Enzymatic pretreatment to enhance anaerobic bioconversion of high strength wastewater to biogas: A review

Journal Pre-proof Enzymatic pretreatment to enhance anaerobic bioconversion of high strength wastewater to biogas: A review

Yuh Xiu Liew, Yi Jing Chan, Manickam Sivakumar, Mei Fong Chong, Siewhui Chong, Timm Joyce Tiong, Jun-Wei Lim, GuanTing Pan PII:

S0048-9697(19)36369-7

DOI:

https://doi.org/10.1016/j.scitotenv.2019.136373

Reference:

STOTEN 136373

To appear in:

Science of the Total Environment

Received date:

29 October 2019

Revised date:

24 December 2019

Accepted date:

26 December 2019

Please cite this article as: Y.X. Liew, Y.J. Chan, M. Sivakumar, et al., Enzymatic pretreatment to enhance anaerobic bioconversion of high strength wastewater to biogas: A review, Science of the Total Environment (2018), https://doi.org/10.1016/ j.scitotenv.2019.136373

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© 2018 Published by Elsevier.

Journal Pre-proof

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Enzymatic Pretreatment to Enhance Anaerobic

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Bioconversion of High Strength Wastewater to

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Biogas: A Review

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Yuh Xiu Liew1, Yi Jing Chan1,*, Manickam Sivakumar1, Mei Fong Chong2, Siewhui Chong1, Timm Joyce Tiong1, Jun-Wei Lim3, Guan-Ting Pan4

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Department of Chemical and Environmental Engineering, University of Nottingham Malaysia, Broga Road, Semenyih 43500, Selangor Darul Ehsan, Malaysia; [email protected] ; [email protected] ; [email protected] ; [email protected] ; [email protected] 28, Jalan Pulau Tioman U10/94, Taman Greenhill, Shah Alam, 40170, Selangor Darul Ehsan, Malaysia; [email protected] Department of Fundamental and Applied Sciences, Centre for Biofuel and Biochemical Research, Institute of Self-Sustainable Building, Universiti Teknologi PETRONAS, 32610 Seri Iskandar, Perak Darul Ridzuan, Malaysia; [email protected] Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Zhongxiao E Rd, Da’an District, 106 Taipei City, Taiwan (R.O.C.); [email protected]

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* Correspondence: [email protected]; Tel.: +60 3 8924 8773

Journal Pre-proof Highlights

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Lignin, carbohydrate, protein, oil and grease cause operational problems in high-rate anaerobic bioreactor

Improving hydrolysis stage using enzymes could enhance overall anaerobic digestion

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Usage of enzymes improved biogas production by 7 – 76%

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pH (7 - 8), temperature (30-55°C), and enzyme dosage (1-2%w/w) are optimum conditions

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for pretreatment

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Abstract

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Oil and grease, carbohydrate, protein, and lignin are the main constituents of high strength

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wastewaters such as dairy wastewater, cheese whey wastewater, distillery wastewater, pulp and paper mill wastewater, and slaughterhouse wastewaters. These constituents have contributed to

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various operational problems faced by the high-rate anaerobic bioreactor (HRAB). During the

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hydrolysis stage of anaerobic digestion (AD), these constituents can be hydrolyzed. Since hydrolysis is known to be the rate-limiting step of AD, the overall AD can be enhanced by improving the hydrolysis stage. This can be done by introducing pretreatment that targets the degradation of these constituents. This review mainly focuses on the biological pretreatment on various high-strength wastewaters by using different types of enzymes namely lipase, amylase, protease, and ligninolytic enzymes which are responsible for catalyzing the degradation of oil and grease, carbohydrate, protein, and lignin respectively. This review provides a summary of enzymatic systems involved in enhancing the hydrolysis stage and consequently improve biogas production. The results show that the use of enzymes improves the biogas production in the range of 7 to 76%. Though these improvements are highly dependent on the operating conditions of pretreatment and the types of substrates. Therefore, the critical parameters that would affect the effectiveness of pretreatment are also discussed. This review paper will serve as a useful piece of

Journal Pre-proof information to those industries that face difficulties in treating their high-strength wastewaters for the appropriate process, equipment selection, and design of an anaerobic enzymatic system. However, more intensive studies on the optimum operating conditions of pretreatment in a larger-scale and synergistic effects between enzymes are necessary to make the enzymatic pretreatment economically feasible.

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Keywords: High Strength Wastewater; Anaerobic Digestion; Biological; Pretreatment; Enzyme

anaerobic digestion

ASBR

anaerobic sequencing batch reactor

BOD

biological oxygen demand

COD

chemical oxygen demand

CSTR

continuously stirred tank reactor

EGSB

expanded granular sludge bed

GHG

greenhouse gas

HRAB

high-rate anaerobic bioreactor

HRT

hydraulic retention time

LiP

lignin peroxide

MnP

manganese peroxidase

OG

oil and grease

OLR

organic loading rate

TOC

total organic content

TSS

total suspended solids

UASB

up-flow anaerobic sludge blanket

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AD

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NOMENCLATURE

UASFF up-flow anaerobic sludge fixed-film

Journal Pre-proof VSS

volatile suspended solids

1. Introduction Wastewater is characterized as high strength due to the elevated concentration of organic matter, biological oxygen demand (BOD) (> 250 mg/L), total suspended solids (TSS) (>140 mg/L), or fats, oils and greases (FOG) (> 40 mg/L) [1]. A high concentration of organic matters may cause a severe impact on the environment if discharged untreated due to the depletion of dissolved oxygen

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level. Biological wastewater treatment appears to be the most promising method among various

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wastewater treatment technologies. Anaerobic digestion (AD) technology has been applied for the treatment of a wide variety of high strength wastewaters, including dairy wastewater [2], cheese

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whey wastewater [3], distillery wastewater [4], pulp and paper mill wastewater [5], and

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slaughterhouse wastewater [6]. AD is well suited for high strength wastewater [7], owing to low

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energy requirement, the potential of biogas renewable energy production, and low surplus sludge production [8]. The reduction in sludge disposal not only can destroy most of the pathogens present

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in the sludge but could also help to reduce possible odor problems. For these reasons, AD can

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minimize the treatment cost [9–14].

AD is a complex biological process where complex organic matters undergo degradation in the absence of oxygen. The degradation occurs through complex sequential, parallel, and interdependent biological reactions by different groups of microorganisms. Overall, AD may be divided into four phases, namely, hydrolysis, acidogenesis, acetogenesis, and methanogenesis [14]. Figure 1 illustrates the overall pathway of AD. (Figure 1 inserted here)

Hydrolysis involves the breakdown of insoluble organic material and high molecular weight compounds (i.e., carbohydrates, lipids, and proteins). Hydrolytic microorganisms such as Streptococci, Bacteriocides, Clostridia, Enterobacteriaceae, and Bifidobacteria release hydrolytic enzymes (i.e., cellulase, amylase, lipase, protease, and xylanase) to break down the complex compounds into

Journal Pre-proof soluble organic substances (sugar, fatty acids, amino acids) that can be used by bacteria to perform fermentation. Most of these bacteria are strict anaerobes [15]. This phase is rather vital as the hydrolytic enzymes are supposed to rupture the cell walls and extracellular polymeric substances to release readily available organic material for the acidogens in the next phase. However, cell walls that contain glycan strands cross-linked by peptide chains cause resistance in the hydrolysis phase [16]. Therefore, hydrolysis is known to be the rate-limiting step. In acidogenesis, soluble organic substances formed are converted into organic acids and

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alcohols by acidogenic (or fermentative) bacteria. Along with these, the acidogens also produce

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volatile fatty acids (VFA), ammonia (NH3), carbon dioxide (CO2), hydrogen sulphide (H2S), and

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other by-products. VFA produced by the acidogens during this phase tends to reduce the pH of the AD system. However, this does not affect this stage significantly as the acidogens are less sensitive

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balanced in the later stage.

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to pH changes and can function in a wide pH range between 4.0-8.5 [17]. The pH of the AD system is

The higher organic acids and alcohols produced during the acidogenesis are further digested in

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the acetogenesis phase. Here, the acetogens mainly convert the higher organic acids and alcohols

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into acetic acid, CO2, and hydrogen (H2). The metabolism of acetogens can be affected by the accumulation of H2 [13]. Therefore, it is crucial to maintain low partial pressure of H 2 for the acidogens.

