Enhancing Biodegradation for Detoxification of Herbicide Waste in

Chapter 19

Enhancing Biodegradation for Detoxification of Herbicide Waste in Soil

Downloaded by CALIFORNIA INST OF TECHNOLOGY on November 1, 2017 | http://pubs.acs.org Publication Date: May 3, 1990 | doi: 10.1021/bk-1990-0426.ch019

A. S. Felsot and Ε. K. Dzantor Center for Economic Entomology, Illinois Natural History Survey, 607 East Peabody Drive, Champaign, IL 61820

P e s t i c i d e s i n soil at high concentrations have been found t o be unusually r e s i s t a n t t o normal biodegradative processes. M i c r o b i a l systems have been proposed as cost e f f e c t i v e techniques s u i t a b l e f o r cleanup of p e s t i c i d e waste. In an attempt t o enhance the d e t o x i f i c a t i o n i n soil of high concentrations of the h e r b i c i d e a l a c h l o r , experiments were designed to t e s t the e f f e c t s of several f a c t o r s on a l a c h l o r p e r s i s t e n c e : soil d i l u t i o n , concentration, formulation, n u t r i e n t amendments, and m i c r o b i a l i n o c u l a t i o n . A l a c h l o r i n soil from a waste s i t e degraded f a s t e r when d i l u t e d with uncontaminated soil by 90% than when d i l u t e d by 10 or 50%. A l a c h l o r was metabolized into water-soluble compounds i n soil at a concentration of 10 ppm but not at 1000 ppm. Amendment of soil with ground corn or soybean stubble stimulated the biodegradation of a l a c h l o r at a concentration of 100 ppm but not at 1000 ppm. When a l a c h l o r was present as a sole carbon source, several b a c t e r i a l i s o l a t e s p a r t i a l l y d e t o x i f i e d concentrations of 10 ppm but not 100 ppm. Fungal i s o l a t e s could cometabolically degrade 100 ppm a l a c h l o r i n pure c u l t u r e ; however, i n o c u l a t i o n of soil with an alachlor-degrading fungus alone d i d not enhance degradation.

The u s e o f p e s t i c i d e s h a s r e c e i v e d i n t e n s e s c r u t i n y f o r generations. The concern about r e s i d u e s i n f o o d and p o t e n t i a l adverse h e a l t h e f f e c t s predates the advent o f s y n t h e t i c organic p e s t i c i d e s i n t h e l a t e 1940 s ( 1 ) . 1

0097-6156/90/0426-0249$06.00/0 © 1990 American Chemical Society

Racke and Coats; Enhanced Biodegradation of Pesticides in the Environment ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT

Since t h e passage o f t h e Resource Conservation Recovery Act ( 1 9 7 6 ) , much c o n c e r n h a s f o c u s e d o n p e s t i c i d e w a s t e , which i s i n e v i t a b l y generated by a g r i c u l t u r a l and urban sectors alike (2-4). In I l l i n o i s , agrochemical retail f a c i l i t i e s have been p a r t i c u l a r l y p r o b l e m a t i c . These f a c i l i t i e s provide farmers with a v a r i e t y o f s e r v i c e s i n c l u d i n g t h e custom a p p l i c a t i o n o f f e r t i l i z e r s and pesticides. Many c h e m i c a l s a r e h a n d l e d a t o n e l o a d i n g l o c a t i o n w h e r e s p i l l a g e i s common, r e s u l t i n g i n t h e accumulation o f high c o n c e n t r a t i o n s o f hazardous chemicals. R i n s i n g o f e q u i p m e n t a n d o f empty c o n t a i n e r s also produces p e s t i c i d e contaminated discharges that can move o f f - s i t e a s r u n o f f i f n o t h a n d l e d p r o p e r l y . Very s i m i l a r c o n d i t i o n s o f s p i l l a g e a n d r i n s e o u t may o c c u r o n i n d i v i d u a l f a r m s , e s p e c i a l l y when t h e same s i t e i s u s e d r e p e a t e d l y f o r l o a d i n g and cleanup. The I l l i n o i s Environmental P r o t e c t i o n Agency has documented problems o f high level pesticide contamination at various agrochemical r e t a i l f a c i l i t i e s around t h e state, and t h e I l l i n o i s Department o f P u b l i c H e a l t h has f r e q u e n t l y d e t e c t e d unusually high concentrations of pesticides i n on-site w e l l s a n d n e a r b y community w e l l s ( ϋ ) . B i o d e g r a d a b l e p e s t i c i d e s c a n be e x t r e m e l y persistent when p r e s e n t i n t h e s o i l a t u n u s u a l l y h i g h c o n c e n t r a t i o n s (6-10), w h i c h c o n t r i b u t e s t o an i n c r e a s e d r i s k o f s u r f a c e and ground water c o n t a m i n a t i o n . When a b u s i n e s s h a s a major contamination i n c i d e n t , s t a t e o r f e d e r a l r e g u l a t o r y a g e n c i e s c a n o r d e r a c l e a n u p , b u t s u c h a c t i o n i s more d i f f i c u l t f o r p r i v a t e farms and r e s i d e n c e s . The n a t u r e o f t h e c l e a n u p i s more p r o b l e m a t i c . Contaminated soil i s e x c a v a t e d and removed t o a " s e c u r e " l a n d f i l l . The e n d r e s u l t i s perhaps a c l e a n e r s i t e , but t h e waste has not been d e t o x i f i e d . Cleanup t e c h n o l o g i e s have been proposed f o r o n - s i t e a n d / o r o f f - s i t e d e s t r u c t i o n o f p e s t i c i d e w a s t e s (4.) . T h e most r e l i a b l e t e c h n o l o g i e s f o c u s on d e t o x i f i c a t i o n o f l i q u i d wastes. D e s t r u c t i o n o f w a s t e s i n s o i l i s more d i f f i c u l t and expensive, e s p e c i a l l y f o r small businesses and i n d i v i d u a l homeowners. New a p p r o a c h e s that are consistent with the ubiquity of p e s t i c i d e waste and m i n d f u l o f t h e c o s t s a r e needed f o r cleanup i n situ, e s p e c i a l l y when s o i l h a s b e e n h i g h l y contaminated by past d i s p o s a l p r a c t i c e s (A) . Decontamination by m i c r o b i a l systems, which i s s u i t a b l e for meeting those needs, i s becoming a f e a s i b l e t e c h n o l o g y t o c l e a n up waste (11-13). S e v e r a l s t r a t e g i e s have been used f o r t h e development o f m i c r o b i a l decontamination systems ( i . e . , b i o r e m e d i a t i o n systems): (1) p r e t r e a t m e n t of contaminants with v a r i o u s reagents t o produce d é g r a d â t e s m o r e a m e n a b l e t o m i c r o b i a l m i n e r a l i z a t i o n ; (2) t r e a t m e n t o f w a s t e s w i t h m i c r o b i a l e n z y m e s ; (3) e n r i c h m e n t of wastes t o s t i m u l a t e indigenous microorganisms


