Biodegradation of Munition Waste, TNT (2,4,6-Trinitrotoluene), and

Chapter 11

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: August 2, 1991 | doi: 10.1021/bk-1991-0468.ch011

Biodegradation of Munition Waste, TNT (2,4,6-Trinitrotoluene), and RDX (Hexahydro– 1,3,5-Trinitro-1,3,5-triazine) by Phanerochaete chrysosporium Tudor Fernando and Steven D. Aust Biotechnology Center, Utah State University, Logan, UT 84322-4430 Rapid b i o d e g r a d a t i o n o f TNT (2,4,6-trinitrotoluene) and RDX ( h e x a h y d r o - 1 , 3 , 5 - t r i n i t r o 1,3,5-triazine) by t h e w h i t e r o t fungus Phanerochaete chrysosporium BKM F-1767 was observed. A t an initial c o n c e n t r a t i o n o f 1.25 nmoles/10 ml cultures o f P. chrysosporium, 50.8 ± 3.2% of the [ C] TNT was degraded to CO in 30 days and only 2.8% of the initial TNT could be r e c o v e r e d . I n RDX contaminated liquid cultures, (1.25 nmoles/10 ml cultures), 66.6 ± 4.1% o f t h e [ C] RDX was converted t o CO over a 30 day p e r i o d and o n l y 4% o f t h e initial RDX was r e c o v e r e d . I n soil, slow but s u s t a i n e d d e g r a d a t i o n , 6.3 + 0.6% o f t h e [ C] TNT initially p r e s e n t , i.e., 57.9 nmoles/10 g soil, was mineralized over a 30 day incubation. In contrast, 76.0 ± 3.9% of the [ C] RDX added, (1.25 nmoles/10 g soil), was degraded t o CO2 in 30 days. Mass balance a n a l y s e s o f liquid and soil cultures contaminated w i t h TNT r e v e a l e d t h a t p o l a r m e t a b o l i t e s were formed, and HPLC a n a l y s i s i n d i c a t e d t h a t not more than 5% o f t h e radioactivity remained as undegraded TNT. However, t h e mass balance a n a l y s e s o f liquid (10 ml) and soil (10 g) cultures contaminated w i t h 1.25 nmoles o f [ C] RDX showed no m e t a b o l i t e s . When t h e c o n c e n t r a t i o n of TNT in cultures (both liquid and soil) was adjusted t o contamination levels in t h e environment, i.e., 10,000 mg/kg in soil and 100 mg/liter in water, 18.4 ± 2.9% and 19.6 ± 3.5% of t h e initial TNT was converted t o CO in 90 days in soil and liquid cultures, r e s p e c t i v e l y . I n both systems, about 85% o f t h e initial TNT 14

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0097-6156/91/0468-0214$06.00/0 © 1991 American Chemical Society In Emerging Technologies in Hazardous Waste Management II; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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disappeared over a 90 day incubation period. These r e s u l t s suggest that P. chrysosporium may be useful for the reclamation of s i t e s contaminated with TNT and RDX. One o f t h e m a j o r e n v i r o n m e n t a l p r o b l e m s a s s o c i a t e d w i t h U.S. m i l i t a r y i s t h e p r e s e n c e o f s o i l , s e d i m e n t and w a t e r c o n t a m i n a t e d w i t h t o x i c e x p l o s i v e r e s i d u e s a t and a r o u n d many m a j o r m i l i t a r y f a c i l i t i e s . In these f a c i l i t y s i t e s t h e s o i l , s e d i m e n t and t h e w a t e r w e r e c o n t a m i n a t e d o v e r s e v e r a l y e a r s by i m p r o p e r d i s p o s a l o f w a s t e and w a s t e w a t e r g e n e r a t e d d u r i n g t h e p r o d u c t i o n of m u n i t i o n s , b u r n i n g o r d e t o n a t i o n o f o f f s p e c i f i c a t i o n m a t e r i a l , and d e m i l i t a r i z a t i o n o f o u t - o f d a t e m u n i t i o n s . TNT ( 2 , 4 , 6 - t r i n i t r o t o l u e n e ) and RDX (hexahydro-1,3,5-trinitro-l,3,5-triazine) are the p r e d o m i n a n t c o n v e n t i o n a l e x p l o s i v e s u s e d by m i l i t a r y f o r c e s ( 1 0 , 1 9 , 2 6 ) . The m a j o r w a s t e w a t e r p r o b l e m s t h a t o r i g i n a t e f r o m TNT and RDX a r e a s s o c i a t e d w i t h t h e f i n i s h i n g p r o c e s s e s , s h e l l w a s h o u t p r a c t i c e s and e q u i p m e n t and b u i l d i n g w a s h o f f o p e r a t i o n s ( 1 3 , 1 6 , 2 2 ) . TNT and RDX a r e o n l y s l i g h t l y s o l u b l e i n w a t e r and t h e r e f o r e l a r g e v o l u m e s o f w a t e r h a v e b e e n u s e d t o wash o f f r e s i d u e s (26,32). F o r example, a s i n g l e m u n i t i o n m a n u f a c t u r i n g p l a n t c a n g e n e r a t e and d i s p o s e a s much a s 500,000 g a l l o n s o f w a s t e w a t e r p e r day ( 2 2 , 3 1 ) . A s a r e s u l t waste water c o n t a i n i n g t h e s e t o x i c contaminants l e a c h t h r o u g h t h e s o i l c o n t a m i n a t i n g t h e s o i l and e v e n t u a l l y t h e ground water (16,22). The t o x i c and m u t a g e n i c e f f e c t s o f t h e s e compounds i n d i f f e r e n t k i n d s o f o r g a n i s m s i n c l u d i n g humans h a v e been w e l l documented (4,11,12,33). E x p o s u r e t o TNT i s known t o c a u s e p a n c y t o p e n i a , a d i s o r d e r o f t h e b l o o d f o r m i n g t i s s u e s c h a r a c t e r i z e d by a p r o n o u n c e d d e c r e a s e i n t h e number o f l e u k o c y t e s , e r y t h r o c y t e s and r e t i c u l o c y t e s i n humans and o t h e r mammals ( 9 ) . T o x i c e f f e c t s i n c l u d i n g l i v e r damage and a n e m i a h a v e b e e n r e p o r t e d i n w o r k e r s engaged i n l a r g e - s c a l e m a n u f a c t u r i n g and h a n d l i n g p r a c t i c e s ( 6 , 8 , 2 5 , 2 9 ) . A l s o , TNT i s t o x i c t o f a t h e a d minnow ( P i m e p h a l e s p r o m e l a s ) and b l u e g i l l s ( L e p o m i s m a c r o c h i r u s ) a t c o n c e n t r a t i o n s o f 2 t o 3 Mg/ml ( 2 0 , 2 7 ) . I t i s a l s o t o x i c t o c e r t a i n green algae (Selenastrum c a p r i c o r n u t u r n . M i c r o c y s t i s a e r u g i n o s a . Chlamydomonas r e i n h a r t i i ) . t i d e p o o l copepods, ( T i q r i o p u s c a l i f o r n i c u s ) , and o y s t e r ( C r a s s o s t r e a g i g u s ) l a r v a e (27,33). F u r t h e r m o r e , TNT i s a mutagen a s a s s a y e d by t h e Ames t e s t ( 3 3 ) . On t h e o t h e r h a n d , RDX h a s b e e n f o u n d t o be a p o t e n t c o n v u l s a n t i n t h e r a t w i t h a n i n t r a p e r i t o n e a l minimum l e t h a l d o s e o f 25 mg/kg ( 3 0 ) . I t s a c u t e t o x i c i t y has been i n v e s t i g a t e d i n mice, c a t s , p i g s and d o g s ( 2 6 , 2 8 , 3 0 ) . RDX r e l a t e d t o x i c e f f e c t s t o humans h a v e b e e n r e p o r t e d f r o m a l l o v e r t h e 1

