Background

Contamination of soil by hydrocarbons is a widespread environmental problem. Hydrocarbon contamination includes a wide range of substances: crude oil; petroleum- derived products such as gasoline, diesel, lubrication oils, bunker fuels; and processing waste products such as American Petroleum Institute (API) separator sludges. There are four technologies currently employed on a routine commercial basis for remediating hydrocarbon contaminated soils: incineration, air stripping, landfill and bioremediation. Bioremediation typically employs one of two techniques: treatment of contaminated soil with nutrients and air to stimulate endogenous bacteria or treatment with pure or enriched cultures of selected bacteria grown ex situ. Cost of incineration and landfill are increasing, and available landfill space decreasing. EPA "land ban" regulations also restrict disposal of some hydrocarbon waste such as petroleum refinery separator sludges. Air stripping in which hydrocarbons are volatized in an air stream is low cost but limited to volatile low molecular weight hydrocarbons such as gasoline. Air stripping does not degrade the hydrocarbon; it simply disperses the contaminant into the air. Bioremediation has been commercially successful in treating sites contaminated with gasoline, diesel and solvents. However, this technology has not been consistently effective at sites contaminated with heavy petroleum hydrocarbons, crude oil, heavy diesel fuels, bunker fuels, petroleum sludges and separator sludges.

Summary of the Invention

The present invention pertains to a bioremediation composition for degrading the aromatic portion of an aromatic ring-containing compound. The composition contains a conidia preparation derived from a strain of Beauveria bassiana , e.g., the strain of Beauveria bassiana identified as GMB6 having an ATCC accession number of 74250. The present invention also pertains to a bioremediation composition which degrades a complex mixture of high molecular weight hydrocarbons including an aromatic ring-containing compound. The composition for degrading complex mixtures also contains a conidia preparation derived from a strain of Beauveria bassiana, e.g., the strain of Beauveria bassiana identified as GMB6 having an ATCC accession number of 74250.

Other aspects of the invention include bioremediation methods using the above- described compositions for degrading contaminants in materials, e.g.,water or soil, and compositions containing conidia in various forms, e.g., concentrated powder, or dry culture. Advantages of GMB6 in remediating hydrocarbon contaminated soils include:

- complete degradation of complex heavy hydrocarbon

- degradation of aromatic rings;

- Inoculation of soil using conidia that are stable in storage and transport and can be economically produced. - Inoculation of soil at much lower rates than used with whole culture white rot fungus treatment processes. White rot fungus treatments typically require 0.25 to one cubic meter of whole culture per cubic meter of soil treated. Approximately ten liters (0.01 cubic meter) of GMB6 culture volume can be used to produce the conidia necessary to treat one cubic meter of contaminated soil.

- Use of a pure culture with defined characteristics and known range of degradable compounds offers more predictable remediation processes than stimulation of indigenous bacteria

-It is an additional advantage that unused starch substrate from cultures is ideally suited as the added starch used in remediation processes for contaminated soil. It is an additional advantage that the conidia of GMB6 produced by this process are stable at room temperature and can be stored for an extended period of time and transported to treatment sites under ambient conditions. It is a further advantage that the production process can produce a highly concentrated conidia preparation containing at least lxlO'O up to 3xlθl 1 conidia per gram. This minimized transportation costs.

Brief Description of the Drawings

Figure 1 shows a chromatograph for the extract of the untreated control in Example 3 below.

Figure 2 shows a chromatograph of an extract from bottles treated with strain GMB6 and incubated for four days in Example 3 below.

Figure 3 shows a chromatograph for sludge treated with strain GMP 8/31 and incubated for four days in Example 3 below. Figure 4 shows a chromatograph for sludge treated with strain GMP 8/31 and incubated for six days in Example 3 below.

Figures 5 A - 5C show chromatograph of extracts from untreated soil (5A), number 2 diesel (5B0 at 3,000 ppm and soil six weeks after treatment with GMB6 (5C) as described in Example 4 below.

Figure 6 shows a chromatograph of an extract for a 25,000 ppm diesel standard in methylene chloride as described in Example 5 below.

