Chapter 16
Algal-Induced Decay and Formation of Hydrogen Peroxide in Water: Its Possible Role in Oxidation of Anilines by Algae 1
Rkhard G. Zepp, Y. I. Skurlatov , and J. T. Pierce
Downloaded by UNIV ILLINOIS URBANA on March 8, 2013 | http://pubs.acs.org Publication Date: December 8, 1987 | doi: 10.1021/bk-1987-0327.ch016
Environmental Research Laboratory, U.S. Environmental Protection Agency, Athens, GA 30613
Studies of the rates of decomposition and photoproduction of hydrogen peroxide(H O )by several green and blue-green algae in water are reported. Results suggest that algae have an important influence on the environmental concentration of H O , widely distributed oxidant in natural waters. The algal-catalyzed decomposition of H O2 in the dark was found to be second-order overall, first-order with respect to H O , and first-order with respect to algal biomass. Exposure of algal suspensions to sunlight resulted in a buildup of H O , indicating that algae can photoproduce as well as decompose this oxidant. Kinetic results for the algal-induced photooxidation of substituted anilines are presented and a mechanism Involving the intermediacy ofH O is discussed. 2
2
a
2
2
2
2
2
2
2
2
2
Recent investigations have shown that hydrogen peroxide (H2O2) i s a common constituent of the hydrosphere, including clouds, rainwater, freshwater, and seawater (1-3). In Figure 1, we have summarized several of the processes l i k e l y to be Important i n the formation and decay of H2O2 i n natural water bodies. Both f i e l d and laboratory studies indicate that the major pathway for production of H2O2 i n natural waters i s photochemical (2-3), although i t also i s introduced to water bodies through r a i n and b i o l o g i c a l processes. The photochemical formation l s most l i k e l y mediated by electron transfer to dioxygen to form superoxide ion, an oxidant that dismutes to form Η2Ο2· The conjugate acid of super oxide ion, the hydroperoxyl r a d i c a l , also rapidly disproportionates to form hydrogen peroxide (4). 'Current address: Institute of Chemical Physics, Academy of Sciences of the Union of Soviet Socialist Republics, Moscow, USSR This chapter not subject to US. copyright. Published 1987, American Chemical Society
In Photochemistry of Environmental Aquatic Systems; Zika, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
216
PHOTOCHEMISTRY OF ENVIRONMENTAL AQUATIC SYSTEMS
Downloaded by UNIV ILLINOIS URBANA on March 8, 2013 | http://pubs.acs.org Publication Date: December 8, 1987 | doi: 10.1021/bk-1987-0327.ch016
S e v e r a l pathways can account f o r t h e d e c o m p o s i t i o n o f hydrogen p e r o x i d e i n n a t u r a l w a t e r s . Some of these decay p r o c e s s e s n o t o n l y remove H2O2, but a l s o r e s u l t i n t h e o x i d a t i o n o f c h e m i c a l s , p o s s i b l y i n c l u d i n g v a r i o u s p o l l u t a n t s , that a r e present i n n a t u r a l waters. These p r o c e s s e s i n c l u d e d i r e c t o x i d a t i o n ( 5 ) , p e r o x i d a s e - c a t a l y z e d o x i d a t i o n ( 6 ) , and f r e e r a d i c a l o x i d a t i o n i n i t i a t e d by p h o t o c h e m i c a l o r m e t a l - c a t a l y z e d d e c o m p o s i t i o n ( 7 ) . L i t t l e i s known about t h e s i g n i f i c a n c e and r a t e s o f t h e s e v a r i o u s p r o c e s s e s under e n v i r o n m e n t a l c o n d i t i o n s , but they have a l l been shown t o o c c u r r a p i d l y w i t h c e r t a i n organic substrates i n the laboratory. Many p r e v i o u s s t u d i e s have shown t h a t t h e l i g h