Emerging Technologies in Hazardous Waste Management III

Chapter 10

Degradation of Cyanides by the White Rot Fungus Phanerochaete chrysosporium Manish M . Shah and Steven D. Aust

Downloaded by MONASH UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: February 22, 1993 | doi: 10.1021/bk-1993-0518.ch010

Biotechnology Center, Utah State University, Logan, UT 84322-4705

Potassium cyanide and various other cyanide salts (Fe, Cu, Zn, Cd and Cr) were mineralized by the white rot fungus Phanerochaete chrysosporium. At 1.5 mM potassium cyanide, the rate of mineralization was about 0.17 ± 0.01 mmoles/lit/day. P. chrysosporium also mineralized [ C]-cyanide contaminated soil (3000 ppm) using ground corn cobs as nutrient (10 ± 0.75 ppm/day). Cyanide was oxidized to the cyanyl radical by a lignin peroxidase from P. chrysosporium. We suggest that the ability of P. chrysosporium to mineralize cyanides may make it useful in the treatment of cyanide contaminated soils, sediments and aqueous wastes. 14

Lignin is a complex three dimensional biopolymer that provides structural support to plants. The structure of lignin is heterogeneous, non-specific and non-stereoselective. The complexity of its structure makes it resistant to most microbes. The white rot fungus Phanerochaete chrysosporium is known to degrade lignin to C 0 (1). The lignin degradative process of P. chrysosporium is free radical based (2,3) which gives the advantages of being non-specific and non stereoselective. Some of the known important components of the lignin degradation system of R chrysosporium include lignin peroxidases, H 0 and vertryl alcohol (2,3). R chrysosporium is also able to degrade variety of structurally diverse organopollutants (4,5). Results of various studies indicate that lignin degrading enzyme system is involved in the degradation of organopollutants by P. chrysosporium (4-6). Recent attention has focused on the possible usefulness of P. chrysosporium forbiodegradationof hazardous and environmentally persistent organopollutants (3-8). Hydrogen cyanide is the form of cyanide used most by industry. It is used as a chemical intermediate in the production of methyl methacrylate, cyanuric chloride, sodium chloride, chelators and other chemicals (9). Potassium cyanide and sodium cyanide have 2

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0097-6156/93/0518-0191$06.00/0 © 1993 American Chemical Society In Emerging Technologies in Hazardous Waste Management III; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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been designated as hazardous substances and priority pollutants by the E P A (9). Cyanide containing wastes are generated by various industries like metal plating, mining, pharmaceutical, refining, paint, electronic and others (9). The presence of cyanides in sewage plant upsets treatment processes and are toxic to fish at very low concentration (0.02 mg/1) (10). The available options for management of cyanide wastes include minimization, recycling and treatment by alkaline chlorination, ozonation, thermal destruction and biodegradation. Of all the treatment methods, alkaline chlorination is most widely used. Biological treatment of cyanide is an attractive process as the products are generally nontoxic and the process is inexpensive. In this paper, we show that R chrysosporium is able to mineralize potassium cyanide and salts of Fe, Cd, Cu, Cd, Cr(VI) in soil and liquid cultures. We also show that a pure lignin peroxidase from R chrysosporium oxidized cyanide to the cyanyl radical.

Toxicity of Cyanide to R chrysosporium Cyanide is an inhibitor of heme proteins and metal containing oxidases which include cytochrome oxidases. Cyanide forms relatively stable complexes with many metals, reacts with keto groups to form cyanohydrin, and reduces thiol groups (10). Therefore, exposure of cells to cyanide results in inactivation of respiration. Figure la and lb shows the effect of addition of cyanide on respiration (glucose metabolisms) of spores and six day old cultures of R chrysosporium (11). Fifty percent inhibition of respiration for spores and six day old cultures occured at about 100 μ Μ and 5 m M of cyanide, respectively. 14

