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||||||III USOO5508194A United States Patent (19) 11 Patent Number: 5,508,194 Lee Et Al ||||||III USOO5508194A United States Patent (19) 11 Patent Number: 5,508,194 Lee et al. 45 Date of Patent: Apr. 16, 1996 54 NUTRIENT MEDIUM FOR THE 4,962,034 10/1990 Khan. BIOREMEDIATION OF POLYCYCLIC 4,992,174 2/1991 Caplanet al.. AROMATIC 5,017,289 5/1991 Ely et al.. HYDROCARBON-CONTAMINATED SOL 5,059.2525,030,591 10/19917/1991 Renfro,Cole et al.Jr. 75 Inventors: Sunggyu Lee; Teresa J. Cutright, both 3.ww. AS SEonlux et. al. of Akron, Ohio 5,114,568 5/1992 Brinsmead et al. 73) Assignee: The University of Akron, Akron, Ohio 5,409,830 4/1995 Lim et al. ............................ 435/252.8 Primary Examiner-Herbert J. Lilling 21 Appl. No.: 444,161 Attorney, Agent, or Firm-Renner, Kenner, Greive, Bobak, Taylor & Weber 22 Filed: May 18, 1995 57 ABSTRACT Related U.S. Application Data A mixed bacteria culture for biodegrading polycyclic aro A matic hydrocarbon contaminants includes Achromobacter 62) Rigif Ser. No. 248,373, May 24, 1994, Pat. No. sp. and Mycobacterium sp. which have been grown together and gradually acclimated to utilize polycyclic aromatic (51 Int. Cl." ........................... C12N 1/20; C12N 1/26 hydrocarbons as a primary food source. The mixed bacteria 52 U.S. Cl. ......................... 435/253.6; 435/42, 435/262: culture can be utilized for in situ or ex situ bioremediation 435/262.5; 435/2524; 435/824; 435/863 of contaminated soil, or in any of various conventional 58 Field of Search ................................. 435/253.6, 262, bioreactors to treat contaminated liquids such as landfill 435/262.5, 42, 252.4, 824, 863 leachates, groundwater or industrial effluents. The bacteria, the nutrients used to sustain growth of the bacteria, and the 56) References Cited products of the biodegradation of the polycyclic aromatic or U.S. PATENT DOCUMENTS other hydrocarbons are all substantially harmless to the environment. The mixed bacteria can be utilized in the 3,300,405 l/1967 Black. presence of oxygen, or hydrogen peroxide can be used alone 3,871,957 3/1975 Mohan et al. or in combination with oxygen as an effective alternative 3,935,067 1/1976 Thayer ................................. 435/253.6 electron acceptor. The mixed bacteria culture of Achromo 4,450,908 5/1984 Hitzman. bacter sp. and Mycobacterium sp. provides an environmen 4,511,657 4/1985 Colarutolo et al. tally safe and affordable means for rapidly and effectively 4.554,0754,655,926 11/19854/1987 Chang et al.al.. eliminating a varietyy of polpolycyclic li aromatictic nyarocarponhyd b 4,761,376 8/1988 Kulpa et al. contaminants from the environment. 4,765,902 8/1988 Ely et al. 4,803,166 2/1989 Kulpa et al.. 4 Claims, No Drawings 5,508,194 1. 2 NUTRENT MEDUM FOR THE using multistep unit operations for separating and recovering BOREMEDIATION OF POLYCYCLIC the soil from the contaminants. Removal, incineration, indi AROMATIC rect thermal treatment and other methods of handling con HYDROCARBON-CONTAMINATED SOL taminated soil which involve complete excavation of the contaminated soil have the advantage that they can be CROSS REFERENCE accomplished in a relatively short amount of time. These methods are particularly attractive in those situations where This is a division of application Ser. No. 08/248,373, filed there is a substantial risk that the contamination will rapidly on May 24, 1994, now U.S. Pat. No. 5,427,944, of Sunggyu spread to adjacent property, however, in most cases such Lee and Teresa J. Cutright, for "Bioremediation of Polycy 10 methods are prohibitively expensive. clic Aromatic Hydrocarbon-Contaminated Soil". Aeration, venting and air sparging are in situ techniques for removing volatile hazardous contaminants from the soil FIELD OF INVENTION while simultaneously drawing oxygen into the contaminated The invention relates generally to the remediation of soil to enhance biodegradation. Effective removal of haz materials which have been contaminated with hazardous 15 ardous materials by evaporation is generally limited to substances. More particularly, the invention relates to biore contaminants having a relatively high vapor pressure. In mediation of soil, groundwater, and various waste effluents general, compounds such as polycyclic aromatic hydrocar containing polycyclic aromatic hydrocarbons. bons, which have a low-vapor pressure, cannot be success fully removed by volatilization. Moreover, conventional BACKGROUND 20 bioremediation techniques utilizing indigenous microorgan isms alone or in combination with naturally occurring or Over the past several decades worldwide production, genetically altered exogenous microorganisms is not always processing, storage, transportation and utilization of syn effective for degrading certain types of recalcitrant contami thetic and naturally