Microbial removal of hazardous organic compounds Within certain broad limitations, many microorganisms not previously considered useful for biological waste treatment could be applied to the removal of anthropogenic substances
Hester Kobayashi Sohio Research Center Cleveland, Ohio 44128 Bruce E. Rittmann Department of Civil Engineering University of Illinois at Urbana-Champaign Urbana, Ill. 61801 The presence of man-made (anthropogenic) organic compounds in the environment is a serious public health problem. Sixty-five classes of such chemical compounds are considered hazardous, and among them, 114 organic compounds have been designated by the U S . Environmental Protection Agency (EPA) as priority pollutants (I). The presence of these compounds appears to be attributable largely to inadequate disposal techniques ( 2 ) , which have caused contamination of water and soil. Accidental generation of such compounds during treatment processes, such as the generation of chloroform during chlorination ( 3 ) , is another source of water pollution. Existing legislation to control and regulate the entry of hazardous chemicals into the environment includes the Safe Drinking Water Act (SDWA), the Clean Water Act (CWA), the Toxic Substance Control Act (TSCA), and the Resource Conservation and Recovery Act (RCRA). The public health danger of anthropogenic compounds and the enforcement of the pertinent laws and regulations require that considerable effort be placed upon reducing or eliminating environmental intrusion and persistence of hazardous organic compounds. In numerous cases, biological treatment can eliminate hazardous compounds by biotransforming them into innocuous forms, degrading them 170A
Environ. Sci. Technol., Vol. 16, No. 3, 1982
by mineralization to carbon dioxide and water, or anaerobically decomposing them to carbon dioxide and methane. Many compounds, however, are not removed efficiently by existing biological treatment techniques, either because they are metabolized very slowly, or because they are resistant to microbial attack under prevailing environmen tal conditions. Under the support of the Advanced Environmental Control Technology Research Center at the University of Illinois and the U.S. EPA, we conducted an in-depth evaluation of the potential for microorganisms to remove anthropogenic organic compounds, mainly priority pollutants and related compounds. The evaluation indicates that use of properly selected populations of microbes, and the maintenance of environmental conditions most conducive to their metabolism, can be an important means of improving biological treatment of organic wastes. One major theme is that microorganisms not normally associated with biological waste treatment have potential advantages when the removal of anthropogenic compounds is the goal. A broadened perspective into what constitutes biological treatment opens promising new areas of research and application. Biodegradability In contrast to naturally occurring compounds, man-made compounds are relatively refractory to biodegradation. One reason is that organisms that are naturally present often cannot produce the enzymes necessary to bring about transformation of the original compound to a point at which the resultant intermediates can enter into common metabolic pathways and be completely mineralized. The required transformation steps
to initiate biodegradation are fairly well known, and can be found in general reviews by Dagley (4), Alexander (9,Evans (6), and Matsumura and Benezet (7). Attempts to generalize the relationships between chemical structure and biodegradability have led to lists of chemical substituents that, when attached to organic parent compounds, make the compounds persistent. These lists include amines, methoxy, sulfonates, and nitro groups; chlorine, substitutions in the meta position in benzene rings, ether linkages, and branched carbon chains (8). In addition, larger molecules are generally considered less degradable than smaller ones. However, so many exceptions to these generalizations exist that such rules should be considered only as broad guidelines. Many environmentally important man-made compounds are halogenated, and halogenation is often implicated as a reason for persistence. The list of halogenated organics includes pesticides, plasticizers, plastics, solvents, and trihalomethanes. Chlorinated compounds are the best known and most studied because of the highly publicized problems associated with DDT, other pesticides, and numerous industrial solvents. Hence, chlorinated compounds serve as the basis for most of the information available on halogenated compounds. Some of the characteristics that appear to confer persistence to halogenated compounds are the location of the halogen atom, the halide involved, and the extent of halogenation (8,9). The first step in biodegradation, then, is sometimes dehalogenation, for which there are several biological mechanisms (IO). Again, simple generalizations do not appear to be applicable. For example, until recently, oxidative pathways were
