Water stress effects on toluene biodegradation by Pseudomonas putida

We quantified the effects of matric and solute waterpotential on toluene biodegradation by Pseudomonasputida mt-2, a bacterial strain originally isolated fromsoil. Across the matric potential range of 0 to – 1.5 MPa,growth rates were maximal for P. putida at – 0.25MPa and further reductions in the matric potentialresulted in concomitant reductions in growth rates.Growth rates were constant over the solute potential range0 to – 1.0 MPa and lower at – 1.5 MPa. First ordertoluene depletion rate coefficients were highest at0.0 MPa as compared to other matric water potentialsdown to – 1.5 MPa. Solute potentials down to – 1.5 MPadid not affect first order toluene depletion ratecoefficients. Total yield (protein) and carbon utilizationefficiency were not affected by water potential, indicatingthat water potentials common to temperate soils were notsufficiently stressful to change cellular energyrequirements. We conclude that for P. putida: (1)slightly negative matric potentials facilitate faster growthrates on toluene but more negative water potentials resultin slower growth, (2) toluene utilization rate per cell massis highest without matric water stress and is unaffected bysolute potential, (3) growth efficiency did not differ acrossthe range of matric water potentials 0.0 to – 1.5 MPa.     

Controls on microbial CO2 production: a comparison of surface and subsurface soil horizons

Although a significant amount of the organic C stored in soil resides in subsurface horizons, the dynamics of subsurface C stores are not well understood. The objective of this study was to determine if changes in soil moisture, temperature, and nutrient levels have similar effects on the mineralization of surface (0–25 cm) and subsurface (below 25 cm) C stores. Samples were collected from a 2 m deep unsaturated mollisol profile located near Santa Barbara, CA, USA. In a series of experiments, we measured the influence of nutrient additions (N and P), soil temperature (10–35°C), and soil water potential (?0.5 to ?10 MPa) on the microbial mineralization of native soil organic C. Surface and subsurface soils were slightly different with respect to the effects of water potential on microbial CO₂ production; C mineralization rates in surface soils were more affected by conditions of moderate drought than rates in subsurface soils. With respect to the effects of soil temperature and nutrient levels on C mineralization rates, subsurface horizons were significantly more sensitive to increases in temperature or nutrient availability than surface horizons. The mean Q₁₀ value for C mineralization rates was 3.0 in surface horizons and 3.9 in subsurface horizons. The addition of either N or P had negligible effects on microbial CO₂ production in surface soil layers; in the subsurface horizons, the addition of either N or P increased CO₂ production by up to 450% relative to the control. The results of these experiments suggest that alterations of the soil environment may have different effects on CO₂ production through the profile and that the mineralization of subsurface C stores may be particularly susceptible to increases in temperature or nutrient inputs to soil.     

Toluene diffusion and reaction in unsaturated Pseudomonas putida biofilms

Biofilms are frequently studied in the context of submerged or aquatic systems. However, much less is known about biofilms in unsaturated systems, despite their importance to such processes as food spoilage, terrestrial nutrient cycling, and biodegradation of environmental pollutants in soils. Using modeling and experimentation, we have described the biodegradation of toluene in unsaturated media by bacterial biofilms as a function of matric water potential, a dominant variable in unsaturated systems. We experimentally determined diffusion and kinetic parameters for Pseudomonas putida biofilms, then predicted biodegradation rates over a range of matric water potentials. For validation, we measured the rate of toluene depletion by intact biofilms and found the results to reasonably follow the model predictions. The diffusion coefficient for toluene through unsaturated P. putida biofilm averaged 1.3 x 10(7) cm(2)/s, which is approximately two orders of magnitude lower than toluene diffusivity in water. Our studies show that, at the scale of the microbial biofilm, the diffusion of toluene to biodegrading bacteria can limit the overall rate of biological toluene depletion in unsaturated systems. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 656-670, 1997.     

