WO2006123081A1 - Bioremediation - Google Patents

Bioremediation Download PDF

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Publication number
WO2006123081A1
WO2006123081A1 PCT/GB2005/001957 GB2005001957W WO2006123081A1 WO 2006123081 A1 WO2006123081 A1 WO 2006123081A1 GB 2005001957 W GB2005001957 W GB 2005001957W WO 2006123081 A1 WO2006123081 A1 WO 2006123081A1
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Prior art keywords
method
grains
treatment agent
coating
soil
Prior art date
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PCT/GB2005/001957
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French (fr)
Inventor
Gary Canny
Michael Broaders
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Randall & Walsh Associates Limited
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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINED SOIL SOIL
    • B09BDISPOSAL OF SOLID WASTE
    • B09B3/00Destroying solid waste or transforming solid waste or contaminated solids into something useful or harmless
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B09DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINED SOIL SOIL
    • B09CRECLAMATION OF CONTAMINATED SOIL
    • B09C1/00Reclamation of contaminated soil
    • B09C1/10Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • B09C1/105Reclamation of contaminated soil microbiologically, biologically or by using enzymes using fungi or plants
    • CCHEMISTRY; METALLURGY
    • C05FERTILISERS; MANUFACTURE THEREOF
    • C05FORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00Preparation of fertilisers characterised by the composting step
    • C05F17/009Elimination of harmful substances, the end product being a fertilizer
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/06Contaminated groundwater or leachate
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00Use of specific compounds during water treatment
    • C02F2305/06Nutrients for stimulating the growth of microorganisms
    • CCHEMISTRY; METALLURGY
    • C02TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02FTREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00Biological treatment of water, waste water, or sewage
    • C02F3/02Aerobic processes
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/10General improvement of production processes causing greenhouse gases [GHG] emissions
    • Y02P20/14Reagents; Educts; Products
    • Y02P20/141Feedstock
    • Y02P20/145Feedstock the feedstock being materials of biological origin
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/10Biological treatment of water, waste water, or sewage
    • Y02W10/15Aerobic processes
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00Technologies for wastewater treatment
    • Y02W10/30Wastewater or sewage treatment systems with climate change mitigation effect characterised by the origin of the energy
    • Y02W10/37Wastewater or sewage treatment systems with climate change mitigation effect characterised by the origin of the energy using solar energy
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02WCLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00Technologies for solid waste management
    • Y02W30/40Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
    • Y02W30/43Aerobic fermentation, e.g. composting

Abstract

A method of bioremediating contaminated substrates (e.g. earth materials or waters) utilises a treatment agent selected from the group consisting of: (a) cereal grains which have been used for the production of, and separated from, a wort suitable for use in an alcoholic beverage brewing process, said grains not having been composted subsequent to being separated from the wort; (b) an at least partially dried form of the cereal grains defined in (a); and (c) a biologically active extract of the grains as defined in (a) or (b). The treatment agent may be provided with a coating which is broken down by the pollutant. In a particular embodiment of the invention, the treatment agent is provided in the form of a barrier that may be incorporated into earth material in the path of travel of a contaminant material but upstream of a location (e.g. a body of water) to be protected.

Description

BIOREMEDIATION

The present invention relates to the bioremediation of substrates containing a contaminant, pollutant or other undesired or toxic substance for the purpose of reducing or eliminating the amount of, and/or hazard posed by, that substance. The invention is applicable particularly (but by no means exclusively) to bioremediation of earth materials (which term is used herein to include soil, sand, clay, mixtures thereof, and like materials) and also bioremediation of waters (which term is used herein to include groundwaters, surface waters, effluents and like aqueous liquids).

Bioremediation may be considered to be the elimination, attenuation or transformation of polluting or contaminating substances by the use of biological processes only to minimise the risk to human health and the environment. This is generally achieved by the use of biological "agents" such as plants, plant materials and/or micro-organisms for directly or indirectly achieving the stated aims. For example, the biological "agents" may effect breakdown of contaminants in the environment into a harmless, or at least less hazardous, form. Bioremediation of earth materials may be used, for example, for the breakdown of organic contaminants such as hydrocarbons, e.g. resulting either from an oil spillage or present on a "brown field" site as a result of previous industrial activity.

Bioremediation of earth materials and waters may be effected either in situ or ex situ. For in situ treatment, the earth material or water (as the case may be) remains in the original location where contamination exists and the biological "agent" is incorporated into the contaminated earth material or waters at that location. For ex situ treatment, the earth material or waters are removed (e.g. by excavation or pumping) from the location where contamination occurred and then transferred to a separate site where bioremediation is effected. Subsequently the decontaminated earth material or water may be moved either to the original or other location. Whichever approach is used, bioremediation of contaminated earth materials and waters has a number of advantages. In particular, it has environmental attractions in that it provides a "natural" way of dealing with the contaminants and also avoids the alternative possibility of simply disposing of the contaminated material in a landfill site, effectively shifting the problem of contamination to an alternative location and requiring high maintenance to avoid leaching into the environment or gassing from the decomposing material.

There have been a variety of proposals for the bioremediation of contaminated earth materials and waters and a number of such proposals that provide background to the present invention are summarised below.

JP 2000-254635 discloses a method of bioremediating soil contaminated with hydrocarbons, including pyrene and benzopyrene, by means of a treatment agent consisting essentially of a compost such as cow dung compost, excrement compost, sewage sludge compost, and beer grains compost. The composted forms of these materials are used because the compost contains a high microbial load whereby the compost may be a used as a microbial inoculate for the soil.

