## Abstract

Wastewater treatment and generated biological sludge provide an alternative source of enzymes to conventional industrial production methods. Here, we present a protocol for extracting enzymes from activated sludge using ultrasonication and surfactant treatment. Under optimum conditions, ultrasound disruption of activated sludge gave recovery rates of protease and cellulase enzymes equivalent to 63.1% and ∼100%, respectively. The extracting of enzymes from activated sludge represents a potentially significant, high-value, resource recovery option for biological sludge generated by municipal wastewater treatment.

## INTRODUCTION

Enzymes are high-value industrial biocatalysts with extensive applications in a wide range of manufacturing and processing sectors. The catalytic efficiency of enzymes can be several orders higher compared with inorganic chemical catalysts (e.g. metals, metal ions and metal oxides) under mild conditions (i.e. ambient temperature, atmospheric pressure and neutral pH) (Hermes et al. 1987). The global market for industrial enzymes has shown a steady increase since 1995 to $5.5 billion in 2018 and it is expected to reach$7.0 billion by 2023 at an annual growth rate of 4.9% (Arun 2018). Among various enzymes, hydrolases (i.e. hydrolytic enzymes, such as proteases, lipases and amylases) are widely used in agriculture, food and the household care industries. However, the culture medium for enzyme production is a major reason for the high cost of industrial enzymes.

Activated sludge (AS) mainly consists of various microorganisms that are capable of degrading organic pollutants in wastewater by producing substantial quantities of hydrolytic enzymes. Thus, AS is potentially a cost-effective alternative raw material for hydrolytic enzyme production. The enzymes are either adsorbed to the cell surface or embedded in the extracellular polymeric substances (EPS) of the cellular biomass in AS. Enzyme extraction following sludge floc disruption has been demonstrated and is technically feasible in bench-scale experiments (Jung et al. 2002; Gessesse et al. 2003; Marin et al. 2018). Ultrasonication is effective in destroying microbial cell membranes, releasing intracellular substances and enzymes embedded in the sludge EPS matrix (Zielewicz 2016) and is widely applied in intracellular and/or extracellular extraction (Capelo et al. 2004; Hong et al. 2017). However, the extraction efficiency is affected by the treatment conditions, including power intensity and treatment duration. Triton X100 (TX100) is a non-ionic surfactant that is widely used in cell lysis processes. It can permeabilise microbial cell membranes and improve the release of cellular proteins (Koley & Bard 2010).

No consistent, optimal approach is available for the enzyme extraction process since the growth of microorganisms and the production of bio-enzymes can be affected by a range of factors. Here, we develop a technique for enzyme extraction from AS. Enzyme activity assays for four different types of hydrolytic enzymes that are typically found in sludge were carried out, including protease, amylase, cellulase and lipase. The performance of ultrasonication in disrupting sludge flocs, applied in combination with TX100 surfactant, was examined. The patterns of enzyme activity and the viability of the sludge biomass was also investigated for the first time.

## METHODS

### Sludge samples

Sludge samples were collected from two major wastewater treatment plants in the UK, WWTP1 and WWTP2, with treatment capacities of 180,000 m3/day and 53,000 m3/day, respectively.

WWTP1 operated a standard AS process and thickened waste activated sludge (WAS) samples were collected from the thickening belts after flocculant dosing (Flopam, 0.24% w/w active) at WWTP1.

Settled sewage was treated with a biological nutrient removal (BNR) process at WWTP2, comprising anaerobic, anoxic and aerobic zones. Mixed liquor (ML) samples were collected directly at five equidistant positions along the aeration tank, which is a plug flow reactor. The mixed liquor suspended solids (MLSS) concentration at WWTP2 was typically in the range of 3,800–4,300 mg/L. The ML samples were collected with a bucket and dosed with polyacrylamide (Flopam, 0.24% w/w active) at a rate of 200 ml/20 L. The mixture was filtered after 30 minutes through a strainer bag and the flocculated sludge was collected. A WAS sample was also collected from the thickening belt, after polymer dosing.

Sludge samples were transported to the laboratory in an ice box on the day of collection and were stored in a fridge overnight at 4°C and enzyme extraction was performed the following day.

### Sludge characterisation

The total solids (TS) and volatile solids (VS) of the sludge samples were measured according to standard methods (Eaton 2005).

