MICROBIAL TRANSFORMATION OF RECALCITRANT PCB COMPOUNDS

Cross-Reference to a Related Application

This is a continuation-in-part of co-pending application Serial No. 07/369,365, filed June 21, 1989.

Background of the Invention

Polychlorinated biphenyls (PCB's) are well-known pollutants in the environment. Though they can be at least partially degraded by some microbes, chemicals, and photochemicals, the PCB's still persist in the environment throughout the world. Their presence has been considered to be a serious health risk to animals and humans.

The inability of microbes and various chemicals to completely degrade PCB's to harmless entities is understandable in view of the complexity of PCB's. It has been reported that PCB molecules consist of a biphenyl nucleus carrying 1 to 10 chlorines. Thus, there are 209 possible PCB congeners that differ in the number and position of the chlorines. Commercial PCB products such as Aroclors

(Monsanto) contain more than 60 to 80 congeners at analytically detectable levels (Bedard, D.L. et al. [1987] Appl. and Environ. Microbiol. 53:1094-1102; and Bedard, D.L. et al. [1987] Appl. and Environ. Microbiol. 53:1103-1112).

Based generally on the work reviewed and described in the above-noted publications, it can be stated that virtually all microorganisms described, both

Gram-negative and Gram-positive, degrade biphenyl by 2,3-diox genation. In their search for novel PCB degraders, the scientists isolated a large number of biphenyl- and mono-chlorobiphenyl-utilizing strains, and their ability to degrade mixtures of PCB congeners was assessed. PCB's containing two or more chlorine groups did not support the growth of any organism isolated, but these chemicals were degraded co-metabolically when biphenyl or mono-chlorobiphenyl was present.

Two bacterial strains were identified with the ability to attack specific PCB congeners where 2,3-dioxygenation was not possible. These microbes have been shown to introduce oxygen functions at positions 3 and 4 of the biphenyl molecule. Although the products appear to be dead-end metabolites, mixtures of microorganisms with broad specificity dioxygenase mechanisms have been shown capable of removing substantial amounts of PCB's from various Aroclors under laboratory conditions. Despite this ability, these microorganisms have shown limited usefulness in the treatment of PCB's in situ.

Thus, there remains a need for more efficient microbial systems which can be used to degrade PCB's in situ. The invention described and claimed herein is directed to such an efficient microbial system and to the use of novel native and recombinant microbes to remediate PCB's in situ.

Brief Summary of the Invention The subject invention relates to the use of novel recombinant microbes to remediate sites contaminated with PCB's. The novel recombinant microbes of the invention contain structural novel genes cloned from a novel microbe designated Pseudomonas paucimobilis strain EPA505sc, and novel microbes designated CRE1, CRE2, CRE3, CRE4, CRE5, CRE6 and CRE7. These recombinant microbes can be used alone or with other microbes, including non-recombinant microbes capable of degrading recalcitrant PCB compounds, as disclosed herein, to remediate sites contaminated with PCB's.

Brief Description of the Drawings Figure 1 presents gas chromatograms of methylene chloride extracts of

MS+PAH broths either un-inoculated (Figure la), inoculated with Pseudomonas putida [NAH7] strain PpG7 (ATCC 17485) (Figure lb), or inoculated with the fluoranthene-induced consortium (Figure lc). With the exception of two formalin- associated peaks at 3.6 and 6.5 min, the gas chromatograms of the killed-cell controls were essentially identical to that presented in Figure la. After 8 days incubation, there were no detectable losses of PAH's from the un-inoculated

controls. In the presence of PpG7, only naphthalene (peak 1) and 2-MN (peak 2) were degraded beyond the limit of detection. The fluoranthene-induced consortium, however, exhibited extensive degradation of all the PAH's present in the defined mixture. Only fluoranthene (peak 13) and pyrene (peak 14) were , 5 present in detectable amounts (Table 3). Though the fluoranthene peak does not appear to have been significantly reduced, the area associated with this peak corresponds to 41.6% recovery which, in turn, corresponds to degradation of 0.25 mg fluoranthene in 8 days.

10 Detailed Disclosure of the Invention

The use of a gene obtainable from a novel Pseudomonas microbe to degrade PCB is a feature of the subject invention. A subculture of the novel microbe has been deposited in the permanent collection of the Northern Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, USA on June 9, 1989.

