ABSTRACT
Excessive nitrate (NO3-) concentration in groundwater raises health and environmental issues that must be addressed by all European Union (EU) member states under the Nitrates Directive and the Water Framework Directive. The identification of NO3- sources is critical to efficiently control or reverse NO3- contamination that affects many aquifers. In that respect, the use of stable isotope ratios 15N/14N and 18O/16O in NO3- (expressed as δ15N-NO3- and δ18O-NO3-, respectively) has long shown its value. However, limitations exist in complex environments where multiple nitrogen (N) sources coexist. This two-year study explores a method for improved NO3- source investigation in a shallow unconfined aquifer with mixed N inputs and a long established NO3- problem. In this tillage-dominated area of free-draining soil and subsoil, suspected NO3- sources were diffuse applications of artificial fertiliser and organic point sources (septic tanks and farmyards). Bearing in mind that artificial diffuse sources were ubiquitous, groundwater samples were first classified according to a combination of two indicators relevant of point source contamination: presence/absence of organic point sources (i.e. septic tank and/or farmyard) near sampling wells and exceedance/non-exceedance of a contamination threshold value for sodium (Na+) in groundwater. This classification identified three contamination groups: agricultural diffuse source but no point source (D+P-), agricultural diffuse and point source (D+P+) and agricultural diffuse but point source occurrence ambiguous (D+P±). Thereafter δ15N-NO3- and δ18O-NO3- data were superimposed on the classification. As δ15N-NO3- was plotted against δ18O-NO3-, comparisons were made between the different contamination groups. Overall, both δ variables were significantly and positively correlated (p < 0.0001, rs = 0.599, slope of 0.5), which was indicative of denitrification. An inspection of the contamination groups revealed that denitrification did not occur in the absence of point source contamination (group D+P-). In fact, strong significant denitrification lines occurred only in the D+P+ and D+P± groups (p < 0.0001, rs > 0.6, 0.53 ≤ slope ≤ 0.76), i.e. where point source contamination was characterised or suspected. These lines originated from the 2-6 range for δ15N-NO3-, which suggests that i) NO3- contamination was dominated by an agricultural diffuse N source (most likely the large organic matter pool that has incorporated 15N-depleted nitrogen from artificial fertiliser in agricultural soils and whose nitrification is stimulated by ploughing and fertilisation) rather than point sources and ii) denitrification was possibly favoured by high dissolved organic content (DOC) from point sources. Combining contamination indicators and a large stable isotope dataset collected over a large study area could therefore improve our understanding of the NO3- contamination processes in groundwater for better land use management. We hypothesise that in future research, additional contamination indicators (e.g. pharmaceutical molecules) could also be combined to disentangle NO3- contamination from animal and human wastes.
Subject(s)
Environmental Monitoring , Nitrogen/chemistry , Water Pollutants, Chemical/analysis , Groundwater , Nitrates , Nitrogen IsotopesABSTRACT
A dense non-aqueous phase liquid sample formed by release of coal tar into the environment was derivatised by trimethylsilylation using the reagent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and extracted in hexane using accelerated solvent extraction. This procedure enables comprehensive extraction of an extensive suite of organic compounds from tar, which has not previously been described. Comprehensive two dimensional gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS) was used for the analysis of the sample for concurrent evaluation of -OH functional group-containing compounds along with aliphatics, polycyclic aromatic hydrocarbons and other typical tar compounds normally determined via classic gas chromatography. Using statistically designed experiments, a range of conditions were tested for complete recovery of four different surrogates. The robustness and repeatability of the optimised derivatisation/extraction method was demonstrated. Finally, more than a hundred and fifty derivatised compounds were identified using mass spectra elucidation and GC×GC logical order of elution.
