Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates.

Critical Reviews in Biotechnology, 12(5/6):357-389 ( I 992)

Microbiological and Biotechnological Aspects of Metabolism of Carbamates and Organophosphates Satya Chapalamadugu Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Universite De Sherbrooke on 11/12/12 For personal use only.

Ph.D. Candidate, Health and Environmental Chemistry, Oakland University, Rochester, MI

G. Rasul Chaudhry Associate Professor, Department of Biological Sciences, and Institute of Biochemistry and Biotechnology, Oakland University, Rochester, MI

ABSTRACT: Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.

KEY WORDS: biodegradation, bioremediation, environmental pollution genetic engineering, pesticides, ~

xenobiotics.

1. INTRODUCTION Synthetic chemicals are used widely for improving crop productivity and public health. Halogenated hydrocarbons were the first group of xenobiotics introduced for the control of insect pests and disease vectors. However, the intensive use of the halogenated compounds resulted in enormous problems of environmental contami-

nation. 36 For example, dichlorodiphenyl trichloroethane (DDT), the best known among the chlorinated hydrocarbons, was used extensively for pest control from the 1930s until its ban in 1979. Metabolites of this pesticide were found to contaminate soil and ground water and were even detected in humans. Dieldrin, heptachlor , benzene hexachloride, chlordane and a number of other chlorinated pesticides have also 13,2031303'93

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caused a widespread contamination of biota.4.12,26.197The recalcitrance and susceptibility to biomagnification, as well as the toxicity, mutagenicity, and carcinogenicity of chlorinated compounds, had raised public health concerns. This led to the development of biodegradable carbamates and organophosphorus compounds. The newly developed compounds have gradually replaced most of the chlorinated pesticides. At present, carbamates and organophosphates are the active ingredients of most of the insecticides and some of the herbicides in use. Their uses include crop protection and control of insects and weeds in recreational facilities, as well as eradication of insect vectors of animal and human diseases. In spite of many useful properties (low persistence and high potency at low doses) of the carbamates and organophosphates, their widespread use has also caused environmental and health problems. First, most of the synthetic carbamates and organophosphates are highly toxic and potent inhibitors of acetylcholinesterase, a vital enzyme involved in neurotransmission. Therefore, concerns have been raised regarding even the judicial commercial usage of these chemicals. Chemical residues of pesticides and their metabolites that accumulate in the food chain cause short- as well as long-term human health p r o b l e m ~ . * * , ~A~national , ~ ~ , ' ~monitoring ~ survey conducted by the U. S. Environmental Protection Agency found that 0.6 and 0.8% of the rural domestic and community water system wells, respectively, were polluted above the health advisory levels with at least one pesticide.195Because of ground water contamination, aldicarb (2- methyl-2- (methy1thio)-propionaldehyde-0(methyl-carbomyo1)oxime) and parathion (0,Odiethyl-O-p-nitrophenyl phosphorothioate) are banned or classified as restricted-use pesticides in many parts of the U.S.68.2"3 Since many carbamate and organophosphate pesticides with properties similar to aldicarb and parathion are still in use, the risk of environmental contamination remains. Second, repeated application of these pesticides has caused enhanced degradation of the pesticide^.^^ The enhanced degradation of pestcontrolling agents results in inefficient pest control and leads to poor crop yields and economic losses. Third, some target insects have been

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shown to develop resistance to these pesticides11'.'30,'96 similar to the drug resistance acquired by human pathogens. This, in turn, warrants application of higher doses of the pesticides in order to achieve the desired level of insect control, which further increases the risk of environmental pollution. Finally, degradation of some pesticides such as aldicarb yield metabolites as toxic as the parent compound.'68 Generally, oxidation products of these compounds are more toxic than the hydrolytic metabolites. Since no new pesticides have been registered for commercial use in recent years (because of high research and development cost), it is important to understand the metabolic fate and the risks of pesticides that are currently in wide use. Extensive work has been carried out on the persistence and fate of carbamates and organophosphates, and the earlier studies have been reviewed in excellent reports.58,85,98,144 In this article, recent investigations on determining the biochemical and genetic basis of degradation of carbamates and organophosphates are reviewed. In addition, the potential for enhancing the catabolic properties of microorganisms is discussed.

II. CARBAMATES Biologically active carbamate chemicals have a long history, with a reported use of physostigmine, a neurotoxin from calabar bean seeds (Physostigma venenosurn), as an ordeal poison in the West African witchcraft trials during the 17th and 18th centuries.95Physostigmine is the only known naturally occurring carbamate ester.29The first group of synthetic carbamate esters to exhibit insecticidal potential were derivatives of dithiocarbamic acids. Carbamic acid, the monoamide of carbon dioxide, is the backbone of all carbamate structures (Table 1). This acid does not exist in free form and its salts, which are more stable, are referred to as carbamates or carbaminates. Carbamates possess the broadest spectrum of biological activity and are used as insecticides, herbicides, fungicides, and nematicides. The major classes of carbamate chemicals used as pesticides include N-methylcarbamates, thiocarbamates, and phenylcarbamates.

TABLE 1 Structure and Toxicity (Rat, Oral) of Carbarnates Common name (trade name) Aldicarb (Temik)

Structure

1

CH3 I

HjCS-F-CHnNOCONHCH3 CH3 OCONHCH3


Aminocarb (Matacil)

Barban (Carbyne)

LD, (mglkg of body wt)

30

CI NHCOOCHz CkCCH2CI

600

Benomyl (Benlate)

9000

Butylate (Sutan)

4000 OCONHCH3

Carbaryl (Sevin)

307 OCONHCH,

Carbofuran (Furadan)

Chlorpropham (CIPC)

a CI

5000

Cycloate (Ro-Neet)

3000

Desmedipham (Betanil AM)

9600

Diallate (Avadex)

395

1367

Phenmedipham (Betanal)

8000

359

TABLE 1 (continued) Structure and Toxicity (Rat, Oral) of Carbarnates Common name (trade name) Promecarb (Carbamult)

H3D Structure

OCONHCH3

LD, (mg/kg of body wt) 74

(H3C)2Hc


5000

90

522

SWEP

Vernolate Nernarn)

H7C3,

0

H7C3

S-C3H7

,N-C:

1170

A. Methylcarbamates These chemicals are primarily used as insecticides. The members of this group include commonly used pesticides such as carbofuran (2,2dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbamate), carbaryl (1-naphthyl N-methylcarbamate), and aldicarb (Table 1). 1, Carbofuran

Carbofuran was introduced in 1967 under the registered trademark of Furadan@by the Niagara Chemical Division of FMC Corporation (Table 1). It is a broad-spectrum, residual insecticide and nematicide effective by contact, ingestion, and systemic action. Carbofuran usually is applied to foliage for insect control and is placed in or above seed furrows or broadcast for nematode control. Some of the pests controlled by carbofuran include corn rootworm, rice water weevil, wire-worm, sugar cane borer, alfalfa weevil, snout beetle, army worms, budworms, corn flea beetle, pea aphid, thrips, and hornworms. This insecticide has also been used in the production of bananas, coffee, and sugar beet^.^^,^^

Although it is highly toxic and effective in controlling many insects, carbofuran has shown poor insect control in certain cases. The loss of efficacy was attributed to rapid degradation of the pesticide by microorganisms when used for controlling phylloxera in a vineyard.2" The rapid degradation of carbofuran was associated with factors including high levels of actinomycetes in the history of the pesticide applicafion,28,59.'2,'92 and soil conditions.151200,202 Carbofuran-degrading microorganisms have been isolated from different terrestrial and aquatic environments (Table 2). Soil enrichment cultures were used to study the degradation of carbofuran under laboratory conditions. The incubation of uniformly ring-labeled [ 14C]carbofuran with an enrichment culture developed at 35°C converted 90% of the radioactivity to I4CO2in 5 d.I5O The culture medium was spiked with 10 pg of cold and lo4 dpm of labeled carbofuran per ml. Likewise, enrichment cultures obtained from three flooded soils previously exposed to carbofuran or its hydrolytic product, carbofuran phenol, rapidly degraded carbofuran compared with enrichment cultures obtained from unexposed soils .45 An Arthrobacter sp .,isolated from an enrichment culture obtained from a pesticide-treated flooded soil, mineralized the ring-labeled [ 14C] carbof1443150

TABLE 2 Microbial Degradation of Carbamates


Compound

Microorganism

Arthrobacter sp. Carbof uran Actinomycetes Azospirillum lipoferum Streptomyces spp. Achromobacter sp. Pseudomonas sp. Arthrobacter sp. Micrococcus sp. Bacillus sp. Pseudomonas cepacia Nocardia sp. Achromobacter sp. Pseudomonas spp. Flavobacterium spp. Pseudomonas spp. Carbaryl Rhodococcus sp. P. aeruginosa strain 50581 Bacillus sp. Micrococcus sp. Several fungal and bacterial isolates P. aeruginosa strain 50552 1-Naphthol Achromobacter sp. Aldicarb Pseudomonas sp. Pseudornonas sp. Nocardia sp. Arthrobacter sp. Fusarium oxysporum EPTC Epicoccum purpurascens Paecilomyces lilacinus Penicillium spp. Diheterospora spp. Bacillus sp. Alcaligenes sp. Micrococcus sp. Pseudomonas sp. Arthrobacter sp. strain TE1 Flavobacterium sp. strain VI.15 Rhodococcus sp. strain JE1 Flavobacterium sp. strain VI.15 Butylate, vernolate Barban, CIPC, IPC, P. alcaligenes and SWEP P. cepacia BIPC, ClPC Achromobacter sp. Propoxur Moraxela sp. strain G Aniline R. erythropolis P. multivorans Alcaligenes faecalis Pseudomonas sp. strain ClTl Pseudomonas sp. strain SB3 P. cepacia 3-Chloroaniline Moraxela sp. P. putida 3,4-Dichloroaniline P. putida Desmedipham, Flavobacterium sp. phenmedipham, Aspergillus versicolor and Dromecarb

