Biotechnology Letters Vol ii No 3 207-210 (1989) Received as revised February 7

CHARACTERIZATION OF POLYSACCHARIDASE ACTIVITY OPTIMA IN THE ANAEROBIC DIGESTION OF MUNICIPAL SOLID WASTE

W.S. Adney*, C.J. Rivard, K. Grohmann, and M.E. Himmel

Applied Biological Sciences Section, Biotechnology Research Branch, Solar Fuels Research Division, Solar Energy Research Institute

1617 Cole Blvd. Golden, CO 80401

SUMMARY Cellulolytic enzymes from a laboratory anaerobic digester fed municipal solid waste were examined with respect to pH and temperature. The pH optimum was pH 6.6, considerably lower than the pH range in which digesters are normally operated (pH 7.2-7.6). The optimum temperature was between 50 and 60~ rather than the 35-37~ range in which most digesters are controlled.

INTRODUCTION

Anaerobic digestion for disposal of municipal solid waste (MSW)

has gained increased attention as a means of producing considerable

amounts of energy, while reducing the organic disposal problem with a

method less energy intensive than aerobic digestion (Gibbs and

Greenhalgh, ]983). Problems include slow rates of conversion, which

increase the retention times, reactor volumes, and capital costs. For

maximum conversion rates in producing methane from MSW feedstocks, the

slow rate of polymer hydrolysis must be enhanced (Horton et al., 1980).

This requires an understanding of the important controlling factors

governing hydrolytic enzyme production and action.

L i t t le information is available on the cel lulolyt ic bacteria found

in anaerobic digesters fed MSW (Bullock et al., 1980) or the digester-

resident hydrolytic enzymes. We recently reported the development of

numerous assays for hydrolytic enzyme activities extractable from

digester sludge (Adney et al., 1988), and now report the temperature

and pH activity optima for three important polysaccharide hydrolases.

MATERIALS AND METHODS

Reactors: The experiments were performed using four anaerobic digesters with 3.5 l i t e r working volumes constructed and operated as

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described by Henson et al. (1986). The reactors were batch-fed daily with a 5% wt/volume feed of a densified municipal solid waste feedstock obtained from Future Fuels Inc., Thief River Falls, MN. This material was processed by knife milling with a Wiley mill (model 4) equipped with a I mm rejection screen. The nutrient solution used for batch- feeding consisted of 8 g/ l i ter yeast extract (Difco), 50 mM K2HPO 4, and a trace mineral/vitamin addition as described by Balch et al. (1979).

Substrates, Enzymes, and Deterqents: The p-nitrophenyl compounds used for enzyme assay were obtained from Sigma Chemical Co. Carboxymethyl- cellulose (CMC) 7LF, obtained from Hercules, Inc. Cultures of Acidothermus cellulolyticus were prepared in-house as described by Mohagheghi et al. (1986). Cultures of Clostridium populeti were grown on 0.1% Solka Floc at 37~ in 5000 mL round-bottom shake flasks following the methods of Sleat and Mah (1985). Clarified culture broth was concentrated 20-fold by hollow-fiber ul t raf i l t rat ion (Amicon DC2, 10,000 MW cutoff membrane), diafiltered against 50 mM acetate buffer pH 5 (A. cellulolyticus) or 100 mM Tris buffer pH 7.5 (C. populeti), and then used directly. Zwittergent was obtained from Calbiochem.

Enzyme assays: Assays for endo-1,4-#-glucanase [EC 3.2.1.4] (CMCase) activities followed partially the method of Ghose et al. (1987). Under the recommended conditions of the CMCase assay, enzyme dilutions must be adjusted so that 0.5 mg glucose is released from 10 mg CMC after 30 min incubation at 50~ The final incubation mixture (1.0 mL) resulted from the addition of 0.5 mL of enzyme solution to 0.5 mL of 2% CMC in buffer. Since, in this study, enzyme activities were found directly from the amount of glucose released and not from the required enzyme dilutions as in the IUPAC recommendations, the enzyme activities from digester samples are referred to as "apparent" cellulase activities. #-Glucosidase [EC 3.2.1.21] was determined according to the method of Wood (1971) as aryl-#-glucosidase by the hydrolysis of p-nitro-phenyl- #-glucopyranoside (pNPG). e-Glucosidase [EC 3.2.1.20] activities were determined in a similar way using p-nitrophenyl-e-glucopyranoside.

Preparation of deterqent extracts from diqester sludqe: The particulates from a 30 mL sample were removed by centrifugation (15,000 x g) at 4~ for 20 min using a Sorval Model RC5B refrigerated superspeed centrifuge and SS34 rotor. The particulates were washed three times with 100 mM Tris buffer pH 7.5 and resuspended in 15 mL of buffer. The extraction procedure consisted of agitating the sample using a Fisher model 346 rotator at 37~ in the presence of the 0.1% Zwittergent detergent for 4 h The particulate material was removed by centrifugation at 15,000 x g o at 4 C for 20 min and the supernatant, after 20-fold concentration, was used to perform the enzyme assays.