In Emerging Technologies in Hazardous Waste Management II; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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world. F o r example, o c c u p a t i o n a l l y r e l a t e d c a s e s o f human RDX i n t o x i c a t i o n h a v e b e e n r e p o r t e d i n many c o u n t r i e s i n c l u d i n g t h e U n i t e d S t a t e s ( 1 1 ) . As i n a n i m a l s t u d i e s , t h e m a i n symptoms o f RDX i n t o x i c a t i o n i n humans w e r e r e l a t e d t o t h e c e n t r a l n e r v o u s s y s t e m (11,1) . The w h i t e r o t f u n g u s , P h a n e r o c h a e t e c h r y s o s p o r i u m h a s shown t o d e g r a d e a w i d e v a r i e t y o f e n v i r o n m e n t a l l y p e r s i s t e n t o r g a n o p o l l u t a n t s , i n c l u d i n g a number o f o r g a n o h a l i d e s and a v a r i e t y o f t o x i c c h e m i c a l s ( 2 , 5 ) . A l s o , t h i s f u n g u s i s one o f t h e r e l a t i v e l y few m i c r o o r g a n i s m s known t o be a b l e t o d e g r a d e l i g n i n , a n a t u r a l l y o c c u r r i n g and r e c a l c i t r a n t b i o p o l y m e r , t o c a r b o n d i o x i d e ( 1 4 ) . R e c e n t s t u d i e s i n a number o f l a b o r a t o r i e s (2,5,14) h a v e shown t h a t t h e a b i l i t y t o d e g r a d e s u c h a d i v e r s e g r o u p o f compounds i s d e p e n d e n t on t h e n o n s p e c i f i c and n o n s t e r e o s e l e c t i v e l i g n i n d e g r a d i n g s y s t e m w h i c h i s e x p r e s s e d by t h e f u n g u s u n d e r n u t r i e n t (nitrogen, carbon, or s u l f u r ) l i m i t i n g c o n d i t i o n s ( 1 4 , 1 5 ) . The l i g n i n d e g r a d i n g s y s t e m c o n s i s t s , i n part, of a family of l i g n i n peroxidases (commonly known a s l i g n i n a s e s ) w h i c h a r e a b l e t o catalyze the i n i t i a l o x i d a t i v e depolymerization of l i g n i n ( 1 4 ) . I t h a s r e c e n t l y b e e n shown t h a t ligninases are also able to catalyze the i n i t i a l o x i d a t i o n o f a number o f e n v i r o n m e n t a l l y p e r s i s t e n t t o x i c chemicals (24). The p u r p o s e o f t h i s i n v e s t i g a t i o n was t o d e t e r m i n e w h e t h e r P^_ c h r y s o s p o r i u m w o u l d grow i n t h e p r e s e n c e o f TNT and RDX and d e g r a d e t h e s e compounds a s i t d i d o t h e r xenobiotics. B e c a u s e P^_ c h r y s o s p o r i u m h a s t h e a b i l i t y to degrade a wide v a r i e t y of e n v i r o n m e n t a l l y p e r s i s t e n t o r g a n o p o l l u t a n t s t o c a r b o n d i o x i d e , we h a v e s u g g e s t e d t h a t t h i s o r g a n i s m may be u s e f u l i n c e r t a i n h a z a r d o u s waste t r e a t m e n t systems ( 2 ) . I n t h i s s t u d y , t h e a b i l i t y o f P^ c h r y s o s p o r i u m t o d e g r a d e TNT c o n t a m i n a t e d w a t e r and s o i l was i n v e s t i g a t e d by a d j u s t i n g t h e TNT c o n c e n t r a t i o n e q u i v a l e n t t o some o f t h e h i g h e s t contamination l e v e l s found i n the environment. The r e s u l t s of t h i s study i n d i c a t e that b i o l o g i c a l t r e a t m e n t s y s t e m s t h a t u t i l i z e P^. c h r y s o s p o r i u m m i g h t be u s e d a s e f f e c t i v e , and e c o n o m i c a l m e t h o d s f o r r e m e d i a t i o n o f TNT and RDX c o n t a m i n a t e d w a t e r and s o i l . Materials

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Organism P. c h r y s o s p o r i u m BKM-F-1767 was o b t a i n e d f r o m t h e F o r e s t P r o d u c t s L a b o r a t o r y , U.S. D e p a r t m e n t o f A g r i c u l t u r e , M a d i s o n , WI. The o r g a n i s m was m a i n t a i n e d a t room t e m p e r a t u r e on 2% (w/v) m a l t a g a r s l a n t s . S u b c u l t u r e s w e r e r o u t i n e l y made e v e r y 30-60 d a y s .


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R a d i o l a b e l e d [ C]TNT ( r i n g l a b e l e d , s p e c i f i c a c t i v i t y 21.58 mCi/mmol) a n d [ C ] R D X ( 2 , 4 , 6 - C , s p e c i f i c a c t i v i t y 7.75 mCi/mmol) w e r e p u r c h a s e d f r o m Chemsyn S c i e n c e L a b o r a t o r i e s , L e n e x a , KS and t h e n o n r a d i o a c t i v e TNT was o b t a i n e d f r o m t h e U.S. Army T o x i c and H a z a r d o u s M a t e r i a l s A g e n c y , A b e r d e e n , MO and f r o m Chem S e r v i c e , I n c . (West C h e s t e r , P A ) . The p u r i t y o f t h e c h e m i c a l s was d e t e r m i n e d b y t h i n l a y e r c h r o m a t o g r a p h y (TLC) and h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) and was f o u n d t o b e g r e a t e r t h a n 98% i n a l l c a s e s . Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: August 2, 1991 | doi: 10.1021/bk-1991-0468.ch011