Figure 7 shows a chromatograph for an extract of the untreated DE, diesel sample after one week incubation as described in Example 5 below.

Figure 8 shows a chromatograph for an extract of a GMB6 treated DE diesel fuel sample described in Example 5 below. Figure 9 is a chromatograph of the untreated control after seven days described in Example 6 below.

Figure 10 is a chromatograph of the substrate control after seven days described in Example 6 below.

Figure 1 1 is a chromatograph of the GMB6 treated sample after seven days described in Example 6 below.

Figure 12 is a graph depicting the degradation of eicosane and pyrene made by GMB6 as described in Example 6 below.

Detailed Description The present invention pertains to a biomediation composition containing the conidia preparation derived from a strain of Beauveria bassiana. The composition is capable of degrading the aromatic portion of an aromatic ring-containing compound. The language "conidia preparation" is intended to include a preparation which contains the conidia of the Beauveria bassiana such that the conidia are capable of performing their intended degradation function. Examples of such conidia preparations include dry culture, wet culture, and concentrated conidia powder.

The language "strain of Beauveria bassiana" is intended to include those strains of Beauveria bassiana which perform the intended degradation function. Preferably, the strain of Beauveria bassiana has the identifying characteristics of the strain GMB6 having an American Type Culture Collection (hereinafter ATCC) accession number of 74250. An example of such a strain is the strain designated GMB6.

The language "has the identifying characteristics of GMB6" (is intended to include those morphological characteristics that make it the same or similar to GMB6 (see Example 1 below) or degradation characteristics of GMB6, e.g., having similar degradation properties as described in the examples below.

The language "aromatic ring-containing compound" is intended to include compounds which contain aromatic rings. The aromatic ring can be contained within a fused ring system within the compound or can be a single ring . Specific examples of aromatic ring-containing compounds are described in the examples below. The aromatic ring-containing compound also can be contained within a complex mixture of high molecular weight hydrocarbons The language "complex mixture of high molecular weight hydrocarbons" is art recognized and is intended to include such mixtures as API separated sludge, diesel fuel, bunker fuel, crude oil, or lubrication oil. The complex mixture of high molecular weight hydrocarbons further can be contained within a material and the material can be contacted with the compositions of the present invention. The material can be any material which typically contains such mixtures such that the mixtures can be degraded by the compositions of the present invention. Examples of such material include soil and water.

The present invention further pertains to bioremediation methods for degrading complex mixture of high molecular weight hydrocarbons in a material. The method involves contacting material containing a complex mixture of high molecular weight hydrocarbons with the conidia preparation derived from a strain of Beauveria bassiana such that the complex mixture of high molecular weight hydrocarbons in the material is degraded. The present invention even further pertains to a bioremediation method for degrading a contaminant in a material. The method involves contacting material containing a complex mixture of high molecular weight hydrocarbons with a conidia preparation derived from a strain of Beauveria bassiana such that the complex mixture of high molecular weight hydrocarbons in the material is degraded. The present invention even further pertains to a bioremediation method for degrading a contaminant in a material. The method involves contacting material containing a contaminant with a conidia preparation derived from a strain of Beauveria bassiana having the identifying characteristics of the strain designated GMB6 such that the contaminant is degraded. The contaminant can be any substance susceptible to degradation by GMB6, e.g., lower hydrocarbons, higher hydrocarbons, or the complex mixtures described above.

The present invention is further illustrated by the following non-limiting examples. The contents of all cited references (including issued patents, published patent applications, publications, and EPA guidelines) are expressly incoφorated by reference.

