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Biology Laboratory, University of Kent,. Canterbury CT2 7NJ, England. SUMMARY. Cyanide hydratase, which converts cyanide to formamide, was induced in.
Biotechnology Letters Vol.3 No.7 363-368 (1981)

CYANIDE DEGRADATION BY IMMOBILISED FUNGI Naheed Nazly and Christopher J. Knowles* Biology Laboratory, University of Kent, Canterbury CT2 7NJ, England.

SUMMARY

Cyanide hydratase, which converts cyanide to mycelia of Stemphylium loti by growth in the ations of cyanide. Mycelia ~ere immobilised most useful system was found to be treatment This technique is applicable to a wide range that ccntain cyanide hydratase.

formamide, was induced in presence of low concentrby several methods. The with flocculating agents. of easily isolated fungi

INTRODL~TION Considerable amounts of cyanide are found in various industrial solid and aqueous wastes (Anonymous, 1976a, 1976b). This is a serious problem due to the high toxicity of cyanide, and many processes have been developed to degrade or detoxify cyanide-containing effluents. These include treatment by alkaline chlorination, hypochlorite, bleach, peroxide or ozone, or via ion exchange or electrolysis (Green & Smith, 1972; Scott & Ingles, 1980). Various attempts have been made to develop a biological process for treating cyanide-containing effluents. For exani01e, seeding acclimated activated sludge with a cyanidedegrading bacteri~n (Fujii & Oshimi, 1973). Many fungi, particularly those species that are pathogenic to cyanogenic plants, are able to degrade cyanide to formamide (Fry & Millar, 1972; Fry & Munch, 1975; Fry & Evans, 1977; Fry & Myers, 1981). The reaction catalysed is: H20 + HCN

+

HCONH 2

It is carried out by an enzyme, cyanide hydratase (formamide hydrolyase), that can be induced in spores and mycelia that have been exposed to cyanide (FqJ & Millar, 1972; Rissler & Millar, 1977). Other fungi and possibly bacteria are known to degrade cyanide to CO 2 , presumably via formamide (Knowles, 1976; Bunch & Knowles, 1980). Cyanide hydratase has been partially purified frcm Stemphylium lot~ (Fry & Millar, 1972) and shown to be fairly stable, with about half the activity retained after 2 days storage at rocm temperature (25O) and 6½ w~eks at 4°C. It has a wide pH optimum (pH 7.0 to 9.0) and half-maximal activity at 15-2OmM cyanide. We felt that organis[ns producing this enzyme might form the basis of a cyanide detoxifying system of cut,t~rcial interest. This ~Duld depend on the lower toxicity of formamide cempared to cyanide (Brinkman & Kuhn, 1975). The development of an irm~bilised system, utilising intact fungal spores, or probably better, mycelia, could offer the

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advantages of convenience, enhanced stability and re-usability, particularly if a continuous reactor were used. METHODS