15 The accession number is as follows:

Pseudomonas paucimobilis strain EPA505sc. — NRRL B-18512 The taxonomy of Pseudomonas paucimobilis strain EP.A505sc is as follows: Gram-negative, aerobic, non-glucose fermenting, motile (weakly) rod (0.5 x 1.5 μm). Forms a 1.0 to 2.0 mm bright yellow colony on nutrient agar plus 0.5%

20 glucose after 5 days at 28°C. Yellow pignment is non-diffusible and non- fluorescent. Oxidizes glucose, D-gluconate, and lactose. Hydrolyses esculin. Does not assimilate arabinose, maltose, mannose, malate, N-acetyl-D-glucosamine, caprate, adipate, TWEEN™80, citrate, or phenylacetate. Does not reduce nitrate of nitrite. Negative reactions for urease, .arginine dihydrolase, gelatinase, and

25 tiyptophanase.

The subject culture has been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposit is

30 available as required by foreign patent laws in countries wherein counterparts of

the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action. Further, the subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of

Microorganisms, i.e., it will be stored with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture. The depositor acknowledges the duty to replace the deposit should the depository be unable to furnish a sample when requested, due to the condition of the deposit. All restrictions on the availability to the public of the subject culture deposit will be irrevocably removed upon the granting of a patent disclosing it. The degradation of PCB congeners in situ can be carried out by the use of various well-known procedures. For example, the degradation process can be carried out by adding liquid culture media of a PCB-degrading microbe to contaminated soil or water wastes. Generally, procedures as disclosed in U.S. Patent Nos. 4,477,570 and 4,483,923 can be used. As any person skilled in this art knows, good growth conditions for the degrading microbes must be employed in order to enable the microbes to degrade contaminated sites effectively. Determination of such optimum growth conditions are routine for the skilled professional.

The novel recombinant microbes of the invention can be made by isolating the gene(s) from the native microbes, as disclosed herein, and transforming suitable hosts with the gene(s). The gene(s) encode enzymes which are capable of degrading recalcitrant PCB compounds.

A wide variety of ways are available for introducing a gene into a microorganism host under conditions which allow for stable maintenance and expression of the gene. One can provide for DNA constructs which include the

transcriptional and translational regulatory signals for expression of the gene, the gene under their regulatory control and a DNA sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system which is functional in the host, whereby integration or stable maintenance will occur.

The transcriptional initiation signals will include a promoter and a transcriptional initiation start site. In some instances, it may be desirable to provide for regulative expression of the gene, where expression of the gene will only occur after contact with PAH's. This can be achieved with operators or a region binding to an activator or enhancers, which are capable of induction upon a change in the physical or chemical environment of the microorganisms.. For example, a temperature sensitive regulatory region may be employed, where the organisms may be grown up in the laboratory without expression of a gene, but upon contact with PAH's, expression would begin. Other techniques may employ a specific nutrient medium in the laboratory, which inhibits the expression of the gene, where the nutrient medium in the environment would allow for expression of the gene. For translational initiation, a ribosomal binding site and an initiation codon will be present.

Various manipulations may be employed for enhancing the expression of the messenger RNA, particularly by using an active promoter, as well as by employing sequences, which enhance the stability of the messenger RNA. The transcriptional and translational termination region will involve stop codon(s), a terminator region, and optionally, a polyadenylation signal.

In the direction of transcription, namely in the 5' to 3' direction of the coding or sense sequence, the construct will involve the transcriptional regulatory region, if any, and the promoter, where the regulatory region may be either 5' or 3' of the promoter, the ribosomal binding site, the initiation codon, the structural gene having an open reading frame in phase with the initiation codon, the stop codon(s), the polyadenylation signal sequence, if any, and the terminator region. This sequence as a double strand may be used by itself for transformation of a microorganism host, but will usually be included with a DNA sequence involving

a marker, where the second DNA sequence may be joined to the expression construct during introduction of the DNA into the host.

