Bioresource Technology 82 (2002) 157±164

Enhancement of proteolytic enzyme activity excreted from Bacillus
stearothermophilus for a thermophilic aerobic digestion process
Young-Kee Kim, Jin-Hye Bae, Byung-Keun Oh, Won Hong Lee, Jeong-Woo Choi


Department of Chemical Engineering, Sogang University, C.P.O. Box 1142, Seoul 100-611, South Korea
Received 18 December 2000; received in revised form 11 September 2001; accepted 15 September 2001

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 classi®ed, and an enhancement of protease activity was achieved
using economical chemical additives for WAS digestion. Proteases excreted from B. stearothermophilus were classi®ed 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. Ó 2002
Elsevier Science Ltd. All rights reserved.
Keywords: Bacillus stearothermophilus; Protease; Thermophilic aerobic digestion; Waste activate sludge (WAS); Activation; Divalent ion

1. 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


Corresponding author. Tel.: +82-2-705-8480; fax: +82-2-711-0439.
E-mail address: (J.-W. Choi).

and e€ective sludge disposal, the TAD process has recently emerged as an e€ective 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 exoenzymes identi®ed as inducible catabolic enzymes
ost, 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 (Haner et al., 1994). However, the hydrolysis of proteins was not successfully achieved by exoenzymes produced by thermophilic aerobic microbes,
and no study on proteolytic activity enhancement in

0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
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0) and 0. it is essential that the activity of the proteolytic enzymes. Fe2‡ . The WAS digestion was conducted in a 250 ml ¯ask with a working volume of 150 ml. be enhanced to the maximum possible. and Zn2‡ of 2000.05 g. protein concentration was determined so as to monitor the inhibition of protease activity and classify the protease (Beynon and Bond. MnCl2 0. containing 20 ll metal ion solution at varying concentrations. namely: serine protease inhibitor ± 100 mM phenylmethylsulfonyl¯uoride (PMSF) solution in DMSO. pH 7. Proteolytic activity enhancement experiments The enhancement of protease activity was investigated by adding Ca2‡ (0±2000 lM). Batch digestion experiments were operated over a period of 80 h. and result in the release of intercellular organic substances to the aqueous medium. Stock solutions of each protease inhibitor were then prepared. the authors studied and characterized the proteases produced by Bacillus stearothermophilus (ATCC 31197). require Ca2‡ for maximum thermostability (Sonnleitiner and Fiechter. The experiment for protease classi®cation was carried out as given below: the culture solution was harvested at the early stationary phase (at 20 h). because the characteristics of WAS ¯uctuated according to the operating conditions of the plant. and bu€er were added. the supernatant was incubated after the addition of Ca2‡ and Zn2‡ for 1 h.5 at a temperature of 25 °C.3. tryptone 0. pH 5) at 55 °C. Bacteria culture and classi®cation of protease B. the 2.158 Y. In order to upgrade the performance of the TAD process. 2. DMSO.0. One gram of B. It is known from previously published research that metal ions play a role in the control of proteolytic activity (Nakamura et al. 2. is to maintain stable substrate characteristics for comparison purposes.05 g. respectively).5 g. and therefore. aspartic protease inhibitor ± 100 lg/ml pepstatin solution in DMSO. a temperature of 55 °C. These digestion conditions were used for all experiments. stearothermophilus (by fresh cell weight). The mixture was incubated for 1 h at various temperatures. These organic substrates are mainly proteins and carbohydrates. industrial WAS was occasionally collected from the wastewater treatment plant. and each of Fe2‡ . were carried out after the addition of divalent ions (Ca2‡ . Methods Industrial WAS was collected from the sludge thickener of a wastewater treatment unit in an oil re®nery plant in Korea.5 g. and the residual activity was measured at 55 °C.1 ml supernatant was incubated for 5 min. and 100 lM. 1992). metallo-protease inhibitor ± 100 mM 1. pH 8. One of the important preconditions for microbial experiments with a heterogeneous substrate. and Zn2‡ on the TAD performance. aliquots were removed from the solution and the casein hydrolysis activities of the residual protease were determined. with ATCC 31197. secreted by thermophilic bacteria. After the addition of casein as a substrate. stearothermophilus (ATCC 31197) was cultivated in a 250 ml ¯ask with 100 ml of medium (soluble starch 1 g. and intracellular and extracellular protein concentrations are slightly di€erent. The 0. Fe2‡ . 1989). After 10 min.10-phenanthroline solution in DMSO. before the residual activity was determined.5±11. In addition. The e€ect of temperature on proteolytic activity was measured in the range 45±85 °C at pH 7 (6:7  10 2 M phosphate bu€er). the initial values of total suspended solids (TSS). Each of the six tubes containing 0. Such enhancement of protease activity would allow proteases to promote the lysis of bacterial cells in WAS by the cell wall decomposition. cysteine protease inhibitor ± 100 mM E64 solution in water. and the enhancement of their activity by the addition of divalent cations. and they can be hydrolyzed to unit molecules by enzymatic activity. / Bioresource Technology 82 (2002) 157±164 WAS digestion has ever been published. such as WAS. Kim et al. The authors also investigated the e€ects of the addition of Ca2‡ .1. 1997). To estimate the e€ect of Ca2‡ and Zn2‡ on the optimum proteolytic activity temperatures. 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. respectively. Zn2‡ . with shaking at 200 rpm (ATCC.-K. and EDTA containing 0±500 lM in the assay bu€er (50 mM Tris±HCl bu€er.5 ml culture supernatant solutions in 50 mM Tris±HCl bu€er. and 10 ll of four inhibitor stock solutions. Thermophilic aerobic digestion experiment .89 ml bu€er solution (phosphate bu€er. many of the extracellular enzymes. pore 2. was inoculated after being concentrated by ®ltration (MFS membrane ®lter. dissolved organic carbon (DOC). a pH between 7. collected from the pure cell culture at the early stationary phase (20 h). and a shaker speed of 300 rpm. after incubation. The e€ect of pH on proteolytic activity was investigated in the pH range 3.1 g. were incubated at 55 °C in closed glass tubes to avoid evaporation.0 and 8. KH2 PO4 0.2.. A series of digestion tests. and crude protease solution was obtained by centrifuging the culture solution at 7500g for 20 min at room temperature. However. In this present study.0). 100. yeast extract 0. and CaCl2 0. to the authors' knowledge. Mg2‡ . Total suspended solids concentrations of the raw sludge were in the range 14±16 g/l. 1983). produced by thermophilic microorganisms.

