Enzyme and Microbial Technology 31 (2002) 419–424 The enzymology of sludge solubilisation utilising sulphate reducing systems Properties of proteases and phosphatases C.G. Whiteley ∗ , P. Heron, B. Pletschke, P.D. Rose, S. Tshivhunge, F.P. Van Jaarsveld, K. Whittington-Jones Goldfields Biotechnology Laboratory, Department of Biochemistry and Microbiology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa Received 13 July 2001; received in revised form 1 March 2002; accepted 11 March 2002 Abstract The first stage in the degradation and recycling of primary sewage sludge (PSS) and particulate organic matter is the solubilisation and enhanced hydrolysis of complex polymeric organic carbon structures associated with the anaerobic sulphidogenic environment. Protease and phosphatase enzyme activities were predominantly associated with the organic particulate matter of the sewage sludge. Sonication of the sludge gave an increase in enzyme activity as the enzymes were released into the supernatant fluid. pH optimisation studies showed a broad range of proteolytic activities with prominent enzyme activity at pH 10, while the phosphatases had greatest activity at pH 4.5. Temperature optimisation studies demonstrated neutral proteases surviving temperatures up to 70 ◦ C, those at pH 5 and 10 with temperature optima at 50 and 60 ◦ C and phosphatases at 60 ◦ C, respectively. All enzymes indicated extensive stability for several hours at their respective optimum temperatures and pH. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Enzymology; Proteases; Phosphatases; Sewage sludge; Sulphate reduction 1. Introduction Sulphate reducing bacteria (SRB) have attracted the atten- tion of biotechnologists due to their fundamental properties and possible residual water treatment process application in acid mine drainage (AMD) remediation. Complex organic molecules are hydrolysed by enzymes from fermentative bacteria to simple organic compounds. These are acted upon by H 2 producing and consuming acetogenic bacteria to yield acetate, H 2 and CO 2 which in turn are acted upon by CO 2 consuming and acetoclastic methanogenic bacte- ria. Under anaerobic conditions the simple organic electron donormolecules, e.g.lactic acid, are used by the SRB’s such as Desulfovibrio and Desulfuromonas to reduce sul- phate to hydrogen sulphide and bicarbonate with a resultant increase in pH (Eq. (1)). Soluble metal salts react with H 2 S in situ to produce insoluble metal sulphides, thereby reducing the concentration of metals and salts to acceptable levels (Eq. (2)). Bicarbonate ions react with protons to form CO 2 and water, thus removing the ‘acidity’ from solution ∗ Corresponding author. Tel.: +27-46-6038085; fax: +27-46-6223984. E-mailaddress: c.whiteley@ru.ac.za (C.G. Whiteley). as CO 2 gas (Eq. (3)): 3SO 4 2− + Lactate → 3H 2 S + 6HCO 3 − (1) H 2 S + M 2+ → MS(s) + 2H + (2) HCO 3 − + H + → CO 2 (g) + H 2 O (3) When sulphate is removed biologically from an effluent such as AMD, the two major problems that are experienced are the availability and cost of the organic carbon substrate. One method of overcoming these problems and obtaining a potentially low-cost, readily available alternative energy carbon source for sulphate reduction, has been forthcoming with the use of municipal sewage sludge [1]. SRB could thus be used for the bioremediation of sulphate-rich in- dustrial effluents, tannery effluents and AMD. The attempt to derive enough electron donors from sewage sludge to support large-scale sulphate reduction in containing AMD waters has met with limited success, probably due to insuf- ficient hydrolysis of the complex substrate. Solubilisation rates for primary sewage sludge (PSS) are slow in conventional anaerobic digestion systems [2] with maximum soluble product formation reported between 8 and 20 days, and atyields of5–10% in the mesophilic 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 1 0 0 - X