Example 1 - The Isolation and Identification of A Beauveria bassiana Isolate (GMB6)

The GMB6 strain was isolated from an infected pupae of a gypsy moth

(Lymantria dispar) collected in Delaware, USA. The strain is a single conidium isolate designated GMB6. The GMB6 strain has been deposited with the American Type

Culture Collection and has been assigned the designation number 74250. A scraping of the GMB6 from the pupae (original source) was placed in ten milliliters of water and the solution was agitated and then serially diluted. GMB6 was derived through a series of three separate dilution, single colony isolations from the original source. The strain was identified as Beauveria bassiana by macroscopic and microscopic examination following the identification keys of De Hoog, G.S. ( Studies in Mycology, No.l,pp 1-41 (September 1972)) and the Commonwealth Mycological Institute (CMI) descriptions of pathogenic fungi and bacteria. Cultures also were compared with known isolates of B. bassiana obtained from the USDA ARS collection of Entomogeneous Fungi (ARSFF), Ithaca, NY. The description is as follows:

Colonies on potato dextrose agar (PDA) or malt agar (MA) at fourteen days at 23 degrees Celsius: velvety to powdery, rarely forming synnemata; white at the edge, becoming pale yellow, sometimes reddish, reverse colourless, yellow or reddish. Conidiophores abundant, arising from the vegetative hyphae, 1 -2 μ wide, bearing groups of clustered conidiogenous cells, 3-6 x 3-5 μ, which may branch to give rise to further conidiogenous cells, globose to flask-shaped with a well-developed rachis up to 20 μ long by 1 μwide, geniculate with denticles up to 1 μ long. Conidia blastic, hyaline, smooth, globose to broadly ellipsoidal, sometimes with an apiculate base, 2-3 x 2-2.5 μ. Chlamydospores absent. Fungus on the host cadaver as in culture, but the conidiogenous cells tend to be more tightly grouped.

Differs from B. brongniartii by the more clustered conidiogenous cells and the globose conidia.

Example 2 - Conidia Production Process for A Beauveria bassiana Strain (GMB6)

The conidia production process for B. bassiana strain GMB6 was the same as that described in published PCTs WO 95/10598 and WO 95/10597. The GMB6 cultures can be used in soil remediation as whole dried culture or whole ground culture in addition to a separated, concentrated conidia preparation. It is an advantage of the GMB6 strain that it can be produced using essentially the same equipment as that used for mycoinsecticide production.

Inoculum Culture Preparation

GMB6 was maintained on nutrient agar slants at -10 degrees Celsius or as dry conidia powder at four degrees Celsius. To prepare inoculum cultures, an aliquot of dry conidia or slant scrape was transferred to a nutrient broth. The composition of the broth is not crucial as a wide range of broths containing common sources of carbon and nitrogen are suitable. Table 1 lists compositions of two types of broth used in preparation of inoculum cultures. For large scale inoculum, the culture was transferred to fresh broth at one- ten percent V/V. In commercial scale production of GMB6, a slant culture was shaken with sterile water to dislodge conidia and the water was transferred to 15 L of Media 2 (Table 1). This was incubated at 20-30 degrees Celsius with agitation and aeration provided by sparging filter sterilized air. The aeration rate is not crucial with suspended cells. After 48 to 96 hours of incubation, the 15 L culture was transferred to 1500 L of sterile Media 2 in a stainless steel tank equipped with air sparging and heat exchange for temperature control. Common configurations of fermentor tank and operating procedures can be employed.

After incubation at 20-30 degrees Celsius for 48 to 96 hours, the culture was sprayed onto a solid culture substrate. The media in Table 1 were designed to promote formation of blastospores in addition to mycelia. Typically blastospore concentrations after 72 hours at 25 degrees Celsius in this media was approximately lxlO^/ml.

Table 1

Nutrient Media for B. bassiana (GMB6) Inoculation Culture

Media 1 (Laboratory) Media 2 (Production)

Glucose 40 g/1 Molasses (sugar beet) 10 g/1

KNO3 10 g/1 K.H2PO4 (technical) 1 g/1

KH2PO 5 g/1 Yeast Extract 1 g/1 Yeast Extract 1 g/1 Trace Elements H2SO4 to pH 3.8

Preparation of Solid Culture Substrate

Barley was an ingredient used to obtain high yields of conidia. Physical substrate configuration can vary provided that barley is the principle component. The composition of added nutrient solutions also can vary. For example, the use of water only is suitable; however, a source of readily utilizable sugar and nitrogen stimulates more rapid growth.

