Enhanced Biodegradation of Pesticides in the Environment - American

Chapter 14

Microbial Adaptation in Aquatic Ecosystems J. C. Spain

Downloaded by UNIV OF ARIZONA on April 2, 2017 | http://pubs.acs.org Publication Date: May 3, 1990 | doi: 10.1021/bk-1990-0426.ch014

U.S. Air Force Engineering and Services Center, Tyndall Air Force Base, Panama City, FL 32403-6001

Aquatic microbial communities can adapt to degrade a novel substrate i f the chemical can serve as a source of nutrients. We have studied biodegradation of p-nitrophenol (PNP) in laboratory test systems and in the field. Adaptation in freshwater microbial communities required from 3 to 10 days and biodegradation rates increased as much as a thousandfold at PNP concentrations as low as 10 mg/liter. Populations of PNP degraders increased dramatically during adaptation and no evidence of genetic change associated with adaptation was detected. Bacteria able to grow at the expense of PNP were detected in microbial communities prior to treatment with PNP. They did not begin to grow and degrade PNP until several days after i n i t i a l exposure. Results indicate that accurate prediction of biodegradation rates requires a better understanding of the factors that control adaptation in the environment. These include: enzyme induction, genetic changes, increases in microbial populations, predation, and nutrient limitation.

F a c t o r s t h a t c o n t r o l t h e f a t e and t r a n s p o r t o f o r g a n i c compounds i n a q u a t i c systems i n c l u d e : photolysis, hydrolysis, s o r p t i o n , hydrodynamics, and biodégradation. Biodégradation i s t h e l e a s t w e l l u n d e r s t o o d o f t h e s e p r o c e s s e s , p r i m a r i l y because i t i s d i f f i c u l t t o p r e d i c t the e f f e c t s o f a d a p t a t i o n , o r a c c l i m a t i o n , o f m i c r o b i a l p o p u l a t i o n s i n r e s p o n s e t o s p e c i f i c compounds. A d a p t a t i o n i s c h a r a c t e r i z e d by an i n c r e a s e i n t h e r a t e o f biodégradation o f a compound as a r e s u l t o f exposure o f t h e m i c r o b i a l p o p u l a t i o n t o t h e compound ( 1 ) . The a b i l i t y t o p r e d i c t s u c h i n c r e a s e s i n biodégradation r a t e s i s c r i t i c a l t o u n d e r s t a n d i n g of the behavior o f chemicals i n the f i e l d . F o r example, a c c u r a c y i n p r e d i c t i o n o f t h e onset o f r a p i d biodégradation would a l l o w

This chapter not subject to U.S. copyright Published 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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a c c u r a t e assessment o f the impact o f a c c i d e n t a l r e l e a s e i n a r i v e r that flows i n t o a s e n s i t i v e estuary. I n groundwater, a c c u r a t e i n f o r m a t i o n about a d a p t a t i o n would a l l o w p r e d i c t i o n o f whether and when a plume o f c o n t a m i n a t i o n might a f f e c t a s o u r c e o f d r i n k i n g w a t e r . A p a r t i a l l i s t o f compounds known to e l i c i t a d a p t a t i o n i n a q u a t i c systems i s p r o v i d e d i n the f o l l o w i n g t a b l e .


Table

1. C h e m i c a l s t h a t cause a d a p t a t i o n i n a q u a t i c systems

Compound

Reference

p-Cresol

L e w i s e t a l . (2)

Nitrilotriacetic

acid

2,4-Dichlorophenoxyacetic

P f a e n d e r e t a l . (3) acid

Audus

(4)

Phenol

Murakami and A l e x a n d e r

4,6-Dinitro-o-cresol

Gundersen and J e n s e n

p-Nitrophenol

Spain et a l .

(7)

Methylparathion

Spain et a l .

(7)

(£)

(6)

The common c h a r a c t e r i s t i c o f compounds t h a t e l i c i t a d a p t a t i o n i s the a b i l i t y to s e r v e as a growth s u b s t r a t e f o r b a c t e r i a . Thus i t seems t o be the r e s p o n s e o f a s p e c i f i c p o p u l a t i o n o f b a c t e r i a t o a growth s u b s t r a t e t h a t i s r e s p o n s i b l e f o r the a d a p t a t i o n . To my knowledge, t h e r e a r e no r e p o r t s o f a d a p t a t i o n to compounds t h a t do n o t s e r v e as growth s u b s t r a t e s . G r a t u i t o u s i n d u c e r s a r e known but t h e y a r e n o t degraded more r a p i d l y as a r e s u l t o f a d a p t a t i o n . Patterns of