1996).0) bu€er was incubated at 55 °C for 10 min. 159 the degree of freedom and the degree of con®dence.10phenanthroline. 0. but other transition metals may be substituted (Christopher and Nicholas. (2) E64 [cysteine protease inhibitor]. The protease classi®cation by inactivation using respective inhibitors as follows: (1) PMSF [serine protease inhibitor]. 3. Tokyo. First. Sodium dodecylsulfate. 1989). . the supernatant contained metal-activated proteases. 1998). Results and discussion 3. Japan) 2. 3. In addition. Standard deviations are represented by error bars. Korea). polyacrylamide gel electrophoresis (SDSPAGE) was used to monitor protein hydrolysis by proteases in18% SDS-acryl amide gels. The serine protease family has a characteristic set of functional amino acid residues. using bovine serum albumin as a standard (Bradford. (5) DMSO [solvent].4.5 ml of the appropriate enzyme solution and the reaction stopped by the addition of 1 ml of 10% TCA solution after 30 min. 0. …1† where x is the mean value. cysteine peptidases. The supernatant was collected by centrifugation at 5000g for 10 min and the absorbance of the supernatant measured at 376 nm versus protease K type XI as a standard (Bomio et al. Classi®cation of proteases Proteases can be classi®ed into four mechanistic groups. A 1 ml volume of the 0. 1989). In this study. indicating that the proteases excreted from B. (4) 1.45 lm. 1. / Bioresource Technology 82 (2002) 157±164 size 0. the precipitates were resuspended with lysis bu€er (50 mM Tris±HCl (pH 8. s is the standard deviation. substrate with supernatant. In order to classify the proteases excreted from B. (3) pepstatin [aspartic protease inhibitor]. serine peptidases. After electrophoresis. Mixtures were centrifuged at 12 500g for 20 min and the supernatant prepared for intracellular protein concentration determination. The results showed that PMSF and 1. The TSS were measured using the Standard Methods 2540D (APHA et al. 1 ml of 10% trichloroacetic acid (TCA) solution was added to stop proteolysis. Japan).10-phenanthroline [metallo-protease]. to form the active site (Beynon and Bond.1. and it was evaluated from a t-distribution table.5 ml of supernatant was added to a tube containing 1 ml of 1% azocasein solution (6:7  10 2 M phosphate bu€er at pH 7).10-phenanthroline inhibited protein hydrolysis. arranged in a particular con®guration. 8.27 mM N -Laurolysacosin). Analytical Protease activity was measured by two di€erent spectroscopic methods. Samples were ®ltered through pre-weighted Whatman GF/C micro®ber ®lters and dried at 90° C for 24 h. This metal is usually zinc.. Shimadzu. Metallo-peptidases utilize a metal ion as a coordinator to exert bond cleavage. and sonicated for 2 min. stearothermophilus are serine proteases.1 mM EDTA. After primary centrifugation. The mixture was kept at 4 °C for 30 min and then centrifuged at 5000g for 10 min (Beynon and Bond. aspartic peptidases. namely.15% casein solution in 50 mM Tris±HCl (pH 8. and metallo peptidases.5. Seoul. as shown in Fig. (6) control [without inhibitor]. protein hydrolysis inhibition was determined using the corresponding inhibitors of each protease class. the degree of con®dence used was 95%. mixed well. and n is the number of replications. t value depends on Fig. and substrate with 2. (7) substrate only. The reaction was initiated by the addition of 0. 1. Vision Scienti®c. stearothermophilus. The second method used was based on the measurement of casein concentration. TSS concentrations were calculated by the di€erence method. Enhancement of proteolytic activity The SDS-PAGE results of samples of substrate only.0). Toyo Roshi Kaisha. Tokyo. This tube was incubated at 55 °C in a shaking incubator (VS8480SR. Statistical analysis The con®dence limits of experimental data (l ) are calculated by the following equation: p l ˆ x  t…s= n†.-K. the gels were stained with coomassie brilliant blue R-250. DOC was assayed with a total organic carbon analyzer (TOC-5050A. since the proteolytic activity was also inhibited by 1. ADVANTEC. 1989). After 1 h.2. Casein concentrations were determined by the Bradford method. 1976).. as follows: Samples were centrifuged at 10 000g and supernatants were analyzed for DOC and extracellular protein. and all experimental results represent the mean of at least three experiments. Kim et al.Y.