The barley substrate used was pearled barley flakes. Barley flakes were prepared by pearling to remove the hull, followed by steam rolling. Alternative substrates include: steam rolled barley, barley flour or whole ground barley coated with water onto corn cob grit, barley flour or whole ground barley coated onto plastic rings, water extracts of barley soaked into porous beads of diatomaceous earth. A wide range of materials and physical shapes also are suitable carriers for a barley substrate (see published PCT WO 95/10598)

The barley substrate was wetted to between 40 and 70% moisture with the nutrient solution shown in Table 1 (leaving out glucose when using Media 1 ) which contains a source of soluble sugar and nitrogen. The nutrient media shown in Table 1 are typical; however, a wide range of common microbiological nutrients are suitable. The wetted barley substrate was sterilized by autoclaving. Alternatively, the wetted substrate can be tyndalized to reduce contamination. After sterilization or tyndalization, the substrate was cooled to between 20 and

35 degrees Celsius and inoculated with inoculum culture. The inoculum was uniformly mixed by agitating the substrate while the inoculum was sprayed.

The inoculated substrate was incubated at 20-37 degrees Celsius for five to fourteen days. Humidified air was circulated through the substrate for aeration and to maintain temperature and substrate moisture content. In large scale cultures, air flow rates of up to 10 volumes 90% or greater RH air per volume of culture per minute can be circulated throughout the bed. After seven to fourteen days culture time, conidia concentrations read 1x10^ to 3x10*0 per gram dry weight culture. The culture was dried by passing warm dry air through the bed. The dry culture can be used whole in soil treatment. Alternatively, the dried culture was milled and the conidia was recovered as a concentrated dry powder containing greater than lxl 0' conidia per gram.

Example 3 - Degradation of API Separator Sludge Filter Cake By A Beauveria bassiana Strain (GMB6)

API separator sludge filter cake was obtained from the Cenex Oil Company refinery in Laurel, Montana. API separator sludge is the residual material or "bottoms" remaining from crude oil distillation. Separator sludge is the fraction of crude oil too heavy or not sufficiently volatile to distill. In the Cenex refinery, this material is filtered onto diatomaceous earth to form a friable solid and disposed of by landfill. API separator sludge contains a complex mixture of high molecular weight hydrocarbons particularly PAH's (fused ring, aromatic compounds) and PAH structures with alkyl side chains. High molecular weight straight and branched chain hydrocarbons, and hydrocarbons containing oxygen, nitrogen, or sulfur groups can also be present in API separator sludge. Analysis of the API separator sludge samples by an independent EPA certified laboratory, (Energy Laboratories, Inc., Billings, Montana) showed a total petroleum hydrocarbon concentration of 160,000 ppm by EPA method 418.1. This experiment compared B. bassiana strain GMB6 to another strain of B

.bassiana (Mycotech GMP 8/31 WC). Conidia of the two B. bassiana strains were produced as previously described above and used as a whole dry culture containing about 1x10** or above conidia per gram.

For the treatment comparison, 50g samples of API separator sludge were placed in a series of one liter bottles forming a layer about two cm deep in the bottom.

B. bassiana conidia preparations were mixed with sludge at the rate of 5g per bottle for a final concentration of about 1x10** conidia per gram. Six bottles were prepared for each fungus and six untreated bottles were prepared as controls. The bottles were covered and incubated at room temperature. At four, six, eight, and twelve days post-treatment, one bottle was randomly selected and extracted for analysis of TPH (total petroleum hydrocarbons). For extraction, the entire contents of each bottle was transferred to an extraction thimble, placed in a soxhlet apparatus and extracted for four hours in 200ml of methylene chloride. The extract was dried by passing it through a column of anhydrous sodium sulfate. The column was washed with one column volume (approximately 50ml) of methylene chloride which was added to the extract. The extract and wash were made up to 250 ml final volume in a volumetric flask. An aliquot of the extract was then passed through a column of Florisil (Supelco Inc., Belleforte, PA) and the first 2 ml was collected and analyzed by gas chromatography. Gas chromatographic conditions were as follows:

Instrument: Varion Model 3700 Detector: FID

Column: Supelco SP 2100 sm x 1/4" ID glass nitrogen carrier gas 40 cc/mis Injector Temperature: 300° F.