Subject(s)
Coal Tar/chemistry , Gas Chromatography-Mass Spectrometry/methods , Organic Chemicals/chemistry , Trimethylsilyl Compounds/chemistry , Environmental Restoration and Remediation , Organic Chemicals/isolation & purification , Reproducibility of ResultsABSTRACT
(15)N and (18)O isotope abundance analyses in nitrate (NO(3)(-)) (expressed as δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) values respectively) have often been used in research to help identify NO(3)(-) sources in rural groundwater. However, questions have been raised over the limitations as overlaps in δ values may occur between N source types early in the leaching process. The aim of this study was to evaluate the utility of using stable isotopes for nitrate source tracking through the determination of δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) in the unsaturated zone from varying N source types (artificial fertiliser, dairy wastewater and cow slurry) and rates with contrasting isotopic compositions. Despite NO(3)(-) concentrations being often elevated, soil-water nitrate poorly mirrored the (15)N content of applied N and therefore, δ(15)N-NO(3)(-) values were of limited assistance in clearly associating nitrate leaching with N inputs. Results suggest that the mineralisation and the nitrification of soil organic N, stimulated by previous and current intensive management, masked the cause of leaching from the isotopic prospective. δ(18)O-NO(3)(-) was of little use, as most values were close to or within the range expected for nitrification regardless of the treatment, which was attributed to the remineralisation of nitrate assimilated by bacteria (mineralisation-immobilisation turnover or MIT) or plants. Only in limited circumstances (low fertiliser application rate in tillage) could direct leaching of synthetic nitrate fertiliser be identified (δ(15)N-NO(3)(-)<0 and δ(18)O-NO(3)(-)>15). Nevertheless, some useful differences emerged between treatments. δ(15)N-NO(3)(-) values were lower where artificial fertiliser was applied compared with the unfertilised controls and organic waste treatments. Importantly, δ(15)N-NO(3)(-) and δ(18)O-NO(3)(-) variables were negatively correlated in the artificial fertiliser treatment (0.001≤p≤0.05, attributed to the varying proportion of fertiliser-derived and synthetic nitrate being leached) while positively correlated in the dairy wastewater plots (p≤0.01, attributed to limited denitrification). These results suggest that it may be possible to distinguish some nitrate sources if analysing correlations between δ variables from the unsaturated zone. In grassland, the above correlations were related to N input rates, which partly controlled nitrate concentrations in the artificial fertiliser plots (high inputs translated into higher NO(3)(-) concentrations with an increasing proportion of fertiliser-derived and synthetic nitrate) and denitrification in the dairy wastewater plots (high inputs corresponded to more denitrification). As a consequence, nitrate source identification in grassland was more efficient at higher input rates due to differences in δ values widening between treatments.
Subject(s)
Nitrates/analysis , Nitrogen Isotopes , Oxygen Isotopes , Denitrification , Environmental Monitoring/methods , Soil/chemistry , Water Pollutants, Chemical/analysisABSTRACT
The Monkstown zero-valent iron permeable reactive barrier (ZVI PRB), Europe's oldest commercially-installed ZVI PRB, had been treating trichloroethene (TCE) contaminated groundwater for about 10 years on the Nortel Network site in Northern Ireland when cores from the reactive zone were collected in December, 2006. Groundwater data from 2001-2006 indicated that TCE is still being remediated to below detection limits as the contaminated groundwater flows through the PRB. Ca and Fe carbonates, crystalline and amorphous Fe sulfides, and Fe (hydr)oxides have precipitated in the granular ZVI material in the PRB. The greatest variety of minerals is associated with a approximately 1-2 cm thick, slightly cemented crust on top (up-gradient influent entrance) of the ZVI section of the PRB and also with the discontinuous cemented ZVI material ( approximately 23 cm thick) directly below it. The greatest presence of microbial communities also occurred in the up-gradient influent portion of the PRB compared to its down-gradient effluent section, with the latter possibly due to less favorable conditions (i.e., high pH, low oxygen) for microbial growth. The ZVI filings in the down-gradient effluent section of the PRB have a projected life span of >10 years compared with ZVI filings from the continuous to discontinuous cemented up-gradient ZVI section (upper approximately 25 cm) of the PRB, which may have a life span of only approximately 2-5 more years. Supporting Information from applied, multi-tracer testing indicated that restricted groundwater flow is occurring in the upper approximately 25 cm of the ZVI section and preferential pathways have also formed in this PRB over its 10 years of operation.