US-A-5 209 851 discloses a bioremediation of materials, e.g. soils, that have been contaminated with PCBs, oils, creosotes and other organic or petroleum based products. The process is an ex situ process and involves mixing the contaminated material with protein nutrients in water so as to entrain air and form a bioactive structure which is then allowed to cure in air until remediation has occurred to a predetermined innocuous level. Examples of protein nutrients that may be used include powdered cows milk, soya bean oil, soya bean meal, fish oil, fish products, and brewery residues and brewery bottoms which are the yeast rich bottoms from the fermentation vessel used in a brewing process.

Applied Soil Ecology 27 (2004) 165-175 (L. Molina-Barahona et al) discloses diesel removal from contaminated soils by biostimulation and supplementation with crop residues. These residues stimulate autochthonous microflora for biodegredation of hydrocarbons. Examples of crop residues used include corn straw and sugar cane bagasse. It is believed that these residues serve primarily as a bulking agent to increase oxygen availability in the soil. JP 2002-224658 discloses purification of soil using "pot ale waste fluids", i.e. waste liquid from the distillation of an alcohol beverage. The waste liquid creates anaerobic conditions in contaminated soil to activate indigenous anaerobic bacteria to decompose organic chlorine compounds.

JP 2003-251331 discloses use of various waste materials including "brewery wastes" to bioremediate soils contaminated with chlorinated wastes. The "brewery waste" is applied to soil to create anaerobic conditions and activate indigenous anaerobic micro-organisms which breakdown organochlorine pollutants. An example of "brewery waste" disclosed is "wine lees".

US-A-5 578 210 relates to the use of bioenhancing agents selected from yeast extract and malt extract to remediate soils contaminated with halogenated hydrocarbons. As with the JP publications summarised in the immediately two preceding paragraphs, this US patent is using an organic rich substance to stimulate anaerobic breakdown or organohalogens in contaminated soils.

According to the present invention there is provided a method of bioremediating contaminated substrates comprising incorporating in said substrate a treatment agent selected from the group consisting of:

(a) cereal grains which have been used for the production of, and separated from, a an aqueous liquid suitable for use in an alcoholic beverage brewing process, said grains not having been composted subsequent to being separated from the wort;

(b) an at least partially dried form of the cereal grains defined in (a); and

(c) a biologically active extract of the grains as defined in (a) or (b), and effecting bioremediation. The invention may be applied to the treatment of substrates that have previously become contaminated or for the treatment of uncontaminated substrates for the purposes of dealing with contamination that is likely to occur. For convenience, all of these possibilities are covered herein by the term "bioremediation of contaminated substrates".

The method of the invention is applicable particularly to the bioremediation of earth materials and waters containing a contaminant, pollutant or other undesired or toxic substance. The bioremediation may be effected under aerobic and/or anaerobic conditions. The contaminating substance may be organic or inorganic in nature. Examples of pollutants that may be treated in accordance with the invention include hydrocarbons, organohalogens, organic compounds containing one or more of nitrogen, oxygen and sulphur, organometallic compounds and inorganic salts incorporating an organic ion and an inorganic counterion. The invention is particularly applicable for the case where the contaminant is a hydrocarbon, e.g. resulting from an oil spill or present in earth material and waters at a brown field site as a result of previous industrial activity but is also applicable to treatment of cyanide, thiocyanate or carbon disulphide.

The present invention has been based on our research which has established that so-called "spent brewery grains" that have not been subjected to a composting operation are eminently suitable for the bioremediation of earth materials and waters contaminated with pollutants. By way of background, "spent brewery grains" are a well known by-product of the brewing industry rather than simply being "crop residues" (e.g. straw) and, more particularly, are the grain materials that have been used to produce the wort which (after separation from the grains) is fermented to produce the alcoholic beverage, e.g. beer. Spent brewery grains are generated in high tonnage quantities by the brewing industry and are mainly used as an animal feed.

By way of further background, a typical brewing process starts with malted grain (usually barley grain) which may then be dry milled (optionally with added "raw", i.e. non-malted, grain) to produce a coarse "grist". The grist is then fed to a "Mash Vessel" where it is mixed with hot water and held for a set period of time (optionally with progressive elevation of the temperature). After this treatment, the liquid (the so-called "wort") is separated from the grain materials and subjected to a separate fermentation reaction to produce the beverage. The remaining grains are the "spent brewery grains" and normally include about 80% water. The spent brewery grains are a hemi-cellulose rich material and contain concentrated levels of protein, fat and fibre.

Spent brewery grains are very effective for the bioremediation of earth materials and waters. Whilst we do not wish to be bound by any theory, we believe that spent brewery grains are effective for the purpose of bioremediation of earth materials and waters not by acting as an inoculating medium (e.g. in the manner of the composted materials employed in JP 2000 254635) nor as a bulking agent (in the manner of the "unprocessed" crop residues disclosed by L. Molina-Barahona et al) but rather due to the "physical and chemical composition" of the spent brewery grains. They are rich in hemi-cellulose and contain concentrated levels of proteins, fat and fibre. The grains stimulate indigenous microbial populations in the earth material and waters, increasing the production of enzymes that assist the microorganisms that act as "pollutant degraders" in the degradation of pollutants (which may be complex and recalcitrant products).

