Efficiency of Indigenous and Inoculated Cold-Adapted Soil Microorganisms for Biodegradation of Diesel Oil in Alpine Soils

Biodegradation of diesel oil (5 g(middot)kg [soil dry weight](sup-1)) was investigated in five alpine subsoils, differing in soil type and bedrock, in laboratory experiments during 20 days at 10(deg)C. The biodegradation activities of the indigenous soil microorganisms and of a psychrotrophic diesel oil-degrading inoculum and the effect of biostimulation by inorganic fertilization (C/N/P ratio = 100:10:2) were determined. Fertilization significantly enhanced diesel oil biodegradation activity of the indigenous soil microorganisms. Biostimulation by fertilization enhanced diesel oil biodegradation to a significantly greater degree than bioaugmentation with the psychrotrophic inoculum. In none of the five soils did fertilization plus inoculation result in a higher decontamination than fertilization alone. A total of 16 to 23% of the added diesel oil contamination was lost by abiotic processes. Total decontamination without and with fertilization was in the range of 16 to 31 and 27 to 53%, respectively.     

Hexadecane mineralization activity in ornithogenic soil from Seabee Hook,Cape Hallett,Antarctica

Ornithogenic soils that form in penguin rookeries contain high levels of organic carbon and nitrogen. On Seabee Hook, Cape Hallett, Antartica, ornithogenic soil was contaminated with hydrocarbons following establishment of a scientific research station. In these soils hydrocarbon biodegradation could be supported by available soil nitrogen. Hexadecane mineralization activity was detected in vitro in ornithogenic soil when incubated at 5 or 15°C. At 5°C the extent of hexadecane mineralization was higher in hydrocarbon-contaminated soil than in uncontaminated soil. Alkane-degrading bacteria isolated from Seabee Hook soil were identified as Rhodococcus or Gordonia spp. or an unclassified Corynebacterineae. The alkane degraders grew on n-alkanes from heptane (C8) to eicosane (C20) and pristane, and utilized uric acid or ammonium nitrate as nitrogen source. All of the isolates possessed urease activity. Results of this study indicate biodegradation of hydrocarbons may contribute to the natural attenuation of oil spills in ornithogenic surface soils in summer.     

Potential of vetiver (vetiveria zizanioides (L.) Nash) for phytoremediation of petroleum hydrocarbon-contaminated soils in Venezuela

Venezuela is one of the largest oil producers in the world. For the rehabilitation of oil-contaminated sites, phytoremediation represents a promising technology whereby plants are used to enhance biodegradation processes in soil. A greenhouse study was conducted to determine the tolerance of vetiver (Vetiveria zizanioides (L.) Nash) to a Venezuelan heavy crude oil in soil. Additionally, the plant's potential for stimulating the biodegradation processes of petroleum hydrocarbons was tested under the application of two fertilizer levels. In the presence of contaminants, biomass and plant height were significantly reduced. As for fertilization, the lower fertilizer level led to higher biomass production. The specific root surface area was reduced under the effects of petroleum. However, vetiver was found to tolerate crude-oil contamination in a concentration of 5% (w/w). Concerning total oil and grease content in soil, no significant decrease under the influence of vetiver was detected when compared to the unplanted control. Thus, there was no evidence of vetiver enhancing the biodegradation of crude oil in soil under the conditions of this trial. However, uses of vetiver grass in relation to petroleum-contaminated soils are promising for amelioration of slightly polluted sites, to allow other species to get established and for erosion control.     

Microorganisms participating in the biodegradation of modified polyethylene films in different soils under laboratory conditions

The degree of biodegradation of low-density polyethylene (LDPE) films modified with Bionolle^® polyester in different soils under laboratory conditions was evaluated. Films were incubated in soils from waste coal, a forest and an extinct volcano crater. Prior to degradation studies, soils underwent chemical and microbiological analysis. Film weight loss and mechanical properties, as well as the surface of the polymeric samples determined via scanning electron microscopy, were evaluated after 75, 150 and 225 days of biodegradation. Important chemical changes in the polymeric chains were detected by Fourier Transform Infrared Spectroscopy (FTIR). Fungal and bacterial species that were able to grow on the film surfaces were monitored in order to see whether the films were easily colonised by autochthonous microorganisms (i.e., typical to each soil). Identification of microorganisms was based on their cellular fatty acid methyl ester (FAME) profiles. Biodegradation of modified polyethylene films in soils led to significant changes (i.e., elongation at brake of 98%) in their mechanical properties that were caused by biochemical modifications of both polyester and polyethylene. Compared to waste coal soil, films underwent rapid biodegradation in soils that were rich in organic matter. Bacteria belonging to the genus, Bacillus, and the fungi, Gliocladium viride, Aspergillus awamori and Mortierella subtilissima, were easily able to colonise both polyethylene and polyethylene modified with Bionolle^®.     