Protein and deoxyribonucleic (DNA) release provide indicators of cell lysis. The protein content of the sludge was determined by the Lowry method (Lowry et al. 1951) using bovine serum albumin as the standard. The DNA content was quantified by the diphenylamine method (Li et al. 2014) using salmon sperm DNA as the standard.

All measurements were completed in triplicate.

### Enzyme activity assays

Enzyme activity were measured based on the product formation during the hydrolysis reactions (Scopes 2002; Bisswanger 2011). One enzyme activity unit (U) was defined to generate 1 μmol of product per minute.

The enzyme activity assays were conducted in triplicate as follows:

• 1.

Protease activity was measured using the Lowry method described by Nabarlatz et al. (2010) with casein and L-tyrosine as the substrate and standard, respectively.

• 2.

α-Amylase activity was determined by the 3,5-dinitrosalicylic acid method with starch as the substrate and glucose as the standard, as described by Kanimozhi et al. (2014). Pre-heating treatment at 68°C was applied to deactivate β-amylase.

• 3.

Cellulase activity was measured according to the carboxymethyl cellulose (CMC) method (Ghose 1987) using CMC as the substrate and glucose the standard.

• 4.

Lipase activity was determined using p-nitrophenol palmitate as substrate and the release of p-nitrophenol was measured by continuous spectrophotometric rate determination (Pencreach & Baratti 1996).

• 5.

Dehydrogenase activity was performed following the method of Yao et al. (2010), with 2,3,5-triphenyltetrazolium chloride as the substrate and triphenyl formazan formation was determined by toluene extraction.

### Enzyme extraction protocol

The sludge was centrifuged at 2,000 g for 15 min to remove excess water content, and the sediment was washed with 10 mM Tris-HCl (pH 7.0) buffer and centrifuged again at 5,000g for 15 min. The sediment was collected and re-suspended in 10 mM Tris-HCl buffer (pH 7.0) to its original volume. The suspension was diluted with buffer and subjected to ultrasonic disruption (VCX130, Sonics & Materials, Inc., UK) for a certain time period (as prescribed by the experimental design – see later), after which the suspension was shaken at 120 rpm for 45 min and centrifuged at 12,000g for 15 min. The supernatant was collected as crude enzyme extract.

To prevent warming of the samples and to preserve biological activity in the extract, ultrasonic disruption was carried out in an ice-water bath and the ultrasound pulse was set to ten seconds on and ten seconds off; other operations including centrifugation and washing of the sludge were carried out at 4 °C.

### Optimisation of enzyme extraction protocol

Thickened sludge samples from WWTP1 were used for the optimisation process.

#### Effect of ultrasonication conditions

The operational parameters of ultrasonication include treatment duration and energy intensity. Sludge samples were diluted with 10 mM Tris-HCl (pH 7.0) buffer to a ratio of 1:5 of the original VS content before ultrasound disruption; the final solids concentration was 10.6 g VS/L.

The sonicator and probe delivered a constant amplitude (AMP) with a corresponding power input of 76 micrometres at 100% AMP. To investigate the impact of treatment duration, the diluted sludge samples were subjected to ultrasonication at 40% AMP for 2, 5, 10 and 15 min. The impact of energy intensity was examined by disrupting diluted sludge samples (a dilution factor of 5 was applied) at 20%, 40%, 60% and 80% AMP for 10 min; the corresponding energy intensities during treatment were 343, 872, 1,547 and 2,312 W/L.

#### Effect of solids content

The sludge sample had a solids content of 69.4 ± 0.42 g TS/L and 53.2 ± 0.10 g VS/L and was diluted with 10 mM Tris-HCl buffer to provide dilution factors (DF) of 2, 3, 5 and 10 before ultrasonication. The sludge disruption was performed at 40% AMP for 10 min. The specific energy input was calculated by the following equation and is shown in Table 1:
Table 1

Specific energy input by the ultrasonicator at different dilution factors for sludge samples

Dilution factor23510
Specific energy input (kJ/g VS) 21.8 30.4 49.9 99.2
Dilution factor23510
Specific energy input (kJ/g VS) 21.8 30.4 49.9 99.2

#### Effect of surfactant addition

Sludge samples were diluted (DF = 5) with 10 mM Tris-HCl (pH 7.0) buffer (containing 1% v/v TX100) and subjected to ultrasound disruption at 40% AMP and 10 min duration.