Temperature and pH studies: Studies of temperature activity optima were conducted by incubating the reaction mixtures at ten temperatures as described above. Studies of pH activity optima were conducted by incubating the reaction mixture described above (including substrate and enzyme or sludge) at the appropriate temperature in a McIlvane universal buffer (McIlvane, 1921) that had been titrated to the desired pH. These buffer systems maintain relatively constant ionic strength over the specified ranges of pH. Assays of digester extracts and C. populeti cultures were performed at a substrate incubation temperature of 37~ Endoglucanase assays performed on A. cellulolyticus cultures were conducted at 65~ Data for relative T. reesei endoglucanase activities versus assay pH were obtained from Genencor (1985); assay temperatures were 50~ Buffers used in this study were not disclosed.

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RESULTS AND DISCUSSION

Examination of the results shown in Figure I clearly i l lustrates

the mismatch in temperature and pH of normal operation of anaerobic

digestion and the activity optima shown by e-glucosidase, #-

glucosidase, and endoglucanase enzymes extracted from digester sludge.

At the temperature of digester operation, #-glucosidase (an aryl-

glucosidase) and endoglucanase assays show 50% of maximum activity, and

e-glucosidase activity was only 35% of maximum. Also, the aryl-

glucosidases display somewhat broader activity-temperature curves than

that shown by the endoglucanase activity. Similarly, all three enzyme

activities from one sludge were maximal at pH 6.3 and were dist inct ly

sub-optimal at the normal digester pH which is 7.3. In this respect

the digester enzymes are markedly different from the endoglucanase

activities in cultures of A. cellulolyticus and T. reesei, for which

the optimum pH is 4.2, and also from the endoglucanase of C. populeti, which has maximal activity at pH 5.5. Maximum eglucosidase and #-

!oo

�9 ~ 8o .r (D

§ 60

40-

20- �9

0

I

D a p h o d a e .... ". f o beta-glucosidGse / / / ~ \ \ endog, ....... / r~ / '\ \ \\ [ ] / v / ,, \\ /

L \ r \

/ / I

/ , ' / i ', / , J ,

Z i digester pH I

~-~ j - " i I i ,

s 6 7 8 2s s's ~'5 s'5 6's Incubotion pH Incubotion temperature (~

Figure I. Effects of assay pH (left) and temperature (right) on three polysaccharidase activities extracted from anaerobic digester sludge.

glucosidase activit ies as a function of pH and temperature were 0.022

and 0.0052 units/mL and 0.035 and 0.015 units/mL for 20-fold

concentrated extract, respectively. Maximum apparent endoglucanase

activit ies as a function of pH for 20-fold concentrated digester

extracts, C. populeti cultures, A. cellulolyticus cultures, and 50-fold

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concentrated T. reesei cultures were 6.9 x 102 , 2.9 x 103 , 4.6 x 103 ,

and 5.2 x 105 pg glucose released min -1 mL -I preparation, respectively.

In the present study, both the temperature and the pH of normal

digester operation (dictated by requirements for optimum growth of the

methanogenic population) were found to be signif icantly far from the

optima of the three important enzymatic act ivi t ies studied. The

temperature and pH mismatch was about 20~ and 0.7 to 1.0 pH units.

Considering the two effects, temperature appears to be the most severe

in inducing act iv i ty loss. Augmentation of digester sludge with fungal

enzymes was shown highly ineffective considering the near zero level of

T. reesei endoglucanase act ivi ty at pH 7.2 (not shown). Eventually,

anaerobic digestion of cellulosic feedstocks may be improved by the in

situ augmentation with cellulases of high activit ies at pH 7.2 (such as

C. populeti), enzyme recycle, or selection oF microbial consortia.

Acknowledgement: This work was funded by the Biochemical Conversion Program of the DOE Biofuels and Municipal Waste Technology Division through FWP BF83.

REFERENCES

Adney, W.S., Rivard, C.J., Grohmann, K., and Himmel, M.E. (]988). "Detection of Extracellular Hydrolytic Enzymes in Anaerobic Digestion of Municipal Solid Waste", abstract no. 053, 1988 American Society for Microbiology Annual Meeting, Miami Beach, FL.

Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R., Wolfe, R.S. (1979). Microbiol. Rev. 43, 260-296.

Bullock, L.D., Higgins, G.M., R.B. Smith, and Swartzbaugh, J.T. (1980). Enzymatic Enhancement of Solid Waste Bioconversion, In: Energy from Biomass and Wastes IV, pp. 319-331. D.L. Klass (Chairman), Institute of Gas Technology, Chicago, IL.

Genencor, Inc. (1985). Bulletin: "Enzyme Products: Genencor Cellulase 150L", 180 Kimball Way, South San Francisco, CA.

Ghose, T.K. (]987). Pure and Appl. Chem. 59, 257-268.

Gibbs, D.F. and Greenhalgh, M.E. (1983). Biotechnology, Chemical Feedstocks and Energy Utilization, p. 49-69, Frances Pinter, London.

Henson, J.M., Bordeaux, F.M., Rivard, C.J., and Smith, P.H. (1986). Appl. Environ. Microbiol. 51, 288-292.

Horton, G.L., Rivers, D.B., and Emert, G.H. (1980). Ind. Eng. Chem. Prod. Res. Dev. 19, 422-429.

McIlvane, T.C. (1921). J. Biol. Chem. 49, 183-186.

Sleat, R. and Mah, R.A. (1985). Int. J. Syst. Bacteriol. 31, 160-163.

Wood, l.M. (1971). Biochem. J. 121, 353-362.

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