Subject(s)
Environmental Restoration and Remediation/methods , Iron/chemistry , Trichloroethylene/isolation & purification , Water Pollutants, Chemical/isolation & purification , PermeabilityABSTRACT
In this case, an individual was suspected of attempting to burn materials potentially relating to a murder case. A number of spent and unspent matches were seized at the scene by police for forensic examination. Coincidentally, a police raid at the suspect's house revealed a number of matchboxes, all of the same brand, containing matches that had a visual similarity to those recovered at the scene. Stable Isotope Profiling (SIP) was used to assess whether matches could either be distinguished or shown to be indistinguishable by 13C and 2H isotopic composition. These results were then compared to those from the X-ray diffraction (XRD) analysis of match heads and microscopy of the wood. SIP showed the scene matches and seized matches to be different, which was confirmed by XRD and microscopy analyses.
Subject(s)
Forensic Sciences/methods , Household Articles , Wood/chemistry , Carbon Isotopes/analysis , Deuterium/analysis , Firesetting Behavior , Humans , Microscopy , X-Ray DiffractionABSTRACT
AIMS: To investigate the distribution of a polymicrobial community of biodegradative bacteria in (i) soil and groundwater at a former manufactured gas plant (FMGP) site and (ii) in a novel SEquential REactive BARrier (SEREBAR) bioremediation process designed to bioremediate the contaminated groundwater. METHODS AND RESULTS: Culture-dependent and culture-independent analyses using denaturing gradient gel electrophoresis (DGGE) and polymerase chain reaction (PCR) for the detection of 16S ribosomal RNA gene and naphthalene dioxygenase (NDO) genes of free-living (planktonic groundwater) and attached (soil biofilm) samples from across the site and from the SEREBAR process was applied. Naphthalene arising from groundwater was effectively degraded early in the process and the microbiological analysis indicated a dominant role for Pseudomonas and Comamonas in its degradation. The microbial communities appeared highly complex and diverse across both the sites and in the SEREBAR process. An increased population of naphthalene degraders was associated with naphthalene removal. CONCLUSION: The distribution of micro-organisms in general and naphthalene degraders across the site was highly heterogeneous. Comparisons made between areas contaminated with polycyclic aromatic hydrocarbons (PAH) and those not contaminated, revealed differences in the microbial community profile. The likelihood of noncultured bacteria being dominant in mediating naphthalene removal was evident. SIGNIFICANCE AND IMPACT OF THE STUDY: This work further emphasizes the importance of both traditional and molecular-based tools in determining the microbial ecology of contaminated sites and highlights the role of noncultured bacteria in the process.
Subject(s)
Bacteria/classification , Ecosystem , Soil Microbiology , Soil Pollutants/metabolism , Water Microbiology , Bacteria/genetics , Bacteria/growth & development , Biodegradation, Environmental , Polycyclic Aromatic Hydrocarbons , Polymerase Chain ReactionABSTRACT
Recent natural catastrophes with large-scale loss of life have demonstrated the need for a new technique to provide information for disaster victim identification when DNA methods fail to yield the identification of an individual, or in other situations where authorities need to determine the recent geographical life history of people. The latter may be in relation to the identification of individuals detained on suspicion of terrorism or in relation to people-trafficking or smuggling. One proposed solution is the use of stable isotope profiling (SIP) using isotope ratio mass spectrometry (IRMS). Exploiting the link between the isotopic signal of dietary components and the isotopic composition of body tissue, the aim of this study was to refine a non-invasive method of analysing human material such as scalp hair and fingernails using SIP and to assess the degree of natural variability in these profiles. Scalp hair and fingernail samples were collected from British and non-British volunteers at Queen's University Belfast every 2 weeks for a minimum of 8 months. Samples were analysed using IRMS to determine their isotopic composition for 13C, 15N, 2H and 18O. The results of this longitudinal study yielded information on the natural variability of the isotopic composition of these tissues. The data demonstrate the relatively low degree of natural variation in the 13C/15N isotopic abundance of scalp hair and fingernails whilst greater variations were recorded in the hydrogen and oxygen values of the same samples. The 15N and 18O values of nail are noticeably more variable than that of scalp hair from the same subject. A hypothesis explaining this trend is put forward based on the faster rate of formation of hair than of nails. This means that there is less time for the compounds forming hair to be affected by biochemical processes that could alter their isotopic signature.
