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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
  • B09C—RECLAMATION OF CONTAMINATED SOIL
  • B09C1/00—Reclamation of contaminated soil
  • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/26—Processes using, or culture media containing, hydrocarbons
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  • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
  • C02F3/00—Biological treatment of water, waste water, or sewage
  • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
• • C—CHEMISTRY; METALLURGY
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/05—Alcaligenes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/38—Pseudomonas
  • C12R2001/40—Pseudomonas putida
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/41—Rhizobium
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/425—Serratia
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/44—Staphylococcus

Definitions

• This invention is directed towards bacterial strains useful for bioremediation and processes for using the bacterial strains.
• it relates to unique bacterial isolates that can degrade polyaromatic hydrocarbons (PAHs) and methods to use these novel bacterial strains for bioremediation. Remediation of lead by reducing bioavailability has also been demonstrated by one of the strains indicating dual use for organic and inorganic remediation.
• PAHs polyaromatic hydrocarbons
• PAHs Polyaromatic hydrocarbons
• US Pat. No. 6,503,746 to Daane describes bacterial strains in the family Bacillaceae which are used in PAH remediation efforts.
• US Pat. No. 5,100,455 to Pickard discloses using indigenous microflora and fauna in combination with humic substrates to biologically treat soil contaminants including petroleum associated hydrocarbons.
• US Pat. No. 4,849,360 to Norris discloses a bioreactor for treating petroleum contaminated soil in which air is forced through the contaminated soil to facilitate the bioremediation. The bioreactor uses indigenous microflora which are supplemented with phosphorus and nitrogen nutrients.
• PAH-degrading bacteria While a variety of PAH-degrading bacteria are known and have been utilized in various applications for remediation, there remains a need for improvement in the art in terms of identifying new and useful species having novel properties which are effective for the rapid degradation of petroleum pollutants. Further, there remains a need for improvements directed to the field of metal remediation such as by reducing the bioavailability of metal present within contaminated soils, metal-containing waste streams, and industrial process materials.
• the present invention relates to methods for the degradation of petroleum pollutants including polyaromatic hydrocarbons (PAHs). Additionally, the present invention relates to a biotreatment process which enhances the removal of heavy metals from soil.
• the present invention uses isolated and purified bacterial strains of bacterial isolates from an oil refinery field. Certain of the isolates having a further ability to produce useful biosurfactants. It is one aspect of at least one of the present embodiments of the present invention to provide isolated bacterial strains that produce biosurfactants under in situ and ex situ remediation conditions. The innate ability of the isolated and purified bacterial strains to produce biosurfactants contributes to the remediation properties of the bacteria.
• the biosurfactant provides increased solubility of PAHs and access of the bacteria to the PAHs, thereby increasing the efficiency of the bioremediation by the bacteria strains.
• the biosurfactant can also reduce soil Pb bioavailability and is believed to have utility in bioremediation of other metal contaminants which also respond to surfactant treatments.
• An additional aspect of at least one of the embodiments of the present invention is related to isolated and purified strains of bacteria in which the surfactant producing properties contribute to enhanced solubilization of petroleum and petroleum-derived products.
• the biosurfactants increase the solubilization of the petroleum products which promotes the aqueous flushing or removal of petroleum products associated with biosurfactant aggregates such as micelles and related structures. Further, the biosurfactants also increase the bioavailability of petroleum products that enhance the microbial ability to degrade contaminants. The enhanced bioavailability is beneficial to the isolated and purified strains as well as other beneficial microorganisms present in the contaminated substrate.
• PAHs such as naphthalene, phenanthrene, and fluoranthene
• PAHs including pyrene and fluoranthene.
• the 4-ring and higher PAHs are much more persistent in the environment and resistant to degradation compared to low molecular weight PAHs. Accordingly, the ability to provide strains of bacteria which degrade 4-ring and higher high molecular weight PAHs is significant.
• the PAH degradation intermediates may further function as metal chelators. This chelating activity or metal complexation may assist remediation in waste containing both PAHs and metals. Additionally, at least some of the bacterial strains identified herein have an ability to degrade several different types of PAHs (including 2-, 3-, and 4-ring PAHs) in addition to the ability to degrade phenanthrene.