As indicated above, the treatment agent employed in the present invention is selected from the group consisting of:

(a) cereal grains which have been used for the production of, and separated from, a wort suitable for use in an alcoholic beverage brewing process (particularly for the case where the alcoholic beverage is beer), said grains not having been composted subsequent to being separated from the wort;

(b) an at least partially dried form of the cereal grains defined in (a); and

(c) a biologically active extract of the grains as defined in (a) or (b). In order to obtain a treatment agent in accordance with (a) above, malted grains may be milled (to form a so-called grist) and then treated with hot water at a temperature of 50-80°C (e.g. 55-750C) for a period of 1-5 hours (e.g. 2-4 hours). For the purposes of this treatment, the milled, malted grain may be admixed with milled "raw" grain. During the treatment, the temperature of the water may be varied stepwise, e.g. temperatures of 55°C, 65°C and 750C may be used.

A preferred treatment agent in accordance with (a) above is spent brewery grains, particularly for the case where the cereal is barley. The abundance and low cost of this by-product material, coupled with its effectiveness for the bioremediation of soil, makes it an ideal choice for use in the present invention. It should however be appreciated that a material as defined in (a) whilst being the exact equivalent of "spent brewery grains" need not actually be produced for the purposes of the brewing industry. Provided that the cereal grain has been treated in a manner similar to that for producing a liquor which is suitable for fermentation into an alcoholic beverage then the ultimate end use of that liquor does not matter. Thus in an embodiment of the invention (which will be less preferred commercially) the liquor might be used for purposes other than conversion into a beverage.

Further possibilities for the treatment agent are as defined under (b) and (c) above. With regard to (b) this may be obtained from (a) by conventional techniques, e.g. by evaporation or mechanical removal of water. Evaporation may be by means of oven-drying (for example in a convection oven), microwave drying, infrared drying, solar drying or mechanical aeration. Mechanical drying ("dewatering") may conveniently be effected by means of a screw press, filter press, centrifuge etc. In accordance with this embodiment of the invention, the moisture content of the grains will most preferably be reduced from about 80% to a maximum of 60%. With a maximum of 60% moisture, the grains are more easily handleable and have a much improved shelf life as compared to the treatment agent (a). Also with a maximum of 60% moisture content the grains do not produce a polluting liquid effluent. Drying the grains also reduces transportation costs and allows coating thereof with a hydrophobic material {see infra). The drying of treatment agent (a) to provide (b) may be such as to provide somewhat less than 60% moisture, e.g. less than 10% moisture.

A number of techniques are available for obtaining a biologically active extract (i.e. one that is bioremedially active in the method of the invention) of either (a) or (b). At the simplest level, the grains (a) or (b) may be treated with boiling water and this water can then be used as a treatment agent. For example, boiling of grains as defined under (a) with water for 4-8 hours (e.g. 6 hours) will produce a suitable liquid extract. A further possibility is to contact the grains (a) or (b) with boiling water for several minutes and then allowing the mixture to cool to ambient temperature with the grains remaining in contact with the water before separation of the liquid extract, e.g. by means of either filtration through muslin cloth or centrifugal separation.

Liquid extracts as produced in accordance with the procedures outlined in the preceding paragraph may be subjected to evaporation to produce either a more concentrated form of the liquid or a powder. This will facilitate transport and application of the treatment agent.

For all of embodiments of the invention, it is particularly preferred that the treatment agent is used in conjunction with inorganic nutrients, e.g. provided by a fertiliser, such as potassium, nitrogen and/or phosphorus. The nutrients (e.g. in the form of a fertiliser) may be admixed with the treatment agent and applied jointly therewith to the contaminated substrate to be treated. Alternatively the nutrients and treatment agent may be added separately to the substrate. The nutrients will most preferably be provided by a fertiliser containing nitrogen, phosphorous and potassium. The nutrients should be non-limiting. Therefore additional nutrients may be added periodically during the bioremediation to prevent them becoming limiting.

Generally, electron acceptors will also be required for the bioremediation process. Such electron acceptors include oxygen (aerobic) and nitrate and sulphate (anaerobic). Oxygen may conveniently be incorporated periodically into the substrate thus, for example, in the case of bioremediation of earth material, the oxygen may be incorporated by periodic "turning" of the earth material. Alternatively, for bioremediation of contaminated waters the oxygen may be incorporated by bubbling air through the water, stirring or otherwise agitating the water. In the case of nitrate, sulphate and other additions for anaerobic micro-organisms these may be added to the substrate either jointly with, or separately from, the treatment agent.

The method of the invention for the treatment of contaminated earth materials and water may be effected either in situ or ex situ. For an in situ method of bioremediating earth material, the treatment agent may be incorporated into the earth material by any convenient technique, e.g. by digging the treatment agent into the ground and possibly also with an homogenisation step. For in situ bioremediation of water, the treatment agent would typically be applied by injection via boreholes installed in the ground, the injection being under gravity feeding or via pressure injection. Alternatively a "sock" set-up could be used within a borehole to provide a longer term, replaceable system supplying the active ingredients and nutrients. For ex situ treatment, the contaminated earth material and waters will be removed to a suitable location and then mixed with the treatment agent by any appropriate technique. Irrespective of whether an in situ or ex situ treatment is used, the amount of the treatment agent (based on the dry weight thereof in, for example form (a) or (b)) will generally be in the range up to 10% w/w, more preferably 0.5% to 10% and even more preferably 0.5% to 5% (e.g. 1% to 5%) on the same basis.

