Biodegradation of S-Triazines: An Approach To Dispose of

C h a p t e r 14 Biodegradation of S-Triazines: An Approach To Dispose of Recalcitrant Wastes

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 1, 2017 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch014

Alasdair M. Cook Microbiology Department, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland Biological treatment of wastes, i f available, is recognized as being less expensive and as producing a better-quality outflow than physico-chemical methods. Up till now, s-triazines (e.g., the herbicide atrazine) and by-products from chemical syntheses were regarded as non-biodegradable and wastes were usually treated physically. We have developed analytical HPLC methods to identify and determine routinely the whole range of by-products (e.g., N,N'-bis(ethyl)-N"-(1methylethyl)-l,3,5-triazine-2,4,6-triamine and 2chloro-4-ethylamino-l,3,5-triazine-6(5H)-one) in wastes from these syntheses. We have also obtained aerobic cultures (e.g., Pseudomonas spp.) that quantitatively utilize the by-products as sole sources of nitrogen for growth. Biochemical pathways of catabolism have been elucidated and no toxic intermediates seem to be involved. A mixed culture has been used to treat real wastes from herbicide syntheses under non-sterile conditions and about 80 % conversion of s-triazines to c e l l material was observed. Small-scale (2 and 25 1) fluidized beds were constructed. The main problems encountered were the costs of the carbon source and oxygen and the low rates of some enzymes. A need e x i s t s f o r t h e a u t h e n t i c treatment o f w a s t e s , whether due t o o u r p l a t o n i c r e s p e c t f o r o u r f e l l o w man a n d t h e r i g h t s o f t h e n e x t generation to a safe environment, o r f o r l e g a l reasons. I stress the word ' a u t h e n t i c ' , i . e . , r e a l c o n v e r s i o n o f waste t o something nontoxic, as a reminder that h i d i n g wastes i s a s e l f - d e c e p t i o n and a n o n - t r e a t m e n t w h i c h we o r o u r c h i l d r e n w i l l h a v e t o c o p e w i t h . T h e r e are good physical, chemical and b i o l o g i c a l treatments a p p r o p r i a t e f o r d i f f e r e n t w a s t e s , b u t b i o l o g i c a l t r e a t m e n t , where a v a i l a b l e , i s l e s s expensive than competing methods. F u r t h e r , b i o l o g i c a l treatment is frequently needed f o r products resulting from p h y s i c a l and chemical treatments (1_). So t h e r e i s a s o u n d e c o n o m i c r e a s o n f o r b i o l o g i c a l waste treatment.

0097-6156/87/0334-0171 $06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


172

B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y

A c t i v a t e d s l u d g e s y s t e m s h a v e b e e n a m a t u r e t e c h n o l o g y f o r some 7 5 y e a r s (2^) b u t many s u b s t a n c e s a r e n o t d e g r a d e d i n a s e w a g e w o r k s . I t may seem t o b e a c o n t r a d i c t i o n , b u t o u r a i m i s t h e b i o d é g r a d a t i o n of these 'non-biodegradable' compounds, which are also termed ' x e n o b i o t i c ' o r ' r e c a l c i t r a n t ' compounds. X e n o b i o t i c s a r e compounds t h a t a r e foreign to the biosphere (see 3_ f o r fuller discussion) and t h a t , i n c o n t r a s t to n a t u r a l p r o d u c t s , are seldom degraded i n n a t u r e . But x e n o b i o t i c s have always been o c c u r r i n g i n n a t u r e , say by the a c t i o n of v o l c a n o e s , and these novelties are now considered to be n a t u r a l products and are biodegradable. The p r o b l e m nowadays i s t h e enormous i n c r e a s e i n t h e p r o d u c t i o n and d i v e r s i t y o f t h e c h e m i c a l i n d u s t r y i n about the last 40 y e a r s . N a t u r e h a s n o t b e e n a b l e t o k e e p u p . So o u r w o r k i s r e a l l y t o d i r e c t n a t u r a l p r o c e s s e s t o d e g r a d a t i o n o f t h e compounds i n w h i c h we a r e i n t e r e s t e d . The critical aspect of this natural process, called b i o d é g r a d a t i o n , i s t h a t i t i s e n z y m i c , and enzymes c a t a l y z e specific r e a c t i o n s . So we h a v e t o w o r k w i t h s p e c i f i c x e n o b i o t i c s a n d n o t w i t h some u n d e f i n e d mixture. T h u s we must have specific analytical chemistry a v a i l a b l e to e l i m i n a t e the a r t i f a c t s arising from using solely indirect assays. Our probem i s t h e x e n o b i o t i c w a s t e ( T a b l e I ) a r i s i n g from the

