Use of Microorganisms and Microbial Systems in the Degradation of

C h a p t e r 13 Use of in

Microorganisms the

and

Degradation

Microbial of

Systems

Pesticides

Downloaded by CALIFORNIA INST OF TECHNOLOGY on September 17, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch013

Jeffrey S. Karns, Mark T. Muldoon, Walter W. Mulbry, Myra K. Derbyshire, and Philip C. Kearney Pesticide Degradation Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 Numerous agricultural chemicals have been shown to be degraded to varying degrees by microorganisms isolated from soil and water. By studying the biochemical and genetic basis of pesticide metabolism in bacteria we hope to be able to maximize the efficiency of these biochemical degradation processes so that feasible biological waste disposal methods can be developed. Hydrolase enzymes which degrade methyl-carbamate insecticides or organophosphorus insecticides have been isolated from several bacteria and have been partially characterized. One of these, parathion hydrolase, has proven useful in waste processing. The gene encoding parathion hydrolase has been shown to be identical in two very different bacteria. In both cases the gene is carried on plasmid DNA. While the genes are identical DNA-DNA hybridization and restriction enzyme mapping of the plasmid DNA have shown that the DNA outside the gene for parathion hydrolase differs markedly between the two bacteria. One bacterium which produces parathion hydrolase has proven useful in the elimination of the insecticide coumaphos, used extensively in cattle dipping operations.

The e f f e c t i v e d i s p o s a l o f s m a l l v o l u m e s o f a q u e o u s p e s t i c i d e w a s t e s i s one o f t h e major p r a c t i c a l p r o b l e m s f a c i n g A m e r i c a n a g r i c u l t u r e today. P u b l i c c o n c e r n f o r t h e s t a t e o f groundwater p u r i t y and t h e broad r e g u l a t i o n s embodied i n t h e Resource C o n s e r v a t i o n and Recovery A c t (RCRA) h a v e s t i m u l a t e d renewed a n d i n c r e a s e d i n t e r e s t i n t h e u s e of microorganisms w i t h unique biodegradative p r o p e r t i e s f o r the safe disposal of agrochemical wastes. As d e m o n s t r a t e d b y t h e b r o a d r a n g e o f t o p i c s p r e s e n t e d d u r i n g the c o u r s e o f t h i s symposium, b i o t e c h n o l o g y o f f e r s tremendous p o t e n t i a l f o r s i g n i f i c a n t a d v a n c e m e n t s i n many a r e a s o f a g r i c u l t u r e . Our l a b o r a t o r y i s i n v e s t i g a t i n g t h e r o l e t h a t b i o t e c h n o l o g y c a n p l a y i n t h e management o f p e s t i c i d e r e s i d u e s i n t h e e n v i r o n m e n t . One s u c h