In a particularly advantageous development of the invention, which is applicable in the case where the treatment agent is a solid, it is preferred that particles of the agent have a coating that is degraded by the contaminant material with which the agent will come into contact. In this way, the treatment agent may be provided in the substrate but only "activated" on contact with the contaminant material (as a result of degradation of the coating). This strategy will increase the life of the treatment agent. Thus, for example, in the case where the contaminant material is an oil, the protective coating for the treatment agent may be of a contaminant-soluble material so that the coating is degraded (i.e. by dissolution) on contact with the oil to expose and thereby "activate" the treatment agent. By way of example, the coating may be a naturally derived biodegradable oleochemical with a melting point in the range 50- 600C and HLB (Hydrophilic Lipophilic Balance) value in the range 2-8. The coating may be a polyolester of a C]2-C-24 fatty acid (preferably saturated). More preferably the fatty acid residue will have 12 to 24 carbon atoms. The polyol component may, for example, be glycerol or a sugar alcohol, e.g. sorbitol. Particular examples of coating materials include glycerol monostearate, sorbitol monostearate and sorbitol laurate.

The invention, may, with particular advantage, be used to prevent (or at least reduce) passage of a contaminant through earth material via transport within groundwater. Thus, for example, a fresh incident of contamination (e.g. an oil spill) may result in contaminant travelling and passing through what was previously uncontaminated earth material. This can give rise to problems, for example, where a body of water is in the path of travel of the contaminant and therefore likely to become polluted. In this case, the treatment agent may be provided in the earth material in the form of a barrier across the path of travel of the contaminant material but upstream of a location (e.g. a body of water) to be protected. In this way, the passage of the contaminant through the previously uncontaminated earth material is arrested, or substantially reduced, by the treatment agent. Such a barrier may be used as part of an overall treatment strategy, another part of which involves bioremediation of earth material and waters present at the original location of contamination using an in situ or ex situ method in accordance with the invention.

The barrier may be one that is provided "simply" by digging the treatment agent into the ground across the path of travel of the contaminant material. However in a particularly preferred embodiment of the invention, the barrier is of a prefabricated "cartridge" construction and comprises an outer porous covering with the treatment agent within the "cartridge". The treatment agent would most preferably be in particulate form and provided with a degradable coating in accordance with the techniques described above. The invention will be further described by the following non-limiting Examples and accompanying drawings in which Figs 1-12 illustrate the results of the Example 1 and Figs 13-15 illustrate the results of Example 2.

Example 1

Introduction

A pilot scale study was carried out to investigate the effect of spent brewery grain (SBG) amendment on the degradation of hydrocarbon contaminants. Three different types of soil contaminated with different hydrocarbons were amended with inorganic nutrients and SBG at different concentrations. The treated soils were incubated in the field at temperatures ranging from -2 to 120C. In all soils the addition of inorganic nutrients (N:P:K) lowered the volatile organic compounds (VOCs) and enhanced the removal of diesel range organics (DRO) compared to unamended soil. The removal of VOCs and DRO was further enhanced in all soil types through the addition of the SBG. The increased removal was linked to a significant increase in the number of indigenous mesophilic bacteria in the soil and a less significant increase in the number of hydrocarbon degrading bacteria.

Materials and methods

Soils.

Soil A, a dark brown organic clay contaminated with relatively fresh diesel was collected from 0.5 and Im below ground level from a location in Cavan, Ireland.

Soil B, a blue-grey silty clay contaminated with partially weathered diesel was obtained from 1.0 to 1.5mBGL at the same location as Soil A.

Soil C, a red-brown sandy clay contaminated with fresh kerosene and diesel was collected from between surface to 2.OmBGL from a site in Cork, Ireland. Spent brewery grain (SBG)

The spent brewery grain, a by product of beer production from Guinness, Ireland, was obtained from KW Forage System, Wexford.

Experimental Set-up.

In this study the soil (approximately 4kg) was dispersed into plastic flower box containers. For the duration of the experiment all reactors were covered with perspex and stored in the field under ambient conditions (-2 to 120C). Weekly, each container was manually mixed and the moisture content adjusted using the tap water to ensure adequate wetting.

Experimental conditions.

The natural attenuation of diesel was investigated by setting up microcosms in which only the moisture content of the soil was adjusted (Soil A (C), Soil B (C) and Soil C (C)). The affect of inorganic nutrients in the form of NPK was studied by applying 1.2g (7:7:7 N:P:K) per kg of wet soil (Soil A (N), Soil B (N) and Soil C (N)). The SBG was added to Soil A and Soil B to give a final concentration of 5% on a dry weight basis, and in all cases, inorganic nutrients were added (Soil A (N+5% SBG), Soil (B (n+ N+ 5% SBG). In Soil C the SBG was added to give final concentrations of 1, 5, and 10 % on a dry weight basis, and again in all cases, inorganic nutrients were added (Soil C (N+1% SBG), Soil C (N+5% SBG), Soil C (N+10% SBG)). In addition Soil C was also amended with just 5% SBG and no inorganic nutrients in order to investigate if nutrients made a difference (Soil C (5% SBG)). The soils were aerated and moisture content adjusted weekly

Enumeration of bacteria.

