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 (14)CO(2) from active versus poisoned soil dosed with 50 mug of the individual TCBs per g gave conclusive proof that both isomers are biodegradable. At 20 degrees 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, (14)CO(2) 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 [(14)C]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.     

Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene.

Hexachlorobenzene (HCB), pentachlorobenzene (QCB), all three isomers of tetrachlorobenzene (TeCB), 1,2,3-trichlorobenzene (1,2,3-TCB), and 1,2,4-TCB were reductively dechlorinated by enrichment cultures in the presence of lactate, glucose, ethanol, or isopropanol as the electron donor. The enrichment cultures originated from percolation columns filled with Rhine River sediment in which dechlorination of TCBs and dichlorobenzenes (DCBs) occurred. A stable consortium obtained by transfer on lactate as the energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-DCB. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best substrates. Optimal temperature and pH for dechlorination were 30 degrees C and 7.2, respectively. The specificity of the enrichment on lactate and 1,2,3-TCB was tested after approximately 60 transfers (after 2.5 years). HCB and QCB were stoichiometrically dechlorinated to 1,3,5-TCB and minor amounts of 1,2,4-TCB. 1,3,5-TCB was the sole product formed from 1,2,3,5-TeCB, while 1,2,3,4-TeCB and 1,2,4,5-TeCB were converted to 1,2,4-TCB. 1,2,4-TCB, 1,3,5-TCB, and the three isomers of DCB were not dechlorinated during 4 weeks of incubation. For further enrichment of the 1,2,3-TCB-dechlorinating bacteria, a two-liquid-phase (hexadecane-water) system was used with hydrogen as the electron donor and 1,2,3-TCB or CO2 as the electron acceptor. Methanogens and acetogens were the major substrate-competing (H2-CO2) microorganisms in the two-liquid-phase system. Inhibition of methanogenesis by 2-bromoethanesulfonic acid did not influence dechlorination, and acetogens which were isolated from the enrichment culture did not have dechlorinating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment and properties of an anaerobic mixed culture reductively dechlorinating 1,2,3-trichlorobenzene to 1,3-dichlorobenzene.

Hexachlorobenzene (HCB), pentachlorobenzene (QCB), all three isomers of tetrachlorobenzene (TeCB), 1,2,3-trichlorobenzene (1,2,3-TCB), and 1,2,4-TCB were reductively dechlorinated by enrichment cultures in the presence of lactate, glucose, ethanol, or isopropanol as the electron donor. The enrichment cultures originated from percolation columns filled with Rhine River sediment in which dechlorination of TCBs and dichlorobenzenes (DCBs) occurred. A stable consortium obtained by transfer on lactate as the energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-DCB. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best substrates. Optimal temperature and pH for dechlorination were 30 degrees C and 7.2, respectively. The specificity of the enrichment on lactate and 1,2,3-TCB was tested after approximately 60 transfers (after 2.5 years). HCB and QCB were stoichiometrically dechlorinated to 1,3,5-TCB and minor amounts of 1,2,4-TCB. 1,3,5-TCB was the sole product formed from 1,2,3,5-TeCB, while 1,2,3,4-TeCB and 1,2,4,5-TeCB were converted to 1,2,4-TCB. 1,2,4-TCB, 1,3,5-TCB, and the three isomers of DCB were not dechlorinated during 4 weeks of incubation. For further enrichment of the 1,2,3-TCB-dechlorinating bacteria, a two-liquid-phase (hexadecane-water) system was used with hydrogen as the electron donor and 1,2,3-TCB or CO2 as the electron acceptor. Methanogens and acetogens were the major substrate-competing (H2-CO2) microorganisms in the two-liquid-phase system. Inhibition of methanogenesis by 2-bromoethanesulfonic acid did not influence dechlorination, and acetogens which were isolated from the enrichment culture did not have dechlorinating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Multiphasic Kinetics of Transformation of 1,2,4-Trichlorobenzene at Nano- and Micromolar Concentrations by Burkholderia sp. Strain PS14

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.5 nM. At low initial 1,2,4-TCB concentrations, a first-order relationship between specific transformation rate and substrate concentration was observed with a specific affinity (a⁰_A) of 0.32 liter · mg (dry weight)⁻¹ · h⁻¹ followed by a second one at higher concentrations with an a^o_A of 0.77 liter · mg (dry weight)⁻¹ · h⁻¹. This transition from the first-order kinetics at low initial 1,2,4-TCB concentrations to the second first-order kinetics at higher 1,2,4-TCB concentrations was shifted towards higher initial 1,2,4-TCB concentrations with increasing cell mass. At high initial concentrations of 1,2,4-TCB, a maximal transformation rate of approximately 37 nmol · min⁻¹ · mg (dry weight)⁻¹ was measured, irrespective of the cell concentration.     