#### Patterns in sludge microbial and enzymic activities

Mixed liquor sludge samples from the aeration tank, and WAS samples from the thickening belt, at WWTP2 were used to investigate the patterns of enzyme activity and the viability of the sludge biomass.

Sludge samples were diluted (DF = 5) before ultrasonic disruption (40% AMP for 10 min, 1% v/v TX100 addition). Dehydrogenase activity was used as an indicator of the general rate of microbial activity.

### Enzyme recovery rate

The sludge sample from WWTP1 was spiked separately with commercial protease and cellulase enzyme products and was subjected to the same extraction protocol under the optimum operational conditions, to determine the enzyme recovery rate. The recovery rate (R) was calculated as follows:
where Background enzyme activity refers to the activity of the crude products extracted from sludge samples without the enzyme spike, and Spiked enzyme activity was obtained from standard enzyme profiles (data not shown).

## RESULTS AND DISCUSSION

### Enzyme extraction efficiency

#### Effect of ultrasonication conditions

Ultrasonication conditions had a profound influence on the enzyme extraction efficiency.

Figure 1 shows the effect of different durations of ultrasonication on protein and DNA release, which provide markers of the effects of the treatment on cell disruption. Protein and DNA release increased with duration, but the magnitude of the response generally declined with increasing treatment time. A similar response was also observed with the enzyme recovery patterns, with maximum enzyme activities being observed at 15 min duration, shown in Figure 2; α-amylase gave the largest overall enzyme activity in AS, followed by cellulase and protease, with maximum enzyme activity units per g VS of approximately 25, 7.5 and 3.0, respectively.

Figure 1

Effect of ultrasonication duration (minutes) on protein and DNA release from activated sludge at 40% amplitude (vertical bars represent the standard deviation, n = 3).

Figure 1

Effect of ultrasonication duration (minutes) on protein and DNA release from activated sludge at 40% amplitude (vertical bars represent the standard deviation, n = 3).

Figure 2

Effect of ultrasonication duration (minutes) on enzyme activity at 40% amplitude (vertical bars represent the standard deviation, n = 3).

Figure 2

Effect of ultrasonication duration (minutes) on enzyme activity at 40% amplitude (vertical bars represent the standard deviation, n = 3).

The ultrasonicator used in this work was AMP controlled, thus, under certain amplitudes, the energy consumption was directly proportional to the processing time. Extending the processing time from 10 to 15 min increased the energy consumption also by 1/3 (data not shown). However, the activities of protease, α-amylase and cellulase were only modestly improved with increasing energy input, by approximately 10%, 17% and 9.0%, respectively. Therefore, 10 min was selected as the optimum duration for ultrasonication treatment, which also offered practical advantages compared with the longer duration period, by reducing the sample processing time and the risk of heating the sample and enzyme denaturation. The activities of protease, α-amylase and cellulase obtained were equivalent to 2.7, 22.4 and 6.8 U/g VS, respectively.

Figure 3 shows the extraction efficiency of sludge sonication treatment at different AMP and 10 min duration; increasing the AMP raises the energy intensity. The specific enzyme activity increased with energy intensity and the maximum activity of protease, α-amylase and cellulase was 2.71 U/g VS at 872 W/L (40% AMP), 29.7 U/g VS at 2,312 W/L (80% AMP) and 7.34 U/g VS at 1,547 W/L (60% AMP), respectively. Amplitudes of 60% and 80% caused rapid heating of the samples, despite the measures adopted to control the sample temperature by completing the cellular disruption step in an iced-water bath. Reduced thermostability and damage to the chemical bonds that maintain enzyme structural conformation may account for the reduced activity of AS protease enzyme at the higher energy intensities (Nadar & Rathod 2017), compared with α-amylase and cellulase (Figure 3). Therefore, 40% AMP (energy intensity = 872 W/L) was selected as the optimum energy intensity level for further experiments.

Figure 3

Effect of ultrasonication energy intensity (for 10 min duration) on enzyme activity; activity of protease and cellulase are shown on the left axis and α-amylase is shown on the right axis (vertical bars represent the standard deviation, n = 3).

Figure 3

Effect of ultrasonication energy intensity (for 10 min duration) on enzyme activity; activity of protease and cellulase are shown on the left axis and α-amylase is shown on the right axis (vertical bars represent the standard deviation, n = 3).

