Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies

Perchlorate is a widespread environmental contaminant having both anthropogenic and natural sources. Stable isotope ratios of O and Cl in a given sample of perchlorate may be used to distinguish its source(s). Isotopic ratios may also be useful for identifying the extent of biodegradation of perchlorate, which is critical for assessing natural attenuation of this contaminant in groundwater. For this approach to be useful, however, the kinetic isotopic fractionations of O and Cl during perchlorate biodegradation must first be determined as a function of environmental variables such as temperature and bacterial species. A laboratory study was performed in which the O and Cl isotope ratios of perchlorate were monitored as a function of degradation by two separate bacterial strains (Azospira suillum JPLRND and Dechlorospirillum sp. FBR2) at both 10 degrees C and 22 degrees C with acetate as the electron donor. Perchlorate was completely reduced by both strains within 280 h at 22 degrees C and 615 h at 10 degrees C. Measured values of isotopic fractionation factors were epsilon(18)O = -36.6 to -29.0% per hundred and epsilon(37)Cl = -14.5 to -11.5% per hundred, and these showed no apparent systematic variation with either temperature or bacterial strain. An experiment using (18)O-enriched water (delta(18)O = +198% per hundred) gave results indistinguishable from those observed in the isotopically normal water (delta(18)O = -8.1% per hundred) used in the other experiments, indicating negligible isotope exchange between perchlorate and water during biodegradation. The fractionation factor ratio epsilon(18)O/epsilon(37)Cl was nearly invariant in all experiments at 2.50 +/- 0.04. These data indicate that isotope ratio analysis will be useful for documenting perchlorate biodegradation in soils and groundwater. The establishment of a microbial fractionation factor ratio (epsilon(18)O/ epsilon(37)Cl) also has significant implications for forensic studies.     

Influence of humic substance adsorptive fractionation on pyrene partitioning to dissolved and mineral-associated humic substances

Changes in pyrene binding by dissolved and mineral-associated humic substances (HS) due to HS adsorptive fractionation processes were examined in model environmental systems using purified Aldrich humic acid (PAHA) and Suwannee River fulvic acid (SRFA). For PAHA, carbon-normalized pyrene binding coefficients for nonadsorbed, residual fractions (Koc(res)) were different from the original dissolved PAHA Koc value (Koc(orig)) prior to contact with the mineral suspensions. A strong positive correlation between pyrene log Koc(res) and log weight-average molecular weight (MWw) for residual PAHA fractions was observed, which was relatively independent of the specific mineral adsorbent used and hypothesized fractionation processes. A strong positive correlation between log Koc(ads) and log MWw was also found for PAHA fractions adsorbed to kaolinite at low mass fraction organic carbon levels, although the relationship was statistically different from the one found with residual PAHA fractions. The same trends and correlations found for PAHA were not observed with SRFA, suggesting that the impacts of HS adsorptive fractionation on changes in hydrophobic organic contaminants binding are also influenced by the source and other biogeochemical characteristics of HS.     

Effect of natural organic substances on the surface and adsorptive properties of environmental black carbon (char): attenuation of surface activity by humic and fulvic acids

Black carbon (BC) plays a potentially important role in the availability of pollutants in soils and sediments. Recent evidence points to the possible attenuation of the high surface activity of raw BC by natural substances. We studied the effects of soil humic (HA) and fulvic (FA) acids on the surface properties and affinity for organic compounds of synthesized wood charcoal. Char powder suspended in a solution of HA or FA was loaded with organic matter via adsorption, evaporation of the water, or coflocculation with Al3+. These treatments were chosen to simulate initial and more advanced stages of environmental exposure. Coevaporation dramatically reduced the N2 Brunauer-Emmett-Teller total surface area of the char, but only moderately the CO2 cumulative surface area up to 1.4 nm. Organic compound adsorption was suppressed in proportion to molecular size, benzene < naphthalene < phenanthrene and 1,2,4-trichlorobenzene < phenanthrene, for humics in the adsorbed and coflocculated states, respectively. Humic substances also increased the linearity of the isotherms. The model we propose assumes that humic substances are restricted to the external surface where they act as pore blocking agents or competitive adsorbates, depending on the temperature and adsorbate size. Nitrogen is blocked from the internal pore space due to stiffness at 77 K of humic strands extending into pore throats, giving an artificially low surface area. Together with previous results, this finding indicates that N2 may not detect BC microporosity in geosorbents. At higher temperatures (CO2, 273 K; organics, 293 K), humic strands are more flexible, allowing access to interior pores. The counterintuitive molecular size dependence of adsorption suppression by humics is due to a molecular sieving effect in pores in which the adsorption space available to the organic compound is more and more restricted to external sites.     