Adaptation

The c o u r s e o f a d a p t a t i o n i n v o l v e s s e v e r a l d i s t i n c t p h a s e s : a) B e f o r e exposure t o the c h e m i c a l , the p o p u l a t i o n o f b a c t e r i a a b l e to degrade the c h e m i c a l i s s m a l l and i n a c t i v e . T h e r e may be no o r g a n isms i n the m i c r o b i a l community t h a t can s y n t h e s i z e the enzymes n e c e s s a r y f o r growth on the compound i n q u e s t i o n , b) Upon a d d i t i o n of the c h e m i c a l , t h e r e i s a v a r i a b l e l a g p e r i o d d u r i n g w h i c h s p e c i f i c b a c t e r i a become a c t i v e . This period involves induction of i n d u c i b l e enzymes and the onset o f growth when s p e c i f i c d e g r a d e r s a r e p r e s e n t . The f a c t o r s t h a t c o n t r o l the l e n g t h o f t h i s l a g p e r i o d a r e n o t c l e a r and w i l l be d i s c u s s e d i n d e t a i l below, c) Growth o f the b a c t e r i a a t the expense o f the added c h e m i c a l r e s u l t s i n the r a p i d d e p l e t i o n o f the c h e m i c a l and an enrichment o f the p o p u l a t i o n t h a t i s a b l e to d e r i v e c a r b o n and energy from the c h e m i c a l . The r e s u l t a n t p o p u l a t i o n can respond r a p i d l y w i t h o u t a l a g p e r i o d t o


14.

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Microbial Adaptation in Aquatic Ecosystems