Table

I.

S t r u c t u r e s of

Abbreviation CCCT CIET£ A IEET-2. A EEOT! A EEAT£ A CEOT! EOOTI 0

s - t r i a z i n e s * - and t h e i r

S u b s t i t u t i o n at p o s i t i o n 4 2 -CI -CI -CI -NHCH(CH )CH -NHCH(CH )CH -NHCH CH -NHCH CH -NHCH CH -NHCH9CH3 -NHCH CH -CI -NHCH CH -NHCH CH -OH 3

3

2

2

3

3

3

2

3

2

3

2

3

2

3

3

abbreviations^..

-CI -NHCH CH -NHCH CH -OH -NH -OH -OH 2

3

2

3

2

— The s^triazines are symmetrical, six-membered rings with a l t e r n a t i n g c a r b o n and n i t r o g e n atoms: e a c h c a r b o n and n i t r o g e n atom n o m i n a l l y c o n t r i b u t e s t h r e e bonds t o t h e r i n g s t r u c t u r e and the r i n g c a r r i e s t h r e e s u b s t i t u e n t s , one on each carbon atom (i. e., at positions 2, 4, and 6). — The f o u r - l e t t e r a b b r e v i a t i o n h a s one l e t t e r f o r each o f t h e t h r e e r i n g s u b s t i t u e n t s and the letter 'T' for t h e j 3 - t r i a z i n e r i n g . — The e d u c t (CCCT) and t h e p r o d u c t , e.g., a t r a z i n e ( C I E T ) do n o t o c c u r i n t h e w a s t e s i n significant amounts. — This is a major component of the waste and r e p r e s e n t s a l s o p o s s i b l e homologues ( e . g . , ΕΕΕΤ). R e p r o d u c e d with permission from R e f . 33. C o p y r i g h t 1986, John W i l e y & Sons.

syntheses of the ^ - t r i a z i n e h e r b i c i d e s , which are not degraded in sewage works (4_) and for w h i c h no simple, single routine d e t e r m i n a t i o n was a v a i l a b l e (5)·


14.

