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ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Review of Enzymatic Sludge Hydrolysis

GUO Jin-song* and Xu Yu-feng
Key Laboratory of the Three Gorges Reservoir Region’s Eco-environment, Ministry of Education, Chongqing University, Chongqing 400030, P. R. China
Corresponding Author : GUO Jin-song
Key Laboratory of the Three Gorges Reservoir Region’s Eco-environment
Ministry of Education
Chongqing University
Chongqing 400030, P. R. China
E-mail: [email protected]
Received October 24, 2011; Accepted November 15, 2011; Published November 17, 2011
Citation: Guo Js, Xu YF (2011) Review of Enzymatic Sludge Hydrolysis. J Bioremed Biodegrad 2:130. doi: 10.4172/2155-6199.1000130
Copyright: © 2011 Guo Js, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Abstract

Biotechnology is the major process in urban sewage treatment, the operation of which generates a large amount of excess sludge. Treatment of excess sludge has become a major environmental problem. The present article reviews the enzymatic cleavage of activated sludge, which involves the promotion of sludge floc cracking by extracellular enzymes. Appropriate filamentous fungi may have a positive effect on strengthening sewage treatment efficiency and recessive growth reduction. Based on the extracellular enzyme distribution in the sludge flocs, extracellular polymeric substances may be an important factor in inhibiting enzymatic sludge cracking. Furthermore, the key to enhancing the enzymatic activity is by breaking the sludge flocs. Several treatment processes that enhance the enzymatic sludge cleavage are included in this paper.

Keywords
Sludge hydrolysis; Extracellular hydrolase; EPS (extracellular polymeric substances); Strengthening process.
Introduction
The activated sludge process is the most widely used method for biological wastewater treatment in the world. It offers several advantages, such as low investment, mature technology, and efficient performance. However, as a result of wastewater purification, a large amount of excess sludge is generated, which must in turn be properly disposed. According to statistical facts, developed Western countries have been experiencing an annual increase in the production of excess sludge since the 1980s. Among European Union member states, the excessive sludge for treatment increased from 9.4 million tons in 2005 to 10 million in 2007. Excess sludge production in the United States alone rose from 7.6 million tons in 2005 to 8.2 million tons in 2010 [1-2]. Within the context of accelerated urbanization in China, which is characterized by population and industrial expansion, expanding sewage treatment capacity, and regulatory standard development, the production of excess sludge experienced unparalleled acceleration. The State Environmental Protection Administration reports that the annual sludge (dry weight) production surged from approximately 5.66 million tons in 2005 to about 11.2 million tons in 2010. This result indicates an annual incremental increase of 20%, which corresponds to the continuing decrease in land resources that could be used for sludge storage or disposal via landfills due to the limited land area per capita. Therefore, sludge treatment and disposal have become an increasingly significant challenge in the field of environmental engineering.
Sludge minimization technologies aim to reduce sludge production in biological wastewater and ensure that treatment remains effective at all times under similar conditions. A number of approaches to minimizing biological sludge production in conventional wastewater treatment plants are available. Different processes are categorized based on the location of the plant where minimization takes place. Two main strategies have been identified, namely, in the waste waterline or in the sludge line. Processes in the sludge line specifically refer to the reduction of excess sludge production via enhanced treatment (such as anaerobic fermentation) of the sludge. Processes in the water line refer to the reduction of sludge production in the biological wastewater treatment by introducing certain technologies (such as physical, chemical, and biological methods). These technologies aim to enhance lysis-cryptic growth, the uncoupling and maintenance of metabolism, and the predation on sludge bacteria to reduce the final stream of sludge for disposal [3-4]. In practical terms, the ideal way of solving the problem is to minimize excess sludge production during wastewater treatment rather than treating the sludge after its generation. However, introducing new technologies typically brings problems common to new discoveries, such as the introduction of physical and chemical methods that would result in additional energy consumption, high cost, secondary pollution, and other economic costs [5]. Introducing new biological methods would result in problems such as dealing with time-consuming techniques and harsher reaction conditions, to name a few. Overall, the introduction of new methods would create further problems in energy consumption, introduce additional cost, and cause environmental harm [6-9]. Thus, an increasing number of studies have been focusing on better alternatives for sludge treatment, such as lysiscryptic growth, uncoupling and maintenance metabolism, and bacterial predation [10-14]. The present paper reviews sludge hydrolysis via an enzymatic reaction to reduce wastewater sludge generation.