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Soil The s o i l u s e d i n t h e c u l t u r e e x p e r i m e n t s was c l a s s i f i e d a s a s i l t l o a m c o n s i s t i n g o f 19% s a n d , 54% s i l t and 27% clay. The o r g a n i c m a t t e r c o n t e n t i n t h e s o i l was 3.62%, t h e o r g a n i c c a r b o n c o n t e n t was 2.10% and t o t a l n i t r o g e n was 0.19%. The s o i l pH was 6.4 and t h e c a t i o n e x c h a n g e c a p a c i t y was 23.6 meq/100 g. T h e c o n c e n t r a t i o n s o f s e l e c t e d e x t r a c t a b l e c a t i o n s were a s follows: Na, 0.1; K, 0.99; Ca, 1 6 . 8 5 ; and Mg, 3.59 meq/100 gm. The c o n c e n t r a t i o n s o f s e l e c t e d d i v a l e n t cations extractable with diethylenetriamine-pentaacetic a c i d were a s f o l l o w s : Zn, 2.3; F e , 35.1; Cu, 1.2; Mn, 29.4; Cd, 1.4; Pb, 0.86; N i , 0.47; and C r , 0.16 mg/kg. L i q u i d Culture Conditions The l i q u i d c u l t u r e medium c o n s i s t e d o f 56 mM g l u c o s e ( w / v ) , 1.2 mM ammonium t a r t r a t e ( n i t r o g e n - l i m i t e d ) , t r a c e m e t a l s ( 1 4 ) , and t h i a m i n e (1 mg/ml) i n 20 mM 2 , 2 - d i m e t h y l s u c c i n a t e (sodium) b u f f e r , pH 4.2 ( 1 5 ) . The c u l t u r e medium was s t e r i l i z e d b y f i l t r a t i o n t h r o u g h a c e l l u l o s e a c e t a t e membrane f i l t e r (0.22 Mm). Culture b o t t l e s w e r e s t e r i l i z e d b y a u t o c l a v i n g a t 121°C and 15 p s i f o r 20 m i n . A l i q u o t s o f t h e c u l t u r e medium (9 m l ) w e r e d i s p e n s e d i n t o e a c h o f s e v e r a l 250 m l Wheaton b o t t l e s e q u i p p e d w i t h a gas e x c h a n g e m a n i f o l d w i t h a T e f l o n s e a l . C u l t u r e s were i n o c u l a t e d w i t h a s p o r e s u s p e n s i o n o f P^_ c h r y s o s p o r i u m BKM F-1767 (1 m l ; o p t i c a l d e n s i t y = 0.5 a b s o r b a n c e u n i t s a t 650 nm; - 2.5 χ 1 0 s p o r e s ) and grown a t 39°C. C o n t r o l i n c u b a t i o n s contained everything except 1 ml of L . chrysosporium spore suspension. The c u l t u r e s w e r e grown u n d e r a m b i e n t a t m o s p h e r e f o r 6 d a y s . On day 6, 1.25 n m o l e s o f [ C] TNT o r [ C] RDX, w e r e a d d e d t o s e p a r a t e c u l t u r e s and a t 3 day i n t e r v a l s t h e r e a f t e r t h e h e a d s p a c e s w e r e f l u s h e d w i t h o x y g e n (99.9%) and t h e l i b e r a t e d CO^ was p a s s e d t h r o u g h a v o l a t i l e o r g a n i c t r a p c o n s i s t i n g o f 10 m l s c i n t i l l a t i o n c o c k t a i l ( S a f e t y Solve, Research Products I n t e r n a t i o n a l Corp., Mt. 1

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P r o s p e c t , I L ) p r i o r t o a v i a l c o n t a i n i n g 10 m l o f C 0 trap. The C 0 t r a p was a m i x t u r e o f e t h a n o l a m i n e i n methanol and s c i n t i l l a t i o n c o c k t a i l (1:4:5, v / v / v ) . The v o l a t i l e o r g a n i c t r a p was u s e d t o e n s u r e t h a t t h e r a d i o l a b e l e d m a t e r i a l t r a p p e d i n t h e C 0 t r a p was n o t contaminated with v o l a t i l e organics as a r e s u l t o f a i r stripping during flushing. The amount o f r a d i o a c t i v i t y i n e a c h t r a p was d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n spectrometry. 2

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Mass Balance Analyses A t t h e e n d o f t h e i n c u b a t i o n p e r i o d , mass b a l a n c e a n a l y s e s w e r e p e r f o r m e d o n l i q u i d c u l t u r e s o f P. c h r y s o s p o r i u m t h a t c o n t a i n e d [ C ] TNT o r [ C ] RDX. The c o n t e n t s o f e a c h 250 m l Wheaton b o t t l e w e r e e x t r a c t e d t h r e e t i m e s w i t h 30 m l o f m e t h y l e n e c h l o r i d e and w a t e r ( 1 : 1 , v / v ) . The m e t h y l e n e c h l o r i d e e x t r a c t s were t h e n p o o l e d and c o n c e n t r a t e d by e v a p o r a t i o n under a g e n t l e stream o f n i t r o g e n . F o l l o w i n g these e x t r a c t i o n s , p a r t i c u l a t e m a t e r i a l , i . e . , f u n g a l mat, was s e p a r a t e d f r o m t h e a q u e o u s f r a c t i o n b y f i l t r a t i o n t h r o u g h g l a s s w o o l . The amount o f [ C ] w h i c h was b o u n d t o t h e f u n g a l mat a n d n o t e x t r a c t a b l e b y o r g a n i c s o l v e n t was d e t e r m i n e d b y c o m b u s t i o n i n a H a r v e y b i o l o g i c a l o x i d i z e r (R.J. Harvey Instrument Corp., H i l l s d a l e , NJ). Measurement o f r a d i o a c t i v i t y i n t h e l i b e r a t e d C 0 was d e t e r m i n e d b y t r a p p i n g C 0 a s d e s c r i b e d a b o v e . The amount o f C 0 i n t h e t r a p was determined by l i q u i d s c i n t i l l a t i o n spectrometry. The a q u e o u s f r a c t i o n was a l s o a s s a y e d f o r r a d i o a c t i v i t y b y l i q u i d s c i n t i l l a t i o n s p e c t r o m e t r y (Beckman LS 5 8 0 1 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 , Beckman I n s t r u m e n t s , I r v i n e , CA). U

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A n a l y s e s f o r [ C ] TNT a n d [ C ] RDX m e t a b o l i t e s w e r e p e r f o r m e d u s i n g a n HPLC s y s t e m e q u i p p e d w i t h a S p e c t r a P h y s i c s m o d e l SP 8810 pump ( S p e c t r a P h y s i c s , S a n J o s e , C A ) , a Rheodyne i n j e c t o r (Rheodyne I n c . , C o t a t i , C A ) , a r e v e r s e p h a s e c o l u m n (5 μ, 4.6 b y 250 mm; R s i l C-18; Beckman I n s t r u m e n t I n c . , S a n Ramon, C A ) , a n d a S p e c t r a P h y s i c s m o d e l SP 8450 ( S p e c t r a P h y s i c s , S a n J o s e , CA) v a r i a b l e wavelength absorbance d e t e c t o r . Isocratic e l u t i o n was p e r f o r m e d i n a s o l v e n t s y s t e m composed o f m e t h a n o l : w a t e r ( 5 0 : 5 0 , v / v ) . The f l o w r a t e was 1.0 ml/min. I n a t y p i c a l i n j e c t i o n , t h e methylene c h l o r i d e e x t r a c t ( o b t a i n e d d u r i n g mass b a l a n c e a n a l y s i s ) was evaporated under g e n t l e stream o f n i t r o g e n and t h e r e s i d u e was r e d i s s o l v e d i n 0.5 - 2.0 m l o f m e t h y l e n e chloride. T w e n t y μΐ a l i q u o t s o f t h i s m a t e r i a l w e r e u s e d f o r i n j e c t i o n i n t o t h e HPLC. The r e t e n t i o n t i m e f o r TNT a n d RDX was d e t e r m i n e d b y m o n i t o r i n g e l u t i o n a t