Soil initial bacterial diversity and nutrient availability determine the rate of xenobiotic biodegradation

Understanding the relative importance of soil microbial diversity, plants and nutrient management is crucial to implement an effective bioremediation approach to xenobiotics-contaminated soils. To date, knowledge on the interactive effects of soil microbiome, plant and nutrient supply on influencing biodegradation potential of soils remains limited. In this study, we evaluated the individual and interactive effects of soil initial bacterial diversity, nutrient amendments (organic and inorganic) and plant presence on the biodegradation rate of pyrene, a polycyclic aromatic hydrocarbon. Initial bacterial diversity had a strong positive impact on soil biodegradation potential, with soil harbouring higher bacterial diversity showing ~ 2 times higher degradation rates than soils with lower bacterial diversity. Both organic and inorganic nutrient amendments consistently improved the degradation rate in lower diversity soils and had negative (inorganic) to neutral (organic) effect in higher diversity soils. Interestingly, plant presence/type did not show any significant effect on the degradation rate in most of the treatments. Structural equation modelling demonstrated that initial bacterial diversity had a prominent role in driving pyrene biodegradation rates. We provide novel evidence that suggests that soil initial microbial diversity, and nutrient amendments should be explicitly considered in the design and employment of bioremediation management strategies for restoring natural habitats disturbed by organic pollutants.     

Mass transfer and hydrocarbon biodegradation of aged soil in slurry phase

Addition of toluene into slurry phase laboratory microcosm is proposed in order to increase desorption rate of hydrocarbons and as an alternative to improve bioavailability of hydrocarbon in aged soils. Our studies showed that toluene has a positive effect on desorption of total petroleum hydrocarbons (TPH). Addition of 14,000 mg toluene/kg of soil, in highly polluted soil, increased the consumption rate of hydrocarbons three times in comparison to control without solvent. In 30 days the initial TPH concentration in soil, 292,000 mg/kg, diminished 45%. Although toluene was able to dissolve complex organic compounds such as asphaltene fraction, it probably yielded a highly toxic toluene-hydrocarbons phase. The inhibitory effect of toluene-TPH was also studied. A substrate inhibition model was used: the k(m) and k(i) constants were 57 and 490 mg TPH/L liquid phase, respectively. Experimental data were well described when the proposed model included sequential desorption and biodegradation phenomena. Damk?hler number evaluation showed that rate of mass transfer was the limiting step in overall biodegradation in nonsolvent control. When high concentration of toluene was added, then bioreaction was the limiting step, but inhibitory effect should be considered. However, toluene addition at low concentrations facilitates the biodegradation of aromatic compounds.     

The rate of microbial degradation of oil in a beach gravel column

An automatic, continuous flow respirometer was used to follow the bio-oxidation of a crude oil in a column of fairly coarse beach material. A number of water percolation rates were employed, with and without inorganic nutrient supplementation (nitrate and phosphate). Initially, nutrient supplementation was required to allow significant oxidation rates, but a capacity for biodegradation in the absence of continued supplementation developed slowly. The increase in oxidation rate with nutrient supplementation was in the proportion 0.23 mg oxygen permol nitrate. This proportionality was similar to previous results in this laboratory (using different systems), as was the effect of temperature. The mean of two Q₁₀ values in this work was 2.7.     

Legacy phosphorus in subtropical wetland soils: Influence of dairy, improved and unimproved pasture land use

