impact structure (Virginia, USA) reveal its geologic, hydrologic, and biologic history. We conducted stratigraphic and petrologic analyses of the cores to elucidate the timing and results of impact-melt creation and distribution, transient-cavity collapse, and ocean-water resurge. Comparison of post-impact sedimentary sequences inside and outside the structure indicates that compaction of the crater fill influenced long-term sedimentation patterns in the mid-Atlantic region. Salty connate water of the target remains in the crater fill today, where it poses a potential threat to the regional groundwater resource. Observed depth variations in microbial abundance indicate a complex history of impact-related thermal sterilization and habitat modification, and subsequent post-impact repopulation.

The distribution and relative abundance of ammonia-oxidizing bacteria in lakes of the McMurdo Dry Valley, Antarctica

Marked differences in the concentrations of major ions and cations, macronutrient chemistry and general trophic status exist among the lakes of the McMurdo dry valleys in Antarctica. These differences have been attributed to both variations in stream inputs and in situ lake processes (Priscu, 1995; Lizotte et al., 1996, Spigel and Priscu, 1996). This study examines the role of nitrifying bacteria in nitrogen transformations in these lakes. Applying two polymerase chain reaction (PCR) assays targeting the 16S rRNA genes of ammonia-oxidizing bacteria and the active site of the ammonia monooxygenase gene (amoA), the distribution of ammonia-oxidizers was examined in six Antarctic lakes: Lake Bonney, Lake Hoare, Lake Fryxell and Lake Joyce in the Taylor Valley, Lake Miers in the the Miers Valley and Lake Vanda in the Wright Valley. Using a two stage amplification procedure, ammonia-oxidizers from both the beta and gamma- subclasses of the Proteobacteria were detected and their relative abundances were determined in samples collected from all sites. Ammonia-oxidizers were detected in all lakes sampled. Members of the gamma subclass were only present in the saline lakes. In general, nitrifiers were most abundant at depths above the pycnocline and were usually associated with lower concentrations of NH4 and elevated concentrations of NO3 or NO2. The distribution of nitrifiers suggests that the primary N2O peak observed in most of the lakes was produced via nitrification. Preliminary data on the rate of nitrification (Priscu et al., 1996) support the occurrence of nitrification and the presence of nitrifiers at the depth intervals where nitrifiers were detected. In all lakes, except Lake Miers, the data indicate that nitrifying bacteria have an important role in the vertical distribution of nitrogen compounds in these systems.

Evidence for iron‐mediated anaerobic methane oxidation in a crude oil‐contaminated aquifer

In a methanogenic crude oil contaminated aquifer near Bemidji, Minnesota, the decrease in dissolved CH(4) concentrations along the groundwater flow path, along with the positive shift in δ(13) C(CH) (4) and negative shift in δ(13) C(DIC) , is indicative of microbially mediated CH(4) oxidation. Calculations of electron acceptor transport across the water table, through diffusion, recharge, and the entrapment and release of gas bubbles, suggest that these processes can account for at most 15% of the observed total reduced carbon oxidation, including CH(4) . In the anaerobic plume, the characteristic Fe(III)-reducing genus Geobacter was the most abundant of the microbial groups tested, and depletion of labile sediment iron is observed over time, confirming that reduced carbon oxidation coupled to iron reduction is an important process. Electron mass balance calculations suggest that organic carbon sources in the aquifer, BTEX and non-volatile dissolved organic carbon, are insufficient to account for the loss in sediment Fe(III), implying that CH(4) oxidation may also be related to Fe(III) reduction. The results support a hypothesis of Fe(III)-mediated CH(4) oxidation in the contaminated aquifer.