Alan G. Seech

Biodegradation of hexachlorocyclohexane (HCH) by microorganisms

The organochlorine pesticide Lindane is the γ-isomer of hexachlorocyclohexane (HCH). Technical grade Lindane contains a mixture of HCH isomers which include not only γ-HCH, but also large amounts of predominantly α-, β- and δ-HCH. The physical properties and persistence of each isomer differ because of the different chlorine atom orientations on each molecule (axial or equatorial). However, all four isomers are considered toxic and recalcitrant worldwide pollutants. Biodegradation of HCH has been studied in soil, slurry and culture media but very little information exists on in situ bioremediation of the different isomers including Lindane itself, at full scale. Several soil microorganisms capable of degrading, and utilizing HCH as a carbon source, have been reported. In selected bacterial strains, the genes encoding the enzymes involved in the initial degradation of Lindane have been cloned, sequenced, expressed and the gene products characterized. HCH is biodegradable under both oxic and anoxic conditions, although mineralization is generally observed only in oxic systems. As is found for most organic compounds, HCH degradation in soil occurs at moderate temperatures and at near neutral pH. HCH biodegradation in soil has been reported at both low and high (saturated) moisture contents. Soil texture and organic matter appear to influence degradation presumably by sorption mechanisms and impact on moisture retention, bacterial growth and pH. Most studies report on the biodegradation of relatively low ( 500 mg/kg) concentrations of HCH in soil. Information on the effects of inorganic nutrients, organic carbon sources or other soil amendments is scattered and inconclusive. More in-depth assessments of amendment effects and evaluation of bioremediation protocols, on a large scale, using soil with high HCH concentrations, are needed.

Monitoring bioremediation in creosote-contaminated soils using chemical analysis and toxicity tests

Three soils with a history of creosote contamination (designated NB, TI and AC) were treated in bench-scale microcosms using conditions (nutrient amendment, moisture content and temperature) which had promoted mineralization of 14C-pyrene in a preliminary study. Bioremediation was monitored using the solid-phase Microtox test, seed germination and earthworm survival assays, SOS-chromotest, Toxi-chromotest and a red blood cell (RBC) haemolysis assay. Contaminant concentrations in the AC soil did not change after 150 days. Polycyclic aromatic hydrocarbon (PAH) concentrations decreased in the NB soil, and toxicity decreased overall according to the earthworm, seed germination and Microtox tests. Although total petroleum hydrocarbons (TPHs) in the TI soil were reduced following treatment, results of the earthworm, seed germination, RBC and Microtox tests suggested an initial increase in toxicity indicating that toxic intermediary metabolites may have formed during biodegradation. Toxicity testing results did not always correlate with contaminant concentrations, nor were the trends indicated by each test consistent for any one soil. Each test demonstrated a different capacity to detect reductions in soil contamination. Journal of Industrial Microbiology & Biotechnology (2000) 24, 132–139.

Colorimetric assay for Lindane dechlorination by bacteria.

A colorimetric microtitre plate-based assay that detects haloalkane dehalogenase activity was modified to detect dechlorination of gamma-hexachlorocyclohexane (Lindane). Dechlorination is indicated by the colour change of phenol red from red to yellow, in a weakly buffered solution, as the solution becomes acidic due to HCl formed during dechlorination. Enzyme activity can be monitored by reading the absorbance of each well at 540 nm. Positive controls for the assay were the known Lindane-degrading microorganisms, Rhodanobacter lindaniclasticus and Sphingomonas paucimobilis UT26. Dechlorination in a scaled-up version of the assay was confirmed by GC/ECD detection of known metabolites of the test microorganisms from which the enzyme extracts were prepared. The assay was used to measure the rate of dechlorination in cell-free extracts of R. lindaniclasticus. It was also used to screen the cell-free extracts of 24 bacterial isolates, from a Lindane-contaminated soil, for Lindane dechlorination activity. Although no isolates tested positive, the assay represents a new inexpensive and rapid screening tool for the detection of Lindane-degrading microorganisms.

Identification and characterization of a soil bacterium with extracellular emulsifying activity

Abstract A gram‐negative bacterial strain (designated UG2) isolated from oil‐contaminated soil was identified as Pseudomonas aeruginosa based on biochemical characterization, total cellular fatty acid analysis, and serotyping. The strain produced high extracellular emulsifying activity when grown on various aromatic and straight chain hydrocarbons such as naphthalene, stearic acid, hexadecane and sunflower oil. The emulsifying activity in crude culture filtrates of UG2 exhibited stability at 80°C. In addition, the presence of the emulsifying agent increased partitioning of 14C‐PCB into the aqueous phase in soil slurries by a factor of 30‐fold over that of the water control. The results suggest that UG2 emulsifying agent may have a potential application in remediation of PCB‐contaminated soil that has also been inoculated with PCB‐degrading organism(s).

