Solving Hazardous Waste Problems - American Chemical Society

Chapter 28

Downloaded by STONY BROOK UNIV SUNY on December 17, 2016 | http://pubs.acs.org Publication Date: April 24, 1987 | doi: 10.1021/bk-1987-0338.ch028

Biodegradation of Chlorinated Organic Compounds by Phanerochaete chrysosporium, a Wood-Rotting Fungus John A. Bumpus and Steven D. Aust Department of Biochemistry, Michigan State University, East Lansing, MI 48824

The white rot fungus, Phanerochaete chrysosporium, is able to mineralize a number of environmentally persistent organochlorides such as 1,1-Bis(4-chlorophenyl)2,2,2-trichloroethane (DDT), polychlorinated biphenyls (PCBs), 2,3,7,8-tetrachlorodibenzo[p]dioxin (2,3,7,8-TCDD), Lindane (1,2,3,4,5,6-hexachlorocyclohexane), and pentachlorophenol (PCP). Studies suggest that the ability to degrade these compounds is dependent upon the lignin degrading system of this fungus. For example, we have shown that, like C-lignin mineralization, mineralization of C-pentachlorophenol ( C-PCP) and C-DDT is promoted in nutrient nitrogen deficient cultures of P. chrysosporium whereas their mineralization is suppressed in nutrient nitrogen sufficient cultures. Also, the temporal onset and disappearance of both C-PCP and C-DDT mineralization appeared similar to that observed for C-lignin, thus suggesting that the same general degradative system may be responsible. It is suggested that the ability of P. chrysosporium to mineralize such a wide variety of organochlorides may make this fungus a useful microorganism for use in the biological treatment of contaminated soils, sediments and aqueous wastes when used in appropriate aerated waste treatment systems. 14

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During the past 40 years l a r g e q u a n t i t i e s of chlorinated organic compounds have been manufactured for use worldwide. Some of these compounds, such as the p e s t i c i d e s DDT, Lindane and pentachlorophenol, were purposely introduced i n t o the environment to c o n t r o l v a r i o u s noxious or harmful p l a n t and animal pests {]). Others, such as p o l y c h l o r i n a t e d b i p h e n y l s , contaminated the environment through use or as waste from manufacturing processes ( ] ) . Some, such as 2,3,7,8-TCDD, were unknowingly i n t r o d u c e d as contaminants of other compounds or preparations {]). 0097-6156/87/0338-0340$06.00/0 © 1987 American Chemical Society

Exner; Solving Hazardous Waste Problems ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


28.

