Effect of environmental parameters on the biodegradation of oil sludge.

A laboratory study was conducted with the aim of evaluating and optimizing the environmental parameters of "landfarming", i.e., the disposal by biodegradation in soil of oily sludges generated in the refining of crude oil and related operations. Oil sludge biodegradation was monitored by CO2 evolution and by periodic analysis of residual hydrocarbons. The parameters studied were soil moisture, pH, mineral nutrients, micronutrients, organic supplements, treatment rate, teratment frequency, and incubation temperature. Oil sludge biodegradation was optimal at a soil water-holding capacity of 30 to 90%, a pH of 7.5 to 7.8, C:N and C:P ratios of 60:1 and 800:1, respectively, and a temperature of 20 degrees C or above. Addition of micronutrients and organic supplements was not beneficial; sewage sludge interfered with hydrocarbon biodegradation. Breakdown of the saturated hydrocarbon (alkane and cycloalkane) fraction was the highest at low application rates, but higher application rates favored the biodegradation of the aromatic and asphaltic fractions. An application rate of 5% (wt/wt) oil sludge hydrocarbon to the soil (100,000 liters/hectare) achieved a good compromise between high biodegradation rates and efficient land use and resulted in the best overall biodegradation rate of all hydrocarbon classes. Frequent small applications resulted in higher biodegradation than single large applications. Two 100,000-liter/hectare (255 barrels per acre) or four 50,000-liter/hectare oil sludge hydrocarbon applications per growing season seem appropriate for most temperate zone disposal sites.     

Effects of mineral nutrients,sludge application rate,and application frequency on biodegradation of two oily sludges

A continuous flow soil respirometer was used to evaluate the effect of nutrient addition, application rate, and application frequency on biodegradation of 2 complex oily sludges in soil. The most rapid biodegradation of the refinery sludge occurred when nitrogen was added to reduce the carbon to nitrogen (C∶N) ratio to 9∶1. The petrochemical sludge was degraded most rapidly when nitrogen, phosphorus, and potassium were added at a rate of 124∶1, C∶NPK; CO₂evolution from both wastes increased with increasing application rates, but the fraction of applied sludge which degraded decreased with increasing application rates. Small frequent applications resulted in a slight increase in respiration rate per unit applied over a single equivalent application, indicating that repeated applications of smaller amounts of sludge result in a more rapid rate of decomposition. The population of total soil bacteria was greatest when 1% of either sludge was added to the soil, whereas 5 and 10% sludge additions resulted in slightly lower microbial populations.     

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.     

Development and Evaluation of an Online CO2 Evolution Test and a Multicomponent Biodegradation Test System

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO₂) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO₂ method with existing CO₂ evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO₂ evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO₂ and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.     

Estimation of Biodegradation Potential of Xenobiotic Organic Chemicals

A method is described to estimate the biodegradation potential of soluble, insoluble, and unknown organic chemicals. The method consists of two stages: (i) generation of a microbial inoculum in a bench scale semicontinuous activated sludge system during which microorganisms are acclimated to test material and the removal of dissolved organic carbon is monitored and (ii) biodegradability testing (CO₂ evolution) in a defined minimal medium containing the test material as the sole carbon and energy source and a dilute bacterial inoculum obtained from the supernatant of homogenized activated sludge generated in the semicontinuous activated sludge system. Removal and biodegradation are measured using nonspecific methods, at initial concentrations of 5 to 10 mg of dissolved organic carbon per liter. Biodegradability data are accurately described by a nonlinear computer model which allows the rate and extent of biodegradation for different compounds to be compared and statistically examined. The evaluation of data generated in the combined removability-biodegradability system allows the biodegradation potential of a variety of xenobiotic organic chemicals to be estimated.     

Microcosm assays and Taguchi experimental design for treatment of oil sludge containing high concentration of hydrocarbons

Microcosm assays and Taguchi experimental design was used to assess the biodegradation of an oil sludge produced by a gas processing unit. The study showed that the biodegradation of the sludge sample is feasible despite the high level of pollutants and complexity involved in the sludge. The physicochemical and microbiological characterization of the sludge revealed a high concentration of hydrocarbons (334,766 ± 7001 mg kg⁻¹ dry matter, d.m.) containing a variety of compounds between 6 and 73 carbon atoms in their structure, whereas the concentration of Fe was 60,000 mg kg⁻¹ d.m. and 26,800 mg kg⁻¹ d.m. of sulfide. A Taguchi L₉ experimental design comprising 4 variables and 3 levels moisture, nitrogen source, surfactant concentration and oxidant agent was performed, proving that moisture and nitrogen source are the major variables that affect CO₂ production and total petroleum hydrocarbons (TPH) degradation. The best experimental treatment yielded a TPH removal of 56,092 mg kg⁻¹ d.m. The treatment was carried out under the following conditions: 70% moisture, no oxidant agent, 0.5% of surfactant and NH₄Cl as nitrogen source.     