Kinetics of microbial and chemical reduction of humic substances: implications for electron shuttling

Humic substances (HS) are redox-active natural organic compounds and serve as electron shuttles between microorganisms and iron(III) minerals. Here we demonstrate that electron shuttling is possible only at concentrations of dissolved HS of at least 5-10 mg C/L. Although such concentrations can be found in many rivers, lakes, and even in some aquifers there are also many marine and freshwater systems with DOC < 5 mg C/L where consequently electron shuttling is not expected to happen. We found that in the case of HS concentrations which do not limit electron shuttling, Geobacter sulfurreducens transfers electrons to HS at least 27 times faster than to Fe(III)hydroxide. Microbially reduced HS transfer electrons to ferrihydrite at least 7 times faster than cells thereby first demonstrating that microbial mineral reduction via HS significantly accelerates Fe(III) mineral reduction and second that electron transfer from reduced HS to Fe(III) minerals represents the rate-limiting step in microbial Fe(III) mineral reduction via HS. Microbial reduction of HS transfers as many electrons to HS as chemical reduction with H2 indicating that all redox-active functional groups that can be reduced at a redox potential of -418 mV (Eh(0) of H2/H+ redox couple at pH 7) can also be reduced by microorganisms.     

Spectrofluorescence of sediment humic substances and historical changes of lacustrine organic matter provenance in response to atmospheric nutrient enrichment

Humic substances were extracted from the sediments of two small alpine lakes in the Colorado Front Range and characterized by three-dimensional fluorescence spectroscopy. The fluorescence index (FI), defined as the ratio of fluorescence emitted at 450 nm to that emitted at 500 nm from an excitation wavelength of 370 nm, was computed and thereafter compared to additional sediment proxies of recent environmental change. Stratigraphic changes in both sediment C:N ratios and diatom assemblages parallel those of Fl, together indicating pronounced increases in the contribution of autochthonous organic matter to lake sediments since the mid-20th century. This result is consistent with increased algal production attributable to nutrient enrichment, given thatthe region undergoes episodic nitrogen saturation in response to anthropogenic N emission. The validation of sediment Fl measurements through comparisons with independent methods demonstrates the utility of this technique for characterizing shifts in the provenance of lake sediment organic matter arising from changing environmental conditions.     

Influence of sorption to dissolved humic substances on transformation reactions of hydrophobic organic compounds in water. I. Chlorination of PAHs

The effect of sorption to dissolved humic acids (HAs) on the chlorination of PAHs in aqueous solution was studied. The addition of HA accelerated the chlorination of fluoranthene and naphthalene in hypochlorite solutions at pH 5, the stronger effect being observed for fluoranthene that is sorbed to a higher extent than naphthalene. Sorption coefficients (K(DOC)) of the analytes were determined by solid-phase microextraction (SPME). The observed rate constant for fluoranthene chlorination is, for example, larger by a factor of 5 in the presence of 10 mg L(-1) of an aquatic HA as compared to HA-free solution (k' = 0.02 h(-1) at 60 mg L(-1) active chlorine, pH 5, without HA). While Cl2 is the dominant reactive species in pure aqueous solution for both PAHs, the reaction of fluoranthene seems to involve an additional pathway of chlorination by HOCl in the presence of HA. It was found that not only did HA not protect PAHs from the electrophilic attack of the chlorinating species, but the sorption of PAHs on the hydrophobic domains of the HA favored instead the extent of the chlorination reaction.     