Enzymatic Sludge Hydrolysis
In the activated sludge process, bacteria tend to aggregate and form sludge flocs, which consist of microbial, prokaryotic (bacteria, archaea), and eukaryotic (algae, fungi) microorganisms kept together by extracellular polymeric substances (EPS). Various studies reported that sludge flocs constitute 60%-70% of the organic fraction [15]. In fact, microbial cells undergo lysis or death, during which the cell contents (substrates and nutrients) are released into the medium and provide an autochthonous substrate that is subsequently used in microbial metabolism. Microbial metabolism and a portion of the carbon are released as products of respiration, resulting in a reduced overall biomass production. This process is known as lysis-cryptic growth, which was first introduced by Ryan [13]. Lysis-cryptic growth involves two stages: lysis and biodegradation. As the first step in cell fractionation, cell lysis refers to the cell destruction of microbial cells as catalyzed by a hydrolytic enzyme (mainly protease).
During this process, the cell contents (substrates and nutrients) are released into the medium and provide an autochthonous substrate that is used in microbial metabolism. The biomass then grows on an organic lysate; however, this growth is different from that on the original substrate and is therefore termed as cryptic. The rate-limiting step of lysis-cryptic growth is the lysis stage [13]. An increase in the lysis efficiency can therefore lead to an overall reduction in sludge production and play an important role in reducing investment and operational costs as well as in optimizing the existing sewage treatment system. Hydrolase can enhance the sludge hydrolysis. Previous studies have shown that some microbial strains, which exhibit extracellular hydrolytic enzyme secretions, or other commercial hydrolytic enzymes are often directly used in reactors to promote sludge lysis. table 1 lists some enzymes that the hydrolysis of complex organic structures in the degradation of biodegradable particulate organic matters heavily depends on hydrolytic enzymes. Hydrolases, including protease, amylase, and lipase enzymes, are produced by a large number of mixed flora. Hydrolysis is the primary agent of deflocculation, hydrolysis, and oxidation of sludge flocs. It is also the first step in breaking down big molecules. The large, insoluble organic molecules in activated sludge flocs can be broken down into simpler carbohydrate molecules by the action of hydrolytic enzymes. The first step in protein degradation involves breaking the protein down into peptides, or into two peptides and amino acids. Amino acids can be further converted into some low-molecular weight organic acids, ammonia, and carbon dioxide. The following sections review some enzymes that can enhance sludge hydrolysis.
Lysozyme
The key to lysis-cryptic growth is lysis as well as the disruption of the microorganism cell structure because the major components of activated sludge are the microbial cells bound in the sludge flocs. Lysozyme, which is an enzyme that can dissolve polysaccharides as substrates in the bacterial cell wall, is also known as muramidase or N-acetylmuramide glycanhydrolase. Its mechanism of action involves the lysis of β-1,4 glycosidic that bond between N-acetyl glucosamine and N-acetylmuramic acid in the bacterial cell wall. The insoluble mucopolysaccharide in the bacterial cell wall can then be dissolved into a soluble glycopeptide. The bacterial cell wall is then ruptured and dissolved, and the cytoplasm is released. Lysozyme not only affects lysis, it can also degrade organic molecules. Yasunori et al. [18] compared the difference between the removal of VSS (Volatile solids) in a concentrated excess sludge inoculated with slime bacteria that secrete lysozyme and that in concentrated excess sludge without any inoculation. They found that the removal of VSS in the inoculated sludge reached 62%, whereas that in concentrated excess sludge without inoculation was only 9.8% after five days of shake cultivation. Masahiko et al. [19] isolated a new thermophilic bacterium, B-acillus stearotherm ophilus SPT2-1 [FERM P-15395], which can secrete lysozyme, and applied it to the liquefaction of the sludge floc. which had yielded highly desirable results. Ogawa et al. [20] activated the bacterial strain B. stearotherm ophilus SPT2-1 [FERM P-15395], which also secretes lysozyme, and inoculated four batches of excess sludge after sterilization. The average VSS removal in the concentrated excess sludge reached 30%-50% with the bacterial strain, whereas that without the bacterial strain was 11.9% after five days of shake cultivation at 30˚C, 40˚C, 50˚C, and 60˚C. At present, bacterial strains that secrete lysozymes belong to major fungal classifications or are parts of viruses. Ogawa et al. [20]. found that aside from the Bacillus species, the B. Brevis, B. Cereus, B. Circulans, B. Coagulans, B. Firmus, B. Licheniformis, B. Macerans, B. Megaterium, B. Mycoides, B. Pumilu, B. sphaericus, B. Subtilis, and B. Thuringiensis species all had the ability to secrete lysozymes.