Response of Microorganisms to an Accidental Gasoline Spillage in an Arctic Freshwater Ecosystem

The response of microorganisms to an accidental spillage of 55,000 gallons of leaded gasoline into an Arctic freshwater lake was studied. Shifts in microbial populations were detected after the spillage, reflecting the migration pattern of the gasoline, enrichment for hydrocarbon utilizers, and selection for leaded-gasoline-tolerant microorganisms. Ratios of gasoline-tolerant/utilizing heterotrophs to “total” heterotrophs were found to be a sensitive indicator of the degree of hydrocarbon contamination. Respiration rates were elevated in the highly contaminated area, but did not reflect differences between moderately and lightly contaminated areas. Hydrocarbon biodegradation potential experiments showed that indigenous microorganisms could extensively convert hydrocarbons to CO₂. In situ measurement of gasoline degradation showed that, if untreated, sediment samples retained significant amounts of gasoline hydrocarbons including “volatile components” at the time the lake froze for the winter. Nutrient addition and bacterial inoculation resulted in enhanced biodegradative losses, significantly reducing the amount of residual hydrocarbons. Enhanced biodegradation, however, resulted in the appearance of compounds not detected in the gasoline. Since the contaminated lake serves as a drinking water supply, treatment to enhance microbial removal of much of the remaining gasoline still may be advisable.     

Effect of concentration and environmental form of tetradecenyl succinic acid on its mineralization in soil

Tetradecenyl succinic acid (TSA) is the major component of a detergent builder (C12-C14 alkenyl succinic acid), which is inherently biodegradable. ¹⁴C-TSA was dosed as a component of sewage sludge into a soil with a history of sludge amendment at final added concentrations of 1.5 and 30 mg (kg soil)^-1. In addition, it was dosed to the soil in an aqueous solution to a final added concentration of 30 mg (kg soil)^-1. Dose and form were found to have a pronouced effect on the mineralization kinetics. When dosed in a realistic form and concentration (i.e. 1.5 mg (kg soil)^-1 as a component of sludge), TSA was mineralized at its highest rate and to its greatest extent, and the mineralization half-life was 2.4 days. When dosed at 30 mg (kg soil)^-1 as a component of sludge, mineralization began immediately, and the half-life was 23 days. In contrast, when dosed at this concentration in aqueous solution, the onset of mineralization was preceded by a 13 day lag period and the mineralization half-life was 69 days. Primary biodegradation and mineralization rates of TSA were very similar. Approximately, half the radioactivity was evolved as ¹⁴CO₂, while the remaining radioactivity became non-extractable, having presumably been incorporated into biomass or natural soil organic matter (humics). This study demonstrated that TSA is effectively removed from sludge-amended soils as a result of biodegradation. Furthermore, it showed the effect that dose form and concentration have on the biodegradation kinetics and the importance of dosing a chemical not only at a relevant concentration but also in the environmental form in which it enters the soil environment.     

Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of 14CO2

Most heterotrophic bacteria assimilate CO₂ in various carboxylation reactions during biosynthesis. In this study, assimilation of ¹⁴CO₂ by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient ¹⁴CO₂ during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of ¹⁴C-labeled organic substrates. Experiments with E. coli showed that ¹⁴CO₂ was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO₂-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “Candidatus Microthrix parvicella” in activated sludge. “Ca. Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [¹⁴C]oleic acid. However, the new HetCO₂-MAR approach indicated that “Ca. Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO₂⁻, whereas the addition of O₂ or NO₃⁻ initiated growth, as indicated by detectable ¹⁴CO₂ assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO₂-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO₂-MAR results were supported by stable isotope analysis of ¹³C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of ¹³CO₂. In conclusion, the novel HetCO₂-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