Ref. 149, 150 206 200 59 147

200 82 34 96

32 147 155 32 82 34 152

100

190 118 50 118 109 199 82 216 10 75 186 9 81 199 216 21 1 94

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uran to I4CO, within 72 to 120 h utilizing the chemical as a sole source of carbon and nitrogen under aerobic conditions.150 No degradation of carbofuran occurred under anaerobic conditions, and the mineralization was more rapid at 35 than at 20°C. In similar studies, an Azospirillum lipoferum and two Streptomyces s p ~ . and species of Achromobacter and Pseudomonas were also implicated in the degradation of c a r b ~ f u r a n . ~ ~ Attempts have been made to understand the biochemical mechanisms of inactivation of this pesticide in soil environments. Camper et a1.28 identified the degradation products of carbofuran

as 3-ketocarbofuran and 3-hydroxycarbofuran (Figure 1). In this case, the pesticide was transformed to the stable intermediates. In another study, it was found that even though carbofuran phenol and 3-hydroxycarbofuran were identified as metabolites of carbofuran, these compounds never accumulated in large quantities, suggesting their further degradation through ring cleavage. I5O Alternatively, in flooded soils, carbofuran phenol was found to be the major product of bacterial metabolism of carbofuran, with 3-hydroxy carbofuran as the minor product.201However, the amount of carbofuran phenol decreased

Catechol

Carbofuran

I I

/ \ bCH i

C02

&:;:

CH3

Carbofuran phenol

NH2 CH3

&

OH

CH3

*'=*\

+

+ HzO

CHI

+ COz

/ \carbon source

Hydroxycarbofuran phenol

Nitrogen source

1 II I I I

I

i

.

'. .

3-Hydroxycarbofuran I I

i

OCONHCHj

@:;:

.

8 ,

..

3-Ketocarbofuran

Ketocarbofuran phenol

FIGURE 1.

362

Proposed pathways for the microbial degradation of carbofuran.


after incubation for 30 d , indicating its slow transformation. Contrary to these studies, carbofuran phenol was found to be persistent in a carbofuran-amended soil. 14' An Arthrobacter sp. isolated from this soil rapidly degraded 3-hydroxy carbofuran and 3-ketocarbofuran, but not carbofuran phenol. These findings suggest that the major pathway involved in the initial breakdown of carbofuran is hydrolysis. However, little is known about the fate of hydrolytic metabolites of carbofuran. Initial studies on the fate of carbofuran in enrichment cultures led to the isolation of microorganisms that were used for investigating the metabolic pathways and respective enzymes involved in the degradation of the pesticide. Venkateswarlu and Sethunathan2misolated P. cepacia and a Nocardia sp. from a flooded alluvial soil. The Nocardia sp. metabolized carbofuran to colored, water-soluble, and nonextractable metabolites. An Achromobacter sp. hydrolyzed the pesticide to carbofuran phenol, and the hydrolytic enzyme was regulated and repressed during growth on nitrogen-rich medium.** It is not clear whether this hydrolase was an esterase (cleaving between the carbonyl group of N-methyl carbamic acid and the phenol) or an amidase (cleaving between the carbonyl and amine moieties of N-methyl carbamic acid). The purified enzyme, having a molecular size of 150 kDa84 also catalyzed the degradation of other N-methyl carbamates, such as carbaryl, aldicarb, baygon (2-isopropoxyphenyl-N-methylcarbamate), but not chloropropham (isopropyl-N-3-chlorophenylcarbamate) or EPTC (S-ethyl diporpylthiocarbamate) .49 Although study of soil enrichment cultures and isolated microorganisms proved useful in defining the fate of carbofuran in soil, the features that could be common in various soils and enrichment cultures, as well as their individual members, have been investigated poorly. Studies of Chaudhry and c o - w o r k e r ~focused ~~ on comparing enrichment cultures and their members obtained from different soils, as well as the mechanisms of pesticide degradation simultaneously in enrichment cultures and in the isolated bacteria. In this study, 17 soil samples collected from different geographical areas yielded 12 enrichment cultures that metabolized carbofuran to a

variable degree. Some cultures hydrolyzed carbofuran to carbofuran phenol only, while no metabolites could be detected in other cultures. Further study of these enrichment cultures led to the isolation of 15 bacteria belonging to either the Pseudomonas or Flavobacterium genus. Six isolates, placed in group 1 , utilized carbofuran as a sole source of nitrogen, while seven isolates, placed in group 2, used carbofuran as a sole source of carbon. Two other isolates, placed in group 3, also utilized the pesticide as a sole source of carbon, but degraded carbofuran completely and more rapidly. When ring-labeled [ "C] carbofuran was used as a carbon source for group 3 isolates, up to 40% of the pesticide was lost as I4CO, in 1 h and no metabolic product was detected in the culture medium. The isolates of this group metabolized carbofuran via an oxidative pathway. The proposed pathways involved in the catabolism of carbofuran are shown in Figure 1 . Isolates of groups 1 and 2 hydrolyzed carbofuran to carbofuran phenol. Crude cell extracts prepared from selected isolates of these groups exhibited hydrolase activity. The enzyme activity was greater in isolates of group 2 compared with isolates of group 1 . The bacteria utilizing carbofuran as a nitrogen source exhibited the mechanism of carbofuran degradation similar to that of the Achromobacter sp. strain WM 1I 1 reported by Karns et a1.82Strain WM111 harbored several plasmids , and the carbofuran-degradation function was found to be plasmid encoded.'" In a manner similar to the hydrolase from Achromobacter sp. the hydrolase enzyme from the isolates of groups 1 and 2 also was able to hydrolyze other methylcarbamates , including carbary 1 and aldicarb. The plasmid-encoded gene (mcd) for the methylcarbamate degradation enzyme (hydrolase) has been cloned. 19' However, the cloned gene expressed poorly in the other microorganisms tested, including A . pestifer, Acinetobacter calcoaceticus, Alcaligenes eutrophus, and P . putida. It is interesting to note that a probe using the cloned mcd gene of Achromobacter sp. failed to hybridize with plasmid or chromosomal DNA (unpublished data) of any of the above 15 bacteria isolated by Chaudhry and Ali.34The above results suggest that the hydrolase gene in these bacteria is different than the hydrolase gene of Achromobacter sp. Members of group 3 also degraded

363


carbaryl and aldicarb. Pseudornonas sp. 50432 of group 3 effectively decontaminated carbamate-polluted water samples, suggesting its potential use for the detoxification of pesticide-polluted environments. 37 This isolate harbored several plasmids. A study on the function of these plasmids may help to elucidate the oxidative pathway involved in the degradation of carbofuran. More recent studies of our laboratory focused on the investigation of genetic determinants involved in the degradation of carbofuran.

2. Carbaryl Carbaryl is the common name for the active ingredient of the insecticide manufactured by Union Carbide Corporation (now Rhone-Poulenc) under the trade name of Sevinm (Table 1). This pesticide is available as a wettable powder, sprayable powder, granule, dust, and other formulations. Carbaryl is the least toxic among the N-methylcarbamates and is by far the most widely used carbamate insecticide. Some of the important uses of carbaryl include applications to cotton, corn, soybeans, various fruit and vegetable crops, bananas, pineapple, olives, cacao, coffee, rice, and sugar cane for the control of various insects. It is also used in forests, range land, livestock, poultry, and buildings for this purpose. Public concern regarding the safety of carbaryl caused the New Jersey Department of Environmental Protection to temporarily suspend the use of this insecticide.'06 Although carbaryl has been effective against several insects, it also has shown reduced efficacy in soils treated previously with the pesticide. Three applications of carbaryl to a submerged soil resulted in its rapid degradation. Studies of other soils showed that the amount of residual pesticide was 28 and 90% after 4 d of incubation with ring-labeled ['4C]carbaryl in samples from the treated and untreated soils, respectively.155 In another study, an untreated soil that failed to degrade carbaryl was induced to rapidly metabolize the pesticide when treated with 1-naphthol, a hydrolysis product of carbaryl. 148 These investigations suggest that, like other carbamates, carbaryl degradation is also enhanced by repeated applications of this pesticide and its metabolites.