Subject(s)
Biomarkers/analysis , Forensic Medicine/methods , Hair/chemistry , Magnetic Resonance Spectroscopy/methods , Nails/chemistry , Radioisotopes/analysis , Female , Humans , Longitudinal Studies , Male , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Isotope ratio mass spectrometry (IRMS) was used to assess what contribution the technique could make towards the comparative analysis of matchstick samples within the 'normal' framework of a forensic investigation. A method was developed to allow the comparison of samples submitted as a result of an investigation, with the added advantage of rapid sample turn-around expected within this field. To the best of our knowledge this is the first time that wooden safety matches have been analysed using IRMS. In this particular case, bulk stable isotope analysis carrried out on a 'like-for-like' basis could demonstrate conclusively that matches seized from a suspect were different from those collected at the scene of crime. The maximum delta13C variability observed within one box was 2.5 per thousand, which, in conjunction with the error of measurement, was regarded to yield too wide an error margin as to permit differentiation of matchsticks based on 13C isotopic composition alone given that the 'natural' 13C abundance in wood ranges from -20 to -30 per thousand. However, from the delta2H values obtained for crime scene matches and seized matches of -114.5 per thousand and -65 per thousand, respectively, it was concluded that the matches seized were distinctly different from those collected at the crime scene.
ABSTRACT
Paints have a dual role in society, to protect materials from environmental agents such as ultraviolet light, moisture and oxygen, and to make painted materials look more attractive. Variability in paint samples is often due to binder and pigment type within the sample. The most common resin used in decorative paints is drying oil alkyd resin, which incorporates soybean oil and vinyl acrylic based latexes. Traditional analytical methods used by forensic scientists may be able to say whether two paint samples are indistinguishable but cannot conclusively say that they both originate from the same source. To find out if isotopic composition can provide an added dimension of information, 28 different white architectural paints were analysed for (13)C abundance using isotope ratio mass spectrometry. In addition, variations in application, drying time and thickness were also investigated to assess the discriminatory power of (13)C data from white paints with an unknown history. Preliminary results indicate that this method could aid screening of paint samples.
Subject(s)
Nitrates/isolation & purification , Nitrates/metabolism , Soil Microbiology , Soil Pollutants/isolation & purification , Soil Pollutants/metabolism , Water Pollutants/isolation & purification , Water Pollutants/metabolism , Biodegradation, Environmental , Porosity , Water Movements , Water SupplyABSTRACT
Chloromethane (CH(3)Cl) is the most abundant halocarbon in the atmosphere. Although largely of natural origin it is responsible for around 17% of chlorine-catalysed ozone destruction. Sources identified to date include biomass burning, oceanic emissions, wood-rotting fungi, higher plants and most recently tropical ferns. Current estimates reveal a shortfall of around 2 million ty(-1) in sources versus sinks for the halocarbon. It is possible that emissions from green plants have been substantially underestimated. A potentially valuable tool for validating emission flux estimates is comparison of the delta13C value of atmospheric CH(3)Cl with those of CH(3)Cl from the various sources. Here we report delta13C values for CH(3)Cl released by two species of tropical ferns and show that the isotopic signature of CH(3)Cl from pteridophytes like that of CH(3)Cl from higher plants is quite different from that of CH(3)Cl produced by biomass burning, fungi and industry. delta13C values for CH(3)Cl produced by Cyathea smithii and Angiopteris evecta were respectively -72.7 per thousand and -69.3 per thousand representing depletions relative to plant biomass of 42.3 per thousand and 43.4 per thousand. The characteristic isotopic signature of CH(3)Cl released by green plants should help constrain their contribution to the atmospheric burden when reliable delta13C values for all other major sources of CH(3)Cl are obtained and a globally averaged delta13C value for atmospheric CH(3)Cl is available.
Subject(s)
Air Pollutants/analysis , Atmosphere/chemistry , Environmental Monitoring/methods , Ferns/chemistry , Methyl Chloride/analysis , Atmosphere/analysis , Biomass , Carbon Isotopes , Ferns/metabolism , Fungi/physiology , Gas Chromatography-Mass Spectrometry/methods , Industry , Methyl Chloride/metabolism , Solanum tuberosum/chemistryABSTRACT
Dehalogenation of trichloroethene (TCE) in the aqueous phase, either on palladium catalysts with hydrogen as the reductant or on metallic iron, was associated with strong changes in delta13C. In general, the delta13C of product phases were more negative than those of the parent compound and were enriched with time and fraction of TCE remaining. For dehalogenation with iron, the delta13C of TCE and products varied from -42/1000 to +5/1000. For the palladium experiments, the final product, ethane, reached the initial delta13C of TCE at completion of the dehalogenation reaction. During dehalogenation, the carbon isotope fractionation between TCE and product phases was not constant. The variation in delta13C of TCE and products offers a new monitoring tool that operates independently of the initial concentration of pollutants for abiotic degradation processes of TCE in the subsurface, and may be useful for evaluation of remediation efficiency.