Bacterial growth based on reductive dechlorination of trichlorobenzenes

An anaerobic mixed bacterial culture was enriched forbacteria dechlorinating 1,2,3- and1,2,4-trichlorobenzene (TCB) to dichlorobenzenes byexclusive use of non-fermentable substrates and theapplication of vancomycin. Growth and dechlorinationoccurred in a purely synthetic medium with formate orhydrogen, acetate, and TCB. Neither acetogenesis normethanogenesis was detected in the culture. Repeatedsubculturing maintaining high dechlorinatingactivities was also achieved when only hydrogen andTCB were supplied. This indicated that reductivedechlorination of TCB was the primary energyconservating process. The number of dechlorinatingbacteria was strictly limited by the amount of TCBsupplied in the medium. In addition, thedechlorinating activity could be maintained only inthe presence of TCB. A most probable number analysisshowed that the dechlorinating species amounted to atleast 6 10⁵ cells per ml at a total cell numberof about 2 10⁶ cells per ml. Vitamin B₁₂significantly stimulated the dechlorinating activity.     

Biodegradation of N-Nitrosodimethylamine in Aqueous and Soil Systems

N-Nitrosodimethylamine (NDMA) was mineralized by microorganisms in aqueous and soil systems. Initial rates of mineralization (micrograms per milliliter per day) were calculated for a wide range of initial concentrations of NDMA (micrograms per milliliter to picograms per milliliter). Log-log plots of the data were fitted with both linear and nonlinear least-squares analyses; however, linear models provided better fits for the kinetic data in all cases. The slopes of the linear fits were not significantly different than 1.0 (P < 0.05); thus, first-order reaction kinetics were in effect over the range of concentrations tested, and saturation kinetics were not achieved. Rate constants (day⁻¹) and total percent mineralized increased with decreasing initial concentrations of NDMA. Rates of mineralization were reduced in aqueous systems when supplemental carbon was available, whereas in soils, percentages of organic matter and supplemental carbon had little effect on rates of mineralization. Implications of these results for predictions of rates and threshold limits of mineralization activity in natural systems are discussed. A laboratory scale simulated trickling filter containing an activated charcoal bed provided a suitable environment for mineralization of NDMA at concentrations of 50 and 100 μg/ml on a continuous basis. NDMA was not toxic to natural populations of microorganisms at concentrations up to 10 mg/ml. Using high-pressure liquid chromatography coupled with radioactivity detection, we identified formaldehyde and methylamine as intermediates produced during the biodegradation of NDMA.     

Reductive dehalogenation of chlorinated benzenes and toluenes under methanogenic conditions.