Methods for Assessment of Biodegradability of Plastic Films in Soil

Traditional and novel techniques were tested and compared for their usefulness in evaluating biodegrad-ability claims made for newly formulated “degradable” plastic film products. Photosensitized polyethylene (PE), starch-PE, extensively plasticized polyvinyl chloride (PVC), and polypropylene (PP) films were incorporated into aerobic soil. Biodegradation was measured for 3 months under generally favorable conditions. Carbon dioxide evolution, residual weight recovery, and loss of tensile strength measurements were supplemented, for some films, by gas chromatographic measurements of plasticizer loss and gel permeation chromatographic (GPC) measurement of polymer molecular size distribution. Six- and 12-week sunlight exposures of photosensitized PE films resulted in extensive photochemical damage that failed to promote subsequent mineralization in soil. An 8% starch-PE film and the plasticized PVC film evolved significant amounts of CO₂ in biodegradation tests and lost residual weight and tensile strength, but GPC measurements demonstrated that all these changes were confined to the additives and the PE and PVC polymers were not degraded. Carbon dioxide evolution was found to be a useful screening tool for plastic film biodegradation, but for films with additives, polymer biodegradation needs to be confirmed by GPC. Photochemical cross-linking of polymer strands reduces solubility and may interfere with GPC measurements of polymer degradation.     

Microbe-aliphatic hydrocarbon interactions in soil: implications for biodegradation and bioremediation

Aliphatic hydrocarbons make up a substantial portion of organic contamination in the terrestrial environment. However, most studies have focussed on the fate and behaviour of aromatic contaminants in soil. Despite structural differences between aromatic and aliphatic hydrocarbons, both classes of contaminants are subject to physicochemical processes, which can affect the degree of loss, sequestration and interaction with soil microflora. Given the nature of hydrocarbon contamination of soils and the importance of bioremediation strategies, understanding the fate and behaviour of aliphatic hydrocarbons is imperative, particularly microbe-contaminant interactions. Biodegradation by microbes is the key removal process of hydrocarbons in soils, which is controlled by hydrocarbon physicochemistry, environmental conditions, bioavailability and the presence of catabolically active microbes. Therefore, the aims of this review are (i) to consider the physicochemical properties of aliphatic hydrocarbons and highlight mechanisms controlling their fate and behaviour in soil; (ii) to discuss the bioavailability and bioaccessibility of aliphatic hydrocarbons in soil, with particular attention being paid to biodegradation, and (iii) to briefly consider bioremediation techniques that may be applied to remove aliphatic hydrocarbons from soil.     

Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil

The main objective of this study was to evaluate the effect of oily sludge concentration on its biodegradability in soil. Oily sludge was collected and applied to microcosms at full-, half-, or quarter-strength concentrations equivalent to 44.2, 22.2, and 11.1 g kg^?1 soil, respectively, of total petroleum hydrocarbons (TPH) contained in oily sludge. The biodegradability of oily sludge was evaluated by measuring CO₂ evolution and by measuring removal of TPH as well as its main composing fractions; namely; alkanes, aromatics, NSO-compounds, and asphaltenes. The collected soil contained 3.63 × 10⁶ cfu g^?1 soil of hydrocarbon-degrading bacteria, which is satisfactory to drive successful biodegradation of hydrocarbons in soil. These numbers increased significantly with oily sludge addition at a rate proportional to the added TPH reaching 3.35 × 10⁷ cfu g^?1 soil in the half-strength treatment. TPH mineralization rate followed the same pattern. However, TPH-mineralization efficiency was the greatest in quarter-strength treatment at 18.3%. TPH-removal efficiency was also highest in quarter-strength treatment at 30.9%. Nutrients addition caused mineralization inhibition. Since nutrients were added as a ratio of the added carbon, inhibition was the greatest with the highest TPH treatment. While alkanes were degraded, aromatics and asphaltenes were not, and NSO-compounds were enriched. Although SDS was completely biodegradable in soil, its addition promoted mineralization and removal of TPH from soil.     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Endogeic earthworms shape bacterial functional communities and affect organic matter mineralization in a tropical soil