19.

FELSOT & DZANTOR

(biostimulation


adapted

Detoxification of Herbicide Waste in Soil

[14.]);

microorganisms

(4) i n o c u l a t i o n (bioaugmentation

o f wastes

251

with

[14.] ) .

R e c e n t r e s e a r c h w i t h p e s t i c i d e w a s t e w a t e r h a s shown t h a t p r i o r t r e a t m e n t w i t h o z o n e a l o n e ( l u ) o r UV l i g h t a n d o z o n e (1£,12) f a c i l i t a t e d t h e m i c r o b i a l m e t a b o l i s m o f t h e resulting degradation products. S i m i l a r l y , exposure o f c h l o r i n a t e d p h e n o l s t o UV l i g h t a n d h y d r o g e n p e r o x i d e a l l o w e d m i c r o b i a l m i n e r a l i z a t i o n t o proceed (Ifi). These emerging t e c h n o l o g i e s , although p r o m i s i n g f o r wastewater, are not e a s i l y applicable t o treatment of contaminated soil. A second s t r a t e g y that might prove u s e f u l f o r d e c o n t a m i n a t i n g p e s t i c i d e wastes i n s o i l has been t h e a d d i t i o n o f h y d r o l y t i c enzymes d e r i v e d f r o m microorganisms (12.,20.) . O r g a n o p h o s p h o r u s i n s e c t i c i d e s s u c h a s p a r a t h i o n and d i a z i n o n a r e most s u s c e p t i b l e t o d e c o n t a m i n a t i o n b y enzymes. Some s u c c e s s h a s b e e n r e a l i z e d w i t h i m m o b i l i z a t i o n o f d e g r a d a t i v e enzymes on i n e r t s u r f a c e s through which wastewater i s passed and d e t o x i f i e d . The t h i r d s t r a t e g y , b i o s t i m u l a t i o n , stimulates d e g r a d a t i v e a c t i v i t i e s o f r e s i d e n t m i c r o f l o r a by e n r i c h i n g the e n v i r o n m e n t w i t h n u t r i e n t amendments o r b y c h a n g i n g the p h y s i c a l c h a r a c t e r i s t i c s of the environment. For example, d e g r a d a t i o n o f o r g a n i c s o l v e n t s and p e t r o l e u m p r o d u c t s c a n be enhanced i n groundwater by a d d i t i o n o f oxygen and n u t r i e n t s (21). C h l o r i n a t e d p e s t i c i d e s such as toxaphene can be p a r t i a l l y decontaminated by a d d i t i o n o f n u t r i e n t s t o s o i l s maintained under a n a e r o b i c c o n d i t i o n s (22.) . Recently, the degradation of a variety of pesticide c l a s s e s , i n c l u d i n g phenoxyacetate, d i n i t r o a n i l i n e , and t r i a z i n e h e r b i c i d e s and organophosphorus i n s e c t i c i d e s was enhanced i n h i g h l y contaminated s o i l by o r g a n i c , n u t r i e n t , a n d m i n e r a l a m e n d m e n t s (22.) . Degradation of p e n t a c h l o r o p h e n o l i n a n a e r o b i c ( f l o o d e d ) s o i l was e n h a n c e d b y t h e a d d i t i o n o f a n a e r o b i c s e w a g e s l u d g e (24.) . DDT d e g r a d e d s i g n i f i c a n t l y f a s t e r i n f l o o d e d s o i l amended w i t h r i c e s t r a w (25.) . The b i o a u g m e n t a t i o n s t r a t e g y i n v o l v e s t h e s e l e c t i o n of adapted m i c r o b i a l s t r a i n s t h a t m e t a b o l i z e a p e s t i c i d e as a carbon o r n u t r i e n t s o u r c e . Under t h e s e circumstances, the rate o f degradation i s enhanced compared t o t h e r a t e n o r m a l l y o b s e r v e d i n s o i l o r water. The d e v e l o p m e n t o f an a d a p t e d m i c r o b i a l s t r a i n u s u a l l y b e g i n s w i t h t h e enrichment and i s o l a t i o n o f p e s t i c i d e degraders from the contaminated environment. Enrichment i s r e l a t i v e l y e a s y w i t h compounds t h a t a r e u s e d a s c a r b o n or n u t r i e n t s o u r c e s . Compounds t h a t a r e n o t r e a d i l y u t i l i z a b l e , however, r e q u i r e n o v e l approaches t o e n r i c h a n d i s o l a t e p o t e n t i a l d e g r a d e r s , w h i c h may b e m a n i p u l a t e d to enhance t h e i r d e g r a d a t i v e c a p a b i l i t i e s . Recently, contaminant-degrading strains of microorganisms have been c o n s t r u c t e d by r e c r u i t i n g i n t o a s i n g l e o r g a n i s m t h e genes c o d i n g f o r d e g r a d a t i v e enzymes