This chapter not subject to U.S. copyright. Published 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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r o l e i s i n the e l i m i n a t i o n of p e s t i c i d e r e s i d u e s i n aqueous wastes g e n e r a t e d a f t e r a p p l i c a t i o n and r i n s i n g o p e r a t i o n s . Another i s i n removing r e s i d u a l p e s t i c i d e from c o n t a i n e r s p r i o r to t h e i r d i s p o s a l . Our h o p e i s t h a t t h e t e c h n i q u e s c o m m o n l y a s s o c i a t e d w i t h b i o t e c h n o l o g y w i l l f a c i l i t a t e the development of e f f i c i e n t , low-cost b i o l o g i c a l methods t h a t f a r m e r s o r p e s t i c i d e a p p l i c a t o r s can u s e o n - s i t e f o r e f f e c t i v e w a s t e management. Yet another area of a c t i v e i n t e r e s t i s i n u s i n g b i o t e c h n o l o g y to c o n t r o l the r a t e of m i c r o b i a l d e g r a d a t i o n of a p e s t i c i d e i n t h e f i e l d so t h a t an e f f e c t i v e c o n c e n t r a t i o n of p e s t i c i d e i s m a i n t a i n e d l o n g enough to c o n t r o l target pests. P e s t i c i d e c o n t a m i n a t i o n o f g r o u n d w a t e r may o c c u r a s a c o n s e q u e n c e o f t w o t y p e s o f s i t u a t i o n . F i r s t , t h e r e may be p r o b l e m s r e s u l t i n g from the a p p l i c a t i o n of p e s t i c i d e s under f i e l d c o n d i t i o n s f o r the c o n t r o l of a c t i v e p e s t s . Most s o i l a p p l i e d p e s t i c i d e s e x e r t t h e i r e f f e c t s i n the root zone. Once a p e s t i c i d e e s c a p e s t h i s z o n e i t becomes a w a s t e m a t e r i a l . Movement o f some c o m p o u n d s o u t o f t h e r o o t z o n e , t h r o u g h t h e v a d o s e zone and i n t o g r o u n d w a t e r i s o f particular concern. In r e a l i t y , o n l y a f r a c t i o n of a percentage of any p e s t i c i d e a p p l i e d t o s o i l a c t u a l l y r e a c h e s groundwater (1). N e v e r t h e l e s s , w i t h modern a n a l y t i c a l c a p a b i l i t i e s , t h e s e s m a l l but detectable concentrations of p e s t i c i d e residues are the b a s i s for the present concern f o r groundwater q u a l i t y . The s e c o n d p r o b l e m i s t h e d i s p o s a l of waste w a t e r s g e n e r a t e d by f a r m e r s , c o m m e r c i a l a p p l i c a t o r s , and i n d u s t r y from e x c e s s o r unused aqueous p e s t i c i d e solutions. I f i m p r o p e r l y h a n d l e d , t h e s e w a s t e s may a l s o l e a d t o groundwater p o l l u t i o n . T h i s s i t u a t i o n i s e s p e c i a l l y a c u t e where u n l i n e d waste d i s p o s a l p i t s are used f o r d i s p o s a l . Here, a c c u m u l a t i o n o f p e s t i c i d e r e s i d u e s may o v e r w h e l m t h e i n h e r e n t b i n d i n g and b i o d e g r a d a t i v e c a p a c i t y of the s o i l s , l e a d i n g t o r a p i d m i g r a t i o n o f r e l a t i v e l y l a r g e amounts o f p e s t i c i d e t h r o u g h t h e s o i l and i n t o the groundwater. T h e r e i s a p r e p o n d e r a n c e o f e v i d e n c e t h a t most a g r i c u l t u r a l p e s t i c i d e s a r e s u b j e c t t o some d e g r e e o f m i c r o b i a l m e t a b o l i s m . It is b e y o n d t h e s c o p e o f t h i s a r t i c l e t o a t t e m p t t o r e v i e w t h e more t h a n t h r e e decades of r e s e a r c h on the r o l e of microorganisms i n p e s t i c i d e metabolism. A number o f e x c e l l e n t r e v i e w s on t h i s s u b j e c t a r e available (2,3,4,5). A m a j o r m e s s a g e t h a t c a n be d e r i v e d f r o m p a s t r e s e a r c h i s t h a t t h e r e e x i s t s i n the s o i l m i c r o b i a l community a w e a l t h o f g e n e t i c m a t e r i a l t h a t c o u l d be e x p l o i t e d f o r t h e c o n t r o l l e d d e g r a d a t i o n of p e s t i c i d e s and t h e i r p r o d u c t s i n w a s t e d i s p o s a l efforts. We w i l l d e s c r i b e o u r e f f o r t s t o e l u c i d a t e some o f t h e b i o c h e m i c a l and g e n e t i c mechanisms of p e s t i c i d e m e t a b o l i s m by s o i l b a c t e r i a and t o e x p l o i t b i o l o g i c a l d e g r a d a t i o n of p e s t i c i d e s i n a waste d i s p o s a l s i t u a t i o n . Biochemical B a s i s of

P e s t i c i d e Degradation i n Microorganisms

Numerous b i o c h e m i c a l r e a c t i o n s d i r e c t l y a f f e c t i n g p e s t i c i d e s have been d e s c r i b e d u s i n g pure c u l t u r e s of b a c t e r i a . Enzymes w h i c h c a t a l y z e t h e c o n v e r s i o n o f p e s t i c i d e s t e n d t o f a l l i n t o two c l a s s e s ; h y d r o l a s e s ( e s t e r a s e s , a m i d a s e s , h a l i d o h y d r o l a s e s ) and o x y g e n a s e s (mono o r dioxygenases).