The number of colony- forming bacteria in the different treatments was determined by the standard pour-plate method using Nutrient agar (Oxoid). The plates were incubated at 220C for 72 hrs. Hydrocarbon analysis.

Representative soil samples were collected from each treatment and photo ionisation detector (PID) field screenings of headspace volatile organic components carried out. The soil samples were then sent to an independent laboratory (SpillGo) for DRO analysis using solid phase micro extraction (SPME) followed by GC-FID analysis.

The peak area of the target compounds, Diesel Range Organics (DRO) which is all chromatographic peaks, both resolved and unresolved, eluting between the peak start of M-nonane (n-Cg) and the peak end of n-pentacosane (n-C25), using forced baseline-baseline integration, were measured relative to that of the external diesel sample.

Results.

PID Field Screening

The effect of the different amendments on VOC removal from Soil A and B could not be reliably analysed as the initial PID readings on Day 0 were quite low, due to the fact that the contamination in these two soils was historical (>3yrs) with the majority of VOC being removed prior to this study. In Soil C, which has recently being contaminated with both kerosene and diesel, VOC readings of between 125 and 155ppm were recorded on Day O. Amending this soil with inorganic nutrients did not significantly enhance the removal of VOCs compared to the unamended soil. However as can be seen from Figure 3, the addition of 1, 5 and 10 % SBG significantly enhanced the removal of VOC in comparison to the soil receiving inorganic nutrients only. The concentration of SBG did not appear to significantly affect the removal of VOCs. The general trend of VOC removal in all SBG amended microcosms followed a similar pattern. A rapid decrease in PRO was observed in the early stages of the experiment, i.e. within 7 days. Following this decrease, the residual VOCs were degraded at a much slower rate. This pattern of high degradation initially followed by a much slower rate is typical of first order degradation, and is the characteristic pattern found in the literature in relation to petroleum bioremediation studies.

DRO removal

In general, amending all three soil types with inorganic nutrients slightly increased the removal of DRO compared to the unamended soils. Further amendment with SBG significantly enhanced the removal of DRO even more in all soils. The effectiveness of SBG appeared to be independent of the concentrations added with 1% as effective as 10%.

The most significant increase in DRO removal was in Soil C, which had been recently contaminated with kerosene and diesel. In this soil, addition of SBG increased the removal of DRO after 7 days from 55% in nutrient only amended soils to greater than 95% in the SBG amended soils. This percentage removal increased to greater than 99% removal after 21 days in the SBG amended soils compared to 68% in the nutrient amended soils. Again, the general trend of DRO removal in the organically amended microcosms was typical of first order degradation. The initial concentration of DRO in Soil C averaged 2,271 mg/kg (wet weight basis) and decreased to 741 mg/kg and 494mg/kg in the control and nutrient amended soil, respectively, and to between 12 and 147mg/kg in the SBG amended soils, after 21 days of incubation.

Abiotic processes can contribute to the overall removal of hydrocarbons from soil. These processes result in the lower molecular weight, highly volatile compounds being removed first followed by the less volatile compounds. Such processes did contribute to VOC and some of the DRO removal in these experiments. However, examination of the chromatographic fingerprints (data not shown) illustrated that in the SBG amended soils not only the low molecular weight, highly volatile compounds were removed but also the higher molecular weight, less volatile compound. In all SBG amended soils, the ratio of straight chain alkanes (the more biodegradable components of diesel and kerosene) decreased significantly compared to the biomarkers pristane and phytane and the more recalcitrant unresolved complex mixture. This demonstrated that bioremediation of the hydrocarbon components of diesel was the more dominant of the processes.

Microbiological patterns.

The enhanced removal of VOC and DRO in the different treatments corresponded with increases in the microbiological parameters measured. Figures 7 to 12 illustrate the effect the different amendments on the total number of microorganisms and the total number of hydrocarbon degrading bacteria present in the soil. The addition of SBG significantly increased the total numbers of microorganisms from an average of 1.8x106 CFU/g soil (dry weight basis) in the nutrient amended soils to greater than 5.8x10 CFU/g soil (dry weight basis) after 21 days, more than a 100 fold increase.

The addition of SBG did not appear to have as significant an affect on the enhancement of hydrocarbon degrading bacteria as it did on total microrganisms. There did appear to be some enhancement in the total hydrocarbon degrading bacteria through SBG amendment, but there was no significant difference between SBG amended and unamended soils, with all soils having between l.lxlO6 and 2.2xlO6 CFU/g soil.

The increase in numbers of total microorganisms following the application of the SBG was not as a result of the introduction of microorganisms present in the organic material itself, but was due to enhancement of the indigenous soil population. This was confirmed through microbial analysis of the two organic substrates prior to addition. Analysis of SBG prior to its use as amendments showed that they contained very low numbers of microorganisms, with an average of 11 CFU/g (dry weight basis). The reason for such low numbers in both organic materials is that prior to application both materials had been oven dried at 1000C for 24 hours which would have killed off most microorganisms. Conclusion

The results of this investigation into the effect of SBG amendment on remediation of diesel and kerosene contaminated soil showed that the SBG acted to enhance degradation of hydrocarbon components compared to controls. This suggests that SBG did not act as a competing energy source for the degrading microorganisms.