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ENHANCED BIODEGRADATION OF PESTICIDES IN T H E ENVIRONMENT

(ZÂf21) . T h e s u c c e s s o f t h i s t e c h n i q u e h a s h i n g e d u p o n t h e k n o w l e d g e t h a t many x e n o b i o t i c - d e g r a d i n g g e n e s a r e r e s i d e n t o n e x t r a c h r o m o s o m a l p i e c e s o f DNA c a l l e d plasmids (22). P e s t i c i d e d e g r a d a t i o n p l a s m i d s were f i r s t d e s c r i b e d f o r 2,4-D a n d MCPA ( 2 9 - 3 1 ) . P l a s m i d s a r e a l s o known t o c o d e f o r e n z y m e s d e g r a d i n g 2 , 4 , 5 - T (13.) a n d t h e OP i n s e c t i c i d e d i a z i n o n (22.) . Several copies of a s p e c i f i c p l a s m i d o c c u r w i t h i n an i n d i v i d u a l c e l l , and p l a s m i d s can be t r a n s f e r r e d f r o m a d o n o r c e l l t o a r e c i p i e n t cell. Thus, t h e d e g r a d a t i v e p o t e n t i a l o f a m i c r o o r g a n i s m can be quickly amplified i n the population. Furthermore, plasmid exchange can o c c u r between s p e c i e s , a l t h o u g h i t o c c u r s more r e a d i l y w i t h i n a s p e c i e s . By t a k i n g a d v a n t a g e o f b a c t e r i a l t r a n s f o r m a t i o n a n d c o n j u g a t i o n , r e s e a r c h e r s have c o n s t r u c t e d s t r a i n s p o s s e s s i n g an e n t i r e pathway f o r x e n o b i o t i c m i n e r a l i z a t i o n by c u l t u r i n g i n c h e m o s t a t s two o r more s t r a i n s p o s s e s s i n g complementary p a r t s o f the pathway. F o r example, m i c r o b i a l i n o c u l a from h a z a r d o u s waste s i t e s were p l a c e d i n a chemostat w i t h m i c r o b i a l s t r a i n s p o s s e s s i n g known p l a s m i d s f o r a r o m a t i c h y d r o c a r b o n and c h l o r o b e n z o a t e metabolism (22). A f t e r c u l t i v a t i o n w i t h 2,4,5-T as a s o l e c a r b o n s o u r c e f o r 8 t o 10 m o n t h s , a n i s o l a t e was produced t h a t c o u l d u t i l i z e 2,4,5-T. Success i n breeding a m i c r o b i a l s t r a i n with the c a p a b i l i t y of metabolizing c h l o r o b e n z e n e has been o b t a i n e d u s i n g a t h r e e - s t a g e chemostat s y s t e m w i t h two known i s o l a t e s h a v i n g complementary c a p a b i l i t i e s of chlorobenzoate metabolism