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BIOTECHNOLOGY IN AGRICULTURAL CHEMISTRY

H y d r o l a s e s have a number of p r o p e r t i e s t h a t make them a t t r a c t i v e f o r use i n waste d i s p o s a l s t r a t e g i e s . They r e q u i r e no c o f a c t o r s f o r a c t i v i t y , are s t a b l e over a wide range o f pH and t e m p e r a t u r e , and have a broad s u b s t r a t e s p e c i f i c i t y . Examples of p r e v i o u s l y d e s c r i b e d h y d r o l a s e s a f f e c t i n g p e s t i c i d e s are the h a l i d o h y d r o l a s e s w h i c h d e h a l o g e n a t e d a l a p o n ( 2 , 2 , - d i c h l o r o p r o p i o n i c a c i d ) and a number o f o t h e r h a l o g e n a t e d a l i p h a t i c a c i d s ( 6 , 7 , 8 ) ; t h e amidase d e s c r i b e d by W a l l n o f e r eit al ( 9 , 1 0 ) w h i c h r a p i d l y degrades the a c y l a n i l i d e h e r b i c i d e p r o p a n i l and s e v e r a l u r e a h e r b i c i d e s ( l i n u r o n , d i u r o n ) ; and p a r a t h i o n h y d r o l a s e ( p h o s p h o d i e s t e r a s e ) ( 1 1 , 1 3 ) which degrades p a r a t h i o n and a number of r e l a t e d 0 , 0 - d i e t h y l t h i o p h o s p h o r a t e i n s e c t i c i d e s . Munnecke ( 1 2 , 1 3 ) has demonstrated t h a t the p a r a t h i o n h y d r o l a s e from Pseudomonas d i m i n u t a can be used e f f e c t i v e l y i n t h e d e g r a d a t i o n o f οrganophosphate i n s e c t i c i d e w a s t e s , r a n g i n g from i n d u s t r i a l p r o c e s s wastewaters t o r e s i d u a l m a t e r i a l l e f t i n containers. Oxygenases tend t o be more complex enzymes and can be d i v i d e d i n t o two groups; mono-oxygenases ( p r e v i o u s l y c a l l e d m i x e d - f u n c t i o n o x i d a s e s ) w h i c h r e q u i r e reduced p y r i d i n e n u c l e o t i d e s as c o f a c t o r s , and d i o x y g e n a s e s w h i c h do not r e q u i r e reduced compounds as c o f a c t o r s . A l l oxygenases r e q u i r e m o l e c u l a r oxygen as a s u b s t r a t e , and tend t o be l e s s s t a b l e t h a n h y d r o l a s e s . An example of an oxygenase which d i r e c t l y a f f e c t s a p e s t i c i d e i s the enzyme w h i c h i s r e s p o n s i b l e f o r 2,4-D ( 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d ) d e g r a d a t i o n i n pure c u l t u r e s of A l c a l i g e n e s and A r t h r o b a c t e r . ( 1 4 , 1 5 ) . Presumably a s i m i l a r enzyme i s r e s p o n s i b l e f o r t h e f i r s t s t e p i n the d e g r a d a t i o n o f 2 , 4 , 5 - T ( 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c a c i d ) by a pure c u l t u r e of Pseudomonas c e p a c i a ( 1 6 ) . A l t h o u g h i t i s u n l i k e l y t h a t oxygenase enzymes themselves w i l l be u s e f u l i n waste d e g r a d a t i o n systems, i t has been demonstrated t h a t t h e 2 , 4 , 5 - T d e g r a d i n g £ . c e p a c i a can e f f e c t i v e l y remove the h e r b i c i d e from h e a v i l y contaminated s o i l s (17,18).

Recent r e p o r t s have demonstrated t h a t the w h i t e r o t fungus Phanerochaete c h r y s o s p o r i u m produces a p e r o x i d a s e - l i k e l i g n i n d e g r a d i n g enzyme which can a t t a c k r e c a l c i t r a n t p e s t i c i d e s such as DDT and p e s t i c i d e r e l a t e d compounds such as d i o x i n (2,3,7,8-tetrachlorodibenzo-p-dioxin) (19). The f u n g i c e r t a i n l y e x h i b i t a broad range of dégradâtive c a p a b i l i t i e s which d e s e r v e a t t e n t i o n i n f u t u r e r e s e a r c h e f f o r t s on b i o l o g i c a l e l i m i n a t i o n o f p e s t i c i d e wastes. U s i n g a m o d i f i e d enrichment t e c h n i q u e , we i s o l a t e d a b a c t e r i u m w h i c h was v e r y p r o f i c i e n t i n d e g r a d i n g t h e i n s e c t i c i d e and nematocide c a r b o f u r a n w h i l e u t i l i z i n g i t as a s o u r c e o f n i t r o g e n ( 2 0 ) . This o r g a n i s m i s c a p a b l e o f r a p i d l y d e g r a d i n g a number o f o t h e r r e l a t e d N-methylcarbamate i n s e c t i c i d e s ( F i g u r e 1 ) . We have i s o l a t e d and p a r t i a l l y p u r i f i e d an enzyme from t h i s Achromobacter s p . which r a p i d l y c l e a v e s the N-methylcarbamate s i d e c h a i n o f c a r b o f u r a n (Figure 2) y i e l d i n g the 7-phenol m e t a b o l i t e ( 2 , 3 - d i h y d r o - 2 - d i m e t h y l - 7 - b e n z o f u r a n o l ) . T h i s enzyme seems t o f a l l i n t o t h e h y d r o l a s e c l a s s o f enzymes. I t was a c t i v e over a broad range of pH and t e m p e r a t u r e , and once p a r t i a l l y p u r i f i e d i t was r e l a t i v e l y s t a b l e a t 4 C . E x t e n s i v e d i a l y s i s of the enzyme d i d not a f f e c t i t s a b i l i t y to cleave carbofuran, i n d i c a t i n g that s o l u b l e