The anaerobic metabolism of chlorinated benzenes and toluenes was evaluated in soil slurry microcosms under methanogenic conditions. A mixture of hexachlorobenzene, pentachlorobenzene, and 1,2,4-trichlorobenzene (TCB) in soil slurries was biotransformed through sequential reductive dechlorination to chlorobenzene (CB). The metabolic pathway for hexachlorobenzene and pentachlorobenzene decay proceeded via 1,2,3,4-tetrachlorobenzene (TTCB)-->1,2,3-TCB + 1,2,4-TCB-->1,2-dichlorobenzene (DCB) + 1,4-DCB-->CB. In a mineral salts medium, the CB-adapted soil microorganisms dehalogenated individual 1,2,4,5-TTCB, 1,2,3,4-TTCB, 1,2,3-TCB, and 1,2,4-TCB but not 1,2,3,5-TTCB or 1,3,5-TCB. Similarly, a mixture of 2,3,6-trichlorotoluene (TCT), 2,5-dichlorotoluene (DCT), and 3,4-DCT was reductively dechlorinated in soil slurries to predominantly toluene and small amounts of 2-, 3-, and 4-chlorotoluene (CT). Toluene was further degraded. When tested individually in a mineral salts medium, the CT-adapted soil microorganisms dechlorinated several TCT and DCT isomers. Key metabolic routes for TCTs followed: 2,3,6-TCT-->2,5-DCT-->2-CT-->toluene; 2,4,5-TCT-->2,5-DCT + 3,4-DCT-->3-CT + 4-CT-->toluene. Among DCTs tested, 2,4-DCT and 3,4-DCT were dechlorinated via the removal of o- and m-chlorine, respectively, to 4-CT and subsequently to toluene via p-chlorine removal. Likewise, 2,5-DCT was dechlorinated via 2-CT to toluene. Evidently, microorganisms capable of removing o-, m-, and p-chlorines are present in the soil system, as reflected by the dechlorination of different isomers of CBs and CTs to CB and toluene, respectively. These findings help clarify the metabolic fate of chlorinated benzenes and toluenes in anaerobic environments.     

Evaluating Biodegradation as a Primary and Secondary Treatment for Removing RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) from a Perched Aquifer

Ground water beneath the U.S. Department of Energy (USDOE) Pantex Plant is contaminated with the high explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). The authors evaluated biodegradation as a remedial option by measuring RDX mineralization in Pantex aquifer microcosms spiked with ¹⁴C-labeled RDX (75 g soil, 15 ml of 5 mg RDX/L). Under anaerobic conditions and constant temperature (16°C), cumulative ¹⁴CO₂ production ranged between 52% and 70% after 49 days, with nutrient-amended (C, N, P) microcosms yielding the greatest mineralization (70%). The authors also evaluated biodegradation as a secondary treatment for removing RDX degradates following oxidation by permanganate (KMnO₄) or reduction by dithionite-reduced aquifer solids (i.e., redox barriers). Under this coupled abiotic/biotic scenario, we found that although unconsumed permanganate initially inhibited biodegradation, > 48% of the initial ¹⁴C-RDX was recovered as ¹⁴CO₂ within 77 days. Following exposure to dithionite-reduced solids, RDX transformation products were also readily mineralized (> 47% in 98 days). When we seeded Pantex aquifer material into Ottawa Sand that had no prior exposure to RDX, mineralization increased 100%, indicating that the Pantex aquifer may have an adapted microbial community that could be exploited for remediation purposes. These results indicate that biodegradation effectively transformed and mineralized RDX in Pantex aquifer microcosms. Additionally, biodegradation may be an excellent secondary treatment for RDX degradates produced from in situ treatment with permanganate or redox barriers.     

Reducing effect of aerobic selector on the toxicity of synthetic organic compounds in activated sludge process

The effects of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP) and 1,2,4-trichlorobenzene (1,2,4-TCB) on the biodegradation kinetics of the conventional activated sludge system (CASS) and the selector activated sludge system (SASS) were investigated. Experiments were carried out using a respirometric method on unacclimated biomass from two lab-scale systems that were operated with the sludge age of 8 days. Toxicity of the test compounds for both reactors were arranged according to EC₅₀ (effective concentration) values in order as: 1,2,4-TCB > 2,4-DCP > 2-CP > phenol. All selected test compounds induced higher inhibition effect in the CASS. The SASS appeared to reduce inhibition effect in comparison to the CASS, by 21.36%, 66.95%, 64.37% and 33.33% for phenol, 2-CP, 2,4-DCP and 1,2,4-TCB, respectively. Consequently, the SASS may be recommended as a promising configuration alternative for the waste streams containing toxic compounds.     