364

Investigation of carbaryl-polluted river samples indicated that both abiotic and biotic degradation processes play an important role in the degradation of carbaryl. '06 The degradation of carbaryl has been reported to be mediated This observation prompted studies to isolate microorganisms capable of degrading carbaryl from soils with a history of the pesticide treatment (Table 2). A Pseudomonas sp . (NCIB 12042) and a Rhodococcus sp. (NCIB 12038) isolated from a garden soil utilized carbaryl as the sole source of carbon and nitrogen." Although both bacteria hydrolyzed the pesticide, the pathway for further metabolism of l-naphthol was found to be different in these bacteria. Strain NCIB 12042 metabolized l-naphthol via salicylic acid only, while strain NCIB 12038 metabolized it via both salicylic and gentisic acids (Figure 2). In contrast, another Pseudomonas sp. (NCIB 12043), isolated from the same soil by perfusion column enrichment, metabolized carbaryl rapidly to 1 naphthol, which was degraded further via gentisic acid alone. The pathway for l-naphthol degradation in NCIB 12042 exhibited similarities to that of naphthalene catabolism in Pseudomonus ~ p pIn. a~separate ~ study, up to 70% of carbaryl was found to be hydrolyzed by a Bacillus sp. isolated by enrichment culture techniques from while the second bacterial strain, a flooded Micrococcus sp., isolated from the same soil hydrolyzed carbaryl poorly. It degraded only 15% of the added carbaryl under similar conditions. These reports indicate the functional diversity among the bacteria in adapting to catabolism of the same substrate. A soil bacterium, P. aeruginosa strain 50581, also hydrolyzed carbaryl to l-naphthol and utilized the pesticide as a sole source of carbon (Figure 3).32 Unlike the carbofuran-hydrolyzing b a c t e ~ i a , ~ this ~ . ~isolate ~ . ' ~ ~did not degrade other carbamates. This indicates that the hydrolase in P . aeruginosa isolate 5058 1 is different from the mcd gene product, which has a wide substrate range. This was confirmed by DNA hybridization studies using the mcd gene as the probe. No homology between the total DNA from the isolate 50581 and rncd gene was observed. Recently, Mulbry and EatonIz0purified a cytosolic enzyme from Pseudornonas strain CRL-

OCONHCHa

aa Carbaryl

i

w

+

f-

1-Naphthol

0

1,4-Napthoquinone Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Universite De Sherbrooke on 11/12/12 For personal use only.

NH2 C H J + C O ~

I I

I 7

Salicylaldehyde

Salicylate

Catechol I I I I

i Ring fission

FIGURE 2. of carbaryl.

Gentisate I

I I I

i Ring fission

Proposed pathways for the microbial degradation

OK isolated from a sewage sludge. The bacterium utilizes carbaryl as a sole source of carbon and energy. The enzyme hydrolyzes carbaryl and other N-methylcarbamates such as carbofuran and aldicarb. However, the purified enzyme did not hydrolyze thiocarbamate, or phenylcarbamate such as EPTC and CIPC (chlorpropham), re-

spectively. This enzyme consists of two identical subunits of 85 kDa, and the size of the native enzyme was determined to be 187 kDa.I2OIt has a pH optimum of 8.5 and a temperature optimum of 60°C. The carbaryl-hydrolyzing strain 50581 isolated in our lab also was found to have a novel enzyme activity. This activity was specific to

365


a

Time (hr)

b

C

l i m e (hr)

Time

(hr)

FIGURE 3. Degradation of carbaryl and 1-naphthol by the P. aeruginosa strains. (A) Isolate 50581 hydrolyzes carbaryl to 1-naphthol; (B) isolate 50552 mineralizes 1-naphthol completely to CO, and H,O. Recombinant 50605 was isolated as a transconjugant in the cross between 50581 and 50552. It slowly mineralizes carbaryl (C). Bacteria were grown in LB medium3, for 16 h, harvested, and washed with minimal media.32Minimal medium containing carbaryl (A, C ) or 1-naphthol (B) was inoculated with the bacteria to an OD,, of 1.O and incubated at 30°C in a rotary shaker. Samples were drawn from the cultures at regular intervals, and the disappearance of carbaryl (H) and 1 -naphthol (El)from the culture media was monitored using HPLC as described previou~ly.~~

carbaryl but not other N-methyl-carbarnate, and was associated with the membrane fraction (Chaudhry, unpublished data). Strain 50581 harbored a 50-kb plasmid, pCDl . Initial attempts to cure the plasmid DNA were not successful. However, conjugation experiments between 50581 and 50552 led to the isolation of derivatives of the latter bacterium that completely metabolized carbaryl (Figure 3). Analysis of these transconjugants should yield information on the gene(s) of 50581 involved in the hydrolysis of carbaryl in isolate 50581.

B. Aldicarb Aldicarb was the first oxime carbamate pesticide introduced under the trade name of TemikB by Union Carbide Corporation in 1967 (Table 1). Aldicarb has been used on tobacco, sugar beets, sugar cane, potatoes, and peanuts for the control of aphids, thrips, mealybugs, white flies, mites, and nematodes. Only the granular formulation of aldicarb is marketed, mostly because of its high mammalian toxicity (Table 1). Although microbial degradation of aldicarb is poorly understood, it has been shown to be oxidized to its sulfoxide and sulfone metabolites,

366

and hydrolyzed to its oxime and nitrile metabolites. 104*114.132,176,179Both the oxidative and hydrolytic products have been detected in the The oxidation products may be as toxic as the parent compound, while the hydrolytic metabolites have low toxicity.68 Therefore, it is important to know the mechanism of its transformation under various environmental conditions. Attempts have been made to isolate microorganisms responsible for aldicarb degradation in soil. Several fungal isolates of Fusarium and Penicillium, and bacterial isolates of Arthrobacter, Pseudomonas, Nocardia, Achromobacter, and Bacillus have been isolated from aldicarbtreated soil. 152 The fungal isolates, Fusarium and Penicillium, slowly metabolized aldicarb. While the bacterial isolates collectively degraded the pesticide rapidly, none of the single bacterial strains degraded aldicarb or its toxic metabolites. The degradation of aldicarb was concentration dependent. Concentrations of the pesticide higher than 800 and 5000 ppm inhibited bacterial and fungal growth, respectively. The microorganisms capable of degrading carbofuran were also found to metabolize aldicarb. Aldicarb was reported to be hydrolyzed under anaerobic conditions as well.92,132 It stimulated methanogenesis, as methylamine, a metabolite of aldicarb, was 34,72,823150

utilized as a source of energy by the methanogenic b a ~ t e r i a ..92 ~'


C. Thiocarbamates This group of carbamates is used as herbicides and fungicides. These compounds differ structurally from other carbamate insecticides in that they are primarily thio- or dithiocarbamates. ' I 3 The most widely used chemicals in this group are butylate (S-ethyl diisobutylthiocarbamate), cycloate (S-ethyl N-ethylthiocyclohexane carbamate), diallate (S-(2,3-dichloro-allyl) diisopropylthiocarbamate), EPTC , and vernolate (Spropyldipropylthiocarbamate) (Table 1). EPTC was the first among the thiocarbamates to be developed and registered for use in alfalfa, corn, cotton, potatoes, fruit crops, ornamentals, and a number of bean crops for the control of weeds. Similar to methylcarbamates, repeated exposure of soils to thiocarbamates results in a reduction in the efficacy of these compounds. 94,156,174,175 The reduced efficacy has been attributed to the rapid microbial degradation of the Microbial populations evolved traits responsible for the degradation of structurally related thiocarbamates, even though the soils were exposed to only one of the herb i c i d e ~ However, .~~~ if the herbicides are structurally different, adaptation of microorganisms to degrade one herbicide may not confer upon them the ability to degrade other herbicides of the same group. Since vernolate and EFTC are similar in structure (Table l), prolonged exposure of soil to either herbicide resulted in an adaptation of the microorganisms that degraded both compounds .207However, microorganisms adapted to the degradation of butylate, which is somewhat structurally different from EPTC and vernolate, failed to degrade EPTC and vernolate. Similarly, cycloate degradation was not influenced by EPTC, vernolate, or butylate because of the presence of a benzene ring (Table 1). Several reports implicated the involvement of microorganisms in the degradation of EPTC. 100-101,131,143 These organisms are listed in Table 2. Lee and co-workers'O' isolated 29 fungal and 9 bacterial isolates capable of degrading EPTC. While all the fungal isolates retained the

ability to degrade EPTC, the bacterial isolates lost this function after 15 months of storage. The authors speculated that the loss of EPTC degradation may be associated with loss of the plasmids harbored by these bacteria. The loss of this plasmid-associated degradative function has also been reported in other herbicide-degrading bact e ~ i a These . ~ ~ findings prompted a search for plasmids involved in the degradation of EPTC. Tam et al. I9O isolated an EPTC-degrading bacterium, Arthrobacter sp. strain TE1, that harbored four plasmids of 65.5, 60.0, 50.5, and 2.5 MDa in size. One of these plasmids (50.5 MDa) was cured (either spontaneously or by acridine orange treatment), and the cured derivatives of the strain TE1 lacked the ability to degrade EPTC. Results of the curing studies suggested that the 50.5-MDa plasmid may be associated with the catabolism of EPTC. The involvement of this plasmid in EPTC degradation was confirmed by conjugal transfer of the 50.5-MDa plasmid back into the mutants of strain TE1, as the transconjugants recovered the ability to degrade EPTC. Dick et al.50 isolated a Rhodococcus strain JEl from a loamy soil that was capable of metabolizing EPTC and exhibited a plasmid profile similar to the above Arthrobacter strain TE1. However, Rhodococcus plasmids could not be cured and no function could be attributed to its plasmids. In another study, a Flavobacterium sp. strain VI. 15 isolated from a soil with vernolateuse history utilized butylate, EPTC, or vernolate as a sole source of carbon. l 8 This bacterium also harbored two plasmids, pSMB1 and pSMB2, but only one of the plasmids, pSMB2, was implicated in the butylate-utilizing ability. The above studies demonstrate that genes for the degradation of thiocarbamates are encoded on plasmids in the isolated bacteria. Information on the catabolic pathways of thiocarbamates in isolated microorganisms is limited. Recent investigations aimed at studying the microbial metabolism of EPTC indicated that the Rhodococcus strain JEl metabolized EPTC to propionaldehyde and N-depropyl EPTC.50Ndepropyl EPTC was further degraded to mercaptan by the same isolate. Behki and Khan" proposed a different pathway for EPTC degradation in the Arthrobacter strain TE1 strain. They reported that dipropyl amine was found in culture