COOK

Biodégradation of S-Triazines


Analytical

chemistry of

173

s-triazines

The i m p o r t a n c e o f m e t h o d o l o g y i s s e e n i n a r e v i e w of work on the degradation of s _ - t r i a z i n e h e r b i c i d e s ( 6 0 ; o n l y 2 o u t o f 12 s e t s o f data are v a l i d because only i n those two c a s e s was thorough analytical chemistry done. The i n t e n s i v e study of triazine m e t a b o l i s m a n d w a s t e s became p r a c t i c a b l e with the advent o f new methodology for the routine determination of these compounds. S e v e r a l groups s i m u l t a n e o u s l y developed s i m i l a r methods t o separate and q u a n t i f y many j s - t r i a z i n e s u s i n g r e v e r s e d - p h a s e HPLC c o l u m n s a n d buffered mobile phases ( 5 , 7 ) : i n the absence of b u f f e r , separation can be s e e n t o b e p o o r ( 8 , 9 , 1 0 ) . A l o w w a v e l e n g t h ( e . g . , 2 2 0 nm) i s necessary f o r high s e n s i t i v i t y i n a UV-detector, because the major peak i n the UV-spectrum of s - t r i a z i n e s i s u s u a l l y i n t h i s r e g i o n ( 5 ) . Whereas t h e methods u s i n g acetonitrile to adjust the polarity of t h e m o b i l e p h a s e may o f f e r b e t t e r r e s o l u t i o n ( 1 0 ) , o u r use o f m e t h a n o l i s b a s e d o n g r o u n d s o f l o w e r t o x i c i t y and much l o w e r price. Our method has proved i t s e l f i n b a s i c and a p p l i e d r e s e a r c h ( 6 , 1 1 , 1 2 , 1 3 , 1 4 ) . To c o m p l e m e n t t h i s r o u t i n e use of analytical HPLC and s e m i - p r e p a r a t i v e r e v e r s e d phase methods t o o b t a i n m a t e r i a l f o r mass s p e c t r a ( e . g . , 6 ) , i d e n t i f i c a t i o n o f i n t e r m e d i a t e s i s a i d e d by GLC o f d e s a l t e d ( 1 5 ) , d e r i v a t i z e d s - t r i a z i n e s ( 1 6 , s e e a l s o 1 7 ) . The a d v e n t o f mass s p e c t r o m e t r y interfaced w i t h HPLC (e.g., 18) should, i f applicable to the mainly n o n - v o l a t i l e intermediates, further simplify the i d e n t i f i c a t i o n of metabolites of s-triazines. An alternative HPLC m e t h o d ( a m i n o p h a s e ) f o r some s - t r i a z i n e s i s a v a i l a b l e (19) but i t does n o t work w e l l i n our hands ( 1 2 ) . And there i s methodology f o r the products of ^ - t r i a z i n e r i n g cleavage (20). The first use of o u r a s s a y was t o c o n f i r m b i o d é g r a d a t i o n o f components o f model waste by n e w l y - i s o l a t e d bacteria (6^). T h e specific rates of degradation were sufficiently high that a p r a c t i c a b l e b a c t e r i a l treatment of the wastes in a sensibly-sized reactor c o u l d b e c a l c u l a t e d . We o b s e r v e d t h a t t h e a n a l y t i c a l m e t h o d f u n c t i o n e d w i t h waste w a t e r from ametryne p r o d u c t i o n ( 1 2 ) and i t was a g r e e d t h a t we s h o u l d e x a m i n e a n d t r e a t t h e s - t r i a z i n e s ( T a b l e I ) i n the wastes from t h e p r o d u c t i o n o f a t r a z i n e ( F i g . 1) and s i m a z i n e . Strategy

of our biodégradation research

We chose to treat wastes directly as they emerged from the p r o d u c t i o n u n i t and before they entered an a c t i v a t e d sludge p r o c e s s . The w a s t e t r i a z i n e s a r e t h u s a t a h i g h c o n c e n t r a t i o n and c o n t a i n e d no other wastes: this simplified the process control and the analytical chemical evaluation of the treatment. We a r e thus proposing small biotreatment u n i t s s p e c i f i c f o r p a r t i c u l a r problem wastes; other groups propose to a d d new o r g a n i s m s to activated sludge plants ( e . g . , 21). T h e p r o b l e m w a s t h a t t h e r e a l w a s t e s h a d l i t t l e i n common with the model wastes - t h e s y n t h e s i s h a d b e e n m o d i f i e d - a n d we c o u l d d e g r a d e o n l y t h e wrong s p e c t r u m o f compounds (13). So we h a d t o restart the research programme ( T a b l e I I ) t o o b t a i n t h e o r g a n i s m s w i t h t h e r e q u i r e d d e g r a d a t i v e c a p a b i l i t i e s . The g e n e r a l a p p r o a c h to the microbiology, physiology and biochemistry of the project i s sketched elsewhere (22, 23) and a d e t a i l e d description is in preparation.


174 Table


II.