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F i g u r e 1· Hydrolysis of several N-methylcarbamate i n s e c t i c i d e s by r e s t i n g c e l l s o f Achromobacter sp WM 1 1 1 . E a c h c o m p o u n d w a s a d d e d t o 100 u g / m l a n d t h e y a n d t h e i r h y d r o l y s i s p r o d u c t s were r e s o l v e d b y HPLC o n a C 1 8 c o l u m n in a solvent of a c e t o n i t r i l e and p h o s p h o r i c a c i d . Key: (•), a l d i c a r b ; ( Δ ) , baygon; ( · ) , carbofuran; ( A ) , carbaryl; (•), 3,5-dimethylphenyl-N-methyl carbamate ; and ( Ο ) , o - n i t r o p h e n y1dimethy1carbamate. (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 20· C o p y r i g h t 1986, Academic P r e s s . )

c "c5 Ε α> k_

hosphate d e g r a d a t i o n ) f r o m £ · d i m i n u t a a n d F l a v o b a c t e r i u m i n d i c a t e d t h a t t h e g e n e s f r o m t h e s e two s o u r c e s were v e r y s i m i l a r , i f not i d e n t i c a l . R e s t r i c t i o n mapping of cloned DNA f r a g m e n t s f r o m b o t h o r g a n i s m s a l s o s u g g e s t e d t h a t t h e DNA e n c o d i n g t h e opd gene i t s e l f i s v e r y s i m i l a r i n t h e s e two o r g a n i s m s . H o w e v e r , t h i s m a p p i n g h a s a l s o shown t h a t t h e p l a s m i d DNA o u t s i d e o f t h e opd c o d i n g r e g i o n i s v e r y d i f f e r e n t i n t h e two o r g a n i s m s ( F i g u r e 4). The o b s e r v a t i o n t h a t t h e p a r a t h i o n h y d r o l a s e i n two t e m p o r a l l y , g e o g r a p h i c a l l y , a n d b i o l o g i c a l l y d i s t i n c t i s o l a t e s o f b a c t e r i a was encoded by i d e n t i c a l genes c a r r i e d on n o n - i d e n t i c a l p l a s m i d s s u g g e s t s t h a t t h e g e n e may be p a r t o f a m o b i l e g e n e t i c e l e m e n t o r t r a n s p o s o n (26). Coumaphos;

A model p e s t i c i d e

degradation

system

The A n i m a l a n d P l a n t H e a l t h I n s p e c t i o n s e r v i c e ( A P H I S ) o f t h e USDA i n c o o p e r a t i o n w i t h the s t a t e of Texas c a r r i e s out e x t e n s i v e c a t t l e - d i p p i n g o p e r a t i o n s a l o n g t h e b o r d e r o f Texas and M e x i c o . The program i s designed to prevent the r e i n t r o d u c t i o n of the c a t t l e f e v e r t i c k (Boophilus) i n t o the United States. The i n s e c t i c i d e c u r r e n t l y u s e d i n t h i s o p e r a t i o n i s coumaphos [ 0 , 0 - d i e t h y l 0 - ( 3 - c h l o r o - 4 methyl-2-oxo-2H-l-benzopyran-7-yl) phosphorothioate]· Annually, this o p e r a t i o n g e n e r a t e s o v e r 5 7 0 , 0 0 0 L o f aqueous w a s t e s c o n t a i n i n g 1500 t o 3000 ug/ml o f coumaphos. C u r r e n t l y t h i s waste i s placed i n concrete l i n e d evaporation p i t s . S i n c e coumaphos h a s a v e r y l o n g



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F i g u r e 3 , Agarose g e l e l e c t r o p h o r e s i s o f DNA o b t a i n e d from the c a r b o f u r a n d e g r a d i n g Achromobacter s p , WM111. Lane 1 - Eco RI d i g e s t e d ; Lane 2 - Bam HI d i g e s t e d ; Lane 3 - Hind I I I d i g e s t e d ; and Lane 4 - u n t r e a t e d . The numbers t o the l e f t i n d i c a t e the m i g r a t i o n d i s t a n c e o f the fragments i n a H i n d I I I d i g e s t o f phage lambda i n c l u d e d i n the same g e l and i n d i c a t e the s i z e o f the fragments i n k i l o b a s e pairs·