Hydration of natural organic matter: effect on sorption of organic compounds by humin and humic acid fractions vs original peat material

Natural organic matter (NOM) hydration is found to change activity-based sorption of test organic compounds by as much as 2-3 orders of magnitude, depending on the compound and the specific NOM sorbent. This is demonstrated for sorption on humin, humic acid, and the NOM source material. Hydration assistance in organic compound sorption correlates with the ability of the sorbate to interact strongly with hydrated sorbents, demonstrating the important role of noncovalent polar links in organizing the sorbent structure. Differences in hydration effect between the sorbents are caused mainly by differences in compound-sorbent interactions in the dry state. For a given compound, hydration of the sorbent tends to equalize the sorption capability of the three sorbents. No correlation was found between the strength of sorbate-sorbent interactions or the type of sorbate functional groups and the extent of sorption nonlinearity. Sorption nonlinearity compared over the same sorbed concentration range is greater on the original NOM than on either of the two extracted fractions. In elucidating sorption mechanisms on hydrated NOM, it is important to explicitly consider the participation of water molecules in organic compound interactions in the NOM phase.     

Absence of mobile carbohydrate domains in dry humic substances proven by NMR, and implications for organic-contaminant sorption models

The mobility and domain structure of various standard humic substances have been investigated by 1H and 1H-13C solid-state nuclear magnetic resonance (NMR) experiments. In four dry humic acids, a fulvic acid, a natural organic matter sample, and a whole peat sample, segments that undergo fast, large-amplitude motions account for <9% of the sample. This disproves a previous suggestion, based on 1H NMR data, that flexible domains, presumably carbohydrates, make up >40% of various humic acids; these putative mobile domains were also linked to dual-mode sorption. In particular, neither the polar alkyl (carbohydrate) nor the aromatic components show any fast, large-amplitude mobility. A small fraction of mobile nonpolar alkyl segments identified by us before is the only component undergoing large-amplitude motions, apart from absorbed water that we observe in humic acids exposed to ambient air. 1H-13C wide-line separation NMR shows that, contrary to previous suggestions, the dipolar couplings in the aromatic regions are smaller than in the polar alkyl segments, most likely due to differences in local 1H densities. Series of 1H-13C heteronuclear correlation experiments with 1H spin diffusion reveal close proximity of aromatic and polar alkyl segments in several humic acids, precluding carbohydrate domains on a scale of > 1-nm diameter. In the standard peat humic acid, nonpolar aromatic segments also do not form sorption domains of significant size, while nonpolar aliphatic domains, which we had previously shown to correlate with sorption capacity, have been confirmed.     

Dual (C, H) isotope fractionation in anaerobic low molecular weight (poly)aromatic hydrocarbon (PAH) degradation: potential for field studies and mechanistic implications

Anaerobic polycyclic aromatic hydrocarbon (PAH) degradation is a key process for natural attenuation of oil spills and contaminated aquifers. Assessments by stable isotope fractionation, however, have largely been limited to monoaromatic hydrocarbons. Here, we report on measured hydrogen isotope fractionation during strictly anaerobic degradation of the PAH naphthalene. Remarkable large hydrogen isotopic enrichment factors contrasted with much smaller values for carbon: ε(H) = -100‰ ± 15‰, ε(C) = -5.0‰ ± 1.0‰ (enrichment culture N47); ε(H) = -73‰ ± 11‰, ε(C) = -0.7‰ ± 0.3‰ (pure culture NaphS2). This reveals a considerable potential of hydrogen isotope analysis to assess anaerobic degradation of PAHs. Furthermore, we investigated the conclusiveness of dual isotope fractionation to characterize anaerobic aromatics degradation. C and H isotope fractionation during benzene degradation (ε(C) = -2.5‰ ± 0.2‰; ε(H) = -55‰ ± 4‰ (sulfate-reducing strain BPL); ε(C) = -3.0‰ ± 0.5‰; ε(H) = -56‰ ± 8‰ (iron-reducing strain BF)) resulted in dual isotope slopes (Λ = 20 ± 2; 17 ± 1) similar to those reported for nitrate-reducers. This breaks apart the current picture that anaerobic benzene degradation by facultative anaerobes (denitrifiers) can be distinguished from that of strict anaerobes (sulfate-reducers, fermenters) based on the stable isotope enrichment factors.     