Wetlands provide various ecosystem services. One of these services includes nutrient storage in soils. Soils retain and release nutrients such as phosphorus (P). This dynamic can be controlled by soil characteristics, overlying water quality, environmental conditions and historical nutrient loading. Historical nutrient loading contributes to a legacy of P stored in soils and this may influence present day P dynamics between soil and water. We quantified P characteristics of wetland soils and determined the availability and capacity of soils to retain additional P loadings. We sampled surface (0-10) and subsurface (10-30) wetland soils within dairy, improved and unimproved pastures. Surface soils had much greater concentrations of organic and inorganic P. Wetland soils in dairy had greatest concentrations of Ca and Mg, probably due to inputs of inorganic fertilizer. They also had much greater total P, inorganic P, and P sorption capacity; however, these soils were P saturated and had little capacity to retain additional P loading. Improved and unimproved pasture wetland soils had greatest amounts of organic P (>84%) and a capacity to store additional P loadings. Using multivariate statistics, we determined that rather than being different based on land use, wetland soils in improved and unimproved pasture were dissimilar based upon organic matter, organic P fractions, residual P, and soil metal (Fe and Al) content. The legacy of stored P in soils, particularly wetland soils from dairies, combined with best management practices (BMPs) to reduce nutrient loading to these systems, could contribute to a short-term release of soil-stored P to overlying wetland water.     

Bioremediation and Biorestoration of a Crube Oil-Contaminated Freshwater Wetland on the St.Lawrence River

Biostimulation by nutrient enrichment and phytoremediation were studied for the restoration of an acutely stressed freshwater wetland experimentally exposed to crude oil. The research was carried out along the shores of the St. Lawarence River at Ste. Croix, Quebec, Canada. The research determined the effectiveness of fertilizer addition in enhancing the biodegradation rates of residual oil. It further examined the rate at which the stressed ecosystem recovered with and without the addition of inorganic fertilizers and the role of nutrients in enhancing wetland restoration in the absence of healthy wetland plants. Chemical analysis of integrated sediment core samples to the depth of oil penetration within the experimental plots indicated that addition of inorganic nutrients did not enhance the disappearance of alkanes or PAHs. In surface samples, however, hydrocarbon disappearance rates were higher when the metabolic activity of wetland plants was suppressed by the removal of emergent plant growth. These results suggest that oxygen limitation plays a major role in preventing rapid biodegradation of hydrocarbons in anoxic wetland sediment.     

Microbiological methods for the cleanup of soil and ground water contaminated with halogenated organic compounds

There is growing interest in the enhancement of microbial degradative activities as a means of bringing about the in situ cleanup of contaminated soils and ground water. The halogenated organic compounds are likely to be prime targets for such biotechnological processes because of their widespread utilisation and the biodegradability of many of the most commonly used compounds. The aim of this review is to consider the potential for microbiological cleanup of haloorganic-contaminated sites. The technologies available involve the provision of suitable environmental conditions to facilitate maximum biodegradation rates either in the subsurface or in on-site bioreactors. Methodologies include the supply of inorganic nutrients, the supply of oxygen gas, the addition of degradative microbial inocula and the introduction of co-metabolic substrates. The potential efficiencies and limitations of the methods are critically discussed from a microbiological viewpoint with respect to substrate degradability and population responses to supplementation.     

Microbiological methods for the cleanup of soil and ground water contaminated with halogenated organic compounds

Abstract There is growing interest in the enhancement of microbial degradative activities as a means of bringing about the in situ cleanup of contaminated soils and ground water. The halogenated organic compounds are likely to be prime targets for such biotechnological processes because of their widespread utilisation and the biodegradability of many of the most commonly used compounds. The aim of this review is to consider the potential for microbiological cleanup of haloorganic-contaminated sites. The technologies available involve the provision of suitable environmental conditions to facilitate maximum biodegradation rates either in the subsurface or in on-site bioreactors. Methodologies include the supply of inorganic nutrients, the supply of oxygen gas, the addition of degradative microbial inocula and the introduction of co-metabolic substrates. The potential efficiencies and limitations of the methods are critically discussed from a microbiological viewpoint with respect to substrate degradability and population responses to supplementation.     