Enhanced crude oil biodegradation in soil via biostimulation

Research on feasible methods for the enhancement of bioremediation in soil contaminated by crude oil is vital in oil-exporting countries such as Kuwait, where crude oil is a major pollutant and the environment is hostile to biodegradation. This study investigated the possibility of enhancing crude oil bioremediation by supplementing soil with cost-effective organic materials derived from two widespread locally grown trees, Conocarpus and Tamarix. Amendments in soils increased the counts of soil microbiota by up to 98% and enhanced their activity by up to 95.5%. The increase in the biodegradation of crude oil (75%) and high levels of alkB expression substantiated the efficiency of the proposed amendment technology for the bioremediation of hydrocarbon-contaminated sites. The identification of crude-oil-degrading bacteria revealed the dominance of the genus Microbacterium (39.6%), Sphingopyxis soli (19.3%), and Bordetella petrii (19.6%) in unamended, Conocarpus-amended, and Tamarix-amended contaminated soils, respectively. Although soil amendments favored the growth of Gram-negative bacteria and reduced bacterial diversity, the structures of bacterial communities were not significantly altered.     

Physical and Biological Treatment of Oil-Contaminated Soil in a Baffled Roller Bioreactor

Physical and biological removal of diesel oil from contaminated soil was studied in a baffled roller bioreactor. Initially, the effects of four factors (soil loading, temperature, pH, and surfactant) on physical removal of diesel oil were investigated. Only the presence of a surfactant (sodium dodecyl sulfate [SDS]) demonstrated a significant effect on diesel oil removal. Diesel oil removal efficiency was increased from 32.0% to 63.9% in the presence of 100 mg/L SDS. Using a microbial culture enriched from contaminated soil, biological treatment of diesel oil polluted soil under different soil loadings (15% to 50%), different diesel oil concentrations (1 to 50 g/L), and different types of soil (sand, silt, and clay) was then investigated in the baffled roller bioreactor. Biodegradation consisted of both fast and slow stages for degradation of light and heavy compounds, respectively. All biodegradation experiments demonstrated significant decreases in diesel oil concentrations (88.3% in 14 days for initial diesel oil concentrations of 1000 mg/L and a wide range of soil loadings). The presence of silty or sandy soils enhanced the biodegradation rate compared to the control bioreactor (without soil). The sandy soil loading had no effect on the biodegradation results. Using the enriched culture, the baffled roller bioreactor was able to biodegrade high diesel concentrations (up to 50 g/L) with biodegradation rates of 112.2 and 39.3 mg/L· h during fast and slow stages, respectively.     

Bioremediation of Crude Oil-Contaminated Soil Using Slurry-Phase Biological Treatment and Land Farming Techniques

Field-scale experiments on bioremediation of soil heavily contaminated with crude oil were undertaken on the territory of the Kokuyskoye oil field (Perm region, West Urals, Russia) owned by the LUKOIL Company. The pollution consisted of the contents of a oil waste storage pit, which mostly received soils contaminated after accidental oil spills and also the solid n-alkane (paraffin) wastes removed from the surface of drilling equipment. Laboratory analyses of soil samples indicated contamination levels up to 200?g/kg of total recoverable petroleum hydrocarbons (TRPH). Average oil composition consisted of 64% aliphatics, 25% aromatics, 8% heterocyclics, and 3% of tars/asphaltenes. Ex situ bioremediation techniques involved the successive treatment of contaminated soil using a bioslurry reactor and land farming cells. An oleophilic biofertilizer based on Rhodococcus surfactant complexes was used in both treatment systems. An aerobic slurry bioreactor was designed, and the biofertilizer applied weekly. Slurry-phase biotreatment of the contaminated soil resulted in an 88% reduction in oil concentration after 2 months. The resulting reactor product, containing approximately 25?g/kg of TRPH, was then loaded into land farming cells for further decontamination. To enhance bioremediation, different treatments (e.g., soil tilling, bulking with woodchips, watering, and biofertilizer addition) were used. The rates of oil biodegradation were 300 to 600?ppm/day. As a result, contamination levels dropped to 1.0 to 1.5?g/kg of TRPH after 5 to 7 weeks. Tertiary soil management involved phytoremediation where land farming cells were seeded with a mixture of three species of perennial grass. The effect of phytoremediation on the residual decontamination and rehabilitation of soil fertility is being evaluated.     