BUMPUS AND AUST

Biodegradation of Chlorinated Organic Compounds

Regardless of the manner by which they were introduced into the environment, organohalides as a group represent an environment a l l y p e r s i s t e n t c l a s s of chemicals, many of which accumulate i n the food chain i n the body f a t of animals occupying higher t r o p h i c l e v e l s ( J ) . Many of these compounds are toxic, mutagenic and/or carcinogenic. Furthermore, many s i t e s e x i s t worldwide that are contaminated with these compounds. Often these s i t e s are old production or use f a c i l i t i e s which contain high c o n c e n t r a t i o n s of the p o l l u t a n t i n q u e s t i o n and represent potential health hazards. Of great concern i n many cases i s the threat of ground water contamination. Thus these s i t e s are candidates f o r decontamination and reclamation. Recent a t t e n t i o n has focused on the possible usefulness of the white rot fungus Phanerochaete chrysosporium for the biodegradation of hazardous and 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 organohalides (2-7). This paper summarizes our present knowledge concerning the a b i l i t y of t h i s fungus to degrade halogenated organic compounds. P h a n e r o c h a e t e chrysosporium and the B i o d e g r a d a t i o n of L i g n i n . Lignin i s an abundant, naturally o c c u r r i n g polymer whose f u n c t i o n i n nature i s to provide s t r u c t u r a l support to woody plants (8). Its formation in vivo i s c a t a l y z e d by the f r e e r a d i c a l o x i d a t i v e p o l y m e r i z a t i o n of cinnamyl a l c o h o l s ( 8 ) . Because the type and quantity of cinnamyl alcohols may vary and because i t s b i o s y n t h e s i s occurs v i a a n o n - s t e r e o s p e c i f i c free r a d i c a l mechanism, the l i g n i n polymer i s a racemic heteropolymer whose s t r u c t u r e v a r i e s from species to species (8). The lack of an ordered and repeating structure coupled with the racemic nature of the polymer (8) combine to make l i g n i n resistant to attack by most enzyme systems. I n i t i a l studies i n our l a b o r a t o r y (9) and by others (10-15) suggested t h a t , l i k e l i g n i n biosynthesis, l i g n i n biodegradation by the white rot fungus P. chrysosporium a l s o proceeded v i a a f r e e r a d i c a l process. I t had been known f o r some time that hydrogen peroxide ( H 2 O 2 ) i s secreted by many wood r o t t i n g f u n g i (16,17) and i t was suspected that H 2 O 2 was required for l i g n i n biodegradation (17). Forney et a l . (£) subsequently presented evidence which showed t h a t , i n cultures of P. chrysosporium, H 2 O 2 gave r i s e to the formation of h y d r o x y l f r e e r a d i c a l s ( F i g u r e 1), a very r e a c t i v e oxygen species that i s able to cause depolymerization of the l i g n i n polymer (13). Furthermore, i t was shown that H 2 O 2 formation c o i n c i d e d with the onset of l i g n i n mineralization i n P. chrysosporium ( F i g u r e 2) (£). Subsequent work i n a number o f l a b o r a t o r i e s (18-25) has confirmed the requirement for H 2 O 2 and that an active oxygen s p e c i e s i s indeed r e q u i r e d f o r l i g n i n d e p o l y m e r i z a t i o n and/or d e g r a d a t i o n . However, these d e t a i l e d s t u d i e s of l i g n i n degradation i n P. chrysosporium have shown that the most important oxygen a c t i v a t i o n step i s enzyme-mediated by a family of H 2 0 2 ~ r e quiring hemeproteins, c o l l e c t i v e l y referred to as l i g n i n a s e s , t h a t are s e c r e t e d by t h i s fungus during i d i o p h a s i c metabolism i n r e sponse to nutrient starvation (18-25). By catalyzing various oxidat i o n s of the l i g n i n molecule these enzymes mediate cleavage of many C-C and C-0 bonds, thus achieving d e p o l y m e r i z a t i o n . Furthermore, Schoemaker et a l . suggest that carbon centered f r e e r a d i c a l s of lignin-derived m e t a b o l i t e s may be a b l e to d i f f u s e away from the


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SOLVING HAZARDOUS WASTE PROBLEMS

HOURS

F i g u r e 1. T h e p r o d u c t i o n o f h y d r o x y l r a d i c a l (-0H) i n l i g n i n o l y t i c c u l t u r e s o f P. c h r y s o s p o r i u m a s d e t e c t e d b y e t h y l e n e formation. T h e 'OH was d e t e c t e d by t h e ' O H - d e p e n d e n t f o r m a t i o n o f ethylene following the addition of a-keto-Y-methiolbutyric a c i d ( K T B A ) ( 3 . 3 mM f i n a l c o n c e n t r a t i o n ) t o c u l t u r e s g r o w n f o r 14 days i n n u t r i e n t n i t r o g e n d e f i c i e n t m e d i a . The e t h y l e n e prod u c e d was d e t e c t e d u s i n g gas chromatography and i s expressed as nanomoles o f ethylene/ml o f headspace. Reproduced w i t h permiss i o n f r o m R e f . 9.