Protease
Proteases are large molecules in a living organism, and their basic structural unit is the aminophenol. Proteases, which primarily exist in the intracellular space and the external EPS of living microorganisms, are abundant in activated sludge. Hong et al. [21] reported that proteins and carbohydrates are the main components of activated sludge and that their proportions vary with the sludge sample. Meanwhile, proteins and carbohydrates are still the main components of EPS in activated sludge [22]. However, which component has the higher proportion remains to be determined [21]. Some studies reported that the highest proportion was that of carbohydrates, whereas others suggested that the protein content was the highest [23-26]. Pei et al. [27] reported that the protein content was the highest at 70.78%, whereas that of polysaccharides was only 8.04%. Protease activity are the primary factors affecting protein hydration and translation. Protease has the peculiarity of catalyzing the hydrolysis of peptide bonds in proteins to produce peptide chains and amino acids, which is prepared for microorganism use. Azize et al. [28-31] reported that the recombination action of protease, lipase, and endoglucanase can significantly enhance the efficiency of sludge lysis. At the same time, Ayol and Parmar [28,29,32] reported that protease, amylase, and cellulase can enhance the efficiency of sludge lysis. Roman et al. [30] reported that the reaction of a procatalyzing peptide bond is the main enzymatic action in surplus sludge assimilation and lysis, and that protease activity is an important factor affecting the efficiency of activated sludge process minimization. Therefore, protease can catalyze and hydrolyze proteins to enhance the lysis efficiency of total sludge, thereby significantly enhancing sludge reduction. At present, the main bacterial types with protease lactation activities are partial bacteria and filamentous fungi. Watson et al. [31] reported that Raoultella and Pandoraea of the Acinetobacter family also exhibit protease lactation activities.
Lipase
Lipids are important components of sludge with larger molecular weights and are also important materials in microorganisms. An extremely high lipid content is present in the wastewater sludge of some special industries, particularly those of oil refineries and restaurants. Thus, lipid hydrolysis in the sludge using lipase plays an important role in sludge degradation [32]. Concurrently, lipase has a relevant function in sludge digestion. It is one of the earliest known enzymes and has some catalytic activities. It can catalyze triacylglycerols and other water-insoluble esters of hydrolysis, esterification, and alcoholysis, as well as transesterification and the contrary synthetic of the ester. Lipase is widely distributed in microorganisms, and the primary producing strains are mildew and bacteria. Fungoids, which are known to secrete lipase, comprise 33 kinds, 18 of which are filamentous fungi, and 7 are bacteria. Dharmsthiti [35] used Pseudomonas aeruginos, which has lipase secretion capacity, to treat restaurant wastewater and obtained good results. Felice [34] used tame Yarrowia lipolytica ATCC 20255 to treat wastewater from an olive oil factory, and the COD was significantly reduced. Palma [37] studied P.Restrictum, which also has the ability to secrete lipase. They found that it not only has the ability to secrete lipase, but it also secretes proteinase and amylase. Therefore, Restrictum has great potential in sludge reduction. Leal [38] also studied the optimization parameters for P. restrictum. Other researchers [33-36] investigated Mucor, Aspergillus, Rhizopus, and Penicillium, all of which have the ability to secrete lipase.
