Stable carbon and nitrogen isotope analysis of TNT: two-dimensional source identification.

Data from a combination of laboratory and fieldwork is presented to initiate testing of stable carbon and nitrogen isotope ratios to trace sources of TNT in contaminated soil and groundwater. Evaluation of these extraction methods resulted in 99.9 and 99.8% recovery of TNT with Soxhlet and solid-phase extraction (SPE), respectively. As a result of the high extraction efficiency, isotope fractionation did not occur, thus providing an accurate stable isotope value on TNT from laboratory and field samples. Subsequent experiments evaluated the stability of isotope signatures through incubations lasting up to four weeks with a 70% decline in the TNT concentration. During these experiments, no significant variation in stable carbon and nitrogen isotope ratios was measured. Five different sources of TNT, compared for stable carbon and nitrogen isotope ratios, showed a range of 4.2 and 15%, respectively. This large range in the isotope ratios suggests excellent potential to trace sources in a complex environment. Finally, a site was surveyed for concentrations and isotope values of TNT extracted from groundwaters. Values from this site were substantially different relative to the variation measured on standards and in laboratory incubation experiments. The data set indicates good potential to use stable isotopes to determine TNT sources and fate in the environment.

Utilization of dissolved nitrogen by heterotrophic bacterioplankton: a comparison of three ecosystems.

The contributions of different organic and inorganic nitrogen and organic carbon sources to heterotrophic bacterioplankton in batch cultures of oceanic, estuarine, and eutrophic riverine environments were compared. The importance of the studied compounds was surprisingly similar among the three ecosystems. Dissolved combined amino acids (DCAA) were most significant, sustaining from 10 to 45% of the bacterial carbon demands and from 42 to 112% of the bacterial nitrogen demands. Dissolved free amino acids (DFAA) supplied 2 to 7% of the carbon and 6 to 24% of the nitrogen incorporated into the bacterial biomass, while dissolved DNA (D-DNA) sustained less than 5 and 12% of the carbon and nitrogen requirements, respectively. Ammonium was the second most important source of nitrogen, meeting from 13 to 45% of the bacterial demand in the oceanic and estuarine cultures and up to 270% of the demand in riverine cultures. Nitrate was taken up in the oceanic cultures (uptake equaled up to 46% of the nitrogen demand) but was released in the two others. Assimilation of DCAA, DFAA, and D-DNA combined supplied 43% of the carbon demand of the bacteria in the oceanic cultures, while approximately 25% of the carbon requirements were met by the three substrates at the two other sites. Assimilation of nitrogen from DCAA, DFAA, D-DNA, NH(4), and NO(3), on the other hand, exceeded production of particulate organic nitrogen in one culture at 27 h and in all cultures over the entire incubation period (50 h). These results suggest that the studied nutrient sources may fully support the nitrogen needs but only partially support the carbon needs of microbial communities of geographically different ecosystems. Furthermore, a comparison of the initial concentrations of the different substrates indicated that relative pool sizes of the substrates seemed to influence which substrates were primarily being utilized by the bacteria.

Utilization of dissolved nitrogen by heterotrophic bacterioplankton: effect of substrate c/n ratio.

The significance of dissolved combined amino acids (DCAA), dissolved free amino acids (DFAA), and dissolved DNA (D-DNA) as sources of C and N for marine bacteria in batch cultures with variable substrate C/N ratios was studied. Glucose, ammonium, alanine, and phosphate were added to the cultures to produce C/N ratios of 5, 10, and 15 and to ensure that phosphorus was not limiting. Maximum bacterial particulate organic carbon production (after 25 h of incubation) was inversely correlated with the C/N ratio: with the addition of identical amounts of carbon, the levels of production were 9.0-, 10.0-, and 11.1-fold higher at C/N ratios of 15, 10, and 5, respectively, relative to an unamended control. The bacterial growth efficiency increased from 22% (control cultures) to 44 to 53% in the cultures with manipulated C/N ratios (C/N-manipulated cultures). Net carbon incorporation from DCAA, DFAA, and D-DNA supported on average 19, 4, and 3% (control cultures and cultures to which only phosphate was added [+P cultures]) and 5, 4, and 0.3% of the particulate organic carbon production (C/N-manipulated cultures), respectively. In the C/N-manipulated cultures, a 2.6- to 3.4-fold-higher level of incorporation of DCAA, relative to that in the control cultures, occurred. Incorporation of D-DNA increased with the substrate C/N ratio, suggesting that D-DNA mainly was a source of N to the bacteria. Organic N (DCAA, DFAA, and D-DNA) sustained 14 to 49% of the net bacterial N production. NH(4) was the dominant N source and constituted 55 to 99% of the total N uptake. NO(3) contributed up to 23% to the total N uptake but was released in two cultures. The studied N compounds sustained nearly all of the bacterial N demand. Our results show that the C/N ratio of dissolved organic matter available to bacteria has a significant influence on the incorporation of individual compounds like DCAA and D-DNA.