The protease activity was in¯uenced by the addition of calcium sulfate.2  2. However. The activity pro®le was found to be bell shaped.5  2. / Bioresource Technology 82 (2002) 157±164 supernatant with various divalent cations are shown in Fig. In particular. The e€ect of metal ion addition on protease activity was not shown clearly enough by Fig.2  2.5. it was noted that higher concentrations of metal ions.2 ZnSO4 0 100 250 500 2000 100.7 NDb FeSO4 0 100 250 500 2000 100. Not determined. disappeared. protein hydrolysis was markedly activated (above 25%) by the addition of Ca2‡ .3. 3. 7. The e€ect of metal ions on protease activity was investigated at various concentrations.8 NDb Mean  standard error (n ˆ 3). 127. such as 5 mM ZnSO4 and 1 mM FeSO4 .1 31. and 100 lM Fe2‡ were 128. E€ective enhancement of protease activity was observed at low concentrations (below 100 lM) of Fe2‡ . and also inhibited by FeSO4 above 250 lM.1  2.3  3. 11. and 1 mM CaSO4 .5  3. quantitatively. it was decided to investigate the proteolytic activity quantitatively.0 102. and 2. and 1 mM FeSO4 . For substrate with supernatant.5 116.9  1.5±11. 50. 10. with an optimum pH of 7.0 126. and 10 or 50 lM of Zn2‡ addition. 100 lM.0  3. ferrous sulfate.3  3.5 101.5 125. The relative activities of the protease at concentrations of Table 1 The e€ect of divalent ions on the protease activity a b Reagent Concentration (lM) Relative activitya (%) CaCl2 0 100 250 500 2000 100. using a spectrophotometer. is similar to those of .5 NDb MgSO4 0 100 250 500 2000 100.3%.9 117. 2. The optimum temperature and thermal stability determined in the presence of Ca2‡ or Zn2‡ were the same as those of the experiment without a divalent ion.6 93. 50 lM. and Zn2‡ and at a relatively higher concentration (2 mM) of Ca2‡ . 10 lM Fe2‡ . 8.7  3. respectively.5 128.0%. (2) no additives. and Zn2‡ .-K.0%. Maximum protease activity was observed using 2 mM of Ca2‡ in the protease solution. 13. and magnesium sulfate.1 120.0  2.1  2. as shown in Fig. 4. 100 lM Zn2‡ . inhibited proteolytic activity.0  2. and 125. 3. and casein completely disappeared in substrates with supernatants containing either 1 mM Ca2‡ . 2. E€ect of temperature and pH on proteolytic activity Fig. 4. The optimum temperature for protease activity was determined by measuring the proteolytic activity at temperatures between 45 and 85 °C. EDTA.0  3.1  2. due to the precipitation of the Fe-enzyme complex. Fe2‡ .6  4. because of its metal chelating capacity. The e€ects of Ca2‡ and Zn2‡ addition on the optimum temperature and thermal stability of the protease are also shown in Fig.0  2. the experiment was repeated in the pH range 3. 14: with 10 lM. 3. Kim et al. EDTA and magnesium sulfate did not enhance protease activity over 15%. SDS-PAGE results of the casein hydrolysis by ATCC 31197 supernatant with the following compositions: (1) substrate only (without ATCC 31197 supernatant). To determine the e€ect of pH on protease activity. the 30 kDa fraction.1 113. when the protease was assayed with azocasein as a substrate. as shown in Table 1. 2. respectively. 100 lM. respectively. casein.0  2. The protease was inhibited by EDTA above 1 mM. stearothermophilus exhibited maximum activity at 75 °C.5. The optimum temperature of 75 °C.5 127. However.5 113. respectively. 9: with 10. zinc sulfate. 5.1  2. 12: with 10. relative to these of the control protease solution.2 123.0 NDb EDTA 0 100 250 500 2000 100. The protease excreted from B. for maximum protease activity. respectively.5 mM MgSO4 . 6: with 10.0  2. and 5 mM ZnSO4 .160 Y.5 115.0  1.7 28. and therefore. 2 mM Ca2‡ .

