5 Denitrification and Removal of Heavy Metals from
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Waste Water by Immobilized Microorganisms J. HOLLÓ and J. TÓTH University of Technical Sciences, Budapest, 1521 Budapest, Hungary R. P. TENGERDY and J. E. JOHNSON Colorado State University, Fort Collins, CO 80523
This paper deals with the problem of removal of heavy metals and nitrate from waste waters by fixed-bed biological reactors. It is quite evident today that contamination by heavy metals increases simultaneously with the development of industry, e.g., the contamination of Lake Saint Claire near Detroit, the fish of the North Sea, the mercury poisoning in Minimata, Japan, or the ailments caused by cadmium in Itai-Itai. Nitrates and nitrites in drinking water may cause methhemoglobin anemia and may also contribute to the formation of nitrosamines which are highly suspected carcinogens. Nitrates and nitrites in effluents can cause eutrophication of the receiving bodies of water, the effusion of plants, seaweed and algae, and can increase BOD to such a level that capacity for self purification stops. Biological denitrification and metal removal may be especially recommended as a treatment for bringing the level of these pollutants below the present stringent water quality standards; the removal of the last traces of these pollutants probably can be done more efficiently by biological than by physical or chemical means. Biological denitrification Many facultative heterotrophic bacteria (e.g., Pseudomonas, Micrococcus, Denitrobacillus, Spirillium, Achromobacter, etc.) are capable of denitrification. Denitrification takes place under anaerobic conditions, a precondition for the formation of a nitrate-reducing enzyme system. Heterotrophic bacteria require the presence of an organic carbon source. Methanol proved to be the most suitable for giving maximal rate of nitrate reduction. The optimal methanol:nitrate ratio is 2.47 (g/g), which corresponds to a C:N mole ratio of 1 (1). In the technology of denitrification with suspension cultures, the main problem is the removal of the suspended 0-8412-0508-6/79/47-106-073$05.00/0 © 1979 American Chemical Society In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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b a c t e r i a by f l o c c u l a t i o n followed by sedimentation and/or filtration. For the e l i m i n a t i o n of these problems, fixed-bed r e a c t o r s have been a p p l i e d which have the a d d i t i o n a l advantage of o p e r a t i n g w i t h high concentrations of m i c r o b i a l mass (2,3,8). The other advantage of t h i s method i s that c a r r i e r m a t e r i a l s w i t h r e l a t i v e l y l a r g e s p e c i f i c s u r f a c e areas i n c r e a s e the adhesion and mass t r a n s f e r i n the heterogeneous phase (3)· Many fixed-bed r e a c t o r s are the extension of the t r i c k l i n g f i l t e r sewage treatment process using v a r i o u s adsorbing s u r f a c e s f o r microbes. S i k o r a and Keeney (2) s t u d i e d the d e n i t r i f i c a t i o n of a s e p t i c tank e f f l u e n t by Pseudomonas at v a r i o u s temperatures i n continuous flow columns packed w i t h limestone chips u s i n g methanol as an energy source. Nearly complete removal of n i t r a t e was a t t a i n e d i n 17 hours at 5°C, 13 hours at 13°C and l e s s than 2 hours a t 20°C; the k i n e t i c s of the system was depicted as f i r s t order; an Arrhenius r e l a t i o n s h i p was shown w i t h a c a l c u l a t e d energy of 12-25 kcal/mole NO3-N between 5-25°C. The optimum pH of Pseudomonas aeruginosa i s 7.0-8.2. One problem w i t h f i n e l y granulated i n o r g a n i c c a r r i e r s i s the high h y d r a u l i c r e s i s t a n c e and hence decreased flow r a t e . To avoid t h i s problem p l a s t i c s are now used r a t h e r e x t e n s i v e l y as c a r r i e r s f o r fixed-bed r e a c t o r s (9,10,12), i n c l u d i n g d e n i t r i f i c a t i o n (8). The shape and pore s i z e of p l a s t i c s can be c o n t r o l l e d to give d e s i r a b l e flow c h a r a c t e r i s t i c s , and the s u r f a c e can be modified f o r b e t t e r m i c r o b i a l attachment, e.g. by plasma treatment. In e a r l i e r a p p l i c a t i o n s no r i g o r o u s c o n d i t i o n s or requirements e x i s t e d f o r true immobilization of the microbes. Consequently, many c e l l s were detached from the c a r r i e r to the e f f l u e n t causing an i n c r e a s e i n the BOD and COD of the e f f l u e n t water (4). B i o l o g i c a l removal of heavy metals The c a p a c i t y of microorganisms, i n c l u d i n g algae, f o r accumulation and metabolism of heavy metals i s w e l l documented. The a p p l i c a t i o n s range from sewage and i n d u s t r i a l waste treatment (5,7) to ore l e a c h i n g (18), and to plutonium removal from h o l d i n g ponds (17). For s i m i l a r reasons as mentioned i n d e n i t r i f i c a t i o n , immobilized microorganisms would have advantages over suspended c u l t u r e s i n c o n t a i n i n g accumulated metals. In some cases, as i t w i l l be shown below, heavy metal uptake and some other m i c r o b i o l o g i c a l f u n c t i o n s , such as d e n i t r i f i c a t i o n , can be performed i n one o p e r a t i o n , using the same fixed-bed bioreactor. Heavy metal uptake i s p r i m a r i l y based on the a b i l i t y of m i c r o b i a l surfaces to complex with metal c a t i o n s . The negat i v e l y charged sugar u n i t s of p o l y s a c c h a r i d e chains, extending from the m i c r o b i a l c e l l w a l l , may complex with metal c a t i o n s .