• the isolated and purified strains of bacteria produce biosurfactant monomers.
• the biosurfactant monomers are produced in sufficient quantity that the monomers aggregate into three-dimensional structures including micelles.
• the biosurfactant micelles define polar head groups which bind with metal ions in the soil.
• the micelles, containing the metal ions, can be removed by aqueous suspensions or flushing, thereby lowering the metal ion content of the substrate.
• the resulting removed metals, contained within the biosurfactant micelles, are then more easily separated and concentrated for efficient disposal or storage.
• TPH total petroleum hydrocarbons
• the ability of certain of the isolates to produce a biosurfactant during bioremediation conditions increases the bioavailability of petroleum hydrocarbons to other microorganisms that may be present within the contaminated soil or other waste product.
• the biosurfactant produced by one or more of the isolates may be used in combination with inorganic phosphates to reduce the bioavailability of lead within a contaminated substrate such as soil.
• aspects of at least one embodiment of this invention include a process of bioremediation of petroleum pollutants from a contaminated environment comprising the steps of providing a supply of a substrate contaminated with a petroleum pollutant; introducing into the supply of contaminated substrate at least one bacteria isolate which metabolizes constituents of the petroleum pollutant and which further produces a biosurfactant; and, providing adequate nutrients for a treatment time sufficient for the petroleum pollutant utilizing isolate to degrade the petroleum pollution to a target concentration of 100 ppm TPH or less.
• Figure 1 sets forth a graph showing the bioavailability of lead with respect to various additives and biological isolates with respect to highly contaminated soil (900mg Pb/Kg soil).
• Figure 2 is a graph setting forth the changes in total organic matter within soil samples following a treatment protocol.
• the present invention is directed to bacterial isolates obtained from a century-old Czechowice oil refinery in Tru.
• the aged sludge from the oil refinery is characterized by its acidic (pH 2) properties and contains high concentrations of PAHs along with heavy metals. Additionally, the sludge is characterized by the presence of spent catalysts, asphaltics, diatomaceous earth, silica gel, and coal fly ash, all containing high background levels of heavy metals (Pb/Cd/Zn) which have been previously deposited at the site.
• the collection site is from an area having approximately 120,000 tons of waste material deposited in unlined lagoons 3 meters deep covering an area of 3.8 hectares. A total of 45 bacteria, 68 fungi, and 7 yeast species were isolated from the sludge on an acidic minimum medium (pH 4) exposed to naphthalene vapor.
• BIOLOGTM taxonomic criteria
• SSU rRNA genes included Proteo bacteria, Ralstonia, Pseudomonas, and Alcaligenes species. Further characterizations of the isolates may be seen in reference to the information provided in Tables 1 and 2.
• BIOLOGTM characterization protocols using minimal nutritional factors along with various organic substrates of interest are described in reference to the publication Use of BIOLOGTM Technology for Hazardous Chemical Screening, Microbiological Techniques 18:329-347, 1993, and which is incorporated herein by reference.
• a total of 45 bacteria, 68 fungi, and 7 yeast species were isolated using a naphthalene vapor acidic mineral salts basal growth medium. While not separately set forth, it is noted that many of the isolates have the ability to metabolize catechol and the bacterial isolates were characterized by the ability to degrade PAHs. Additionally, it is noted that the isolated and purified organisms having the ability to degrade PAHs also have the ability to degrade a variety of petroleum pollutants associated with measurements of total petroleum hydrocarbons.
• CZORL1B three bacterial species designated CZORL1B, BP20, and CZORUBsm, and which correspond to isolates 1 through 3 in Table 2, were observed to produce a surfactant when grown in a minimal medium containing naphthalene, phenanthrene, or fluoranthene.
• Nine additional strains identified in Table 1 were observed to degrade a range of PAHs indicating the isolates have a catalytic or enzymatic ability to degrade the contaminants although the additional isolates do not demonstrate an ability to produce a surfactant.
