This invention relates to a method for the remediation of contaminated solid material such as soil, sludge, spent biomass etc.

Background of the Invention

Traditionally, biore ediation projects have been conducted in a number of ways. For example, the activity of degradative microorganisms already present in the environment has been enhanced by the addition of suitable nutrients and by improving other physico-chemical conditions (bioaugmentation) . Alternatively, suitable microorganisms or their enzymes have been introduced to the contaminated material. A further procedure has been to allow the icrobial activity to take place in a specially designed vessel under ideal conditions.

Many common environmental pollutants have been found to be resistant to biodegradation, for example the high molecular weight polyaromatic hydrocarbons (PAHs) and some halogenated aromatics. Some of these compounds, especially PAHs with more than three fused rings, tend to be very stable and may only be degraded in the presence of another source of carbon and energy.

The fungus Phanerochaete chrvsosporium has been widely used as a model system to understand the process of lignin biodegradation. This fungus has been shown to generate free radicals, which allows it to catalyse numerous non¬ specific cleavage reactions, resulting in breakdown of macrostructures into small fragments capable of being metabolised by more conventional enzymes.

The ability to produce these potent oxidative enzymes has resulted in numerous studies of the ability of P. chrysosporium to degrade aromatic environmental pollutants. In fact the system has a very broad substrate specificity and acts on virtually all aromatics, including PAHs, polychlorinated biphenyls (PCBs) and dioxins. See Bumpus et al. Science (1985) 228.

Although the ability of E> chrvsosporium to degrade a wide range of chemicals of environmental concern is now well established, attempts to remediate contaminated soil by direct inoculation with this organism have achieved little success, mainly because soil is not its normal habitat. See Glaser, in: Biotechnology and Biodegradation (Advances in Biotechnology series, vol. 4), ed. Kamely et al (1990)). Competition from other strains of fungi and bacteria severely limits growth although survival and growth are poor even in sterile soils.

Bacteria can under certain circumstances grow well in soil and cause the destruction of organic compounds by direct enzyme action. In this case, the enzyme binds to the molecule (substrate) and, by the introduction of other chemical species, e.g. H ₂ 0, begins the microbial degradation pathway. However, many complex compounds are resistant to such enzyme attack since their stereochemistry prevents the binding of the molecule to the enzyme or passage of the substrate into the cell for subsequent intracellular action. This is an important part of any enzyme reaction. Summary of the Invention

The present invention is based on the use of free radicals, i.e. a non-enzymatic reaction. As indicated above, this has a number of advantages, including a non¬ specific mode of attack which does not occur in true enzyme systems. This non-specificity provides a wide application. However, before the technology can be applied on a large scale, a number of problems need to be solved. These may be any or all of:

1. How to present a large number of radicals to the material to be decontaminated.

2. How to satisfy the conditions required for radical production, e.g. temperature, moisture content, oxygen etc.

3. How to ensure that the radical-generating system is protected from the possible toxic effects of contaminants to be degraded.

4. How to ensure the continued production of radicals over a prolonged period, to ensure that the degradation proceeds to the required level of acceptable contamination or below.

5. How to add adequate nutrient, since excessive amounts of readily-available carbon, nitrogen or sulphur will inhibit the production of radicals and therefore reduce or eliminate the degradation of the target compound. 6 How to maintain biological activity, since the use of free radicals can result in the production of toxic intermediates of the contaminating compound. These intermediates may even prove to be more toxic than the original compound. 7. How to make the system effective in a wide variety of soil types. For example, problems may occur with oxygen availability, water content and temperature where soil contains an excessive amount of clay. According to the present invention, a method for remediating material, e.g. soil, sand or spent biomass containing organic contaminants, comprises intimately mixing the material with a biomass including a micro¬ organism capable of producing free radicals that initiate break-down of the contaminants.

More specifically, the microbial biomass is pre-grown on a suitable substrate before it is combined with the contaminated material. The established mycelium is then able to withstand both the effects of lethal contaminants and competition from the indigenous microflora. Concentration of pollutants normally thought of as being too high for biological processing can then be degraded. The system is not classically enzymatic and non¬ specific. Thus, a range of aromatic compounds is degraded. This is especially important, for example, where a soil is

contaminated with PAHs which tend to be present in a wide range of high molecular weight forms normally regarded as being recalcitrant. Description of the Invention The novel method is applicable to the treatment of particulate solids allowing intimate admixture, e.g. sewage sludge, silt and soil. For the purpose of illustration only, the invention will be described below with reference to the treatment of soil. The present invention is based on the discovery of techniques by means of which organisms of the type exemplified by the fungus Phanerochaete chrysosporium can be prepared and grown on material containing both cellulose and lignin in such a manner to enable the organisms to be introduced into soil in a viable condition with sufficient substrate to allow a considerable level of free radical production, on and after the initial introduction. Sufficient supporting material is added to allow free radical production of the fungus to continue over the period of time required for decontamination. Additional nutrients may be added but are preferably of a low nitrogen composition.