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254 nm . F r a c t i o n s ( 1 m l ) w e r e c o l l e c t e d i n scintillation vials. S a f e t y S o l v e (9 m l ) was t h e n a d d e d t o e a c h f r a c t i o n a n d r a d i o a c t i v i t y 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 s p e c t r o m e t r y a s d e s c r i b e d previously. S o i l Cultures 14

The a b i l i t y o f P^. c h r y s o s p o r i u m t o d e g r a d e [ C ] TNT and [ C ] RDX i n s o i l was a l s o d e t e r m i n e d . An a g r i c u l t u r a l s i l t l o a m s o i l was u s e d i n t h i s s t u d y . S o i l s a m p l e s (10 gm) w e r e p l a c e d i n 250 m l Wheaton b o t t l e s a n d t h e n c o n t a m i n a t e d s e p a r a t e l y , w i t h 57.9 n m o l e s a n d 1.25 n m o l e s o f [ C ] TNT a n d [ C ] RDX, r e s p e c t i v e l y , d i s s o l v e d i n 100-160 μΐ o f a c e t o n e . The a c e t o n e was a l l o w e d t o e v a p o r a t e a n d t h e s o i l was t h e n m i x e d w i t h 6.7 gm o f g r o u n d c o r n c o b s ( M t . P o l a s k i , I n c . , M t . P o l a s k i , I L ) t h a t h a d b e e n p r e v i o u s l y (10 days) i n o c u l a t e d w i t h L . chrysosporium. P r e i n o c u l a t e d c o r n c o b s w e r e made b y a u t o c l a v i n g t h e g r o u n d c o r n c o b s (121°C a n d 15 p s i ) f o r 30 m i n a n d a s e p t i c a l l y i n o c u l a t i n g w i t h a s p o r e s u s p e n s i o n o f P^_ c h r y s o s p o r i u m a s d e s c r i b e d a b o v e . The m o i s t u r e c o n t e n t o f t h e s o i l c u l t u r e s was b r o u g h t up t o t h e o p t i m a l m o i s t u r e c o n t e n t r e q u i r e d f o r t h e o r g a n i s m , 4 0 % w/w ( 7 ) , b y a d d i n g u n s t e r i l i z e d water. Unlike the liquid cultures, the s o i l was n o t b u f f e r e d o r s u p p l e m e n t e d w i t h t r a c e m e t a l s or o t h e r n u t r i e n t s o r g l u c o s e as a carbon source. Most i m p o r t a n t l y , s t e r i l e c o n d i t i o n s were n o t o b s e r v e d t o simulate environmental conditions. C u l t u r e s were i n c u b a t e d a t 39°C f o r 30 d a y s o r l o n g e r . C o n t r o l c u l t u r e s w e r e i d e n t i c a l e x c e p t t h e y c o n t a i n e d 6.7 gm o f u n i n o c u l a t e d c o r n cobs. During t h e f i r s t 3 days o f i n c u b a t i o n , c u l t u r e s w e r e grown u n d e r a m b i e n t a i r . Every t h r e e days t h e r e a f t e r , head spaces o f t h e c u l t u r e b o t t l e s w e r e f l u s h e d w i t h o x y g e n f o r 25 m i n , a n d l i b e r a t e d C 0 was t r a p p e d a n d t h e amount o f r a d i o a c t i v i t y i n t h e COp a n d i n t h e v o l a t i l e o r g a n i c t r a p was d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n s p e c t r o m e t r y a s d e s c r i b e d above.


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Mass Balance Analyses Mass b a l a n c e a n a l y s i s w e r e p e r f o r m e d o n s o i l c u l t u r e s u s i n g e x t r a c t i o n procedures p r e v i o u s l y d e s c r i b e d by J e n k i n s and Walsh (10). A t t h e end o f t h e i n c u b a t i o n p e r i o d , t h e c o n t e n t s o f e a c h 250 m l Wheaton b o t t l e was e x t r a c t e d t h r e e t i m e s w i t h 30 m l a c e t o n i t r i l e . The s o i l , c o r n cob and a c e t o n i t r i l e m i x t u r e s were d i s p e r s e d by u s i n g a v o r t e x m i x e r f o r 10 m i n f o l l o w e d b y s o n i c a t i o n i n a Branson u l t r a s o n i c bath (Branson I n c . , S h e l t o n , CT) f o r 18 h r . The a c e t o n i t r i l e e x t r a c t s w e r e p o o l e d and c o n c e n t r a t e d by e v a p o r a t i o n under a g e n t l e s t r e a m o f n i t r o g e n . The c o n c e n t r a t e d e x t r a c t was t h e n



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c e n t r i f u g e d f o r 5 m i n a t 1500 rpm a n d t h e c l e a r s u p e r n a t a n t was removed u s i n g a v o l u m e t r i c p i p e t a n d mixed w i t h an e q u a l volume o f w a t e r i n a g l a s s s c i n t i l l a t i o n v i a l ( 1 0 ) . The c o n t e n t s o f t h e v i a l w e r e t h o r o u g h l y m i x e d a n d a l l o w e d t o s t a n d f o r 15 m i n a n d f i l t e r e d t h r o u g h a Gelman S c i e n c e s 0.45 μπι ARCO LS-25 d i s p o s a b l e f i l t e r a s s e m b l y . T w e n t y μΐ a l i q u o t s o f t h i s m a t e r i a l w e r e u s e d f o r HPLC a n a l y s e s a s d e s c r i b e d a b o v e . F r a c t i o n s (1 m l ) w e r e c o l l e c t e d i n s c i n t i l l a t i o n v i a l s a n d t h e r a d i o a c t i v i t y was determined as described p r e v i o u s l y . Radioactivity w h i c h was r e t a i n e d i n t h e s o i l / c o r n c o b / f u n g a l m a t r i x and n o t r e c o v e r e d by o r g a n i c s o l v e n t e x t r a c t i o n was d e t e r m i n e d by c o m b u s t i o n t o C0 i n a Harvey b i o l o g i c a l o x i d i z e r f o l l o w e d by measurement o f C 0 b y l i q u i d s c i n t i l l a t i o n spectrometry. 2

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Degradation o f TNT a t L e v e l s Found i n the Environment I n a s e p a r a t e e x p e r i m e n t , P_j_ c h r y s o s p o r i u m was t e s t e d f o r i t s a b i l i t y t o m i n e r a l i z e t h e [ C ] TNT i n b o t h l i q u i d and s o i l c u l t u r e s a t l e v e l s e n c o u n t e r e d i n t h e TNT c o n t a m i n a t e d s i t e s , i . e . , 100 m g / l o f w a t e r a n d 10,000 mg/kg i n s o i l ( p e r s o n a l c o m m u n i c a t i o n , U.S. N a v a l C i v i l E n g i n e e r i n g L a b o r a t o r y , P o r t Hueneme, C A ) . C u l t u r e c o n d i t i o n s were as d e s c r i b e d above e x c e p t f o r t h e c o n c e n t r a t i o n o f TNT a n d t h e p e r i o d o f i n c u b a t i o n . U n l e s s o t h e r w i s e s t a t e d , C0 e v o l u t i o n s t u d i e s were performed i n quadruplicate. Rates of m i n e r a l i z a t i o n w e r e o b t a i n e d a n d mass b a l a n c e a n a l y s e s w e r e p e r f o r m e d a s d e s c r i b e d a b o v e a f t e r 30, 60, a n d 90 d a y s f o r t h e l i q u i d a n d t h e s o i l c u l t u r e s . One c u l t u r e b o t t l e was e x t r a c t e d a t t h e end o f each i n c u b a t i o n p e r i o d e x c e p t f o r d a y 90 when two c u l t u r e s w e r e u s e d . 14