• the above identified bacterial strains are grown and maintained on 1 percent peptone, trypticase, yeast extract, glucose (PTYG) plates.
• the bacteria were grown aerobically at 30° C and maintained on a minimal medium at 4° C or long-term storage in a frozen medium maintained at -7O 0 C or in liquid nitrogen (- 196 0 C).
• the identification of the bacteria was made using rDNA or Fatty Acid
• PB15 indigo NAP was designated BP20
• indigo production of indigo from indole; meta fission, 2,3 catechol dioxygenase activity
• NAP naphthalene
• PHE phenanthrene
• ANT anthracene
• FLE fluorene
• ACE acenanphthene
• FLA fluoranthene
• PYR pyrene
• the above identified bacteria isolates have been established as distinct species. Each of the identified isolates has PAH-degrading properties and have demonstrated an ability to reduce TPH in soil as well. In addition, certain isolates have the ability to produce a biosurfactant. Each isolate is believed novel, based upon the rDNA characterization and variations noted in Tables 1 and 2 with respect to physiological growth characteristics.
• PTA-5579 (Ralstonia pickettii SRS); and ATCC PTA-5581 ⁇ Psuedomonas putida Biotype B SRS) identified above, all demonstrate the ability to produce a biosurfactant, the formation of which was noted during culturing conditions.
• the biosurfactant exudate was evaluated for each isolate and determined to have a surface tension altering property consistent with a surfactant.
• Isolates 4-12 all demonstrate the ability to biodegrade a variety of PAHs (Table 1 ).
• Example 1 the use of a consortium of the twelve isolates identified in Table 2 to remediate petroleum hydrocarbons contained in soil in a bioreactor remediation study results in visible quantities of biosurfactants being produced under the bioremediation conditions.
• the ability of certain of the isolates to produce bioreactants is believed to enhance remediation through several different mechanisms.
• the production of the biosurfactant increases the biological availability of PAHs and other hydrophobic petroleum compounds.
• the increased biological availability includes the ability of the produced surfactant to solubilize and make available to the isolate the PAHs and other petroleum compounds.
• the isolates' ability to produce surfactants increases the efficiency of the isolates to degrade and metabolize PAHs.
• Biosurfactants are generally known to have a chemistry consisting of a polar head and a non-polar tail.
• biosurfactants serve to reduce liquid surface tension and to facilitate the formation of an emulsion between liquids of different polarities. This ability facilitates the biosurfactants' usefulness in that hydrophobic, non-polar tail regions of the biosurfactants and biosurfactant micelles may trap oils and other petroleum compounds.
• the trapped oils and petroleum compounds have greater bioavailability to bacteria for biodegradation.
• micelles containing trapped oils and petroleum compounds may be periodically removed or flushed from the system, thereby providing an ability to further isolate and separate petroleum compounds from the soil substrate.
• micelles formed by the biosurfactants promote the removal of metals from the soil.
• the hydrophilic polar head groups of micelles will bind metal and metal ions present within the soil. Once bound, the soluble nature of the micelles allows the micelles and bound metals to be collected. Once collected, the now concentrated volume of micelles and contained metals can be further treated to separate the metals from the biosurfactant.
• selected isolates which produce biosurfactants can be used to reduce the bioavailability of certain metals such as lead present within the soil.
• the surfactants are not used to remove a contaminant, but rather provide a method of long term stabilization of a contaminated site. Stabilization includes reducing the bioavailability of lead along with preventing the migration of lead in surface runoff to adjacent, uncontaminated sites and groundwater.
• a mobile bioreactor was constructed and was supplied with a four ton volume of soil contaminated with low level cesium-137 and 26,000 ppm petroleum hydrocarbons.
• the contaminated soil was weathered material obtained from the Savannah River Site (Aiken, SC).
• the source and make up of the petroleum products is unknown but believed to be a mixture of used motor oil and diesel fuel.
• the soil was amended with a 7% bulking agent of aged compost.
• a three liter culture in log growth phase was added and distributed within the four tons of mixed waste soil.
• the bioreactor is equipped with a raised, secondary, perforated floor having bottom feed aeration lines which provide a continuous supply of ambient air to the bioreactor.