Characterisation of biodegradation capacities of environmental microflorae for diesel oil by comprehensive two-dimensional gas chromatography

In contaminated soils, efficiency of natural attenuation or engineered bioremediation largely depends on biodegradation capacities of the local microflorae. In the present study, the biodegradation capacities of various microflorae towards diesel oil were determined in laboratory conditions. Microflorae were collected from 9 contaminated and 10 uncontaminated soil samples and were compared to urban wastewater activated sludge. The recalcitrance of hydrocarbons in tests was characterised using both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GC×GC). The microflorae from contaminated soils were found to exhibit higher degradation capacities than those from uncontaminated soil and activated sludge. In cultures inoculated by contaminated-soil microflorae, 80% of diesel oil on an average was consumed over 4-week incubation compared to only 64% in uncontaminated soil and 60% in activated sludge cultures. As shown by GC, n-alkanes of diesel oil were totally utilised by each microflora but differentiated degradation extents were observed for cyclic and branched hydrocarbons. The enhanced degradation capacities of impacted-soil microflorae resulted probably from an adaptation to the hydrocarbon contaminants but a similar adaptation was noted in uncontaminated soils when conifer trees might have released natural hydrocarbons. GC×GC showed that a contaminated-soil microflora removed all aromatics and all branched alkanes containing less than C₁₅. The most recalcitrant compounds were the branched and cyclic alkanes with 15–23 atoms of carbon.     

Biodegradation of Dinoseb (2-sec-Butyl-4,6-Dinitrophenol) in Several Idaho Soils with Various Dinoseb Exposure Histories

We examined the ability of native microorganisms in various Idaho soils to degrade dinoseb and studied some physical and chemical soil characteristics which might affect the biodegradation process. Dinoseb biodegradation rates were higher in silt-loam soils than in loamy-sand soils. Biodegradation rates were not influenced by previous exposure of the soils to dinoseb. Bacterial numbers, measured by standard plate counts on soil extract agar, were the best predictors of biodegradation rates, accounting for 53% of the variability between soils. Soil nitrate-N inhibited dinoseb biodegradation and accounted for 39% of the variability. Sorption of dinoseb to soil surfaces also appeared to influence biodegradation rates. No other soil parameter contributed significantly to the variability in biodegradation rates. Persistence of dinoseb in one soil was due to inhibition of biodegradation by nitrate, while in another soil persistence appeared to be due to lack of native degradative microorganisms.     

Effects of Zeolite-Containing Material and Ammonium Nitrate on Biodegradation of n-Tridecane in Heavy Clay-Loam Leached Chernozem with High Organic Matter Content

Biostimulation based on usage of soil amendments is growing due to their efficiency in removing different petroleum hydrocarbons (PHC) from contaminated sand or loam-sand soils. However, the research on clay-rich soils with higher organic carbon content, in which PHC biodegradation may proceed differently and which are more difficult to clean up, has been less extensive. In a pot experiment, we studied and compared the effects of two soil amendments, natural zeolite-containing material (ZCM, 50 g kg^?1) as a bulking agent and ammonium nitrate (0.3 g N kg^?1) as a nitrogen fertilizer, on biodegradation of n-tridecane (1 wt.%) in a weakly acidic heavy clay loam leached chernozem with fairly high organic carbon content (3.71%). After 48 days, the nitrogen-amended contaminated soil showed enhancement of both respiratory activity (basal and substrate-induced respiration rates) and the number of n-tridecane- degraders. As a consequence, the extent of n-tridecane biodegradation (86.5%) was essentially higher in the presence of added nitrogen than that in the non-amended soil (73.7%). In contrast, due to the partial retention of n-tridecane molecules in its pores, ZCM retarded biodegradation to 56.0%, showed no significant effect on the number of n-tridecane-degraders and, moreover, enhanced the decomposition of the soil intrinsic organic matter. The obtained data indicate that more precautions should be considered when using porous sorbents such as ZCM for remedial arrangements in PHC-contaminated soils.     