Phenanthrene degradation by estuarine surface microlayer and bulk water microbial populations

Paired surface microlayer and bulk water samples from five sites in the Great Bay Estuary, New Hampshire, were examined with regard to numbers of bacteria,¹⁴C-phenanthrene biodegradation potentials, and organic and inorganic chemical characteristics. Microlayer samples were generally enriched in nutrients (N and P), dissolved organic matter, and culturable heterotrophic bacteria compared with their corresponding bulk waters. Microlayer samples from marina environments were also enriched in aromatic hydrocarbons, as determined by UV spectrophotometric and fluorometric analyses, and demonstrated substantial phenanthrene biodegradation activity in the assay employed. Biodegradation activity of marina bulk water samples ranged from nil to levels exceeding those exhibited by microlayer samples. No diminution of biodegradation activity was observed after filtration (1.2 m effective retention) of microlayer water, indicating that the responsible organisms were not particle-associated. Phenanthrene-degrading bacteria, enumerated by counting clearing zones in a crystalline phenanthrene overlay after colony development on a phenanthrene/toluene agar (PTA) medium, were superior to epifluorescence direct counts or standard plate counts on PTA or estuarine nutrient agar in predicting¹⁴C-phenanthrene biodegradative activity.     

Nutrient-limited biodegradation of PAH in various soil strata at a creosote contaminated site

The effects of nutrient addition on the in situ biodegradation of polycyclic aromatic hydrocarbons in creosote contaminated soil were studied in soil columns taken from various soil strata at a wood preserving plant in Norway. Three samples were used: one from the topsoil (0–0.5 m), one from an organic rich layer (2–2.5 m) and one from the sandy aquifer (4.5–5 m). The addition of inorganic nitrogen and phosphorous stimulated the degradation of polycyclic aromatic hydrocarbons (PAHs) in the top soil and the aquifer sand. These two soils, which differed strongly in contamination levels, responded similarly to nutrient addition with the corresponding degradation of 4-ring PAHs. The ratio between available nitrogen (N) and phosphorous (P) might explain the degree of degradation observed for the 4-ring PAHs. However, the degree of degradation of 3-ring PAHs did not significantly increase after nutrient addition. An increase in the respiration rate, after nutrient addition, could only be observed in the topsoil. In the aquifer sand, 4-ring PAH degradation was not accompanied by an increase in the respiration rate or the number of heterotrophic micro-organisms. PAH degradation in the organic layer did not respond to nutrient addition. This was probably due to the low availability of the contaminants for micro-organisms, as a result of sorption to the soil organic matter. Our data illustrate the need for a better understanding of the role of nutrients in the degradation of high molecular weight hydrocarbons for the successful application of bioremediation at PAH contaminated sites.     

Soil biological activity and their seasonal variations in response to long-term application of organic and inorganic fertilizers

The objectives of this study were to explore the effects of long-term and continued application of fertilizers and manures on microbial biomass, soil biological activity and their seasonal variations in surface and subsurface soils in relation to soil fertility. For this, soils were sampled in spring, summer and autumn from Shenyang Long-term Experimental Station, northeastern China. The results showed that soil total nitrogen (N), organic carbon (C), basal respiration, microbial biomass and enzymatic activity increased in manure-amended surface soils, but decreased with soil depth. Long-term application of inorganic fertilizers significantly decreased soil pH value, sucrase activity and microbial biomass C, but increased soil metabolic quotient (qCO₂). However, no significant effect of inorganic fertilizers on soil total N, urease activity and microbial biomass N was observed in comparison with CK0 (neither tillage nor fertilization) and CK (no fertilizers). There was no significant difference between CK0 and CK in soil total N, organic C and microbial activity in surface soil layer (0–20 cm), but these parameters in subsurface soil layer (20–40 cm) were higher in CK than in CK0. Moreover, seasonal changes were observed in terms of soil nutrient contents, enzymatic activity, microbial biomass and soil respiration. There were significant correlations between soil microbial biomass C and N, between organic C and sucrase activity and between total N and urease activity, respectively. It is recommended that combined use of organic manure with inorganic fertilizers should be considered to maintain higher microbial biomass, soil biological activity and soil fertility. Considering considerably high nutrients reserve and microbial activity in subsurface layers of soil and wind-erosion-caused nutrient loss in spring in north China, we also propose that low tillage should be considered to make use of nutrients in soils.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in vadose sediments