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Results Phanerochaete chrysosporium m i n e r a l i z e d 51% o f t h e [ C ] TNT o v e r a p e r i o d o f 30 d a y s i n c u b a t i o n ( F i g u r e 1). A mass b a l a n c e a n a l y s i s o f t h e c u l t u r e s r e v e a l e d t h a t 50.8 ± 3.2% o f t h e t o t a l r a d i o a c t i v i t y was e v o l v e d a s COp, 2 1 . 9 % was p r e s e n t a s w a t e r s o l u b l e m e t a b o l i t e s , 10.7% was f o u n d i n t h e m e t h y l e n e c h l o r i d e f r a c t i o n , a n d 9.3% was a s s o c i a t e d w i t h t h e m y c e l i a l fraction. A t o t a l mass r e c o v e r y o f 9 2 . 7 % was a c h i e v e d . HPLC a n a l y s i s ( F i g . 2) o f t h e m e t h y l e n e c h l o r i d e e x t r a c t r e v e a l e d t h a t o n l y a b o u t 2.8% o f t h e [ C ] TNT i n i t i a l l y p r e s e n t be i d e n t i f i e d a s u n d e g r a d e d TNT. The r e m a i n i n g 9% r e p r e s e n t e d u n i d e n t i f i e d m e t a b o l i t e s f o r m e d d u r i n g t h e 30 d a y i n c u b a t i o n p e r i o d . Almost a l l of t h e u n i d e n t i f i e d m e t a b o l i t e s formed i n t h e i n c u b a t i o n p e r i o d w e r e more p o l a r t h a n TNT. In control c u l t u r e s i n c u b a t e d u n d e r t h e same c u l t u r e c o n d i t i o n s b u t n o t i n o c u l a t e d w i t h P^ c h r y s o s p o r i u m . 9 7 - 9 8 % o f t h e 14

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F i g u r e 1. M i n e r a l i z a t i o n o f [ C ] TNT i n n u t r i e n t n i t r o g e n l i m i t e d c u l t u r e s o f P^. c h r y s o s p o r i u m . E a c h c u l t u r e c o n t a i n e d 1.25 n m o l e s o f [ C ] TNT. D a t a p o i n t s r e p r e s e n t t h e mean ± s t a n d a r d d e v i a t i o n (N = 4 ) . 14


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80000 64000 Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date: August 2, 1991 | doi: 10.1021/bk-1991-0468.ch011

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Figure 2. HPLC e l u t i o n p r o f i l e of a methylene chloride extract of a nutrient nitrogen l i m i t e d culture of Ρ. chrysosporium that was incubated with t"C] TNT (1.25 nmoles) f o r 30 days. Authentic [ C ] TNT (top) eluted from the column about 12-16 minutes. DPM = disintegrations per minute. (Reprinted with permission from r e f . 34. Copyright 1990 American Society f o r Microbiology.) U


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r a d i o a c t i v i t y a d d e d was f o u n d i n t h e m e t h y l e n e c h l o r i d e f r a c t i o n and was u n m e t a b o l i z e d [ C] TNT. I n [ C] RDX c o n t a m i n a t e d l i q u i d c u l t u r e s , P. c h r y s o s p o r i u m m i n e r a l i z e d 67% o f t h e i n i t i a l [ C] RDX o v e r a p e r i o d o f 30 d a y s i n c u b a t i o n ( F i g . 3 ) . Mass b a l a n c e was done a t t h e end o f t h e 30 day i n c u b a t i o n p e r i o d and a t o t a l o f 66.6 + 4.1% o f t h e i n i t i a l r a d i o a c t i v i t y a d d e d t o t h e c u l t u r e s was l i b e r a t e d a s C 0 , 20.2% was r e c o v e r e d a s w a t e r s o l u b l e m e t a b o l i t e s , 4.8% was p r e s e n t i n t h e m e t h y l e n e c h l o r i d e f r a c t i o n and 2.1% was associated w i t h the m y c e l i a l fragments. A t o t a l of 93.7% r a d i o a c t i v i t y added t o t h e c u l t u r e s were r e c o v e r e d . HPLC a n a l y s i s o f t h e m e t h y l e n e c h l o r i d e f r a c t i o n d e m o n s t r a t e d t h a t o n l y a b o u t 4% o f t h e [ C] RDX a d d e d t o t h e c u l t u r e s was u n d e g r a d e d . No m e t a b o l i t e s w e r e found i n the methylene c h l o r i d e f r a c t i o n . In control c u l t u r e s , w h e r e t h e P^ c h r y s o s p o r i u m was n o t a d d e d b u t i d e n t i c a l c u l t u r e p a r a m e t e r s were i n c l u d e d , 97-98% o f t h e r a d i o a c t i v i t y i n i t i a l l y a d d e d was r e c o v e r e d i n t h e m e t h y l e n e c h l o r i d e f r a c t i o n and was i d e n t i f i e d a s u n d e g r a d e d [ C] RDX. Biodégradation was a l s o e x a m i n e d i n a s y s t e m i n w h i c h [ C] TNT and [ C] RDX was s e p a r a t e l y a d s o r b e d o n t o s o i l t h a t was amended w i t h c o r n c o b s p r e v i o u s l y i n o c u l a t e d w i t h P^_ c h r y s o s p o r i u m (10 d a y s e a r l i e r ) . In t h i s s o i l - c o r n cob m i x t u r e , 6.3 ± 0.6% o f t h e [ C] TNT i n i t i a l l y p r e s e n t was d e g r a d e d t o C 0 o v e r a 30 day incubation period (Fig. 4). Mass b a l a n c e a n a l y s i s o f c u l t u r e s o f P_j_ c h r y s o s p o r i u m i n c u b a t e d w i t h [ C] TNT i n a s o i l - c o r n cob m a t r i x f o r 30 d a y s r e v e a l e d t h a t 6.3 + 0.6% o f t h e r e c o v e r e d r a d i o a c t i v i t y was l i b e r a t e d as C0 , 63.6% was f o u n d i n t h e a c e t o n i t r i l e f r a c t i o n , and 25.2% was n o t e x t r a c t a b l e by t h e o r g a n i c s o l v e n t s and was f o u n d i n t h e s o i l - c o r n cob m i x t u r e . The m a t e r i a l c o u l d n o t be i d e n t i f i e d a s i t c o u l d n o t be e x t r a c t e d from the m a t r i x . A t o t a l mass r e c o v e r y o f 95.1% was o b s e r v e d . HPLC a n a l y s i s o f t h e r a d i o l a b e l e d m a t e r i a l i n the a c e t o n i t r i l e e x t r a c t r e v e a l e d t h a t only about 2.2% o f t h e [ C] TNT i n i t i a l l y a d d e d t o t h e c u l t u r e s was i d e n t i f i e d as u n d e g r a d e d TNT. Mass b a l a n c e a n a l y s i s o f c u l t u r e s o f P. c h r y s o s p o r i u m i n c u b a t e d w i t h [ C] RDX i n a s o i l / c o r n cob m a t r i x f o r 30 d a y s r e v e a l e d t h a t 76.0 + 3.9% o f t h e r e c o v e r e d r a d i o a c t i v i t y was e v o l v e d as C 0 , 4.5% was p r e s e n t i n t h e a c e t o n i t r i l e e x t r a c t and 9.7% was u n e x t r a c t a b l e and p r e s e n t i n t h e s o i l / c o r n cob m a t r i x (Fig. 3). A t o t a l mass r e c o v e r y o f 90.2% was a c h i e v e d . A n a l y s i s of the r a d i o l a b e l e d m a t e r i a l i n the a c e t o n i t r i l e e x t r a c t r e v e a l e d t h a t o n l y 4% o f t h e [ C] RDX i n i t i a l l y p r e s e n t was i d e n t i f i e d a s u n d e g r a d e d RDX. In c o n t r o l u n i n o c u l a t e d c u l t u r e s , incubated under t h e same n o n - s t e r i l e c o n d i t i o n s , g r e a t e r t h a n 99% o f t h e r a d i o a c t i v i t y a d d e d was f o u n d i n t h e a c e t o n i t r i l e f r a c t i o n and was u n m e t a b o l i z e d [ C] RDX. The a s s a y 14