Oil bioremediation in a high and a low phosphorus soil

Phosphorus (P) content may influence bioremediation of soils contaminated with crude oil. A soil testing high in plant available P (Weswood, 194 mg P kg^?1 soil) and one testing low in plant available P (Lufkin, 2 mg P kg^?1 soil) were selected for laboratory experiments on oil biodegradation. Plant available P content was determined using acidified ammonium acetate at pH 4.2 as the soil extractant. Soils were amended with 3, 6, and 9% crude oil by weight and incubated for 120 d at 25°C. Treatments consisted of a factorial arrangement, with soil, N, P, and oil concentration as factors. Addition of P without N generally did not enhance biodegradation. Addition of N without P approximately tripled the quantity of oil degraded. Addition of P and N together did not increase biodegradation of oil more than addition of N alone when oil concentration was 3%. At 6 and 9% oil concentrations, CO₂ evolution increased for both soils by adding P and N together in comparison to adding N alone, and total petroleum hydrocarbon (TPH) bio‐degradation increased by 30% for the Weswood soil by 60 d and at least 25% for the Lufkin soil by 30 d. The quantity of plant‐available P or total P in soil was not very useful in predicting need for supplemental P. Addition of P to soil to enhance oil degradation was only beneficial for oil concentrations above 3% and the positive effect for higher concentrations was transitory.     

Soil lipase activity – a useful indicator of oil biodegradation

The evaluation of soil lipase activity as a tool to monitor the decontamination of a freshly oil-polluted soil was tested in a laboratory study. An arable soil was experimentally contaminated with diesel oil at 5 mg hydrocarbons g^–1 soil dry weight and incubated with and without fertilization (N-P-K) for 116 days at 20°C. Lipase activity and counts of oil-degrading microorganisms were measured at regular time intervals, and the correlations with the levels of hydrocarbon concentrations in soil were investigated. The residual soil hydrocarbon concentration correlated significantly negatively with soil lipase activity and with the number of oil-degrading microorganisms, independent of fertilization. The induction of soil lipase activity is a valuable indicator of oil biodegradation in naturally attenuated (unfertilized) and bioremediated (fertilized) soils.     

改变施肥管理后不同肥力稻田土壤CO2排放特征

利用一个长达30a水稻土长期定位试验,在保证原有定位试验继续正常开展的前提下,将原化肥处理改施有机肥,原有机肥处理改施化肥或者增施有机肥。通过观测田间试验2012—2013年双季稻轮作周期内不同肥力水平稻田土壤施肥管理改变后的土体CO2排放通量(FCO2),研究不同后续施肥管理对不同肥力红壤性水稻土CO2排放的影响。结果表明:变更施肥能明显改变CO2排放动态变化,其中长期施用有机肥处理改施化肥后其FCO2明显减小,长期施用化肥或有机肥处理增施有机肥后其FCO2显著增大。有机肥和土壤有机碳均可促进土体CO2排放,有机肥处理有机物料碳添加量与CO2-C年排放量呈极显著的正相关关系(r=0.9015**,n=21),单施化肥处理土壤有机碳含量与土体CO2-C年排放量符合线性方程:y=10.962x-68.86(R2=0.7507,n=9,P0.01)。长期施用有机肥土壤改施化肥会导致其有机碳矿化损失,土壤有机碳含量越高,矿化损失量越多,最终其有机碳水平将与长期施用化肥的土壤有机碳平衡值一致;长期施用化肥或有机肥土壤改施或增施有机肥可促进土壤有机碳积累,外源添加碳越多,土壤积累碳越多;相同有机肥施用量下土壤有机碳含量越高,有机物料表观分解率越大,积累于土壤中的有机碳越少,不同有机碳水平土壤在相同有机肥管理下其有机碳最终会达到相同的平衡值。在有机碳水平较低(20.46 g/kg)红壤稻田上增施有机肥是提升已培肥水稻土有机碳含量的可持续发展措施,而在有机碳水平较高(14.45 g/kg)红壤稻田上应避免改施化肥。总之,在有机碳含量较高或者较低的中国南方红壤性水稻土上,持续的有机肥施用是保持或者提高其有机碳水平的必要措施。     