367

media as one of the metabolites of EPTC. And mutants of the TE1 strain deficient in EPTC degradation metabolized dipropyl amine or propyl amine as efficiently as the parent strain. This finding demonstrated that the 50.5-MDa plasmid of TEl codes for the determinants involved only in the initial cleavage of the thioester linkage in EPTC.


D. Phenylcarbamates Phenylcarbamates, or carbanilates, include both herbicidal and insecticidal carbamates (Table 1). Phenylcarbamateswere among the earliest herbicides developed.85 Like other carbamates, phenylcarbamates are degraded rapidly in soils, particularly those with a history of herbicide treatment. Again, the rapid degradation or loss in efficacy of these herbicides has been shown to be associated with the adaptation of microbial populations (Table 2). Vega et a1.Iw isolated Pseudomonas cepacia, which utilized two phenylcarbamate herbicides, BIPC (1-methyl-prop-2ynyl-3-chlorophenyl carbamate) and chlorpropham (isopropy l-N-3-chloropheny l carbamate), as the sole source of carbon and energy. Similarly, a P. alcaligenes was found to hydrolyze four phenylcarbamate herbicides -BIPC, chlorpropham, propham (isopropyl-N-phenylcarbamate), and SWEP (methyl 3,4-dichloro-phenylcarbamate) - to the corresponding anilines: aniline from propham, 3-chloroaniline from BIPC and chlorpropham, and 3,4-dichloroaniline from SWEP@ '' (Figure 4). This isolate also metabolized barban (4-chlorobutynyl 3-chlorocarbanilate), but without the production of 3-chloroaniline, even though barban has the same 3chlorophenyl carbamate group as BIPC and chloroprophamlo8 (Table 1). In both the above isolates, the herbicide-degrading function was found to be stable and inducible. The isolated microorganisms were used to study the biochemistry of phenylcarbamate degradation. P. alcaZigenes was used to purify the enzyme responsible for the hydrolysis of phenylcarbamates. 'I0 The isolated enzyme, an amidase, catalyzed the hydrolysis of all of the structurally related herbicides, but failed to use barban and carbetamide as substrates.

368

Aniline and its ring-substituted derivatives were also found to be the degradative products of other herbicides that accumulate in soil and represent important pollutants. Since spontaneous transformation of anilines in soil is very slow,187studies have been conducted on the microbial degradation of aniline and substituted anilines. 10,75,93,97,186,211,214,215,2l6 A MoraeZZa sp. strain G, isolated from a chemostat inoculated with soil, utilized aniline, 2-chloroaniline, 3chloroaniline, and 4-chloro-, 4-fluoro-, and 4bromoanilines but not 3,4-dichloroaniline as a sole source of carbon and nitrogen ,215*216 while in another study, rapid degradation of 3,4-dichloroaniline was observed in soils treated with aniline.2 1 1 3,4-DichloroaniIine was also mineralized rapidly in soil samples incubated with P. putida isolated from sewage by enrichment on propionanilide. Degradation of 3,4-dichloroaniline occurred through 4,5-dichlorocatechol, 3,4dichloromuconate, 3-chlorobutenolide, 3-chloromaleylacetate, and 3-chloro-4-ketoadipate to succinate and Biochemical characterization of the isolated microorganism^^^^ 1,81 revealed that anilines are generally degraded to catechols, which in turn are subjected to well-established ortho- or metacleavage pathway^^^.^^ except 4-chloroaniline, which was degraded via a modified ortho-cleavage ~ a t h w a y ~(Figure ~.~'~ 4). The ability of microorganisms to degrade the phenylcarbamate herbicides depends on the presence of broad-specificity hydrolases and oxygenases. These enzymes are involved in the transformation of herbicides first to their corresponding anilines and then to the catechols, which are further metabolized to tricarbaxylic acid (TCA)-cycle intermediates. Lack of one or more of these enzymes in the microorganisms may be the cause of incomplete degradation of phenylcarbamate herbicides. E. Other Carbamates Microbial degradation of other carbamates such as desmedipham, phenmedipham, and promecarb (Table 1) was investigated by Knowles and Benezet.94Both bacterial and fungal species that degraded these pesticides were isolated from

NHCooCmQY

NHCOOCHQY

0

61

Propham

Chlorpropham

I

I

6

0-

CI

Aniline


SWEP

ci

3-Chloroaniline

3,4-Dichloroaniline

,

I

I I

0

i

i

(y

t

0 [::a]

\

Catechol

3-Chlorocalechol

3,4-Dichloromuconate * Acetate

Succinale c 2-Chlorosuccinate

-

,.-*

/’ 7

I

-.-. - -.

k*

CI

3-Chlorobutenolide

+ - - -CI O L * 3-Chlorolevulinale

FIGURE 4. Proposed pathways for the microbial degradation of propharn, chlorpropharn, and SWEP.

soil. Metabolites resulting from the cleavage of Yardon et al. 209 investigated the degradation of the ester linkage of the three carbamates were methyl-benzimidazol-2-ylcarbamate (MBC) in identified as ethyl-N-(3-hydroxyphenyl)carbafive soils. MBC is the hydrolytic product of bemate, methyl-N-( 3-hydroxyphenyl)carbamate, nomyl (methyl- 1-(butylcarba-moy1)benzimidaand isothymol from desmedipham, phenmedizole-2-ylcarbamate). MBC was degraded rapidly pham, and promecarb, respectively. In three other in soils previously treated with benomyl. Howindependent studies, benfuracarb, bufencarb, ever, the degradation was delayed in soils treated carbosulfan, cleothocarb, furathiocarb, prowith the fungicide, tetramethylthiuram disulfide. poxur, and trimethacarb were found to be inefBased upon these observations, they proposed fective against cabbage and sorghum pests in soils that fungal species may be involved in the rapid treated previously with c a r b ~ f u r a n . ’ The ~ ~ ’ ~ ~ degradation ~~~~ of MBC. Degradation of another carloss of efficacy was attributed to the rapid degbamate, aminocarb (4-dimethylamino-3-methylradation of these pesticides by microorganisms. phenyl-N-methylcarbamate), was reported in cul369


tures of human intestinal bacteria using nutrient broth supplemented with 250 to lo00 pg of aminocarb per ml.23Only traces of aminocarb were detected in aerobic cultures of bacteria after 5 d. Most of the studies on the degradation of carbamate pesticides are concerned with aerobic microorganisms. Very little is known about the degradation of these compounds by anaerobic microorganisms. Since anaerobic conditions may be present in waterlogged soils, aquatic sediments, and subsurface soil, efforts should be focused on degradation of carbamates under anaerobic conditions.

F. Environmental Contamination by Carbamates Use of the pesticides in the production of agricultural commodities has increased with the worldwide demand for food and fiber, which in turn increased the potential for contamination of terrestrial and aquatic environments by agricultural chemicals. In recent years, several pesticides have been reported to contaminate ground water.33.3y*'y4 Aldicarb residues ranging from 1 to 50 ppb have been found in ground water in Arizona, California, Florida, Maine, Missouri, New York, North Carolina, Oregon, Virginia, Washington, and Wisconsin.15,33-159*213 Similarly, carbofuran has also been found in ground water in New York and Wisconsin at I - to 50-ppb levels (EPA data). Pesticide residues have been detected in vegetables and fruits that were sprayed with these chemicals for insect control. Three outbreaks of illnesses associated with aldicarb sulfoxide-contaminatedwatermelons and cucumbers in California and one in Nebraska have been reported .68 Although the concentrations of these chemicals required to cause acute toxicity in humans would generally be higher than those found in the contaminated environment, conditions present in the human gut favor the formation of N-nitrosocarbmates .56*154 The potential for the formation of such mutagens in the gut cautions that carbamate contamination might pose health problems. Therefore, it is important to investigate the enzymology and biochemistry of microbial degradation of these pesticides to help design strategies for detoxifying pollutants in the environment. 370

111. ORGANOPHOSPHATES

Numerous organic compounds of phosphorus occur naturally and are essential for the maintenance of life. However, many synthetic organophosphates such as lubricants, plasticizers, and pesticides are produced for commercial uses. These compounds are relatively less persistent but more effective compared with the chlorinated compounds used for similar purposes. 'I3 Thus, they replaced some of the recalcitrant chlorinated compounds and are at present one of the widely used classes of pesticide^.^^ Approximately 140 organophosphate compounds are being used currently as pesticides and plant growth regulators worldwide, and over 60,000 tons of these chemicals are produced annually in the U.S. alone. Microbial degradation of the major organophosphate pesticides are discussed below.