The fungus relies on the material used for physical support and on which it grows for some of its nutrients. The support material offers some protection against toxic effects and other bacterial competition. The fungal mycelium is however able to grow out into the soil in search of other nutrients. Mineral salts are absorbed from the soil. Again high nitrogen levels in the soil will inhibit radical production and the degradation process. It can therefore be expected that the amounts of undegraded chemical contaminants will reach an asymptote at a point at which the fungus dies and radical production ceases. The means of maintaining degrading activity is therefore preferably calculated either experimentally or empirically before treatment begins.

This mixture of soil and actively-growing fungus is covered, and moisture and aeration are controlled. The resulting activity of the fungus on the substrate results in further radical production capable of cleaving complex ring structures. The types of compounds that can be degraded in this way are exemplified by chlorophenols and polyaromatic hydrocarbons.

The support material is ultimately degraded, and the introduced microorganisms gradually decline in numbers due to competition from the natural population. This should occur only when degradation is substantially complete.

The microorganisms used are non-pathogenic naturally- occurring isolates which need not have been manipulated in any way to produce unusual enzymes. This means it is highly unlikely that they will lose their ability to degrade the targeted compounds, as sometimes occurs with bacterial systems.

The temperature is preferably held at 5 to 40°C. This may be achieved by ensuring that secondary biological activity occurs, and thus raises the temperature. In conditions when the presence of a secondary fermentation is uncertain, e.g. in very sandy soils or in the presence of high contamination, this may be achieved by the addition of suitable bacteria. The production of secondary and toxic metabolites is a potential problem that may be resolved when the fungus is left undisturbed. However, other micro-organisms may be involved in the final degradation process to mineralisation, i.e. to carbon dioxide and water, and in degradation of secondary metabolites. It may therefore be desirable to add suitable strains of bacteria to achieve or enhance this process. Suitable bacteria for this purpose, and for maintaining temperature, are available to, or can be selected by, the ordinarily skilled man. Various steps will now be described, that should be adopted for optimum operation of the remediation process (specific amounts and materials are given by way of

illustration only) . These steps are all capable of being operated by the ordinarily skilled man. Laboratory Determination

Before the start of any bioremediation scheme, it is necessary to undertake careful laboratory tests on samples from the contaminated site itself. From these tests, it should be possible to determine the rate-limiting factors affecting biodegradation of contaminants within a particular soil. The major factors affecting biological treatment of soils include temperature, pH, moisture content, oxygen availability, particle size distribution, soil type, nutrient concentrations, the presence of inhibitory compounds and the levels of the contaminants and their effect on radical generation. These factors, which are often inter-related, can be altered to improve microbial activity. The effects of these adjustments are best judged by performing experiments under carefully controlled conditions on quantities of soil to be degraded, and not on spiked samples of similar soil. Spore production

Viable spores are produced using a medium which includes the preparation of spores on kraft lignin waste. Spore production is promoted at the expense of biomass. An acrylic resin (e.g. Junlon) can be included to prevent the tendency to pellet.

Viable spores can be prepared for transportation by centrifugation and stabilisation with a suitable agent such as carboxymethylcellulose. When stored at 4°C, the spores are viable for up to six months. Concentrated preparations of spores and fungal propargules are prepared for application by dilution in clean but not necessarily distilled water. Preparation of fungal biomass

A large biomass, usually at least 50 m , preferably at least 500 m , e.g. up to 1000 or 5000 m , but possibly more, is prepared. All materials must be mixed to homogeneity.

It is essential that the growth of the organism be well established before mixing with the contaminated material. Straw or similar material (approx. 150 m ³ ) should be processed to an average length of 5 cm using a crushing machine. This material is thoroughly soaked with clean water and mixed. This is sprayed with the diluted liquid spore inoculum, covered and left for 10 days. It is then uncovered and mixed with a further 700 m of material, which then becomes the primary biomass pile. This primary biomass pile is spread in thin or shallow layers of not greater than 1 m depth and then thoroughly sprayed with a total of 16 m ³ of diluted inoculum.

The material is covered, e.g. with black HDPE sheeting, to maintain moisture and exclude light. Two to three weeks later, the covers are removed and 1 m thick layers are sprayed again with 8 m dilute inoculum and covered as before.