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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E x o c e l l u l a r polyphosphate groups, excreted by b a c t e r i a , can a l s o complex metal ions through c h e l a t i o n (7_) . Membrane l i p i d s complex l e a d and probably t r a n s u r a n i c s (14,15).
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Results Immobilization of Pseudomonas aeruginosa. Seeking an improvement on some e a r l i e r fixed-bed b i o l o g i c a l r e a c t o r s , the primary c r i t e r i o n i n s e l e c t i n g a p l a s t i c c a r r i e r was the f i r m attachment of the microbe to the p l a s t i c . T h i s can be achieved i f the microbe i s e i t h e r u s i n g the p l a s t i c as a carbon source and thereby burrows i t s e l f i n t o the p l a s t i c , or i f the p l a s t i c s u r f a c e i s t r e a t e d , e.g., by plasma treatment, to f a c i l i t a t e chemical bonding. A f t e r screening a wide range of p l a s t i c m a t e r i a l s , the two most s u i t a b l e ones were s e l e c t e d : p o l y v i n y l c h l o r i d e (PVC) f i l m s and melt blown polypropylene (PP) webs. The s o f t e n e r s i n PVC are the carbon source that f a c i l i t a t e s f i r m embedment and continued metabolic a c t i v i t y i n long d u r a t i o n continuous operations (20,21). The melt blown PP has an unusually l a r g e s u r f a c e which a f t e r plasma treatment has a l a r g e c a p a c i t y f o r c e l l l o a d i n g and very s t a b l e h o l d i n g of b a c t e r i a , d e s i r a b l e f o r heavy metal removal. Immobilization of P. aeruginosa on PVC. A g l a s s column of 3 X 150 cm was l o o s e l y packed w i t h PVC sheets cut i n t o small ( l e s s than 1 cm ) pieces and s t e r i l i z e d with dry steam f o r 20 minutes. The v o i d volume was f i l l e d with a s t e r i l e mineral solution* (MS) o f : ( N H 4 N O 3 , 0.1%; ΚΗ Ρ0ί+, 0.1%; Νβ ΗΡ0ι>, 0.1%; NaCl, 0.1%; MgS0i «7H 0, 0.1%; C a C l ' 2 H 0 , 0.01%; F e C l * 6 H 0 , 0.01%. A 24 hour beef-broth c u l t u r e of P. aeruginosa (ATCC 13388) c o n t a i n i n g about 1.0 X 10 v i a b l e c e l l s / m l was i n o c u l a t e d to the MS, 2 ml/1. Temperature was kept constant at 25°C. In F i g u r e 1 i t can be seen that the change i n v i a b l e c e l l numbers was r a t h e r slow, developing i n the column r e a c t o r on the p l a s t i c s u r f a c e i n an order of magnitude of 10 c e l l s / c m i n 14 days. I f an a d d i t i o n a l Csource, glucose or methanol, was added, growth a c c e l e r a t e d . I t f o l l o w s from the f i g u r e that the p l a s t i c may serve as s o l e carbon source r e q u i r e d f o r the m u l t i p l i c a t i o n of b a c t e r i a but an a d d i t i o n a l carbon source promotes the i n i t i a l phase of attachment, due to the b e t t e r adhesion of a l a r g e number of q u i c k l y m u l t i p l y i n g b a c t e r i a , which become attached as a new l a y e r to the l a y e r of b a c t e r i a f i x e d on the p l a s t i c s u r f a c e . The same holds true f o r the a d d i t i o n of v a r i o u s t r a c e elements as n u t r i e n t supplements, e.g., calcium, magnesium, i r o n , manganese and phosphate s a l t s , which not only s t i m u l a t e the m u l t i p l i c a t i o n of c e l l s , but by s u r f a c e charge a l t e r a t i o n s a l s o a f f e c t p h y s i c a l attachment between n e g a t i v e l y charged b a c t e r i a and p o s i t i v e l y charged s u r f a c e groups of the p l a s t i c 2
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In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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Growth of Pseudomonas aeruginosa on PVC with or without added carbon source
In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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(6). Scanning e l e c t r o n micrographs (Figures 2 and 3) of the immobilized b a c t e r i a c l e a r l y show the mass of r o d - l i k e v i a b l e b a c t e r i a i n the process of c e l l - d i v i s i o n on the s u r f a c e of the p l a s t i c . T h i s proves immobilization of a considerable amount of b a c t e r i a on the s o f t PVC s u r f a c e as they have not become detached from the s u r f a c e even during p r e l i m i n a r y treatment r e q u i r e d f o r e l e c t r o n microscopy. No b a c t e r i a could be detected, on the other hand, on the s u r f a c e of l e s s s u i t a b l e p l a s t i c beds. The t o t a l number of b a c t e r i a attached to the p l a s t i c s u r f a c e was determined by the Lowry t e s t (16), the number of v i a b l e b a c t e r i a was determined c o l o r i m e t r i c a l l y by the t r i p h e n y l - t e t r a z o l i u m - c h l o r i d e (TTC) r e d u c t i o n t e s t (13). Immobilization of P. aeruginosa on PP. Melt blown, f i n e PP f i l a m e n t s (