Subject(s)
Carbon/chemistry , Solvents/chemistry , Trichloroethylene/chemistry , Halogens/chemistry , Iron/chemistry , Oxidation-Reduction , Palladium/chemistry , Soil Pollutants/analysis , Water Pollutants/analysisABSTRACT
Gas-phase photocatalysis of 1,4-dichlorobut-2-enes and 3,4-dichlorobut-1-ene (DCB) has been studied using TiO2 and 3% WO3/TiO2 supported on SiO2. DCB was found to oxidize efficiently over these catalysts; however, only low rates of CO2 formation were observed. With these chlorinated hydrocarbons, the catalysts were found to deactivate over time, probably via the formation of aldol condensation products of chloracetaldehyde, which is the predominant intermediate observed. The variation in rate and selectivity of the oxidation reactions with O2 concentration is reported and a mechanism is proposed. Using isotope ratio mass spectrometry, the initial step for the DCB removal has been shown not to be a carbon bond cleavage but is likely to be hydroxyl radical addition to the carbon-carbon double bond.
Subject(s)
Hydrocarbons, Chlorinated/chemistry , Oxidants, Photochemical/chemistry , Ozone/chemistry , Catalysis , Gases , Oxidation-Reduction , Photochemistry , Semiconductors , Titanium/chemistry , Waste Disposal, Fluid/methodsABSTRACT
A water balance study was used for determining recharge rate and mechanisms in the Enler Catchment, Northern Ireland. Here spatially limited data for the water balance resulted in varied calculation of the annual and monthly net infiltration rate. This paper outlines a method whereby high-resolution soil profiles (1 to 2 cm) were obtained from field cores in the upper 2 m of the unsaturated zone using delta 18O of water. These profiles show changes in isotopic composition that range from individual rainfall events to annually integrated cycles of rainfall. Recharge rates were calculated from stable isotope profiles for each of the four main soil types in the study catchment and summed over each area resulting in an average recharge in the range 55 to 70 mm/a, which is comparable with previous findings. Applied isotopic tracer tests were also conducted to evaluate the extent of preferential flow through the two main soil types in the catchment. Rates of water movement found from these experiments show good agreement with natural isotopic profiles; however, evidence suggests that preferential flow is not the dominant process controlling water movement in this catchment. This type of data provides valuable information about recharge rates and mechanisms and may facilitate better prediction of contaminant transport pathways in the vadose zone.
Subject(s)
Oxygen Isotopes/analysis , Soil , Water Movements , Environmental Monitoring , RainABSTRACT
The largest biological fractionations of stable carbon isotopes observed in nature occur during production of methane by methanogenic archaea. These fractionations result in substantial (as much as approximately 70 per thousand) shifts in delta(13)C relative to the initial substrate. We now report that a stable carbon isotopic fractionation of comparable magnitude (up to 70 per thousand) occurs during oxidation of methyl halides by methylotrophic bacteria. We have demonstrated biological fractionation with whole cells of three methylotrophs (strain IMB-1, strain CC495, and strain MB2) and, to a lesser extent, with the purified cobalamin-dependent methyltransferase enzyme obtained from strain CC495. Thus, the genetic similarities recently reported between methylotrophs, and methanogens with respect to their pathways for C(1)-unit metabolism are also reflected in the carbon isotopic fractionations achieved by these organisms. We found that only part of the observed fractionation of carbon isotopes could be accounted for by the activity of the corrinoid methyltransferase enzyme, suggesting fractionation by enzymes further along the degradation pathway. These observations are of potential biogeochemical significance in the application of stable carbon isotope ratios to constrain the tropospheric budgets for the ozone-depleting halocarbons, methyl bromide and methyl chloride.