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h a l f - l i f e (A/ 300 d a y s ) i n s o i l a n d w a t e r ( 2 7 ) , a d i s p o s a l m e t h o d w h i c h a s s u r e s t h e s a f e and e f f e c t i v e c o n v e r s i o n o f coumaphos t o l e s s e n v i r o n m e n t a l l y h a r m f u l p r o d u c t s was s o u g h t . The p r o c e s s o f U V - o z o n a t i o n , w h e r e b y a q u e o u s p e s t i c i d e suspensions are p r e t r e a t e d w i t h i n t e n s e u l t r a - v i o l e t r a d i a t i o n i n the p r e s e n c e o f o x y g e n p r i o r t o s o i l d i s p o s a l , was s h o w n t o be e f f e c t i v e i n a c c e l e r a t i n g t h e d e g r a d a t i o n o f s e v e r a l commonly u s e d h e r b i c i d e s (28). Coumaphos was r e s i s t a n t t o a n y d e g r a d a t i o n b y U V - o z o n a t i o n , w i t h o n l y 25% d e g r a d e d o v e r a 5 h p e r i o d ( 2 9 ) . E x p o s u r e o f coumaphos c a t t l e - d i p suspensions to l a r g e concentrations of mechanically g e n e r a t e d o z o n e i n d i c a t e d t h a t t h i s compound was v e r y r e s i s t a n t t o o x i d a t i o n by o z o n e . The p a r a t h i o n h y d r o l a s e enzyme p r o d u c e d by F l a v o b a c t e r i u m s p ATCC 2 7 5 5 1 was a b l e t o r a p i d l y h y d r o l y z e c o u m a p h o s y i e l d i n g chlorferon (3-chloro-4-methyl-7-hydroxycoumarin) and d i e t h y l t h i o p h o s p h o r i c a c i d as p r o d u c t s ( F i g u r e 5 ) . Resting c e l l s u s p e n s i o n s of the F l a v o b a c t e r i u m were v e r y e f f e c t i v e i n d e g r a d i n g coumaphos ( F i g u r e 6 A ) . These s m a l l s c a l e experiments used a dense s u s p e n s i o n of c e l l s (10^ c e l l s / m l ) to a c c o m p l i s h t h e d e g r a d a t i o n of t h e coumaphos w i t h i n a s h o r t p e r i o d o f t i m e . T h i s p r a c t i c e may be i m p r a c t i c a l i n the a c t u a l use of organisms f o r p r o c e s s i n g l a r g e volumes of waste. The c h l o r f e r o n p r o d u c e d a s a r e s u l t o f t h e m i c r o b i a l h y d r o l y s i s o f c o u m a p h o s was v e r y s u s c e p t i b l e t o f u r t h e r d e g r a d a t i o n by UV-ozonation (Figure 6B). The e n t i r e combined p r o c e s s o f m i c r o b i a l h y d r o l y s i s f o l l o w e d by U V - o z o n a t i o n r e q u i r e d l e s s t h a n 7 h o u r s t o a f f e c t the s a f e d e s t r u c t i o n of coumaphos. Although the c h l o r f e r o n had c o m p l e t e l y d i s a p p e a r e d a f t e r t h i s p r o c e s s , most o f t h e ^ C l a b e l o r i g i n a l l y p r e s e n t i n t h e b e n z e n e m o e i t y o f c o u m a p h o s was s t i l l p r e s e n t i n aqueous s o l u t i o n ( F i g u r e 6 B ) . Gas c h r o m a t o g r a p h y / m a s s s p e c t r o m e t r y of the o r g a n i c p r o d u c t s i n s o l u t i o n has i n d i c a t e d t h a t 2 , 4 - d i h y d r o x y a c e t o p h e n o n e and s h o r t c h a i n a l k a n o i c a c i d s a r e detectable products. These more p o l a r o r g a n i c s were v e r y s u s c e p t i b l e to c o m p l e t e d e g r a d a t i o n by s o i l m i c r o o r g a n i s m s ( F i g u r e 7 ) . In c o n t r a s t , n e i t h e r coumaphos n o r c h l o r f e r o n were a p p r e c i a b l y d e g r a d e d by i n d i g e n o u s s o i l m i c r o o r g a n i s m s . M o r e o v e r , coumaphos s u b j e c t e d t o U V - o z o n a t i o n w i t h o u t p r i o r m i c r o b i a l h y d r o l y s i s underwent very l i m i t e d metabolism. The U V - o z o n a t i o n p r o c e s s r a p i d l y k i l l e d t h e F l a v o b a c t e r i u m c e l l s t h a t were added t o t h e d i p - v a t w a s t e . T h i s was e x p e c t e d s i n c e many European communities use U V - o z o n a t i o n or d i r e c t o z o n a t i o n to t r e a t d r i n k i n g water ( i n s t e a d of c h l o r i n a t i o n ) f o r the c o n t r o l of h a r m f u l microbes. T h u s , the end r e s u l t o f t h i s p r o c e s s i s an aqueous p r o d u c t t h a t c o n t a i n s o n l y r e a d i l y biodegradable organic m a t e r i a l . T h i s p r o c e s s was f i e l d - t e s t e d o n 2 4 7 0 L o f c o u m a p h o s c a t t l e - d i p w a s t e a t t h e APHIS v a t s i n L a r e d o , T e x a s . To o v e r c o m e t h e n e e d t o d e l i v e r a n e x t r e m e l y l a r g e number o f F l a v o b a c t e r i u m c e l l s t o t h e s i t e , t h e o r g a n i s m s w e r e a d d e d a s a 1% i n o c u l u m ( 2 2 . 8 L o f c u l t u r e grown i n n u t r i e n t b r o t h p l u s x y l o s e ) a l o n g w i t h 9 . 5 k g o f x y l o s e as a c a r b o n s o u r c e a n d 4 . 5 k g o f ammonium s u l f a t e f e r t i l i z e r a s a n i t r o g e n s o u r c e , i n o r d e r to a l l o w growth of the organisms i n t h e w a s t e . The p H o f t h e m a t e r i a l i n t h e t a n k was a d j u s t e d t o b e t w e e n 6 . 8 a n d 7 . 0 b y t h e a d d i t i o n o f 1.4 k g o f m o n o b a s i c p o t a s s i u m p h o s p h a t e a n d 1.8 k g o f



