Chlorinated hydrocarbons: oxidation in the biosphere Here is a survey of metabolic pathways, problems, and limitations involved, and a suggestion that genetic engineering might help to expand microbial capabilities to perform this function
Jot* M . Wood Gray Freshwater Biological
Institute University of Minnesota Navarre, Minn. 55392 Modern techniques for the synthesis of organic compounds add approximately 200 000 new chemicals each year to the millions already used by advanced industrial nations ( / ) . Microorganisms, which took billions of years to evolve their metabolic diversity, are assumed to have the ability to degrade these synthetic compounds, many of which bear no resemblance to those natural products used as nutrients in the microbial world. Since most of the synthetic chemicals, used on a large scale, have been added to the biosphere only in the last 30 years, it seems reasonable to ask the question, "How long will it take for microorganisms to acquire the ability to degrade all the new synthetic chemicals introduced into the environment by modern technology?'* It should be recognized that there are economic and technical factors that conflict with applying the basic principles of biodcgradability in the design of new chemicals for agricultural use. For example, farmers know that persistent pesticides are much more effective than their biodegradable substitutes. Obviously, the longer an insecticide remains in the soil, the more insects it kills: this is why DDT is so effective. Indeed, economic considerations usually dictate the selection of the more persistent chemicals over safer biodegradable counterparts. It can be argued that with the in-
creasing world population, there is a greater need for insecticides to control insect-borne diseases, and for the provision of agricultural chemicals to protect the world food supply. However, this increased use of chemical technology on a global scale increases the risk to the public health because of the toxicity, persistence, and bioconcentration of many substances currently in use. At this time, we need a more systematic approach to understand those basic principles pertinent to the removal of synthetic chemicals in the biosphere. Although photochemical reactions play a role in the removal of certain compounds (2). biodégradation by microorganisms is of primary importance. If a synthetic chemical is biodegradable in a reasonable time frame, then that compound is unlikely to pose a threat to the public health ( J , 4). Nevertheless, while microorganisms can adapt to remove many toxic substances, the great variety of synthetic chemicals used today may disrupt the balance of the carbon cycle. In the future it will be necessary to develop microbial systems that can change this trend. This review will explore the limitations imposed on the biological oxidation of some synthetic compounds, as determined from recent studies with enzymes that catalyze the oxidation of aromatic compounds (5-9). It will furnish examples of how existing metabolic pathways can adapt to degrade, or partially degrade, a number of widely used synthetic chemicals. Special emphasis will be given to the problems confronting microorganisms
0013-936X/82/091 β-0291 At01.25/0 © 1982 American Chemical Society
Biological oxidation: some pitfalls • Microorganisms do not "view" the oxidation ol organic compounds from the same perspectives as does the talented industrial chemist: theretore, predictions for biodégradation cannot generally be based on concepts of chemical substituents or chemical reactivity. • Νtodangerous to assume that the degradation of a given synthetic compound will follow the same meta bolic pathway to that already discov ered for a structurally similar natural product. • The microbial ecology of the specific ecosystem receiving the pollutant is more critical to its degra dation than are the degradative path ways determined with either pure cultures or microbial consortia grown in laboratories. • Even though the biodégradation of a synthetic chemical pollutant may be complete, quite often partial metabolites are more toxic than the primary pollutant. • While plasmid technology has been used successfully to improve the metabolic capabilities of a number of microbial strains to degrade organic poHutants (for example. 2.4.5-τ). those mutants lacking stability will not be effective in carrying out such tasks, because they are likely to lose their plasmids in complex environmental situations. Some: Papw* by Oagley ( M- ret.
Envron Sci Technol. Vol. 16. No 5. 1982
291A
in the oxidative degradation of the most persistent chlorinated aromatic compounds such as PCBs. This wellknown problem will be treated from an evolutionary perspective. In addition, the potential application of genetic engineering to the selection of mutants for the purpose of degrading specific pollutants in industrial effluents will be discussed. For instance, plasmid technology has the potential to produce "super-bugs," which have better degradative capabilities than do natural populations of microorganisms. This technology involves transfers of genetic material from one organism to anoth er, by isolating extra chromosomal material in the form of circular D N A or R N A . T h e use of mixed cultures of microorganisms to effect the total degradation of synthetic compounds is an old idea. More than 50 years ago, Gray and Thornton (10) recognized that mixed cultures of bacteria (now called microbial "consortia") can contain organisms that partially de grade synthetic compounds, without deriving any nutritional benefits, to give partial metabolites that function as growth substrates for other organ isms in the consortium. Also, in 1959, Leadbetter and Foster ( / / ) showed that methane oxidizing bacteria (now called methylotrophs) are capable of the "cooxidation" of a wide variety of synthetic chemicals, all of which fail to function as growth substrates. In the early 1970s, this concept was resur rected and called "cometabolism" (12, 13). Mixed-culture technology is now rapidly developing in the private sec tor, with several companies claiming successes in the improved treatment of both domestic and industrial wastes. They claim that some of their products are remarkably versatile, degrading everything from grease to dioxin her-
FIGURE 1
Nature's bleach, "chloroperoxidase": how it works3
ROOH
Fe (lll
+
>
R = H, CH3, φ, and so on
Α^Α0
2
]
2. [ 0 2 + A + 2 H + + 2e~ *=* A-OH + H20] 3.
I Fe (III)
RO O-R
OH OH
+
Fe (III)
_ H+
"Compound X" X" OCI
ι
|
Fe Fe (III) (III)
++
A-H A-H Ha
A A--C CII
++
Fe Fe (ill) (III)
++
O OH ~ "
A = organic substrate 3 A heme-containing protein thai reacts with hydrogen peroxide, as well as organoperoxides, to yield "Compound I." Note: L.P. Hager, et al- (T7-27) have shown that a "Compound II" precedes the active halogenating complex.
bicides. Even PCB-degraders are of fered, but very little experimental data is given in support of such claims. To be sure, this technology has a good future, but it is in its infancy, with very little investment in both fundamental research and product quality control. Any qualified microbiologist will say that mixed cultures are difficult to standardize and preserve. Biological oxidations The evolution of oxygen-based photosynthesis provided the necessary energy for the creation of the earth's biomass. Photolysis of water by photosynthetic organisms introduced oxygen to the earth's atmosphere, which in turn led to the evolution of a
Electrons transferred
Process
Enzymes
0
Oxygen fixation
Dioxygenases
2
Hydroxylation
Mixed function oxygenases
2
Dismutation
Superoxide dismutases
4
Respiration
Cytochrome oxidases
4. 0 2 + 4 H + + 4e - ^ 2H 2 0
292A
+
I
0 2 + e~ *=* 0 2 0 2 + 2 H + + e~ *=* H 2 0 2
Ν ote:
:
"Compound I"
Biological oxidation: enzyme reactions
1. [ 0 2 +
OH Fe(IV) Fe(lV)
TABLE 1
Biological '"activation" of oxygen
X)-0-R t 1 Fe ^=°
C O O H
C I ^ \\
HCI COOH
6=0 Π COOH HO-1^ •"Dehalogenation by lactonizabon. followed by HCI elimination, is energetically the most favored mechanism.
OH
COOH
V
HCI
CI H2O
Ο
H2O
COOH
"φ0»
COOH
CI-C-o
cVc*°
Cl
C |
H20 H
HC,
'°
COOH -