Subject(s)
Bacteria/metabolism , Carbon Isotopes/isolation & purification , Hydrocarbons, Brominated/metabolism , Methyl Chloride/metabolism , Bacteria/enzymology , Gas Chromatography-Mass Spectrometry , Oxidation-Reduction , SoilABSTRACT
Gas chromatography/mass spectrometry/isotope ratio mass spectrometry (GC/MS/IRMS) methods for delta(13)C measurement of the halomethanes CH(3)Cl, CH(3)Br, CH(3)I and methanethiol (CH(3)SH) during studies of their biological production, biological degradation, and abiotic reactions are presented. Optimisation of gas chromatographic parameters allowed the identification and quantification of CO(2), O(2), CH(3)Cl, CH(3)Br, CH(3)I and CH(3)SH from a single sample, and also the concurrent measurement of delta(13)C for each of the halomethanes and methanethiol. Precision of delta(13)C measurements for halomethane standards decreased (+/-0.3, +/-0.5 and +/-1.3 per thousand) with increasing mass (CH(3)Cl, CH(3)Br, CH(3)I, respectively). Given that carbon isotope effects during biological production, biological degradation and some chemical (abiotic) reactions can be as much as 100 per thousand, stable isotope analysis offers a precise method to study the global sources and sinks of these halogenated compounds that are of considerable importance to our understanding of stratospheric ozone destruction.
Subject(s)
Hydrocarbons, Brominated/analysis , Hydrocarbons, Chlorinated/analysis , Hydrocarbons, Iodinated/analysis , Biodegradation, Environmental , Carbon Isotopes , Fungi/metabolism , Hydrocarbons, Brominated/metabolism , Hydrocarbons, Chlorinated/metabolism , Hydrocarbons, Iodinated/metabolism , Kinetics , Mass SpectrometryABSTRACT
Chloromethane (CH3Cl) with a global atmospheric burden of 5.3 million t is the most abundant halocarbon in the atmosphere. However, the origin of ca. 50% of the estimated annual global input of 4 million t of the gas to the atmosphere has yet to be determined. As the oceanic contribution to the global CH3Cl flux is now tightly constrained, an important terrestrial source is either underestimated or unrecognized. It has recently been proposed that higher plants may represent a CH3Cl source of sufficient magnitude to resolve the global budget imbalance. A potentially useful tool in validating CH3Cl emission flux estimates is comparison of the carbon isotope ratio of atmospheric CH3Cl with those of CH3Cl originating from various sources. Here we report the first measurements of delta13C for CH3Cl produced biologically. The CH3Cl released by the higher plant species Batis maritima and Solanum tuberosum was dramatically depleted in 13C with respect to plant tissue (delta13C = -36.8/1000 and -34.5/1000, respectively); CH3Cl released by the fungus Phellinus pomaceus also showed significant 13C depletion with respect to the wood growth substrate (delta13C = -17.9/1000). When reliable delta13C values for the other major sources of atmospheric CH3Cl become available, the distinctive isotopic signature of plant-derived CH3Cl should help constrain the contribution to the atmospheric burden from this source.
Subject(s)
Air Pollutants/analysis , Carbon Isotopes/analysis , Environmental Monitoring/methods , Methyl Chloride/analysis , Brassicaceae/physiology , Carbon Isotopes/chemistry , Fungi/physiology , Models, Theoretical , Solanum tuberosum/physiologyABSTRACT
Degradation experiments of benzoate by Pseudomonas putida resulted in enzymatic carbon isotope fractionations. However, isotopic temperature effects between experiments at 20 and 30 degrees C were minor. Averages of the last three values of the CO(2) isotopic composition (delta(13)C(CO2(g))) were more negative than the initial benzoate delta(13)C value (-26.2 per thousand Vienna Pee Dee Belenite (VPDB)) by 3.8, 3.4 and 3.2 per thousand at 20, 25 and 30 degrees C, respectively. Although the maximum isotopic temperature difference found was only 0.6 per thousand, more extreme temperature variations may cause larger isotope effects. In order to understand the isotope effects on the total inorganic carbon (TIC), a better measure is to calculate the proportions of the inorganic carbon species (CO(2)(g), CO(2)(aq) and HCO(3)(-)) and to determine their cumulative delta(13)C(TIC). In all three experiments delta(13)C(TIC) was more positive than the initial isotopic composition of the benzoate at a pH of 7. This suggests an uptake of (12)C in the biomass in order to match the carbon balance of these closed system experiments.