(22) . Our i n t e r e s t i n m i c r o b i a l s y s t e m s f o r d e c o n t a m i n a t i o n of p e s t i c i d e waste e v o l v e d from our attempts t o remediate h i g h l y c o n t a m i n a t e d s o i l a t an a g r o c h e m i c a l retail f a c i l i t y i n P i a t t C o u n t y , I L (2A). Soil containing high levels of alachlor (2-chloro-2',6'-diethyl-N[methoxymethyl] a c e t a n i l i d e ) ( 2 4 , 0 0 0 ppm i n t h e t o p 7.5 cm o f o n e l o c a t i o n ) was e x c a v a t e d a n d l a n d - a p p l i e d t o c o r n and s o y b e a n p l o t s i n an e f f o r t t o s t i m u l a t e n a t u r a l b i o d e g r a d a t i v e mechanisms. Other contaminants i n c l u d e d metolachlor (2-chloro-N-[2-ethyl-6-methylphenyl]-N-[2methoxy-l-methylethyl] acetamide), atrazine (2-chloro-4[ethylamino]-6-[isopropylamino]-S-triazine), trifluralin (α,α,α-trifluro-2,6-dinitro-N-N-dipropyl-p-toluidine), and nitrogen f e r t i l i z e r . In c o r n and soybean p l o t s receiving c o n t a m i n a t e d s o i l , a l a c h l o r and m e t o l a c h l o r p e r s i s t e n c e was s i g n i f i c a n t l y g r e a t e r t h a n i n p l o t s f r e s h l y t r e a t e d with h e r b i c i d e sprays comprised of equivalent c o n c e n t r a t i o n s (1Û). S o i l t h a t h a d b e e n l e f t i n p i l e s on t h e waste s i t e seemed t o have a r e d u c e d m i c r o b i a l p o p u l a t i o n a n d a d e p r e s s e d e n z y m e a c t i v i t y t h a t may have accounted f o r the p e r s i s t e n c e of the h e r b i c i d e s . In laboratory experiments, simulated s p i l l s of alachlor ( 1 0 , 0 0 0 ppm s o i l ) reduced m i c r o b i a l b i o a c t i v i t y , and the p e s t i c i d e d i d n o t d e g r a d e a f t e r o n e y e a r (2Λ) .


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In an e f f o r t t o e n h a n c e d e t o x i f i c a t i o n o f h i g h c o n c e n t r a t i o n s o f a l a c h l o r , we d e s i g n e d s t u d i e s t o t e s t the e f f e c t s of s o i l d i l u t i o n , concentration, formulation, a n d n u t r i e n t amendments on p e r s i s t e n c e . Additionally, we d e v e l o p e d a p r o t o c o l t o e n r i c h , i s o l a t e , and screen b a c t e r i a and f u n g i f o r enhanced c a p a b i l i t i e s o f a l a c h l o r degradation. Our s t u d i e s r e p r e s e n t t h e i n t i t a l s t a g e s o f t h e b i o s t i m u l a t i o n and b i o a u g m e n t a t i o n strategies for waste cleanup. Materials

