Elsevier

Bioresource Technology

Volume 82, Issue 2, April 2002, Pages 157-164
Bioresource Technology

Enhancement of proteolytic enzyme activity excreted from Bacillus stearothermophilus for a thermophilic aerobic digestion process

https://doi.org/10.1016/S0960-8524(01)00177-8Get rights and content

Abstract

Proteolysis is one of the main enzymatic reactions involved in waste activated sludge (WAS) digestion. In this study, proteases excreted from Bacillus stearothermophilus (ATCC 31197) were classified, and an enhancement of protease activity was achieved using economical chemical additives for WAS digestion. Proteases excreted from B. stearothermophilus were classified into two families: serine and metallo-proteases. Various metal ions were investigated as additives which could potentially enhance protease activity. It was observed that Ca2+ and Fe2+ could markedly activate these enzymes. These results were applied to thermophilic aerobic digestion (TAD) of industrial WAS using B. stearothermophilus. The addition of these divalent ions enhanced the degradation performance of the TAD process in terms of reducing the total suspended solids (TSSs), the dissolved organic carbon (DOC) content, and the intracellular and extracellular protein concentrations. The best result, with respect to protein reduction in a digestion experiment, was obtained by the addition of 2 mM Ca2+. Therefore, a proposed TAD process activated by calcium addition can be successfully used for industrial and municipal WAS digestion to the upgrading of TAD process performance.

Introduction

The volume of waste activated sludge (WAS) produced from biological wastewater treatment processes has drastically increased as a result of the quantitative and qualitative expansion of wastewater treatment, and stringent environmental regulations. This has resulted in a water pollution problem becoming a solid waste disposal problem. WAS is the main by-product of biological wastewater treatment processes and usually consists of 70% organic matter (Christopher and Nicholas, 1996). Biological stabilization is considered as one of the more attractive methods of reducing the major portion of the organic fraction in WAS. Of the biological stabilization approaches, thermophilic aerobic digestion (TAD) has some advantages compared to conventional mesophilic anaerobic digestion; faster reaction, shorter retention time, and stability against shock loads or toxic materials (WEF and ASCE, 1998). Also, from the viewpoint of pathogenic microorganism inactivation and effective sludge disposal, the TAD process has recently emerged as an effective process for WAS treatment (Hamer and Zwiefelhofer, 1986).

Thermo-stable proteases, produced from thermophilic bacteria, are essential for the degradation of WAS in the TAD process, and several researchers have reported that thermophilic aerobic bacterial culture supernatants are able to hydrolyze various soluble proteins (Bomio et al., 1989; Hamer and Mason, 1987). Also it has been reported that aquatic microbes synthesize exo-enzymes identified as inducible catabolic enzymes (Chröst, 1990), and Bacillus spp., which are known to produce heat stable extracellular proteases; these degrade vegetative bacterial cells (Kume and Fujio, 1990). Proteolytic cleavage of peptide bonds, by protease, is the main enzymatic activity of the sludge digestion process. Furthermore, considerable attention has been paid to the enzymatic hydrolysis of proteins for the improvement of sludge digestibility (Bomio et al., 1989), since protein hydrolysis is regarded as the rate limiting step during WAS digestion (Häner et al., 1994). However, the hydrolysis of proteins was not successfully achieved by exo-enzymes produced by thermophilic aerobic microbes, and no study on proteolytic activity enhancement in WAS digestion has ever been published, to the authors' knowledge.

In order to upgrade the performance of the TAD process, it is essential that the activity of the proteolytic enzymes, secreted by thermophilic bacteria, be enhanced to the maximum possible. Such enhancement of protease activity would allow proteases to promote the lysis of bacterial cells in WAS by the cell wall decomposition, and result in the release of intercellular organic substances to the aqueous medium. These organic substrates are mainly proteins and carbohydrates, and they can be hydrolyzed to unit molecules by enzymatic activity. It is known from previously published research that metal ions play a role in the control of proteolytic activity (Nakamura et al., 1997). Thermophilic Bacilli's proteolytic-neutral protease and several related bacterial metallo endo-peptidases form a family in which Zn and Ca-binding sites are well conserved in their structure. In addition, many of the extracellular enzymes, produced by thermophilic microorganisms, require Ca2+ for maximum thermostability (Sonnleitiner and Fiechter, 1983).

In this present study, the authors studied and characterized the proteases produced by Bacillus stearothermophilus (ATCC 31197), and the enhancement of their activity by the addition of divalent cations. The authors also investigated the effects of the addition of Ca2+,Fe2+, and Zn2+ on the TAD performance.

Section snippets

Bacteria culture and classification of protease

B. stearothermophilus (ATCC 31197) was cultivated in a 250 ml flask with 100 ml of medium (soluble starch 1 g, tryptone 0.5 g, yeast extract 0.5 g, MnCl2 0.05 g, KH2PO4 0.1 g, and CaCl2 0.05 g, pH 5) at 55 °C, with shaking at 200 rpm (ATCC, 1992).

The experiment for protease classification was carried out as given below: the culture solution was harvested at the early stationary phase (at 20 h), and crude protease solution was obtained by centrifuging the culture solution at 7500g for 20 min at

Classification of proteases

Proteases can be classified into four mechanistic groups, namely, serine peptidases, cysteine peptidases, aspartic peptidases, and metallo peptidases. In order to classify the proteases excreted from B. stearothermophilus, protein hydrolysis inhibition was determined using the corresponding inhibitors of each protease class. The results showed that PMSF and 1,10-phenanthroline inhibited protein hydrolysis, as shown in Fig. 1, indicating that the proteases excreted from B. stearothermophilus are

Conclusions

The use of B. stearothermophilus, for thermophilic aerobic digestion, did enhance WAS reduction. It is a relatively simple and inexpensive method employed for industrial WAS treatment. Divalent ions, such as Ca2+ and Fe2+ in the operating system, affect the state of the microbial communities. Protease activity is a critical factor in terms of upgrading the performance of sludge digestion. From these experimental results, Fe2+ and Zn2+ inhibited protease activity at high concentrations but

Acknowledgments

This work was supported by Korea Research Foundation Grant (KRF-2000-041-E00394).

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