Microbial trophic interactions in aquatic microcosms designed for testing genetically engineered microorganisms: A field comparison.

Microcosms may potentially be used as tools for evaluating the fate and effects of genetically engineered microorganisms released into the environment. Extrapolation of data to the field, however, requires that the correspondence between microcosm and field is known. Microbial trophic interactions within the microbial loop were compared quantitatively and qualitatively between field and microcosms containing estuarine water with and without intact sediment cores. The comparison showed that whereas proportions between trophic levels in microcosms were qualitatively similar to those in the field, rates of microbial processes were from 25 to 40% lower in microcosms. Nitrogen cycling was disrupted in microcosms incubated in the dark to eliminate primary production. Examination of the microbial parameters further suggests that sediment in microcosms may be an important factor regulating the bacterial trophic level. These results demonstrate that analysis of microbial trophic interactions is a sensitive method for the field comparison of aquatic microcosms and a potentially useful tool in the risk assessment of genetically engineered microorganisms.

Stable carbon isotope analysis of nucleic acids to trace sources of dissolved substrates used by estuarine bacteria.

The natural abundance of stable carbon isotopes measured in bacterial nucleic acids extracted from estuarine bacterial concentrates was used to trace sources of organic matter for bacteria in aquatic environments. The stable carbon isotope ratios of Pseudomonas aeruginosa and nucleic acids extracted from cultures resembled those of the carbon source on which bacteria were grown. The carbon isotope discrimination between the substrate and total cell carbon from bacterial cultures averaged 2.3% +/- 0.6% (n = 13). Furthermore, the isotope discrimination between the substrate and nucleic acids extracted from bacterial cultures was 2.4% +/- 0.4% (n = 10), not significantly different from the discrimination between bacteria and the substrate. Estuarine water samples were prefiltered through 1-micron-pore-size cartridge filters. Bacterium-sized particles in the filtrates were concentrated with tangential-flow filtration and centrifugation, and nucleic acids were then extracted from these concentrates. Hybridization with 16S rRNA probes showed that approximately 90% of the nucleic acids extracted on two sample dates were of eubacterial origin. Bacteria and nucleic acids from incubation experiments using estuarine water samples enriched with dissolved organic matter from Spartina alterniflora and Cyclotella caspia had stable carbon isotope values similar to those of the substrate sources. In a survey that compared diverse estuarine environments, stable carbon isotopes of bacteria grown in incubation experiments ranged from -31.9 to -20.5%. The range in isotope values of nucleic acids extracted from indigenous bacteria from the same waters was similar, -27.9 to -20.2%. Generally, the lack of isotope discrimination between bacteria and nucleic acids that was noted in the laboratory was observed in the field.(ABSTRACT TRUNCATED AT 250 WORDS)

Measuring microzooplankton grazing on planktonic marine bacteria by its impact on bacterial production.

Grazing on planktonic bacteria by microzooplankton was estimated by separating bacteria from the larger plankton with 1µm pore Nuclepore filtration and measuring changes in bacteria in filtered and unfiltered samples over 24 hours. In the absence of grazers, bacteria increased linearly. The regression coefficient of linear increase was used to estimatein situ bacterial production. When grazers were present, the changes in bacteria concentration usually took the form of a linear decline, and grazing was estimated by subtracting the regression coefficient of the unfiltered sample from that of the 1µm filtrate. Results from the Essex estuary-coastal system of northern Massachusetts show grazing and production at rates that indicate a daily turnover of the standing crop of bacteria, with highest values in mid-estuarine waters. Experiments on the size distribution of grazing showed that microzooplankton from 1-3µm were responsible for most of the observed decrease in bacteria. It was suggested that the basic pattern of linear increase of the bacteria in the absence of grazing reflects density-dependent limitation by substrate present at the outset of the incubation and is indicative of a population that has been maintained around the mid-point of the logistic growth curve by grazing.