F o l i a Microbiol.

29, 104-- 107 (1984)

Effect of Aluminium and Nickel on Aflatoxin Production by Aspergillus flavus

R. M_~LrNIa, K.G. MUKERJI a and T.A. VENKITASUBRAMANIAN b
a Mycology Laboratory, Department of Botany, University of Delhi, Delhi -- 110 007, India b Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi -- 110 007, india Received July 13, 1983

ABSTIIACT. Effect o f nickel a n d a l u m i n i u m was s t u d i e d on a f l a t o x i n a n d lipid p r o d u c t i o n b y two s t r a i n s of Aspergillus flavus in a s u c r o s e - - a s p a r a g i n e - - s a l t s m e d i u m . I n c l u s i o n o f a l u m i n i u m in t h e m e d i u m e s t a b l i s h e d a n inverse r e l a t i o n s h i p b e t w e e n a f l a t o x i n a n d lipid p r o d u c t i o n . A t lower c o n c e n t r a t i o n s a l u m i n i u m s t i m u l a t e d a f l a t o x i n p r o d u c t i o n , w h e r e a s a t h i g h e r concent r a t i o n s it s t i m u l a t e d t o t a l lipid p r o d u c t i o n . Nickel a t h i g h e r c o n c e n t r a t i o n s r e s u l t e d in a n increase in t o t a l a f l a t o x i n p r o d u c t i o n . H o w e v e r , no definite c o r r e l a t i o n w a s o b s e r v e d b e t w e e n t o t a l a f l a t o x i n a n d t o t a l lipid p r o d u c t i o n w h e n nickel was included in t h e m e d i u m .

Trace elements play an important role in the regulation of biosynthesis of secondary metabolites. Although several hypotheses have been put forward to explain the mineral element control of secondary metabolism, the mechanism of action has not yet been established (Weinberg 1978). There are several reports regarding the role of trace elements on alfatoxin production by the A. flavus group of moulds in either a synthetic medium or on natural substrates (Mateles and Adye 1965; Lee et al. 1966; Davis et al. 1967; Reddy et al. 1972; Maggon et al. 1973; Marsh et al. 1975; Bhatnagar et al. 1979; Rabie and Meyer 1981). Most striking among these is the stimulatory effect of zinc and inhibitory effect of barium (Mateles and Adye 1965; Lee et al. 1966). Further, the susceptibility or resistance of natural substrates to alfatoxin formation was shown to depend on their trace metal content (Bassir and Adekunle 1972; Maggon and Venkitasubramanian 1973; Lillehoj et al. 1974). Nickel and aluminium are known to affect the function of several metabolic pathways (Underwood 1962). Moreover, an inverse correlation has been demonstrated between aflatoxin and lipid production (Detroy and Hesseltine 1969; Shih and Marth 1975a, b). In the present investigation an attempt was made to study the effect of nickel and aluminium on alfatoxin and lipid production by two toxigenic strains of A. flavus.
MATERIALS AND METHODS

Organism. Two strains of Aspergillusflavus were used: A. flavus NRRL 5520 obtained from the Northern Regional Research Laboratory, Peoria, Illinois (USA) and A. flavus DU/KM/502 isolated from soil collected from Delhi (India).

1984
25
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METALS AND AFLATOXINPRODUCTION

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FIG. 1. Effect of aluminium (closed symbols) and nickel (open symbols) (concentration c, mg/L) on biomass (B, g/L), total aflatoxin (A, mg/L; triangles) and total lipid (L, mg/L; circles) production by A. flavus NRRL/5520 (left) and DU/KM/502 (right). The strains were maintained on glucose--peptone--agar medium (Raper and Fenne]l 1965). Spores from 5-6-d old cultures were transferred to sucr o s e - l o w salts medium (Reddy et al. 1971) prepared in deionized water. :Nickel nitrate and aluminium chloride were used at concentrations of 5, 10, 20 and 50 mg/L of the medium. Shake cultures were grown at 28 =~ 1 ~ in dark for 4 d. At the end of the incubation period mycelial d r y mass was determined b y drying the mycelium at 110 ~ for 2 h. Aflatoxins were extracted from the medium and mycelium as described previously (Reddy et al. 1971) and estimated spectrophotometrically by the method of :Nabney and :Nesbitt (1965). The total lipids were extracted from the mycelium as described by Folcb et al. (1957) and were estimated gravimetrically. Total alfatoxins a n d total lipids were expressed as m g / L of the medium. The values reported are an average of three separate sets of experiments done in duplicats.
RESULTS AND DISCUSSION

Both nickel a n d aluminium had little or no effect on vegetative growth (expressed as mycelial dry mass) (Fig. 1). However, a concentration of 50 mg

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R. M A L I N I et al.

