Enzymatic reactions in the production of biomethane from organic waste

Journal Pre-proof Enzymatic reactions in the production of biomethane from organic waste ´ Dufour, Joanna Lecka, Brar Topwe Milongwe Mwene-Mbeja, Amelie ´ Satinder Kaur, Celine Vaneeckhaute

PII:

S0141-0229(19)30148-6

DOI:

https://doi.org/10.1016/j.enzmictec.2019.109410

Article Number:

109410

Reference:

EMT 109410

To appear in:

Enzyme and Microbial Technology

Received Date:

7 April 2019

Revised Date:

6 July 2019

Accepted Date:

15 August 2019

Please cite this article as: Mwene-Mbeja TM, Dufour A, Lecka J, Kaur BS, Vaneeckhaute C, Enzymatic reactions in the production of biomethane from organic waste, Enzyme and Microbial Technology (2019), doi: https://doi.org/10.1016/j.enzmictec.2019.109410

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Enzymatic reactions in the production of biomethane from organic waste Topwe Milongwe Mwene-Mbejaa, Amélie Dufourb,c, Joanna Leckad, Brar Satinder Kaurd, Céline Vaneeckhauteb,c* a

Department of Chemistry, Faculty of Sciences, University of Lubumbashi, D.

R.

Congo,

E-mail

address:

[email protected]

/

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Lubumbashi,

[email protected] b

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BioEngine - Research team on green process engineering and biorefineries,

Department of Chemical Engineering, Faculty of Science and Engineering,

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Université Laval, 1065 avenue de la Médecine, Pavillon Adrien-Pouliot, Québec,

[email protected]

CentrEau, Centre de recherche sur l'eau, Université Laval, 1065 Avenue de la

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c

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QC, Canada, G1V 0A6, E-mail address: [email protected],

Médecine, Québec, QC, Canada, G1V 0A6 d

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Centre Eau Terre Environnement, Institut national de la recherche scientifique, 490

rue de la Couronne, Québec, QC, Canada, G1K 9A9, E-mail address: [email protected], [email protected] *Corresponding author.

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Highlights 

Enzymatic reactions refer to organic reactions catalyzed by enzymes



Enzymatic reactions relative to the production of biomethane are reviewed



The structure of organic compounds and reaction mechanisms are represented

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

Understanding of enzymatic reactions can be used for biomethanation optimization



Understanding of enzymatic reactions can lead to the development of new bioproducts

Abstract

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Enzymatic reactions refer to organic reactions catalyzed by enzymes. This review

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aims to enrich the documentation relative to enzymatic reactions occurring during the anaerobic degradation of residual organic substances with emphasis on the

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structures of organic compounds and reaction mechanisms. This allows to

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understand the displacement of electrons between electron-rich and electron-poor entities to form new bonds in products. The detailed mechanisms of enzymatic

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reactions relative to the production of biomethane have not yet been reviewed in the scientific literature. Hence, this review is novel and timely since it discusses the

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chemical behavior or reactivity of different functional groups, thereby allowing to better understand the enzymatic catalysis in the transformations of residual proteins, carbohydrates, and lipids into biomethane and fertilizers. Such understanding allows to improve the overall biomethanation efficiency in industrial applications.

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Keywords: Biomethane; Carbohydrates; Enzymatic reactions; Lipids; Mechanisms; Proteins

1. Introduction

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Biomethanation is an established technology to convert organic waste into biomethane and digestate fertilizer products under the action of microorganism’s enzymes. The valorization of organic waste through biomethanation contributes to the improvement of hygiene, reduces bad odors as well as the use of synthetic fertilizers and pesticides. It also helps to avoid incineration and the dumping of

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organic waste in landfills, thereby reducing greenhouse gas emissions such as carbon dioxide and nitrous dioxide [1,2]. Energy from biogas is very important

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especially in regions lacking energy infrastructure, for instance in Africa, because it

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reduces the use of fossil fuels, limits deforestation and improves people's livelihoods. The aim of this article is to enrich the documentation relative to different