Priming effect (PE) is defined as a stimulation of the mineralization of soil organic matter (SOM) following a supply of fresh organic matter. This process can have important consequences on the fate of SOM and on the management of residues in agricultural soils, especially in tropical regions where soil fertility is essentially based on the management of organic matter. Earthworms are ecosystem engineers known to affect the dynamics of SOM. Endogeic earthworms ingest large amounts of soil and assimilate a part of organic matter it contains. During gut transit, microorganisms are transported to new substrates and their activity is stimulated by (i) the production of readily assimilable organic matter (mucus) and (ii) the possible presence of fresh organic residues in the ingested soil. The objective of our study was to see (i) whether earthworms impact the PE intensity when a fresh residue is added to a tropical soil and (ii) whether this impact is linked to a stimulation/inhibition of bacterial taxa, and which taxa are affected. A tropical soil from Madagascar was incubated in the laboratory, with a ¹³C wheat straw residue, in the presence or absence of a peregrine endogeic tropical earthworm, Pontoscolex corethrurus. Emissions of ¹²CO₂ and ¹³CO₂ were followed during 16 days. The coupling between DNA-SIP (stable isotope probing) and pyrosequencing showed that stimulation of both the mineralization of wheat residues and the PE can be linked to the stimulation of several groups especially belonging to the Bacteroidetes phylum.     

Biodegradation, sorption, and transport of 2,4-dichlorophenoxyacetic acid in saturated and unsaturated soils.

The fate of an organic contaminant in soil depends on many factors, including sorption, biodegradation, and transport. The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was used as a model compound to illustrate the impact of these interacting factors on the fate of an organic contaminant. Batch and column experiments performed with a sandy loam soil mixture under saturated and unsaturated conditions were used to determine the effects of sorption and biodegradation on the fate and transport of 2,4-D. Sorption of 2,4-D was found to have a slight but significant effect on transport of 2,4-D under saturated conditions (retardation factor, 1.8) and unsaturated conditions (retardation factor, 3.4). Biodegradation of 2,4-D was extensive under both batch and column conditions and was found to have a significant impact on 2,4-D transport in column experiments. In batch experiments, complete mineralization of 2,4-D (100 mg kg-1) occurred over a 4-day period following a 3-day lag phase under both saturated and unsaturated conditions. The biodegradation rate parameters calculated for batch experiments were found to be significantly different from those estimated for column experiments.     

Ratio [13C]/[12C] as an index for express estimation of hydrocarbon-oxidizing potential of microbiota in soil polluted with crude oil

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO₂ production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO₂. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.     

Survey of the Anaerobic Biodegradation Potential of Organic Chemicals in Digesting Sludge

The degradation potential of 77 organic chemicals under methanogenic conditions was examined with an anaerobic digesting sludge from the United Kingdom. Degradation was assessed in terms of net total gas (CH₄ plus CO₂) produced, expressed as a percentage of the theoretical production (ThGP). The compounds tested were selected from various chemical groups and included substituted phenols and benzoates, pesticides, phthalic acid esters, homocyclic and heterocyclic ring compounds, glycols, and monosubstituted benzenes. The results obtained were in good agreement with published surveys of biodegradability in U.S. digesting sludges and other methanogenic environments. In general, the presence of chloro or nitro groups inhibited anaerobic gas production, while carboxyl and hydroxyl groups facilitated biodegradation. The relationship between substituent position and susceptibility to methanogenic degradation was compound dependent. The following chemicals were completely degraded (≥80% ThGP) at a concentration of 50 mg of carbon per liter: phenol, 2-aminophenol, 4-cresol, catechol, sodium benzoate, 4-aminobenzoic acid, 3-chlorobenzoic acid, phthalic acid, ethylene glycol, diethylene glycol, triethylene glycol, sodium stearate, and quinoline. 3-Cresol, 4-chlorobenzoic acid, dimethyl phthalate, and pyridine were partially degraded. Although the remaining chemicals tested were either persistent or toxic, their behavior may differ at more environmentally realistic chemical-to-biomass ratios. Our findings suggest that biodegradability assessments made with sludge from one source can be extrapolated to sludge from another source with a reasonable degree of confidence and should help in predicting the fate of an organic chemical during the anaerobic digestion of sewage sludge.     