Mineralization of [ C]KCN by R chryosporium The ability of six day old cultures of R chrysosporium to tolerate cyanide could be due in part to its ability detoxify cyanide. Figure 2 shows that R chrysosporium mineralized cyanide to C 0 and the rate of mineralization of cyanide followed first order kinetics (11). The inhibition of mineralization at 10 m M of cyanide could be partly due to its toxicity as this concentration inhibited fungal respiration by 65% (Figure lb). 2

Degradation of Cyanide in the Presence of Metals Table I shows that R chrysosporium mineralized cyanide and its salts in the presence of Fe, Cu, Zn, Cd and Cr(VI) in liquid cultures. The rate of mineralization of cyanide was about 30% ±_ 10% in 30 days and was not effected significantly by various concentration (0-1 mM) of Cu, Cd, and Zn. In the case of iron, some (-10%) stimulation was observed at lower concentration (12 μ Μ ) while higher concentration of iron inhibited mineralization of cyanide with I occurring at about 200 μ Μ (Figure 3). However, higher concentration of cyanide could reverse the inhibition of cyanide mineralization (Figure 4). It can be seen that the rate of mineralization increased with increasing in concentration of cyanide. In the case of Cr(VI), mineralization was also 50

In Emerging Technologies in Hazardous Waste Management III; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


10. SHAH AND AUST

Degradation of Cyanides by White Rot Fungus

[Cyanide]mM

Figure la. Toxicity of cyanide to spores of IL chrysosporium. Sodium cyanide and [UL- C]-glucose were added to cultures of P. chrysosporium at day 0, and 3 days later evolved [ C]-C0 was trapped and its radioactivity quantitated using liquid scintillation spectrometry. The controls were without cyanide. Glucose metabolism in the controls was considered as 100 percent. All incubations were carried out in quadruplicates in a closed system at room temperature and in stationary cultures. The data are average values with standard deviations, some of which are within the data points. Reproduced with permission from Reference 11. Copyright 1991 Academic. 14

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Figure lb. Toxicity of cyanide to six day old cultures of P. chrysosporium. Conditions were the same as in Figure la except sodium cyanide and [ULC]-glucose were added to six day old ligninolytic cultures of R chrysosporium. Reproduced with permission from Reference 11. Copyright 1991 Academic. 14



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[KCN] mM

Figure 2. Mineralization of cyanide by six day old cultures of R chrysosporium. [ C]-KCN was added to six day old cultures of P. chrysosporium. 3 days later evolved [ C]-C0 was trapped using 1M Ba(OH) . [ C]-BaC0 was separated from [ C]-Ba(CN) bycentrifugation. Radioactivity of [ C]-BaC0 was quantitated using liquid scintillation spectrometry. All incubations were carried out in quadruplicate in a closed system at room temperature and in stationary cultures. Open squares show the percent of the cyanide mineralized in three days and closed symbols show the rate of mineralization in mmoles per liter per day. The error bars (standard deviations) are within the data points in some cases. Reproduced with permission from Reference 11. Copyright 1991 Academic. 14

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Ζ Ο I

ϋ

•ο Φ Ν


Φ C

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[Fe] μΜ

Figure 3. Effect of iron on the mineralization of cyanide by R chrysosporium. Solutions of ferrocyanide [ C]-K Fe(CN) were added to 6 day old liquid cultures of P. chrysosporium and the amount of [ C]-KCN converted to [ C]-C0 determined as described in ref. (11). Data are the mean percent mineralization in 3 days ± standard deviations for triplicate incubations. 14

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Figure 4. Effect of cyanide concentration on mineralization of cyanide at 200 μΜ Fe. Reaction conditions were similar to those described in Figure 3. In Emerging Technologies in Hazardous Waste Management III; Tedder, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Table I. Mineralization of [ C]-KCN in the Presence of Metals