• periodic nutrient supplements of nitrogen, potassium, and phosphorus fertilizers (10-10-10) were applied to enhance the biological activity within the bioreactor.
• Influent and effluent water couplings were attached. Air compressors, vacuum pumps, and a liquid pump were used to control and regulate the air and liquid flows through the bioreactor and control moisture content in the bioreactor.
• the present isolates may, either individually or as a consortium, be used with conventional bioremediation techniques to improve the efficiency of degradation.
• the action of the biosurfactants creates a greater zone of petroleum solubility for each individual bacterium.
• a greater availability of petroleum products occurs.
• the surfactants are believed to substantially increase the bioavailability of the petroleum substrates for the bacterial aggregates.
• the surfactants also increase the solubilization of heavy metals that may be present and provide an ability to reduce the heavy metal concentration by removal of the produced surfactants.
• the consortium of isolates provides for a bioremediation process which can achieve significant reductions in petroleum from contaminated soil. This property is particularly useful with respect to formulating disposal strategies for mixed waste in which petroleum contaminated soil and low- level radioactive material are present together.
• soil contaminated with low-level radioactive waste and having additional petroleum contaminants must be below regulatory limits of 1 ppm for BTEX (benzene, toluene, ethylbenzene, xylene, and 100 ppm TPH (Total Petroleum Hydrocarbons) before the soil can be classified and disposed of as a low-level radioactive waste.
• BTEX benzene, toluene, ethylbenzene, xylene, and 100 ppm TPH (Total Petroleum Hydrocarbons)
• the use of the present inoculants is further advantageous in that, unlike some prior art techniques, the volume of amendments to the soil is kept at a minimum. Keeping the volume of soil amendments to a minimum reduces the eventual disposal costs, particularly for soil containing low-level radiation.
• the present isolates are also believed useful for in situ remediation projects.
• the consortium of isolates may be supplied to contaminated soil using any number of conventional techniques. As needed, nutritional supplements along with the supply of oxygen in either a physical or chemical form facilitates the bioremediation activity. Given the isolates' ability to degrade PAHs as well as the desired ability to degrade petroleum hydrocarbons generally, in situ remediation using the isolates is advantageous.
• biosurfactant enhances the ability to physically entrap petroleum products and heavy metals as well as providing for increased solubilization and access of petroleum hydrocarbons to both the bacterial isolates as well as native microorganisms present within the soil environment.
• biosurfactants can bring about useful results in a variety of contaminated substrate conditions.
• biosurfactants have useful metal-complexing properties that can both reduce metal toxicity while allowing enhanced biodegradation of other waste in sites contaminated with both organic and metal pollutants.
• One such publication entitled “A Rhamnolipid Biosurfactant Reduces Cadmium Toxicity during Naphthalene Biodegradation", by Sandrin et al, Applied and Environmental Microbiology, Oct. 2000, pp 4585-4588, and which is incorporated herein by reference, describes the ability of biosurfactants to reduce metal toxicity in co- contaminated systems having both organic and metal pollutants.
• Certain isolates disclosed herein have demonstrated an ability to reduce the bioavailability of lead within a contaminated soil.
• Lead contaminants in soil may occur as a result of numerous activities including mining, presence of lead- based paint, ordnance from firing ranges, contamination from gasoline and petroleum additives, lead battery recycling centers, and residue from lead-based explosives.
• Lead exposure is associated with numerous disorders human nervous and reproductive systems. Lead usually enters the body through inhalation or ingestion of lead-containing dust.
• lead containing soils can be treated with the use of soil amendments to reduce the bioavailability of lead.
• Some such amendments include apatites, HR, and other calcium phosphate containing materials.
• Apatites are known to bind with lead and other metals within soil and thereby reduce the tendency of lead to migrate. The binding interaction also reduces the bioavailability of lead.
• Selected surfactants may also be used to reduce the bioavailability of lead as demonstrated by isolate 3.