The organism used in this study was S. loti Graham, which was kindly supplied by Dr. W.E. Fry (Fry & Millar, 1972). Cultures were maintained on V-8 juice agar plates (2COral Campbell's V-~ juice, 3g CaCO 3 and 15g Oxoid purified agar in 800ml H20) at 25 to 29-C under fluorescent light (about i0 lux, frQm an Atlas 13W warm-lite, 0.5m above the plates). Still cultures of mycelia, which w~re inoculated by a 5ram agar plug, were grown for 7 days in 5Oral V-8 juice broth (200ml centrifuged V-8 juice, 50mmoles sodit~n phosphate buffer, pH 7.0, in 80Oral H20) in 250mi conical flasks. The mycelia from 2 flasks were harvested, pooled, washed twice in sterile H20 and resuspended in iOml chilled minimal median minus C- and N-sources. They were blended in an Ultra-Turrax hcmogeniser for 1 minute and 4ml (about 6mg dry wt m1-1) were used to inoculate lOOml minimal medium (7.2g glucose, 2.7g alanine, 4.1g KH2PO~, 0.5g MgSO4, O.ig NaCI, O.13g CaCI2.2H20 and iml trace elements (Beadle & Tatum, 1945) in iI H20, plus KI to give O.Olmg I/-I medium and finally adjust~g the pH to 6.0) in 250mi conical flasks. They were incubated at 30vC in an orbital shaker at 200 r.p.m, for 48 hours. Where indicated, cyanide hydratase was induced by addition of KCN (0.5mM) to the mycelium 38 hours after inoculation. Mycelia were harvested by filtration, washed thoroughly with water and resuspended in 50~M Tris.HCl (pH 8.0). The mycelia were then fragmented in a hand hcmogeniser. Fragmented mycelia were inm~bilised by the method of Lee & Long (1974), using polyelectrolyte flocculating agents AIO and C7 (Rohm & Haas (U.K) Ltd., Croydon). Cyanide degradation was tested either batchwise or continuously. For batch use, 15ml of water-washed flocculated mycelia, in a lO0ml flask, were incubated with lOml of O.IM KCN in 50raM Tris. HCl (pH 8.0) and shaken at 200 r.p.m, at 28 to 30°C. Samples were withdrawn and assayed for cyanide (Lambert et al., 1975) and formamide (Snell & Snell, 1954). For continuous use, flocculated mycelia were poured into a lan x 15cm c ~ t o g r a p h y column and washed in 50raM Tris. HCI (pH 8.0). KCN (50raM) in 50raM Tris.HCl (pH 8.0) was pumped continuously at 7.5mi hour -l , and the eluate assayed for cyanide and fonman~ ide. RESULTS AND DISCUSSION The optimal condition for induction of cyanide hydratase by growing mycelia of S. loti was shown to be 0.5mM cyanide added to the growth median I0 to 12 hours before harvesting. Both higher and lower concentrations of cyanide and longer or shorter periods of exposure to cyanide gave less activity. The enzyme could be induced by addition of cyanide to a wide variety of g r ~ media. When measured in intact mycelia, cyanide hydratase had a wide pH optimum (7.0 to 9.0). The apparent K,, was 27mM and the optimal V was about 600wholes hour -I (n~ protein~ ~ . These values are in max reasonable agreement with those of Fry & Millar (1972). Activity was totally lost by incubation of the mycelia for 16 hours at 22-24-C. Storage at 4°C resulted in loss of 50% activity after 4 days.

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Mycelia were irm~bilised with the flocculating agents C7 and AIO. A wide range of pH values, mycelial protein and CT/AIO concentrations could be used for immobilisation. The optimal concentrations of C7 and AIO were 2% (w/v), added at pH 7.O and 6.5, respectively. The degradation of cyanide was linear with time with mycelial protein concentrations up to at least 9mg ml -I , and the retention of activity ccrmpared to intact, non-inlnobilised mycelia was about 35%. Fig.l shows the initial rate of degradation of cyanide and formation of formamide by a batch of immobilised mycelia. Up to at least IOCmM cyanide could be ccmpletely degraded in 2 hours by this technique. The inrnobilised mycelia were much more stable than non-immobilis~d mycelia; about 55% and 15% activity r~nained after 3 and 6 days at 24VC(Fig.2). The pH optimum was 6.5 to 9.0 and the apparent ~ was 43mM. Cyanide degradation by inmobilised mycelia was also measured in a column reactor. Cyanide was either fed periodically (continuously during the day, and the coltm~n maintained in buffer overnight) or continuously. When run continuously all the cyanide was degraded to fo~amide for a period of 50 hours, when a sudden total loss of activity occurred (Fig.3). When fed periodically the immobilised mycelia degraded cyanide for a longer period, but loss of activity was observed after a time equivalent to the same period of exposure to cyanide as the continuously fed column. Addition of a nutrient, e.g. glucose (0.5 to 50raM),to the colt~n together with cyanide resulted in an extension of the period during which cyanide was maximally degraded to about 90 hours. A gradual decrease in activity was then noted, and about 20% of the initial activity remained for another i00 hours. The c o l ~ n containing "immobilised mycelia could be used in the pH range 6.5 to 9.0, at flow rates up to at least 15ml hour -I and cyanide concentrations up to at least lOOmM. Heat treatment wastes containing NaCN or KCN often contain bari~n salts and carbonates (Anonymous, 1976a). The activity of cyanide degradation by S. loti was essentially unaffected by trace concentrations of a range of metals and by higher concentrations of barium salts and/or Na2CO 3 (up to nearly saturation). Scale-up of this system could therefore be useful in treating many cyanide-containing effluents, including heattreatment wastes. However, it was inactivated by metals such as nickel ar~ probably cannot be used for detoxifying metal finishing wastes. Inm~bilisation of S. loti by other methods (see Cheetham, 1980) was also used. Alginate could not be used due to the cc~plexing ability of cyanide. Inmobilisation by polyacrylamide and carrageenan was readily achieved but the stability was poor in ~ i s o n to mycelia irm~Dbilised with flocculating agents. Spores of S. loti, in which cyanide hydratase had been induced by exposure to 50raM glucose and O. 5mM KCN for 2 hours, were also readily immobilised. However, it is much more difficult to prepare large quantities of conidiospores than mycelia. Other fungi also form cyanide hydratase (Fry & ~{ers, 1981), scrae of which contain more of the enzyme than S. loti. The above techniques are applicable to many of these species. For example, we have recently immobilised mycelia of Gloeocercospora sorghi D. Bain & Edg. (Fry & Munch, 1975) and obtained very high activities of cyanide degradation which are particularly stable.