and 69. and 44.5 mg/ml in experiments with no metal addition (control). 100 lM Fe2‡ . 3. DOC.0. / Bioresource Technology 82 (2002) 157±164 161 value. respectively.0. 12. respectively. 1987). quoted for maximum overall extracellular endoproteolytic activity. Therefore.. The optimum pH of 7. Final extracellular protein concentrations.. these induced 35%. 5(a). However. stearothermophilus. The WAS decomposition capacity of the B. The initial concentrations of extracellular proteins were 9.4. The DOC concentration was rapidly increased by WAS destruction during the early stage of the experiment.2 mg/ml. 1998). This result di€ers from that of previous research on this topic. and extracellular and intracellular protein concentration changes. and these increased to 71. Standard deviations are represented by error bars.6. respectively. respectively.6%. with or without the addition of metal ions. were determined to characterize the thermostable protease produced by B. other thermostable proteases produced from Bacillus sp. 46. 5(b). respectively. 5(a)±(d). since this was a convenient way of representing organic carbon degradation by bacterial activity. 17%.1. In the control experiment without additives. but the temperature The enhancement of sludge digestion rate and eciency in terms of TSS. higher DOC reductions of 38% and 50% were observed in experiments involving the addition of 100 lM Fe2‡ and 2 mM of Ca2‡ . involving the addition of 100 lM FeSO4 . 100 lM ZnSO4 . using cellfree supernatant (Sonnleitiner and Fiechter.5 and 75 °C.† with 100 lM ZnSO4 . expressed as a percentage of the respective maximum concentrations. 100 lM Zn2‡ . respectively. It appears that 100 lM of Fe2‡ and 2 mM . is not claimed to be optimal for TAD process operation (Haner et al. The optimum pH and temperature of 7.4. due to the addition of metal ions.1%.-K.5 and 60 °C. were 63. 3. WEF and ASCE. … † with 2 mM CaSO4 .6%. However. The DOC reduction eciencies are quoted as the DOC percent reduction expressed as a percentage of the maximum DOC concentration. Fig. compared with the other digestion results that involved the addition of 2 mM CaCl2 . 1983). they were drastically increased over the initial 3 h and then gradually increased for a further 17 h. The thermal stability of the protease in the presence of Ca2‡ did not change when compared with that of the control solution. stearothermophilus was also estimated by DOC concentration changes and the e€ect of metal ions on DOC reduction in batch experiments. 4. The pH dependency on protease activity using ATCC 31197 supernatant without metal ions. The optimum temperature for process operation is usually 10±20 °C lower than the optimum as determined by laboratory scale experiments. The temperature dependency on protease activity by additives using an ATCC 31197 supernatant: …† without metal ion. as shown in Fig. where it was observed that calcium ions enhanced the thermal stability of bacterial proteases (Burg et al. 3. and 2 mM Ca2‡ . a reduction of DOC was achieved to 17% of the maximum DOC concentration. 75. The time course behaviors of extracellular and intracellular protein concentration are shown in Figs. 59. and 1. and B. The digestion experiment. 78. It might be postulated that Ca2‡ existing in raw WAS was sucient to support the thermal stability of the proteases. Kim et al. respectively. 1999). Extracellular protein concentrations were similar in all digestion experiments. 5(c) and (d). The relative activity was obtained by measuring the absorbance of supernatant at 376 nm. 1994. WAS digestion experiments were carried out at a pH of 7. and this was degraded by bacterial activity. Digestion experiments Fig.5 showed that the protease had high activity in neutral conditions. The time course behavior of TSS concentration is shown in Fig. is shown in Figs. Standard deviations are represented by error bars. stearothermophilus F1 (Hamer and Mason. exhibited the best decomposition eciency (approximately 37%).Y. The relative activity was obtained by measuring the absorbance of supernatant at 376 nm. TSS concentrations decreased during all digestion experiments. …. and no metal ions. and 36% reductions in the initial TSS concentration.7.8%.