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F i g u r e 3. M i n e r a l i z a t i o n o f [ C ] RDX i n l i q u i d and i n s o i l / c o r n c o b c u l t u r e s o f chrysosporium. E a c h c u l t u r e c o n t a i n e d 1.25 n m o l e s o f [ C] RDX. D a t a p o i n t s r e p r e s e n t t h e mean + s t a n d a r d d e v i a t i o n (N = 4 ) .

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Figure 4. M i n e r a l i z a t i o n of [ C] TNT i n s o i l / c o r n cob c u l t u r e s of P. chrysosporium. Each c u l t u r e contained 57.9 nmoles of [ C] TNT i n 10 g of s o i l and 6.7 g corn cobs. Data points represent the mean - standard d e v i a t i o n (N=4). (Reprinted with permission from r e f . 34. Copyright 1990 American Society f o r Microbiology.) 14


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for r a d i o a c t i v i t y i n the v o l a t i l e organic t r a p revealed t h a t l e s s t h a n 0.5% o f t h e [ C] RDX was v o l a t i l i z e d o r a i r s t r i p p e d during the f l u s h i n g of c u l t u r e s w i t h oxygen. A t t h e end o f 30 and 60, o r 90 d a y s , l i q u i d and s o i l c u l t u r e s c o n t a m i n a t e d w i t h 100 mg/l and 10,000 mg/kg TNT, r e s p e c t i v e l y , w e r e e x t r a c t e d and mass b a l a n c e a n a l y s e s p e r f o r m e d as d e s c r i b e d above. The r e s u l t s o f mass b a l a n c e a n a l y s i s o f 100 mg/l TNT c o n t a m i n a t e d l i q u i d c u l t u r e s showed t h a t 19.0 ± 3.0% o f t h e r e c o v e r e d r a d i o a c t i v i t y was e v o l v e d a s C 0 , 19.5% was f o u n d i n t h e m e t h y l e n e c h l o r i d e e x t r a c t , 51.6% was p r e s e n t a s w a t e r s o l u b l e compounds, and 5.1% was bound t o t h e f u n g a l mat a f t e r a p e r i o d o f 60 d a y s i n c u b a t i o n . A t o t a l mass r e c o v e r y o f 95.2% was a c h i e v e d . When t h e m e t h y l e n e c h l o r i d e f r a c t i o n was a n a l y z e d by HPLC ( F i g . 5 ) , t h e amount o f u n m e t a b o l i z e d [ C] TNT r e m a i n i n g i n l i q u i d c u l t u r e s was 22.1% and 14.9%, r e s p e c t i v e l y , o v e r a p e r i o d o f 30 and 60 d a y s o f i n c u b a t i o n . In control c u l t u r e s , w h i c h w e r e i n c u b a t e d u n d e r t h e same c o n d i t i o n s b u t n o t i n o c u l a t e d w i t h P_2_ c h r y s o s p o r i u m , g r e a t e r t h a n 99% o f t h e r a d i o a c t i v i t y was f o u n d i n t h e m e t h y l e n e c h l o r i d e e x t r a c t and i d e n t i f i e d a s TNT by HPLC. The r e s u l t s o f mass b a l a n c e a n a l y s i s o f s o i l c u l t u r e s c o n t a m i n a t e d w i t h 10,000 mg/kg TNT showed t h a t 18.4 + 2.9% had b e e n e v o l v e d a s C 0 , 62.6% was f o u n d i n t h e a c e t o n i t r i l e e x t r a c t , and 11.5% was bound t o s o i l / c o r n c o b / f u n g a l m a t r i x a f t e r 90 d a y s . The t o t a l mass r e c o v e r y was 92.5% a f t e r a p e r i o d o f 90 d a y s incubation. When t h e a c e t o n i t r i l e e x t r a c t s o f day 30, 60, and 90 c u l t u r e s w e r e a n a l y z e d by HPLC, t h e y showed t h a t t h e amount o f r e s i d u a l C-TNT t h a t was not d e g r a d e d t o C 0 o r i n t e r m e d i a t e s was a p p r o x i m a t e l y 50, 30, and 15%, r e s p e c t i v e l y ( F i g . 6 ) . A m a j o r m e t a b o l i t e e l u t e d f r o m t h e HPLC c o l u m n a t a b o u t 3 m i n u t e s i n a l l c a s e s . The amount o f t h i s m e t a b o l i t e i n c r e a s e d and then decreased w i t h time ( F i g . 6)· A l s o , t h e HPLC e l u t i o n p r o f i l e o f b o t h l i q u i d and s o i l c u l t u r e s r e v e a l e d t h a t most o f t h e m e t a b o l i t e s f o r m e d d u r i n g t h e i n c u b a t i o n p e r i o d s w e r e more p o l a r t h a n TNT i t s e l f . In c o n t r o l c u l t u r e s , i n c u b a t e d u n d e r t h e same n o n - s t e r i l e c o n d i t i o n s b u t n o t i n o c u l a t e d w i t h P^_ c h r y s o s p o r i u m . g r e a t e r t h a n 99% o f t h e r a d i o a c t i v i t y a d d e d was f o u n d i n t h e a c e t o n i t r i l e f r a c t i o n and was u n m e t a b o l i z e d [ C] TNT. The a s s a y f o r r a d i o a c t i v i t y i n t h e v o l a t i l e o r g a n i c t r a p r e v e a l e d t h a t l e s s t h a n 0.5% o f t h e CTNT was v o l a t i l i z e d o r a i r s t r i p p e d d u r i n g t h e f l u s h i n g of c u l t u r e s w i t h oxygen. I n some c o n t r o l e x p e r i m e n t s i n w h i c h C0 was t r a p p e d i n B a ( O H ) r a t h e r t h a n t h e e t h a n o l a m i n e b a s e d s c i n t i l l a t i o n c o c k t a i l , i t was shown t h a t t h e C 0 was q u a n t i t a t i v e l y p r e c i p i t a t e d a s B a C 0 ( d a t a not shown). 14

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F i g u r e 5. HPLC e l u t i o n p r o f i l e s o f m e t h y l e n e chloride extracts of nutrient nitrogen limited l i q u i d c u l t u r e s o f P_s_ c h r y s o s p o r i u m t h a t w e r e i n c u b a t e d w i t h [ C ] TNT (100 m g / l ) f o r 30 a n d 60 days. TNT e l u t e d a t a p p r o x i m a t e l y 15 m i n u t e s . U

F i g u r e 6. HPLC e l u t i o n p r o f i l e s o f a c e t o n i t r i l e e x t r a c t s o f s o i l - c o r n cob c u l t u r e s incubated with P. c h r y s o s p o r i u m a n d 10,000 mg/kg TNT f o r 3 0 , 60 and 90 d a y s . TNT e l u t e d a t a p p r o x i m a t e l y 15 minutes.