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enzymatic reactions which occur in the anaerobic degradation (biomethanation) of

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residual organic substances, with emphasis on the structures of organic compounds and reaction mechanisms to explain the formation of different products (Figure 1). The detailed mechanisms of enzymatic reactions relative to the production of

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biomethane and biofertilizer have not yet been reviewed in the scientific literature. However, the structures of the organic compounds show different functional groups at which the organic reactions can take place. In order to better understand the

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formation of the various products (biomethane and biofertilizer) in the reaction media, it is important to know which functional groups of enzymes and substrates react with each other. On top of that, the mechanisms of electron displacement and bond formation through anaerobic degradation must be understood. All of this, in

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turn, can guide future research efforts in terms of biomethanation optimization, for example, by maximizing biomethane production or biofertilizer quality. Concretely, this review briefly describes the function of enzymes (Section 2), after which it presents a collection of organic reactions occurring upon anaerobic digestion, such as hydrolysis, acidification, synthesis of acetate (ethyl acetate or

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methyl acetate) and synthesis of methane catalyzed by enzymes (Section 3). Various substrates (proteins, carbohydrates, and lipids) are metabolized or

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degraded in an anaerobic fashion by specific enzymes, while intermediate products

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serve as starting materials for subsequent enzymatic conversion to produce methane and fertilizers. Examples of current industrial applications to improve the

2. Enzymes

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efficiency of biomethanation by use of enzymes are provided in Section 4.

Enzymes are proteins or organic catalysts present within the cells of

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microorganisms and capable of catalyzing reactions inside or outside their cell. The active center of an enzyme contains amino acids, which help to stabilize the enzyme’s transition state through hydrogen bond formation either with substrates or

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with other amino acid residues. As such, a triad composed of serine, histidine, and aspartate forms a catalytic center of certain proteases and these three amino acids work together to carry out the expected catalytic activity. In the catalytic triad, histidine plays the role of a base (Figure 2) [3,4]. Indeed, amino acids can act as acids, bases, nucleophiles or electrophiles.

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Proteins are build from amino acids, which are small molecules composed of an amine group, a carboxylic acid group and an organic R group (mostly levorotatory). Those R groups can posses an ionized polar character that influences the shape of the enzyme’s active sites. They facilitate the structural changes of the

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protein in different physicochemical conditions and are crucial in the process of catalysis. (Figure 3). Indeed, these polar amino acids release protons as acids and

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capture protons as bases and being nucleophiles, they transfer electrons to

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electrophiles during catalysis (Figure 3) [3,4]. In the active center, the side chains of the polar amino acids participate in the acid and base chemical and covalent

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catalysis. In other words, they can donate protons in alkaline solution (Figure 3,

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reaction 1) or accept protons in acidic solution (Figure 3, reaction 2). In neutral solution (pH 7), the side chains of the amino acids are neutral (Figure 3). The active

[3,4].

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center is thus the biological equivalent of a strong acidic or strong basic solution

3. Enzymatic reactions

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3.1 Hydrolysis

The residual organic substances consist mainly of proteins, sugars, and

lipids. The enzymatic hydrolysis of these macromolecules is the first step in the production of methane and fertilizers. Nucleophilic N-terminal hydrolases constitute a family of enzymes specialized in the breakdown of amide bonds, for instance,

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serine proteases characterized by the presence of serine, histidine, and aspartate in the active center. The strategic step in the serine protease mechanism is the nucleophilic attack at the carbonyl group of the polypeptide (protein) by the oxygen ion from the hydroxyl group of the serine (Figure 4, reaction 2). The role of aspartate is to remove the proton bound to the nitrogen atom of histidine, while histidine

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removes the acid proton from the hydroxyl group of serine (Figure 4, reaction 1) [5,6]. Sugars or carbohydrates are organic compounds that act as an energy

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reservoir at the level of higher plants. Because humans consume plants, sugars turn

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out to be essential constituents of human food. In general, carbohydrates are one of the major chemical groups containing carbon, hydrogen, and oxygen, such as

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starch, which is an energy reserve substance in the plant cell [7,8]. Starch is a long

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chain made up of nonlinear glucose molecules (amylopectin) and linear glucose molecules (amylose) [7,8]. The glucose molecules within the starch can range from a few hundred to a few thousand linked together by alpha-glycoside linkages [7,8].