A marker structural gene is used to provide for the selection of the host microbe which has acquired the desired nucleotide sequence (via, for example, transformation, electroporation, conjugation, or phage mediated). The marker will normally provide for selective advantage, for example, providing for biocide resistance, e.g., resistance to antibiotics or heavy metals; complementation, so as to provide prototropy to an auxotrophic host, or the like. Preferably, complementation is employed, so that the modified host may not only be selected, but may also be competitive in the field. One or more markers may be employed in the development of the constructs, as well as for modifying the host. The organisms may be further modified by providing for a competitive advantage against other wild-type microorganisms in the field. For example, genes expressing metal chelating agents, e.g., siderophores, may be introduced into the host along with the structural gene. In this manner, the enhanced expression of a siderophore may provide for a competitive advantage for the host, so that it may effectively compete with the wild-type microorganisms.

Where no functional replication system is present, the construct will also include a sequence of at least 50 basepairs (bp), preferably at least about 100 bp, and usually not more than about 1000 bp of a sequence homologous with a sequence in the host. In this way, the probability of legitimate recombination is enhanced, so that the gene will be integrated into the host and stably maintained by the host. Desirably, the gene will be in close proximity to the gene providing for complementation as well as the gene providing for the competitive advantage. Therefore, in the event that a gene is lost, the resulting organism will be likely to also lose the complementing gene and/or the gene providing for the competitive advantage, so that it will be unable to compete in the environment with the gene retaining the intact construct.

A large number of transcriptional regulatory regions are available from a wide variety of microorganism hosts, such as bacteria, bacteriophage, cyanobacteria, algae, fungi, and the like. Various transcriptional regulatory regions

include the regions associated with the trp gene, lac gene, gal gene, the lambda left and right promoters, the Tac promoter, the naturally-occurring promoters

. associated with the gene, where functional in the host. The termination region may be the termination region normally associated with the transcriptional

# 5 initiation region or a different transcriptional initiation region, so long as the two regions are compatible and functional in the host.

Where stable episomal maintenance or integration is desired, a plasmid will be employed which has a replication system which is functional in the host. The replication system may be derived from the chromosome, an episomal element

10 normally present in the host or a different host, or a replication system from a virus which is stable in the host.

The gene can be introduced between the transcriptional and translational initiation region and the transcriptional and translational termination region, so as to be under the regulatory control of the initiation region. This construct will be

15 included in a plasmid, which will include at least one replication system, but may include more than one, where one replication system is employed for cloning during the development of the plasmid and the second replication system is necessary for functioning in the ultimate host. In addition, one or more markers may be present, which have been described previously. Where integration is

20 desired, the plasmid will desirably include a sequence homologous with the host genome.

The transformants can be isolated in accordance with conventional ways, usually employing a selection technique, which allows for selection of the desired organism as against unmodified organisms or transferring organisms, when present.

25 The transformants then can be tested for degradation of PAH's.

Suitable host cells can be Gram-negative bacteria, including Enterobacteriaceae, such as Escherichia. and Pseudomonadaceae, such as

' Pseudomonas.

The recombinant cellular host containing the gene may be grown in any

30 convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the

cells retain the gene. These cells may then be harvested in accordance with conventional ways.

Examples of recalcitrant PAH's are as follows: 2,6-dimethylnaphthalene, 2,3-dimethylnaphthalene, acenaphthene, fluorene, phenanthrene, anthracene, 2- methylanthracene, anthraquinone, fluoranthene, pyrene, 2,3-benzo[b]fluorene, chrysene, benzo[a]pyrene, and the like.

Examples of solubilizing agents which can be used in the subject invention are the many well-known and commercially available non-ionic and anionic surface active agents, emulsifying agents, and detergents. Some examples are TWEEN™80 (a non-ionic surfactant available from Fisher Chemical Co.), DMSO,

Merpol (a non-ionic ethylene oxide condensate produced by E.I. duPont de Nemours and Co., Inc.), Consowet (a dioctylsulfosuccinate anionic detergent produced by Consos, Inc., Charlotte, NC), and Astrowet (a dioctylsulfosuccinate anionic detergent produced by Astro American Chemical Co., Greenville, SC). Generally, the basic chemical structure or nature of these solubilizing agents is not limiting so long as they can be considered to be non-ionic or anionic surface active agents, emulsifying agents, or detergents.

The concentration of the solubilizing agent used in the culture medium should be, advantageously, the least amount which will place the PAH into aqueous solution. This can be done routinely by procedures well known in the art.