0013-936X/82/0916-17OA$01.25/0 @ 1982 American Chemical Society
-I
mostly bclieved to bc the typical means by which halogenated compounds are dehalogenated. At present, anaerobic. reductive dehalogenation, either biological or nonbiological, is now recognized as the critical factor in the transformation or biodegradation of certainclassesofcompounds ( 1 1 - 1 5 ) . Compounds that require reductive dechlorination are common among the pesticides, as well as halogenated oneand two-carbon aliphatic compounds. Chlorinated benzenes and PCBs, however, appear to be attacked only under aerobic conditions ( / 6 - / 8 ) . Reductive dehalogenation involves removal of a halogen atom by oxidation-reduction, as illustratcd in Figure I , whish is a modification of the scheme presented by Esaac 3nd Matsumura ( / / ) . In essence, the mechanism involvcs the transfer of electrons from reduced organic substances via microorganisms or a nonliving (abiotic) mediator, such as inorganic ions (for example, Fe’’), and biological products (for example, SAD(P), fla-
vin, flavoproteins, hemoproteins, porphyrins, chlorophyll, cytochromes, and glutathione). The mediators are responsible for accepting electrons from reduced organic substances and transferring them to the halogenated compounds. The major requirements for the process are believed to be available free electrons and direct contact between the donor, mediator, and acceptor of electrons. Significant reductive dechlorination is reported to occur only when the oxidation-reduction potential (En) of the environment is 0.35 V and lower ( 1 1 ) ; the exact requirements appear to depend upon the compound involved. Evidence for abiotic mediators of environmental significance were shown by Matsumura (19). who used flavoproteins derived from blue-green algae, and Miskus ( 2 0 ) ,who found a relationship between plankton concentration and reductive dechlorination. The flavoproteins are produced in all living cells, and can become available as the cells die. It is proposed that
chlorinated pesticides sorb onto cells in the water column, and in the sediment with them. As thecells decay, the proximity of the free electrons from the decaying organic matter, the mediators, and the chlorinated compounds make conditions ideal for reductive dechlorination to occur. Examples of biological reductive dehalogenation are well documented, especially for pesticides and halogenated aliphatics. A particularly interesting case is the reductive dechlorination that occurs in algae. In algae, the dechlorination process may be a nonenzymatic photochemical transformation, and may occur through absorbance of light energy by photosensitive compounds, which then transfer electrons to the insecticide molecule (7). Numerous cases of apparent photochemical transformation are reported in the literature (7, 21, 22). The emphasis in studies of biodegradation of compounds arising from human activity has been largely on aerobic, oxidative processes because they are best known, and because aerobic techniques are relatively simple compared to anaerobic culture methods. Another reason for their‘ widespread use is that aerobic processes previously have been considered the most efficient and generally applicable ( 4 ) . However, aerobic treatment requires transfer of oxygen to the water, and can create copious amounts of sludge; both aspects often are energy-intensive and expensive. By comparison, anaerobic processes reduce or eliminate both of the major operating expenses of an aerobic system. Thus, anaerobic microbial processes are energy-efficient, and can enhance certain critical reactions, such as reductive dehalogenation, nitroreduction, and reduction of sulfoxides (11). Although simple studies using pure cultures of microorganisms and single substrates are valuable, if not essential, for determining biochemical pathways, they cannot always be used in predicting biodegradability or transformation in more natural situations ( 5 , 8, 16,23,24). The interactions among environmental factors, such as dissolved oxygen, oxidation-reduction potential, temperature, pH, availability of other compounds, salinity, particulate matter, competing organisms, and concentrations of compounds and organisms, often control the feasibility of biodegradation (16, 23, 25-28). The compound‘s physical or chemical characteristics, such as solubility, volatility, hydrophobicity, and
Environ. Sci. Technol., VoI. 16. No. 3, 1982
171A