The extracellular enzymes of bacteria are either accumulated in the gel-like EPS matrix, which has a three-dimensional structure with an extremely large surface area that holds microbial cells together to form the sludge floc, or tightly bound to cell membranes via hydrogen and/or ionic bonding (Yu et al. 2007; Lin et al. 2014). A possible hypothesis for the mechanism of enzyme release from AS by ultrasound treatment is that the cavitation generated by ultrasound disrupts the EPS matrix that acts as a protection layer of enzymes, after which the enzymes in the EPS are exposed to the surrounding aqueous solution and are readily detached by shear forces induced through shaking within the solution (Wingender et al. 1999; Karn et al. 2013; Nadar & Rathod 2017). Cavitation generated by ultrasound also produces pores on the cell membrane, releasing periplasmic enzymes and cellular proteins (Loustau et al. 2018). In this study, DNA, which is mainly located in the nucleoid of bacteria, was detected in the crude enzyme extract, indicating that the disruption of cell membranes had occurred. Consistent exposure to cavitation, which, in the case of this study, was provided through extended ultrasound treatment time, causes more severe damage of the weakened cell membrane and the release of intracellular substances to solution.

The results from the ultrasonication extraction performance analysis showed that short duration or low amplitude (corresponding to low energy intensity) were not effective at cellular disruption and enzyme release. Therefore, it is necessary to provide suitable conditions that achieve the ‘lysis threshold’ when using ultrasound for cell disruption, and this is specific to the type of cells under investigation (Rubin et al. 2018). Our results were consistent with results by Zhang et al. (2007) and demonstrated effective AS cell lysis after 10 min ultrasonication. Ras et al. (2008) found that protein release increased with ultrasound treatment time, from 100 mg protein/g VSS after 2 min to 160 mg protein/g VSS after 10 min. Hong et al. (2017) also found enhanced cell lysis, and greater EPS release, occurred with higher ultrasound power intensities and longer treatment times.

#### Effect of solids content

The fundamental principle of sonication treatment is the conversion of electrical energy to mechanical vibrations. Extensive micro-bubbles (cavities) are produced, which expand and implode violently within a certain dispersion radius, generating extremely high pressures that destroy microbial cells. Consequently, the ultrasonication probe has an effective range over which the mechanical energy is gradually consumed, depending on the operational conditions and solids content. Therefore, an optimum solids content to ensure efficient cellular disruption of the sludge sample is necessary to maximise enzyme release per unit mass of sludge.

Figure 4 shows the enzyme activities of protease, α-amylase and cellulase with increasing sludge dilution; the maximum activities were obtained at DF = 5 and were equivalent to approximately 3.0, 31 and 10.4 U/g VS activity, respectively. Increasing the DF from 5 to 10 almost doubled the energy input from 49.9 kJ/g VS to 99.2 kJ/g VS (see Table 1) but had no effect on enzyme activity. Therefore, the solids content of sludge samples for sonication were prepared at the optimum DF = 5 (approximately 10 g VS/L) in subsequent experiments.

Figure 4

Effect of solids content on enzyme activity after ultrasonication treatment for 10 min duration and 40% amplitude (vertical bars represent the standard deviation, n = 3).

Figure 4

Effect of solids content on enzyme activity after ultrasonication treatment for 10 min duration and 40% amplitude (vertical bars represent the standard deviation, n = 3).

#### Effect of surfactant addition

TX100 increased the release of extracellular proteins, which would be mainly associated with the EPS fraction of the sludge flocs, and more than twice the amount of protein was measured with TX100 compared with the control treatment without surfactant addition (Table 2). However, no effect of TX100 was observed on DNA release or cell lysis. This behaviour was consistent with results reported by Glauche et al. (2017) showing that the addition of TX100 (2% v/v) for cell disruption of E. coli maximised the concentration of soluble proteins.