Lastly, methane is produced in the last stage (methanogenesis phase) by two groups of methanogenic bacteria. The first group utilizes acetate as a substrate to produce methane and CO2, while the other group of bacteria produces methane by using hydrogen as an electron donor and CO2 as acceptor. Methanogenic bacteria are susceptible to pH with an optimum between 6.5-7.2 [13]. The CO2 and bicarbonate (HCO3)-alkalinity produced by the methanogens can increase the pH of the system in the gas and liquid phases, respectively [18]. High strength wastewaters are excellent sources of renewable energy. Taking palm oil industry as an example, it is projected that the total power output in terms of electricity is around 480 MW if the biogas produced from anaerobic digestion from all palm oil mills in Malaysia could be

Journal Pre-proof successfully captured and used. This estimation is based on the gas engine’s electrical conversion efficiency of 40% and a biogas power plant operation of 7,000 hours per year. Nevertheless, the conventional anaerobic digesters require long retention time (hydraulic retention times, HRT of 20-200 days) and large treatment areas to ensure complete digestion of treated influent [14,19]. Furthermore, the biogas produced from the conventional treatment is directly emitted to the atmosphere, contributing to greenhouse gas (GHG) emission. Therefore, these led to the investigation of high-rate anaerobic bioreactors (HRAB) to reduce GHG emission while

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harvesting the produced biogas to be reused. The HRAB investigated comprises of continuously

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stirred tank reactor (CSTR), anaerobic sequencing batch reactor (ASBR), up-flow anaerobic sludge

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blanket (UASB), up-flow anaerobic sludge fixed-film (UASFF), anaerobic contact digester and expanded granular sludge bed (EGSB), anaerobic filter, and fluidized bed [20,21].

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The high-rate anaerobic digestion removes more than 80% of chemical oxygen demand (COD)

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[22]. Moreover, HRAB is robust and handles waste at high organic loading rate (OLR) and high up-flow velocity at low HRT [23,24]. However, they do face operational problems. For instance,

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anaerobic filters and UASFF may face clogging of the fixed bed attributed to the high concentration

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of solid content [14]. While UASB and EGSB face sludge floatation, foaming and scum formation problems due to the high concentration of lipid present in the high strength wastewater [25,26]. Overall, anaerobic microorganisms can degrade all organic materials, mostly except for stale woody materials such as lignin [13].

Overall, the operational problems faced by the conventional AD and high-rate AD are associated with the rate-limiting step of AD and hydrolysis. Hence, AD can be enhanced by favoring the hydrolysis step through the pretreatment approach [27]. The types of pretreatment approaches include physical, chemical, and biological. Physical and chemical pretreatment may be applied to improve the digestibility of the solid substrates. Physical pretreatment such as milling, chipping, and grinding could reduce the particle size, which would then lead to an increase in the available specific surface and a reduction in the degree of polymerization [28]. Consequently, total hydrolysis yield can be increased with a

Journal Pre-proof minimum enzyme loading [29]. Delgenés, Penaud, & Moletta [30] reported that an increment of 5-25% methane yield could be achieved. However, physical pretreatments require high energy demands and are not economically viable [28]. Conventional chemical pretreatment includes acid and alkaline pretreatment, where both help to remove hemicelluloses or lignin, which improves the glucose recovery yield from cellulose and hence promote better hydrolysis [31]. However, acid pretreatment favors bioethanol production instead of biogas production as the use of strong acids such as sulphuric or nitric acids reduces

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sulphate and nitrate to H2S and N2 which result in the reduction of methane production [28]. On the

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other hand, alkali pretreatment has shown to be favorable to biogas production as cellulose

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concentration can be maintained while having good lignin removal [32]. He et al. [33] showed an increase in biogas yield by pre-treating rice straw with solid sodium hydroxide (NaOH). The main

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reason for this is the significant change in chemical structures due to the destruction of linkage

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between lignin, cellulose, and hemicelluloses, causing the rice straw to become more biodegradable thereby enhancing the biogas production. Bruni, Jensen, & Angelidaki [34] and Zinatizadeh,

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Mohamed, & Mashitah [35] have also shown similar results on biofibers and POME.

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On the other hand, the biological pretreatment approach where biomass is pre-treated with degrading microorganisms or with the addition of extracellular enzymes has been conducted on a wide variety of high strength wastewater [36]. The result was promising as the microorganisms or enzymes were of high capability in degrading the recalcitrant compounds and degrading complex polymers into simpler forms. This could, in turn, ease the accessibility of useful substrates for the anaerobes in the latter stage, consequently improving the overall AD. The pretreatment methods, as mentioned above, can indeed enhance biogas production. However, the physical and chemical pretreatments are considered expensive due to high energy demand and operating costs [37]. On the contrary, the biological pretreatment provides a system with low energy requirement, no chemical requirement, and mild environmental conditions as it uses microorganisms to treat lignocelluloses and enhance enzymatic hydrolysis [38]. The usage of enzymes during pretreatment is known to improve hydrolysis, which is the rate-limiting step in

Journal Pre-proof anaerobic digestion [39]. Studies have reported notable improvements in the solubilization of organic matter, methane yield, and biogas production [40–43]. However, majority of the experiments were conducted in lab-scale and not in pilot-scale. Besides, the operating conditions and the type of wastewater being treated would also affect the effectiveness of enzymatic pretreatment. Hence, it is crucial to carry out thorough studies to investigate and obtain the optimum parameters that would provide the best results before scaling up the process to pilot-plant scale or even industry scale. This review provides an overview of the recent studies on biological pretreatment for biogas production,

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focusing on critical pretreatment parameters affecting the effectiveness of biological pretreatment,

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enzymes involved in the degradation of biomass feedstock, and sugar and biogas yields resulting

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from biological pretreatment. Hence, this review only focuses on biological pretreatment.

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2. Biological pretreatment

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Biological pretreatment provides a system with low energy requirement, no chemical requirement and mild environmental conditions as it uses microorganisms with high ability in

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degrading complex polymer or the addition of enzyme that would catalyze specific reactions [38].

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An effective pretreatment method should be able to meet the following requirements [44,45]: 1.

Formation of sugars directly or subsequently by hydrolysis

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Avoids loss and/or degradation of sugars formed

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Limits the formation of inhibitory products

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Reduces energy demands

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Minimizes costs As mentioned earlier, improving the rate-limiting step (hydrolysis) may enhance the overall

AD and biogas production. Hence, this can be done by increasing the microbial activity per unit of surface area through substrate inoculation or the use of extracellular enzyme [27]. Therefore, the primary purpose of biological pretreatment is to render the structure of the complex matrix to prepare substrates for the enzymatic degradation [46], which fulfils the requirements for an effective pretreatment mentioned.

Journal Pre-proof Carbohydrate, lignin, oil and grease and protein have found to be the four constituents mainly found in high strength wastewater that contribute to various operational problems to HRAB as well as hindering the AD. These constituents are mainly hydrolyzed during the hydrolysis stage in the AD. Hence, the enzyme can be used to catalyze the degradation of these constituents. Enzymes are proteins that have active sites of unique structure for specific substrates. Therefore, the enzyme can only act on specific substrates. Hence, ligninolytic enzyme, lipase, amylase, and protease are the enzymes that could catalyze the degradation of lignin, oil and grease, carbohydrate, and protein,

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respectively. The following section discusses more on the enzymes that could catalyze the

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degradation of each of the constituents to enhance the overall AD of various high strength

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wastewaters.

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3. Types of ligninolytic enzymes and their catalytic cycle

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Lignin is one of the main components found in the plant cell wall that could inhibit the biodegradation due to its cross-linking with cellulose and hemicellulose fibers. Hence, reducing the

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lignin content of the biomass helps to ease accessibility to sugars by hydrolytic enzymes [47].

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Various studies on pretreatment associated with fungi have been widely investigated in other high-strength wastewater due to the capability of fungi to produce the ligninolytic enzyme that could selectively degrade lignin, hemicellulose, and polyphenols. However, it is crucial to understand

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mechanism

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delignification.

The

degrading

microorganisms

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delignification through the production of enzymes that could degrade lignin known as degrading enzymes or ligninolytic enzymes. There are three major oxidative enzymes secreted by white-rot fungi, namely lignin peroxide (LiP), manganese peroxidase (MnP) and laccase [48]. LiP is capable of catalyzing both phenolic and non-phenolic units, while MnP and laccase can only oxidize phenolic compounds to produce phenoxy radicals and quinones [49].