183


subsequent a d d i t i o n s o f the same c h e m i c a l . I f the c o n c e n t r a t i o n o f the added compound i s low, t h e r e may n o t be an i n c r e a s e i n the t o t a l number o f h e t e r o t r o p h i c b a c t e r i a p r e s e n t , whereas the number o f s p e c i f i c d e g r a d e r s may i n c r e a s e s e v e r a l o r d e r s o f magnitude. Very low c o n c e n t r a t i o n s might cause i n c r e a s e s i n d e g r a d a t i o n r a t e s without detectable increases i n population s i z e . A v a r i e t y o f f a c t o r s i n f l u e n c e the l e n g t h o f the l a g p e r i o d n o t e d above. V e r y l o n g and v a r i a b l e l a g p e r i o d s ( F i g u r e 1A) have been t a k e n t o i n d i c a t e t h a t no organisms a b l e t o degrade the c h e m i c a l were p r e s e n t i n the i n i t i a l i n o c u l u m . The l a g would be the time r e q u i r e d f o r the d e g r a d a t i v e a b i l i t y t o e v o l v e t h r o u g h m u t a t i o n o r g e n e t i c exchange. Wyndham (&) d e s c r i b e d a c l a s s i c example o f a g e n e t i c change t h a t a l l o w e d an A c i n e t o b a c t e r t o e v o l v e the a b i l i t y to degrade h i g h c o n c e n t r a t i o n s o f a n i l i n e i n r i v e r w a t e r . Wiggins and A l e x a n d e r (9) s u g g e s t e d a s i m i l a r e x p l a n a t i o n f o r the v a r i a b l e l a g p e r i o d i n v o l v e d i n a d a p t a t i o n o f sewage and l a k e water communities t o b i o d e g r a d e n i t r o s a l i c y l a t e s . Lag p e r i o d s o f up to a y e a r have been n o t e d i n enrichment c u l t u r e s s e t up f o r i s o l a t i o n o f b a c t e r i a a b l e t o grow on c h l o r i n a t e d a r o m a t i c compounds (10, 11, 12, and 1 1 ) . K e l l o g g e t a l . ( l f i ) s u g g e s t e d t h a t g e n e t i c exchange d u r i n g t e n months i n a chemostat was r e s p o n s i b l e f o r the e v o l u t i o n o f a pseudomonad a b l e t o degrade 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 . K r o c k e l and F o c h t (14) have r e c e n t l y d e m o n s t r a t e d more r a p i d e v o l u t i o n o f the a b i l i t y t o degrade c h l o r o b e n z e n e s t h r o u g h g e n e t i c exchange i n a n o v e l chemostat system. S h o r t e r and more r e p r o d u c i b l e l a g p e r i o d s ( F i g u r e IB) seem t o r e f l e c t the time r e q u i r e d f o r growth o f a s m a l l p o p u l a t i o n o f b a c t e r i a . W i g g i n s e t a l . (1£) n o t e d t h a t even i f the p o p u l a t i o n s t a r t s t o use the c h e m i c a l f o r growth i m m e d i a t e l y , t h e r e may be a s i g n i f i c a n t a p p a r e n t l a g b e f o r e enough s p e c i f i c d e g r a d e r s accumulate to cause a d e t e c t a b l e l o s s o f the c h e m i c a l . A v a r i e t y o f as y e t p o o r l y u n d e r s t o o d f a c t o r s can a f f e c t the growth o f the i n i t i a l s m a l l p o p u l a t i o n s and thus extend o r s h o r t e n the l a g p e r i o d . W i g g i n s and A l e x a n d e r (16) p r o v i d e d e v i d e n c e t h a t prédation by p r o t o z o a extended the a c c l i m a t i o n p e r i o d b e f o r e biodégradation o f p - n i t r o p h e n o l (PNP), 2,4-dichlorophenoxyacetic a c i d (2,4-D), and 2 , 4 - d i c h l o r o p h e n o l . In a n o t h e r s e r i e s o f e x p e r i m e n t s ( 1 5 ) , the same a u t h o r s found t h a t the a d d i t i o n o f i n o r g a n i c n u t r i e n t s s h o r t e n e d the l a g p e r i o d f o r biodégradation o f PNP i n l a k e water and sewage. S i m i l a r l y , L e w i s e t a l (2) d e m o n s t r a t e d t h a t p - c r e s o l was degraded much more q u i c k l y i n pond w a t e r supplemented w i t h n i t r o g e n and p h o s p h o r o u s . Low r a t e s o f p - c r e s o l t r a n s f o r m a t i o n were d e t e c t a b l e i m m e d i a t e l y i n t h e i r e x p e r i m e n t s but added n u t r i e n t s d r a m a t i c a l l y i n c r e a s e d the r a t e . Z a i d i e t a l (17) c l e a r l y d e m o n s t r a t e d t h a t a d d i t i o n o f i n o r g a n i c n u t r i e n t s s t i m u l a t e d growth o f the s p e c i f i c dégrader p o p u l a t i o n when d e g r a d a t i o n r a t e s were enhanced. These r e s u l t s s u g g e s t t h a t any f a c t o r t h a t a f f e c t s the growth o f the s p e c i f i c p o p u l a t i o n i n v o l v e d i n d e g r a d a t i o n can a f f e c t the l e n g t h o f the a p p a r e n t l a g period. I n some i n s t a n c e s t h e r e i s an extended l a g p e r i o d b e f o r e a p o p u l a t i o n o f s p e c i f i c d e g r a d e r s b e g i n s to grow and m e t a b o l i z e the c h e m i c a l o f i n t e r e s t ( F i g u r e 1C). The a p p r o p r i a t e b a c t e r i a a r e p r e s e n t but do n o t respond to the p r e s e n c e o f the added c h e m i c a l . T h i s s i t u a t i o n has been o b s e r v e d s e v e r a l times w i t h PNP i n our


ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT


4

6

8

MONTHS 100h

< UJ

ο ζ ο ο

100

< UJ

ο ζ ο ο

Figure 1. Patterns of substrate disappearance related to mechanism of adaptation. Genetic changes such as mutation, plasmid exchange, or recombination (A). Growth of a small population of bacteria able to degrade the test chemical immediately (B). Delayed induction of enzymes or activation of specific organisms (C).


14.