A. Parathions Parathion (Table 3) was first synthesized in 1944. Parathion and its methyl analog, methyl parathion (U,U-dimethyl-0-p-nitrophenyl phosphorothioate) (Table 3), have been used widely for controlling insects of agricultural and public health importance. Although parathions are considered to be less persistent, there have been instances when these pesticides and their metabolites, particularly p-nitrophenol, have caused environmental pollution problems. 18,90 p-Nitrophenol imparts odor problems in water resources. Several studies have reported the fate of parathions in various environments and the role of microorganisms in their degradation (Table 4). Although parathion can be hydrolyzed chemically at high pH and the hydrolytic product can escape to the atmosphere by volatilization, the bulk of applied pesticide is removed from the environment by microbial degradation.85,127 Microorganisms capable of utilizing parathion as a sole source of carbon were isolated from pesticide-treated soils.69 Nelson et al.Iz9 observed a proportional increase in the bacterial population of a loamy soil with an increase in the concentration of parathion added. These observations suggested that microbial growth was stimulated, and parathion was used as a carbon source. In another study, enrichment cultures from flooded

TABLE 3 Structure and Toxicity (Rat, Oral) of Organophosphates Common name (trade name)

Structure

866

Acephate (Orthene)

Carbophenothion (Trithion)


LD, (mg/kg of body wt.)

6

Chlorpyriphos (Dursban)

97

Coumaphos (Co-Ral)

13

Diazinon

66

Dichlorovos (Vapona)

EHI

FI (CH3O)z P-O-CHICCI~

0 & n (CHJO)~P-S-CH~C-NH-CHS

25 250

7

EPN

61

Fenitrothion (Sumithion)

yp

(H3CO)z P-0

250

Fensulfothion (Dasanit)

2

Fonofos (Dyfonate)

8

lsofenphos (Oftanol)

38

Malathion (Cythion)

S It

:

CH2-C-OC2Hs

885

I

(H3C0)2 P-S-CH-C-OC~HS II

0

371

TABLE 3 (continued) Structure and Toxicity (Rat, Oral) of Organophosphates Common name (trade name)

Structure

25

Methidathion (Supracide)


LD, (mglkg of body wt.)

Methylparathion

9

Monocrotophos (Azodrin)

21

3

Parathion (Niram) S

Phorate (Thimet)

(H&20)2 P - S - C H ~ - S - C ~ H S

soils, water, and sediment from river, lake, and pond samples were found to hydrolyze parathion. I S Flooded soil conditions favored the hydrolysis of parathion. More 14C0, was evolved from ring-labeled [ 14C]parathion in the rhizosphere of rice seedlings under flooded conditions compared with nonflooded conditions, indicating that soil planted with rice under flooded conditions permits significant ring cleavage by microorganisms. 153 Similar to carbamates, enhanced degradation of parathions was reported in soils previously exposed to the pesticide. Ferris and LichtensteiP used soil samples treated with parathion or p-nitrophenol for 4 d to investigate the fate of ring-labeled [I4C]parathion. On the average, 37 and 2% of the spiked [14C]parathion was released as I4CO2in treated and untreated samples, respectively, after 24 h of incubation. NelsonI2*isolated 50 bacteria from a loamy soil with a history of parathion use, and found that eight of the isolates hydrolyzed 75% of the added 10 pg of parathion per gram of dry soil in 5 d and appeared to be Bacillus strains. Ten of the isolates of the Arthrobacter genus hydrolyzed all of the parathion after 5 d. Arthrobacter sp. degraded parathion even in rich medium, suggesting that the enzymes are expressed consti-

372

2

tutively. The Bacillus sp. lost the degradation capability upon subculturing, while Arthrobacter sp. did not, suggesting that the genes for hydrolysis of parathion may be plasmid-borne in Bacillus sp. In another study, two Pseudomanas strains isolated from soil and sewage utilized parathion and malathion [S-(1,2-dicarboxy-ethyl)0,O-dimethyl dithiophosphate]as sources of carbon. Is8 These bacteria hydrolyzed parathion to pnitrophenol. A Flavobacterium sp. ATCC 2755 1 isolated from flooded soil hydrolyzed both diethy1 (parathion and diazinon) and dimethyl (methyl parathion and fenitrothion) phosphorothioates, while another bacterium, Pseudomonas sp. ATCC 29353, isolated from the same soil hydrolyzed only diethyl (parathion and diazinon) phosphorothioates .’ Similar observations were recorded by Chaudhry et aL3’ They isolated two mixed bacterial cultures that utilized both methyl parathion and parathion as a sole source of carbon. A Pseudomonas sp. isolated from these mixed cultures degraded the pesticides to p-nitrophenol only, while the mixed cultures degraded the pesticides completely. An investigation of methyl parathion degradation by aufwuchs, an aquatic microbial growth attached to submerged surfaces and suspended in streamers or

TABLE 4 Microbial Degradation of Organophosphates Compound


Parathion

Methyl parathion

p-Nitrophenol

DETP Diazinon

lsofenphos Fenetrothion Dichlorovos Cournaphos Methidathion

Microoraanism Bacillus strains Arthrobacter strains Flavobacterium sp. ATCC 27551 Pseudomonas sp. ATCC 29353 Pseudomonas spp. Pseudomonas sp. Pseudomonas strain CTP-01 P. diminuta MG Pseudomonas sp. Strain SC Pseudomonas sp. Pseudomonas sp. isolate 50541 Flavobacterium sp. ATCC 27551 Aufwuchs Flavobacterium sp. Pseudomonas spp. PNP-1, 2, and 3 Pseudomonas sp. isolate 50445 Moraxella sp. Pseudomonas sp. strain 24 P. acidovorans Flavobacterium sp. ATCC 27551 Pseudomonas sp. ATCC 29353 Pseudomonas spp. Pseudomonas sp. Arthrobacter spp. SB3 and SB4 Arthrobacter sp. Pseudomonas sp. Flavobacterium sp. ATCC 27551 P. aeruginosa Pseudomonas sp . Isolates B-1, B-2, and 8-3 B. coaaulans

Ref. 128 2 158 33 31 166 126 121 35 44 2 105 35 76 44

180 165 42 2 158 16 19 142 141 2 103 172 64

trations by immobilized bacteria in an aqueous mats, demonstrated a rapid transformation of the pesticide. I o 5 These and other s t u d i e ~ ’ ~ , ” . waste ~~~ A Moruxella sp. isolated from showed that parathions are generally first hydroactivated sludge utilized p-nitrophenol as the sole lyzed to p-nitrophenol and diethylthiophosphate source of carbon releasing nitrite. I8O Similarly, a (DETP), which are further metabolized (Figure Fluvobucteriurn sp. , degrading p-nitrophenol , released nitrite, which was assimilated into the bac5). terial cells.35 Recently, Spain and Gibson17*inThe fate of p-nitrophenol in the environment vestigated the enzymology and pathway of phas been investigated extensively. Several microorganisms have been isolated that readily denitrophenol degradation in Moruxella sp. They grade p-nitrophenol in soil, 1 0 7 , 1 8 8 sedifound that membrane bound p-nitrophenol oxymerit, 139,173,178,18 1 , 198 activated ~ l u d g e , ~ ~genase , ~ ~ mediated the conversion of p-nitrophenol water,80~’33,182,198 and ground water.3 The degrato hydroquinone, which was further metabolized to P-ketoadipate via r-hydroxymuconic semidation of p-nitrophenol has also been examined aldehyde and maleyl acetate (Figure 5 ) . in the presence of inorganic nutrients, 161,188,204 by bacteria in granular activated-carbon columns, The second metabolite of parathions, DETP, at low concentrations, 164.204 and at high concenwas found to be metabolized by enrichment cul-

373

Parathion

-..

s. Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Universite De Sherbrooke on 11/12/12 For personal use only.

I I

Melhylparaihion

pNiirophenol

H&zO-f-OCzH,

/

o(

/

Diethylthlophoaphate

Dimethylthiophosphata

CH,

t

+ Phosphate +

Sulfate

0

J

Hydroquinone

4-Nitrocatechol

I

0

i

t

Ring fission

TCA cycle

-------

B-Keioadipaie 0

FIGURE 5. parathions.

Proposed pathways for the microbial degradation of

tures obtained from cattle dip solution containing coumaphos. These cultures mineralized DETP to sulfate and phosphate while utilizing ethyl moieties as a carbon and energy source. Cook et al.42isolated P . acidovorans from sewage sludge that utilized DETP as a sole source of sulfur. These studies suggest that the hydrolysis of parathions and subsequent degradation of p-nitrophenol and DETP by soil microorganisms may be responsible for the rapid removal of these chemicals from the environment.