(c) extracellular protein.3%. and the next involved a gradual . it is dicult to state the precise TSS degradation capacity. 5(d). … † with 2 mM CaCl2 . (b) DOC. Several stages are apparent during the course of DOC concentration change. but this comparison must be made with the sum of the extracellular and intracellular protein. Time course behavior of WAS degradation in batch digestion experiments. 5. and 2 mM Ca2‡ . the DOC reduction eciencies in experiments. (a) TSS. The ®rst stage involved a rapid increase in DOC concentration. (d) intracellular protein concentration [symbols: …† control. 3. During the early operation period. …. it appears that digestion with 100 lM Fe2‡ is superior to the other options. 5(b). the rapid decrease in TSS concentration was due to the solubilization of solid particles by the temperature increase. the extracellular protein is derived from the intracellular protein and is smaller than intracellular protein in terms of total weight. including the addition of divalent ions (except Zn2‡ ). in comparison with the DOC reduction eciency (17%) in the control experiment. demonstrated that a signi®cant TSS concentration decrease occurred during the ®nal 30 h of the experiment. of Ca2‡ were superior to control and 100 lM Zn2‡ in terms of protein degradation eciency.4% in the experiments with no metal ions added (control). WAS digestion. The reduction of TSS in the later period was dominated by lytic activity. as shown in Fig.6%.† with 100 lM ZnSO4 .-K. respectively. and 52. involving the additions of 100 lM Fe2‡ . were found to be 38% and 50% of the maximum values. Standard deviations are represented by error bars. and the solubilization of the solid particulate material. Comparison of TAD performance TSS concentration degradation was similar in all experiments. In terms of TSS concentration. TSS concentrations decreased rapidly over 12 h and then reduced slowly until the end of the digestion period.3%. with 2 mM of Ca2‡ added. The degradation eciencies of intracellular protein were 38. as shown in Fig. …O† with 100 lM FeSO4 ]. proved to be the most e€ective at degrading proteins in WAS. 38. In conclusion. This is shown in Fig. 100 lM Fe2‡ . 100 lM Zn2‡ . 42. and 2 mM Ca2‡ . Industrial WAS digestion experiments. Kim et al.162 Y. 5(a). / Bioresource Technology 82 (2002) 157±164 Fig. except for the experiment with 100 lM Zn2‡ . since the di€erence in TSS degradation eciencies was not suciently large. respectively. this might have been due to be the dual e€ects of thermal destruction and the increased proteolytic activity of enzymes produced by the thermophilic aerobic microbes. however.5. The WAS decomposition ability was also estimated by DOC concentration behavior.