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F i g u r e 4. R e s t r i c t i o n endonuclease maps o f c l o n e d Eco R l fragments c o n t a i n i n g t h e opd genes from diminuta (from Serdar and G i b s o n [ 3 3 ] ) , and F l a v o b a c t e r i u m s p . ATCC 27551. The t h i c k l i n e s r e p r e s e n t t h e a d j a c e n t p o r t i o n s o f v e c t o r DNA; t h e t h i n l i n e s r e p r e s e n t i n s e r t e d p l a s m i d DNA from F l a v o b a c t e r i u m sp. (top) and J?, d i m i n u t a (bottom). The u n d e r l i n e d area d e l i n e a t e s t h e 2.1 k i l o b a s e r e g i o n o f t h e two c l o n e d fragments where t h e r e s t r i c t i o n maps a r e i d e n t i c a l . R e s t r i c t i o n endonucleases : B. Bam H I ; E. Eco R I ; H. H i n d I I I ; P. P s t I ; S. S a l I ; X. Xho I . (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 25. C o p y r i g h t 1985, American S o c i e t y f o r M i c r o b i o l o g y . )

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F i g u r e 5. H y d r o l y s i s o f coumaphos t o y i e l d c h l o r f e r o n and d i e t h y l t h i o p h o s p h o r i c a c i d as c a t a l y z e d by p a r a t h i o n h y d r o l a s e enzymes from F l a v o b a c t e r i u m sp. ATCC 27551 and Pseudomonas d i m i n u t a .



164


Time (hours) Figure

6.

S p e n t c o u m a p h o s d i p - v a t s o l u t i o n was s u p p l e m e n t e d w i t h U - B e n z y l - ^ C - c o u m a p h o s and F l a v o b a c t e r i u m c e l l s w e r e a d d e d t o a d e n s i t y o f 10^ c e l l s / m l ( P a n e l A). The h y d r o l y s e d m a t e r i a l was t h e n s u b j e c t e d to U.V.-ozonation (Panel B). Samples were t a k e n at v a r i o u s t i m e s and remaining i n the m a t e r i a l was d e t e r m i n e d by l i q u i d s c i n t i l l a t i o n c o u n t i n g . Samples were d i l u t e d 1:10 w i t h m e t h a n o l and s u b j e c t e d t o HPLC i n a s o l v e n t s y s t e m o f m e t h a n o l i n p h o s p h o r i c a c i d t o d e t e r m i n e coumaphos (A) and c h l o r f e r o n (B) levels.



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S o i l metabolism of the products r e s u l t i n g from U . V . - o z o n a t i o n o f coumaphos a n d m i c r o b i a l l y hydrolyzed/U.V. ozonated coumaphos. ^CC>2 r e l e a s e d f r o m U - b e n z y l - ^ C - c o u m a p h o s was measured i n biometer f l a s k s by t r a p p i n g i n potassium hydroxide and c o u n t i n g i n a l i q u i d s c i n t i l l a t i o n c o u n t e r . C o n t r o l s c o n t a i n e d s o i l t h a t h a d been a u t o c l a v e d one time but were not k e p t s t e r i l e .


166



d i b a s i c potassium phosphate. The t e m p e r a t u r e o f t h e l i q u i d v a r i e d b e t w e e n 36°C a n d 3 3 ° C . T h e t a n k was v i g o r o u s l y a e r a t e d . Samples were t a k e n a t v a r i o u s t i m e s and d i l u t e d w i t h m e t h a n o l f o r l a t e r a n a l y s i s o f coumaphos and c h l o r f e r o n . O n - s i t e d e t e r m i n a t i o n of c o u m a p h o s l e v e l s i n t h e t a n k was p e r f o r m e d u s i n g a c o l o r i m e t r i c a s s a y k i t ( B a y v e t , Shawnee, K S ) . The m i c r o b i a l h y d r o l y s i s o f coumaphos i n t h e t r e a t e d c o m p a r t m e n t was e s s e n t i a l l y c o m p l e t e w i t h i n 4 8 h r s ( F i g u r e 8 ) . As e x p e c t e d , the chlorferon hydrolysis product accumulated. At t h i s p o i n t a e r a t i o n of t h e t a n k was s t o p p e d a n d o z o n e ( g e n e r a t e d u s i n g a G r i f f i n o z o n e g e n e r a t o r ) was i n t r o d u c e d i n t o t h e t a n k f o r a b o u t 20 h r s . Subsequent a n a l y s e s i n d i c a t e d t h a t o v e r 50% o f t h e c h l o r f e r o n was d e g r a d e d during ozonation. L a b o r a t o r y a n a l y s e s on s m a l l e r volumes o f m a t e r i a l h a d i n d i c a t e d t h a t c h l o r f e r o n was v e r y s u s c e p t i b l e t o o x i d a t i o n b y m e c h a n i c a l l y generated ozone i n the absence of U . V . l i g h t . The r e s u l t s o f t h e f i r s t L a r e d o f i e l d t r i a l i n d i c a t e t h a t t h e m e t h o d o f m i c r o b i a l h y d r o l y s i s - o z o n a t i o n was v e r y e f f e c t i v e i n t h e e l i m i n a t i o n of waste coumaphos. A s e c o n d , more e x t e n s i v e f i e l d trial i s planned f o r the l a t e s p r i n g of 1986. Among t h e i t e m s t o be t e s t e d a r e a l t e r n a t i v e methods f o r t h e d e l i v e r y of a c t i v e c u l t u r e s t o a remote l o c a t i o n . The d e l i v e r y o f a d e q u a t e amounts o f o z o n e i n t o t h e h y d r o l y z e d m a t e r i a l was a l s o a p r o b l e m d u r i n g t h e f i r s t f i e l d t r i a l . However, t h i s i s s t r i c t l y a problem of e n g i n e e r i n g e x i s t i n g t e c h n o l o g y t o f i t t h i s u n i q u e p u r p o s e a n d s h o u l d p o s e no g r e a t d i f f i c u l t y to the development of the o v e r a l l p r o c e s s . As n o t e d above, l a r g e s c a l e U . V . - o z o n a t i o n u n i t s f o r the treatment of e x t r e m e l y l a r g e volumes of d r i n k i n g water a l r e a d y e x i s t . The t e c h n o l o g y b e h i n d s u c h s y s t e m s s h o u l d be d i r e c t l y a p p l i c a b l e t o o u r purposes. D i s c u s s i o n and