% mineralization


KCN KCN KCN KCN KCN KCN

+ FeS0 + CuS0 + ZnS0 4- CdS0 + Cr0

25 33 30 35 35 40

4 4 4 4

3

±2 ±4 ±3 ±2 ±3 ±5

"Culture conditions were as described inref. 11. Solution of cyanide complexes with metals were prepared by mixing various amounts of metals (0-1 mM) with 1.5 mM [ C]-KCN and they were added to six day old cultures of R chrysosporium. There was no significant change in mineralization of cyanide at different concentration of Cu, Zn and Cd. At 200 μΜ and 500 μΜ of Fe and Cr(VI), respectively, mineralization of cyanide was inhibited about 50%. 14

inhibited at higher concentration with I occurring at about 500 μΜ. Cyanide might be partly oxidized by Cr(VI) as it is a good oxidizing agent. This might help in enhancing cyanide mineralization. Iron and Cr(VI) inhibited fungal respiration (glucose metabolism) at 1200 μΜ and 100 μΜ, respectively. The mechanism of inhibition of cyanide mineralization by iron needs more study due to the complexity of iron chemistry and reactivity of cyanide. 50

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Mineralization of [ C]-KCN from Contaminated Soil Table II shows the properties of the soil used in studies of cyanide mineralization in soil. Mineralization of cyanide was observed when soil contaminated with cyanide at a final concentration of 3000 ppm was incubated with P. chrysosporium grown on corn cobs (1:1) as a nutrient (Figure 5). The rate of mineralization was 10 ppm ±_ 0.75 ppm/day. It can be seen that degradation of cyanide was continuous. Volatilization of cyanide was about 4% of the total cyanide mineralized over 30 days. Oxidation of Cyanide by Lignin Peroxidases Lignin peroxidases are the enzymes which are proposed to components of the lignin degrading system of P. chrysosporium produced during idiophasic metabolism and the production of H at the same time (3). Lignin peroxidases are activated by H 0 2

2

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be important (3). They are 0 also occurs as are typical 2


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10

oH 0

1

•

10

1

•

1

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30

Time(days)

Figure 5. Mineralization of cyanide in soil by R chrysosporium. Five grams of corn cobs previously (15 days) inoculated with P. chrysosporium were incubated with 5 grams of an agricultural silt loam soil containing 3000 ppm [ C]-KCN. The amount of [ C]-KCN converted to [ C]-C0 was determined every 3 days and plotted as a percent of the cyanide mineralized versus time. The data are means ±_ standard deviations for triplicate incubations. In most cases the standard deviation bars are within the data points. 14

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Table II. Properties of the Agricultural Silt Loam Soil in Which the Metabolism of Cyanide by R chrysosporium was Studied pH Nitrogen (ppm N0 ) Sulfur (ppm S0 ) Total nitrogen (%) Organic matter (%) Organic carbon (%) Diethylenetriaminepentoacetic acid extractable metals (ppm) Zn Fe Cu Mn Pb Ni Cr Water extractable ions (meq/100 g) Na Κ Ca Mg 3


4

6.4 18.4 6.7 0.19 4.0 2.3

2.3 35.1 1.2 29.4 0.86 0.47 0.16 0.10 0.99 16.85 3.59

peroxidases (13) and the resultant activated enzyme intermediate is called as compound I. In the presence of suitable electron donors (ie. veratryl alcohol), compound I can undergo two single electron reductions to bring the enzyme to its resting state (3). On incubation of lignin peroxidase with cyanide in the presence of H 0 , cyanyl radicals were formed suggesting that lignin peroxidase is capable of oxidizing cyanide through a free radical based mechanism (11). Figure 6a shows the PBN (*-tert-butyl-N-tert-butyl nitrone) cyanyl radical adduct observed by ESR spin trapping upon incubation of lignin peroxidase isoenzyme H2 with H 0 and cyanide. The formation of the cyanyl radical was confirmed by experiments with [ C]-KCN (Figure 6b). 2

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Advantages of P. chrysosporium for the Degradation of Cyanide Containing Wastes Cyanide containing wastes usually contain various metals like Fe, Cd, Cu, Zn and Ni. The results of our present research suggest that R chrysosporium has the ability to degrade cyanide in the presence of moderate concentrations of such metals. In addition to metals, the wastes sometimes also contain a variety of hazardous organopollutants and some of them are highly insoluble and recalcitrant (ie. polyaromatic hydrocarbons). The extracellular degradative