Visual examination of the gas chromatographic fingerprints of the DRO at different time periods, clearly demonstrated that the SBG material had a significant affect on the degradation of hydrocarbon components compared to nutrient only. The pattern of eluted peaks changed significantly with time, with the disappearance of the large resolvable peaks (straight chain alkanes), leaving behind the unresolvable fraction referred to as the "hump" or unresolved complex mixture (UCM).

It is hypothesised that the enhanced bioremediation of the diesel was due to the increase in microbial numbers of the indigenous soil population. Hydrocarbon degrading microbes are capable of using a wide range of substrates, and are not reliant on hydrocarbons as a sole source of carbon and energy. It is believed that the organic amendments acted as an easily metabolisable alternative carbon and energy source for the hydrocarbon degrading microorganisms to use whilst degrading the diesel components by co-metabolism. A further possibility is that the grains stimulate indigenous microbial populations in the earth material and waters, increasing the production of enzymes that assist the micro-organisms that act as "hydrocarbon degraders" in the degradation of the hydrocarbons.

Example 2

Materials and methods

Materials.

A sandy soil was collected from the top 15cm of the soil surface from a location in Sligo, Ireland. It was allowed to air dry for 1 week (moisture content, 0.54%) and then passed through a 2mm sieve. The moisture holding capacity (MHC) of the soil was determined by weighing a known amount of air-dried soil before and after saturation with deionised water. The hydrocarbon retention capacity (HRC) was determined by a similar method using fresh diesel.

The spent brewery grain used in this Example was a by product of beer production from Guinness Ireland, Dublin, Ireland. This material, rich in protein, fat and fibre, is currently used as a low-grade animal feed ingredient. Prior to use in the present Example, the spent brewery grain was oven dried at 1000C for 24 hours.

Experimental apparatus.

In this Example the sandy soil was artificially contaminated with fresh diesel. The diesel was spiked at 5% v/w on a dry weight basis. In order to maximize distribution of diesel, an appropriate amount of diesel was spiked to every 200 - 300 g of dry sample. The diesel was distributed at five points on the surface of the sample and then mixed thoroughly to give a well homogenized sample. Soil was dispersed into pilot scale compost reactors (approximately 3kg), consisting of a PVC tube 25cm in diameter and 40cm in length. For the duration of the experiment all reactors were stored at room temperature (18 to 250C). Weekly, the moisture content was maintained at 60-70% of the MHC using the tap water.

Experimental conditions.

The natural attenuation of diesel was investigated by setting up microcosms in which only the moisture content of the soil was adjusted. The affect of inorganic nutrients in the form of NH4NO3 and KH2PO4 was studied by placing appropriate volumes to give a final concentration of approximately 1000:10:1.5 (C:N:P). Biweekly, similar amounts of nutrients were added to ensure that nutrients did not become limiting. The dried SBG was added to give a final concentration of 5% on a dry weight basis, and in all cases, inorganic nutrients were added. In order to maximize distribution, the organics were added to the dry soil prior to the addition of diesel and were mixed thoroughly using the cone-and-quarter method. Nutrient and moisture content were adjusted biweekly In order to minimize sampling error, duplicate samples were taken from two locations within each microcosm using a sterile corer (100mm x 12mm diameter). The two sub-samples were then integrated to one sample by mixing. The combined sample was then analysed for moisture content, total mesophilic bacteria count and residual hydrocarbon content. The moisture content was measured at each sampling point and used to convert analytical data from wet weight basis to dry weight basis. This measurement was also used to calculate the amount of water required in order to maintain the require moisture level (i.e. 60-70%) moisture holding capacity

Enumeration of bacteria.

The number of colony-forming bacteria in the different treatments was determined by the standard pour-plate method using Nutrient agar (Oxoid). The plates were incubated at 220C for 72 hrs.

Hydrocarbon analysis.

Diesel extraction was carried out using solvent extraction with n-hexane as the solvent and an ultrasonic extraction procedure adapted from EPA method 355Ob (USEPA, 1996b). A weighed sample of soil (5-1Og, weighed to four decimal places) was transferred to a 100ml Erlenmeyer flask for extraction. MgSO4 (dried overnight at 1020C) was added to the sample in order to remove water (Approximately 3g of MgSO4 was added to every 1Og soil). The contents were mixed with a spatula to form a free- flowing powder. At this stage the surrogate standard was added (ImI naphthalene stock standard to give a final concentration of 0.05% w/v) along with 20ml of w-hexane, the contents of the flask were mixed, placed in an Ultrasonic water bath and extracted for 10 minutes. The extract was decanted and filtered through Whatmann No.41 filter paper (or equivalent), into a pre-weighed round bottom flask. The extraction procedure was repeated twice with 20ml portions of w-hexane. The extracts were combined together in one flask, and the solvent evaporated in a rotary evaporator operating at 5O0C. Hydrocarbon extracts recovered from soil were re- dissolved in approximately 3ml of n-hexane and transferred to a 10ml volumetric flask. The sample container was rinsed a further two times with 1-2 ml of n-hexane and the solvent mixture transferred to the volumetric flask. One ml of both internal standards, 1-phenylhexane and 1-phenyltridecane, was added to give final concentrations of 0.05% and 0.1% v/v, respectively.