Recommended

r e s e a r c h s t r a t e g y f o r the biodégradation x e n o b i o t i c compounds

of


0) a n a l y t i c a l methodology 1) e n r i c h m e n t c u l t u r e s 2) g r o w t h p h y s i o l o g y 3) b i o c h e m i s t r y 4) e n g i n e e r i n g 5) e v a l u a t i o n

T h i s method of l e a r n i n g how t o approach a similar project correctly, causes a bias i n the a p p r a i s a l of the r e l a t i v e e f f o r t s r e q u i r e d f o r t h e r e s e a r c h . O f some 2 0 r e s e a r c h y e a r s i n t h e project (about half by d o c t o r a l students) 20 % of the time was for analytical chemistry, 70 % f o r microbiology, physiology and b i o c h e m i s t r y , and 10 % f o r e n g i n e e r i n g . F o r t h i s p r e s e n t a t i o n o f t h e w a s t e t r e a t m e n t i t i s o n l y n e c e s s a r y t o know that the s-triazines a r e d e g r a d e d q u a n t i t a t i v e l y t o N H ^ a n d CO2 ( a n d , w h e r e a p p r o p r i a t e , t o C l " and an u n i d e n t i f i e d alkyl group) v i a defined, converging pathways of hydrolytic reactions i n w h i c h no t o x i c intermediates o c c u r ( 6 , 1 1 , 2 4 , 2 5 , 2 6 , 2 7 ) . The s - t r i a z i n e s s e r v e o n l y a s s o u r c e s o f n i t r o g e n (25) so a c a r b o n s o u r c e must be added t o a l l o w g r o w t h . +

Waste

treatment

The wastewater from syntheses o f c h l o r o - s ^ t r i a z i n e h e r b i c i d e s i s a c l e a r , c o l o u r l e s s m i x t u r e of wastes from the c h e m i c a l reaction and from washing the p r o d u c t . I t has a t o t a l organic carbon content of a b o u t 1 2 0 0 mg L . a t o t a l Kjeldahl nitrogen concentration of about 810 mg o f Ν L , a b i o l o g i c a l o x y g e n demand a f t e r 5 d a y s o f a b o u t 5 3 0 mg o f O o L , a n d c o n t a i n s N a C l a t a b o u t 35 g L ; t h e pH i s a b o u t 12 ( 1 4 ) . The w a s t e t r e a t m e n t i s b a s e d o n t h e b i o c h e m i c a l a c t i v i t y o f an aerobic mixed culture which i s used under n o n - s t e r l i e c o n d i t i o n s throughout the work. The c u l t u r e consists almost entirely of bacteria and does n o t grow i n t h e absence o f a s o u r c e o f combined n i t r o g e n . The w a s t e s a r e e s s e n t i a l l y s t a b l e (13) and c o n t a i n only sj-triazines as a source of n i t r o g e n ( 1 4 ) . P r e l i m i n a r y experiments (14) i n batch culture used mineral g r o w t h medium c o n t a i n i n g a c a r b o n s o u r c e and r e a l w a s t e s a s n i t r o g e n s o u r c e . The c u l t u r e grows b i p h a s i c a l l y ( F i g . 2 ) , t h e h i g h e r s p e c i f i c rate (ji) being < 0.2 I T and the lower r a t e being < 0.05 h . The s - t r i a z i n e s d i s a p p e a r s e q u e n t i a l l y ( F i g . 3) w i t h E00T (representing the behaviour of 000T, EEAT a n d E00T (see Table I)) being m e t a b o l i z e d f i r s t , CEOT l a t e r , and IEET a n d EEOT (a catabolic p r o d u c t from IEET) s l o w l y . The d i s a p p e a r a n c e o f s j - t r i a z i n e s i s about 7 5 - 8 0 %, a v a l u e t h a t i s c o n f i r m e d by d e t e r m i n a t i o n s of Kjeldahl nitrogen; the s ^ t r i a z i n e - n i t r o g e n i s converted into c e l l m a t e r i a l . The p r o c e s s h a s a t e m p e r a t u r e optimum o f about 35-40 °C a n d t h e culture c a n t o l e r a t e up t o about 50 % (v/v) w a s t e w a t e r . The w a s t e s do n o t s u p p l y s i g n i f i c a n t amounts o f c a r b o n f o r g r o w t h . sj-Triazine wastes c a n be t r e a t e d i n continuous c u l t u r e (14) ( n o t shown) and r e q u i r e m e n t s f o r m i n e r a l components i n the growth 1


14.