374

Biochemical studies of microorganisms capable of degrading parathions showed the presence of a parathion hydrolase enzyme.27,35,'27,166,167 P . diminuta cells grown for 48 h exhibited 3400 units of parathion hydrolase activity per liter of culture. Cell-free extracts of another Pseudomonas strain hydrolyzed parathion at a rate of 1 X 104 nmol/min/mg of protein.3' In another study, crude cell extracts prepared from a methyl parathion-hydrolyzing Pseudomonas sp. showed an optimum pH range


of 7.5 to 9.5 for the enzymatic hydrolysis of methyl p a r a t h i ~ n .Mulbry ~~ and KarnsI2' conducted extensive studies to characterize the parathion hydrolase from three bacteria isolated from different locations. The hydrolase from a Flavobacterium strain' was found to be membrane bound, having a single subunit of 35 kDa in size. This enzyme was inhibited by sulfhydryl reagents such as dithiothreitol (DTT) and by metal salts such as copper chloride (CuCI,). The enzyme from the SC strain121was also membrane bound, but was composed of four identical subunits of 67 kDa. It was inhibited by DTT, but stimulated by CuCl,. Unlike the above, parathion hydrolase was found in the cytosol of the B-1 It is composed of a single subunit of about 43 kDa and was stimulated by DTT but inhibited by CuC1,. The relative affinities of the enzymes to parathion and 0-ethyl-0-4-nitrophenyl phenylphosphonothioate (EPN), another organophosphate insecticide, also differed. The hydrolase from the B- 1 strain showed equal affinity for both insecticides, while the Flavobacterium enzyme displayed twofold lower affinity for EPN than for parathion. However, the hydrolase for strain SC exhibited no affinity for EPN. The hydrolase from all of the tested strains was produced constitutively and had a similar temperature optima at about 40°C. I 2 I In another independent study, the yield of parathion hydrolase was found to be improved 22-fold by growing Pseudomonas sp. on complex medium instead of on parathion in minimal medium.126Since the enzymes, esterase, aryl esterase or phosphotriesterase, involved in the inactivation of parathions from the isolated bacteria appeared to be isofunctional, their spread among the microorganisms may have a common ancestral origin. Although methyl parathion has also been known to be metabolized by oxidation, little is known about the biochemistry of the oxidative pathway. A Pseudomonas sp. strain 50541 isolated from the pesticide-waste disposal site was found to oxidize methyl parathion rapidly and completely.44 Since mineralization of parathion in this bacterium is complete without accumulation of intermediate metabolites, it may have greater potential for detoxification of pesticidecontaminated sites compared with the microorganisms that hydrolyze parathions, where p-ni-

trophenol accumulates as the hydrolysis product. Further investigation of this isolate may be helpful in further defining the pathway for the degradation of parathions.

1. Parathion Hydrolase Gene (opd) In bacteria, genes encoding unique degradative functions are often plasmid encoded, which range in size from a few to several hundred kilobases of DNA.30.136 Initial studies on parathionhydrolyzing P. diminuta strain MG showed that expression of parathion hydrolase activity from this strain was lost at a high frequency (9 to 12%) after treatment of the cells with mitomycin C.L66 The hydrolase-negative derivatives were found to be missing a plasmid, pCMS1, that was present in the wild-type organism, indicating that parathion hydrolase is encoded by the pCMS1 plasmid. Similarly, a plasmid pPDL2 from Flavobacterium sp. ATCC 27551 was found to be involved in the hydrolysis of parathion.Iz3The parathion hydrolase gene from both the P. diminuta and the Flavobacterium sp. has been cloned71.123.165.I67 and sequenced. 1 1 2 . 1 2 2 . I68 The cloned gene was located on a 1.5-kb BamHIL6s and a 7.3-kb EcoRILZ3fragment in P . diminuta and Flavobacterium sp., respectively. Hybridization studies demonstrated that the opd gene from both sources was h o r n o l o g o u ~and ~~~~~~ showed homology with total DNA from a Pseudomonas sp. that hydrolyzed methylparathi~n.~~ Mulbry et d.124 characterized the plasmids pCMS 1 and pPDL2 to determine the regions of homology other than the opd gene. The sizes of the pCMS 1 and pPDL2 were reported to be 70 and 39 kb, respectively. Further, the opd genes of both plasmids were located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids was found outside of this region. To characterize the cloned gene in detail, Mulbry and K a r n ~ ' ~ ~ sequenced a 1.6-kb BamHI-Pstl fragment containing the opd gene of plasmid pPDL2. This fragment contained only one open reading frame large enough to encode the 35-kDa protein. The amino acid composition of the purified protein

375


corresponded well with that predicted from the nucleotide sequence, and the data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Although a promoter region with perfect match at - 35, but a less favorable match at - 10 consensus sequences was identified upstream of the opd coding region, it failed to express in P. putida.IZ2However, the cloned gene fused with ZacZ expressed at higher levels in Escherichia coli compared with the parent strain. Similarly, the cloned opd gene from P . diminuta expressed poorly in E. coli and in a Pseudomonas sp., even though a niftype promoter sequences1was present upstream of the opd coding region.'z2 However, high levels of hydrolase activity were obtained when the opd gene was placed under the control of the lambda P, promoter in E. coli,168 indicating that the opd gene in E. coli, and P . putida requires an exogenous promoter. DNA sequencing studies showed that the nucleotide sequence of the two opd genes is identical. 1z2,168 The opd gene has also been cloned in various systems and its product has been characterA comparison of the cloned and native hydrolases showed that the hydrolase synthesized in E. coli was larger in size than the Flavobacterium enzyme. Iz2 In contrast, the cloned gene in Gram-positive S. lividans had a gene product similar in size to the wild-type enzyme.Ix4In addition, the enzyme produced in s. lividans was excreted into the culture medium, suggesting that the enzyme required processing. In fact, the DNA sequencing results suggest the presence of a signal sequence in the precursor protein. 122~168Rowland et al. purified parathion hydrolase to homogeneity from the recombinant S. lividans. The recombinant hydrolase and the native hydrolase had similar characteristics, including molecular weight, temperature optima, and K, values except that the recombinant enzyme had a higher affinity for ethyl parathion (K, value of 46 pmol/min/mg of protein) than the native enzyme (K, of 211 pmol). Both proteins appeared to have been processed similarly in the native Flavobacterium sp. and recombinant strain of S. lividans, resulting in the same Nterminal amino acid sequence. Analysis of the native membrane-bound parathion hydrolases

376

from Flavobacterium sp. and P . diminuta and the recombinant hydrolases from E. coli and S . lividans indicate that the S. lividans system can be used effectively to produce high levels of enzyme. Since the enzyme is secreted into the culture medium, it offers more potential uses for toxic waste treatment strategies, as purification of the enzymes secreted into culture medium is relatively easier than purifying from the whole cell. Further, the excreted enzyme may have greater accessibility to chemicals that are not actively taken up by the bacterium or metabolites that are toxic to the host. It should also be interesting to compare the properties of this enzyme with the isofunctional enzyme from another Grampositive bacterium, S. pilosus .63

B. Diazinon Diazinon (O,O-diethyl-O-2-isopropyl-4methyl-6-pyrimidyl phosphorothioate (Table 3) is used as a soil and foliar insecticide and is effective against a broad range of insect pests of crops and ornamental plants. It provides a good residual treatment for control of flies in barns and is also used in household sprays and dusts for ant and cockroach ~ o n t r o l . " It ~ has also been used widely on golf courses and sod farms and nurseries. Two Pseudomonas spp. isolated from sewage sludge degraded diazinon, producing diethy1 phosphorothioate as the metabolite in the culture medium.158A Pseudomonas sp. that hydrolyzed several organophosphates, including parathion, methyl parathion, dursban, paraxon, aminoparathion, and other methoxy- or ethoxysubstituted organophosphates, was found to hydrolyze diazinon as we11.16The crude cell extracts of this bacterium exhibited an enzyme activity that hydrolyzed diazinon at an optimal pH of 9.0. The specific activity of the enzyme in the crude extract was 0.44 pmoUmg of protein per min, and the optimum temperature for this activity was determined to be between 35 and 47°C. In another study, Barik et al." found that the cell-free extracts from two strains of Arthrobacter spp., SB3 and SB4, hydrolyzed diazinon, and the K, values for the enzyme activity for the two strains were 1.3 and 2.0 pmoUmg of protein per min,


respectively. This enzyme activity had a broad pH and temperature optima of 6 to 9 and 25 to 36"C, respectively. Because of the potential toxicity of diazinon to birds, its use on golf courses and sod farms has been suspended twice since 1988.7In spite of the concerns regarding the use of diazinon, little is known about the microbial degradation of this pesticide. Although Flavobacterium sp. ATCC 27551 was isolated originally as a diazinon-degrading baterium, it was well characterized with respect to parathion hydrolysis. Clearly, additional studies are needed to elucidate the metabolic fate and biochemical pathways involved in the degradation of diazinon.

aeruginosa,a Pseudomonas sp., and a Gram-positive, spore-forming bacterium individually had less activity than the enrichment culture responsible for the degradation of dichlorovos.