Detector Temperature: 300° F.

Oven Temperature Profile: Initial temperature 100° F. four minutes, Increase 4° F/minute, final temperature 280° F., hold five minutes

Figure 1 shows a chromatograph for the extract of the untreated control. Figure

2 shows a chromatograph of extracts from a bottles treated with strain GMB6 and incubated for four days. Figures 3 and 4 show the same data from sludge treated with strain GMP 8/31 at four and six days. Fungus growth was visible throughout the filter cake at four days in GMB6 treated bottles. By twelve days, API separator sludge filter cake treated with strain GMB6 showed nearly complete absence of all peaks in the chromatograph. Chromatographs from GMP 8/31 treated sludge were essentially unchanged between day four and day six. After twelve days treatment with GMB6, the original black "tar-like" appearance of the filter cake was changed to a brown colored material.

Example 4 - Treatment of Hydrocarbon Contaminated Soil With A Strain of B. Bassiana (GMB6)

Hydrocarbon contaminated soil was obtained from a rail car storage yard in

Snoqualmie,WA. This soil was contaminated by seepage from waste pits containing a mixture of hydrocarbons associated with railroad maintenance and storage operations including bunker fuels, grease, lubricating oils, etc. Total petroleum hydrocarbon concentration as determined by EPA method 418.1 ranged from about 600 ppm to more than 50,000 ppm over the contaminated soil at the site.

A study was conducted with a composite soil sample representative of the site. Portions of soil were taken from a plurality of areas within the site and combined to form the composite soil sample. TPH concentration in this sample and in experimental treatments were determined by extraction as described above and gas chromatographic analysis as described above; except that silica gel rather than Florisil was used for sample clean up. Soil was mixed and screened through a 10 mesh (US std) screen to remove rocks. Screened composite soil samples contained 7,800 ppm TPH by the gas chromatographic method described above using Number 2 diesel fuel as a standard.

Two kilograms of soil were blended with 25 grams of GMB6 whole culture. Water was added to achieve a beginning moisture content of 25%. The treatment was monitored by removing 50g samples and assaying for TPH as described above. Figures 5A-5C show chromatographs of extracts from untreated soil (FIG 5A), number 2 diesel (FIG 5B) at 3,000 ppm and soil six weeks after treatment with GMB6 (FIG 5C). The ratios of integrated area under the chromatographs cover show soil was reduced from 7,800 ppm to about 30 ppm in comparison with the 3,000 ppm diesel standard.

Example 5 - The Degradation of Diesel Fuel By A Strain of B. Bassiana (GMB6) Aliquots of diatomaceous earth (20 g) were weighed into one liter glass jars and sterilized by autoclaving. Thirty grams of sterile filtered water and 0.5 g of number 2 diesel fuel (Conoco, Butte, Montana) were added to each jar to yield a TPH concentration of 25,000 ppm on a dry weight basis in the diatomaceous earth. Five grams of barley flour were mixed with a conidia concentrated powder derived from GMB6 and added to the diesel spiked diatomaceous earth sample in each jar. The final conidia concentration was 5.3 x 10 ⁸ per gram of DE mix. Thirty ml of water was added and mixed and the jars were incubated at room temperature for seven days. Untreated controls were run in parallel. After seven days, the entire contents of each jar was extracted in 200 ml methylene chloride for four hours in a soxhlet. The extract was dried through a column of anhydrous sodium sulfate and made up to 250 ml final volume. An aliquot was passed through Florisil and analyzed by gas chromatography under the following conditions:

Instrument: Varian model 3700 Detector: FID

Column: Supelco gp2100 nitrogen carrier gas

Injector temperature: 300 degrees Celsius

Detector temperature: 300 degrees Celsius

Temperature profile: 70 degrees Celsius five minutes, 70 degrees to 230 degrees Celsius at four degrees Celsius per minute

Figure 6 shows a 25,000 ppm diesel standard in methylene chloride. Figure 7 shows an extract of the untreated DE, diesel sample after one week incubation and Figure 8 shows an extract of the GMB6 treated DE diesel fuel sample. Diesel fuel concentration as determined by ratio of integrator counts compared with the standard were reduced from 2,500 ppm to 300 ppm in seven days.