l i g n i n a s e s and c a t a l y z e f u r t h e r oxidation of l i g n i n at sites that a r e r e m o t e f r o m t h e a c t i v e s i t e o f t h e enzyme ( 2 6 ) . Once t h e i n i t i a l o x i d a t i v e d e p o l y m e r i z a t i o n o f l i g n i n o c c u r s , t h e s m a l l e r and more s o l u b l e l i g n i n d e r i v e d m e t a b o l i t e s t h e n u n d e r g o f u r t h e r m o d i f i c a t i o n and, u l t i m a t e l y , metabolism t o carbon d i o x i d e . A l t h o u g h t h e most i m p o r t a n t f a c t o r i n t h e i n i t i a l o x i d a t i v e depolymerization o f l i g n i n i s undoubtedly the catalytic action a t t r i b u t e d t o t h e l i g n i n a s e s , t h e h y d r o x y l r a d i c a l may s t i l l p l a y a r o l e i n l i g n i n degradation (26). A d d i t i o n a l l y , a manganese depend e n t p e r o x i d a s e , w h i c h i s a l s o s e c r e t e d b y t h i s f u n g u s may h a v e a n i m p o r t a n t f u n c t i o n i n l i g n i n degradation (24,27-29). Regardless of the p r e c i s e mechanism o f l i g n i n d e g r a d a t i o n , i t i s a p p a r e n t that t h i s system i s a very 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 biodegrad a t i o n system. I t i s t h i s l a c k o f s p e c i f i c i t y t h a t makes t h i s f u n gus a p o t e n t i a l l y u s e f u l microorganism f o r t h e treatment o f h a z a r d -


28.

BUMPUS A N D AUST



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DAYS F i g u r e 2. R e l a t i o n s h i p b e t w e e n t h e s p e c i f i c a c t i v i t y f o r H C>2 production i n cell e x t r a c t s ( A ) ,t h e metabolism o f 2' ^C-labeled synthetic lignin to C0 ( 0 ) , and m y c e l i a l dry w e i g h t ( • ) . P. c h r y s o s p o r i u m w a s g r o w n i n l o w n i t r o g e n m e d i u m . R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 9. 2

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ous c h l o r i n a t e d o r g a n i c s . Furthermore, would a l l o w c l e a v a g e o f C - C l bonds.

a free

radical

mechanism

D e g r a d a t i o n o f C h l o r i n a t e d Compounds b y P. c h r y s o s p o r i u m ; A n O v e r view. T h e f i r s t u s e o f P. c h r y s o s p o r i u m i n t h e t r e a t m e n t o f c h l o r i n a t e d o r g a n i c compounds f o c u s e d on t h e t r e a t m e n t o f c h l o r i n a t e d l i g n i n (30) and c h l o r i n a t e d l i g n i n - d e r i v e d low m o l e c u l a r weight compounds t h a t a r e b y - p r o d u c t s o f the bleaching procedure that i s o f t e n used w i t h t h e K r a f t p u l p i n g p r o c e s s ( 6 / 7 ) . In these s t u d i e s , i t was d e m o n s t r a t e d t h a t e v e n h i g h l y c h l o r i n a t e d compounds l i k e t e t r a c h l o r o g u i a c o l c o u l d be d e g r a d e d b y t h e l i g n i n d e g r a d i n g system of t h i s organism (6,7). Other s t u d i e s showed t h a t c h l o r i n a t e d l i g n i n s , l i k e t h e i r n o n c h l o r i n a t e d p r e c u r s o r s , c o u l d be d e g r a d e d t o carbon dioxide ( 3 £ ) . A s o m e w h a t s u r p r i s i n g f i n d i n g was t h a t t h e c h l o r i n a t e d l i g n i n was m i n e r a l i z e d f a s t e r t h a n u n m o d i f i e d lignin (30).


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SOLVING HAZARDOUS WASTE PROBLEMS