3.1. Lignin Peroxidase (LiP)

Journal Pre-proof LiP has a high redox potential due to its heme pocket architecture where Fe(II) is pentacoordinated to the four heme tetrapyrrole nitrogens and a histidine residue. This enables LiP to oxidise benzenic rings of lignin directly [50]. The catalytic cycle of LiP is illustrated in Figure 2. (Figure 2 inserted here)

First and foremost is the oxidation of resting LiP enzyme with H2O2 to produce compound I (two electron-oxidized intermediates) in which iron is present as Fe(IV) and a free radical sojourns on the tetrapyrrole ring. Then, Compound I oxidizes the lignin substrate by one-electron to produce

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compound II (one-electron oxidized intermediate) and a free radical substrate. At this stage, the iron

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is still present as Fe(IV); however, no radical is present on the tetrapyrrole. Lastly, compound II

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oxidizes a second molecule of lignin substrate to return to the resting state of the peroxidase and also

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giving another substrate-free radical.

3.2 Manganese peroxidase (MnP)

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A commonly faced problem is that not all white-rot fungi produce LiP. Hence, MnP is the most

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widespread enzyme that can be found in most of the white-rot fungi. Similar to LiP, MnP is also a potent oxidizing agent. The only difference is that MnP could not oxidize nonphenolic lignin-related

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structure [51]. The catalytic cycle of MnP is essentially similar for LiP with the exception that Mn(II) is required to complete the cycle. As shown in Figure 3, the resting enzyme reacts with H2O2 forming compound I. Then, compound I reacts with Mn(II) where one-electron is transferred to form Mn(III) and also producing compound II. Compound II then reacts with another Mn(II) to return to the resting enzyme state. (Figure 3 inserted here)

3.3 Laccase Laccases fall under the category of phenol oxidases. Unlike LiP and MnP, laccases have a low redox potential that prevents a direct attack on lignin [50]. It can only attack the phenolic subunits of lignin. Laccases contain 4 copper (Cu) atoms distributed in three redox sites (T1, T2, and T3). The first step in laccase catalysis cycle is the one-electron reduction of lignin substrate by Cu in site T1,

Journal Pre-proof resulting in a free radical. The electrons are then transferred to the T2/T3 trinuclear site resulting in the conversion of the resting enzyme to a fully reduced state. Laccases operate as a battery that stores electrons from individual oxidation reactions to reduce oxygen molecule [52]. Therefore, 4 electrons from 4 lignin substrates are necessary to reduce the enzyme completely. Then, the reduction of dioxygen to form peroxide intermediate takes place where the dioxygen molecule first binds to the T2/T3 site, and at the same time, 2 electrons are rapidly transferred from Cu T3. It is then followed by the decaying of peroxide intermediate to oxy radical intermediate and releases a water

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molecule through a 2 electron reductive cleavage of the O-O bond. Lastly, O2- is released as a second

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water molecule when all four Cu centers are oxidized, which returns to the resting enzyme state [53].

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This catalytic cycle is illustrated in Figure 4.

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(Figure 4 inserted here)

As for the case of lignin degradation by laccases, the phenoxy radicals are formed after

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one-electron reduction occurs at site T1. This product is unstable and hence leads to Cα-oxidation or

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cleavage between Cα-Cβ and aryl-alkyl bonds. The Cα-oxidation results in an oxygen-centered free radical that can be converted into quinone through a second enzyme-catalyzed reaction. Quinone

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and the free radicals can then be subjected to polymerization. As mentioned previously, not all of the white-rot fungi would produce all 3 types of ligninolytic enzymes. Most of them would provide at least 2 of the 3 types. In the study of Dias et al. [54], it was observed that Euc-1 produced all 3 types of enzymes; however, Irpex lacteus only produced MnP and LiP. Similarly, Arora et al. [55] conducted a study to investigate the ligninolytic enzymes produced by 7 different white-rot fungi. It was reported that only 4 out of the 7 fungi studied produced all 3 ligninolytic enzymes (Tinea versicolor, Phlebia fascicularia, Phlebia floridensis, Phlebia radiata) while the remaining 3 fungi produced only 2 types of ligninolytic enzymes (Daedalea flavida –LiP & Laccase; Dichomitus squalens - MnP & Laccase; Phaner-ochaete chrysosporium - LiP & MnP). LiP and MnP have been described as true lignin degraders due to their high redox potential [56]. Jayasinghe et al. [57] conducted a study in evaluating the performance of different peroxidase

Journal Pre-proof enzymes on methane production. They reported that MnP showed the best performance, which resulted in an increase in the methane yield from 5.7 to 200 mL CH 4/g VS and an increase in the delignification from 6.2 to 68.4%. Similarly, Frigon et al. [58] have reported that MnP resulted in better improvement in methane production (222.9 ± 22.5 mg/gVS) when compared to LiP (202.1 ± 9.8 mg/gVS). However, Fackler et al. [59] reported that there is no correlation between peroxidase activity and the decrease of lignin. It was shown that even though Dichomitus squalens excreted a much higher concentration of MnP than Phlebia brevispora, the lignin content at the end of the

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3.4 Production and performance of ligninolytic enzymes

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delignification could take place in the absence of laccase.

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experiment by the former fungus was not lower than that of the latter. It was also reported that

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Various fungi produce ligninolytic enzymes. The investigated fungi include brown-rot, white-rot, and soft-rot fungi. Brown-rot fungi primarily attack cellulose, whereas white and soft-rot

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fungi attack both lignin and cellulose through the production of ligninolytic enzymes that degrade

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lignin [60]. Among the fungi mentioned, white-rot fungi were found to be the most active during biological pretreatment [47] as it selectively degrades lignin, thereby minimizing cellulose loss. This

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is the most important to fungal pretreatment for enhancing AD. Table 1 shows a summary of recent studies carried out based on white-rot fungi pretreatment such as Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Pleurotus ostreatus, Stereum hirsutum, and Echinodontium taxodi over a wide range of biomass feedstocks. (Table 1 inserted here) Most of the studies shown in Table 1 have reported high delignification efficiency of white-rot fungi (7.2 to 45.6%). Highest delignification of 45.6% was shown in the study reported by Yu et al. [66] as compared to others. This can be attributed to the long pretreatment time (120 days). The pretreatment time has been observed to be proportional to the release in total sugar yield. Moreover, a high dosage of fungi has been used (40% w/v). Besides, the selection of fungi has contributed to the high percentage of delignification. The selected fungus, E. taxodii has shown a high selectivity value

Journal Pre-proof (28.9). The selectivity value (lignin/cellulose ratio) indicates that E. taxodii can selectively degrade lignin. The selectivity is crucial as holocellulose is one of the digestible sugars that could contribute to the production of biogas. It is worth noting that lignin degradation has led to the increment in the release of sugar in the pretreatment step (21 to 60%) (Table 1) [61,63,64,67,68]. Delignification renders the structure of a complex matrix to ease accessibility of hydrolytic enzymes to the digestible sugar. Even though the release if total sugar increases with pretreatment time, Taniguchi et al. [61] have shown that the total

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release of sugar did not increase until the concentration of lignin decreased to about 15% of the total

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weight of the sample.

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As mentioned earlier, LiP and MnP play an essential role in the degradation of lignin due to its high redox potential as compared to Laccase. The result shown by Fackler et al. [59] complies to this

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as delignification sustained without the presence of laccase activity. While comparing LiP and MnP,

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MnP was seen to surpass LiP [54,57]. This is mainly attributed to the generation of Mn3+ by MnP, which is capable of oxidizing both phenolic and non-phenolic lignin units while LiP can only oxidize

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non-phenolic lignin units by one-electron transfer mechanism [80].

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The removal of lignin is crucial in enhancing the release of sugars to improve AD as the sugar serves as a substrate for the methanogens in the production of biogas. The enzymatic digestibility and methane yield showed a positive linear relationship. In other words, as the total sugar yield increases, the methane yield also increases (25 to 60%) [58,62]. This can be attributed to the increment in the substrates available for the anaerobes for AD. Hence, this shows that the application of pretreatment of white-rot fungi could enhance delignification, which in turn improved the total sugar yield and then enhances the AD. Consequently, it is crucial to pre-treat wastewater that contains a high concentration of lignin before AD.

4. Lipase Wastewater containing high concentrations of oil and grease (OG) may result in lower biodegradation in the system. This is attributed to the tendency of OG to accumulate on the surface

Journal Pre-proof of sludge. This would restrict the transport of soluble substrates to the biomass and consequently reducing the conversion rate of substrates. Besides, this also causes clogging, development, and flotation of sludge with reduced activity, and the emergence of unpleasant odors [81]. Thus, a hydrolytic step before AD can be taken by using Lipase which catalyzes the hydrolysis of triacylglycerol into free long-chain fatty acids (LCFA) and glycerol. This pretreatment could degrade OG to improve the biological degradation of lipids-rich wastewaters, accelerating the process and improving the efficiency of the process. The mechanism of lipase follows the ping-pong

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bi-bi mechanism, as illustrated in Figure 5. Firstly, the enzyme combines with an acyl donor (A), in

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this case, a triglyceride to form a lipase-triglyceride complex (E.A). Isomerization converts E.A into

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an intermediate complex (F). The isomerization generates the first product, glycerol (P). Then, 3 molecules of water (B) attach to F, resulting in a binary complex (F.B3). Finally, F.B3 undergoes

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unimolecular isomerization, where fatty acids (Q) are released, and the enzyme is regenerated [82].