COOK

175

Biodégradation of S-Triazines CI

X X

iPrHN^NT^NHEt


Fig. 1. The s - t r i a z i n e herbicide a t r a z i n e . I n t h e homologue, simazine, both aminoalkyl substituents are ethylamino; i n the analogue, ametryne, the chloro s u b s t i t u e n t i s r e p l a c e d by t h e thiomethyl group.

10r

Ί

9

Hours F i g u r e 2 . Growth and s u b s t r a t e u t i l i z a t i o n o f the mixed c u l t u r e i n medium c o n t a i n i n g S - t r i a z i n e w a s t e s . G r o w t h medium a t pH 7 . 5 a n d 3 0 °C a n d c o n t a i n i n g 33% ( v / v ) w a s t e w a t e r a n d 10 g o f g l u c o s e / L was i n o c u l a t e d from a c o n t i n u o u s c u l t u r e u s i n g w a s t e water. (A) Growth ( O ) was measured t u r b i d i m e t r i c a l l y . (B) Glucose c o n c e n t r a t i o n s ( • ) are a b s o l u t e whereas the c o n c e n t r a t i o n s o f S - t r i a z i n e s a r e p l o t t e d as a f r a c t i o n o f t h e i n i t i a l v a l u e s . (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 1 4 . C o p y r i g h t 1 9 8 5 , John Wiley & Sons.)