and

Methods

S o i l used i n t h e a l a c h l o r p e r s i s t e n c e s t u d i e s and i n t h e enrichments f o r a l a c h l o r - d e g r a d i n g o r g a n i s m s was derived f r o m two s o u r c e s : a waste s i t e a t an agrochemical f a c i l i t y i n P i a t t Co., IL and a soybean p l o t near the w a s t e s i t e t h a t was d i v i d e d i n t o r e p l i c a t e d b l o c k s f o r a land a p p l i c a t i o n study (2A). T h e s o i l t y p e was a m i x t u r e of Ipava s i l t loam ( f i n e , m o n t m o r i l l o n i t i c , mesic, Aquic A r g i u d o l l s ) and S a b l e s i l t y c l a y loam ( f i n e s i l t y mixed, m e s i c , T y p i c H a p l a q u o l l s ) w i t h pH 5 . 4 - 5 . 5 . The s o i l a t t h e waste s i t e had been e x c a v a t e d and s t o r e d i n p i l e s w h i c h were sampled as n e e d e d f o r l a b o r a t o r y s t u d i e s (waste-pile s o i l ) . Untreated check p l o t s i n the soybean f i e l d s e r v e d as s o u r c e s o f u n c o n t a m i n a t e d s o i l (CHECK soil). S o i l s w e r e s t o r e d a t 2 - 4 ° C a n d p a s s e d t h r o u g h a 3mm screen before use. S o i l D i l u t i o n Experiment. W a s t e - p i l e s o i l was m i x e d w i t h CHECK s o i l i n l a r g e p l a s t i c b a g s t o p r o d u c e d i l u t i o n s o f 0, 10, 50, a n d 90%, w h i c h y i e l d e d m e a n a l a c h l o r c o n c e n t r a t i o n s o f 46.8, 47.4, 29.4, a n d 6.52 ppm, respectively. T h i r t y - g r a m p o r t i o n s (oven-dry weight) of s o i l w e r e d i s p e n s e d i n t o 250 mL E r l e n m e y e r flasks and a d j u s t e d t o 30% m o i s t u r e (w/w). The f l a s k s were c o v e r e d w i t h P a r a f i l m and i n c u b a t e d a t 25°C. Immediately a f t e r m i x i n g , a n d a t p e r i o d i c i n t e r v a l s d u r i n g t h e n e x t 42 days, t r i p l i c a t e f l a s k s were removed f o r e x t r a c t i o n o f a l a c h l o r . A c t i v i t y o f d e h y d r o g e n a s e s o i l e n z y m e was a s s a y e d (25.) in s o i l s s i m u l t a n e o u s l y i n c u b a t e d i n companion f l a s k s . E f f e c t o f C o n c e n t r a t i o n and F o r m u l a t i o n . M o i s t CHECK s o i l ( 2 6 . 4 % m o i s t u r e , 30 g o v e n - d r y w e i g h t ) was t r e a t e d w i t h t e c h n i c a l g r a d e a l a c h l o r ( p r e p a r e d i n a c e t o n e ) o r an e m u l s i f i a b l e c o n c e n t r a t e f o r m u l a t i o n ( L a s s o 4EC, 45.1% a . i . , p r e p a r e d i n w a t e r ) t o y i e l d a p p l i c a t i o n r a t e s o f 10, 100, a n d 1000 ppm soil. Stock s o l u t i o n s o f a l a c h l o r were p r e p a r e d b y m i x i n g t h e a p p r o p r o p i a t e amount o f e i t h e r t h e t e c h n i c a l g r a d e o r e m u l s i f i a b l e a l a c h l o r w i t h 2.6 \lCi o f uniformly ring-labelled C a l a c h l o r (Monsanto Co., s p e c i f i c a c t i v i t y = 1 3 . 7 4 mg/mCi, r a d i o c h e m i c a l p u r i t y = 9 5 % ) . 1 4

One h u n d r e d m i c r o l i t e r s o f s t o c k s o l u t i o n s w e r e a p p l i e d to the s o i l . After s i t t i n g for approximately


6

254


h o u r s u n d e r a fume h o o d , t h e f l a s k s w e r e s w i r l e d b y h a n d to mix t h e s o i l and c l o s e d o f f w i t h r u b b e r s t o p p e r s from w h i c h h u n g p l a s t i c c e n t e r w e l l s c o n t a i n i n g 0.5 mL o f 2 Ν KOH, w h i c h s e r v e d as t r a p s f o r C 0 2 . One s e t o f t r e a t e d s o i l s was capped immediately to determine i f C02 was e v o l v e d d u r i n g t h e 6-hour a e r a t i o n p e r i o d . A set of u n t r e a t e d s o i l s s e r v e d as c o n t r o l s t o c o r r e c t f o r background r a d i o a c t i v i t y . A t h i r d set of s o i l s was t r e a t e d w i t h 80 μ g o f u n i f o r m l y r i n g - l a b e l l e d C-glucose t o e n s u r e t h a t CO2 was b e i n g t r a p p e d i n t h e c e n t e r w e l l s . 1 4