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During the binding of the enzyme with either acid (E.C) or alcohol (E.Ac.A), this reaction is

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irreversible and would lead to dead-end inhibition [83]. (Figure 5 inserted here)

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Table 2 summarizes the recent studies done based on lipase pretreatment on lipids-rich wastewater. Overall, the studies showed that the use of lipase in pre-treating the lipids-rich wastewater (OG content > 100 mg/L) manage to improve the concentration of fatty acids (3 to 8 times) required for the AD in the latter stage despite varying dosage of enzyme used in each of the studies [84–87]. The release of fatty acids indicates that the lipase enzyme degraded OG substrate in the wastewater sample. However, the concentration of free fatty acids released does not increase with the pretreatment time as expected. A decline in the concentration of fatty acid was observed after a particular time of pretreatment. This is mainly attributed to the presence of other microorganisms in the reaction medium that would have consumed the released fatty acid [81,88]. Hence, it is crucial to carry out a thorough investigation to obtain the optimum pretreatment time for different wastewaters.

Journal Pre-proof (Table 2 inserted here) Lipase enzyme has shown good OG degradation (37 to 55%) even for high OG content wastewater [90,94,99]. The degradation of OG substrate to fatty acid and glycerol has led to an improvement in COD (5.4 to 90%) and BOD (10%) reduction [91,92,95]. The effective COD removal was due to the enhanced biodegradation of lipids. Since lipolytic activity itself does not remove COD, the reduction in COD during the pretreatment indicates that the bacteria or fungi had used up a significant amount of organic matter present in the sample. Besides, an improvement in the

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BOD/COD ratio (from 0.19 to 0.55) has been observed in the study conducted by de Oliveira

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Zawadzki et al. [98]. The high BOD/COD ratio indicates that the sample has sufficiently high

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biodegradability for easier AD.

The lipid removal also enhanced the biogas formation (15% to 93%) [89,93,96,97]. This can be

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attributed to the increase in fatty acids released that could be used by anaerobes. Another reason is

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that the enzymatically pre-treated samples were not subjected to scum flotation, unlike for the raw wastewater. The most common operational problem encountered by high OG content wastewater

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treatment is scum formation that could prevent the liberation of the formed gas. Hence, pre-treating

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the samples enzymatically could prevent scum formation and hence ease release of gas formed. The reduction in OG content could prevent scum formation by converting OG to other useful substrates [92,96]. Hence, lipase pretreatment not only could enhance AD but also could solve the operational problems of HRAB.

Generally, high enzyme dosage would result in better biodegradability, COD reduction, and biogas production. However, using a lower enzyme dosage would produce slightly lower results [91,96,97]. Therefore, from an economic point of view, a lower dose may be a better option.

5. Amylase and Protease Carbohydrate is the other useful constituent that cannot be degraded easily. Carbohydrate, also known as polysaccharides, are polymers made up of a large chain of simple sugars (monomers). Starch is a form of carbohydrates stored in plants where it is made up of a large number of glucose

Journal Pre-proof units joined by glycosidic bonds. It is composed of 10-20% amylose and 80-90% amylopectin. Amylose molecules typically consist of 200 to 20,000 glucose units and can form colloidal dispersion in hot water. On the other hand, amylopectin is completely insoluble as it consists of highly branched 20 million (~) glucose units. Hence high hydrolysis rate of carbohydrate would result in the release of more readily available, reducing sugars for the anaerobes in the latter stage. Amylase is the key enzyme in catalyzing the hydrolysis of carbohydrates that could breakdown the bonds in both amylose and amylopectin molecules. 2 critical residues (a proton donor and a

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nucleophile base) are required for this hydrolysis to take place via general acid catalysis. The

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hydrolysis occurs through 2 major mechanisms, namely retaining and inverting mechanism. The

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proton donor is situated within hydrogen-bonding distance of the glycosidic oxygen in both

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mechanisms. Figure 6 and Figure 7 illustrate both the mechanisms. (Figure 6 inserted here)

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(Figure 7 inserted here)

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In retaining mechanism, the nucleophilic catalytic base is in a shorter distance of the sugar anomeric carbon. This shorter distance is required for direct attack of the nucleophile. Retaining

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mechanism involves a two-step double displacement mechanism where the 2 active-site carboxylic acid residues act as an acid/base catalyst. First, the glycosidic oxygen is protonated by the acid catalyst (AH) with the assistance of a nucleophilic base to form a covalent glycosyl-enzyme intermediate. Then the glycosyl enzyme intermediate is hydrolyzed by a water molecule to generate glycosyl-enzyme species. Unlike the retaining mechanism, there is a further distance between the sugar anomeric carbon and the nucleophilic catalytic base for the inverting mechanism. This distance is likely just right to accommodate a water molecule to bind to the substrate simultaneously. In the inverting mechanism, the 2 active-site carboxylic acid residues are suitably oriented so that one served as a general base to the attack of water, while the other serves as a general acid to the cleavage of the glycosidic bond. Hence the 2 active-site carboxylic acid residues serve as the nucleophile that attacks at the sugar

Journal Pre-proof anomeric center to form the glycosyl-enzyme species. Therefore, this is known as the direct displacement mechanism [107]. The inorganic forms of nitrogen (ammonium, nitrite, and nitrate) commonly found in wastewater are relatively easy to remove using nitrification/ denitrification process; however, the organic compounds are relatively recalcitrant and tend to persevere in wastewater effluent. Proteins constitute a significant fraction of total organic carbon (TOC) and organic matter [108]. Hence, biodegradation of protein not only can reduce the TOC but also could increase the release of readily

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available sugar required by methanogens for AD. Indirectly, an enhancement in biogas production

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can be achieved. Proteins are natural polymers composed of amino acid units joined one to another

constituent polypeptides and amino acids.

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by peptide bonds. Proteases are the extracellular enzymes that can hydrolyze proteins into their

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Table 3 shows a summary of recent studies done based on the use of amylase and protease

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(Table 3 inserted here)

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pretreatment before AD on a wide range of wastewater.

The use of amylase and protease for pretreatment increased the sugar yield (377%) [111]. This

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indicates that the enzymes are capable of converting most of the carbohydrates to glucose (for the case of amylase) and protein to amino acids (for the case of protease). Similar to lipase, the sugar released is crucial as it can be used as a substrate for the anaerobes in the latter stage. Besides, the enzymatic pretreatment has also increased sCOD [113]. This indicates that more readily biodegradable substrates being released in the sample. There was a drastic increase in the sugar yield as well as sCOD in the study conducted by Yu et al. [113] as compared to others as the enzyme dosage and temperature used were higher. An increase in the sugar yield has then led to an increase in the biogas yield (14 to 26%) [109,110,112]. Besides, it is worth noting that the use of amylase and protease in the pretreatment phase can reduce VSS (22 to 58.43%) and TSS (19%) concentration [111,114]. This is attributed to the

Journal Pre-proof ability of enzyme in enhancing the solubilization of the particulate organic matters into soluble organic matters. Studies have been conducted using the mixture of the enzyme on the effect of hydrolysis. In terms of VSS reduction and methane yield, it was observed that a mixture of enzymes showed better results as compared to treating the sample with individual enzymes [111,112]. Hence, mixed-enzyme exhibits a synergistic effect that could enhance the overall hydrolytic activity. However, intensive studies are still required to obtain information on the optimum operating

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conditions (the type of mixture, ratio, dosage, treatment time, temperature, pH, and mixing rate) to

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6. Prospects, bottlenecks, and perspectives

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achieve the best results.

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The use of enzyme during pretreatment has the potential to overcome the operational problems

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of anaerobic digestion. As most of the recalcitrant components in high-strength wastewater has been degraded during pretreatment, this could result in lower viscosity of feed into the anaerobic

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digestor. This can improve the pumpability of the feed which helps to decrease wear and tear of

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involved equipment and hence less maintenance and repair required. A more commonly faced problem in high-rate anaerobic digestor is the formation of floating layer due to high oil and grease which could hinder the production of biogas and also cause pipe clog. This problem could be solved by the application of lipase pretreatment as shown in Table 2. The pretreatment can be very effective when using the correct enzyme type under its optimum operating conditions such as dosage. When compared to the usage of microbes in pretreatment, enzymes do not require acclimatization phase to the biomass, and hence there is no delay in the process. Moreover, enzymes are biodegradable proteins. Therefore, the enzyme degrades itself without causing new biomass generation. The pretreatment of organic-rich wastes using enzymes can be conducted under a mild operating condition. Besides, enzymes can work under a wide range of environmental conditions such as pH and temperature.