Vol. 29

aluminium per L resulted in an increase in mycelial dry mass in A. flavus DU/KiVI/502. Aluminium at a concentration of 5 and 10 mg/L resulted in m a x i m u m stimulation of total aflatoxin production in A. flavus DU/KM/502 and N R R L 5520, respectively. Higher concentrations of nickel (10, 20 and 50 mg/L) resulted in an increase in total aflatoxin production in both strains. With both nickel and aluminium, no uniformity regarding the ratio of ariatoxin B to G was observe (data not shown). The stimulatory effect of nickel and aluminium on aflatoxin production m a y be attributed to the activation of pyruvate carboxylase (EC 6.4.1.1) and acetyl-CoA carboxylace (EC 6-4.1.2) by these two trace metals (Dixon and Webb 1958; Underwood 1962). Activation of pyruvate carboxylase m a y result in an increased production of intermediates of the citrate cycle which are known to stimulate aflatoxin production (Gupta et al. 1977). The two elements may also result in increased aflatoxin production through an increased production of malonyl-CoA by activating acetyl-CoA carboxylase. Malonate has been suggested to be an intermediate in aflatoxin biosynthesis (Gupta et al. 1975). Further, aluminium is report to activate phosphoglucomutase (EC 2.7.5.1) (Dixon and Webb 1958) which m a y result in increased levels of glycolytic intermediates and hence increased aflatoxin production (Tyagi and Venkitasubramanian 1981). With the addition of aluminium, total lipids showed a trend opposite to that shown by total aflatoxins. Aflatoxin and lipid production have an inverse relationship and share a common precursor, acetate (Detroy and Hesseltine 1969; Shih and Marth 1974a, b). It can therefore be suggested t h a t aluminium acts at an early step common to aflatoxin and lipid synthesis and stimualates aflatoxin production preferentially. With nickel, however, no correlation was obtained between total aflatoxin and total lipid production.
The first author is grateful to the National Council of Educational Research and Training, New Delhi, for the award of Junior Research Fellowship. REFERENCES ]~ASIR 0., ADEKUI~LE A. A.: Production of aflatoxin B from defined natural cultures of Aspergilluaflavus (Link.). Mycopathol.Mycol.Appl. 46, 141 (1972). BHAT~CAUa_a R.K., MgKERJI K.G., MAOOO~ K.K., VE~KXTAStrS~AM,*~IA~T.A. : Effect of lithium on aflatoxin biosynthesis by certain strains of Aspergillus flavus group. Eur.J.Appl.MicrobioLBiotechnol. 7, 99 (1979). DAVIS N.D., DIEWSR U.L., Ao~ri~toT~I V.P.: Production of aflatoxin B1 and G1 in chemically defined medium b y Aspcrgillus flavus. Mycopathol.Mycol.Appl. 31, 251 (1967). ~)ETROY R.W., I-IEssELTII~E C.W.: Not synthesis of 14C-labelled lipid and aflatoxins in resting cells of Aspergillu8 parasiticu8. Dev.Ind.Microbiol. 10, 127 (1969). DIxoN M., WEBB E.C.: Enzymes and cofactors, p. 451 in Enzymee. Longmans, Green and Co., New York 1958. FOLCH J., LEES M., SLOA~E-STA~L~.Y G.H.: A simple method for the isolation and purification of total lipids from animal tissue. J.Biol.Chem. 225, 479 (1957). GUPTA S.K., MAOGO~ K.K., V~.~rKIT,~SUS~AM~IA-~ T.A.: Regulation of aflatoxin biosynthesis. II. Comparative study of tricarboxylie acid cycle in aflatoxigenic and nonaflatoxigenic strains of AspergiUusflavus. Microbios 19, 7 (1977). GuPTA S.R., PBASA~X H.R., VisWANA~HA~r L., V~.~ITASUBRA~A~rIA~r T.A.: Malonate as a precursor in the biosynthesis of aflatoxins. J.Gen.Microbiol. 88, 317 (1975). LE~. E.G.H., Tow~csL~.Y P.M., WALX)E~ C.C.: Effect of bivalent metals on the production of aflatoxins in submerged cultures. J.Food Sci. 31,432 (1966). LIImEHOJ E.B., GARCIA W.J., LAM~ROWM. : Aspergitlusflavus infection and aflatoxin production in corn: Influence of trace elements. Appl.Microbiol. 28, 763 (1974).

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