Biodegradation process and the nature of metabolism of metalaxyl in soil

The enhanced biodegradation of metalaxyl was studied in tobacco, citrus, avocado and corn soils. The most rapid degradation of metalaxyl occurred in a tobacco soil in which the half-life (50% degradation) of metalaxyl was 6 days. The main breakdown product of metalaxyl in all soils was the acid metabolite. Ring labelled [¹⁴C]metalaxyl incubated for 4 wk in 6 soils demonstrated a low rate of ¹⁴CO₂ evolution ranging from 2.1% to 11.3% which was unrelated to the biodegradation properties of the soil. A relationship between the concentration of metalaxyl and the subsequent rate of biodegradation was found in the tobacco soils. Higher concentrations of metalaxyl resulted in faster biodegradation rates. A single exposure of tobacco and corn soils to metalaxyl (100 μg/ml or 200 μg/g dry weight of soil) significantly increased their subsequent capacity to degrade the fungicide. Addition of the fungicide thiram or the antibiotics streptomycin and chloramphenicol to an avocado soil resulted in 75% and 51% inhibition of metalaxyl degradation, respectively. A combination of the fungicide and antibiotics resulted in 89% inhibition. The results indicate that enhanced microbial degradation of metalaxyl can occur in a wide range of soils. Under experimental conditions using soil solutions or soil systems, a single application of the fungicide may trigger this event. A wide range of fungi and bacteria appear to take part in degrading metalaxyl.     

Utility of lipid biomarkers in support of bioremediation efforts at army sites

Lipid biomarker analysis has proven valuable in testing the hypothesis that attributes of the extant microbiota can directly reflect the occurrence of contaminant biodegradation. Two past research efforts have demonstrated this utility and are described here. A 4.5 m vertical core was obtained from a diesel fuel oil contamination plume. Core material was assayed for total petroleum hydrocarbons (TPH) and bacterial membrane phospholipids (PLFA) via a single solvent extraction. Microbial viable biomass and the relative abundance of Gram-negative bacterial PLFA biomarkers were found to be significantly correlated with TPH concentration. The core TPH profile also revealed two distinct areas where the average TPH level of 3,000 microg g(-1) fell to near detection limits. Both areas were characterized by a three-fold decrease in the hexadecane/pristane ratio, indicating alkane biodegradation, and a distinct PLFA profile that showed a close similarity to the uncontaminated surface soil. Low-order, incomplete detonations can deposit hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) into training range surface soils. Since surface soils are exposed to temporal and diurnal moisture cycles, we investigated the effect two very different soil moisture tensions had on the in situ microbiota and RDX biodegradation. Saturated soils were characterized by rapid RDX biodegradation, 4 day half-life, a decrease in number of species detected and increase in PLFA biomarkers for Gram-negative proteobacteria (n16:1omega7c, n18:1omega9c, and n18:1omega7c) and Gram-positive firmicutes (i15:0 and a15:0). Terminal restriction fragment length polymorphism (T-RFLP) profiles of endpoint microbial communities indicated a shift from 18 to 36% firmicutes, the loss of gamma-proteobacteria and the emergence of alpha-proteobacteria. These two past research efforts demonstrated the utility of the lipid biomarker analysis in identifying microbial community characteristics that were associated with two very different soil contaminants. Lipid biomarkers defined areas of TPH biodegradation and identified community shifts as a result of soil conditions that affected explosives fate. Information like this can be used to enhance the predictive power of ecological models such as the Army Training and Testing Area Carrying Capacity for munitions model [ATTACC].     