Journal Pre-proof Concerns have been raised on the cost and feasibility of the use of enzymes for pretreatment due to very long pretreatment time. As compared to mechanical pretreatment and chemical pretreatment, biological pretreatment has lower energy requirement as enzymes require mild temperature and pH operating conditions [122]. Ben Yahmed et al. (2017) showed that the use of Aspergillus fumigatus SL1 in pretreating green macroalgae Ulva sp. reduced the pretreatment cost when compared to chemical pretreatment [123]. In the study conducted by Ziemiński and Kowalska-Wentel (2017), mechanical pretreatment required 50.5% higher energy input when

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compared to biological pretreatment [124]. However, enzymes could not survive and function

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outside of their range. Therefore, it is important to maintain the optimum operating condition

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during the pretreatment. Another way to reduce the cost of enzymatic pretreatment is by controlling the cost of the enzyme used as it takes up approximately 50% of the total cost [48]. Purified lipase

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purchased from market roughly cost MYR 1049 per 500 g. In the study conducted by Mutuoong et al.

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(2017), 6 mg/g of lipase was required to achieve 97.78% of hydrolysis [125]. If a small-scale pretreatment has a capacity of 200 kg, 1.2 kg of lipase is required to achieve similar hydrolysis yield

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where the enzyme required cost around MYR 2518. Therefore, the cost can be reduced by

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substituting purchased purified enzymes with enzymes produced on-site [126]. For on-site production, the enzymes can be produced at the wastewater treatment plant itself. Hence, sharing resources such as land, buildings and utilizes can further reduce the pretreatment cost [127]. Besides, the use of agricultural waste as carbon and nutrient source for fungi or bacteria culture can help to reduce the cost too. In addition to that, the spent agricultural waste can be recycled and reused for other purposes such as fertilizer and animal feed [128]. Combined pretreatment with other pretreatment methods has been shown to improve enzyme digestibility synergistically and thus has the potential to shorten pretreatment time. As the municipal wastewater is made up of a complex matrix of substances, a mixture of enzymes must be used for efficient pretreatment. Therefore, the enzyme mixture and optimized operating condition for the pretreatment need to be customized for different types of wastewater. Most of the studies have been conducted on lab-scale, and thus there

Journal Pre-proof is a need to study intensively in pilot and full-scale to have a better understanding of the challenges and prospects of the enzymatic pretreatment for commercialization.

7. Critical parameters affecting pretreatment Enzymes are optimally active at a specific pH and temperature. Therefore, it is crucial to optimize the pretreatment condition to achieve optimal hydrolysis of substrates. Higher enzyme loadings may be required to achieve the same level of hydrolysis efficiency when enzymes are not

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operating under optimal conditions. This would then affect the overall cost of the process. The study

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of factors affecting the pretreatment should first be understood before optimization could be

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conducted.

7.1. pH

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Faulds et al. [129] investigated the effect of pH on the solubilization of brewer’s spent grain over

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a pH range of 3.2 - 11.2. An enzyme mixture from Trichoderma (Depol 686, Biocatalysts) was efficient at low pH. In the Depol 686 mixture, side-chain cleaving enzymes such as arabinofuranosidase lost

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activity at higher pH levels, and cellulase activity was absent at pH 7.5. On the contrary, an enzyme

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mixture from Humicola (Depol 740, Biocatalysts) was effective over the entire pH range. In the Humicola mixture (Depol 740), optimum activities were found out between pH 6 and 8, with maximum solubilisation occurring at pH 9. Hence, this shows that enzymes work best at their optimum pH condition, but it varies from each type of enzyme. Besides, Neves Petersen et al. [130] observed that at the optimum pH range where lipase shows maximum activity, and negative potential in the active site causes electrostatic repulsion of the ionized fatty acids, thus indicating a fast release of lipolysis reaction products from the interface. Based on Tables 1 – 3, it could be observed that enzymes have a wide range of working pH. Generally, the optimum pH for most of the enzymes falls in the range of pH 7-8. Hence, it is crucial to conduct enzymatic pretreatment at an optimum pH of the respective enzymes.

7.2. Agitation

Journal Pre-proof The optimum agitation speed for effective enzymatic pretreatment varies from the type of wastewater being treated and the types of the enzyme being used. When dealing with insoluble substrates, suspension and mixing of the substrate in the reactor may have an impact on hydrolysis. Chundawat et al. [131] indicated that the agitation improves the hydrolysis of crystalline cellulose. At lower agitation speed, lower hydrolysis was observed due to the inhomogeneity in mixing. Besides, Al-Zuhair et al. [132] showed similar results in the hydrolysis of palm oil using lipase. An increase in agitation speed led to an increase in the shear rate of the oil droplets causing larger oil

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droplets to break into a smaller size. Hence, the interfacial area for the enzyme to react was

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most of the enzymes falls in the range of 100-200 rpm.

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increased. Generally, it could be observed from Tables 1 – 3 that the optimum agitation speed for

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7.3. Temperature

The temperature of the pretreatment environment is also another crucial factor affecting the

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efficiency of pretreatment. Enzymes will only be active at their optimum temperature. Temperature

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below the optimum would cause enzymes to be inactive, while temperature above the optimum will cause instability in the enzyme activity and may lead to thermal denaturation of the enzymes.

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Kempka et al. [86] observed a higher release in free fatty acid when swine slaughterhouse wastewater was treated with lipase at a higher temperature. Besides, a positive effect on saccharification was observed with an increase in the temperature until an optimum temperature of 55 °C. A decline in the saccharification then occurred beyond the optimum [133]. A generic optimum temperature of 30-55 °C was observed from Tables 1 – 3. Although enzymes are capable of working in a wide range of temperatures, beyond the range, enzymes tend to denature. Hence, it is crucial to obtain an optimum temperature for different enzymes.

7.4. Enzyme dosage Enzymatic dosage showed a positive effect on hydrolysis. This is attributed to the increase in the number of active sites available for the hydrolysis to occur. Mendes et al. [91] showed an increase in free fatty acid when a higher concentration of lipase was used in the hydrolysis of dairy

Journal Pre-proof wastewater. However, an overdose of the enzyme would not help in the hydrolysis but will bring about an adverse effect [134]. Yang et al. [111] showed an increase in enzyme dosage from 3-6 % (w/w) improved the reduction in volatile suspended solids. However, a further increase in dosage after 6 % (w/w) did not show any improvement in the reduction of volatile suspended solids. The general optimum enzyme dosage of 1-2 % (w/w) could be observed from Tables 1 – 3. Hence, it is important to first examine the effect of each factor on the degree of hydrolysis before conducting

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optimization to obtain a maximum hydrolysis rate.

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8. Conclusion

The treatment of high-strength wastewater through anaerobic digestion generates biogas,

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which is a renewable source of energy. There are various high-rate anaerobic bioreactors in the

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current market that have been developed to harvest the biogas for energy generation. However,

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these high-rate anaerobic bioreactors face various operational problems which are directly related to the hydrolysis step in the anaerobic digestion, causing poor biogas yield. Thus, the use of enzymes,

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including ligninolytic enzyme, lipase, amylase, and protease to pre-treat high-strength wastewaters

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has been critically reviewed in this study. The use of enzymes has clearly shown a substantial improvement in the biogas production up to 76%. While enzymes are substrate specific, using the correct mixture of enzymes are crucial to target different organic matters present in the wastewater for effective pretreatment. Although enzymes can work in a wide range of environment, they tend to denature and do not function beyond their working range. Therefore, it is important to maintain the pH, temperature, and dosage at a range of 7-8, 30-55 °C and 1-2 % (w/w), respectively to ensure maximum efficiency of the pretreatment. The development of low-cost enzymes is of high interest in the treatment of high-strength wastewater as the use of readily available commercial enzymes would make the pretreatment economically infeasible. There is also a need to conduct more research in pilot and full-scale to explore the potential of enzymatic pretreatment for commercialization.

Acknowledgements

Journal Pre-proof This work was supported by the University of Nottingham Malaysia and Early Career Research and Knowledge Transfer Award.

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acid fermentation of food waste. J. Chem. Technol. Biotechnol. 2006, 81, 974–980.