1 4


1 4

S o i l s w e r e e x t r a c t e d o n t h e same d a y a s a p p l i c a t i o n a n d a f t e r 28 d a y s o f i n c u b a t i o n a t 2 5 ° C . E v e r y 2-3 days d u r i n g t h e i n t e r i m , f l a s k s were o p e n e d and t h e c e n t e r w e l l s were removed and p l a c e d d i r e c t l y i n l i q u i d scintillation cocktail (Biosafe II) f o r d e t e r m i n a t i o n of radioactivity. S o i l s were e x t r a c t e d t w i c e by stirring w i t h e t h y l a c e t a t e f o l l o w e d by a t h i r d e x t r a c t i o n w i t h a 1:1:1 mixture of hexane/acetone/methanol. A f t e r the last e x t r a c t i o n , t h e s o i l was f i l t e r e d through glass m i c r o f i b e r f i l t e r paper. The s o l v e n t s were c o m b i n e d and evaporated t o d r y n e s s u n d e r vacuum a t 35°C. A f t e r e v a p o r a t i o n , t h e e x t r a c t was partitioned between water and methylene c h l o r i d e (1:1). The water p h a s e was r e e x t r a c t e d w i t h CH2CI2. The aqueous and o r g a n i c p h a s e s w e r e e v a p o r a t e d t o d r y n e s s a n d made t o a 2 mL v o l u m e w i t h a c e t o n e a n d m e t h a n o l , r e s p e c t i v e l y . Five h u n d r e d m i c r o l i t e r s o f e a c h phase were c o u n t e d by liquid s c i n t i l l a t i o n spectrometry. Parent a l a c h l o r was determined i n t h e p a r t i t i o n e d e x t r a c t s by GLC. Enrichment and I s o l a t i o n o f P o t e n t i a l A l a c h l o r - D e g r a d i n a Microorganisms. The p r o t o c o l f o r e n r i c h i n g , i s o l a t i n g , and s c r e e n i n g a l a c h l o r - d e g r a d i n g microorganisms is s u m m a r i z e d i n F i g u r e 1. A modified s o i l p e r f u s i o n system (3£) a n d s o i l s f r o m a s i m u l a t e d a l a c h l o r s p i l l c o n t a i n i n g 1 0 , 0 0 0 ppm a l a c h l o r s e r v e d a s p r i m a r y e n r i c h m e n t s for s e l e c t i n g p o t e n t i a l degraders. I n o c u l a from the primary s o u r c e s were u s e d t o f u r t h e r e n r i c h f o r a l a c h l o r - d e g r a d e r s i n c h e m o s t a t a n d b a t c h s h a k e f l a s k s ( F i g u r e 1, secondary enrichment). The p e r f u s i n g medium c o n t a i n e d m i n e r a l s a l t s m e d i u m (MSM), a l a c h l o r (100 m g / L ) , d e x t r o s e (500 m g / L ) , a n d 100 mg/L of yeast extract, chloroacetate, benzoic a c i d and p - c h l o r o a n i l i n e . The m i n e r a l s a l t s medium was c o m p o s e d o f ( g p e r L f i n a l c o n c e n t r a t i o n ) : MgS04-7H20 (0.2 g) ; N a C l ( 0 . 1 g ) ; C a C l . 2 H 0 ( 0 . 1 g ) ; KNO3 ( 0 . 5 g ) , a n d K2HPO4 ( 1 . 0 g ) . T h e s o u r c e o f s o i l was a s o y b e a n p l o t t o which a l a c h l o r - c o n t a m i n a t e d s o i l had been l a n d - a p p l i e d . 2

2

T h e c h e m o s t a t v e s s e l was a m o d i f i e d 500-mL V i r t i s fermentor with a t e f l o n impeller. The medium c o n s i s t e d o f 350 mL MSM c o n t a i n i n g a l a c h l o r (100 m g / L ) , g l u c o s e (100 m g / L ) , a n d y e a s t e x t r a c t (50 m g / L ) . The c h e m o s t a t was i n o c u l a t e d w i t h 20 mL o f s o i l p e r f u s a t e , t h e i n o c u l u m was a l l o w e d t o grow t o s t a t i o n a r y phase as a b a t c h c u l t u r e ,


19.

F E L S O T & DZANTOR


Secondary Enrichments


Primary Enrichments

Soil Perfusion Simulated Column Chemical Spill I

Chemostat

1 _ ,

Shake Flasks

1

1

ISOLATION

t

Pour plate serial dilutions on MSAI (100 ppm) agar for bacteria SDAI agar for fungi

I

PURIFICATION Streak colonies on DYAI (100 ppm) agar Restreak pure cultures on MSAI agar; select 'actively growing' colonies for storage and continued study Preliminary characterization

SCREENING Inoculate and incubate in different liquid media containing alachlor; determine loss of alachlor Select efficient degraders Continued characterization and identification

Mixed culture study in chemostat Figure

1.

Protocol f o r enrichment, i s o l a t i o n , and study o f microbial c u l t u r e s .