176


medium a r e met from components i n t a p water and i n the wastes t h e m s e l v e s . T h i s s i m p l i f i e d system i s s e n s i t i v e t o excess carbon s o u r c e , presumable t o m e t a b o l i t e s from the g l u c o s e used, and much f l u c t u a t i o n o f biomass can be o b s e r v e d . N e v e r t h e l e s s , t r e a t m e n t e f f i c i e n c i e s o f 75-80 % a r e o b s e r v e d , but the d i l u t i o n r a t e i s low (0.025 h" ) . A l l components o f t h e waste a r e degraded, but whereas e.g., Ε00Τ d i s a p p e a r s t o t a l l y , IEET i s o n l y p a r t i a l l y degraded u n l e s s the d i l u t i o n r a t e i s d e c r e a s e d . The low v o l u m e t r i c l o a d i n g r a t e p o s s i b l e i n the c o n t i n u o u s c u l t u r e and the ease w i t h w h i c h washout i s i n d u c e d , c o u p l e d w i t h a r e q u i r e m e n t f o r a s m a l l u n i t , l e d us t o seek a compact r e a c t o r t h a t ( a ) y i e l d s h i g h v o l u m e t r i c l o a d i n g r a t e s , (b) guarantees retention of t h e mixed c u l t u r e , ( c ) p r o v i d e s a l a r g e s u r f a c e a r e a ( p e r u n i t o f r e a c t o r volume) f o r the attachment o f c e l l s , and, (d) shows l e s s danger o f c l o g g i n g t h a n a f i x e d bed r e a c t o r . I m m o b i l i z e d c e l l f l u i d i z e d bed r e a c t o r s (FBR) can meet t h i s r e q u i r e m e n t (28), t h e i r a p p l i c a t i o n t o waste t r e a t m e n t i s known (29, 30, 31) and l a r g e u n i t s have been i n i t i a t e d i n d u s t r i a l l y f o r waste d i s p o s a l ( 3 2 ) . Each o f our FBRs (2-L ( F i g . 3) and 25 L ) c o n s i s t s o f a v e r t i c a l column f i l l e d w i t h c a r r i e r ( q u a r t z s a n d ) , on w h i c h most o f the b i o l o g i c a l a c t i v i t y o c c u r s , and a s t i r r e d tank f o r a e r a t i o n o f the c u l t u r e (gas b u b b l e s i n t h e column d i s r u p t the f l u i d i z a t i o n by causing the particles t o f l o a t ) . The apparatus i n c l u d e s a t h e r m o s t a t , a p H - s t a t and r e g u l a t i o n o f the oxygen p a r t i a l p r e s s u r e by a u t o m a t i c a l l y s u p p l m e n t i n g the a e r a t i o n w i t h 0 as r e q u i r e d . The r e c i r c u l a t i o n r a t e t h r o u g h t h e f l u i d i z e d bed i s about 7 0 0 - f o l d h i g h e r than the d i l u t i o n r a t e o f t h e r e a c t o r ( 3 3 ) . The mixed c u l t u r e r e a d i l y and s p o n t a n e o u s l y adheres t o the sand p a r t i c l e s w h i l e growing under b a t c h c o n d i t i o n s . When s h i f t e d t o c o n t i n u o u s c u l t u r e c o n d i t i o n s , t h e i m m o b i l i z e d biomass s t a b i l i z e s a t 15-17 g/L a f t e r about a month. Excess biomass must then be removed to a v o i d i n a c t i v e zones. T h i s c o n c e n t r a t i o n o f biomass i s t e n f o l d h i g h e r than observed i n suspended c u l t u r e and the s p e c i f i c growth r a t e i s improved by about 50 % t o 0.04 h : t h i s r a t e i s about s e v e n f o l d lower than the v a l u e used t o e s t i m a t e the e f f i c a c y o f c u l t u r e s d e g r a d i n g model wastes (see above). The d e g r a d a t i v e b e h a v i o u r o f c e l l s i m m o b i l i z e d i n a FBR i s shown i n F i g . 4. A l l compounds a r e degraded, but not a l l t o 100 %; about 75-80 % d e g r a d a t i o n i s p o s s i b l e ( c l o s e d symbols) and IEET (open symbols) i s a good marker o f g e n e r a l performance. T h i s c u l t u r e has an i n i t i a l d e g r a d a t i o n r a t e o f 1.6 mg of N/L.h and a t t a i n s a maximum r a t e o f about 26 mg of N/L.h (about days 33 and 5 0 ) . That t h i s i s t h e maximum v a l u e i s seen when the n i t r o g e n c o n c e n t r a t i o n i s f u r t h e r i n c r e a s e d (day 40) and the system c o l l a p s e s . Recovery on r e d u c t i o n of the n i t r o g e n c o n c e n t r a t i o n i s r a p i d . S e v e r a l experiments a r e shown i n F i g . 4. I n days 13-20 the c a r b o n i n p u t i s a t a reduced l e v e l and the e f f i c i e n c y d r o p s ; r e c o v e r y i s a g a i n r a p i d . An attempt t o o p e r a t e t o t a l l y w i t h o u t a c a r b o n source (day 23) l e a d s t o c o l l a p s e o f t h e system; t h e c e l l s r e q u i r e 10-12 mol of C/mol of N. One major problem i s masked i n the performance d a t a near day 40. Our s u p p l y o f waste aged and a component (CEOT) h y d r o l y z e d s p o n t a n e o u s l y t o Ε00Τ. On a d d i t i o n o f f r e s h waste (day 40) the a b i l i t y t o degrade CE0T i s m i s s i n g and i s r e s t o r e d by a d d i n g new c e l l s . The o b s e r v a t i o n o f l o s s o f d e g r a d a t i o n of CEOT i s r e p r o d u c i b l e , so we must r e t a i n s e l e c t i v e c o n d i t i o n s i n 2


14.