Example 6 - The Degradation of Straight Chain and Aromatic Hydrocarbons By A Strain of B. Bassiana (GMB6)

This example demonstrates the ability of GMB6 to degrade eicosane ( 20 carbon straight chain hydrocarbon), phenanthrene (a polynuclear hydrocarbon containing three rings), and pyrene (a polynuclear hydrocarbon containing four rings).

Diatomaceous earth (DE) was used as the solid media on which to test the degradation of eicosane, phenanthrene, and pyrene by GMB6. In the first series of samples, the hydrocarbons were added to the DE by dissolving 0.153 grams of eicosane,

0.166 grams of phenanthrene, and 0.155 grams of pyrene in 75 milliliters of hexane. Five milliliters of the solution were added to 20 grams of DE to achieve concentrations of 510, 553, and 517 milligrams of eicosane, phenanthrene, and pyrene per kilogram of DE. The samples were placed in a 60 °C oven for approximately 40 hours to allow the hexane to evaporate.

Thirty milliliters of autoclaved water were added to each of the samples. Four of the samples were then set aside as untreated controls. Four grams of autoclaved starch were added to the remaining eight samples, four of which were set aside as substrate controls. Four grams of GMB6 conidia powder containing approximately lxl 0^ conιdιa/g _{rarn W} ere added to the remaining four "treated" samples.

After seven days, two each of the untreated controls, substrate controls, and treated samples were dried, extracted and analyzed. Because water interferes with the extraction procedure, the samples were dried over night in a 60 °C oven. The samples were then extracted with 100 milliliters of methylene chloride in shaker bottles for sixteen hours. The extracts were then cleaned by passing them through a column of silica gel. The cleaned samples were then analyzed on a HP5890 GC with a J&W -DB5 column. The program was 40 °C for 0.5 min, 40 to 320 °C at 15 °C/min, hold for 15 min, with an injector and detector temperatures at 300°C.

Figure 9 represents the gas chromatograph of the untreated controls after seven days. Figure 10 is a chromatograph representing the substrate control after seven days, and Figure 1 1 is a gas chromatograph representing the treated samples after seven days. Phenanthrene comes off the column at approximately 10.8 minutes, eicosane at

12.2 minutes, and pyrene at 13.2 minutes. The small peak representing phenanthrene indicates that the drying procedure may drive off part of the phenanthrene. Even with the small phenanthrene peak, the results of adding GMB6 were significant. GMB6 reduced the levels of all three compounds below their detection limits within seven days. There also was some reduction in compound concentrations in the substrate control samples. These reductions may have been due to stray fungi and bacteria stimulated by the added starch. The 14 day samples supported the seven day results. All three

compounds showed up in all of the control samples and none of them showed up in any of the treated samples, indicating that GMB6 degrades both straight chain and aromatic hydrocarbons.

A second set of experiments were conducted in a manner similar to the first set of experiments. In the second set of experiments, the samples were analyzed on the second, fifth, and seventh days, instead of the seventh and fourteenth days. The purpose of the experiment was to see how fast the GMB6 actually degraded eicosane and pyrene. The initial concentrations of eicosane and pyrene were 590 and 580 milligram per kilogram of DE respectively. The results are summarized in Table 2 set forth below and Figure 12.

The results demonstrate that GMB6 degrades both eicosane and pyrene and that the degradation process occurs within a few days.

TABLE 2 Degradation of Eicosane & Pyrene by GMB6

Peak Areas

Day Sample Eicosane Pyrene

2 Untreated Control 684387 1020422

2 Substrate Control 627424 739203

2 Treated Sample 1 222043 446344

2 Treated Sample 2 470772 860439

5 Untreated Control 620283 818512

5 Substrate Control 730522 776203

5 Treated Sample 3 127694 415091

5 Treated Sample 4 73614 322406

7 Untreated Control 282106 514335

7 Substrate Control 474902 560357

7 Treated Sample 5 14064 1 16679

7 Treated Sample 6 1 1292 179440

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.