The unsaturated subsurface (vadose zone) receives significant amounts of hazardous chemicals, yet little is known about its microbial communities and their capacity to biodegrade pollutants. Trichloroethylene (TCE) biodegradation occurs readily in surface soils; however, the process usually requires enzyme induction by aromatic compounds, methane, or other cosubstrates. The aerobic biodegradation of toluene and TCE by indigenous microbial populations was measured in samples collected from the vadose zone at unpolluted and gasoline-contaminated sites. Incubation at field moisture levels showed little activity on either TCE or toluene, so samples were tested in soil suspensions. No degradation occurred in samples suspended in water or phosphate buffer solution; however, both toluene and TCE were degraded in samples suspended in mineral salts medium. TCE degradation depended on toluene degradation, and little loss occurred under sterile conditions. Studies with specific nutrients showed that addition of ammonium sulfate was essential for degradation, and addition of other mineral nutrients further enhanced the rate. Additional studies with vadose sediments amended with nutrients showed similar trends to those observed in sediment suspensions. Initial rates of biodegradation in suspensions were faster in uncontaminated samples than in gasolinecontaminated samples, but the same percentages of chemicals were degraded. Biodegradation was slower and less extensive in shallower samples than deeper samples from the uncontaminated site. Two toluene-degrading organisms isolated from a gasoline-contaminated sample were identified as Corynebacterium variabilis SVB74 and Acinetobacter radioresistens SVB65. Inoculation with 10⁶ cells of C. variabilis ml^–1 of soil solution did not enhance the rate of degradation above that of the indigenous population. These results indicate that mineral nutrients limited the rate of TCE and toluene degradation by indigenous populations and that no additional benefit was derived from inoculation with a toluene-degrading bacterial strain. Correspondence to: K.M. Scow     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

Environmental factors influencing the rate of hydrocarbon oxidation in temperate lakes.

Rates of hydrocarbon biodegradation were estimated by following oxygen uptake during mineral oil oxidation or oxidation of [1-14C]hexadecane to 14CO2, when these substrates were added to natural water samples from Wisconsin lakes. A lag phase preceded hydrocarbon oxidation, the length of which depended on population density or on factors influencing growth rate and on the presence of nonhydrocarbon organic compounds. Hydrocarbon oxidation was coincident with growth and presumably represented the development of indigenous hydrocarbon-degrading microorganisms in response to hydrocarbon additions. In detailed studies in Lake Mendota, it was found that, despite the continued presence of hydrocarbon-degrading microorganisms in water samples, seasonal variations in the rates of mineral oil and hexadecane oxidation occurred which correlated with seasonal changes in temperature and dissolved inorganic nitrogen and phosphorus. The temperature optimum for oil biodegradation remained at 20 to 25 C throughout the year, so that temperature was the main limiting factor during winter, spring, and fall. During summer, when temperatures were optimal, nutrient deficiencies limited oil biodegradation, and higher rates could be obtained by addition of nitrogen and phosphorus. The rates of hydrocarbon biodegradation were thus high only for about 1 month of the ice-free period, when temperature and nutrient supply were optimal. Nutrient limitation of oil biodegradation was also demonstrated in 25 nutrient-poor lakes of northern Wisconsin, although in almost every case oil-degrading bacteria were detected. Knowledge of temperature and nutrient limitations thus will help in predicting the fate of hydrocarbon pollutants in freshwater.     

Toluene biodegradation rates in unsaturated soil systems versus liquid batches and their relevance to field conditions