History-dependent sorption in humic acids and a lignite in the context of a polymer model for natural organic matter

We examined sorption of two apolar compounds in three samples of macromolecular natural organic matter (NOM) in order to test whether history-dependent ("irreversible") behaviors, including sorption hysteresis and the conditioning effect, agree with a pore deformation/creation hypothesis applicable to the glassy organic solid state as proposed in the polymer literature. The compounds are 1,2,4-trichlorobenzene (TCB) and naphthalene (Naph). The NOM samples are a soil humic acid (H-HA), an Al3+-exchanged form of the same humic acid (Al-HA), and a low-rank coal (Beulah-Zap lignite, BZL). The HAs, at least, are believed free of environmental black carbon. The degree of nonlinearity in the isotherm and the ratio of hole-filling to solid-phase dissolution increased in the order of hardness (stiffness) of the solid: H-HA < Al-HA < BZL. Independent of solid, solutes show a 14-18 kJ/mol preference for hole "sites" as compared to dissolution "sites", which we attribute to the free energy needed in the dissolution domain to create a cavity to accommodate the solute. All solids exhibited hysteresis and the conditioning effect, which refers to enhanced re-sorption after pretreatment with a conditioning agent (in this case, chlorobenzene). Conditioning the sample results in increased sorption and increased contribution of hole-filling relative to dissolution. The effects of original hole population, matrix stiffness, and solute concentration on the hysteresis index and on the magnitude of the conditioning effect are consistent with a pore-deformation mechanism as the underlying cause of sorption irreversibility. This mechanism involves concurrent processes of irreversible hole expansion and the creation of new holes by the incoming sorbate (or conditioning agent). The results show that nonlinear and irreversible behavior may be expected for macromolecular forms of NOM that are in a glassy state and emphasize the case that NOM is not a passive sorbent but may be physically altered by the sorbate.     

The rate of 2,2-dichloropropane transformation in mineral micropores: implications of sorptive preservation for fate and transport of organic contaminants in the subsurface

Nanometer scale pores are ubiquitous in porous geologic media (soils and sediments). Sorption of organic contaminants in micropores (< or = 2 nm) can inhibittheir hydrolytic transformation due to the limited availability of reactive water within hydrophobic micropore spaces. As a test case, we studied the dehydrohalogenation of 2,2-dichloropropane (2,2-DCP) sorbed in the micropores of several model mineral solids. In the micropores of a hydrophobic dealuminated Y zeolite, CBV-780, 2,2-DCP dehydrohalogenation proceeded significantly slower than in bulk aqueous solution and eventually stopped. This was attributed to the depletion of reactive water molecules in the micropore spaces. The 2,2-DCP sorbed in the micropores of more hydrophilic solids (aquifer sediment, aquifer sand, and silica gel) also transformed slower than in aqueous solution, and the reaction no longer followed first-order kinetics. Results of transport modeling support that reactive contaminants sorbed in microporous minerals can be preserved over geological time scales under conditions that limit desorption. This study shows that hydrophobic micropores in geological media may act as an important sink for anthropogenic organic contaminants in the subsurface, and that sorption in micropores may significantly increase the persistence of the sorbed contaminants.     

