Robert J. Watkinson

Hydrocarbon Degradation in Soils and Methods for Soil Biotreatment

The cleanup of soils and groundwater contaminated with hydrocarbons is of particular importance in minimizing the environmental impact of petroleum and petroleum products and in preventing contamination of potable water supplies. Consequently, there is a growing industry involved in the treatment of contaminated topsoils, subsoils, and groundwater. The biotreatment methodologies employed for decontamination are designed to enhance in situ degradation by the supply of oxygen, inorganic nutrients, and/or microbial inocula to the contaminated zone. This review considers the fate and effects of hydrocarbon contaminants in terrestrial environments, with particular reference to the factors that limit biodegradation rates. The potential efficiencies, advantages, and disadvantages of biotreatment techniques are discussed and the future research directions necessary for process development are considered.

Physiology of aliphatic hydrocarbon-degrading microorganisms.

This paper reviews aspects of the physiology and biochemistry of the microbial biodegradation of alkanes larger than methane, alkenes and alkynes with particular emphasis upon recent developments. Subject areas discussed include: substrate uptake; metabolic pathways for alkenes and straight and branched-chain alkanes; the genetics and regulation of pathways; co-oxidation of aliphatic hydrocarbons; the potential for anaerobic aliphatic hydrocarbon degradation; the potential deployment of aliphatic hydrocarbon-degrading microorganisms in biotechnology.

Comparison of abilities of white-rot fungi to mineralize selected xenobiotic compounds

SummaryThe abilities of the white-rot fungi Chrysosporium lignorum, Trametes versicolor, Phanerochaete chrysosporium and Stereum hirsutum to mineralize 3,4-dichloroaniline, dieldrin and phenanthrene were investigated. S. hirsutum did not mineralize any of the test compounds but the other strains partly mineralized them all to varying degrees. The relative degradation rates per unit biomass were T. versicolor > C. lignorum > P. chrysosporium. Evidence was obtained for the production of water-soluble metabolic intermediates but no attempt was made to characterize these. It was found that mineral salts-glucose medium supplemented with trace mineral nutrients, vitamins and 1.5 mm 3,4-dimethoxybenzyl alcohol (veratryl alcohol) resulted in the highest mineralization rate. At no time in these experiments was there detectable extracellular ligninase (lignin peroxidase) activity.

Factors limiting the supply and efficiency of nutrient and oxygen supplements for the in situ biotreatment of contaminated soil and groundwater

Abstract In situ biotreatment of contaminated soil and groundwater requires the provision of optimal conditions for biodegradation in the subsurface. The supply of inorganic nutrient solutions and oxygen in the form of dilute H 2 O 2 was investigated using a number of soils in order to determine limitations of injection and infiltration technologies. It was found that migration of phosphate was limited by the precipitation of insoluble salts and that this reduced soil permeability. Sodium tripolyphosphate was found to reduce partially the amount of precipitation but disrupted soil structure. The addition of inorganic nitrogen to an oil-contaminated soil was found to inhibit mineralization of glucose and phenanthrene. The use of H 2 O 2 as an oxygen source at concentrations above approx. 100 mg H 2 O 2 l −1 was restricted by decomposition reactions. Precipitation of oxidation products and bubble formation owing to degassing resulted in significant reductions in soil permeability. Sodium tripolyphosphate also reduced chemically-catalysed cleavage of H 2 O 2 but extensive biologically-mediated breakdown still occurred. The results demonstrate that significant difficulties may be encountered when using inorganic nutrient and H 2 O 2 solutions for site bioremediation but the effects are site-specific. Detailed assessments of individual sites are a necessary pre-requisite to any in situ biotreatment programme.

Growth and biodegradation by white-rot fungi inoculated into soil

Abstract The colonization of sandy loam soil following inoculation with spore suspensions of the white-rot fungi Phanerochaete chrysosporium ATCC 24725 and Chrysosporium lignorum CL1 was confirmed by an epifluorescence microscopy-image analysis method. These fungi and Trametes versicolor PV1 mineralized 3,4-dichloroaniline and benzo(a)pyrene in soil at concentrations up to 250 μg g −1 . Successful inoculation and biodegradation required supplementary carbon sources. Addition of inorganic nutrients had no stimulatory effect. Glucose, hay, wood chips, pine bark, loam and peat all promoted growth and degradation but chopped wheat straw was the best substrate. Increasing the content of straw in the soil led to increased biomass and mineralization. The optimum ratio of straw: soil for mineralization was 1:4. Both strains sporulated within 7 days of inoculation before a further increase in hyphal growth but this had no effect on the mineralization rate. These results indicate that use of white-rot fungi in biotechnological soil treatment may be feasible.