E. Coumaphos

Coumaphos (O,O-diethyl-O-(3-chloro-4methyl-2axo-2H- 1 -benzo-pyran-7-yl)phosphorothioate) (Table 3 ) is used as an acaricide for the control of the southern cattle tick (Boophilus microplus) and the cattle tick (B. annulatus). It is used mostly by the Animal and Plant Health Inspection Service (APHIS), U. S . Department of Agriculture, in its tick eradication program. The APHIS dips several hundred thousand cattle annually for tick control along the U.S.-Mexican C. lsofenphos border. Each of the 42 vats contains about 12,000 1 of flowable solution (42% coumaphos, Isofenphos (O-ethyl-O-(2-[isopropoxycar58% inert ingredients), 172 presenting a considbonyl1phenyl)N-isopropyl phosphoroamidoerable waste disposal problem. Since the halfthioate) (Table 3 ) is a systemic insecticide with life of coumaphos in soil and water is about 300 nematicidal properties . 6 1 Isofenphos was ded,88 safe and effective methods for disposal of graded more rapidly in soils with a history of the coumaphos waste are required. insecticide use compared with unexposed soils. '42 Soils with enhanced isofenphos degradation conShelton and Karnsl" observed rapid degratained an adapted population of soil microorgandation of coumaphos in several cattle-dipping vats that resulted in the loss of its efficacy against isms. Two bacterial isolates, an Arthrobacter and ticks. In a separate study, three bacteria were a Pseudomonus species, isolated from the adapted isolated from enrichment cultures developed uscultures metabolized the pesticide in pure ing the dip vat solutions as an inoculum. 172 These culture. 141,142 isolates, B-1, B-2, and B-3, hydrolyzed coumaphos to chlorferon (3-chloro-4-methyl-7-hyD. Dichlorovos droxy-coumarin) and diethylthiophosphate. Chlorferon was further metabolized by B-1 and Dichlorovos (2,2-dichlorovinyl-O,O-di- B-2 strains to a-chloro-P-methyl-2,3,4-trihymethyl phosphate (Table 3 ) , is used extensively droxy-trans-cinnamic acid. Parathion hydrolase in Vapona strips a preparation in which the inenzyme produced by the Flavobacterium sp. secticide is impregnated in a resin and volatizes ATCC 27551 was also found to hydrolyze couat a fairly uniform rate to give control of housemaphos, yielding chlorferon and DETP.83,88 hold pests, especially flies. ' I 3 Dichlorovos is efChlorferon thus generated was further degraded fective against ectoparasites and is used in flea by a UV-ozonation process, where pesticide suscollars for dogs and cats and a number of vetpensions are pretreated with UV light in the preserinary applications. Information on the microence of oxygen prior to soil disposal.89However, bial degradation of dichlorovos is limited. In a UV ozonation of coumaphos resulted in only limsingle study, a microbial enrichment was found ited degradation of the pesticide.88 When the to convert dichlorovos to dichloroethanol and method of microbial hydrolysis was followed with dichloroacetic acid and ethyl dichloroacetate. ' 0 3 ozonation on large volumes of coumaphos waste This enrichment culture was obtained from sewunder field conditions, complete hydrolysis of age. Three members of the mixed culture, P . coumaphos was achieved in 48 h and more than

377

20% of the chlorferon produced was degraded in 20 h, indicating that the combined treatment was very effective in eliminating coumaphos


F. Other Organophosphates Some other organophosphates have been used extensively for insect and weed control. Glyphosate (N-phohsphonomethyl glycine), the active ingredient of Roundup@, is a broad-spectrum organophosphate herbicide. This herbicide was used as a sole source of phosphorus by Pseudomonnas spp. ,116,189 and an Alcaligenes sp. 189 One of the Pseudomonas spp.'16 completely degraded glyphosate, and equivalent cellular yields were obtained with equimolar amounts of either inorganic phosphate or glyphosate as the phosphorus source. Methidathion ([S-(S-methoxy-20x0- 1,3,4-thiadiazol-3-(2H)-yl)methyl]O, 0dimethyl phosphorodithioate) (Table 3) is used for the control of insects on alfalfa, cotton, and fruit crops. It is also used in greenhouses, mainly for rose cultures, against thysanopterae and lepidopterae, and in vegetable nurseries. A sewage bacterium, Bacillus coagulans, degraded methidathion.64Desmethyl methidathion was identified as one of the major metabolites found in the culture medium supplemented with the pesticide. This suggested that methidathion is converted by demethylation. Soil and sewage microorganisms were found to degrade several organophosphates, including aspon, azodrin, dasanit, orthene, trithion, dimethoate, and dy10x.I~~ Two isolated Pseudomonas strains utilized these compounds as sole sources of phosphorus. Cell-free extracts of these bacteria catalyzed the degradation of azodrin, trithion, and dasanit, releasing dimethyl phosphate, diethyl phosphorodithioate, and diethyl phosphorothioate, respectively. Another group of workers studied the microbial degradation of chloropyrifos, fonofos, ethoprop, terbufos, and phorate. 142 These chemicals were degraded rapidly in soils with a previous history of pesticide application. As stated above, enhanced degradation of these pesticides was also dependent upon the structural specificity of the compound applied to the soil.

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IV. BIOTECHNOLOGICAL ADVANCEMENT IN DEGRADATION OF CARBAMATES AND ORGANOPHOSPHATES Microorganisms exhibit remarkable metabolic diversity and ability to grow rapidly. Therefore, they have been used for producing a variety of biochemical products, including industrially important metabolite^,'^^ amino antibiotic~,'~~ and pharmaceuticals. They have also been employed to produce recombinant proteins such as insulin,66growth horm ~ n eand , ~ vaccines'62 ~ more economically. Microorganisms play a major and indispensable role in the breakdown of oils and industrial and degradation of sewage as well as environmental pollutants, including pesticides .27.78,79,83.87,88, 125,126 The observations that microorganisms can adapt and evolve new metabolic functions relatively rapidly to degrade hazardous x e n o b i o t i ~ s ~opened ~ * ' ~ ~ up yet another avenue for their practical use in the remediation of polluted environments. Since some of the synthetic chemicals are recalcitrant, while others are not completely metabolized, seeding of toxic wastes and polluted environments with microorganisms could promote bioremediation. Intensive commercial uses of xenobiotics and agricultural practices result in contamination of soil and ground watef'3.39,208and generate pesticide wastes. The options for dealing with the environmental pollution problems include chemical treatment, incineration, and physical removal of polluted soil. These methods are expensive, inefficient, and generate additional problems, whereas the biological methods of treatment are potentially more efficient, inexpensive, and can result in the complete breakdown of contaminants. However, success of the biological methods depends upon the efficacy of microorganisms and the stability of their degradative traits in the polluted environments. Recent advancements in the knowledge of the molecular basis of biodegradation and biotechnology techn i q u e ~provide . ~ ~ promising ways to devise strategies for control of pollution caused by the use of synthetic chemicals.


However, unlike halogenated hydrocarbons, limited attempts have been made to study the catabolic traits of microorganisms involved in the degradation of carbamate and organophosphate pesticides. Two bacteria, P. dirninuta166"67and a Flavobacterium sp. were shown to exhibit plasmid-encoded enzyme activity responsible for the hydrolysis of parathions. The opd gene encoding this activity was first cloned from P. d i m i n ~ t a land ~ ~ then from the Flavobacterium sp." Characteristics of the opd gene isolated from these bacteria have been discussed above. The opd gene product converts parathions to p-nitrophenol. Since p-nitrophenol is considered to be hazardous to biota, to achieve complete degradation of parathion, the opd gene was introduced into a p-nitrophenol-degrading bacterium, Pseudornonas sp. The recombinant derivatives of Pseudomonas sp. failed to metabolize parathion, suggesting that either the opd gene was not expressed or its product was inactive in the new host.16s However, the opd gene was expressed efficiently in a Gram-positive bacterium, S. livi d ~ n sTherefore, .~~ it should be interesting to test the expression of the opd gene in other bacteria, particularly those capable of degrading p-nitrophenol.44 Alternatively, the catabolic genes of microorganisms such as Pseudornonas sp. strain 5054 1, which completely metabolizes organophosphates, should be cloned and characterized. Information on the catabolic genes of these bacteria may lead to the improvement and expansion of the substrate range of microorganisms. Like the opd gene, a hydrolase gene, mcd, involved in the hydrolysis of methylcarbamates was isolated from Achromobacter sp. strain WM111 as described above. Whereas the opd gene from the two bacteria had complete nucleotide sequence homology, the mcd gene from Achromobacter sp. encoding a cytosolic enzyme appeared to be different from similar genes present in other bacteria. The cloned rncd gene did not hybridize to the DNA of other carbamatehydrolyzing bacteria isolated in our, as well as other, laboratories. The hydrolase activity in P. aeruginosa strain 50581 was found to be associated with the membranes, as mentioned above, and the results of the study of Tomasek and KarnsI9' showed that the cloned rncd gene did not express in other Gram-negative bacteria.