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Journal Pre-proof

Figure 1. Anaerobic digestion pathway [12] Figure 2. Catalytic cycle of LiP

Figure 3. Catalytic cycle of MnP

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Figure 4. Catalytic cycle of laccases

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Figure 5. Ping-Pong bi-bi mechanism for lipase

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Figure 6. Retaining mechanism

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lP

Figure 7. Inverting mechanism

Journal Pre-proof Table 1. Summary of lab-scale fungal pretreatment on delignification and enzymatic hydrolysis

Substrat e

Degra ding enzym e

Degradi ng microorg anism

N/A

Pleurotus ostreatus ATCC 66376

10:1 weight ratio of the rice straw to culture

Ceriporiop sis subvermis pora FPL105.7 52

3 mL mycelium suspension (1 MEA plate/70 mL 1.5%, w/v, corn steep liquor)

Ceriporiop sis subvermis pora ATCC 90467

4 pellets of inocula from precultures added to 10 g Japanese cedar wood chip media with 30 mL Milli-QTM water and 1 g of wheat barn

Stereum hirsutum

0.1 g of dry weight mycelium added to 50 g wood chips, 80 mL distilled water

Effects



Refere nces

41% Klason lignin degradation  33% total soluble sugar from holocellulose and 32% glucose from cellulose after hydrolysis with cellulase (100 mg/L enzyme, 10 g/L pretreated rice straw, 40 °C, pH 5.0, 48 h) 7.2% delignification

[61]

Incubat ed at 28 °C for 4-8 weeks 70% relative humidi ty



[62]

Incubated at 30 °C for 8 weeks



 

N/A

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Japanes e cedarwo od

Japanes e red pine (Pinus denisflor a)





Lac cas e Mn P

-p

Lac cas e LiP Mn P

Incubated at 30 °C for 2 weeks

[59]

re



lP

Spruce wood shaving s

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of

Rice straw

Dosage

Operating condition of pretreatme nt 60 days









28.2% delignification Maximum methane yield of 25% with the fermentation system of: o 12 g pre-treated and untreated wood chips o 400 mL digested sludge (SS: 2.2%) o 35 °C o 60 days 14.5% lignin degradation Improved sugar yield (21.01%) after saccharification with Celluclast 1.5 L (80 EGU/g, 0.25 g pretreated woody biomass in 20 mL of 50 mM sodium acetate buffer, pH 5.0, 50 °C, 150 rpm, 72 h)

[63]

Journal Pre-proof Bamboo clums

N/A

Echinodo ntium taxodii 2538

A plug cut from the margins of the PDA culture added to 10 g ground bamboo culms and 20 mL distilled water

Incubated at 25 °C for 30 to 120 days

Cotton stalks (variety Deltaphi ne DP5415 RR)

N/A

Phaneroch aete chrysospo rium ATCC 24725

Submerged cultivation, SmC (5% solid loading):  1 mL spore inoculum (5x106 spores per ml)  1 g cotton stalk (air dried)  18 mL acetate buffer (20 mM, pH 4.5)



[64]



[66]

[65]

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29.14% lignin degradation after 120 days  8.76-fold increase in fermentable sugar yields after hydrolysis with cellulase (20 FPU/g substrate, 2.5% substrate concentration in 50 mM sodium acetate buffer, pH 4.8, 50 °C, 6-120 h) SSC pretreatment demonstrated better lignin degradation (35.53%) than SmC (19.38%).

na

lP

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-p



Incubat ed at 39 °C for 14 days Flasks flushed with oxygen (125 mL.min -1) for 10 min every 3 days



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Solid-state cultivation, SSC (75% wet basis moisture content):  3 mL spore inoculum  3 g cotton stalks  4.8 mL acetate buffer 5 g ground woods and 12.5 ml (for hardwood) or 15 ml (for softwood) distilled water

Chinese willow (Salix babylonic a, hardwo od) & China-fi r (Cunnin ghamia

N/A

Echinodo ntium taxodii 2538

Incubated at 25 °C for 30 to 120 days



Cellulose was barely degraded in comparison with the removal of great lignin o Hardwood: 45.6% o Softwood: 24.1% Selectivity value decreases with the

Journal Pre-proof lanceolat a, softwoo d)

Pycnopor us cinnabari nus ATCC 2004378

7.5 mg fungal mass (dry weight) per gram substrate added to 10 g oven dried substrate moistened with mineral salt solution (moisture ratio of 1:2.5)

Incubated at 30 °C for 25 days

Purchase d from Sigma Aldrich Canada Ltd and Jena Bioscienc e GmbH

40 U LiP (1 U.ml-1) added to 2 g of wet mulched switchgrass, sodium tartrate (pH 4.5, 15 mmol/l), veratryl alcohol (2.5 mmol/l), H2O2 (0.33 mmol/l), oxalate (0.2 mmol/l) 80 U MnP (2 U.ml-1) added to 2 g of wet mulched switchgrass, sodium malonate (pH 4.5, 50 mmol/l), MnCl2 (2 mmol/l), H2O2 (0.33 mmol/l), 0.5% Tween 80 20 U Laccase (10 U.mg-1) added to 0.5 g dried



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lP

LiP Mn P

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 

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Switchg rass (Panicu m vergatu m)

Palm tree fronds

Laccas e

Purchase d from Sigma

[67]

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

-p

N/A

re

Lantana camara and Prosopic juliflora

time of pretreatment o Hardwood: 28.9 (30 days); 1.7 (120 days) o Softwood: 7.1 (30 days); 3.2 (120 days) Delignification after 25 days o P. juliflora: 13.3% o L. camara: 8.87% Sugar release improved by 21.4-42.4% after hydrolysis with cellulase (24 FPU/g substrate, 5.0 w/v% substrate concentration in 50 mM citrate phosphate buffer, pH 5.0, 50 °C, 150 rpm, 48 h) Reduction of more than half of VSS concentration in all controls o LiP: 11.8-4.5 g/l o MnP: 11.8-4.9 g/l Increase in final methane production o LiP: 157-202.1 ml/g VS o MnP: 157-222.9 ml/g VS

Incubated at 22 °C and 37 °C for 8 h, respectivel y Agitated at 2.5Hz

Incubated at 45 °C for 24 h





Increase in total reducing sugar yield (5.6 to 60%) after

[58]

[68]

Journal Pre-proof

Wood fiber

N/A

Phaneroch aete flavido-alb a ATCC 12679

samples, 50 mL distilled water, 1 mM acetate buffer (pH 4.7), 1 mM 1-hydroxybenzo triazole hydrate 240 mL of culture fungus to 200 g of autoclaved substrate

Yard trimmin gs

N/A

Ceriporiop sis subvermis pora ATCC 96608

20 mL of fungus “solution” to 100 g of autoclaved substrate (moisture content at 60%)

Paddy straw

N/A

hydrolysis with cellulase (0.6 U) and xylanase (5 U)

Incubated at 30 °C for 21 days

 Reduction improved by o TOC: 9% o Hemicellulose: 37% o Cellulose: 8% o Lignin: 23%  Increased biogas production (124 NL biogas per dry wood fiber) with 61% methane.  12.4% of dry matter loss  7.4% of cellulose degradation  27.6% of hemicellulose degradation  20.9% of lignin degradation  154% increase in methane yield (from 17.6 to 44.6 L/kg VS).  Degradation in: o Cellulose: 0.6% o Hemicellulose: 20.5% o Lignin: 19.5%  13.9% increase in total sugar production  20.8% increase in biogas production (989.8 ± 20.8 L/kg VS)

[69]

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Aldrich Canada Ltd.