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Discussion P r e v i o u s s t u d i e s o f TNT biodégradation showed t h a t t h i s compound i s r e l a t i v e l y r e s i s t a n t t o biodégradation b y microorganisms i n t h e environment (3,13,17). However, some m i c r o o r g a n i s m s , s u c h a s b a c t e r i a , b i o t r a n s f o r m TNT i n t o h y d r o x y l amino compounds b y s t e p w i s e r e d u c t i o n o f n i t r o g r o u p s a n d a z o x y compounds b y c o n j u g a t i o n o f amino compounds, b u t f u r t h e r biodégradation o f t h e s e b i o t r a n s f o r m e d p r o d u c t s h a s n o t been o b s e r v e d (13,17,33). A c c o r d i n g t o Carpenter e t a l . (1978), s o l u b i l i t y p r o p e r t i e s and i n f r a r e d s p e c t r a c o n f i r m t h a t t h e s e TNT b i o t r a n s f o r m a t i o n p r o d u c t s r e a c t w i t h m i c r o b i a l l i p i d s , f a t t y a c i d s and p r o t e i n c o n s t i t u e n t s t o form polyamide type macromolecular s t r u c t u r e s . A l s o , C a r p e n t e r e t a l . (1978), f u r t h e r c o n f i r m e d t h i s hypothesis by c o r r e l a t i n g t h e i n f r a r e d spectrum o f t h e l i p i d component w i t h t h a t o f a m o d e l compound s y n t h e s i z e d f r o m TNT t r a n s f o r m a t i o n p r o d u c t s a n d l i p i d precursors. The r e c a l c i t r a n c e n a t u r e o f t h e s e p o l y m e r s to f u r t h e r m i c r o b i a l degradation i s p a r a l l e l e d with the d o c u m e n t e d r e s i s t a n c e o f some a l k y l a n d a r y l p o l y a m i d e s c o n t a i n i n g s i m i l a r l i n k a g e s ( 2 3 ) . A l s o , most o f t h e p r e v i o u s i n v e s t i g a t i o n s r e g a r d i n g t h e biodégradation o f TNT d i d n o t p r o v i d e a n y e v i d e n c e t o i n d i c a t e t h a t t h e TNT a r o m a t i c n u c l e u s was c l e a v e d a n d u l t i m a t e l y degraded (3,13,17,18,21,32,33). I n s t e a d i n most s t u d i e s i t h a s b e e n shown t h a t t h e TNT was d e g r a d e d b y a r e d u c t i o n mechanism ( 3 , 1 3 , 2 2 , 2 7 ) . The r e d u c t i o n mechanism appeared t o i n v o l v e a s t e p w i s e r e d u c t i o n o f t h e n i t r o g r o u p s , t h r o u g h t h e n i t r o s o a n d h y d r o x y amino t o t h e amino g r o u p ( 3 , 1 3 , 2 2 , 2 7 , 3 3 ) . However, i n t h e s e r e d u c t i v e d e g r a d a t i o n s , N0 groups t h a t a r e reduced t o h y d r o x y l amine f o r m d i m e r s s u c h a s 2 , 2 , 6 , 6 ' tetranitro-4,4 -azoxytoluene, 4,4 ,6,6 -tetranitro2,2·-azoxytoluene, a n d 2 , 4 , 6 , 6 , - t e t r a n i t r o - 2 , 4 a z o x y t o l u e n e (13,17,33). Thus, t h e r e d u c t i o n mechanism represent only a m o d i f i c a t i o n o f t h e molecule and n o t decomposition o f t h e aromatic nucleus (3,13,17,33). F u r t h e r m o r e , t h e TNT r e d u c t i o n p r o d u c t s may a l s o p o s e e n v i r o n m e n t a l h a z a r d s s i n c e t h e y have been r e p o r t e d t o be t o x i c a n d m u t a g e n i c ( 4 , 1 2 ) . However, i n c o n t r a s t t o TNT biodégradation, M c C o r m i c k e t a l . (19) o b s e r v e d t h e biodégradation o f RDX u n d e r a n a e r o b i c c o n d i t i o n s y i e l d i n g a number o f m e t a b o l i t e s , e.g., h e x a h y d r o - l - n i t r o s o - 3 , 5 - d i n i t r o 1,3,5-triazine, l-hydroxylamino-3,5-dionitroso-l,3,5t r i a z ine, N-hydroxymethylmethy1-enedianitramine, f o r m a l d e h y d e , m e t h a n o l , e t c . T h e biodégradation o f RDX o c c u r r e d v i a s u c c e s s i v e r e d u c t i o n o f t h e N0 groups t o a p o i n t where d e s t a b i l i z a t i o n a n d f r a g m e n t a t i o n o f t h e r i n g occurred. A c c o r d i n g t o McCormick e t a l . , t h e non c y c l i c degradation products a r i s e v i a subsequent r e d u c t i o n and rearrangement o f t h e fragments. Although 2