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1 — Source of PAH-Degrading Microorganisms

Soil highly contaminated with coal-tar creosote was freshly obtained from a nearby creosote-waste site in Pensacola, FL. At one location at this site a former evaporation pond for creosote-contaminated waste water resulted in the formation of a 2-inch layer of tar-like sludge heavily contaminated with more than

50% (weight) methylene chloride-extractable organics. This layer was located approximately 6 inches below the soil surface. Soil immediately adjacent to this sludge was collected from depths of 4 to 8 inches and was used as the source of microorganisms. Detailed reports on the history of creosote use, type and amount of pollutants present, and the extent of environmental contamination at this site are available (Godsy, E.M., and D.F. Goerlitz [1986] In A.C. Mattraw, Jr., and B.J. Franks [eds.], USGS survey of toxic wastes - groundwater contamination program. USGS Water Supply Paper No. 2285, Chapter H, pp. 55-58; Pereira, W.E., and C.E. Rostad [1986] USGS Water Supply Paper No. 2285, Chapter E, pp. 33-40; Troutman, D.E., E.M. Godsy, D.F. Goerlitz, and G.G. Ehrlich [1984] "Phenolic contamination in the sand and gravel aquifer from a surface impoundment of wood treatment wastes, Pensacola, Florida," USGS Water Resources Invest. Report No. 84-4231, 36p.)

Example 2 - Mineral Salts + PAH Medium

The mineral salts (MS) medium used consisted of (mg L): (NH ₄ ) ₂ S0 ₄ = 1000; KH ₂ P0 ₄ = 200; MgS0 ₄ -7H ₂ 0 = 200; CaCl ₂ -2H ₂ 0 = 100; FeCl ₃ -6H ₂ 0 = 5; (NH ₄ ) ₆ Mo ₄ 0 ₂₄ *4H ₂ 0 = 1. To achieve the aqueous PAH concentrations reported in Table 1, TWEEN™80 (Fisher Chemical Co.) was added at 200 mg/L. The pH was adjusted to pH=7.0 with 0.1 N HC1 and the medium was sterilized (104 kPa,

121°C, 20 min.) prior to the addition of organic substrates. Polycyclic aromatic hydrocarbons (Sigma Chemical Company) used were of the highest purity (>98%) available).

To prepare an aqueous solution containing a defined mixture of PAH's closely related to the PAH composition of creosote, the appropriate amount of each compound (Table 1) was added to a sterile flask and dissolved in 5.0 ml methylene chloride to effect sterilization. Methylene chloride was removed under a stream of dry nitrogen passed through a 0.25 μm filter and the PAH's were dissolved by mixing with a magnetic stir bar into an appropriate amount of sterile MS medium. After mixing for 6 hours at room temperature, the medium designated MS+PAH was filtered through a layer of sterile glass wool to remove

undissolved solids. Medium was stored at 1°C in sterile, 1.0 L Wheaton bottles fitted with Teflon-lined screw-caps. The concentration of each compound was determined by capillary gas chromatography of extracted samples as described in following sections.

Table 1. Composition of a defined polycyclic aromatic hydrocarbon (PAH) mixture and its relationship to predominant PAH's found in coal-tar creosote.

Aqueous PAH concentration in

Peak abbreviation solubility ² defined PAH coal tar number ¹ compound (if used) (25°C) mixture ³ creosote^

- mg/L mg/L - range % -total PAH

17.1 3.0 - 15.8

17.1 2.1 - 14.2

16.0 2.1 - 14.2

5.8 2.3 - 2.8

2.1 2.0 - 2.3

1.9 2.0 - 2.4

3.8 4.1 - 9.0

3.9 8.6 - 10.0

7.0 4.6 - 21.0

2.7 1.5 - 2.0

0.2 0.5 - 2.6

0.9 0.1 - 1.0

8.7 6.8 - 10.4

2.3 2.2 - 8.5

0.4 2.0 - 4.6

0.3 2.8 - 3.0 1.2 0.1 - 1.0

TOTAL: 91.4

¹ order of elution through capillary column SBP-5 (Supelco)

² Baker, RJ., W.E. Acree, Jr., and C.C. Tsai (1984) Quant StrucL-AcL Relat.3:10-16; Mackay _. D., and W.Y. Shiu (1977) J. Chem. Eng. Data 22:399-402.