Biodegradation of benzene, toluene, ethylbenzene and xylenes in gas-condensate-contaminated ground-water

The rate and extent of biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) in ground-water was studied in samples from a contaminated site which contained total BTEX concentrations of up to 20 000 microg litre(-1). All compounds were rapidly degraded under natural aerobic conditions. Elevation of incubation temperature, supply of organic nutrients or addition of inorganic fertiliser did not increase the rate or extent of biodegradation and it appeared that oxygen supply was the factor limiting BTEX degradation at this site. Attempts to increase the dissolved oxygen concentration in the ground-water by the addition of hydrogen peroxide to give a final concentration of 200 mg litre(-1) resulted in the complete inhibition of biodegradation. No biodegradation occurred under anaerobic conditions except when nitrate was provided as a terminal electron acceptor for microbial respiration. Under denitrifying conditions there was apparent biodegradation of benzene, toluene, ethyl-benzene, m-xylene and p-xylene but o-xylene was not degraded. Degradation under denitrifying conditions occurred at a much slower rate than under oxygenated conditions.

Biodegradation of components of petroleum

Petroleum can be defined as a naturally-derived organic material that is composed primarily of hydrocarbon compounds and held in geological traps. The liquid fraction of this is crude oil. Crude oils are essentially produced by high pressure and temperature action on biological material over geological time-scales. The variability of all of these factors means that there is great variability in the chemical composition and properties of different crude oils. Furthermore, refined products from different crudes will have different chemical composition even though the products meet the relevant physical and performance standards. The aim of this paper is to review the biochemical and physiological mechanisms involved in the biodegradation of components of crude oil and petroleum products that are not gaseous under physiological conditions.

Automated image analyss method to determine fungal biomass in soils and on solid matrices

Abstract A procedure has been developed for determining fungal biomass in soil and on inert surfaces by fluorescent staining and fully-automated image analysis. Soil samples were homogenised, filtered and stained with Calcofluor M2R (for total hyphal biomass measurements) or fluorescein diacetate (for viable biomass measurements). Fungi on inert surfaces were stained with Calcofluor M2R. Samples were examined by epifluorescence microscopy and images analysed using a Teragon-Contextvision GOP-302 system. Hyphal length and biovolume were calculated in a totally automated process and novel software routines were developed to differentiate fungal hyphae from other stained material. The principles of the software operations should be applicable to many other image analysis systems. Testing of the system against manual microscoplc determination of length and known dry weights of mycelium demonstrated excellent correlation between the automated image analysis and other techniques. The method is rapid, accurate and minimises operator fatigue. Being wholly deterministic, the results obtained do not depend on the judgement of the operator. Application of the technique is illustrated with reference to experiments studying the growth of fungi inoculated into soils and the fungal colonisation of plastics.

The Use of Gel-stabilized Model Systems for the Study of Microbial Processes in Polluted Sediments

SUMMARY: Gel-stabilized model sediment ecosystems were prepared using agar and colloidal silica as gelling agents and were employed in studies of the effects and degradation of xenobiotics in marine and freshwater sediments. All models produced physicochemical and microbiological profiles characteristic of sediments. The fate of 3-nitrophenol was studied in a freshwater system and the compound was found to be readily distributed through the gel column and to be rapidly degraded. The release of the nitro-group during metabolism resulted in the accumulation of nitrate in the aerobic portion of the gel column. The fate of a 1:1 hexadecane/naphthalene mixture was investigated using a seawater model system. The metabolism of these compounds resulted in oxygen depletion in the gel column and in a decrease in the population size of aerobic heterotrophic bacteria. Conversely, the populations of anaerobic heterotrophic bacteria and sulphate-reducing bacteria were significantly increased. The data are discussed with particular respect to the practical uses of gel-stabilized model ecosystems in research into the microbiology of sediments.