Cellulase
The chemical structure of cellulose was first confirmed in 1930 [37]. It is composed of high-molecular weight linear polymers mainly polymerized by p-β-glucose monomers through β-1- and β-4- glycosidic bonds. Its polymers vary significantly. The molecular weight of cotton polymers and other plants can reach 10,000 or more, whereas those of some industrial material handlings are only about 500 [38]. Cellulose does not dissolve in water and in common organic solvents. It is widely distributed in plants and some microorganisms and is the most abundant type of polysaccharide in nature. In urban sewage treatment plants, particularly in the paper and textile industries, the activated sludge contains large amounts of cellulose and other organic substances. Meanwhile, cellulase can efficiently enhance sludge hydrolysis [28,29,32]. The use of cellulase to hydrolyze cellulose in sludge floc treatment is an important development. Cellulase is a highly efficient recombination enzyme, and filamentous fungi are considered as its main source. At present, cellulase is believed to be mainly composed of endoglucanase (EG, Ee3.2.1.4.), exoglucanase (CBH), and β-glucosidase (glucosidase, Ee3.2.1.21) [39-42]. Domingues et al. [45] studied the RutC-30 cellulase production and mycelial morphology of Trichoderma reesei as influenced by the composition of the culture medium and the inoculum size using shake flask experiments. Velkovska et al. [44] investigated the cellulase produced by T. reesei RutC-30 under environmental conditions and established the enzyme kinetic model for batch fermentation. In addition, studies have been made on Aspergillus, Penicillium, Rhizopus, Myrothecium, and others, all of which have the ability to produce cellulose [47].
Sludge floc hydrolysis heavily depends on hydrolytic enzymes. Furthermore, this process can be enhanced by the combination of enzymes, as demonstrated by Roman et al. [30,48] .The combination of protease, lipase, and endoglycanases can accelerate the solubilization of municipal sludge. According to Molla and Fleury [49,50], mixed fungal cultures can enhance substrate utilization through the combination of enzymes, and that the symbiotic association of a mixed fungal culture can improve the fungal colonization of the substrate. The mixed fungal culture of Aspergillus niger and Penicillium corylophilum reportedly degraded sludge more efficiently (COD, 92%) compared with the control (uninoculated) after six days of sludge fungal treatment (TSS, 4% w/w) [51-53]. Wawrzynczyk [34] confirmed the enhancement of sludge floc hydrolysis through a combination of enzymes in batch experiments, and a direct correlation between the hydrolase dose and the sludge dissolution efficiency was found. Although hydrolytic enzymes play an important role in sludge dissolution, the organisms in the traditional activated sludge secrete smaller amounts of enzymes that can enhance the sludge dissolution efficiency. Therefore, sludge minimization technologies are typically accomplished by adding bacteria with hydrolytic enzyme secretory function, commercial enzymes, or antibiotics, among others. However, such an approach is relatively expensive. Therefore, an effective and inexpensive solution for sludge minimization in water treatment facilities is needed. An appropriate and effective way of addressing this problem is to culture microorganisms with hydrolytic enzyme secretory function. Filamentous fungi, which are naturally present in sewage sludge either as spores or vegetative cells, were the selected species. Filamentous fungi have an exceptionally high capacity to express and secrete proteins, enzymes, organic acids, and other metabolites and can produce secondary metabolites in large quantities. Furthermore, the degradation of the refractory organic substrate in sludge can be enhanced by some enzyme excretion of these fungi [48]. Alam et al. [51] offered a similar conclusion using liquidstate bioconversion, which can be used to treat domestic wastewater sludge using fungal biomass to reduce the amount of organic materials. Many academics supported this finding, and a number of studies were conducted. According to Fleury et al. [50], the amount of dry matter of the sludge treated with the microfungi is reduced by approximately 10% to 50%, typically by approximately 20% to 30%, compared with that of the untreated sludge. Depending on the control parameters, this degradation can be greater. According to Gutierrez-Correa and Tengerdy [32,52], a mixed culture leads to higher enzyme production with comparatively little increase in their cell biomass. Cameron et al. [55] reported that the white rot fungus Phanerochaete chrysosporium degrades a variety of persistent environmental pollutants. Mannan et al. [48,56] found that the filamentous fungi P. corylophilum and A. niger comprised almost 95%~8% of the existing microorganisms in the sludge treatment process, with 10%, 15%, and 20% of inoculum treatment dose in two days. However, at 5% fungal inoculum treatment dose, no significant fungus dominance over other existing microorganisms was observed. The 5% fungal inoculum dose was insufficient to adapt in the sludge under natural conditions. The filamentous fungi Mucor hiemalis broth reportedly removed 87% of the COD in treated sludge, with a 98% removal of suspended solids after six days [57]. Mannan et al. [58] reported that P. corylophilum is suitable for the biodegradation of domestic activated sludge. In general, the extracellular enzyme of filamentous fungi has a potent capacity to degrade domestic activated sludge. Thus, a filamentous fungus culture can enhance the biodegradation of sludge floc as well as cryptic growth.