Degradation of 1,2,4-Trichloro- and 1,2,4,5-Tetrachlorobenzene by Pseudomonas Strains

Two Pseudomonas sp. strains, capable of growth on chlorinated benzenes as the sole source of carbon and energy, were isolated by selective enrichment from soil samples of an industrial waste deposit. Strain PS12 grew on monochlorobenzene, all three isomeric dichlorobenzenes, and 1,2,4-trichlorobenzene (1,2,4-TCB). Strain PS14 additionally used 1,2,4,5-tetrachlorobenzene (1,2,4,5-TeCB). During growth on these compounds both strains released stoichiometric amounts of chloride ions. The first steps of the catabolism of 1,2,4-TCB and 1,2,4,5-TeCB proceeded via dioxygenation of the aromatic nuclei and furnished 3,4,6-trichlorocatechol. The intermediary cis-3,4,6-trichloro-1,2-dihydroxycyclohexa-3,5-diene (TCB dihydrodiol) formed from 1,2,4-TCB was rearomatized by an NAD⁺-dependent dihydrodiol dehydrogenase activity, while in the case of 1,2,4,5-TeCB oxidation the catechol was obviously produced by spontaneous elimination of hydrogen chloride from the initially formed 1,3,4,6-tetrachloro-1,2-dihydroxycyclohexa-3,5-diene. Subsequent ortho cleavage was catalyzed by a type II catechol 1,2-dioxygenase producing the corresponding 2,3,5-trichloromuconate which was channeled into the tricarboxylic acid pathway via an ordinary degradation sequence, which in the present case included 2-chloro-3-oxoadipate. From the structure-related compound 2,4,5-trichloronitrobenzene the nitro group was released as nitrite, leaving the above metabolite as 3,4,6-trichlorocatechol. Enzyme activities for the oxidation of chlorobenzenes and halogenated metabolites were induced by both strains during growth on these haloaromatics and, to a considerable extent, during growth of strain PS12 on acetate.     

Induction of hydroxyl radical production in Trametes versicolor to degrade recalcitrant chlorinated hydrocarbons

Extracellular hydroxyl radical (OH) production via quinone redox cycling in Trametes versicolor, grown in a chemically defined medium, was investigated to degrade trichloroethylene (TCE), perchloroethylene (PCE), 1,2,4- and 1,3,5-trichlorobenzene (TCB). The activity of the enzymes catalyzing the quinone redox cycle, quinone reductase and laccase, as well as the rate of OH production, estimated as the formation of thiobarbituric acid reactive substances (TBARS) from 2-deoxyribose, increased rapidly during the first 2–3 days and then remained at relatively constant levels. Under quinone redox cycling conditions, TCE degradation was concomitant to TBARS production and chloride release, reaching a plateau after 6 h of incubation. Similar results were obtained in PCE, 1,2,4- and 1,3,5-TCB time course degradation experiments. The mole balance of chloride release and 1,2,4-TCB and TCE degraded suggests that these chemicals were almost completely dechlorinated. Experiments using [¹³C]-TCE confirmed unequivocal transformation of TCE to ¹³CO₂. These results are of particular interest because PCE and 1,3,5-TCB degradation in aerobic conditions has been rarely reported to date in bacterial or fungal systems.     

Effects of Soil Water Content on Biodegradation of Phenanthrene in a Mixture of Organic Contaminants

Phenanthrene biodegradation was investigated at different soil water contents [0.11, 0.22, 0.33, 0.44 g H₂O (g soil)^?1] to determine the effects of water availability on biodegradation rate. A subsurface horizon of Kennebec silty loam soil was used in this study. [9-¹⁴C] phenanthrene was dissolved in a mixture of organic contaminants that consisted of 76% decane, 6% ρ-xylene, 6% phenanthrene, 6% pristane, and 6% naphthalene, and then added to the soil. The highest rate of mineralization, in which 0.23% of the [9-¹⁴C] phenanthrene degraded to ¹⁴CO₂ after 66 days of incubation, was observed at the soil water content of 0.44 g H₂O/g dry soil. Most of the ¹⁴C remained in the soil as the parent compound or as nonextractable compounds by acetonitrile at the highest water content. Concentrations of nonextractable compounds increased with water content, but residual extractable phenanthrene decreased significantly with increasing water content, which presumably indicates that bio-transformation occurred. The mineralization analysis of radiolabeled 9th carbon in phenanthrene underestimated phenanthrene biodegradation. The strong adsorption and low solubility of phenanthrene contributed to the low mineralization of phenanthrene 9th carbon. The other components were subject to higher biological and abiotic dissipation processes with increasing soil water content.     