t - i n d u c e d t r a n s formation of c e r t a i n p o l l u t a n t s i s g r e a t l y accelerated i n n a t u r a l waters compared t o d i s t i l l e d water. O x i d a t i o n s v i a photoproduced H2O2 may account f o r t h e r e s u l t s o f some o f t h e s e i n v e s t i g a t i o n s . I n t h i s paper, we r e p o r t r e s u l t s t h a t q u a n t i f y t h e r a t e s o f photop r o d u c t i o n and d e c o m p o s i t i o n o f H2O2 by s e v e r a l pure c u l t u r e s o f a l g a e i n water. R e s u l t s a l s o a r e p r e s e n t e d w h i c h i n d i c a t e t h a t H2O2 o r a p r e c u r s o r t h e r e o f may be i n v o l v e d i n t h e a l g a l - i n d u c e d photooxidation of a n i l i n e s . Experimental M a t e r i a l s . A n a l y t i c a l grade m - t o l u i d i n e and _p_-butyl a n i l i n e were o b t a i n e d c o m m e r c i a l l y from ChemService and used as r e c e i v e d . A n i l i n e ( A l d r i c h ) was p u r i f i e d by t r e a t i n g an aqueous s o l u t i o n w i t h a c t i v a t e d c h a r c o a l ( 0 . 1 % by w e i g h t ) . The c h a r c o a l was removed by f i l t r a t i o n through a 0.2 μπι M i l l i p o r e f i l t e r . The f i l t r a t e was e x t r a c t e d by e t h e r and, a f t e r d r y i n g over anhydrous sodium s u l f a t e , t h e l a y e r was e v a p o r a t e d under vacuum. The a n i l i n e f i n a l l y was d i s t i l l e d under vacuum. H o r s e r a d i s h p e r o x i d a s e (HRP) ( a c t i v i t y 152 p u r p u r o g a l l i n u n i t s / m g ) , c a t a l a s e (from b o v i n e l i v e r , a c t i v i t y 17,600 units/mg p r o t e i n ) and l e u c o m a l a c h i t e green (LMG) [ 4 , 4 * b e n z y l i d e n e - b i s (Ν,Νd i m e t h y l a n i l i n e ] were o b t a i n e d from Sigma C h e m i c a l s . The LMG was p u r i f i e d by f i r s t washing a benzene s o l u t i o n (0.1 g/ml) w i t h 5% aqueous sodium h y d r o x i d e . The benzene l a y e r was s e p a r a t e d , d r i e d over anhydrous sodium s u l f a t e , then e v a p o r a t e d t o d r y n e s s . The r e s u l t i n g r e s i d u e was r e c r y s t a l l i z e d t w i c e from hexane. The a l g a e used i n t h e s t u d i e s were e i t h e r o b t a i n e d on s l a n t s from t h e S t a r r c o l l e c t i o n ( 8 ) a t t h e U n i v e r s i t y o f Texas, A u s t i n , Texas, o r from a c o l l e c t i o n o f Dr. Gary K o c h e r t , Botany Department, U n i v e r s i t y of Georgia, Athens, Georgia. The b a c t e r i a - f r e e a l g a e from t h e S t a r r c o l l e c t i o n were handled and grown under a s e p t i c c o n d i t i o n s . UTEX 76, UTEX 26, UTEX 89, Chiamydomonas s p . , Chlorococcum hypnosporum, Anabaena v a r i a b i l i s , and Chlorogonium s p . were grown i n medium and under c o n d i t i o n s s p e c i f i e d by Zepp and S c h l o t z h a u e r ( 9 ) . UTEX 625 and UTEX 1444 were grown a t 39°C i n t h e media t h a t i s d e s c r i b e d by K r a t z and Myers (10) as m o d i f i e d by Van B a a l e n (11) u s i n g an 8:16 h r l i g h t - d a r k c y c l e . Equipment. The a l g a e were grown i n a New Brunswick S c i e n t i f i c Co. c o n t r o l l e d environment i n c u b a t o r s h a k e r . K i n e t i c s t u d i e s were
In Photochemistry of Environmental Aquatic Systems; Zika, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
16.
217
Decay and Formation of Hydrogen Peroxide
ZEPP ET AL.
conducted u s i n g a l g a e s u s p e n s i o n s t h a t were a g i t a t e d on a Brunswick G-10 g y r o t a r y s h a k e r .