A Comparison of Five Bioassays to Monitor Toxicity During Bioremediation of Pentachlorophenol-Contaminated Soil

Five bioassays were used to measure toxicity during bioremediation of a soil contaminated with pentachlorophenol (PCP; 335 ppm), polycyclic aromatic hydrocarbons (PAHs; 1225 ppm) and petroleum hydrocarbons (19 125 ppm). Different bioremediation treatments were tested in soil microcosms including amendment with phosphorus and/or PCP-degrading Pseudomonas sp. UG30, either as free cells or encapsulated in κ-carrageenan. Soil toxicity was monitored using the solid-phase Microtox test, SOS-chromotest, lettuce seed germination, earthworm survival and sheep red blood cell (RBC) haemolysis assays. PCP levels were reduced in all treatments after 210 days. The RBC lysis assay, Microtox test and SOS-chromotest indicated reduced toxicity in most of the microcosms by day 210. Trends depicted by lettuce seed germination and earthworm survival LC50 values varied with each treatment. For example, in soil amended with phosphorus, both the seed germination and earthworm survival LC50 data suggested increased soil toxicity. However, for soil treated with encapsulated Pseudomonas sp. UG30 cells, the earthworm survival LC50 data indicated reduced toxicity while seed germination LC50 values showed little change from values obtained prior to bioremediation. Our results show that toxicity trends in a contaminated soil during bioremediation differ according to the assay used.

Monitoring biodegradation of creosote in soils using radiolabels, toxicity tests, and chemical analysis

Microbial acclimation to, and mineralization of polycyclic aromatic hydrocarbons (PAHs), was studied using four uncontaminated soils (designated HS, HC, GP, MS) spiked with creosote and 14C‐phenanthrene. The effects of nutrient amendments (nitrogen and phosphorus), temperature (10°C and 22°C), and moisture content (50 or 85% of water‐holding capacity, WHC) on mineralization were monitored by measuring evolution of 14CO2 in microcosms. Acclimation and mineralization occurred more rapidly at 22°C than at 10°C and were enhanced by the P amendment, at 50% of WHC for GP soil and 85% of WHC for HC and MS soils. These conditions were applied to microcosms containing 1500 g soil and monitored for biodegradation of total petroleum hydrocarbons (TPHs) and PAHs using chemical analysis and six soil toxicity tests. Although 40% 14C was recovered as CO2 within 35–45 days in the smaller microcosms, analytical and toxicity test data indicated that bioremediation in the larger microcosms was not successful after 130 days. The soil depth and high moisture content may have restricted oxygen diffusion into the soil, which was not stirred during the experiment. Variations in toxicity and contaminant concentrations were observed but were considered a consequence of the sampling protocol and insufficient mixing during spiking, which may have produced pockets of “hot” soil. The mineralization experiment was useful for confirming that indigenous soil microorganisms could degrade PAHs, but was not indicative of the success of the bioremediation protocol on a larger scale. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 99–106, 2000

Survival and transport of lac-lux marked Pseudomonas fluorescens strain in uncontaminated and chemically contaminated soils

Summary Survival of a lac ZY-lux AB strain and rifampicin, ampicillin resistant strain of Pseudomonas fluorescens UG16 in uncontaminated soil and soils containing polycyclic aromatic hydrocarbons (PAHs) and chlorinated phenols was conducted over 7 weeks at 10 and 22°C. Survival of cells containing lac ZY-lux AB reporter gene system determined by viable plating and detection with a charge coupled camera was generally better or similar to the rifampicin, ampicillin resistant strain. Microcosms containing remediated soil with decreased levels of PAHs and chlorinated phenols incubated at 10°C allowed better survival of introduced cells than soil incubated at 22°C, containing higher concentrations of PAHs and chlorinated phenols. Bacterial transport in soil columns of contaminated, bioremediated and uncontaminated control soils was carried out using the lac ZY-lux AB strain of P. fluorescens UG16. Soil columns were flushed with non-sterile rainwater and microorganisms enumerated in the soil profile and leachate. Non-remediated soil containing chemical contaminants displayed lower transport of the inoculated strain. Remediated and uncontaminated soils allowed transport of the introduced bacterial cells through the soil column in into leachate samples.

Bioremediation in Field Box Plots of a Soil Contaminated with Wood-Preservatives: A Comparison of Treatment Conditions using Toxicity Testing as a Monitoring Technique

A soil contaminated with polycyclic aromatic hydrocarbons, petroleum hydrocarbons and chlorophenols was bioremediated in field box plots. Three different bioremediation treatments (tillage and irrigation alone (box plot 2) or in addition to amendment with nitrogen and phosphorus (box plots 3 and 4) and additional organic amendment composed ofagricultural crop residues (box plot 4)) were comparedusing chemical analysis for target contaminants andsix toxicity tests (seed germination, earthwormsurvival, SOS Chromotest, Toxi-Chromotest, solid-phaseMicrotox® andred blood cell (RBC) haemolysisassay). Degradation was enhanced, and toxicity wasgenerally the most reduced, in box plots 3 and 4. Although chemical analysis indicated that the twoamendment protocols were equally effective, soiltoxicity was generally the most reduced in box plot 4. The earthworm survival and seed germination assayswere the most reliable and relevant toxicity tests. Difficulties arising with the other tests includedinsensitivity to changes in soil contaminant levels,inconsistency and interference by soil particles andother soil constituents. Because of the lack ofagreement between toxicity tests, these resultssupport the use of a battery of toxicity tests inconjunction with chemical analysis, when assessing theefficacy of bioremediation.