Comparison of bioremediation strategies for soil impacted with petrochemical oily sludge

Different bioremediation techniques (natural attenuation, biostimulation and bioaugmentation) in contaminated soils with two oily sludge concentrations (1.5% and 6.0%) in open and closed microcosms systems were assessed during 90 days. The results showed that the highest biodegradation rates were obtained in contaminated soils with 6% in closed microcosms. Addition of microbial consortium and nutrients in different concentrations demonstrated higher biodegradation rate of total petroleum hydrocarbons (TPH) than those of the natural attenuation treatment. Soils treated in closed microcosms showed highest removal rate (84.1 ± 0.9%) when contaminated at 6% and bacterial consortium and nutrients in low amounts were added. In open microcosms, the soil contaminated at 6% using biostimulation with the highest amounts of nutrients (C:N:P of 100:10:1) presented the highest degradation rate (78.7 ± 1.3%). These results demonstrate that the application of microbial consortium and nutrients favored biodegradation of TPH present in oily sludge, indicating their potential applications for treatment of the soils impacted with this important hazardous waste.     

Evaluation of the performance of two commercial starters on the bioremediation of soils contaminated with mineral oil and HCH (Hexachlorocyclohexane)

Summary The effect of the addition of two commercial bacterial starters on mineral oil degradation in two soils was studied: CS-O soil (10% mineral oil) and CS-OH soil (10% mineral oil + 45 mg/Kg HCH). Respiratory activity as well as degradation was determined. The two starters enhanced biodegradation in both soils without any significant difference due to HCH (Hexachlorocyclohexane) presence in CS-OH soil.     

Long-term fertilization regimes change soil nitrification potential by impacting active autotrophic ammonia oxidizers and nitrite oxidizers as assessed by DNA stable isotope probing

Chemoautotrophic ammonia-oxidizers and nitrite-oxidizers are responsible for a significant amount of soil nitrate production. The identity and composition of these active nitrifiers in soils under different long-term fertilization regimes remain largely under-investigated. Based on that soil nitrification potential significantly decreased in soils with chemical fertilization (CF) and increased in soils with organic fertilization (OF), a microcosm experiment with DNA stable isotope probing was further conducted to clarify the active nitrifiers. Both ammonia-oxidizing archaea (AOA) and bacteria (AOB) were found to actively respond to urea addition in soils with OF and no fertilizer (CK), whereas only AOB were detected in soils with CF. Around 98% of active AOB were Nitrosospira cluster 3a.1 in all tested soils, and more than 90% of active AOA were Nitrososphaera subcluster 1.1 in unfertilized and organically fertilized soils. Nitrite oxidation was performed only by Nitrospira-like bacteria in all soils. The relative abundances of Nitrospira lineage I and VI were 32% and 61%, respectively, in unfertilized soils, and that of Nitrospira lineage II was 97% in fertilized soils, indicating long-term fertilization shifted the composition of active Nitrospira-like bacteria in response to urea. This finding indicates that different fertilizer regimes impact the composition of active nitrifiers, thus, impacting soil nitrification potential.     

Biodegradation of a dilute waste oil emulsion applied to soil

Summary The use of land treatment for disposal of a dilute waste oil emulsion generated by an aluminum rolling industry was investigated. Major components of the waste, identified by gas chromatography and mass spectrometry, were linear and branched (C₁₂–C₂₅) and fatty acid emulsifiers (primarily, isomers of oleic acid). Hexadecane and pristane were readily biodegraded in vitro when added to soil collected from the waste disposal site. Hydrocarbons and fatty acids extracted from the waste were similarly, biodegraded, however, the rate of decomposition may have depended on the history of waste applications to soil collected from the land treatment site. The apparent half-life of resolvable waste hydrocarbons and fatty acids was 9.5 days in soil which had received waste applications averaging 25.4l m^–2 wk^–1. In contrast, soil receiving either 50.8l m^–2 wk^–1 or no waste application during summer 1987 apparent exhibited half-lives of 28.1 and 60.3 days, respectively. Waste components were restricted to the upper 48 cm of the soil cores collected from the disposal site. Core samples also provided evidence for biodegradation of hydrocarbons and fatty acids as well as an accumulation of other compounds not readily resolvable by gas chromatographyPublished with the approval of the Director of the West Virginia University Agriculture and Forestry Experiment Station as Scientific Article # 2122.     

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.