Table 2

Effect of surfactant addition on sludge disruption and enzyme activity

Protease (U/g VS)α-Amylase (U/g VS)Cellulase (U/g VS)Protein release (mg/g VS)DNA release (mg/g VS)
Without TX 2.71 ± 0.11 22.39 ± 1.53 6.83 ± 0.02 54.70 ± 0.42 127.63 ± 15.57
With 1% TX 5.62 ± 0.04 24.64 ± 0.59 7.72 ± 0.25 128.66 ± 8.22 126.43 ± 8.82
Improvement +107.6% +10.0% +13.1% +135.2% −0.94%
Protease (U/g VS)α-Amylase (U/g VS)Cellulase (U/g VS)Protein release (mg/g VS)DNA release (mg/g VS)
Without TX 2.71 ± 0.11 22.39 ± 1.53 6.83 ± 0.02 54.70 ± 0.42 127.63 ± 15.57
With 1% TX 5.62 ± 0.04 24.64 ± 0.59 7.72 ± 0.25 128.66 ± 8.22 126.43 ± 8.82
Improvement +107.6% +10.0% +13.1% +135.2% −0.94%

The results from the surfactant experiments (Table 2) showed that the addition of 1% (v/v) TX100 significantly improved protease release, almost doubling the activity of this enzyme. Relative to protease, surfactant addition had a comparatively smaller effect on the activity of α-amylase and cellulase. The effect of TX100 addition on the enzymic activity of AS extracts may be explained by the cellular distribution of the enzymes. For example, according to Yu et al. (2007), the majority of protease enzymes was found attached to the cell wall, whilst amylase and other, related, sugar-degrading enzymes were mainly found in loosely bound EPS (LB-EPS). Therefore, surfactant treatment increases protease extraction by removing the protective EPS layer. By contrast, amylase and cellulase are present in LB-EPS and are readily extracted directly without TX addition.

### Patterns in sludge microbial and enzymic activities

Specific enzyme activities measured at the different sampling points of the AS aeration tank at WWTP2 are shown in Figure 5. Sludge microbial activity was determined based on dehydrogenase activity, and increased from the inlet of the aeration tank to the centre position (Point 3), and the maximum rate was equivalent to 5 U/g VS. As may be expected, the rate of microbial activity decreased at the end of the aeration tank, by approximately 35%, reflecting substrate exhaustion. Dehydrogenase is an oxidoreductase that plays an important role in catalysing biochemical reactions for microbial dissimilation. It is involved in transferring electrons from the substrate to an electron acceptor, usually NAD+/NADP+, in the cell. The enzyme activity is indicative of the rate of electron transport when active microorganisms oxidise organic pollutants in wastewater, and there is a strong correlation between dehydrogenase activity and microbial oxygen uptake rate (OUR) (Awong et al. 1985; Bohacz 2018). Thus, higher OUR indicates higher rates of microbiological activity. Consequently, dehydrogenase is frequently used for measuring, and provides an effective indicator of, the activity of the microbial biomass in AS (Goel et al. 1998; Feng et al. 2016; Robledo-Mahón et al. 2019). Indeed, the results reported here are consistent with the microbial growth observed in plug-flow aerobic biological sewage treatment reactors (Tchobanoglous et al. 2014), where returned AS is activated when combined with the incoming substrate stream in settled sewage, and reduced microbial activity and growth rates are observed due to endogenous respiration as substrates are exhausted.

Figure 5

Enzyme and sludge microbial activity (as dehydrogenase activity) at different sampling points along the length of an activated sludge aeration reactor; sample point 6 was taken from the activated sludge thickening belt (vertical bars represent the standard deviation, n = 3). *Note: preheat treatment was omitted from the enzyme assay and total amylase activity is reported (including α- and β-amylase).

Figure 5

Enzyme and sludge microbial activity (as dehydrogenase activity) at different sampling points along the length of an activated sludge aeration reactor; sample point 6 was taken from the activated sludge thickening belt (vertical bars represent the standard deviation, n = 3). *Note: preheat treatment was omitted from the enzyme assay and total amylase activity is reported (including α- and β-amylase).

By contrast, hydrolytic enzyme activities in the AS extract did not follow the same patterns observed in dehydrogenase activity and were generally relatively consistent at the different sampling locations and there was no statistically significant correlation between them (P-values of Pearson correlations for protease, amylase and cellulase activity relative to dehydrogenase activity were 0.34, 0.60 and 0.26, respectively). The maximum activities of the extracted enzymes from the aeration tank were: 8.2 U/g VS, 52.2 U/g VS and 9.9 U/g VS for protease, amylase and cellulase, respectively, which were also comparable to those observed for sludge sampled from the thickening belt (Point 6). The apparent maintenance of hydrolytic enzyme activities even under conditions of severe nutrient deprivation may be related to a microbial ecological strategy to survive extreme conditions. Indeed, Kovárová-Kovar & Egli (1998) showed that hydrolytic enzymes involved in bacteria carbon catabolism are active not only when organic substrates are sufficient at the inlet to the AS process, but also when organic carbon sources are not available. The enzymes are maintained within the sludge flocs, mainly in the EPS fraction of the cell, and allow the hydrolysis and rapid assimilation of new substrates when they become available in the surrounding environment, without the need to divert resources to enzyme synthesis.