Prospects

I n d i g e n o u s s o i l a n d w a t e r m i c r o b i a l p o p u l a t i o n s c o n t a i n many members w h i c h have the p o t e n t i a l f o r c a r r y i n g out the b i o c o n v e r s i o n of p e s t i c i d e molecules to n o n t o x i c p r o d u c t s . The i s o l a t i o n o f i n d i v i d u a l c u l t u r e s c a p a b l e of a l t e r i n g p e s t i c i d e s and the b i o c h e m i c a l c h a r a c t e r i z a t i o n o f p e s t i c i d e d e g r a d i n g enzymes a r e t h e f i r s t steps i n d e v e l o p i n g b i o t e c h n o l o g y f o r the s a f e d i s p o s a l of waste p e s t i c i d e s . The m o l e c u l a r c h a r a c t e r i z a t i o n o f t h e g e n e s encoding p e s t i c i d e degradation provide background i n f o r m a t i o n for f u t u r e m o l e c u l a r g e n e t i c m a n i p u l a t i o n o f these genes f o r use i n waste disposal techniques. I t a l s o p r o v i d e s b a s i c i n f o r m a t i o n on t h e o r i g i n , e v o l u t i o n , and t r a n s m i s s i o n o f p e s t i c i d e d e g r a d a t i v e genes i n microorganisms· One r e c e n t a g r i c u l t u r a l p h e n o m e n o n t h a t may be e l u c i d a t e d b y s u c h b a s i c r e s e a r c h i s t h e manner i n w h i c h p r o b l e m s o i l s a r i s e . In these s o i l s , the m i c r o b i a l p o p u l a t i o n degrades a p p l i e d p e s t i c i d e s so r a p i d l y that p e s t i c i d e efficacy i s l o s t (30). Presumably, the r e p e a t e d a p p l i c a t i o n of a p a r t i c u l a r p e s t i c i d e a c t s as an e n r i c h m e n t p r o c e d u r e , c o n s t a n t l y s e l e c t i n g f o r i n c r e a s e d numbers o f the p a r t i c u l a r m i c r o o r g a n i s m s t h a t c a n m e t a b o l i z e t h e p e s t i c i d e and d e r i v e some n u t r i t i o n a l b e n e f i t f r o m i t . Several basic questions t h a t n e e d t o be a n s w e r e d i n o r d e r t o u n d e r s t a n d t h i s p r o c e s s . First, w h e r e do t h e g e n e s t h a t e n c o d e t h e p e s t i c i d e d e g r a d a t i o n e n z y m e s



13.

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3.0 A

Time (hours) Figure 8.

M e t a b o l i s m a n d o z o n a t i o n o f coumaphos i n a f i e l d t r i a l on 2470 L o f m a t e r i a l i n L a r e d o , T e x a s . Coumaphos a n d c h l o r f e r o n m e a s u r e m e n t s w e r e made b y H P L C a s i n l e g e n d to Figure 7 . E r r a t i c c h l o r f e r o n measurements a t 4 0 h and 48h were due t o p r e c i p i t a t i o n o f t h e c h l o r f e r o n a n d s e t t l i n g o u t when a e r a t i o n w a s s t o p p e d .