SPAIN

Microbial Adaptation in Aquatic Ecosystems

185


laboratory (18). A v a r i e t y o f e x p l a n a t i o n s have been o f f e r e d f o r the f a i l u r e o f PNP d e g r a d e r s t o respond i m m e d i a t e l y . Induction of the a p p r o p r i a t e enzymes i s g e n e r a l l y n e c e s s a r y b e f o r e m e t a b o l i s m and growth a t the expense o f a new s u b s t r a t e can b e g i n . Induction of c a t a b o l i c enzymes has been s t u d i e d e x t e n s i v e l y i n the l a b o r a t o r y w i t h l a r g e p o p u l a t i o n s o f b a c t e r i a and w i t h h i g h c o n c e n t r a t i o n s o f substrate. I n d u c t i o n o f enzymes under s u c h l a b o r a t o r y c o n d i t i o n s t y p i c a l l y o c c u r s w i t h i n m i n u t e s to h o u r s . However, l i t t l e i s known about the i n d u c t i o n o f enzymes under n a t u r a l c o n d i t i o n s where s m a l l p o p u l a t i o n s o f s t a r v e d c e l l s might be exposed t o low concentrations o f a s u b s t r a t e under l e s s t h a n i d e a l c o n d i t i o n s . Hoover e t a l . (19) have d e s c r i b e d s e v e r a l a n o m a l i e s i n m i n e r a l i z a t i o n o f o r g a n i c compounds p r e s e n t a t low concentrations. B o e t h l i n g and A l e x a n d e r (20) r e p o r t e d the e x i s t e n c e o f t h r e s h o l d c o n c e n t r a t i o n s below w h i c h m i n e r a l i z a t i o n c o u l d n o t be d e t e c t e d . S p a i n and Van V e l d (1) r e p o r t e d a t h r e s h o l d o f 10 ug p e r l i t e r f o r a d a p t a t i o n o f r i v e r water communities to PNP and the e x t e n t o f i n d u c t i o n was p r o p o r t i o n a l t o PNP c o n c e n t r a t i o n s above the threshold. W i g g i n s and A l e x a n d e r (9) n o t e d t h a t the l e n g t h o f the a c c l i m a t i o n p e r i o d f o r PNP m i n e r a l i z a t i o n i n sewage was d i r e c t l y p r o p o r t i o n a l t o the c o n c e n t r a t i o n o f PNP. I n c o n t r a s t , Schmidt e t a l (21) and B u t t o n and R o b e r t s o n (22) have s u g g e s t e d t h a t i n d u c t i o n o f enzymes can l e a d to a d a p t a t i o n a t s u b s t r a t e c o n c e n t r a t i o n s t h a t a r e too low t o s u p p o r t growth o f the b a c t e r i a . I t has a l s o been s u g g e s t e d t h a t the p r e s e n c e o f i n h i b i t o r s might d e l a y i n d u c t i o n and the o n s e t o f biodégradation ( £ ) . An i n h i b i t o r might be t o x i c t o the s p e c i f i c p o p u l a t i o n under c o n s i d e r a t i o n , o r i t might s e r v e as an alternate, preferred substrate. Enhanced D e g r a d a t i o n o f

PNP

PNP i s the c l a s s i c example o f compounds t h a t e l i c i t a d a p t a t i o n o f a q u a t i c m i c r o b i a l communities a f t e r a l a g p e r i o d o f s e v e r a l d a y s . The t y p i c a l d e g r a d a t i o n c u r v e i s s i m i l a r t o the one shown i n F i g u r e 1C. We became i n t e r e s t e d i n PNP because i t i s a h y d r o l y s i s p r o d u c t o f m e t h y l p a r a t h i o n i n a q u a t i c systems. When we s t u d i e d the biodégradation o f PNP i n l a b o r a t o r y t e s t systems c o n t a i n i n g r i v e r water and sediment ( Z ) , no d e g r a d a t i o n was d e t e c t a b l e f o r 3-5 days, a f t e r w h i c h the PNP was m i n e r a l i z e d r a p i d l y ( F i g u r e 2 ) . When PNP was added a g a i n , i t was degraded r a p i d l y and w i t h o u t a l a g . M i c r o b i a l p o p u l a t i o n s i n c o n t r o l samples r e c e i v i n g PNP f o r the f i r s t time underwent a l a g p e r i o d s i m i l a r to t h a t i n samples exposed a t the b e g i n n i n g o f the e x p e r i m e n t . I n c l u s i o n of such c o n t r o l s i n l a b o r a t o r y t e s t s i s e s s e n t i a l i f the i n c r e a s e d d e g r a d a t i o n r a t e i s t o be r i g o r o u s l y a t t r i b u t e d to the p r e s e n c e o f PNP. It i s clear from t h i s example t h a t a d a p t a t i o n can e f f e c t a d r a m a t i c i n c r e a s e i n biodégradation r a t e s ; the r a t e i n the adapted system i s o v e r a t h o u s a n d f o l d h i g h e r t h a n t h a t i n the c o n t r o l . Subsequent e x p e r i m e n t s ( F i g u r e 3) i n d i c a t e d t h a t m i c r o b i a l communities i n the l a b o r a t o r y remained adapted to PNP f o r up t o s i x weeks a f t e r exposure to PNP. A l l f r e s h w a t e r communities t e s t e d showed the a b i l i t y to a c c l i m a t e to PNP, whereas none o f the marine communities c o u l d ( 1 ) . T h i s s u g g e s t s t h a t PNP r e l e a s e d i n t o a r i v e r


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Enhanced Biodegradation of Pesticides in the Environment - American

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