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t h e r i n g was c l e a v e d and n o n - c y c l i c d e g r a d a t i o n p r o d u c t s were formed, a c c o r d i n g t o McCormick e t a l . , s e v e r a l of t h e p r o d u c t s were mutagenic o r c a r c i n o g e n i c or both. However, t h i s s t u d y shows t h a t t h e wood r o t t i n g ( w h i t e r o t ) f u n g u s P_s_ c h r y s o s p o r i u m BKM F-1767 i s a b l e t o d e g r a d e [ C] TNT and [ C] RDX i n a r e a s o n a b l y s h o r t p e r i o d of time. D e g r a d a t i o n was d e m o n s t r a t e d by m i n e r a l i z a t i o n o f [ C] TNT and [ C] RDX, metabolite f o r m a t i o n i n t h e c a s e o f [ C] TNT, and mass b a l a n c e analyses. Biodégradation o f [ C] TNT and [ C] RDX was a l s o shown t o o c c u r i n a s o i l and c o r n c o b m a t r i x i n o c u l a t e d w i t h P±. c h r y s o s p o r i u m . Compared w i t h l i q u i d c u l t u r e s s u b s t a n t i a l l y l e s s [ C] TNT was c o n v e r t e d t o C 0 i n s o i l c u l t u r e s . However, i t i s w o r t h n o t i n g t h a t i n l i q u i d c u l t u r e s , m i n e r a l i z a t i o n o f [ C] TNT v i r t u a l l y c e a s e d a f t e r 15 d a y s w h e r e a s i n s o i l c u l t u r e s , i t c o n t i n u e d t h r o u g h o u t t h e 30 day i n c u b a t i o n p e r i o d a t a n e a r l y continuous a l b e i t r e l a t i v e l y slow rate. Most i m p o r t a n t l y , even i n s o i l - c o r n cob c u l t u r e s , a l t h o u g h t h e m i n e r a l i z a t i o n was s l o w , t h e amount o f TNT d e g r a d e d o r c o n v e r t e d t o o t h e r m e t a b o l i t e s was v e r y h i g h . A l s o , t h i s e x p e r i m e n t suggests t h a t the extent of m i n e r a l i z a t i o n i n a s o i l c o r n c o b m a t r i x c o u l d be e x t e n d e d by i n c r e a s i n g t h e incubation period. In c o n t r a s t t o o t h e r m i c r o b i a l d e g r a d a t i o n s y s t e m s , i n w h i c h d e g r a d a t i o n o c c u r s by a s t e p w i s e r e d u c t i o n o f N0 g r o u p s , chrysosporium appears t o d e g r a d e TNT by an o x i d a t i v e m e c h a n i s m . The f a m i l y o f enzymes i n v o l v e d i n t h e s e o x i d a t i o n s c o u l d be l i g n i n peroxidases, which are produced under n u t r i e n t l i m i t i n g ( c a r b o n , n i t r o g e n and s u l f u r ) c o n d i t i o n s . C o n t r a r y t o o t h e r r e p o r t s (17,27,33) c h r y s o s p o r i u m was a b l e t o c l e a v e t h e r i n g as evidenced by t h e l i b e r a t i o n o f C 0 f r o m t h e C - r i n g l a b e l e d TNT and RDX. The o x i d a t i o n o f t h e r i n g was e x t e n s i v e i n t h a t 5 1 % and 67% o f [ C] TNT and [ C] RDX, r e s p e c t i v e l y , was m i n e r a l i z e d i n l i q u i d c u l t u r e s w i t h i n 30 d a y s o f i n c u b a t i o n . M i n e r a l i z a t i o n o f [ C] RDX i n s o i l was h i g h e r t h a n [ C] TNT. Furthermore, as a r e s u l t o f t h e d e g r a d a t i o n o f TNT and RDX by an o x i d a t i v e m e c h a n i s m , t h e p o s s i b i l i t y of conjugation of metabolites f o r the f o r m a t i o n o f aζoxycompounds, i . e , d i m e r s a s o b s e r v e d i n b a c t e r i a l d e g r a d a t i o n , s h o u l d be r e c o n s i d e r e d i n P. c h r y s o s p o r i u m m e d i a t e d s y s t e m . The p o s s i b i l i t y o f f o r m a t i o n o f t o x i c and m u t a g e n i c m e t a b o l i t e s by s y n t h e s i s o f r e d u c e d n i t r o compounds and c o n j u g a t e d a z o x y compounds d u r i n g t h e d e g r a d a t i o n o f TNT by P. c h r y s o s p o r i u m may n e e d t o be r e e v a l u a t e d . A major o b j e c t i v e of our r e s e a r c h i s the d e v e l o p m e n t o f b i o r e m e d i a t i o n s y s t e m s u s i n g P. c h r y s o s p o r i u m t o t r e a t w a t e r , s o i l s , s e d i m e n t s and o t h e r m a t e r i a l s t h a t a r e c o n t a m i n a t e d w i t h t o x i c and 14

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p e r s i s t e n t o r g a n o p o l l u t a n t s . I n t h i s s t u d y we h a v e shown t h a t Ρ±. c h r y s o s p o r i u m i s a b l e t o d e g r a d e [ C ] TNT o r [ C ] RDX e x t e n s i v e l y . A particular interest was t h e f a c t t h a t s u b s t a n t i a l amounts o f t h e c h e m i c a l s w e r e c o n v e r t e d t o C 0 b y t h i s f u n g u s . The c o n c e n t r a t i o n o f [ C ] TNT u s e d i n t h e l i q u i d c u l t u r e e x p e r i m e n t s w e r e o f t h e same m a g n i t u d e a s i s f o u n d i n w a t e r s c o n t a m i n a t e d b y TNT i n t h e e n v i r o n m e n t (20,22). The c o n c e n t r a t i o n o f TNT i n e f f l u e n t s f r o m m a n u f a c t u r i n g p r o c e s s e s a r e , o n a v e r a g e , a b o u t 20 mg/L (20) a n d t h e c o n c e n t r a t i o n o f TNT i n c o n t a m i n a t e d s o i l may b e a s h i g h a s 10,000 mg/kg. H i g h c o n c e n t r a t i o n s o f TNT w e r e n o t l e t h a l t o t h e f u n g u s i n t h e s e e x p e r i m e n t s and c o n s i d e r a b l e q u a n t i t i e s o f TNT w e r e d e g r a d e d . We h a v e s u g g e s t e d t h a t P_j_ c h r y s o s p o r i u m may be u s e f u l i n t h e biodégradation o f h a z a r d o u s w a s t e s i n w a s t e t r e a t m e n t s y s t e m s ( 2 ) . When compared t o c o s t l y a n d tedious p h y s i c a l decontamination processes, t h i s study s u g g e s t s t h a t a v e r s a t i l e o r g a n i s m s u c h a s P. c h r y s o s p o r i u m may p r o v i d e a more e c o n o m i c a l , b i o l o g i c a l t r e a t m e n t system t h a t c o u l d be a p p l i e d t o i n s i t u d e c o n t a m i n a t i o n p r o c e s s e s when t h e c o n d i t i o n s a r e s u i t a b l e f o r t h e growth o f t h e fungus. 14

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Acknowledgments T h i s r e s e a r c h was s u p p o r t e d b y t h e U.S. N a v a l C i v i l E n g i n e e r i n g L a b o r a t o r y , P o r t Hueneme, CA a n d b y g r a n t No. ES04922 f r o m t h e N a t i o n a l I n s t i t u t e o f E n v i r o n m e n t a l H e a l t h S c i e n c e s . We t h a n k S h e l l y Rasmussen f o r t e c h n i c a l a s s i s t a n c e a n d T e r r i Maughan for her expert s e c r e t a r i a l assistance during the preparation of t h i s manuscript.

Literature Cited 1. Barsotti, M.; Crotti, G.; "Epileptic Attacks as Manifestations of Industrial Intoxication caused by Trimethylene Trinitroamine (T ) "; Med. Lavoro. 1949, 40, 107-112. 2. Bumpus, J.Α.; Tien, M.; Wright, D.; Aust, S.D.; "Oxidation of Persistent Environmental Pollutants by a White Rot Fungus"; Science 1985, 228, 14341436. 3. Carpenter, D.F.; McCormick, N.G.; Cornell, J.H.; Kaplan, A.M.; "Microbial Transformation of C– labeled 2,4,6-Trinitrotoluene in an ACtivated– sludge System"; Appl. Environ. Microbiol. 1978, 35, 949-954. 4. Dilley, J.V.; Tyson, C.A.; Newel, G.W. Mammalian toxicolocrical evaluation of TNT waste waters. VIII. Acute and subacute mammalian toxicity of condensate water. SRI International: Menlo Park, CA, 1979; Vol. 111, pp 1-115. 4

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