³ Increased solubility in the presence of 200 mg/L TWEEN™80

⁴ Ranges based on analyses by Andersson, K., J.O. Levin, and CA. Nilsson (1983) Chemosphere 12:197-207; Becker, G. (1977) Proceed. .Am. Wood-Preservers' Assoc. 73:16-25; Borowitzky, H., and G. Schomburg (1979) J. Chrom. 170:99-124; Lorenz, L.J., and L.R. Gjovik (1972) Proceed. Am. Wood-Preservers' Assoc. 68:32-41; Nestler, F.H.M. (1974) Fuel 60:213-220; Novotny, M., J.W. Strand, S.L. Smith, D. Wiesler, and FJ. Schwende (1981) Fuel 60:213-220.

Example 3 - Fluoranthene (A Recalcitrant Chemical^ Enrichment Cultures

A MS + fluoranthene nutrient medium plus 0.5 % glucose was prepared with the following modifications: (1) an excess of fluoranthene (approximately 500 * 5 mg/L) was supplemented for the other organic components shown in Table 1; (2) suspended solids were not removed; and (3) TWEEN™80 was not added. Fifty ml of this medium were transferred to a 250 ml screw-cap Erlenmeyer flask and inoculated with 1.0 g (wet weight) creosote-contaminated soil passed through a 50- mesh sieve. Flasks were incubated in the dark (28+TC, 175 cycles/min) under

10 controlled conditions. After 5 days incubation, a 5.0 ml aliquot was diluted 1:10

(vol vol) with fresh MS+fluoranthene broth and incubated for 3 days. Subsequent samples were diluted 1:50 with the same medium every 3 days. Following several such transfers, disappearance of undissolved fluoranthene crystals was visually apparent. Fluoranthene-utilizers were maintained by regularly diluting established

15 cultures 1:50 with fresh MS+fluoranthene broth every 14 days.

Example 4 - Partial Characterization of Fluoranthene-Utilizing Microbe Consortium

Numerous aliquots from fluoranthene-enrichment cultures of various ages

20 were streaked for isolation on Nutrient Agar (Difco, Detroit, MI) amended with

0.5% glucose (NAG agar). After 5-14 days incubation at 28°C, colonies representative of each of the different morphological types were removed and the single colonies repeatedly purified on NAG agar. Ultimately, 7 morphologically distinct, Gram-negative bacteria were isolated in pure culture. These organisms

25 were designated EPA50FAE 1, 2, 3, 4, 5, 5b, and 6.

To ensure that all organisms essential for fluoranthene-utilization had been isolated, MS+fluoranthene broth was inoculated with all seven isolates to reconstitute the consortium, and fluoranthene degradation was assessed. The consortium was reconstituted by removing single colonies of each organism from

30 NAG plates and suspending them in sterile MS medium to uniform density

(%T ₆₀₀ =50±2.0). Fifty ml of MS+fluoranthene broth were inoculated with 0.2 ml of each suspension and incubated at 28°C with aeration (175 cycles/min). Fluoranthene utilization was qualitatively assessed by recording visually apparent increases in microbial biomass, spectral (color) changes, and disappearance of fluoranthene crystals. The sequence of color changes in the medium was from colorless to bright orange to bright yellow to a light brown which was maintained after fluoranthene crystals were no longer visible. Exhausted cultures to which additional fluoranthene was added completed this sequence in two days. Qualitative increases in microbial biomass were evident. However, since the fluoranthene cultures exhibited a strong tendency to form rapidly settling clumps, increases in microbial biomass could not be measured quantitatively.

When plated on a complex medium such as NAG agar (Nutrient Agar, Difco, amended with 0.5% glucose), a total of seven morphologically distinct, Gram-negative bacteria were isolated. This 7-membered consortium (CRE1-7) maintained its integrity for creosote PAH's remediation throughout the enrichment procedure and through repeated serial transfers, thereby reflecting stability. When MS+fluoranthene broth was inoculated with the reconstituted consortium, fluoranthene degradation was again obvious. However, there was an initial lag of 5 to 7 days before fluoranthene degradation became apparent. After this period, fluoranthene degradation was rapid (<2 days).

Table 2 summarizes percent recovery from MS+PAH broths of 17 PAH's present in the defined mixture 3 days after inoculation with either the fluoranthene-induced consortium or with P. putida PpG7. Extraction efficiencies and losses attributable to abiotic processes were accounted for by comparing recovery values for each compound with that obtained from the killed cell controls.