Sorption-induced effects of humic substances on mass transfer of organic pollutants through aqueous diffusion boundary layers: the example of water/air exchange

This study examines the effect of dissolved humic substances (DHS) on the rate of water-gas exchange of organic compounds under conditions where diffusion through the aqueous boundary layer is rate-determining. A synthetic surfactant was applied for comparison. Mass-transfer coefficients were determined from the rate of depletion of the model compounds by means of an apparatus containing a stirred aqueous solution with continuous purging of the headspace above the solution. In addition, experiments with continuous passive dosing of analytes into the water phase were conducted to simulate a system where thermodynamic activity of the chemical in the aqueous phase is identical in the presence and absence of DHS. The experimental results show that DHS and surfactants can affect water-gas exchange rates by the superposition of two mechanisms: (1) hydrodynamic effects due to surface film formation ("surface smoothing"), and (2) sorption-induced effects. Whether sorption accelerates or retards mass transfer depends on its effect on the thermodynamic activity of the pollutant in the aqueous phase. Mass transfer will be retarded if the activity (or freely dissolved concentration) of the pollutant is decreased due to sorption. If it remains unchanged (e.g., due to fast equilibration with a sediment acting as a large source phase), then DHS and surfactant micelles can act as an additional shuttle for the pollutants, enhancing the flux through the boundary layer.     

Effect of natural organic substances on the surface and adsorptive properties of environmental black carbon (char): pseudo pore blockage by model lipid components and its implications for N2-probed surface properties of natural sorbents

Black carbon (BC; char and soot) particles emitted to the environment typically are formed with high microporosity and surface area, properties that are responsible for their presumed important role in adsorption of anthropogenic organic compounds in soils and sediments. An issue that has received little direct attention is the possibility that naturally occurring organic matter attenuates the surface activity of BC. We found that simulated "aging" of prepared wood char particles in a soil-water suspension leads to a strong decline in char total surface area (TSA) by N2 adsorption at 77 K with BET analysis and a more modest decline in affinity for dissolved benzene. To help determine the underlying cause, we measured the effects of adsorbed natural lipids or lipid fractions of humic substances, modeled by triglycerides of a commercial vegetable oil. With increasing lipid loading (up to 40% by char weight) from aqueous mixtures, N2 TSA was strongly suppressed (up to 100-fold), while CO2 cumulative surface area (CSA, 0-1.4 nm) at 273 K and benzene adsorption at 293 K were hardly affected. In addition, the rate of CO2 adsorption was retarded. We propose that externally adsorbed lipid molecules occupy pore throats with access to interior pore networks. At 77 K, as opposed to the higher temperatures, lipid chains are too inflexible to allow passage of adsorbate. It is concluded that benzene adsorption to char predominates at interior pore sites and does not correlate with N2-probed micropore properties when the char accrues pore-blocking substances from the surroundings. The findings question the suitability of N2 for probing hydrophobic microporosity of BC in soils and sediments.     

Variability in carbon isotopic fractionation during biodegradation of chlorinated ethenes: implications for field applications

Stable carbon isotopic analysis has the potential to assess biodegradation of chlorinated ethenes. Significant isotopic shifts, which can be described by Rayleigh enrichment factors, have been observed for the biodegradation of trichloroethlyene (TCE), cis-dichloroethylene (cDCE), and vinyl chloride (VC). However, until this time, no systematic investigation of isotopic fractionation during perchloroethylene (PCE) degradation has been undertaken. In addition, there has been no comparison of isotopic fractionation by different microbial consortia, nor has there been a comparison of isotopic fractionation by consortia generated from the same source, but growing under different conditions. This study characterized carbon isotopic fractionation during reductive dechlorination of the chlorinated ethenes, PCE in particular, for microbial consortia from two different sources growing under different environmental conditions in order to assess the extent to which different microbial consortia result in different fractionation factors. Rayleigh enrichment factors of -13.8@1000, -20.4@1000, and -22.4@1000 were observed for TCE, cDCE, and VC, respectively, for dechlorination by the KB-1 consortium. In contrast, isotopic fractionation during reductive dechlorination of perchloroethylene (PCE) could not always be approximated by a Rayleigh model. Dechlorination by one consortium followed Rayleigh behavior (epsilon = -5.2), while a systematic change in the enrichment factor was observed over the course of PCE degradation by two other consortia. Comparison of all reported enrichment factors for reductive dechlorination of the chlorinated ethenes shows significant variation between experiments. Despite this variability, these results demonstrate that carbon isotopic analysis can provide qualitative evidence of the occurrence and relative extent of microbial reductive dechlorination of the chlorinated ethenes.     