Location of the Hydrolytic Enzymes and its Influencing Factors
Most prokaryotic microorganisms and some eukaryotic ones in the activated sludge floc can directly absorb only low-molecularweight (<1000) compounds because of the permselectivity of cell membranes [59]. Therefore, most of the substrate subjected to process of metabolism process of living matter in the activated sludge floc need to be hydrolysis, induced by hydrolytic enzymes.
Location of the hydrolytic enzymes
In the study of the macromolecular substrate hydrolysis sites and the distribution of the extracellular hydrolase, Frølund et al. [60]. reported the accumulation of enzymes in the sludge flocs by comparing the the difference existed in the dynamic change of enzyme activity among different positions of sludge. In addition, the exoenzymes were found immobilized in the sludge resulting from adsorption in the EPS matrix, while a very small fraction of the exoenzymes was released into the water. Goel et al. [61] conducted a study via enzyme assays to compare the changes in the enzymatic activity using an activated sludge mixed liquor (F1), the fraction obtained by filtering the supernate from the centrifugation of the activated sludge (F2), and the fraction obtained via filtration of the supernate from the sonication of the activated sludge (F3) (25ml for 5min at 25W to 30W output). The results indicate that the maximum alkaline phosphatase, acid phosphatase, and protease activity of the F3 fraction was 33%, 47%, and 98% of the total, respectively. The F3 fraction for á-glucosidase was not tested. A comparison of the activities in F1 and F2 shows that a major fraction of the total enzyme activity is associated within the flocs. This finding is similar to those of previous studies, indicating that EPS mainly contains polysaccharides, proteins, and humic acid. Cadoret et al. [59] found that 17% of l-aminopeptidase, 5% of á-glucosidase, 23% of protease, and 44% of á-amylase activities were those of cell-free enzymes associated with EPS from the flocs. Further studies by later scholars [62,63] found three types of enzyme activities, namely, (1) enzyme activity associated with the cells, (2) enzyme activity in the bulk solution, and (3) enzyme activity due to the enzymes loosely associated with the cells or entrapped within the floc. Nielsen et al. [64] reported that lipase and phosphatase are associated with the cells exhibiting enzyme-linked fluorescence, indicating that aside from EPS, some hydrolase are also associated with the cells. Van et al. [65] investigated the sampling distribution of the fractions with enzyme activity and found that 5%-44% of the enzyme activity is associated to EPS from the flocs, whereas 56%-95% were located around the cells. This pattern of distribution of hydrolytic enzymes has a significant advantage associated with the degradation of organic substrates, the protection of the organism, and the improvement of the degradation rate. Therefore, (1) because ectoenzymes are bound to EPS, the hydrolase retention time is not lower than the sludge retention time; (2) EPS creates a good environment for enzyme stability; and (3) EPS can provide hydrolysis sites for complex macromolecules. However, this distribution model does not allow a sufficiently close approach of the hydrolase to the sludge floc, thereby weakening the enzymatic sludge hydrolysis and ultimately reducing the efficiency of sludge minimization.