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O

FIG. 1

O

6O 120 MD~ES The cun~_rsion of cymLide to formm~de by an immobllised batch of S. loti

0

!

00

2

4

6

8

i0

FIG. 2 The con~_rsion of cyanide (6OmM) to formamtlde by an imm~billsed batch of S. Zoti. The stability of the system, when incubated fromdaycme ( O ~ t~rough t o d a y s e ~ n ( ~ ) at room temperature (22 to 24~C)

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~-.a I-.4 ~,°

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FOI~MAMIDE

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This work was supported by ~

a

grant from the Science Research Couhcil.

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Anonymous (1976a). Waste Management Paper No.8. Heat-treatment cyanide wastes. London: Her Majesty's Statione~7Office. Anonymous (1976b). Waste Management Paper No.11. Metal finishing wastes. London: Her Majesty's Stationary Office. Beadle, G.W. & Tatum, E.L. (1945). American J. Bot. 32, 678-686. Brinkman, G. & Kuhn, R. (1975). Water Research 14, 231-241. Bunch, A.W. & Knowles, C.J. (1980).

J. Gen. Microbiol. 116, 9-16.

Cheetham,P.S.J. (1980). In Topics in Enzyme and Fermentation Biotechnology, A. Wiseman, ed. Vol.4, pp. 189-238. Fry, W.E. & Evans, P.H. (1977). Fry, W.E. & Millar, R.L. (1972). Fry, W.E. & Munch, D.C. (1975).

Phytopathology, 67, 1001-1006. Arch.Biochem.Biophys. 151, 468-474. Physiol. Plant Pathol. 7, 23-33.

Fry, W.E. &Myers, D.F. (1981). In Cyanide in Biology, B. Vennesland, E.E. Conn, C.J. Knowles, J. Westley & F. Wissing, eds. London: Academic Press, in press. Fujii, Y. & Oshimi, T. (1973). Green, J. & Smith, D.H. (1972).

U.S. Patent No. 3756,947. Metal Finishing Journal, 229-232.

Knowles, C.J. (1976). Bacteriol. Rev. 40, 652-680. Lee, C.K. & Long, M.E. (1974). U.S. Patent No. 3,821,086. Rissler, J.F. & Millar, R.L. (1977). Plant Physiol. 60, 857-861. Scott, J.S. & Ingles, J.C. (1980). Canadian Mining Journal

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57-61,