From a practical perspective, the results also demonstrated that thickened WAS was a suitable source of biomass for enzyme extraction recovery that was easy to access and collect and had a comparable enzyme activity to mixed liquor from the aeration tank.

### Enzyme recovery rate

The activities of four different hydrolytic enzymes (protease, lipase, amylase and cellulase), representing the enzymes responsible for hydrolyzing the main organic constituents in urban wastewater (protein, fat, starch and cellulose, respectively) were measured in AS samples collected from the thickening belt at WWTP1 following the optimum ultrasonication extraction protocol (40% AMP, 10 min duration, DF = 5, 1% v/v TX100 addition) (Table 3). The activities of the extracted enzymes were similar between different batches of sludge (data not shown), indicating the developed protocol for enzyme extraction was consistent and not influenced by variations in treatment conditions at the WWTP.

Table 3

Average activities of major hydrolytic enzymes extracted from activated sludge under optimal conditions (n = 3)

ProteaseLipaseAmylaseaCellulase
Specific activity (U/g VS) 8.40 ± 0.19 21.72 ± 1.88 39.4 ± 0.31 13.48 ± 0.08
ProteaseLipaseAmylaseaCellulase
Specific activity (U/g VS) 8.40 ± 0.19 21.72 ± 1.88 39.4 ± 0.31 13.48 ± 0.08

aNote: preheat treatment was omitted from the enzyme assay and total amylase activity is reported (including α- and β-amylase).

Protease and cellulase were selected to examine the recovery rate of enzymes from sludge using the proposed protocol. The recovery rate was 63.1% and 115.3% for protease and cellulase, respectively. Similar recovery rates for protease have been reported. For example, Ni et al. (2017) obtained a recovery rate of 66.7% by stirring sludge with TX100 for 60 min. Interestingly, the activity of cellulase in the sludge extract was apparently increased with the addition of commercial enzymes (see Methods section), thus a recovery rate >100% was given by the activity assay. Such hyperactivation of enzymes may be explained by conformational changes of the enzyme structure caused by ultrasonication under certain circumstances: ultrasound treatment can cause the breakage of covalent bonds within the enzyme (Ladole et al. 2017), releasing more active sites from inside the protein structure, resulting in higher apparent enzymatic activity when exposed to the substrate (CMC in this case). Ladole et al. (2017) found the hyperactivation for cellulase by ultrasonication was linked to significant changes in the α-helix and β-sheet ratio within the secondary protein structure of the enzyme. Ultrasound treatment can also improve activity through increased mixing and diffusion of the reactive components of both substrate and enzyme (Capelo et al. 2004).

## CONCLUSIONS

Ultrasonication was effective at disrupting AS flocs and releasing hydrolytic enzymes and the results demonstrated that the developed protocol was a suitable approach for extracting enzymes from WAS. The optimum operational parameters were: 40% AMP (equivalent to an energy intensity of 872 W/L) and 10 min duration. The solids content of sludge samples was also an important parameter influencing ultrasonic disruption and optimisation experiments indicated that a solids content of approximately 10 g VS/L (DF = 5) provided the maximum enzyme activity. Surfactant addition (1% v/v TX100) enhanced protein release as well as enzyme activity in the AS extract. Under optimum conditions, the recovery rates of protease and cellulase were 63.1% and ∼100%, respectively.

No correlation was found between sludge microbial activity and the activity of hydrolytic enzymes. Therefore, thickened WAS, collected directly following secondary clarification, is a viable and practical source of biomass for enzyme extraction.

Hydrolytic enzymes play a significant role not only in the water industry (e.g. pre-treatment of wastewater and sludge digestion), but also in other industries (e.g. agriculture and house-care). Therefore, future work will focus on the purification and concentration of hydrolytic enzymes from AS to improve the industrial utility and applications of the enzyme products.

## ACKNOWLEDGEMENTS

The authors thank the China Scholarship Council and Yorkshire Water for financial support. We also thank Thames Water for supplying samples of activated sludge for the project.

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