octanol-water partition coefficient, contribute to the compound’s availability in solution (23, 28-32). Compounds not soluble in the water are not readily available to organisms for biodegradation. Simple culture studies are similarly inadequate for predicting the fate of substances in the environment if there are many interactions between different organisms. First, substances that cannot be changed significantly in pure-culture studies often will be degraded or transformed under mixed culture conditions (16,33-35). A good example of this type of interaction is cometabolism, in which a compound, the cometabolite, is not metabolized as a source of carbon or energy, but is incidentally transformed by organisms using other similar compounds. Second, products of the initial transformation by one organism may be subsequently broken down sequentially by a series of different organisms until compounds that can be metabolized by normal metabolic pathways are formed (5,34,36-38). An example is the degradation of DDT, which is reportedly mineralized directly by only one organism, a fungus; other organisms studied appear to cometabolize only the compound, resulting in numerous transformation products that subsequently can be used by other organisms. Pfaeander and Alexander ( 3 9 ) illustrated the point by showing that Hydrogenomonas can metabolize DDT only as far as p-chlorophenylacetic acid (PCPA), while Arthrobacter sp. can then remove the PCPA. One limitation encountered in biodegradation studies is that antagonistic interactions between organisms can inhibit biodegradation. Bacteria, for instance, are known to be the antagonist to fungi (26, 27). Another limitation is that they were performed with high concentrations of the organic substrate. In many instances involving contaminated water, hazardous compounds are already present at trace concentrations (for example, yg/L), and effluent concentrations still lower may be desired. Very low substrate concentrations pose two problems for biological treatment. The first problem is that the slow substrate utilization kinetics that occur with very low concentrations provide too little energy flux to sustain the microorganisms. Rittmann and McCarty (40-42) demonstrated that steady-state bacterial mass and substrate utilization declined to negligible quantities when the substrate concentration in a biofilm reactor ap-
172A
Environ. Sci. Technol., Vol. 16, No. 3, 1982
proached a threshold value, Smin. Typical Sminconcentrations for aerobic systems typically have been in the 0.1-1.0 mg/L range (42, 43), while the desired effluent concentrations are often 1 yg/L or less. A second problem with trace concentrations is that they may be insufficient to induce the production of necessary enzymes. Tentative evidence for such behavior has been observed in three studies (25, 25a, 42a). Specific groups of organisms In this section we consider groups of microorganisms that might be useful for treating specific types of man-made compounds. Table 1 lists examples of species or groups of organisms and the compounds they have been found to transform or attack. The conditions that prevailed during the observation or experiment (such as aerobic or anaerobic) are also noted in relative terms. In some cases, “aerobic” refers to incubation in air without added aeration; in such cases, it is possible that culture conditions varied from aerobic at the top of the culture vessel to microaerobic or even anaerobic at the bottom. The term “anaerobic culture” is also variable. It sometimes refers to culture in vessels filled to the top and simply capped; at other times it refers to anoxic conditions using a nitrogen atmosphere. In still other cases it refers to cultures grown under strictly (“fastidious”) anaerobic conditions with reduced media. Table 1 demonstrates that members of almost every class of man-made compound can be degraded by some microorganism. The table also illustrates the wide variety of microorganisms that participate in environmentally significant biodegradation reactions. Table 2 is a list of characteristics that might be used for the selective culture of specific types of organisms. In other words, Table 2 indicates how the conditions can be made most favorable for the development of a desired type of microorganism. Actinomycetes. Actinomycetes, a group of organisms morphologically similar to bacteria and fungi, are found in environments in which unusual compounds are encountered. They are known to attack a wide variety of complex organic compounds, including phenols, pyridines, glycerides, steroids, chlorinated and nonchlorinated aromatic compounds, paraffins, other long-chain carbon compounds, and even lignocellulose, which very few organisms can attack. The most commonly found actinomycete in aquatic