255

256



a n d t h e n f r e s h m e d i u m was p u m p e d i n a t t h e r a t e o f 0.1 mL/min. T h e c h e m o s t a t was s a m p l e d p e r i o d i c a l l y f o r m i c r o b i a l i s o l a t i o n and chemical assay o f a l a c h l o r . B a t c h c u l t u r e s y s t e m s c o n s i s t e d o f 125-mL E r l e n m e y e r flasks containing 50 mL MSM a n d 100 mg/L a l a c h l o r . The f l a s k s w e r e i n o c u l a t e d d i r e c t l y w i t h 0.5 g o f s o i l from s i m u l a t e d c h e m i c a l s p i l l e x p e r i m e n t s o r w i t h 1 mL aliquots o f s o i l s u s p e n s i o n s (1 g s o i l / 1 0 mL H 2 O ) . The f l a s k s were i n c u b a t e d o n a r o t a r y s h a k e r a t 25°C f o r u p t o 4 weeks with p e r i o d i c sampling f o r microbial i s o l a t i o n . Cultures f r o m t h e e n r i c h m e n t systems were subsampled p e r i o d i c a l l y a n d p l a t e d e i t h e r o n a g a r c o n t a i n i n g MSM plus alachlor (MSAI) t o i s o l a t e b a c t e r i a o r o n S a b a r o u d dextrose with alachlor (SDA1) t o i s o l a t e f u n g i ( F i g u r e 1, isolation step). T h e a g a r p l a t e s w e r e i n c u b a t e d a t 25°C and examined p e r i o d i c a l l y f o r m i c r o b i a l growth. Bacterial c o l o n i e s t h a t f o r m e d o n MSAI a g a r were p u r i f i e d b y s t r e a k i n g on d e x t r o s e - y e a s t e x t r a c t - a l a c h l o r (DYA1) a g a r ( F i g u r e 1, p u r i f i c a t i o n s t e p ) . Purified bacterial c o l o n i e s were r e s t r e a k e d o n MSAI a n d i s o l a t e s t h a t g r e w w e r e s t o r e d i n DYA1 f o r f u r t h e r c h a r a c t e r i z a t i o n . Fungi w e r e p u r i f i e d b y h y p h a l t i p p i n g o n SDA1. A l l pure i s o l a t e s were m a i n t a i n e d on t h e i r r e s p e c t i v e agar p r e p a r a t i o n s a n d s t o r e d a t 5°C. Screening Potential A1achlor-Degrading Bacteria. Twentye i g h t b a c t e r i a l i s o l a t e s were p r e l i m i n a r i l y s c r e e n e d f o r the a b i l i t y t o degrade a l a c h l o r c o m e t a b o l i c a l l y o r as a sole carbon source. From t h i s t e s t , n i n e i s o l a t e s were selected f o r further screening. F i r s t , t h e b a c t e r i a were i n o c u l a t e d i n t o 20 mL o f f i l t e r - s t e r i l i z e d M S A I containing g l u c o s e ( 1 0 0 mg/L) a n d y e a s t e x t r a c t (500 m g / L ) . After incubation f o r 24 h o n a r o t a r y s h a k e r , 0.2 mL o f c u l t u r e was i n o c u l a t e d i n t o t e s t t u b e s c o n t a i n i n g 5 mL o f o n e o f t h e f o l l o w i n g f i l t e r - s t e r i l i z e d m e d i a : (1) a l a c h l o r (10 o r 100 ppm) i n MSM ( M S - A 1 ) ; (2) a l a c h l o r (10 o r 100 ppm) i n MSM + y e a s t e x t r a c t (50 mg/L) + d e x t r o s e ( 0 . 1 o r 1.0 g / L ) (YA1-D o r Y A 1 - D + ) ; (3) a l a c h l o r (10 o r 100 ppm) i n s o i l e x t r a c t + d e x t r o s e ( 0 . 0 , 0.1 o r 1.0 g / L ) ( S E A 1 , S E A 1 - D o r SEA1-D+). S o i l e x t r a c t was p r e p a r e d b y a u t o c l a v i n g a mixture of 1 k g C H E C K s o i l a n d 1.5 L o f H 0 f o r 30 m i n a t 1 2 1 ° C . A f t e r c o o l i n g a t 5°C o v e r n i g h t , t h e s o i l s u s p e n s i o n was f i l t e r e d through glass m i c r o f i b e r f i l t e r s and centrifuged a t 5523 x g f o r 5 m i n . T h e e x t r a c t was b u f f e r e d a t p H 6.8 using K HP0 (0.5 g / L ) . 2

2

4

A f t e r i n o c u l a t i o n , d u p l i c a t e c u l t u r e s were i n c u b a t e d i n t h e d a r k w i t h o u t a g i t a t i o n a t 2 5 ° C f o r 10 d a y s . FourmL a l i q u o t s o f t h e c u l t u r e s w e r e e x t r a c t e d t w i c e w i t h ethyl acetate f o r analysis of alachlor. Controls c o n s i s t e d o f u n i n o c u l a t e d media.



19.