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lP

re

-p

Incubated at 28 °C for 30 days

Trichoder ma reesei MTCC 164

Coriolus versicolor MTCC 138

25 g of inoculum to 250 g of paddy straws

Incubated at 25 °C ± 2 for 10 days

Incubated at 27 °C ± 2 for 5 days

 Degradation in: o Cellulose: 3.7% o Hemicellulose: 6.3% o Lignin: 7.5%  53.4% increase in total sugar production

[70]

[71]

Journal Pre-proof

N/A

Ceriporiop sis subvermis pora ATCC 96608

50 mL of inoculum to 65g of autoclaved miscanthus (moisture content at 60%)

Incubated at 28 °C under 50% humidity for 28 days

Sweet chesnut and hay

N/A

182.5g TS substrate

Incubated at 37 °C for 3 – 4 weeks

Albizia biomass

N/A

Auricular ia auricula-j udae Ceriporiop sis subvermis pora ATCC 96608

32 mL inoculum to 166 g of autoclaved albizia chips with 199 mL of sterilized DI water

Incubated at 28 °C for 48 days

Wheat straw

N/A

Wheat straw

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lP

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Miscanth us sinensis

 26.2% increase in biogas production (743.9 ± 18.6 L/kg VS)  2.36 ± 1.01% cellulose degradation  13.71 ± 0.90% hemicellulose degradation  25.69 ± 0.77% lignin degradation  25% increase in methane yield  15% increase in biogas production

Polyporus brumalis BRFM 985

1 agar disc of 7-day-old mycelia (5mm diameter) to 100mg of straw

N/A

Polyporus brumalis BRFM 985

Raw champo st

MnP

Pleurotus Sajor Caju MES 03464

10 cm3 of fungal inoculum suspension to 20 g TS of chopped straw 10g of spawn to 100 g autoclaved champost

Corn stover

N/A

Cyathus stercoreus NRRL-65 73

5 mL seed culture to 10 g of autoclaved corn stover with 23 mL tap water

Incubated in water-satur ated air for 12 days at 25 °C Incubated in water bath at 26.36 °C for 19.84 days Incubated at 25 °C for 6 weeks

Incubated at 28 °C for 30 days

[72]

[73]

 10.5% cellulose degradation  15% hemicellulose degradation  24% lignin degradation  4 – 4.6-fold increase in glucose (38%) and xylose (29%) yield  3.7-fold increase in the cumulative methane yield (123.9 L/kg VS)  43% more methane per gram of pretreated volatile solids

[74]

 52% increase in methane production (182 dm3 CH4/kg TS)

[76]

 60% higher in VFA yield (203±9 mg COD/g VSadded) as compared to control  Total sugar yield of 394 ± 13 mg/g  46.2 ± 0.7% of lignin loss  51.8 ± 1.8% of

[77]

[75]

[78]

Journal Pre-proof hemicellulose loss  32.1 ± 1.6% of cellulose loss

Pycnopor us sanguineu s FP-10356 -Sp

 Total sugar yield of 393 ± 17 mg/g  51.0 ± 1.2% of lignin loss  50.7 ± 2.1% of hemicellulose loss

Phlebia brevispora NRRL-13 108

 25.4 ± 0.3% of cellulose loss

Incubated at 28 °C for 25 days, followed by incubation at 28 °C for 7 days

lP

re

2 mL of WRF inoculum to 5 g of ground corn cobs (moisture content at 75%); 1.2 mL of BRF inoculum to 3 g of autoclaved pretreated residues solid and 9mL distilled water.

na

Trametes orientalis (Cui6300) − Fomitopsi s pinicola (Cui1233 0)

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Corn cobs

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of

 Total sugar yield of 383 ± 13 mg/g  39.6 ± 1.6% of lignin loss  42.1 ± 1.0% of hemicellulose loss  16.1 ± 1.8% of cellulose loss  6.4% decrease in hemicellulose  1% decrease in cellulose  glucose yield of 83.0%

[79]

Journal Pre-proof Table 2. Summary of recent studies on the enzymatic pretreatment of lipids-rich wastewater

Dairy wastew ater

1200 mg/L

Lipase (43 U/gds) Lipase

Penicilliu m restrictum CBR 11 and BR10

Lipase (20 U/g)

Penicilliu m sp.

5.0% (w/v)

1:1 (inoculum gVS to substrate (FPW) gVS) 0.1% (w/v)

 

28 °C Shakin g at 75 rpm 24 days 30 °C Stirred at 120 rpm pH 7.0 24 h

  

 

Lipase (33 U/gds)

Rhizopus microspore s CPQBA 312-07 DRM

0.3% (w/v)



3100 ± 150 mg/L

Porcine pancrea s lipase (1,770 U/mgs)

Purchase d from Nuclear (São Paulo,

 



0.05% (w/v)

[84]

[89]



[85]

35 °C Stirred at 150 rpm 72 h







Dairy industri es wastew ater

High yield of free fatty acid o Lipolase 100T: 39.5 µmol/mL o Lipase: 100.1 µmol/mL

Refere nces

76% improvement in CH4 yield



na 1300 mg/L

Jo ur

High-fat dairy wastew ater

Effects

of

-

Purchase d from Novozym es

lP

Fish processi ng wastew ater

Lipolase 100T (2800 U/gds)

Dosage

ro

10 g/L

Source of enzyme

-p

Swine meat industry wastew ater

Enzyme

Operating condition s of pretreatm ent  45 °C  pH 6  100 rpm

re

Substrat e

OG Cont ent

 



37 °C Stirred at 200 rpm pH 8.0



8 times increment in the final concentration of free acid (from 1.71 to 14.46 µmol FA/mL) Improvement in the removal efficiencies of COD o Control: 32.4 ± 9.7% o Lipase: 90.5 ± 3.4% Improvement in BOD reduction o Control: 82% o Lipase: 92% Improvement in COD reduction o Control: 31% o Lipase: 47% Improvement in OG reduction o Control: 38% o Lipase: 80% Increase in the removal of reducing sugar o Control: 89.9 ± 1.7%

[90]

[91]

Journal Pre-proof 

Brazil)

4h 

Penicilliu m simplicissi mum

0.5% (w/v)

 

30 °C Stirred at 150 rpm 8h



lP

Lipase

Purchase d from Sisco Research laboratori es

0.75 g/7.5g of VS

na

3±1.4 % on wet basis





Jo ur

Fleshing s

Dairy wastew ater

219±2 7 mg/L

Lipase (9.75 U/mL)

Aeromonas sp.



ro

Lipase

-p

1500 mg/L

re

Fish processi ng plant wastew ater

of



5% (v/v)

 



o Lipase: 98.0 ± 1.8% Improvement in the reduction of COD o Control: 38.2 ± 2.7% o Lipase: 62.8 ± 4.3% Increase in the production of biogas o Control: 209 ± 27mL o Lipase: 346 ± 30mL High total COD removal efficiencies (85.3%) compared to control (79.9%) No formation of scum throughout AD 15% increment in biogas generation compared to control (from 2839 to 3300 mL) 30% reduction in digestion time o Time required for the production of 2839 mL of biogas – control (42 days); lipase (29 days) Significant and fast reduction in COD, TSS and OG 75% improvement in COD reduction o Final concentratio n - control (600±59



Anaer obic condit ion 42 days reside nce time



37 °C stirred at 200 rpm 4 days







[92]

[93]

[94]

Journal Pre-proof

1000 mg/L

Wastew ater treatme nt plant sewage sludge Poultry industry lipid-ric h wastew ater

22.7 g/L

Lipase (0.3 U/ml)

Pseudomo nas aeruginosa KM110

10% (v/v)

 

45 °C 48 h

2005 mg/L

Jo ur

na

Syntheti c dairy wastew ater

lP

re

-p

ro

of

mg/L); Lipase (168±10 mg/L)  37.5% improvement in OG reduction o Final concentratio n - control (88±25 mg/L); Lipase (55±7 mg/L)  15% improvement in TSS reduction o Final concentratio n - control (276±8 mg/L); Lipase (234±19 mg/L)  Improved COD removal efficiency from 66 to 90%  Increase in biogas production from 2330 to 4710 ml after 13 days Yielded 469.5±4.4 mlCH4/g VS

Lipase (50,000 U/g)

Purchase d from Biocon S.A.

0.83% (v/v)

 

35 °C magne tically stirred

Porcine pancreat ic lipase (1,770 U/mg)

Purchase d from Nuclear (São Paulo, Brazil)

3.0 g/L



35±1 °C pH 8.0 gently stirred at 100 rpm 30 days AD



36 °C pH 8.5



 



Swine slaughte

1,445. 80

Lipase

Aspergillu s niger

1.1%

 



Increase in methane production from 569 ± 95 (crude) to 1,101 ± 10 mL (pretreated) 3-fold increment in COD removal of when compared to crude. Increase in fatty acids

[95]

[96]

[97]

[86]

Journal Pre-proof rhouse wastew aters



mg/L Phosph olipase

12 h

porcine pancreatic





P. aquatica

  

10 mg/L

40 °C pH 8.0 Incuba tion time of 90 min



Biorea ctor tempe rature maint ained at 20 °C Hydra ulic reside nce time of 24 h



Maint ained in water bath of 30 °C Agitat ion of 200 rpm Period of 70

Increase in hydrolysis from 17 to 72%

of

Lipase

-p

ro

-



Syntheti c dairy wastew ater

Lipase (33±2 U/g)

Rhizopus microsporu s CPQBA 312-07 DRM

1.25 kg of moist fermented solids added to 0.06 to 0.3 L/h continuou s flow of wastewate r into a packed-be d bioreactor 0.2% (w/v)



na

600 mg/L

Jo ur

Meat & sausage processi ng factory high-fat wastew ater

lP

re

Poultry processi ng industry wastew ater

2000 mg/L

Lipase Z (360,000 U/g)