UV photolysis of nitrate: effects of natural organic matter and dissolved inorganic carbon and implications for UV water disinfection

Nitrite (NO2-) formation during ultraviolet (UV) photolysis of nitrate was studied as a function of pH and natural organic matter (NOM) concentration to determine water-quality effects on quantum yields and overall formation potential during UV disinfection of drinking water with polychromatic, medium-pressure (MP) Hg lamps. Quantum yields measured at 228 nm are approximately 2 times higher than at 254 nm under all conditions studied. In the absence of NOM, NO2- quantum yields decrease with time. With addition of NOM, initial quantum yields increase, and the time-dependent decrease is eliminated. At 15 ppm dissolved organic carbon (DOC) as NOM, the quantum yield increases with time. Dissolved inorganic carbon significantly decreases NO2- yields at pH 8 but not pH 6, presumably by reaction of CO2(aq) with peroxynitrite, a major intermediate in NO2- formation. The results indicate important and previously unrecognized roles for NOM and CO2(aq) in nitrate photolysis. When photolysis was carried out using the full spectrum MPUV lamp and germicidally relevant UV doses, NO2- concentrations remained well below the U.S. maximum contaminant level of 1 ppm N, even with nitrate initially present at 10 ppm N. Under current U.S. regulations, NO2- formation should not pose a significant problem for water utilities during UV disinfection of drinking water with MP Hg lamps.     

Sorption of heterocyclic organic compounds to reference soils: column studies for process identification

In this study, the sorption behavior of a wide variety of N-, S-, and O-heterocyclic compounds (NSOs) to reference soils (Eurosoils 1-5) was characterized by a soil column chromatography (SCC) approach. The major goal was to identify the compound specific and environmental factors influencing sorption processes. The sorption of S- and O-heterocyclic compounds (thiophene, benzothiophene, 5-methylbenzo[b]thiophene, benzofuran, 2-methylbenzofuran, and 2,3-dimethylbenzofuran) was generally controlled by nonspecific interactions with soil organic carbon (OC). With regard to non-ionizable N-heterocyclic compounds, pyrrole, 1-methylpyrrole, and pyrimidine were hardly retarded in any soil. The sorption of indole, 2-hydroxyquinoline, and benzotriazole was dominated by specific interaction (e.g., complexation of surface-bound cations) rather than partition to soil OC. The sorption of ionizable N-heterocyclic compounds (quinoline, isoquinoline, quinaldine, 2-methylpyridine, and pyridine) can be described by a conceptual model including partitioning to soil OC, cation exchange, and an additional sorption process (probably surface complexation of the neutral species). Cation exchange was usually the dominant mechanism in the sorption of ionizable compounds if the protonated fraction of the compound exceeded 5%. Otherwise, surface complexation became dominant. Soil pH was the most important factor influencing the sorption of ionizable NSOs. Our study suggests that a fairly precise assessment of sorption in most soils can be expected for N-, S-, and O-heterocyclic compounds if the three sorption mechanisms are taken into accountwhere appropriate. Deviations from this behavior indicated special cases where additional soil specific properties (e.g., accessible surface, CEC, charge density) need to be considered such as for 2-methylpyridine and pyridine sorption to Eurosoil 1.