After a further 4 weeks the covers are again removed, and this secondary biomass is mixed with a further 500-1000 m of material. This may now include bark, sawdust and similar material. This secondary biomass is thoroughly soaked with clean water, laid out in layers and sprayed with a further 32 dilute inoculum. The new pile of not more than 1 m depth is again covered and after a further period of not less than 4 weeks becomes the tertiary biomass preparation that will be used for treatment. Soil preparation

Soil needs to be screened or sieved to a reasonable, defined particle size, e.g. 100 mesh, suitable for mixing. The active biomass is mixed with the soil in a suitable ratio, preferably from 1 to 10 parts soil per part biomass, e.g. 2:1 or 4:1, and the treatment begins. Soil beds of no deeper than 0.6 m are ideal for natural aeration; where deeper by necessity, a network of pipes is incorporated into the bed before covering with heavy duty sheeting.

Where soil types are dense, suitable preparations may be obtained by the addition of inert materials such as sand or artificial materials having similar properties. Further, as desired or necessary, a suitable bacterial preparation may be added at this stage to ensure correct temperature generation and to ensure metabolite degradation. Suitable organisms include bacilli and rhodococci.

This material is mixed with the tertiary biomass in a ratio of 2 soil to one biomass and the treatment begins. Soil beds of no deeper than 0.6 m may not require artificial aeration; however, where deeper by necessity, a network of pipes should be incorporated into the bed before covering with heavy duty sheeting. Maintenance of moisture level

The correct moisture level, e.g. less than 20%, depends on soil type. The HDPE covering minimises evaporation from the soils surface. Gas monitoring points are installed in the beds to measure the effect of aeration on the levels of O _z and C0 ₂ on the soil air. Minimum daily aeration is applied using a timer to maintain these gases at optimal levels and to avoid unnecessary moisture loss. If required, an irrigation device consisting of a network of perforated hose may be placed on the surface of the bed beneath the covers. Alternatively, humidified air can be supplied to avoid excessive evaporation. Maintenance of temperature

Optimal temperature for free radical degradation under these conditions appears to be approx. 30°c. However at temperatures of 10-15°C some degradation will occur. The temperature reached is dependent on the the soil type and the proportion of the cellulosic material added. Sandy soils reach higher temperatures than clay soils. Adding more carrier results in higher temperatures being obtained as the activity of added or endogenous bacteria increases. Temperatures generally rise 10-15 ^β C above ambient. Deep treatment beds (2-3 m) have a greater capacity for heat

storage and fluctuations are minimal which is beneficial to the fungal process. In winter lowering of the temperature means less biological activity so aeration is reduced accordingly. This avoids unnecessary cooling due to the pumping of cold ambient air through the beds. Treatment may be accelerated, however, by pumping warm humidified air.

By carefully observing those various parameters, complete degradation can be obtained. It is particularly important to ensure homogeneity, to avoid high and low spots of residual contamination, to maintain moisture levels and to avoid physical disturbance. Failure to ensure suitable moisture levels will result in microbial death from which the fungus will not recover as will the introduction of anaerobic conditions or the lack of sufficient aeration. Physical disturbance will also destroy or seriously damage the fungus and its ability to produce radicals.

The following Example illustrates the invention. Example

1. Preparation of viable spores

Spores of potentially suitable strains are prepared for use on a laboratory scale and for later scale-up should they prove suitable. When required for inoculation purposes, P. chrysosporium is grown in a medium designed for maximum sporulation and minimal biomass. The greatest spore count has been recorded in a medium based on acidic waste from the pulp and paper industry (kraft lignin waste) : Kraft lignin solution 10 ml/1

Yeast extract 5 g/1

Ammonium tartrate 0.44 g/1

Junlon 0.3 g/1

Tween 80 0.5 g/1 Concentrated trace elements solution 1 ml/1

Each 1 litre flask contains 500 ml of the above medium, adjusted to pH 4.5, which is sterilised by

autoclaving. Junlon is an acrylic resin which acts as an anti-pelleting agent as well as having the effect of reducing mycelial growth and encouraging production of spores. Inoculation of the growth medium is by introduction of spores which have previously been produced on 2% malt extract agar (30°C, 5-7 days) . Flasks are incubated at 30°C with agitation (150-200 rpm) . Spores are produced after 4 days' growth.

The trace elements solution is made up as a stock solution and diluted before use. The concentrated solution contains the following:

Na ₂ ETDA.2H ₂ 0 12 g/1

NaOH 2 g/1

MgS0 ₄ . 7H ₂ 0 1 g/1 ZnS0 ₄ .7H ₂ 0 0.4 g/1

CuS0 ₄ . 5H ₂ 0 0. 1 g/1

Na ₂ S0 ₄ 10 g/1

Na ₂ HP0 ₄ .2H ₂ 0 0.1 g/1

FeS0 ₄ .7H ₂ 0 2 g/1 MnS0 ₄ .4H ₂ 0 0.4 g/1 concentrated H ₂ S0 ₄ ca . 0.5 ml/ 1 , to pH 7

An alternative medium, also developed for high sporulation with low biomass , contains the following: Malt extract 10 g/1 Glycerol 10 g/1

Junlon 0.3-1.2 g/1

Tween 80 0.5 g/1

Trace elements solution 10 ml/ 1

K ₂ HP0 ₄ 1. 5 g/1

KH ₂ P0 ₄ 0. 50 g/ 1

MgS0 ₄ . 7H ₂ 0 0. 20 g/ 1 PH to 4 . 5

Junlon has the effect of increasing the viscosity of the medium. High concentrations may give problems later during centrifugation (due to high viscosity) . If the biomass is to be concentrated down to a very small volume, use minimum Junlon. If viscosity is still a problem.