168


o r i g i n a t e ? Second, a r e raacromolecular events such as m u t a t i o n , gene d u p l i c a t i o n , and gene movement between s p e c i e s necessary f o r t h e development o f problem s o i l s ? The r o l e s p l a y e d by p l a s m i d s and transposons i n the o r i g i n and p r o p a g a t i o n o f p e s t i c i d e d e g r a d a t i o n genes a l s o need t o be i n v e s t i g a t e d . As the coumaphos d e g r a d a t i o n process shows, b i o l o g i c a l p e s t i c i d e d e g r a d a t i o n agents can be i n c o r p o r a t e d i n t o v i a b l e waste d i s p o s a l schemes. T h i s p a r t i c u l a r process combines b i o l o g i c a l h y d r o l y s i s w i t h c h e m i c a l o x i d a t i o n f o l l o w e d by c h e m i c a l o x i d a t i o n t o a c c o m p l i s h the complete d e g r a d a t i o n o f a p e s t i c i d e m o l e c u l e . The combination o f s i n g l e - s t e p (or l i m i t e d step) b i o l o g i c a l degradation with a p h y s i c a l / c h e m i c a l process such as U.V. i r r a d i a t i o n , U.V.-ozonation o r o z o n a t i o n can p r o v i d e s a f e , easy, and c o s t e f f e c t i v e d e g r a d a t i o n o f e x t r e m e l y r e c a l c i t r a n t p e s t i c i d e s . The use o f h y d r o l a s e enzymes such as p a r a t h i o n h y d r o l a s e o r c a r b o f u r a n h y d r o l a s e as a f i r s t s t e p i n waste e l i m i n a t i o n i s p a r t i c u l a r l y p r o m i s i n g . These enzymes a r e a c t i v e over a wide range o f e n v i r o n m e n t a l c o n d i t i o n s and t h e r e a c t i o n s they c a t a l y z e g r e a t l y d i m i n i s h the acute t o x i c i t i e s o f t h e i n s e c t i c i d e s . Thus, t h e i r use i s c o n v e n i e n t and c a n render waste s a f e t o s t o r e u n t i l i t can be f u r t h e r p r o c e s s e d . T h i s may be p a r t i c u l a r l y i m p o r t a n t s i n c e the c a p i t a l c o s t s o f d e v i c e s such as U.V.-ozonators c o u l d be p r o h i b i t i v e f o r i n d i v i d u a l farmers. H y d r o l y z e d wastes c o u l d be s t o r e d u n t i l a c o o p e r a t i v e l y owned o r p r i v a t e l y r e n t e d p r o c e s s o r c o u l d be brought around t o f i n i s h the j o b . An a d d i t i o n a l s a f e t y f a c t o r i n c o r p o r a t e d i n t o such a m u l t i s t e p process i s t h a t the organisms t h a t produce these enzymes d e r i v e l i t t l e o r no n u t r i t i o n a l b e n e f i t from the metabolism o f t h e p e s t i c i d e . S i n c e they cannot grow on the p e s t i c i d e they s h o u l d n o t propagate i n f i e l d s i t u a t i o n s where some p e s t i c i d e " s t a y i n g power" i s r e q u i r e d . Thus, use o f such microorganisms does not t h r e a t e n t o c r e a t e new problem s o i l s s i t u a t i o n s . B i o t e c h n o l o g y o f f e r s the p o t e n t i a l f o r many advances i n p e s t i c i d e waste d i s p o s a l . Gene c l o n i n g t e c h n i q u e s o f f e r the methodology by which the genes encoding p e s t i c i d e d e g r a d a t i v e genes can be moved i n t o i n d u s t r i a l l y u s e f u l microorganisms. Thus, important d e g r a d a t i v e enzymes can be produced i n g r e a t q u a n t i t i e s and p r o v i d e d a t r e a s o n a b l e p r i c e s . Enzyme o r organism p r e p a r a t i o n s might be i m m o b i l i z e d t o c r e a t e c a r t r i d g e type d i g e s t o r s t h a t can have l o n g s h e l f - l i v e s and l o n g u s e f u l l i v e s . Knowledge o f gene sequences, p r o t e i n s t r u c t u r e s and r e a c t i o n mechanisms w i l l enable r e s e a r c h e r s t o use t e c h n i q u e s such as s i t e d i r e c t e d mutagenesis (31) t o a l t e r o r i n c r e a s e the c h e m i c a l s u b s t r a t e range o f p e s t i c i d e d e g r a d i n g enzymes. I t i s v e r y c l e a r , t h a t i f pursued v i g o r o u s l y , and u t i l i z e d i n t e l l i g e n t l y , b i o t e c h n o l o g y can o f f e r v i a b l e s o l u t i o n s f o r t h e treatment o f a g r i c u l t u r a l wastes and e n v i r o n m e n t a l h a z a r d s .

Literature Cited 1. 2.

Helling, C. S. and Gish, T. J . Soil Characteristics Affecting Pesticide Movement into Groundwater. ACS Symposium Series. (In press) American Chemical Society, Washington, D.C. 1986. Munnecke, D. M . , L . M. Johnson, H. W. Talbot and S. Barik. "Microbial Metabolism and Enzymology of Selected Pesticides" in;


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3. 4.


5. 6.

7. 8.

9. 10.

11. 12. 13. 14. 15.

16.

17.

Use ofMicroorganisms and Microbial Systems

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