occurring chemical substances has led to nants, such as polycyclic aromatic hydrocarbons, which are the introduction of significant quantities of hazardous mate 25 strongly resistant to biodegradation on account of their rials into the environment. Unintentional spillage of petro polynuclear chemical structure and low concentration in the leum distillates, industrial solvents and other chemical sub natural environment. stances has been caused, for example, by weathering, Thus, while various known techniques are available for chemical corrosion and accidental damage to pipes, storage the disposal or reclamation of contaminated soil, such meth vessels, processing equipment, transportation vehicles, etc. 30 ods do not generally provide a practical, affordable technol Deliberate acts and carelessness have also contributed to the ogy for remediating soil which has been contaminated with release of hazardous substances into the environment. The significant quantities of polycyclic aromatic hydrocarbons. spillage of such materials has resulted in large numbers of Accordingly, there is a need for an inexpensive, environ polluted sites and enormous volumetric quantities of soil and mentally acceptable method and means for remediating soil groundwater which have been contaminated with hazardous 35 substances. Soil contamination can cause extensive damage which has been contaminated with polycyclic aromatic to the local ecosystem by accumulating in the tissue of hydrocarbons. animals and plants, and by causing death thereto and/or mutation to the progeny thereof. Such contamination can SUMMARY OF THE INVENTION also present a serious health threat to humans, and, in 40 extreme cases, can render the contaminated area unsuitable In accordance with the present invention, there is pro for human habitation. In many cases, contaminated sites can vided a mixed culture of Achromobacter sp. and Mycobac pose a danger to adjacent property, such as by entrainment terium sp. which has been found to be especially efficient for of hazardous substances by local groundwater flow, and effecting rapid biodegradation of polycyclic aromatic hydro local laws frequently mandate remediation prior to the sale 45 carbons. The invention also relates to methods of cultivating or lease of property wherein the soil has been contaminated and acclimating a mixed culture of Achronobacter sp. and with hazardous materials. Mycobacterium sp. for biodegradation of polycyclic aro Various methods have been utilized for the treatment, matic hydrocarbons. In accordance with another aspect of remediation or disposal of contaminated soil. These methods the invention, there are disclosed nutrient solutions which generally include permanent removal of the contaminated 50 have been found to be effective for cultivating the mixed soil to a secure landfill, incineration, indirect thermal treat culture of the invention for preparation of a suitable inocu ment, aeration, venting, air sparging and bioremediation. lum, and for sustaining growth of the microorganisms after Removal of contaminated soil to landfills is no longer an they have been introduced into soil containing polycyclic attractive alternative on account of the high excavation, aromatic hydrocarbon contaminants. In accordance with a transportation and disposal costs, and because of the poten 55 further aspect of the invention, hydrogen peroxide is used as tial for residual liability. Incineration and indirect thermal an alternative electron acceptor or oxidant in place of, or to treatment can be achieved either on-site or off-site, but in supplement, oxygen. either case involves excavation, handling and treatment of The mixed culture of the invention can be used for substantially all of the contaminated soil as well as signifi treating soil in situ utilizing any of the well known methods cant amounts of soil adjacent to the contaminated soil. The 60 for introducing exogenous microorganisms into the soil soil must then either be transported to the treatment facility along with nutrients and an optional alternative electron or else the treatment apparatus must be installed on-site. In acceptor compound. Conventional methods of introducing either case, these methods generally involve enormous bioremediating organisms into contaminated soil are transportation and handling costs, and require large amounts described, for example, in U.S. Pat. No. 4,477 570, incor of energy to combust or volatilize the contaminants. Other 65 porated by reference herein. Once established, the organisms elaborate and expensive techniques which have been utilized can utilize the polycyclic aromatic hydrocarbons contained involve excavation and treatment of the contaminated soil in the soil as a source of carbon and energy, thereby 5,508,194 3 4 decontaminating the soil in situ. After
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