Contaminant biodegradation in unsaturated soils may reduce the risks of vapor intrusion. However, the reported rates show large variability and are often derived from slurry experiments that are not representative of unsaturated conditions. Here, different laboratory setups are used to derive the biodegradation capacity of an unsaturated soil layer through which gaseous toluene migrates from the water table upwards. Experiments in static unsaturated soil microcosms at 6–30 % water-filled porosity (WFP) and unsaturated soil columns at 9, 14, and 27 % WFP were compared with liquid batches containing the same culture of Alicycliphilus denitrificans. The biodegradation rates for the liquid batches were orders of magnitude lower than for the other setups. Hence, liquid batches do not necessarily reflect optimal conditions for bacteria; either oxygen or toluene mass transfer at the cell scale or the absence of soil–water–air interfaces seemed to be limiting bacterial activity. For the column setup, the rates were limited by mass supply. The microcosm results could be described by apparent first-order biodegradation constants that increased with WFP or through a numerical model that included biodegradation as a first-order process taking place in the liquid phase only. The model liquid phase first-order rates varied between 6.25 and 20 h^?1 and were not related to the water content. Substrate availability was the primary factor limiting bioactivity, with evidence for physiological stress at the lowest water-filled porosity. The presented approach is useful to derive liquid phase biodegradation rates from experimental data and to include biodegradation in vapor intrusion models.     

Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil.

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 micrograms ml-1) and toluene (1.0 to 20 micrograms ml-1) were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 micrograms ml-1, TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 microgram of TCE ml-1 and 20 micrograms of toluene ml-1 were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 microgram of TCE ml-1 was degraded, whereas 100% of the toluene disappeared. A second addition of 20 micrograms of toluene ml-1 to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18 degrees C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.     

Morphometry and sedimentation as regulating factors for nutrient recycling and trophic state in coastal waters

The aim of this work is to quantify the importance of morphometry and sedimentation/resuspension on nutrient recycling and trophic characteristics in coastal waters. Extensive field work has been carried out in 23 coastal areas in the Swedish and Finnish part of the Baltic Proper. Sediment traps were deployed for two one-week periods in all areas. On average, 56% of the total sedimentation in sediment traps 3 m below the water surface (SedS) and 62% of the total sedimentation on sediment traps 1 m above the bottom (SedB) was resuspended material. Coastal morphometric parameters, surface water retention time and bottom dynamic conditions were determined for all areas. There is a marked relationship between SedS and inorganic-N concentration in the surface water. The relationship was improved significantly by using sedimentation of the resuspended fraction at 3 m water depth (SedR) instead of SedS.This led to the hypothesis that increased concentration of inorganic nitrogen in the surface water results from increased mineralisation of resuspended organic particles. A model describing SedS is presented where inorganic nitrogen concentration, the water surface area and the surface water retention time can explain 82% of the variation in SedS. In another model inorganic nitrogen and water surface area can explain as much as 93% of the variation in SedR.These results emphasise the importance of resuspension for nutrient recycling and trophic state in coastal waters. The importance of coastal morphometry and surface water retention time on total sedimentation and nutrient recycling makes it possible to classify coastal areas in terms of potential nutrient recycling capacity/trophic state from these simple sensitivity parameters.     

Outgassing Losses of Toluene and m-Xylene Evaluated by 14C-Based Mass Balances for Laboratory Bioventing Simulations

Mixtures of toluene, ethylbenzene, and the xylenes spiked with ¹⁴C-labeled toluene or m-xylene were added to bench-scale bioventing simulation columns filled with hydrocarbon-contaminated subsurface soils. After 2 to 4 weeks of incubation during which air was pumped through the column at rates of at least 2?ml·min^?1·kg^?1 between 54 and 84% of the radiolabel was recovered in traps as outgassed parent compound from four columns sterilized with gamma-irradiation. In contrast, seven nonsterilized but otherwise identically treated (except for inorganic nitrogen addition) columns lost less than 0.4% (and one column lost 0.7%) of the radiolabel through outgassing of the parent compound. Nonsterilized columns lost 40 to 61% of the radiolabel as ¹⁴CO₂, whereas gamma-irradiated columns usually lost only trace amounts of ¹⁴C in this form. Biologically active columns also retained much larger fractions than sterilized columns of the radiolabel in the subsoil in forms, possibly microbial biomass, from which it could be recovered by wet oxidation. Addition of 10 or 40?mg/kg of mineral nitrogen had no consistent effect on bioventing performance.