FELSOT & DZANTOR


257

Screening Potential Alachlor-Dearadina Funai. Two f u n g a l i s o l a t e s (CCF-1 a n d CCF-2) w e r e t e s t e d i n b a t c h s h a k e f l a s k c u l t u r e s i n a m e d i u m (PYA1) c o n t a i n i n g (g/L): p e p t o n e (1.0), y e a s t e x t r a c t (3.0), d e x t r o s e (20.0), K 2 HP0 4 (1.0), CaS04 (0.014) a n d a l a c h l o r (0.1). Inocula w e r e p r e p a r e d b y g r o w i n g t w o i s o l a t e s i n PYA1. After 5 d a y s , g r o w t h was h a r v e s t e d b y c e n t r i f u g a t i o n , a n d t h e f u n g a l p e l l e t s were a s e p t i c a l l y m a c e r a t e d . One-mL a l i q u o t s o f t h e homogenized c u l t u r e s were i n o c u l a t e d i n t o 50 mL PYA1 o r MSA1. Controls included alachlor-fortified, uninoculated f l a s k s and inoculated f l a s k s containing p e p t o n e - y e a s t e x t r a c t medium w i t h o u t a l a c h l o r . At i n t e r v a l s o f 3, 7, a n d 14 d a y s , c u l t u r e s w e r e f i l t e r e d a n d washed; t h e f i l t r a t e a n d w a s h i n g s were c o m b i n e d a n d extracted twice with ethyl acetate f o r alachlor analysis. C h l o r i d e r e l e a s e d f r o m a l a c h l o r was a n a l y z e d i n p a r t o f the f i l t r a t e by using a modified f e r r i c y a n i d e c o l o r m e t r i c m e t h o d (22.) . E f f e c t o f N u t r i e n t Amendments a n d M i c r o b i a l I n o c u l u m . B a t c h e s o f CHECK s o i l (2-mm m e s h , 2 5 % m o i s t u r e ) w e r e m i x e d w i t h g r o u n d (2-mm m e s h ) c o r n (CS) o r s o y b e a n (SB) s t u b b l e a t a r a t e o f 20 g / k g o v e n - d r i e d s o i l . T h i r t y grams o f amended s o i l s were w e i g h e d i n t o f l a s k s ; h a l f o f t h e f l a s k s c o n t a i n i n g SB w e r e t r e a t e d w i t h a s t o c k s o l u t i o n o f NH4NO3 a t a r a t e o f 1 mg N / g ( S B + N ) . Aliquots of formulated a l a c h l o r ( L a s s o 4EC) w e r e p i p e t t e d o n s o i l t o y i e l d concentrations o f 100 o r 1000 ppm. A b o u t 1-2 h o u r s a f t e r p e s t i c i d e treatment, h a l f o f the f l a s k s assigned t o each t r e a t m e n t w e r e i n o c u l a t e d w i t h f u n g a l i s o l a t e CCF1 (0.5 mg f u n g a l u n i t s / 0 . 2 8 mL/g o v e n - d r y s o i l ) , w h i c h h a d b e e n b l e n d e d i n p h o s p h a t e - b u f f e r e d w a t e r (pH 7) c o n t a i n i n g a l a c h l o r (100 p p m ) . Unamended, u n i n o c u l a t e d soils f o r t i f i e d w i t h a l a c h l o r were c o n t r o l s . A l l f l a s k s were c l o s e d with P a r a f i l m and incubated a t 25°C. O n c e a week t h e f l a s k s were opened f o r a e r a t i o n . On d a y s 0, 1 4 , 2 8 , a n d 56, f l a s k s were r e m o v e d f o r e x t r a c t i o n a n d a n a l y s i s o f a l a c h l o r , s o i l d e h y d r o g e n a s e (2à) a n d s o i l e s t e r a s e . (2S.) . r

E x t r a c t i o n and A n a l y s i s o f A l a c h l o r . S o i l was s l u r r i e d w i t h 12 mL o f w a t e r a n d e x t r a c t e d t w i c e b y s t i r r i n g w i t h 50 mL o f e t h y l a c e t a t e f o r 45 m i n u t e s . T h e s o l v e n t was d e c a n t e d a f t e r each e x t r a c t i o n a n d c o n c e n t r a t e d on a steam bath. T h e e x t r a c t was r e d i l u t e d w i t h e t h y l a c e t a t e a n d a n a l y z e d b y GLC w i t h n i t r o g e n - p h o s p h o r u s s p e c i f i c detection. R e s i d u e s w e r e s e p a r a t e d o n a 9 0 - c m χ 0.2 mm i . d . g l a s s c o l u m n p a c k e d w i t h 5% A p i e z o n + 0.125% DEGS m a i n t a i n e d i s o t h e r m a l l y a t 190°C. I n j e c t o r and detector were h e l d a t 250°C, a n d g a s f l o w r a t e s were a d j u s t e d a s n e e d e d t o o b t a i n maximum s e n s i t i v i t y a n d r e s o l u t i o n . R e s i d u e s were q u a n t i t a t e d b y t h e method o f e x t e r n a l standards.


258



Results

and

Discussion

E f f e c t s of S o i l D i l u t i o n . i n p r e v i o u s s t u d i e s a t an a g r o c h e m i c a l f a c i l i t y c o n t a m i n a t e d w i t h h e r b i c i d e waste, a l a c h l o r had not t o t a l l y d e g r a d e d a f t e r two y e a r s i n s o i l t h a t had been e x c a v a t e d and s t o r e d i n p i l e s on t h e g r o u n d (1Û.). Land a p p l i c a t i o n has been s t u d i e d as a method f o r s t i m u l a t i n g b i o d é g r a d a t i o n by d i l u t i n g t h e s o i l on c r o p p e d l a n d (U). Under l a b o r a t o r y c o n d i t i o n s , a l a c h l o r d e g r a d e d s l o w e r i n w a s t e - p i l e s o i l t h a n i t d i d i n s o i l d i l u t e d by 90% (w/w) ( T a b l e I ) . S e v e n t y - p e r c e n t o f t h e a l a c h l o r was d e t o x i f i e d i n 42 days compared t o

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