New
A P e r k i n Elmer Lambda 3 u v - v i s i b l e spectrophotometer was used i n the p e r o x i d e a s s a y s . The a l g a e s u s p e n s i o n s were f i l t e r e d u s i n g Micromate 5-cm g l a s s s y r i n g e s equipped w i t h M i l l i p o r e f i l t e r h o l d e r s c o n t a i n i n g Whatman GF/C f i l t e r pads. 3
Downloaded by UNIV ILLINOIS URBANA on March 8, 2013 | http://pubs.acs.org Publication Date: December 8, 1987 | doi: 10.1021/bk-1987-0327.ch016
A n i l i n e s o l u t i o n s were a n a l y z e d u s i n g a M i c r o m e r i t i c s Model 7170 h i g h p r e s s u r e l i q u i d chromatograph equipped w i t h a column packed w i t h ODS-3; m i x t u r e s of a c e t o n i t r i l e and water were used as m o b i l e phase. K i n e t i c P r o c e d u r e s . The a l g a e c u l t u r e s u s p e n s i o n (100ml) was p l a c e d i n a s t e r i l e 200-ml Erlenmeyer f l a s k . The f l a s k was c o v e r e d w i t h aluminum f o i l t o b l o c k out l i g h t . The temperature of the r e a c t i o n was k e p t a t 18°C throughout the experiment. The k i n e t i c run was s t a r t e d by adding s u f f i c i e n t H2O2 t o r e s u l t i n an i n i t i a l c o n c e n t r a t i o n of 5.0 μΜ. The r e a c t i o n m i x t u r e was then p l a c e d on the shaker a t a shaker speed of 100 rpm. A f t e r one minute of s h a k i n g , a 4.5-ml a l i q u o t of the s o l u t i o n was f i l t e r e d and mixed w i t h 0.5 ml of a peroxidase/LMG assay s o l u t i o n (12 uM LMG, 40 mg/L p e r o x i d a s e i n pH 4.0 a c e t a t e b u f f e r ) . A b l u e c o l o r formed i m m e d i a t e l y when the H2O2 and the assay s o l u t i o n were mixed. The sample was then a l l o w e d a t l e a s t 20 minutes t o d e v e l o p c o m p l e t e l y . More samples were t a k e n a t i n t e r v a l s throughout the r u n u n t i l about two h a l f l i v e s were complete. U s i n g the s p e c t r o p h o t o m e t e r , the absorbance of the samples a t 617 nm was determined v e r s u s d i s t i l l e d water. The absorbance a t 750 nm a l s o was determined and s u b t r a c t e d from the 617 nm absorbance to y i e l d a c o r r e c t e d absorbance. A f i r s t - o r d e r r a t e c o n s t a n t f o r t h e c o r r e c t e d absorbance v e r s u s time t h e n was c a l c u l a t e d u s i n g an HP41C c a l c u l a t o r programmed t o f i t the d a t a by l e a s t squares analysis. A f t e r c o m p l e t i o n of the k i n e t i c r u n , 30 ml of the r e m a i n i n g a l g a e s u s p e n s i o n then was a n a l y z e d f o r i t s c h l o r o p h y l l j i c o n t e n t , C ( 1 2 ) . Experiments on the p h o t o p r o d u c t i o n of H2O2 and the a l g a l induced o x i d a t i o n of a n i l i n e s were conducted i n f u l l s u n l i g h t as d e s c r i b e d by Zepp and S c h l o t z h a u e r (9) u s i n g a p - n i t r o a n i s o l e a c t i n o m e t e r (13) t o measure l i g h t i n t e n s i t y . a
E f f e c t s of H2O2 upon the o x i d a t i o n of a n i l i n e i n dark a l g a l c u l t u r e s were s t u d i e d . To a s u s p e n s i o n (100 ml) of the a l g a e ( C , 2000-5000 mg c h l a/m ) i n phosphate b u f f e r c o n t a i n i n g 5.0 μΜ a n i l i n e was added s u f f i c i e n t H2O2 t o make the i n i t i a l c o n c e n t r a t i o n 20 μΜ. The r e s u l t i n g m i x t u r e was i n c u b a t e d on the shaker f o r t h r e e h a l f l i v e s of the H2O2, then a p o r t i o n (2.0 ml) was removed, f i l t e r e d and a n a l y z e d f o r a n i l i n e and Η2Ο2· T h i s procedure was r e p e a t e d f i v e t i m e s . No r e a c t i o n (