With the exception of naphthalene (84.3%) and 2,3-DMN (78.9%), percent recovery from abiotic controls was greater than 85%.

The ability to detect selective utilization of individual components of the defined mixture was demonstrated with the culture inoculated with PpG7. After 3 days incubation, only naphthalene and 2-MN were extensively degraded. These

data were identical to those obtained after 5, 8, and 14 days incubation (data not shown).

When the consortium was grown on fluoranthene and subsequently exposed to fluoranthene plus 16 other PAH's, the fluoranthene-induced consortium exhibited the ability to degrade all of the PAH's present in the defined mixture

(Table 2). After 3 days incubation, 13 of the original 17 PAH's were degraded below the limits of detection (10 ng/L). Additionally, greater than 90% degradation of anthracene and anthraquinone was evidenced by their percent recoveries, 1.5 (±1.5) and 6.5 (±6.5), respectively. The remaining 2 compounds, fluoranthene and pyrene, were also degraded as demonstrated by respective recoveries of 69.5 (±13.6) and 44.3 (±8.5).

Table 2. Biode radation of 17 PAH's b a 7-membered fluoranthene-induced bacterial

^See Table 1 for abbreviations used. fluoranthene-induced bacterial consortium killed with 5% formaldehyde (37% formalin solution) at the time of inoculation.

³ ND = not detected (<0.01 mg/L).

With continued incubation, further degradation of the 4 compounds which were still present at 3 days was observed (Table 3). Following 5 days of incubation, anthracene and anthraquinone were no longer recoverable. The amount of fluoranthene extractable after 5, 8, and 14 days incubation decreased

from 52.1 to 41.6 to 16.8%, respectively. Similarly, recovery of pyrene after 5, 8, and 14 days incubation decreased from 43.9 to 17.4 to 12.0%, respectively.

The relatively high recovery of fluoranthene from tubes inoculated with the

« fluoranthene-induced control requires clarification. Consortium biomass for _i 5 inoculation was generated in MS+fluoranthene broth which contained an excess of insoluble fluoranthene (500 mg/L). It was later determined that there was a significant carry-over of fluoranthene from the cultures. It could be calculated that those tubes inoculated with the fluoranthene consortium received an additional 0.38 mg of fluoranthene (75.1 mg/L) resulting in an initial fluoranthene 10 concentration of 83.8 mg/L. Therefore, after 3 days incubation, the fluoranthene- induced consortium had degraded 30% of the total amount of fluoranthene originally present or 0.13 mg fluoranthene.

Table 3. Continued loss of PAH's remaining after 3 days incubation.

15

Compound % recovery of PAH's after extended incubation with the fluoranthene- induced consortium

Day 3 Day 5 Day 8 Day 14

20

25

1N NDΓ» = = not detected (<0.01 mg L).

30

The following Table 4 gives a partial characterization of the bacterial consortium:

35

Table 4. Partial characterization of the bacteria comprising the fluoranthene- utilizing community.

Strain designation Colony morphology ¹ Gram reaction

FAE1 ² white, 1-2 mm mucoid negative rods

FAE2 light brown, 3-4 mm, mucoid negative cocci FAE3 colorless, 1-2 mm, mucoid negative cocci

FAE4 white, 3-4 mm, slime producing negative rods

FAE5 bright yellow, 1-2 mm, mucoid negative rods

FAE5b opaque yellow, <1 mm, mucoid negative rods

FAE6 white, 4-5 mm, spreading positive cocci

¹ Colony morphology after 5 days incubation at 28°C on NAG agar.

² These strains are also designated CREl-7.

Example 5 —Preparation of Fluoranthene-induced Cell Suspensions of Consortium Fifty ml of MS+fluoranthene broth were transferred aseptically to a clean, sterile 250 ml Erlenmeyer flask fitted with Teflon-lined screw-caps, inoculated with the reconstituted bacterial consortium, and incubated (28°C, 175 cycles/min) for 10 days. After 10 days incubation, cultures were diluted 1:100 (vol vol) in fresh MS+fluoranthene broth. Fluoranthene degradation was visually apparent after 2 days incubation at which time the consortium was diluted 1:25. Following 3 days incubation, fluoranthene-induced cells were concentrated (10,000 g, 10 min, 4°C) and resuspended in 1/10 vol MS medium.