Microbial Decomposition of Synthetic 14C-Labeled Lignins in Nature: Lignin Biodegradation in a Variety of Natural Materials

Lignin biodegradation in a variety of natural materials was examined using specifically labeled synthetic ¹⁴C-lignins. Natural materials included soils, sediments, silage, steer bedding, and rumen contents. Both aerobic and anaerobic incubations were used. No ¹⁴C-labeled lignin biodegradation to labeled gaseous products under anaerobic conditions was observed. Aerobic ¹⁴C-labeled lignin mineralization varied with respect to type of natural material used, site, soil type and horizon, and temperature. The greatest observed degradation occurred in a soil from Yellowstone National Park and amounted to over 42% conversion of total radioactivity to ¹⁴CO₂ during 78 days of incubation. Amounts of ¹⁴C-labeled lignin mineralization in Wisconsin soils and sediments were significantly correlated with organic carbon, organic nitrogen, nitrate nitrogen, exchangeable calcium, and exchangeable potassium.     

Comparison of Gas Chromatography and Mineralization Experiments for Measuring Loss of Selected Polychlorinated Biphenyl Congeners in Cultures of White Rot Fungi

Two methods were used to compare the biodegradation of six polychlorinated biphenyl (PCB) congeners by 12 white rot fungi. Four fungi were found to be more active than Phanerochaete chrysosporium ATCC 24725. Biodegradation of the following congeners was monitored by gas chromatography: 2,3-dichlorobiphenyl, 4,4′-dichlorobiphenyl, 2,4′,5-trichlorobiphenyl (2,4′,5-TCB), 2,2′,4,4′-tetrachlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and 2,2′,4,4′,5,5′-hexachlorobiphenyl. The congener tested for mineralization was 2,4′,5-[U-¹⁴C]TCB. Culture supernatants were also assayed for lignin peroxidase and manganese peroxidase activities. Of the fungi tested, two strains of Bjerkandera adusta (UAMH 8258 and UAMH 7308), one strain of Pleurotus ostreatus (UAMH 7964), and Trametes versicolor UAMH 8272 gave the highest biodegradation and mineralization. P. chrysosporium ATCC 24725, a strain frequently used in studies of PCB degradation, gave the lowest mineralization and biodegradation activities of the 12 fungi reported here. Low but detectable levels of lignin peroxidase and manganese peroxidase activity were present in culture supernatants, but no correlation was observed among any combination of PCB congener biodegradation, mineralization, and lignin peroxidase or manganese peroxidase activity. With the exception of P. chrysosporium, congener loss ranged from 40 to 96%; however, these values varied due to nonspecific congener binding to fungal biomass and glassware. Mineralization was much lower, ≤11%, because it measures a complete oxidation of at least part of the congener molecule but the results were more consistent and therefore more reliable in assessment of PCB biodegradation.     

Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis

Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB. Chromatography coupled to mass spectrometric detection, especially GC–MS, is typically used to determine HCH-isomer metabolites. The important enzymes involved in HCH isomer degradation metabolic pathways include hexachlorocyclohexane dehydrochlorinase (LinA), haloalkane dehalogenase (LinB), and alcohol dehydrogenase (LinC). The metabolic versatility of these enzymes is known. Advances have been made in the identification of actinobacterial haloalkane dehydrogenase, which is encoded by linB. This knowledge will permit future improvements in biodegradation processes using Actinobacteria. The enzymatic and genetic characterizations of the molecular mechanisms involved in these processes have not been fully elucidated, necessitating further studies. New advances in this area suggest promising results. The scope of this paper encompasses the following: (i) the aerobic degradation pathways of hexachlorocyclohexane (HCH) isomers; (ii) the important genes and enzymes involved in the metabolic pathways of HCH isomer degradation; and (iii) the identification and quantification of intermediate metabolites through gas chromatography coupled to mass spectrometry (GC–MS).     