 
Relationship between hydrolase and EPS
EPS significantly affects the degradation of sludge flocs using hydrolytic enzymes because the distribution of hydrolase is mainly associated with the sludge flocs and the microbial cell surface. In addition, a large amount of complex macromolecules are hydrolyzed into readily biodegradable molecules in the EPS matrix. EPS are important integral components of the sludge floc matrices. They consist of a variety of organic substances, such as polysaccharides, proteins, humic acids, uronic acids, lipid compounds, and other undetermined molecules [66-68]. In addition, microorganisms reside in the EPS matrix. EPS play a key role in the formation of activated sludge flocs and provide stability to flocs in high-shear environments. However, they have poor biodegradability, with a BOD/COD ratio of only 0.1 [68,69]. Nielsen et al. [70] reported that EPS can be divided into “bound EPS” and “soluble EPS” Given their association with flocs, bound EPS settle with the activated sludge, whereas the soluble part of EPS is the component of effluent. Lansky et al. [71] further subdivided the bound EPS into two types, namely, “sheath” and “slime”, “Sheath” is closely associated with the cell, whereas “slime” has no direct contact with the cell. The name “slime” is also sometimes used for loosely bound EPS, whereas the name “sheath” is used for firmly bound EPS [71,72]. EPS play significant roles in the hydrolysis of macromolecular organic compounds by hydrolase. First, the release of enzymes by microorganisms into the external environment forms the basis for the interaction between the cells and the high-molecular weight exogenous substrates. In addition, a large proportion of the exoenzymes is immobilized in the activated sludge because of adsorption in the EPS matrix. Thus, exoenzymes should be considered an integrated part of the EPS matrix, allowing the organisms to take up the refractory macromolecular substrates that are too large to diffuse into the cell [73]. Second, extracellular enzymes are either released in freeform (exoenzymes) in the medium or bound to the cell surface (ectoenzymes) [74,75]. Furthermore, the formation of complexes allows the electrostatic interaction to occur between enzymes and EPS and prevents the enzymes from being washed out [75]. Third, EPS serve as the intermediate medium between cells and exogenous substrates and play an important role in the mass transfer fluxes. Given the special mechanisms of EPS, the contact between the enzyme and the substrate is low, whereas molecules within the floc are protected from enzymatic degradation. Nonetheless, the enzymes are entrapped by, adsorbed by, or bound to the sludge. The entrapment decreases the enzyme action on the sludge but does not affect the activity on the added chromogenic soluble substrates [48]. Cadoret et al. [59] proposed that the sludge solubilization rates also depend on the diffusion of the enzyme surface active site into the sludge matrix particles. As such, they suggest that EPS resistance can reduce the contact between the enzyme and the substrate as well as the diffusion efficiency of the substrate in the EPS matrix. Thus, the disruption of the EPS matrix inevitably leads to the enhancement of sludge floc solubilization. By increasing the frequency of floc cleavage, Wawrzynczyk et al. [32] assert that the contact between the enzyme and the substrate can be increased and the sludge hydrolysis can be facilitated. Cadoret et al. [59] dispersed the sludge via ultrasonication and cation-binding and found that sludge deflocculation allows hydrolytic bacteria and their associated enzymes to penetrate into the floc matrix. The results showed that the hydrolysis rate of the straightchain starch increased fivefold. Estera et al. [77] suggested that sludge treatment using cation-binding agents would disintegrate the sludge, consequently making the sludge a better substrate for enzymes. Other studies considered the possibility of enhancing the mass-transfer efficiency of EPS by changing the EPS characteristics to improve the enzymatic sludge floc hydrolysis [28-30].
Element of the Enhanced Enzymatic Sludge Hydrolysis
Based on the aforementioned studies, enzymes are found inefficient for sludge treatment because EPS reduces the contact between the enzyme and the substrate and decreases the diffusion efficiency of the substrate in the EPS matrix. Therefore, sludge pretreatments (such as the use of ultrasonication, radiation, pyrolysis, and surfactants) are necessary. This approach should be applied to the sludge prior to the hydrolase treatment to enhance the degradation performance of the system.