Example 6 - Action of Consortium Cells Towards PAH's Suspensions of fluoranthene-induced cells of the consortium (100 μϊ) were used to inoculate 5.0 ml MS+PAH broth in clean, sterile 50.0 ml test tubes fitted

with Teflon-lined screw-caps. Killed cell controls were generated by adding 250 μ\ of a 37% formalin solution to 8 of the 16 tubes inoculated with the fluoranthene-induced consortium. Uninoculated controls were also incorporated. In addition, 8 tubes containing MS+PAH broth were inoculated with 100 μ\ of cell ) 5 suspension of Pseudomonas putida PpG7 (a gift from Dr. I.C. Gunsalus, University of Illinois) which, in preliminary studies, demonstrated the ability to selectively utilize only 2 compounds (naphthalene and 2-methylnaphthalene) present in the defined PAH mixture. After 3, 5, 8, and 14 days incubation (28°C, 200 cycles/min), duplicate tubes of each treatment were removed and extracted from 10 determination of PAH's present.

Example 7 - Methylene Chloride Extraction Procedure

At selected times, MS+PAH broth in a given tube was transferred to a clean, methylene chloride-rinsed, 15 ml glass conical extraction tube fitted with a

15 Teflon-lined screw-cap. The original incubation tube was rinsed with 2.0 ml methylene chloride which was added to the extraction tube. Tubes were shaken for 1.0 min to facilitate the extraction of unmetabolized PAH's into the organic phase. Methylene chloride was separated from the aqueous phase after centrifugation (2500 g, 5 min). The entire separated organic phase (2.0 ml) was

20 removed employing a methylene chloride-rinsed, 1.0 ml glass syringe fitted with a blunt-end needle, and transferred to a clean, solvent-rinsed concentration tube. The extraction procedure was repeated 2 more times with 0.5 ml methylene chloride. The final volume of methylene chloride (3.0 ml) was reduced to <1.0 ml under a stream of dry nitrogen. After the final volume of methylene chloride

25 was adjusted to 1.0 ml, each extract was spiked with 10 μl of a 1 -naphthoquinone solution (10 mg/ml methylene chloride) as a marker and transferred to a GC vial for subsequent analysis.

30

Example 8 — Capillary Gas Chromatography

Gas chromatographic analysis of methylene chloride extracts and of individual PAH standards was performed on a Hewlett-Packard model 5710A gas chromatographic equipped with a flame ionization detector. Hydrogen was used as carrier gas (0.5 ml/min) while air (240 ml/min) and hydrogen (30 ml/min) was supplied for the flame ionization detector. Polycyclic aromatic hydrocarbons in replicate 1.0 μl injections were separated on a 15.0 m x 0.32 mm LD. SPB-5 (Supelco, Bellefonte, PA) capillary column with a 0.25 μm coating phase. Oven temperature was programmed at 80°C for 2 min followed by a linear increase of 8°C/min to 280°C where it was held for 4 min (30 min run). Injector and detector temperatures were maintained at 270°C. Percent recovery of each PAH was calculated by comparing peak area with that of standards for each compound.

Example 9 - Cloning of Structural Genes A genomic library of partial digestion of a specified endonuclease such as the CRE isolates CRE 1 through CRE 7 as well as EPA 505sc are constructed using BamHl or Sau3A yielding 23 kb or 45 kb nucleotide fragments. The cloning vectors similar to pcoslEMBL, containing the col El origin or other gram-negative replication origins with antibiotic resistant markers are used in the construction of the genomic libraries. The BamHl endonuclease site, as well as other endonucleases that insertionally inactivate an antibiotic structural gene, are used in the construction of the cosmid library. The libraries are constructed in a gram- negative host (initially in E. coli K12) deficient in recombination (rec A~) as well as host restriction modification minus (hsd ^~ ), and endonuclease one deficient (end 1 ^~ ).

Identification of the cosmid derivative plasmid bearing strains for creosote, coal tar, fossil fuel, PCB (biphenols) and other heterocyclic pollutants are verified and amplified for the nucleotide sequences associated with bioremediation of the above-mentioned pollutants. Specific DNA sequences are subcloned into specified shuttle vectors and placed into the appropriate host for expression of the desired enzyme products for degradation of the organic hazardous waste.