Biodegradation of pentachlorophenol in natural soil by inoculatedRhodococcus chlorophenolicus

Rhodococcus chlorophenolicus PCP-1, a mineralizer of polychlorinated phenols, was inoculated into natural sandy loam and peaty soils with pentachlorophenol (PCP) at concentrations usually found at lightly and heavily polluted industrial sites (30 to 600 mg PCP/kg). A single inoculum of 10⁵ to 10⁸ cells per g of peat soil and as little as 500 cells/g sandy soil initiated mineralization of¹⁴C-PCP. The mineralization rates of PCP were 130 to 250 mg mineralized per kg soil in 4 months in the heavily (600 mg/kg) polluted soils and 13 to 18 mg/kg in the lightly (30 mg/kg) polluted soils. There were no detectable PCP mineralizing organisms in the soils prior to inoculation, and also there was no significant adaptation of the indigenous microbial population to degrade PCP during 4 months observation in the uninoculated soils. The inoculum-induced mineralization continued for longer than 4 months after a single inoculation. Uninoculated, lightly polluted soils (30 mg PCP/kg) also showed loss of PCP, but some of this reappeared as pentachloroanisol and other organic chlorine compounds (EOX). Such products did not accumulate in theR. chlorophenolicus-inoculated soils, where instead EOX was mineralized 90 to 98%.R. chlorophenolicus mineralized PCP unhindered by the substrate competition offered by the PCP-methylating bacteria indigenously occurring in the soils or by simultaneously inoculated O-methylatingR. rhodochrous.     

Assessing mineralization rates of petroleum hydrocarbons in soils in relation to environmental factors and experimental scale

Mineralization rates of non-volatile petroleum hydrocarbons (HCs) in five different oil-contaminated soils with initial HC contents ranging from 0.1 to 13 g kg^-1 are estimated as a function of environmental factors. The aim of the study is threefold, (i) to study the relevance of environmental factors that may influence the mineralization rate, (ii) to compare mineralization rates estimated in two experiments at different scales, after standardizing them to environmental reference conditions, (iii) to evaluate the CO₂ production rate as a measure for the mineralization rate of HCs. Experiments were performed at laboratory scale (30–50 cm³ soil volume) in closed-jars under constant environmental conditions and in lysimeters (0.81 m³ soil volume) under dynamic climatic and hydrological conditions. A biodegradation model, coupled to transport models for soil heat, water, and gas dynamics is employed for data interpretation. The transport models are used to simulate the environmental conditions that influence the mineralization rate in the non-steady lysimeter experiments. The results show that temperature, O₂ concentration and HC content have an effect on the mineralization rates. Water content could not be identified as a direct governing environmental factor. However, an indirect effect of water content is that it influences the effective gas diffusion coefficient in soils. The CO₂ production rate seems to be a good quantity to express the mineralization rate of HCs for HC contents>1 g kg^-1. Measured CO₂ production rates standardized to reference conditions are similar for the two different experimental scales. This demonstrates that the usage of biodegradation rates obtained in the laboratory to predict the biodegradation rates under field conditions is sound, as long as the differences in environmental conditions have been taken into account.     

Effect of soil/contaminant interactions on the biodegradation of naphthalene in flooded soil under denitrifying conditions

Summary The mineralization of ¹⁴C-labelled naphthalene was studied in pristine and oil-contaminated soil slurry (30% solids) under denitrifying conditions using a range of concentrations from below to above the aqueous phase saturation level. Results from sorption-desorption experiments indicated that naphthalene desorption was highly irreversible and decreased with an increase in the soil organic content, thus influencing the availability for microbial consumption. Under denitrifying conditions, the mineralization of naphthalene to CO₂ occurred in parallel with the consumption of nitrate and an increase in pH from 7.0 to 8.6. When the initial substrate concentration was 50 ppm (i.e. close to the aqueous phase saturation level), about 90% of the total naphthalene was mineralized within 50 days, and a maximum mineralization rate of 1.3 ppm day^–1 was achieved after a lag period of approx. 18 days. When added at concentrations higher than the aqueous phase saturation level (200 and 500 ppm), similar mineralization rates (1.8 ppm day^–1) occurred until about 50 ppm of the naphthalene was mineralized. After that the mineralization rates decreased logarithmically to a minimum of 0.24 ppm day^–1 for the rest of the 160 days of the experiments. For both of these higher concentrations, the reaction kinetics were independent of the concentration, indicating that desorption of the substrate governs the mineralization rate. Other results indicated that pre-exposure of soil to oil contamination did not improve the degradation rates nor reduce the lag periods. This study clearly shows the potential of denitrifying conditions for the biodegradation of low molecular weight PAHs. Offprint requests to: R. Samson