The f a c t that P. chrysosporium degraded chlorinated l i g n i n and l i g n i n by-products, coupled with the f i n d i n g that l i g n i n degradat i o n proceeded v i a a non-specificfree r a d i c a l process, suggested to us that even more r e c a l c i t r a n t organohalides might be degraded by t h i s microorganism ( 2 - 4 ) . We subsequently demonstrated that a number of normally persistent organohalides (DDT, Lindane, 2,3,7,8TCDD, 3,4,3 4'-tetrachlorobiphenyi (3,4,3 ,4'-TCB), 2,4,5,2',4',5'hexachlorobiphenyl (2,4,5,2',4',5'-HCB)) as well as the polyaromati c hydrocarbon benzo[a]pyrene,can be m i n e r a l i z e d by n i t r o g e n starved cultures of t h i s fungus (Table l a ) . Nearly c o i n c i d e n t a l degradation s t u d i e s of a p o l y c h l o r i n a t e d biphenyl (PCB) mixture (Aroclor 1254) by Eaton (5) using t h i s microorganism produced s i m i l a r r e s u l t s and c o n c l u s i o n s . I t i s i n t e r e s t i n g t o note that l i g n i n , chlorinated l i g n i n , and the overwhelming majority of l i g n i n d e r i v e d low molecular weight by-products are already p a r t i a l l y oxidized. In c o n t r a s t , most of the compounds examined i n our s t u d i e s ( 3 , 4 ) and Eaton's ( 5 ) were highly reduced, having only an aromatic p i electron cloud, a hydrogen or a t y p i c a l l y u n r e a c t i v e c h l o r i n e s u b s t i t u e n t available for attack. This demonstrates that P. chrysosporium i s able to catalyze the i n i t i a l o x i d a t i v e step i n the b i o d e g r a d a t i o n of many highly reduced compounds. This step i s important because the i n i t i a l oxidation of an environmentally pers i s t e n t x e n o b i o t i c i s often (though not always) the most d i f f i c u l t step and a rate l i m i t i n g step i n i t s o v e r a l l biodegradation. Thus, unlike many microorganisms which t y p i c a l l y c a t a l y z e one or a few r e a c t i o n s during the microbial degradation of organopollutants, P. chrysosporium i s able to i n i t i a t e degradation of many o r g a n o p o l l u t a n t s and to c a t a l y z e a l l of the steps i n their complete degradation pathways to carbon dioxide.


f

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P o t e n t i a l Advantages to the use of White Rot Fungi i n Waste Treatment Systems. The potential usefulness of the white r o t fungus P. chrysosporium i n waste treatment systems has been d i s c u s s e d i n greater d e t a i l elsewhere (3,). B r i e f l y the p o t e n t i a l advantages to u s i n g t h i s microorganism are: ( 1 ) The system normally attacks an insoluble substrate. (2) The l i g n i n degrading system i s r e l a t i v e l y n o n - s p e c i f i c , thus i t may be useful i n the treatment of mixtures of organopollutants. (3) P. chrysosporium i s a successful competitor, e s p e c i a l l y when wood based products are a major carbon source. (4) Biodegradation i s not dependent on prior exposure to l i g n i n or to a s p e c i f i c organopollutant. (5) Biodegradation of l i g n i n proceeds to zero l e v e l s and the end product i s carbon dioxide. This i s precisel y the type of degradation one would t r y to achieve for unwanted xenobiotics. Perhaps the most interesting aspect concerning the use of t h i s microorganism i n the b i o l o g i c a l destruction of organopollutants, i s the f a c t that i t i s able to dechlorinate chlorinated organic compounds. Unfortunately, dechlorination of organochlorides per se by P. chrysosporium has not been well studied. Our information concerning t h i s process comes from the f o l l o w i n g : ( 1 ) In s t u d i e s of the d e c o l o r i z a t i o n of the a l k a l i n e e x t r a c t (E-j) obtained from a Kraft bleach plant, i t was demonstrated that many of the organochlor i d e s o r i g i n a l l y present were removed by treatment with P. chrysosporium i n a rotating b i o l o g i c a l contactor ( 6 , 7 ) . Furthermore, most


28.

BUMPUS AND AUST Table l a .


Mineralization of Selected '^C-labeled Phanerochaete chrysosporium.

345

Compounds by

a

Lindane Benzo(a)pyrene DDT 2,3,7,8,-TCDD 3,4,4\4 -TCB 2,4,5,2',4 ,5'-HCB° f


f

Table l b .

% Mineralization

0

21.4 13.8 9.3 4.0 2.0 1.7

Mineralization of ^C-Labeled Biphenyl and Two Polychlorinated Biphenyl Mixtures. % Mineralization

Mineralization (pmoles) 455.8 256.3 224.4

Biphenyl Aroclor 1242 Aroclor 1254

Table Ic.

Mineralization (pmoles) 267.6 171.9 116.4 49.5 25.1 86.0

36.5 20.5 18.0

Mineralization of C-Labeled Hexachlorobenzene and Pentachlorophenol.

Pentachlorophenol Hexachlorobenzene

l4

% Mineralization

Mineralization (pmoles) 570.4

45.6

Solving Hazardous Waste Problems - American Chemical Society

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