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177

Biodégradation of S-Triazines

0 -stat


2

F i g . 3 Schematic r e p r e s e n t a t i o n o f the s m a l l FBR. (Reproduced w i t h p e r m i s s i o n f r o m r e f e r e n c e 2 3 . C o p y r i g h t 1985, P e r g a m o n Press.)

u m r π r

I Ο

ι 10

ι 20

ι 30

ι 40

ι50

Days

F i g . 4. R e m o v a l o f d i s s o l v e d £ - t r i a z i n e s b y t h e m i x e d c u l t u r e in a c o n t i n u o u s F B R r e a c t o r . T h e e x p e r i m e n t w a s d o n e a t 30 ° C a n d p H 7.5. A t t h e numbered a r r o w s , t h e f o l l o w i n g changes were made: 1) mean residence time reduced, nitrogen and carbon inputs i n c r e a s e d ; 2) c a r b o n i n p u t r e d u c e d ; 3) c a r b o n i n p u t i n c r e a s e d ; 4) c a r b o n i n p u t s t o p p e d ; 5) c a r b o n i n p u t r e s t o r e d ; 6) mean r e s i d e n c e time reduced, n i t r o g e n and carbon i n p u t s i n c r e a s e d ; 7) nitrogen i n p u t i n c r e a s e d ( n e w w a s t e w a t e r w i t h h i g h e r n i t r o g e n c o n t e n t ) ; 8) n i t r o g e n i n p u t c o r r e c t e d , mean r e s i d e n c e t i m e i n c r e a s e d ; 9) mean residence time reduced. Total s-triazine (•), IEET (Q). 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 . 33. C o p y r i g h t 1986, J o h n Wiley & Sons.


178


t h e r e a c t o r o r r i s k l o s i n g a c t i v i t i e s . As i n o t h e r cases, i s r a p i d , b u t t h i s t i m e due t o a d d i t i o n o f f r e s h b i o m a s s .


