the regulatory climate which he feels is responsible for suppressing the use of genetically engineered microorganisms (GEMs) for cleaning-up environmental contamination.However, stringent regulations are not the issue here.The real

Vegetable oils, synthetic esters (including transesterified oils) and mineral oils are the main classes of oil used in pesticide formulations. Biodegradation is a major route for the removal of oils from soil systems. Most of the oils used in pesticide formulations are degraded substantially in the laboratory tests that are used to assess aquatic biodegradability. The susceptibility of different oils to biodegradation can be explained in terms of the metabolic capacity of common microorganisms.Fewer soil biodegradability tests have been carried out with oils, but the available data suggest that the mode of degradation is not very different from that in aquatic systems. Supplements of inorganic nutrients (in particular nitrogen) are needed to stimulate microbial activity in soils containing the high concentrations of oil that can be encountered in the event of a spill. However, oils are applied at such low rates in pesticide formulations (maximum of 5 g oil m-2 soil) that nutrient availability is unlikely to affect the rate of biodegradation in the field.

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 μg 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.

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 mug 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.

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 H2O2 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 H2O2 as an oxygen source at concentrations above approx. 100 mg H2O2 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 H2O2 but extensive biologically-mediated breakdown still occurred. The results demonstrate that significant difficulties may be encountered when using inorganic nutrient and H2O2 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.

Using a data set for 39 base oils, formulated oil products and pure compounds it was demonstrated that there was a good positive relationship between biodegradation in CEC L-33-T-82 and mineralisation to CO2 in the modified Sturm test. A mathematical model was developed which described this correlation for most of the materials tested. One outlier from the model was di-iso tridecyl adipate (DITA), the well-degradable calibration oil for the CEC test. The measured mineralisation of DITA was much lower than that predicted by the model based on the compound's high biodegradability in the CEC test. A possible reason for this is given and the implications of this result discussed.

The 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.

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.

Enhanced insitu biotreatment is a recent technology for the cleanup of contaminated soil and ground water but it has not yet been tested for many contaminants. This report describes the assessment of three hydrocarbon-contaminated sites, one contaminated with crude oil, one with lubricating oil and one with gasoline, with respect to their potential for biotreatment. All locations were permeable, sandy soils which contained low concentrations of extractable inorganic macronutrients. Degradative microbial populations were present, although their numbers were reduced in the most highly contaminated portions of the soil. Hydrocarbon analysis demonstrated that vertical penetration of contaminants into the soil was poor for the crude oil but had occurred at the other sites. There was some evidence that biodegradation at the crude and lubricating oil-contaminated sites may have occurred. The available data suggested that biotreatment of the lubricating and gasoline-contaminated sites by the provision of inorganic nutrients and oxygen to the soils might prove viable. However, it was found that the addition of inorganic nutrients resulted in an inhibition of mineralisation in the soils.

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.

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.

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.

There is growing interest in the enhancement of microbial degradative activities as a means of bringing about the in situ cleanup of contaminated soils and ground water. The halogenated organic compounds are likely to be prime targets for such biotechnological processes because of their widespread utilisation and the biodegradability of many of the most commonly used compounds. The aim of this review is to consider the potential for microbiological cleanup of haloorganic-contaminated sites. The technologies available involve the provision of suitable environmental conditions to facilitate maximum biodegradation rates either in the subsurface or in on-site bioreactors. Methodologies include the supply of inorganic nutrients, the supply of oxygen gas, the addition of degradative microbial inocula and the introduction of co-metabolic substrates. The potential efficiencies and limitations of the methods are critically discussed from a microbiological viewpoint with respect to substrate degradability and population responses to supplementation.

Several attempts have been made to carry out fate of oil studies directly in the environment. Such studies are necessary to validate the measurements and observations made in laboratory simulations. The problems associated with studies carried out directly in the environment are that there are so many interactions taking place the interpretation of data is extremely difficult, particularly when the environment itself is varying with time. The converse is true for laboratory simulations, since one controls many environmental parameters and limits the number of interactions in order to be able to interpret the data.