Candida rugosa-fre e form









concentration o lipase: 31.50 μmol/mL o phospholipas e: 31.13 μmol/mL Maximum biogas yield of 89.65 mL COD reduction of 90.01% concerning COD of raw wastewater 7.4 times increment in free fatty acid concentration (0.23±0.07 to 1.7±0.35 mM) Stimulated hydrolysis of approximately 10% of the fats content 96% reduction of OG content Increase in BOD/COD ratio from 0.19 to 0.55



[87]

[98]

[99]

Journal Pre-proof

N/A

Lipase (20 U/mL)

Candida rugosa

Swine slaughte rhouse waste

83.56 %

Lipase

Porcine pancreas

Food waste

27.3%

Lipase (50 U/mg)

Aspergillu ms niger

Lipase (15 U/mL)

Staphyloco ccus xylosus

16 mL of lipase suspensio n to 0.7808 g of butter 0.2% (w/v)

  

min pH 6.6 40 °C 16 h

 

37 °C 24 h

0.5% (w/v)



pH 7.4 – 7.6 40 °C 24 h

N/A

Desiccat ed coconut wastew ater

3.748 g/L

Coconut oil mill effluent

3582 mg/L

Lipase (100 U/g)

re

lP

Animal fat, vegetabl e oil and floatable grease

N/A

Aspergillu s and Candida

0.5 g substrate to 1000 µL enzymatic solution

na

17 g/L

Jo ur

Slaught erhouse wastew ater

-p

ro

 

Lipase (100 – 500 units/m g protein)

Porcine pancreas

Lipase (254.43 U/g)

Staphyloco ccus pasteuri COM-4A

  

   

 0. 1% (w/v)

  



1% (w/v)

  

84% increase in methane production (417.9 mL CH4@STP/g CODadd)

[100]

 Biomethane yield of 851.6 mL/g VSadded  72.7% of biodegradability  1.8 – 75.1% increase in total fatty acids  biomethane yield of 500.1 mL/g VSadded  maximum biodegradability of 73.8%  Lipid content reduced from 17 g/L to 1.12 g/L  Biogas yield of 0.6 L/g COD

[101]

 Increase in total LCFA in all substrates  Maximum daily biomethane production of 180 ± 22 mL  Initial biogas generation rate of 25.43 mL/day  Highest average gas production rate of 7.16 mL/day  The highest cumulative biogas production of 68.5 mL  52% O&G reduction  41% VFA production  28% LCFA production  Maximum

[104]

[102]

of

Butter

37 °C pH 7.0 stirred at 200 rpm 5 – 8 days 40 °C pH 7.0 stirred at 200 rpm 24 h 37 °C pH 7.0 stirred at 100 rpm 24 h

50 °C pH 9.0 orbital shake n at 120 rpm

[103]

[105]

[106]

Journal Pre-proof 24 h

methane gas production of 0.86 L CH4/g VSSadded

Jo ur

na

lP

re

-p

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

Journal Pre-proof Table 3. Summary of recent studies on the lab-scale enzymatic pretreatment on carbohydrate and protein-rich wastewater

Cellulase (0.98 U/mgs), Amylase (0.8 U/mg), Protease (0.013 U/mg) Amylase

Rhizocloniu m

0.001% (w/w), 0.05% (w/w), 0.2% (w/w)

Purchased commercia lly

0.001% (w/w)

  

35 °C pH 8.0 300 h

re

Purchased commercia lly

6% (w/w) (1 protease to 3 amylase)



Lipase, Cellulase, Xylanase, Amylase, Protease

Effects

7 to 14% enhancement in methane production as compared to control

50 °C







lP

Amylase, Protease

na

Sludge from secondary sedimentati on tank of the second municipal wastewater treatment plant

Purchased commercia lly

Jo ur

Simulated food waste (fresh potato TVS: 19.5%)

Dosage

of

Wheat grains

Source of Enzyme

ro

Enzyme

-p

Sample

Operating conditions of pretreatm ent  37 °C  24 h

Purchased from Novozyme





1% (w/w)

  

53 °C pH 7.0 2 days





21% increment in methane yield Methane composition on biogas was 55-73% VSS reduction significantly increased from 10 to 68.43% 377% improvement in reducing sugar 201% improvement in NH4+-N 31% increase in cumulative CH4 produced when compared to control 9% increase in cumulative CH4 with the use of amylase enzyme alone

Referenc es [109]

[110]

[111]

[112]

Journal Pre-proof 

 

Sludge from the wastewater treatment plant

Amylase (0.47 U/mg VSS)

Bacillus subtilis

0.1% (v/v)

Protease

Aeromonas hydrophila

Synthetic raw sewage (550±50 mg COD/L)

Protease, Lipase, Amylase

Purchased from BioCat Microbials, USA

0.9 mg/L, 3.6 mg/L, 18 mg/L



Lucerne pellets and birch leaves pellets

Alpha-amyl ase (EC 3.2.1.1)

N/A

0.5 mL to 10 g of substrate



[113]

Jo ur

na

lP

re

-p

ro

of



37 °C Stirred at 120 rpm 28 h

20% increase in cumulative CH4 with the use of cellulase enzyme alone  SCOD release increased with incubation time o Amylase: 78.2% o Protease: 29.5% o Mixture: 30.2%  Pretreatment with amylase resulted in highest VFA production (133 to 1719 mg/L).  Improved biogas production o Amylase: 18.6% o Protease: 15.6% o Mixture: 20.2%  22% reduction of VSS  19% reduction of TSS  26% increase in biogas production Lucerne pellets  19.95% increase in methane yield (0.481 ± 0.025 L/gDOM)  0.7% increase in average

 

30-32 °C pH 7.0 120 days

38 ± 0.5 °C

[114]

[115]

Journal Pre-proof methane content in biogas (51.43%)

Novozyme s

1.0% (g TS/g TS)

Amylase

na

0.6% (g TS/g TS)

Glucoamyla se (10 U/g dry solids)



directl y added to bioreac tor pH 5 – 9 directl y added to bioreac tor pH 5 – 9 60 °C 24 h 100 rpm

Aspergillus awamori

 

Jo ur Mixed food waste

55 °C 18 h pH 5 – 6

lP

0.6% (g TS/g TS)

Protease

  

-p

Cellulase

re

Cow manure and corn straw

ro

of

Birch leaves pellets  22.9% increase in methane yield (0.263 ± 0.018 L/gDOM)  4.2% increase in average methane content in biogas (42.8%)  103.2% increase in methane yield (364.04 mL CH4/g VS)

 50% (w/v)

  



110.79% increase in methane yield (377.63 mL CH4/g VS)



1.47% increase in methane yield (181.78 mL CH4/g VS) 89.1 g/L glucose 2.4 g/L free amino nitrogen 165 g/L soluble chemical oxygen demand (SCOD) 64% reduction in volatile

 





[116]

[117]

Journal Pre-proof



Dairy plant waste activated sludge

Protease

B. licheniformi s

  

2 g dry cell weight /L

55 °C 24 h 150 rpm

 

Municipal sludge

250 mg of immobiliz ed wet bacterial cells to 500 mL of deflocculat ed sludge

  

ro

Protease

35 °C 36 h 150 rpm

Aspergillus niger

Alcalase 2.5 L (Protease)

Novozyme s

4.2 mL

na

α-amylase (131.3 IU/mL), β-glucosidas e (6 IU/mL) and CMCase (3.7 IU/mL)

Jo ur

Organic Fraction of Municipal Solid Waste

lP

re

-p

Municipal sludge

Microalgae biomass

 



   

50 °C pH 4.5 2h 100 rpm







N/A

  

[118]

of



solids 3.5-times higher in biomethane yield 27% solid reduction 24% COD solubilizatio n 310.6% increase in biogas production potential (2.5211 L/g VS) 17.14% SS reduction 20% increase in COD solubilizatio n Methane yield of 0.24 gCOD/gCOD 34.6% change of soluble COD 40.6% change of reducing sugar 255% increase in methane potential (672 mL/g VS) Organic matter hydrolysis efficiency of 54.7% ± 5.6 Methane yield of 136.9 mL CH4/g CODin

50 °C pH 8 3h





[119]

[120]

[121]

Journal Pre-proof Highlights

Lignin, carbohydrate, protein, oil and grease cause operational problems in high-rate anaerobic bioreactor



Improving hydrolysis stage using enzymes could enhance overall anaerobic digestion



Usage of enzymes improved biogas production by 7 – 76%



pH (7 - 8), temperature (30-55°C), and enzyme dosage (1-2%w/w) are optimum conditions for pretreatment

Jo ur

na

lP

re

-p

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7