These observations suggest that, unlike the opd gene, the isofunctional mcd genes are much more diverse in microbial populations and perhaps distinctly related, whereas the opd genes might have been evolved recently with little or no diversity. P . aeruginosa strain 50581, capable of hydrolyzing carbaryl, and a 1-naphthol-degrading P. aeruginosa strain 50552 grown together mineralized both carbaryl and l -naphthol rapidly and completely32 (Figure 6). However, the potential problems of compatability of the mixed culture could limit the use of the consortium for in situ remediation of contaminated environments. Since l-naphthol is toxic6 another approach was used for achieving complete degradation of carbaryl by a single microorganism. This approach involved the mating of carbarylhydrolyzing and 1-naphthol-degrading bacteria (P. aeruginosa strains 50581 and 50552, respectively), which yielded derivatives of 50552 capable of metabolizing carbaryl completely (Figure 3). Even though microorganisms can be isolated to degrade most of the xenobiotics under controlled laboratory conditions, they often fail to perform well under field conditions for several reasons, including competition with the microbial community and the presence of mixtures of pollutants. Such problems could be dealt with by enhancing and expanding the degradation potential of indigenous microorganisms using recombinant DNA techniques. The steps involved in improving the biodegradation potential of microorganisms using genetic engineering techniques include (1) cloning and characterization of the degradative genes, (2) development of suitable cloning and expression systems, and (3) selection and transformation of indigenous bacterial species that are abundant in soil and water environments. Although gene cloning systems for the Gram-negative soil bacteria have been well developed, 13.14.55.1 17,163 detailed information on the genes involved in the metabolism of carbamates and organophosphates is lacking. This limits the potential application of genetic engineering techniques for constructing microbial strains with novel combinations of genes for the complete and rapid remediation of polluted environments. In addition, the regulation of expression of degradative genes could also limit the application of

379

125 125

6 F

X

4

100

Y

z

0

C

75 751

/”

I’ V

50581 50581 + 50552 50552


50

the degradation of naphthalene to salicylate, and nahGHIJK, coding for the oxidation of salicyalte, are organized as separate operons.210 Likewise, genes tfdCDEF, involved in the degradation of 2,4-D53,and xylABC and xylDEGF, encoding the upper and lower pathway, respectively, of toluene and xylene catabolism, are also arranged in clusters.62If the genes involved in the oxidation of carbamates and organophosphates are organized in operons, they can be cloned and used to equip native bacteria with new catabolic functions. (3) Synthetic portable gene cassettes containing novel combinations of genes can be constructed and recruited (see Figure 10 of Chaudhry and Chapalamad~gu~~) to generate isolates capable of degrading mixtures of carbamate and organophosphate pesticides. Another feasible approach could be to use enzymes, rather than viable microorganisms, for the detoxification of contaminated environments. The enzymes can be more stable and functional than microorganisms under environmental extremes. 134 They can also be immobilized for wider applications in water systems and in bioreactors to decontaminate polluted waters and soils, respectively. This may, however, require large amounts of purified enzyme. Genetic engineering offers the possibility of producing such enzymes in large quantities. Although E. coli is the most commonly used prokaryotic host for producing recombinant proteins, the cloned gene products are generally retained in the cell in this system.zo3 Sometimes, high-level expression of recombinant molecules is deleterious to the host sysIn addition, their purification could be cumbersome. Therefore, Gram-positive bacteria can be used to excrete the recombinant enz y m e ~ . ~S.. ’lividans ~~ was exploited for extracellular excretion of the parathion hydrolase,Is4 and the enzyme preparation was evaluated for the in situ treatment of toxic organophosphate waste by Copella et al.43The recombinant enzyme effectively hydrolyzed coumaphos (waste in cattle dip solution) to chlorferon. Similar studies with the cloned mcd gene could be carried out, and the potential of methylcarbamate hydrolase enzyme in the remediation of carbamate-polluted point sources can be evaluated. The microbial potential to degrade pesticides provides solutions not only to the problems of environmental contamination, but might also be

255 25

0

0

1

2

3

4

6

Incubation timo (hr)

FIGURE 6. Release of ’“CO, from cultures of the consortium and individualP. aeruginosa isolates grown in minimal medium containing [“Clcarbaryl. The amount of ’“CO, released at various intervals from the suspensions was estimated using a scintilation

microorganisms for the purpose of bioremediation. If regulatory elements are needed for gene expression, then the degradativegenes alone may not be expressed in new host systems. The undetectable expression of opd and mcd in foreign h o ~ tis ~indicative ~ ~ ~ of this * ~problem. ~ ~ A few examples of improving the degradation capabilities of microorganisms include (1) the cloning of genes coding for enzymes with broader substrate specificity into native microorganisms. For example, the xylD (toluate 1,2dioxygenase), and x y L (dihydro-dihydroxybenzoic acid dehydrogenase) genes of TOL plasmid and the nahG gene of the NAH7 plasmid were recruited into a 3-chlorobenzoate-degrading Pseudomonas sp. B13(WR1) to expand its catabolic properties.lo2 The recombinants of WR1 were able to degrade 4-chlorobenzoate and 3 3 dichlorobenzoates, salicylate, and its 3-, 4-, and 5-chloro-substituted derivatives as well. Similarly, the opd and mcd genes can be recruited to expand the substrate range of microorganisms. (2) The degradative genes are often clustered in operons that can be cloned to equip the microorganisms with a whole new metabolic pathway. For instance, the genes nahABCDEF, specifying

380


useful in developing crop plants resistant to herbicides. Herbicides are commonly used to control weeds for increasing the crop yields. However, most of the herbicides do not distinguish between weeds and crop plants. Therefore, if crop plants are made resistant to herbicides, many broadspectrum herbicides such as glyphosate can be used more effectively in plant protection. This can be achieved by transferring the useful microbial traits into plants, using genetic engineering techniques. Three approaches have been pursued to develop herbicide tolerance in plants: (1) altering the target enzyme for the herbicide, (2) overproduction of the enzyme, and (3) incorporation of gene coding for an enzyme that degrades the herbicide. In the first case, resistance to sulfonylurea compounds in canola and cotton plants was achieved by introduction of the acetolactate synthase gene. Comai et al.40 isolated the mutant gene aroA from Salmonella tyhimurium that confers resistance to glyphosate. The mutant aroA, overexpressing the 5-enol-pyruvyl-shikimate-3 phosphate synthase, was introduced into glyphosate-sensitive tobacco plants, which yielded herbicide-resistant transgenic cultivars .41 In the third case, the bar gene from S . hygroscopicus, coding for an enzyme that detoxifies phosphinothricin (a potent new herbicide), was transferred into tobacco, tomato, and potato plants. The transgenic plants were completely resistant to high doses of commercial formulation of the herbicide.48 Similarly, Stalker et al.Is3 transferred the bxn gene, encoding a nitrilase enzyme, into tobacco plants. The gene was isolated from the soil bacterium. Klebsiella ozaenae, which converts the herbicide bromoxynil (3,5dibromo-4-hy droxybenzonitrile) to 3,5-dibromo4-hydroxybenzoic acid. Expression of this gene in tobacco plants conferred resistance to bromoxynil. The few examples cited above demonstrate the application of the catabolic traits of microorganisms to the remediation and mitigation of environmental pollution as well as the improvement of genetic traits in crop plants.

alization of xenobiotic compounds, including pesticides, is largely responsible for the removal of the bulk of these compounds from soil and aquatic environments. Like other biological processes, biodegradation is governed by physical parameters such as oxygen, pH, temperature, and nutrients present in a particular environment, and also to a large extent by the nature of the microhabitat with respect to the microbial community and xenobiotic compound. Prior exposure of microbial populations to pesticides is also important, as it often is responsible for the adaption of microorganisms, which evolve new genetic functions under stress such as limited nutrient conditions. In general, microorganisms responsible for the degradation of xenobiotics belong to a few genera, such as Achromobacter, Arthrobacter, Bacillus, Flavobacterium, Nocardia, and Pseudomonas. Numerous microorganisms belonging to these genera have been isolated that can eliminate carbamate and organophosphate compounds from the environment. These microorganisms have been used to study the enzymology of biodegradation of a few selected compounds only. The pathways and genes involved in the degradation of these chemicals are less understood. An advancement in our knowledge about these aspects of microbial degradation of carbamates and organophosphates should be helpful in developing recombinant derivatives of indigenous microorganisms with a broad substrate range. Such microorganisms may degrade mixtures of pollutants, which are often found in the environment, efficiently. Once the recombinant microorganisms are characterized, laboratory studies should be supplemented with smallscale field trials to evaluate the biodegradation potential of the xenobiotic-degrading microorganisms in real-world systems. This knowledge should help in developing strategies for ensuring the safety, while improving the efficacy, of pesticide application. In addition, the catabolic traits of microorganisms can be used for developing transgenic plants tolerant to herbicides.

V. CONCLUSIONS

VI. ACKNOWLEDGMENTS

Microorganisms are ubiquitous, and the range of their activities is enormous. Microbial miner-

We thank Frank Butterworth, S . A . Ansari, and M. J. Anstett for their comments and helpful


discussion during the preparation of the manuscript. Work in our laboratory is supported by National Science Foundation grant DMB-9020525, Florida DER grant WM 301, Public Service Health grant BSRG 50RR713 from the National Institutes of Health, and the Michigan State Research Excellence Fund.

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Biotechnological and environmental microbiological research in the Baltic region.

Biotechnological aspects of cytoskeletal regulation in plants.

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Microbiological and technological aspects of cassava-starch fermentation.

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