E v a l u a t i o n of

the

recovery

project

An evaluation of the positive aspects of the project i s quite s i m p l e . S t r a i g h f o r w a r d a n a l y t i c a l c h e m i s t r y was developed and we enjoyed good cooperation with Ciba-Geigy t o o b t a i n , a n a l y z e and t r e a t r e a l w a s t e s . The a v a i l a b i l i t y o f t h e a n a l y s e s n o t o n l y proved the biology t o be sound b u t a l s o a l l o w e d t h e c h e m i c a l e n g i n e e r s a n a l t e r n a t i v e a p p r a i s a l of the r e a c t i o n s i n the syntheses. All the components of t h e w a s t e s a r e now b i o d e g r a d a b l e - a c o m p l e t e c h a n g e f r o m a few y e a r s a g o , when a l m o s t a l l o f the compounds were nonbiodegradable. Further, the d e g r a d a t i v e pathway c o n t a i n s no t o x i c i n t e r m e d i a t e s t o be e x c r e t e d d u r i n g a d i s r u p t i o n o f t h e s y s t e m . And all the compounds c a n be degraded quantitatively. These are necessary p r e l i m i n a r i e s to applied work. The development of a p r e - p i l o t treatment u n i t has a l s o been demonstrated, so i t i s p o s s i b l e to start with only the idea of biological treatment o f r e c a l c i t r a n t waste and c r e a t e a f u n c t i o n a l system. The f a c t t h a t t h e s y s t e m i s f u n c t i o n a l , h o w e v e r , d o e s n o t mean that i t i s f i n a n c i a l l y competitive with alternative methods. With hindsight we c a n n o t e where the a p p r o a c h must be i m p r o v e d . T h i s i s seen f i r s t i n the low degradation rates for some of the waste components. The chemical components in the real waste were i d e n t i f i e d l a t e i n t h e p r o j e c t a n d we h a d t o o l i t t l e t i m e t o i m p r o v e the degradation o f t h e s e compounds. F o r t h i s r e a s o n I a g a i n s t r e s s the need for analytical chemistry as the first step in biodégradation research, i n order to a l l o w time f o r the b i o l o g y to be developed and quantified before committing oneself to engineering. A s e c o n d p r o b l e m i s t h a t o f t h e c a r b o n s o u r c e . A s b i o l o g i s t s we c h o s e compounds w h i c h f a v o u r e d o u r e n r i c h m e n t c u l t u r e s , b u t w h i c h i n p r a c t i c e a r e e x p e n s i v e . The f a c t o r y c o m p l e x , h o w e v e r , a l s o h a s w a s t e carbon that requires disposal, but it is not u t i l i z e d by our s t r a i n s . T h e s e two p r o b l e m s s h o u l d have been combined from the initiation of the p r o j e c t t o a c h i e v e i n t e g r a t e d and thus economic w a s t e d i s p o s a l . Our i n i t i a l i d e a t o o p e r a t e t h e t r e a t m e n t s y s t e m f o r long periods under n o n - g r o w i n g c o n d i t i o n s ( c f . 6) d i d n o t s u r v i v e p r a c t i c a l t e s t i n g . Another property of the wastes, w h i c h was not considered in t h e e n r i c h m e n t c u l t u r e s , was t h e s a l t c o n c e n t r a t i o n : i f s a l t - t o l e r a n t o r g a n i s m s had been s e l e c t e d , t h e system c o u l d have been operated w i t h o u t d i l u t i o n of the w a s t e s . A f u r t h e r s o u r c e of expense i s 0 , w h i c h must be supplemented to the fluidized bed r e a c t o r d u r i n g t h e r a p i d a e r o b i c g r o w t h w i t h t h e w a s t e s . T h i s c o s t i s i n e v i t a b l e w i t h a e r o b i c g r o w t h b u t w o u l d be e l i m i n a t e d i n anaerobic growth. A l l r e a c t i o n s at the s - t r i a z i n e r i n g and a f t e r i t s cleavage are hydrolytic, so anaerobic growth of appropriate o r g a n i s m s c o u l d a l s o be a n t i c i p a t e d w i t h j s - t r i a z i n e s a s s o u r c e s o f n i t r o g e n , a n d a t l e a s t two s - t r i a z i n e s s u p p o r t anaerobic growth (11, 25). I t must be r e c o g n i z e d t h a t h i g h biomass c o n c e n t r a t i o n a l o n e is insufficient for adequate t r e a t m e n t . The b i o m a s s must c o n t a i n t h e n e c e s s a r y e n z y m e s , w h i c h c a n be l o s t u n d e r c e r t a i n conditions, and 2


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it may b e n e c e s s a r y t o maintain reserves of representative wastewater o r o f , e . g . , f r o z e n a c t i v e biomass i n case there i s an i n t e r u p t i o n i n t h e production rhythm. A very d e l i c a t e problem i s t h e c o o p e r a t i o n between t h e b i o l o g i s t and t h e e n g i n e e r . The p r o j e c t will fail unless both workers a r e n o t o n l y good b u t a l s o r e s p e c t one another. This project reached i t s s c i e n t i f i c goals but d i d not a t t a i n the commercially r e q u i r e d r e a c t i o n r a t e i n our l a b . D i s c u s s i o n o f the reasons f o r t h e l o w r a t e shows them t o b e e s s e n t i a l l y t r i v i a l and t h a t biodégradation o f r e c a l c i t r a n t wastes i s a f e a s a b l e option i n waste treatment ( c f . 3 4 , 3 5 , 3 6 ) . Acknowledgments I thank M s . Annemarie Schmuckle f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . My t h a n k s t o P r o f . R . H l i t t e r and Prof. T. Leisinger f o r making a v a i l a b l e t h e f a c i l i t i e s o f t h i s Department and t o H . Grossenbacher, W. H o g r e f e , a n d P . B e i l s t e i n . T h e w o r k was s u p p o r t e d i n part by grants from t h e Swiss Federal I n s t i t u t e o f Technology, Z u r i c h , and from C i b a - G e i g y AG, B a s e l , S w i t z e r l a n d .

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RECEIVED June 11,1986


Biodegradation of S-Triazines: An Approach To Dispose of

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