Using rt-hexane, five diesel calibration standards ranging from 0.1% to 5% were made up in 10ml volumetric flasks each containing the surrogate standard (naphthalene 0.05% w/v final concentration) and the two internal standards (1- phenylhexane 0.05% v/v and 1-phenyltridecane 0.1% v/v final concentration). Each standard was injected into the GC under the same conditions, i.e. injection volume and technique, to be used during sample analysis and analysed in triplicate. The peak area of the target compounds, Diesel Range Organics (DRO) which is all chromatographic peaks, both resolved and unresolved, eluting between the peak start of M-nonane (/J-C9) and the peak end of n-pentacosane (M-C25), using forced baseline- baseline integration, were measured relative to that of the internal standard 1- phenylhexane. Ratios of the peak areas were tabulated and standard curves plotted of peak area ratios against concentrations.

Results.

DRO removal. hi the unamended soil the first order degradation rate constant for DRO was calculated to be 0.0024/day and after 108 days incubation 27% of the DRO had been removed. Amending the soil with inorganic nutrients increased the first order degradation rate constant to 0.0057/day and resulted in a significantly higher removal of DRO compared to the unamended soil. After 108 days of incubation amendment of the diesel contaminated soil with inorganic nutrients increased the removal of DRO to 51 % compared to 25 % in the unamended soil (Figure 13)

As can be seen from Figure 14, the SBG amendment significantly (PO.05) enhanced the removal of DRO in comparison to the soil receiving inorganic nutrients only. A rapid decrease in DRO was observed in the early stages of the experiment, i.e. within 40 days. Following this decrease, the residual DRO was degraded at a much slower rate. This pattern of high degradation initially followed by a much slower rate is typical of first order degradation, and is the characteristic pattern found in the literature in relation to petroleum bioremediation studies.

The initial concentration of DROs in the freshly contaminated soils was 45,345 and 48,756 mg/kg (dry weight basis) for the control and SBG amended soils, respectively. After 108 days of incubation the DRO concentrations were reduced to 26,754 mg/kg and 5,363 mg/kg, respectively. This represented a 41 and 89% degradation of DRO over the test period. At the end of the 108-day test period SBG had enhanced the overall removal of diesel by 47 % compared to the control, which had received inorganic nutrients only. This indicated that the addition of SBG significantly enhanced the removal of diesel from the sandy soil.

Abiotic processes can contribute to the overall removal of diesel from soil. These processes result in the lower molecular weight, highly volatile compounds being removed first followed by the less volatile compounds. It is possible that such processes may have contributed to some of the DRO removal in this experiment. However, examination of the chromatographic fingerprints (data not shown) illustrated that in the early stages of the experiment not only the low molecular weight, highly volatile compounds were removed but also the higher molecular weight, less volatile compounds. This demonstrated that bioremediation of the diesel is the more dominant of the processes and it was therefore concluded that the contribution of abiotic process towards the removal of diesel under these conditions was insignificant.

Microbiological patterns.

The enhanced removal of the diesel in the different treatments corresponded with increases in all the microbiological parameters measured. The increase in total mesophilic bacteria appeared to mirror closely the removal of diesel from the systems, with a rapid increase in the numbers during the first 7 days of the experiment. Figure 15 illustrates the effect the SBG materials had on the total number of microorganisms present in the soil. The addition of SBG increased the total numbers of microorganisms from 1.6x105 CFU/g soil (dry weight basis) to 1.3x105 after 7 days, after which the total number of microorganisms remained fairly constant for the duration of the experiment ranging from 7.6x108 to 4.OxIO9 CFU/g soil (dry weight basis). As can be seen from the results, the numbers of total bacteria in the control test were statistically significantly lower (P<0.05) than in the SBG amended soils. At each sampling point except for the final day the total mesophilic bacteria in the control system were approximately 100 fold less than those in the SBG treated soil. These result demonstrated that addition of SBG significantly enhanced (P<0.05) the numbers of microorganisms in the diesel contaminated soil.

The increase in numbers of microorganisms following the application of the SBG was not as a result of the introduction of microorganisms present in the organic material itself, but was due to enhancement of the indigenous soil population. This was confirmed through microbial analysis of the SBG substrates prior to addition. Analysis of the SBG showed that they contained very low numbers of microorganisms, the SBG contained an average of 11 CFU/ g (dry weight basis). The reason for such low numbers in the SBG material is that prior to application both materials had been oven dried at 1000C for 24 hours which would have killed off most microorganisms.

The results of this investigation into the effect of different SBG amendment on remediation of diesel contaminated soil showed that it acted to enhance degradation of diesel components compared to controls. This suggests that the organic material tested did not act as a competing energy source for the degrading microorganisms.

Visual examination of the gas chromatographic fingerprints of the DRO at different time periods, clearly demonstrated that the SBG material had a significant affect on the degradation of diesel compared to nutrient only. The pattern of eluted peaks changed significantly with time, with the disappearance of the large resolvable peaks, leaving behind the unresolvable fraction referred to as the "hump" or UCM.

It is hypothesised that the enhanced bioremediation of the diesel was due to the increase in microbial numbers. Even though the proportion of hydrocarbon degrading microorganisms present was not known, the results showed that by stimulating the growth of the overall microbial population, the numbers of hydrocarbon degrading microorganisms increased concomitantly. Hydrocarbon degrading microbes are capable of using a wide range of substrates, and are not reliant on hydrocarbons as a sole source of carbon and energy. It is believed that the organic amendment acted as an easily metabolisable alternative carbon and energy source for the hydrocarbon degrading microorganisms to use whilst degrading the diesel components by co-metabolism.