• isolate 1 Alcaligenes piechaudii
• isolate 3 Ps ⁇ udomonas putida
• the lead contaminated soil was obtained from a small arms firing range where the lead bullets had accumulated in the soil over a number of years. 50 gm samples of contaminated soil were mixed with 3 different calcium phosphate amendments.
• the calcium phosphate amendments included a naturally occurring rock calcium phosphate from North Carolina (USA) hereinafter designated as NCA, a naturally occurring rock calcium phosphate from Florida (USA) hereinafter designated as FA, and a biological apatite obtaining from ground fish bones, hereinafter designated as BA. 10% and 5% by weight additions of the NCA, SA, and BA were made to the respective samples of the lead contaminated soils. Additionally, certain of the amended soil samples were further treated by the introduction of 5 ml samples of the isolate number 1 , Alcaligenes piechaudii in a 1% PTYG nutrient broth. Density of the isolate was 3.11 E + 08 cells per ml.
• Additional samples were also treated with isolate number 3 Pseudomonas putida using a similar 5 ml addition and a density of 3.43 E + 08 cells per ml containing samples for the microbial amendment included a 1% PTYG sterile broth.
• the contaminated soil samples and various amendments and cultures were mixed together in sealed containers which were connected by tubing to a Micro-Oxymax Respirometer (Columbus Instruments, Columbus, OH) for determination of metabolic rates includes oxygen consumption and CO 2 evolution.
• Microbial density was obtained through conventional plating techniques for determining colony and sample density.
• the addition of isolate 3 achieves a significant decrease in lead bioavailability as measured through a toxicity characteristic leaching procedure (TCLP) using protocols identified in the U.S. Environmental Protection Agency Method 6010.
• TCLP toxicity characteristic leaching procedure
• the microbial amendments brought about significant reductions in lead bioavailability when used with the 5% soil amendments of NCA or BA.
• use of the biological apatite in combination with the isolate 3, or combinations thereof offers an excellent reduction for the bioavailability of lead within soil.
• the combination of the biological apatite with the naturally producing biosurfactants from isolate 3 bacteria brings about other noted improvements to the contaminated soil samples.
• the use of the isolates described above bring about a decrease in bioavailability of lead due at least in part to the surfactant producing properties of the isolates.
• the biosurfactants are believed to accentuate the inherent tendencies of the apatite with respect to increasing metabolic rates, nutrient availability, and favorable increases in pH and lowering of lead bioavailability.
• the biosurfactants bring about favorable soil conditions that improve nutrient conditions and availability of micronutrients for micro-organisms.
• the increase in biological activity and associated biomass within the soil is believed to further decrease the lead bioavailability.
• the P. putida activity of lowering soil lead bioavailability is synergistic with BA at the 5% concentration.
• the exponential increase in respiration rate, microbial density and increase in biomass is clear evidence that the activity is related to specific biological activity.
• the isolates are useful for reduction of PAHs as well as facilitating the removal and/or sequestration of mineral ions from contaminated soils and substrates. Accordingly, for contaminated soils and waste streams having a variety of contaminants including PAHs, metal ions, and other metals such as lead, use of one or more of the present isolates, including isolates producing a biosurfactant, can effectively treat multiple contaminants within a given site. While the above examples were given in the context of remediation of a highly contaminated soil (900mg Pb/Kg soil), it is believed that the utility of individual isolates or combinations of isolates is not limited to soil remediation efforts perse.
• the isolates producing biosurfactants can be used as part of a waste stream treatment process of a mine or industrial facility where heavy metals, metal ions, lead, copper, cadmium, zinc, other metals, and other contaminants are generated.
• Many sites i.e., landfills and brownfields
• the use of inoculants which are incorporated into an initital waste stream and which include one or more of the above isolates can bring about a reduced volume of contaminants within the waste stream and introduce a microbial flora population which can persist as part of a longer term treatment protocol for generated and/or stored waste.
• mine tailings can be initially treated to reduce certain contaminants prior to being deposited onto a disposal area. Afterwards, the presence of the isolates within the mine tailings can be used to advantage by further treatment designed to promote the useful biological activity so as to bring about a continuing reduction and/or stabilization of the contaminants present in the waste stream.

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