R. 1985). References APHA. because Zn-binding proteases may be unstable in the TAD system.S. Fiechter. Kim et al. 100 lM FeSO4 . with respect to the initial values. Rockville. 356±362. and 52. From the result of the digestion experiments. ATCC.. Protease activity is a critical factor in terms of upgrading the performance of sludge digestion. Divalent ions. From the results of the control experiments. Both the hydrolysis and solubilization of released compounds might result in an increase in the DOC concentration. respectively (Fig. 5(b). as it was caused by lyses and hydrolysis of WAS. Also. B. which can improve the performance of the digestion process. AWWA. this compares with 38..7%. stearothermophilus. It is believed that these di€erences in the extracellular protein concentration are due to the metabolic state of the microorganisms. see also pp. 4.. / Bioresource Technology 82 (2002) 157±164 decrease over a period of 80 h. 5(d)). 20th ed. especially biological treatment of industrial wastewater produced from agricultural product manufacture. 100 lM Fe2‡ .. In: Proteolytic Enzymes: A Practical Approach.2%. But this considerable reduction in the intracellular protein concentration did not result in the accumulation of extracellular proteins. were markedly di€erent from each another.6% in the control experiment. and that this is responsible for the decomposition of soluble particulate forms. depending on the source of WAS. it was observed that protease activity was not a€ected by Zn2‡ addition. the digestion experiment with 2 mM of added Ca2‡ represented the highest protein degradation eciency. 36. 100 lM Zn2‡ .. New York. A.. it appears that the addition of Ca2‡ ions might present an e€ective method for improving the speed and eciency of the TAD process. Conversely.3%. and 2 mM CaCl2 . and the thermal stability of the extracellular enzymes involved in this proteolysis was stimulated by metal ions (Sierecka. such as Ca2‡ and Fe2‡ in the operating system. It is believed that 163 this particular protein source was further hydrolyzed and utilized by thermophilic microorganisms. Beynon. Compared to extracellular proteins. in particular. the experimental results indicate that the DOC change was induced by the release of soluble organic components caused by the lysis of microbial cells. were 40. and Bacillus neutral proteases.J. respectively. In Fig. did enhance WAS reduction. the increasing ratios of DOC concentration. However. a€ect the state of the microbial communities. 42. used in this work. respectively. And. Fe2‡ and Zn2‡ inhibited protease activity at high concentrations but stimulated protease activity at low concentrations.4%..4%. it is believed that 100 lM of FeSO4 enhanced the cellular activity. 18th ed. M. Fe2‡ is known as a general cell activator (Kendrick et al. the removed extracellular proteins. Washington. since industrial WAS. require Ca2‡ as the most important enzyme-stabilizing factor (Nakamura et al. 32. the total protein degradation eciencies were calculated as 15. p. . 1997). Ca2‡ stimulated protease activity at high concentrations. On the other hand.4%. 1998. From these experimental results. Standard Method for the Examination of Water and Wastewater. Many extracellular enzymes produced by thermophilic microorganisms require Ca2‡ for their thermo-stability (Sonnleitner and Fiechter. and 2 mM Ca2‡ .9% in the experiments with no metal ion addition (control).-K. 53. It is a relatively simple and inexpensive method employed for industrial WAS treatment.. Moreover. 27. Appreciable proteolysis activity was detected throughout the batch process. and the removed intracellular proteins expressed as a percentage of the respective initial value were 38. for thermophilic aerobic digestion. 1989. Bond. MD.4%. Acknowledgements This work was supported by Korea Research Foundation Grant (KRF-2000-041-E00394). WEF. Biotechnol. a proposed TAD process is applicable to municipal WAS treatment. intracellular proteins are present in much higher concentrations and their reduction is achieved over 30% of initial concentration. Bomio. 1989. DC. In series of batch experiments. Conclusions The use of B. and 55. expressed as a percentage of the respective maximal value at a certain stage. and 32. A proposed TAD activated by Ca2‡ addition can be successfully used in industrial treatment with upgrading performance. In the experiments with 100 lM ZnSO4 . this protease activation method can be applied to the biological wastewater treatment. Growth and biocatalytic activities of aerobic thermophilic populations in sewage sludge. From these results. 1992.Y.3%. Appl. 1992). J. 14. 26. Therefore.4%. which is dependent upon the metal ion concentration. On the basis of these results. Catalogue for Bacteria and Bacteriophages. Sonnleitner. Oxford University Press.9% of the maximum extracellular protein was removed. The intracellular protein concentration decrease followed a similar trend to the reduction in the TSS concentration. This result might be due to the different activities of the extracellular enzymes secreted from the thermophilic microbes. the long-term stability of the enzyme can be improved by the addition of Ca2‡ at elevated temperatures. is not essentially di€erent from municipal WAS in characteristics. without divalent ion addition (Fig. 1998). Microbiol. the best protein degradation eciency was achieved with an addition of 2 mM of Ca2‡ . 57±79. 5(c)). American Type Culture Collection (ATCC).

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