Claims

1. A method of bioremediating contaminated substrates comprising incorporating in said substrate a treatment agent selected from the group consisting of:
(a) cereal grains which have been used for the production of, and separated from, a wort suitable for use in an alcoholic beverage brewing process, said grains not having been composted subsequent to being separated from the wort;
(b) an at least partially dried form of the cereal grains defined in (a); and
(c) a biologically active extract of the grains as defined in (a) or (b), and effecting bioremediation.
2. A method as claimed in claim 1 wherein nutrients are added to the substrate in conjunction with the treatment agent.
3. A method as claimed in claim 2 wherein the nutrients comprise nitrogen, phosphorous and/or potassium.
4. A method as claimed in claim 3 wherein the nutrients are provided by a fertiliser.
5. A method as claimed in any one of claims 1 to 4 effected under aerobic conditions.
6. A method as claimed in claim 5 wherein an electron acceptor is provided in conjunction with the treatment agent.
7. A method as claimed in claim 6 wherein the electron acceptor is oxygen.
8. A method as claimed in any one of claims 1 to 4 effected under aerobic conditions.
9. A method as claimed in claim 8 wherein an electron acceptor is provided in conjunction with the treatment agent.
10. A method as claimed in claim 9 wherein the electron acceptor is nitrate and/or sulphate.
11. A method as claimed in any one of claims 1 to 10 wherein the substrate is earth material.
12. A method as claimed in any one of claims 1 to 10 wherein the substrate is water.
13. A method as claimed in any one of claims 1 to 12 effected in situ.
14. A method as claimed in any one of claims 1 to 12 effected ex situ.
15. A method as claimed in any one of claims 1 to 14 wherein the cereal grains comprise barley.
16. A method as claimed in any one of claims 1 to 15 wherein the earth material or waters is contaminated with at least one hydrocarbon.
17. A method as claimed in any one of claims 1 to 16 wherein the treatment agent has a coating that is capable of being broken down by the pollutant.
18. A method as claimed in claim 17 wherein the coating material is a polyol ester of a Cj2 to C24 fatty acid.
19. A method as claimed in claim 17 or 18 wherein the coating has a melting point of 50-60°C and an HLB value in the range 2-8.
20. A method as claimed in any one of claims 1 to 19 wherein the treatment agent is provided in the form of a barrier across the path or potential path of travel of the pollutant through the earth material.
21. The method as claimed in any one of claims 1 to 20 wherein the cereal grains are spent brewery grains.
22. The use of spent brewery grains or an at least partially dried form thereof or a biologically active extract thereof for the bioremediation of earth material and waters contaminated with organic and inorganic pollutants.
23. A method of preventing or reducing passage of pollutant through a body of earth material comprising providing in the path of travel of the contaminant through the earth material a treatment agent selected from the group consisting of:
(a) cereal grains which have been used for the production of, and separated from, a wort suitable for use in an alcoholic beverage brewing process, said grains not having been composted subsequent to being separated from the wort;
(b) an at least partially dried form of the cereal grains defined in (a); and
(c) a biologically active extract of the grains as defined in (a) or (b).
24. A method as claimed in claim 23 wherein the treatment agent has a coating that is capable of being broken down by the pollutant.
25. A method as claimed in claim 24 wherein the coating material is a polyol ester of a Cj2 to C24 fatty acid.
26. A method as claimed in claim 24 or 25 wherein the coating has a melting point of 50-60°C and an HLB value in the range 2-8.
27. A barrier assembly for use in preventing or reducing passage of a contaminant through a body of earth material, the assembly comprising a porous covering material containing a treatment agent selected from the group consisting of:
(a) cereal grains which have been used for the production of, and separated from, a wort suitable for use in an alcoholic beverage brewing process, said grains not having been composted subsequent to being separated from the wort;
(b) an at least partially dewatered form of the cereal grains defined in (a); and
(c) a biologically active extract of the grains as defined in (a) or (b).
28. An assembly as claimed in claim 27 wherein the treatment agent has a coating that is capable of being broken down by a contaminant to expose the treatment agent.
29. An assembly as claimed in claim 28 wherein the treatment agent has a coating that is capable of being broken down by the pollutant.
30. An assembly as claimed in claim 29 wherein the coating material is a polyol ester of a C12 to C24 fatty acid.
31. An assembly as claimed in claim 29 or 30 wherein the coating has a melting point of 50-60°C and an HLB value in the range 2-8.
PCT/GB2005/001957 2005-05-20 2005-05-20 Bioremediation WO2006123081A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB800935A (en) * 1955-01-11 1958-09-03 Robert Alexander Grigor Young A method of treating residual liquors obtained by the distillation of alcohol from mash
DE4424574A1 (en) * 1994-02-04 1995-08-10 Weissheimer Friedr Malzfab Fertiliser contg. malt sprouts from brewing
FR2751344A1 (en) * 1996-07-17 1998-01-23 Elf Aquitaine New biodegradation additive

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB800935A (en) * 1955-01-11 1958-09-03 Robert Alexander Grigor Young A method of treating residual liquors obtained by the distillation of alcohol from mash
DE4424574A1 (en) * 1994-02-04 1995-08-10 Weissheimer Friedr Malzfab Fertiliser contg. malt sprouts from brewing
FR2751344A1 (en) * 1996-07-17 1998-01-23 Elf Aquitaine New biodegradation additive

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