systems is Nocardia. N. aramae organisms occasionally are found proliferating in activated-sludge units where they appear to feed on lipids at the surface (44, 45). They are also found to grow under low nutrient conditions (oligotrophically), such as in distilled water (44-46). These organisms provide several advantages that make them attractive for use in wastewater treatment: sludge production lower than bacteria and fungi; wide temperature range, from psychrophilic to thermophilic; resistance to desiccation; and wide pH range. Organic decomposition brought about by actinomycetes generally results in various metabolites that can be mineralized in the presence of other organisms. Thus, mixed-culture systems are a necessity when actinomycetes are used. The number of nitrogenous compounds actinomycetes can use is limited, and because their cell synthesis is low, most of the nitrogen in the substrate is liberated as ammonia. Also, low cell synthesis makes their population size generally small under natural conditions. Actinomycetes might be especially useful in treatment of contaminated soil where a composting technique would be practical. Fungi. Selective cultures of some forms of filamentous fungi are of potential value in certain cases, since the fungi appear to have greater ability to degrade or transform hydrocarbons of complex structure or long chain length. Bacteria and yeasts, on the other hand, show decreasing abilities to degrade alkanes with increasing chain length (24). Organisms in two orders of fungi-Mucorales (such as Cunninghamella) and Moniliales (Fusarium, Aspergillus, Penicillium)-show the best potential (23).An example of this ability is the complete degradation of DDT by Fusarium oxysporum, a feat never observed with other microorganisms (see Table 1). Because they have nonspecific enzyme systems for aromatic structures, fungi (yeasts and filamentous) are believed to be capable of biodegrading PCBs better than bacteria can (23). However, fungal metabolism, in general, often results in incomplete metabolism. Hence, subsequent bacterial association for complete mineralization is necessary. Bacteria. Examples of bacteria that have been reported to attack different kinds of artificial compounds are listed in Table 1. In many studies,&species were not defined, but were given only by the source of the original inoculum, such as sewage or soil. The most com-
TABLE 1
Examples of anthropogenic compounds and microorganisms that can attack the I
:ompound
OIsanim(*)
Aliphatics (nonhalogenated)
Trichloromethane Trichlwoethane. b~chloromethane.methyl ChlwidB. chloroemane. dichloroethane. vmylidiene chlwide. trichloroethylene. tebachlwoethylene. methyrenschlwide. aibromocnloromethane. bromochlwomethane
Mixed culture of yeast mold. protozoa bacteria: activated sludge
ae
Marine bacteria
ae ae an
Sewage sludge Soil bacteria
Anoxic cmdliln
1 80 81
Trichlwomethanes. iricnloroethylene. Methanogenic (7) culture tesachloroethylene Trichlwoethane. trichloromethane. Sewage sludge tetrachloromthane. dichloroethane. dibromochloromethane, 1.1.2.2lebachlwoelhane. bisi2chloroisopropyl) e h r . bromoform.Dromcdichloromelhane. biChlorOlluOrMethane. 1.1dichlwoethylene. 1.2dichloroethhylene. 1.3dichlwopropylene. 1 .2-transdichloroemylene b-nalic compounds (nonhalogenatec" mene
e )benzene 40initrotoluene Winitrotoluene, di-n-butylphthalate. diphenylhydrazine
reosol
wnol
Pseudomonas put& (1) Sewage sludge Stabilization pond microbes Bacillus sp. (1) P. putida Stabilization pond microbes Stabilization pond microbes Activated sludge Sewage sludge Pseudomonas sp. (1) Aureobasidium pullulans (4)
i
i
i i
ae ae(7) i i
i
seudamonas. Vibrio, Spirillum; af FlavobacteriumChromobacfer, Bacillus, Nocardia ( 5 ) Chiamydamnas ulvaensis (2) ae Phoridium fuveolarum, Scenedesmus basiliensis (2) Euglena gracilus (2) Coiynebacteriumsp. (1) i umen microorganisms i
Light also required
pAmhophenol by nibaeductlon 11, 90
Ammatic wmpnlnds (halogenated). 1.2-; 2.5; l.+Dichlaabenm: p; m: chlorobenroete; 3 . e 3,Midrlaobenzoate. 3-methyl benzoete: cdrlwophenol
Pseudomonas sp. (1). sewage
80 ae attack a nwnber of mese
sole energy and carbon 8 pseudDmonassp.B13(WR1)
ae
richlombenrene 1.2,3- and 12.4-Trichlaobenrene
Sewage sldge Soil microbes
ae ae
pemachlorophenol
Soil microbes
an
2 , 5 2.3Dlchlorobenzene; 2,C and 2,5dlchlaobenzene: CO, slow tetra-. bC. di-, and m 1 Chlorophenol 18) cometabolim
24. 75, 98
1-NapMhol; cis-1,Zdihydroxyl1,ZdihydronapMhalene: 4hyboxy-I-tetralens
16 23 24 21. 97. 98
a+&pMhoi, fl-naphmol, bans- 23 19dihydroxy-1.Zdihydronaph. thalene: 4hydroxy-1-tetralene: 1,cnaphmoguinone 21 tight required; ability to breakdown this wmpwnd is c o m m among algae
18
stream at mi coking site Under static conditions Stream at coal cdcing site cis3.4-Dihydroxy-3.4dihydrp phenanWacene 3,c: 8.9: 10.11Dlhybols Very sbw, insignificant breakdown
100 18
cls-2.3.CJihydro-2.3dihydroxybi- 23
PhanYl
I
. . . . iF.:. . . . .,,:... . ... .. .. .^.' ..~ . ........... . . . . . .".'.. :...'..-. . . . . . . .., . . : . . ... .. . . . ... i:.. ..,:
