1、API PUBL*4b27 95 O732290 0548280 5L9 HEALTH AND ENVIRONMENTAL SCIENCES DEPARTMENT API PUBLICATION NUMBER 4627 JULY 1995 ln Situ and On-Site Biodegradation of Refined and Fuel Oils: A Review of Technical Literature 1988 = 1991 American Petroleum P Institute API PUBLx4b27 95 0732290 0548281 455 In Sif
2、u and On-Site Biodegradation of Refined and Fuel Oils: A Review of Technical Literature 1988 - 1991 Health and Environmental Sciences Department API PUBLICATION NUMBER 4627 PREPARED BY: RONALD J. BAKER AND ARTHUR L. BAEHR DREXEL UNIVERSITY PHILADELPHIA, PENNSYLVANIA FEBRUARY 1994 American Petroleum
3、P institute API PUBLr4627 95 W 0732290 0548282 391 FOREWORD API PUBLICATIONS NECESSARILY ADDRESS PROBLEMS OF A GENERAL NATURE. WITH RESPECT TO PARTICULAR CIRCUMSTANCES, LOCAL, STATE, AND FEDERAL, LAWS AND REGULATIONS SHOULD BE REVIEWED. API IS NOT UNDEKIAKING n MEET THE DUTIES OF EMPLOYERS, MA“FAC-
4、TURERS, OR SUPPLIERS TO WARN AND PROPERLY TRAIN AND EQUIP THEiR EMPLOYEES, AND OTHERS EXPOSED, CONCERNING HEALTH AND SAFETY RISKS AND PRECAUTiONS, NOR UNDFRTAKING THEIR OBLIGATIONS UNDER LOCAL, STATE, OR FEDEML LAWS. NOTHING CONTAINED IN ANY API PUBLICATION IS TO BE CONSTRUED AS GRANTING ANY RIGHT,
5、BY IMPLICATION OR mRWISE, FOR THE MANU- FACTURE, SALE, OR USE OF ANY METHOD, APPARATUS, OR PRODUCT COV- ERED BY LETERS PATENT. NEITHER SHOULD ANYTHING CONTAINED IN ITY FOR INFRINGEMENT OF LE“ERS PATENT. THE PUBLICATION BE CONSTRUED AS INSURING ANYONE AGAINST LIABIL- API PUBLX4627 95 0732290 05q8283
6、228 D ACKNOWLEDGMENTS THE FOLLOWING PEOPLE ARE RECOGNEED FOR THEIR CONTRIBUTIONS OF TIME AND EXPERTISE DURING THIS STUDY AND IN THE PREPARATION OF THIS REPORT - Bruce Bauman, Ph.D., Health and Environmental Sciences Department Tmothy E. Buscheck, Chevron Research Jhaveri et al. 1983). 1.3 Related Li
7、terature Reviews Several reviews of hydrocarbon biodegradation literature have been published since 1987. A literature review of biorestoration of aquifers contaminated with organic compounds was published by Lee et al. (1988), and covered all aspects of microbially-mediated remediation of environme
8、ntal contaminants. Their literature reviewed is divided into three sections: in situ remediation, withdrawal and treatment, and hydrological considerations and mathematically modeling. Battersby (1 990) reviewed the literature related to biodegradation kinetics in the aquatic environment. Rate expre
9、ssions are described, and relevant literature is used to show how to choose the most appropriate kinetic model for a set of biodegradation data. Biodegradation in soil is also reviewed. Alexander and Scow (1989) reviewed the subject of biodegradation kinetics in soil, using a textbook-style presenta
10、tion. Kinetic models are developed for growing and nongrowing organisms, and for Monod and first-order kinetics. Diffusion and adsorption effects are covered, and the special case of fungal metabolism kinetics is described. Thomas and Ward (1989) discussed in situ biorestoration of organic contamina
11、nts in the subsurface as part of a five-article series on remedial actions and technologies. Other articles in the series dealt with field instrumentation for assessing hazardous waste sites; advantages and limitations of pump-and-treat technology; technologies for treating aqueous streams, sludges
12、and solids; and waste minimization. The article by Thomas and Ward (1989) discusses the need for subsurface characterization prior to implementing in situ bioremediation, and the site-specific nature of the technology. Examples of pilot-scale and field investigations are presented, including the use
13、 of endogenous and applied microorganisms. A summary description and evaluation of 13 remedial methods for soil and groundwater cleanup was prepared by Pres10 et al., (1989) for the electric utility industry. The review is 4 API PUBL*4627 95 W 0732290 0548290 468 divided into two main sections, cove
14、ring in situ technologies and non-in situ technologies. In addition to physical and chemical remediation technology, biodegradation (in situ, land treatment and bioreactor technologies) are described. Economic and environmental feasibility are considered for each remediation method. Leahy and Colwel
15、l (i 991) reviewed literature on microbial degradation of hydrocarbons in the environment. Physical and chemical characteristics of petroleum hydrocarbon molecules which control biodegradation rates were discussed. The physical state (separate phase product, emulsion or dissolved) and concentration
16、are of primary importance in determining degradation rates. The effects of temperature, oxygen concentration, nutrients, salinity, pressure and pH are discussed. The microbial species shown to degrade hydrocarbons are reviewed. Bacteria are thought to be much more important than fungi in marine hydr
17、ocarbon degradation, but the relative importance of these two groups in soil and freshwater hydrocarbon biodegradation is not yet known. Literature on microbial adaptation and microbe seeding to increase degradation rates was reviewed. A general discussion of remediation options for hydrocarbon-cont
18、aminated groundwater was presented by Thomas and Stover (1989). Air stripping, steam stripping, activated carbon adsorption, biodegradation, membrane processes, electrodialysis and ion exchange processes are discussed. Conditions under which each process is potentially suitable are presented. Thayer
19、 (1 991) wrote a general discussion of bioremediation. He described the regulatory climate, which is the driving force behind most contaminant remediation. He divided bioremediation into three broad categories: land treatment, bioreactors, and in situ treatment. Each is described, and examples are g
20、iven. Barker and Mayfield (1988) divided their descriptive review of aromatic hydrocarbon biodegradation into four categories, depending upon the characteristic oxidant used (O2, NO;, SO:- or CO,). Biological processes using each of these oxidants are described, and examples of aromatic biodegradati
21、on are given. They cited degradation rates from recent literature and their own work. They concluded that monoaromatic hydrocarbons can be biodegraded in all groundwater environments. Dragun (1 988) wrote a general discussion of petroleum-degrading microbial populations in soil, and described how de
22、gradation is effected by 5 API PUBLx4627 95 0732270 0548293 3T4 soil factors and chemical structure of contaminant components. Genera of hydrocarbon- degrading bacteria and fungi are listed. Microbial transformation reactions are tabulated. Biodegradable organic molecular fractions (e.9. aldehydes,
23、esters, etc.) are also listed. The author points out the tools for predicting biodegradation rates are absent or primitive, and this will be an active research area in the future. Dragun (1989) presented an overview of recovery and treatment technologies for petroleum products in soil and groundwate
24、r. Natural degradation, land treatment, composting and in situ biodegradation were the microbiological technologies discussed. Bauman (1989) stated current issues in management of motor fuel contaminated sites. Current soil cleanup standards and accuracy problems inherent in currently practiced anal
25、ytical methods, were reviewed. The relationship between cleanup objectives, cost, and relative risk to human health and the environment is addressed. Raymond et al. (1990) presented an overview of in situ bioremediation of petroleum hydrocarbons in the unsaturated and saturated zones. Case studies a
26、re given, and relative costs of remediation options are discussed. Fournier (1988) wrote a descriptive history and introduction to in situ bioremediation from the perspective of the pulp and paper industry. Essential preliminary site evaluation steps, and commonly practiced remediation strategies ar
27、e presented. A review of iron and manganese reducing organisms was published by Lovely et a/. (1991). A complete discussion of the various electron acceptors known to contribute to degradation of organic matter in the environment is included. Types of organisms involved (Fe and Mn reducers that are
28、fermentative, sulfur-oxidizing, hydrogen-oxidizing, organic acid oxidizing and aromatic compound oxidizing) were reviewed. The effects of anaerobic organisms in mobilizing and immobilizing metals in soil were discussed. Government agencies, e.g. state or federal transportation departments, are often
29、 required to remediate hydrocarbon-contaminated sites in the course of completing highways or other public projects. Orokunle (1990) prepared a report for the Georgia Department of Transportation in which state of the art remediation methods for organic contaminated soil are described. Advantages an
30、d limitations of excavation and disposal; utilization in asphalt manufacturing; in situ 6 API PUBL*4627 95 m 0732290 0548292 230 m soil washing with surfactant solutions; in situ volatization; in situ vitrification; and in situ biodegradation of contaminated soil were discussed and tabulated. Mobili
31、ty and transport of petroleum-derived hydrocarbons was reviewed by Ptacek and coworkers (1987). Mechanisms that control the fate of benzene, toluene and xylenes (BTX) and other petroleum hydrocarbons are described. A case study is used to demonstrate retardation of BTX by sorption, and to show that
32、BTX compounds can be mobile in groundwater. 7 API PUBLlk4b27 95 0732290 0548293 I177 = CHAPTER 2 PETROLEUM HYDROCARBON MICROBIOLOGY 2.1 Genetics and Metabolic Pathways Biodegradation of petroleum hydrocarbons requires specialized, microbially-produced enzymes. Production of these enzymes is genetica
33、lly controlled, and biodegradation pathways are determined by the genetic makeup of the microorganisms involved. This chapter presents some recent microbiological genetics research and recent information about the organisms responsible for petroleum hydrocarbon biodegradation. The waste treatment in
34、dustry has utilized microbes for degradation of organic substances for several decades (Metcalf and Eddy, Inc., 1979). Domestic and industrial wastewater treatment plants commonly use aerobic degradation (e.g. activated sludge) and anaerobic degradation (e.g. anaerobic digestion of sludge). Informat
35、ion about the responsible organisms, methods of determining rates of degradation and biomass accumulation, and energy requirements have been worked out. However, in situ and on-site biodegradation reaction rates are more difficult to measure, particularly in underground contaminant remediation, and
36、the wide variety of subsurface organisms have not all been identified and characterized. Although progress is being made toward measuring and modeling rates of degradation, this is still a relatively new research area. Biodegradation of hydrocarbons is a multistep process involving a series of enzym
37、es. Examples of degradation pathways for benzene and toluene are shown in Figure 2. Genetic control of degradation metabolism and microbe viability are the two principal areas of concern. The organisms must be equipped to metabolize one or more problem contaminants and be viable in the in situ or on
38、-site environment. If engineered microbes are introduced into a natural system, such as an aquifer, it may be desirable to genetically “program“ them to be viable for a limited time, to avoid unlimited proliferation of the engineered genetic material into the natural microbial gene pool. As recombin
39、ant DNA research is relatively new, there is little material available on using engineered organisms for bioremediation at this time. 9 API PUBLx4627 95 0732290 0548294 003 4 B 1- Figure 2. Bacteria Oxidation Pathways for Toluene and Benzene 10 API PUBL*4627 95 0732290 0548295 T4T Burlage et al. (19
40、89) reviewed literature pertaining to the TOL (pWW0) catabolic plasmid. Plasmids, as defined by Crosa and Falkow (1981), are autonomously replicating extrachromosomal DNA within bacterial cells. They are not essential to the survival of the organism, but may enable it to adapt to a wider variety of
41、conditions. The TOL plasmid encodes the enzymes that initiate degradation of toluene, m and p xylene and related compounds. This plasmid occurs in Pseudomonas and Alcaligenes bacteria species. A complete sequence of enzymes and intermediates has been determined since the plasmid was first described
42、by Williams and Murray (1974). In toluene degradation, the methyl group is oxidized to carboxyl, then removed as benzoate is converted to catechol, at which point the ring is broken, and a series of intermediates leads to the formation of pyruvate and acetaldehyde, which are easily metabolizable com
43、pounds. The literature review of Burlage et al. (1989) describes the genetic composition of TOL plasmids, and lists the enzymes involved. An alternative toluene catabolism pathway was discovered by Shields et al. (1989). The strain 64 organism isolated from a waste treatment lagoon (not othetwise id
44、entified) can grow on toluene, phenol, and o. and mcresol. However, it cannot convert indole to indigo, as can the enzyme toluene dioxygenase. Therefore, a different toluene degrading enzyme system (and pathway) was suspected. A strain of G4 that cannot grow on toluene was generated by mutagenesis.
45、This strain (64 102) can partially degrade toluene to , NO, reduction microcosms, aerobic and denitnfying niicFocosms,sandy- microcosms, riparian soil microcosms,sandy- microcosms, riparian soil References: 1. Payne and Floyd, 1990; 2. Barker and Major, 1987; 3. Kernblowski, 1987; 4. Karlson and Fra
46、nkenberger, 1989; 5. Awin et ai., 1989; 6. CouareIli et ai., 1989; 7. Evans, et ai., 1991; 8. Gersberg, et ab, 1989; 9. Kuhn, et ai., 1988; 10. Major, et ai., 1988; 11. Wawood et al., 1991. 16 Metabolic adaptation of a microbial population to a petroleum hydrocarbon substrate is often required for p
47、opulations not previously exposed to the substrate. Aamand et al. (1989) determined the adaptation periods, or lag times of bacteria with different degrees of previous exposure to test substrates. They listed four possible explanations for lag times: 1. Time needed for induction of substrate-specifi
48、c enzymes in individual organisms; 2. Exchange of genetic material mediated by plasmids; 3. Genetic changes leading to new metabolic capabilities; and 4. Growth of the segment of the microbial population already able to utilize the substrate. Groundwater from contaminated and uncontaminated areas of
49、 a leaking fuel tank site (Gassehaven, Denmark), and from the site of a two-year-old gasoline leak were used. An aqueous solution of 2 mg/L each of toluene, *xylene, 1,3,5-trirnethylbenzene, naphthalene, I - methylnaphthalene, biphenyl, 2-ethylnaphthalene, and 1 ,4-dimethylnaphthalene was used to prepare microcosms. In Experiment 1 groundwater samples were diluted 1:lO with distilled water in 5.5 liter bottles, which were continuously stirred and kept at 12C. Lag times and times for complete removal of hydrocarbons was determined for unpolluted, slightly polluted and heavily polluted wa