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ι

CAUSS

1

b ΓΑλΛΑ

Figure 6. ESR Spectrum of PBN-cyanyl radical adduct formed by lignin peroxidase H2 and H 0 . Reaction mixtures contained 10 μ Μ lignin peroxidase H2, 40 mM sodium cyanide, 500 μΜ Η Ο , and 100 mM PBN in 100 mM sodium phosphate buffer, pH 6.0. (b) [ C]-KCfc was substituted for NaCN. The receiver gain was 4 χ 10 in (a) and 2.5 χ 10 in (b). Reproduced with permission from reference 11. Copyright 1991 Academic. 2

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Degradation of Cyanides by White Rot Fungus 201


system of R chrysosporium may make it suitable for the degradation of insoluble chemicals which might be present in cyanide containing wastes. Also the nonspecific nature of the degradation system may also be useful in the case of mixtures of organopollutants. Also, under nitrogen limiting condition, the organism may compete well with other microorganisms. And biodegradation is not dependent on prior exposure to the pollutants as the lignin degradation enzymes are expressed in response to nitrogen starvation, not prior exposure to the chemicals. Degradation of organochemicals can procède to essentially nondetectable levels as there is no true Km due to the free radical based nonspecific degradative process and degradation can result in mineralization. Furthermore, degradation can be supported by inexpensive lignocellulose based nutrients such as corn cobs. Summary The white rot fungus P. chrysosporium was shown to mineralize cyanide and cyanide complexes of Fe, Cu, Cd, Zn and Cr(VI). The fungus was also able to mineralize cyanide in contaminated soil continuously using ground corn cobs as nutrient. The results also suggest that lignin degrading enzymes might be important in degradation of cyanides by R chrysosporium as cyanide was oxidized to cyanyl radical by a pure lignin peroxidase. The degradation of pollutants by white rot fungi is a free radical process so the rate of reaction would be expected to be first order with respect to concentration of chemical. The rate of mineralization by the fungus was directly proportional to the concentration of cyanide unless it became toxic. Further, cyanide wastes generally contain metal cyanides, especially in the case of metal plating industries. In the present study, we showed that R chrysosporium mineralized cyanide complexes of Fe, Cd, Cu, Zn and Cr(VI). It might be possible to use a white rot fungus based biological treatment system for detoxification of cyanide containing wastes under moderate concentration of cyanide and metals. Acknowledgements This work was supported by NIEHS grant no. ES04922. The authors would like to thank Terri Maughan for assistance in the preparation of this manuscript.

Literature Cited

1. 2. 3. 4.

Kirk, T.K.;Farrel, R.L.. Ann. Rev. Microbiol. 1987, 41, 465-505. Shoemaker, H.E., Recl. Trav. Chim. Pays - Bas 1990, 109, 255-272. Tien, M..Crit. Rev. Microbiol. 1987, 15, 141-168. Bumpus, J.Α.; Tien, M.; Wright, D.S.; Aust, S.D., Science. 1985, 228, 1434-1436.


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5. 6. 7. 8. 9. 10. 11.


Fernando, T.; Aust, S.D., In Biological Degradation and Bioremediation of Toxic Chemicals: Chaudhry, G.R.,Ed.; 1991, in press. Bumpus, J.A.; Aust, S.D., BioEssays 1986, 6, 166-170. Aust, S.D..Microb. Ecol. 1990, 20, 197-209. Eaton, D.C., Enzyme Microb. Techn. 1985, 7, 194-196. Palmer, S.A.K.;Breton, M.A.;Nunnon, T.J.; Sullivan, D.M.; Surprenant, Ν.F., Pollution Technology Review No. 158 1988, 11-45, Noye Data Corp., Park, Ridge, NJ. Knowles, C.J., Bacteriol. Rev. 1976, 40, 652-680. Shah, M.M.; Grover, T.A.; Aust, S.D.,Arch. Biochem. Biophys. 1991, 290, 173-178. October 2, 1992


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