Junlon can be omitted, and the culture prevented from pelleting by adding a large number of spores to each flask (5-6 loopfuls from a Petri dish) . The trace elements solution is as described previously. 2. Spore preservation

Cultures are harvested after the fourth day of growth. The medium is centrifuged so that typically 20 litres of cultures is reduced to around 1.5 litres. Relatively high quantities of Junlon, or large amount of biomass, may not allow it to be concentrated to such a small volume. Low viscosity carboxymethylcellulose (CMC) , at 0.2%, is used as a stabiliser. The biomass is then stored at 4°C, preferably in a foil sachet. Spores should remain viable in this form for up to six months. 3. Biomass preparation

Small-scale decontamination studies are carried out in 1 litre Kilner jars. Due to the inability of p_. chrysosporium to compete with indigenous soil microorganisms, it is pregrown on a carrier material from which the established mycelium is able to penetrate the surrounding soil. The carrier material comprises:

10 g straw (after being soaked with water overnight)

30 g sawdust

7 g sugarbeet waste (or paper pulp, to retain moisture)

The biomass is grown in liquid culture as described previously. Four-day old cultures are centrifuged and the pellets resuspended, to their original volume, in fresh tap water. The resuspended biomass is used to rehydrate the sugar beet (15 ml) and the sawdust (45 ml) . The blended carrier material is placed in a Kilner jar and incubated at 30°C until good growth is seen (2-3 weeks) .

The contaminated soil is sieved, thoroughly mixed to disperse hotspots, and the moisture content adjusted to around 17%. At higher moisture contents (max. 25%) , the fungus will not grow. 150 g are then added to a Kilner jar containing well-established P. chrysosporium and stirred

thoroughly. After several days at 30°C, the fungus can be clearly seen on the dispersed carrier materials, with mycelium penetrating the soil particles. The jars are stirred weekly and tap water is added, if necessary, to maintain moisture content. Soils are sampled by sacrificing one whole jar for each analysis: the contents are dried, ground and solvent-extracted. For each time point, up to 5 jars are analysed, overcoming problems with soil variability, and making the results more statistically significant.

Before excavation of the soil could begin, it was necessary to pregrow the biomass on a larger scale. The above process was used. 4. Remediation Chlorophenol-contaminated soil from around a former timber treatment plant was excavated immediately prior to forming the treatment beds. Screening of the soil and mixing with the inoculated organic material was carried out in a one-stage process using a rotating drum screen with a small, e.g. 100 mm mesh. Processed soil was then transported to the treatment area and laid out over a gravel base enclosed by a HDPE liner. This allowed for leachate collection and removal to a storage tank. Nutrient additions were made at this stage, to remedy the deficiencies discovered during the initial laboratory investigation.

The beds incorporated a network of perforated pipes which, when connected to a pump, allows forced aeration. This is necessary due to the depth of the beds (2-2.5 m) and to avoid turning the bed material during the treatment period, e.g. of up to 2 years. Much of the soil was of a heavy silt-clay consistency which was made workable by adding appropriate quantities of sand/gravel at the screening/mixing stage. Heavy machinery was not allowed to travel onto the beds, to avoid unnecessary compaction which would restrict airflow. Each treatment bed (size approx. 50 m x 30 m x 2 m) was finally covered with HDPE sheeting.

5. Results

On examination in the laboratory, an 80% reduction in total chlorophenol was found after 15 weeks treatment. Both tetrachlorophenol (TECP) and pentachlorophenol (PCP) , the main contaminants, were substantially reduced in concentration, from more than 300 to less than 100 mg/kg, over this period. The average starting concentration of this potent fungicide was around 750 mg/kg, suggesting the organic carrier material was offering some level of protection against the toxic effects of these chemicals. In large-scale field work, the following results were obtained, for chlorophenol degradation in 6,000 m of soil. Values are total chlorophenol (mg/kg) and the means of ten samples each from five sub-samples. Bed time zero 3 months 6 months 9 months 12 months A 203.1 141.5 72 57.6

B 173.2 106.9 27.5 13.6

D 83.7 12

C 37.6 10.9