The response of a natural phytoplankton community from the Godavari River Estuary to increasing CO2 concentration during the pre-monsoon period

This paper reports for the first time upon the effects of increasing CO₂ concentrations on a natural phytoplankton assemblage in a tropical estuary (the Godavari River Estuary in India). Two short-term (5-day) bottle experiments were conducted (with and without nutrient addition) during the pre-monsoon season when the partial pressure of CO₂ in the surface water is quite low. The results reveal that the concentrations of total chlorophyll, the phytoplankton growth rate, the concentrations of particulate organic matter, the photosynthetic oxygen evolution rates, and the total bacterial count were higher under elevated CO₂ treatments, as compared to ambient conditions (control). δ¹³C of particulate organic matter (POM) varied inversely with respect to CO₂, indicating a clear signature of higher CO₂ influx under the elevated CO₂ levels. Whereas, δ¹³C_POM in the controls indicated the existence of an active bicarbonate transport system under limited CO₂ supply. A considerable change in phytoplankton community structure was noticed, with marker pigment analysis by HPLC revealing that cyanobacteria were dominant over diatoms as CO₂ concentrations increased. A mass balance calculation indicated that insufficient nutrients (N, P and Si) might have inhibited diatom growth compared to cyanobacteria, regardless of increased CO₂ supply. The present study suggests that CO₂ concentration and nutrient supply could have significant effects on phytoplankton physiology and community composition for natural phytoplankton communities in this region. However, this work was conducted during a non-discharge period (nutrient-limited conditions) and the responses of phytoplankton to increasing CO₂ might not necessarily be the same during other seasons with high physicochemical variability. Further investigation is therefore needed.     

Inversely Estimating the Vertical Profile of the Soil CO2 Production Rate in a Deciduous Broadleaf Forest Using a Particle Filtering Method

Carbon dioxide (CO₂) efflux from the soil surface, which is a major source of CO₂ from terrestrial ecosystems, represents the total CO₂ production at all soil depths. Although many studies have estimated the vertical profile of the CO₂ production rate, one of the difficulties in estimating the vertical profile is measuring diffusion coefficients of CO₂ at all soil depths in a nondestructive manner. In this study, we estimated the temporal variation in the vertical profile of the CO₂ production rate using a data assimilation method, the particle filtering method, in which the diffusion coefficients of CO₂ were simultaneously estimated. The CO₂ concentrations at several soil depths and CO₂ efflux from the soil surface (only during the snow-free period) were measured at two points in a broadleaf forest in Japan, and the data were assimilated into a simple model including a diffusion equation. We found that there were large variations in the pattern of the vertical profile of the CO₂ production rate between experiment sites: the peak CO₂ production rate was at soil depths around 10 cm during the snow-free period at one site, but the peak was at the soil surface at the other site. Using this method to estimate the CO₂ production rate during snow-cover periods allowed us to estimate CO₂ efflux during that period as well. We estimated that the CO₂ efflux during the snow-cover period (about half the year) accounted for around 13% of the annual CO₂ efflux at this site. Although the method proposed in this study does not ensure the validity of the estimated diffusion coefficients and CO₂ production rates, the method enables us to more closely approach the “actual” values by decreasing the variance of the posterior distribution of the values.     

Biodegradation of 1,2,3- and 1,2,4-Trichlorobenzene in Soil and in Liquid Enrichment Culture

The biodegradation of radiochemically pure (99%) 1,2,3- and 1,2,4-trichlorobenzene (TCB) in soil was investigated. Experimental difficulties posed by the high volatility and slow biodegradation rate of the TCBs were partially overcome by using a specially designed incubation and trapping apparatus. Evolution of ¹⁴CO₂ from active versus poisoned soil dosed with 50 μg of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20°C, 1,2,4-TCB was mineralized at an approximate rate of 1 nmol/day per 20 g of soil sample, and 1,2,3-TCB was mineralized at one-half to one-third that rate. Mineral fertilizers or cosubstrates failed to increase TCB mineralization rates in soil. Anaerobic conditions had a negative effect on mineralization, and increased temperatures had a positive effect. With increasing 1,2,4-TCB concentrations, ¹⁴CO₂ evolution exhibited saturation kinetics with an apparent K_m of 55.5 nmol per g of soil. Recovery of total radioactivity was good from soil containing high organic matter concentrations. From low-organic-matter soil, some of the radioactivity was recovered only on combustion, and overall recovery was lower. In soil-inoculated liquid culture, the cosubstrates glucose and benzene caused a slight stimulation of 1,2,4-TCB mineralization. Cochromatography of known standards with the extracts of soil pretreated with [¹⁴C]TCBs indicated that 3,4,5-trichlorophenol, 2,6-dichlorophenol and, to a lesser degree, 2,3-dichlorophenol were present in soils incubated with 1,2,3-TCB. 2,4-, 2,5-, and 3,4-dichlorophenol were present in soils incubated with 1,2,4-TCB.