Pretreatments via low-intensity ultrasonic enhancement
Ultrasonic treatment is one of the most promising recent technologies for reducing sludge production in wastewater treatment plants. It mechanically disrupts the cell structure and floc matrix by the shear force and the extreme temperature (4000 K) and pressure (40 MPa) conditions locally produced by cavitation [78-79]. Moreover, OH-, HO2-, and H- radicals can be formed under extreme conditions, and these radicals can enhance sludge disintegration [80,81]. Compared with high-intensity ultrasound, low-intensity ultrasound has lower energy losses and can enhance the enzymatic activity; hence, it is more suitable for use as a pre-treatment technology. Bougrier et al. [82] found that the solid surfaces are washed by the cavitation, thereby increasing the solid-liquid transfer efficiency and diffusion in EPS as well as the hydrolase activity. Yu et al. [81] adopted the ultrasonic technology on the sludge to release the enzyme to the liquid phase, which enhances the sludge digestion rate to a large extent. Similar studies have been conducted by Vassilakis [83], Yoshio [84], Bien [85], Schlafer [86], and Zhang [87].
Radiation technology pretreatments
Radiation technology is considered a promising alternative because of its high efficiency in pathogen inactivation, organic pollutant oxidation, and odor nuisance elimination, which facilitate the downstream process of sludge treatment and disposal [88]. As a pretreatment process, gamma-ray irradiation has been used to release soluble carbohydrates from an activated sludge. It can destroy cell walls as well as flocs, release intracellular material, and enhance the solid-liquid transfer efficiency. Although radiation may inhibit the enzyme activity and cause some minor losses, on the whole, the liquefied efficiency of the sludge is improved. Chu et al. [89] found that gamma irradiation can disintegrate sludge flocs and release proteins, polysaccharides, and extracellular enzymes into the bulk solution. Gamma irradiation of sludge is similar to ultrasonic treatment. A study by Kim et al. [90] resulted in similar outcomes.
Low-temperature thermal pretreatment
Thermal pretreatment is a simple and practical approach to sludge degradation. This process essentially destroys the cell walls and allows access into the cellular interior for biological degradation. The behavior of sludge during low-temperature thermal treatment (≤100˚C) is considered a low-power pretreatment, which can generate a release of organic components from the particulate to the soluble fraction. However, only a deflocculation of the macroflocs structure occurs, with no significant floc breakage, whereas the Low-temperature thermal pretreatment for sludge degradation is sufficient [91]. Morever, thermophilic conditions generally result in an increase in hydrolytic activity.
Andreasan et al. [92] studied the effect of temperature on digestion and found that thermal pretreatment enhances biogas production by waste-activated sludge. Protease was also reported to have an optimum temperature of 60˚C [61,93].
Yan et al. [94] investigated the sludge behavior during lowtemperature thermal pretreatment and found that the enhancement of sludge floc hydrolysis by enzymatic activity can separate out the sludge. Nielsen [95], Ferrer [96] and Borges [97] conducted similar studies and reached the same conclusions. In Japan, some full-scale operating plants use low-temperature thermal pretreatment to enhance the hydrolysis.
Outlook and Conclusions
Enzymatic reactions are the basis of biological processes. Hydrolytic reactions by hydrolase primarily control the excess sludge solution. Filamentous fungi have a powerful extracellular enzyme system. The extracellular enzyme of these fungi can significantly enhance the degradation of the sludge floc as well as the hydrolysis of refractory organic macromolecules. Therefore, this process can increase the treatment efficiency and cryptic growth using measurable cultures of filamentous fungi in domestic activated sludge.
The distribution of hydrolase is mainly associated with the sludge flocs and the microbial cell surface. The formation of complexes allows the electrostatic interaction between enzymes and EPS to occur and also prevents enzyme wash out. Therefore, the special mechanisms of the extracellular enzymes complexed by EPS decrease the enzyme action on the sludge. However, they did not affect the activity on soluble substrates.
The sludge solubilization rates depend on the diffusion of the enzyme surface active site into the sludge matrix particles. Therefore, a disruption of the EPS matrix would result in enhanced solubilization of the sludge floc.
Upon completion of this review, knowledge on the degradation of the sludge floc enhanced by an enzyme offers great potential. However, certain things remain unclear, such as the mechanism of hydrolysis, the recombination action of the enzyme, and the appropriate bacteria with enzyme secretion. Furthermore, the instructive significance of the theoretical knowledge to the actual design and use of such mechanism remains to be determined. Therefore, further study must be conducted to better understand the mechanism of degradation of sludge floc enhanced by the enzyme and determine its significance to actual production
 
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