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During the hydrolysis of starch, it was found that the alpha stereochemistry is maintained in the final product and the plausible catalytic mechanism involves the protonation of the glycosidic oxygen by the proton donor glutamate amino acid

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(Figure 5, reaction 2) [9,10]. This step is followed by the ejection of the leaving group, due to an electron lone pair oriented antiperiplanar to the leaving group, to produce a corresponding cyclic oxocarbonium ion (Figure 5, reaction 2). The latter reacts with a water molecule to generate an intermediate compound, which yields a proton to

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aspartate and this results in the formation of glucose molecules in the reaction medium (Figure 5, reaction 3) [9,10]. Cellulose is an organic polymer formed by several units of glucose molecules linked together by beta-(1-4)-glycosidic bonds (Figure 6) [9,10]. It is associated with lignin and hemicellulose, which form a protective layer or a shield of the cellulose. It has

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been observed that lignin and hemicellulose are difficult to hydrolyze with enzymes. For this reason, the cellulosic waste must be treated beforehand, for instance, in

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using dilute acids to degrade lignin and hemicellulose and thus promote the

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efficiency and rapidity of the enzymatic hydrolysis of cellulose (Figure 6) [9,10]. As hemicellulose is built from different polymers, such as pentoses, hexoses, and

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sugar/uronic acids, it requires a wide variety of enzymes to be fully hydrolyzed into

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free monomers [11,12,13]. In this perspective, it has been reported that the enzyme derived from the fungus Trichoderma viride was capable of transforming the cellulosic waste into fermentable sugars (Figure 6) [9,10]. The authors mentioned

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that the fungus Trichoderma viride produces cellulase, which reacts with a crystalline fraction of a cellulose molecule (Figure 6). The authors also reported that the hydrolysis of cellulosic residues from sugar cane with cellulase produced glucose

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with excellent yield (Figure 6) [9,10].

Cellulase represents a group of different enzymes, in particular, the

endoglucanase enzymes which hydrolyze beta-(1-4)-glycosidic internal bonds, exoglucanase enzymes that hydrolyze beta-(1-4)-glycosidic external bonds and the

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cellobiase that hydrolyzes the disaccharide cellobiose to produce two molecules of glucose. Experimental studies showed that two amino acids (glutamate and aspartate) constitute the root of the catalytic activity in the hydrolysis of cellulose. In this regard, the glutamate amino acid yields a proton to glycosidic oxygen and the aspartate amino acid acts as a base by accepting the proton of a water molecule,

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which acts as a nucleophile (Figure 7) [9,10]. According to the theory of stereoelectronic control, the carbon-hydrogen bond of beta-glycoside (equatorial isomer)

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is activated by two ion pairs of electrons oxygen antiperiplanar to the carbon-

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hydrogen bond (two electronic effects) [9,10]. The proposed hydrolysis mechanism involves the formation of a twist-boat conformation, which allows the substrate to line

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up an ion pair of oxygen electrons in an antiperiplanar way to the leaving group,

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before adopting a chair-like transition state (cyclic oxocarbonium ion) (Figure 7) [9,10]. The nucleophilic attack at the oxocarbonium ion leads to the production of an intermediate adduct, which donates a proton to aspartate to produce the axial isomer

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(alpha-glycoside) and the equatorial isomer (beta-glycoside) (Figure 7) [9,10].

The best-known disaccharide is sucrose obtained from sugarcane juice or

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sugar beet. During enzymatic hydrolysis, sucrose degrades to generate D-(+)glucose and D-(-)-fructose (Figure 8, reaction 1) [14,15]. Concerning the reaction mechanism, Reddy and Maley (1996) [14] have demonstrated the role of the aspartate amino acid as well as that of the glutamate amino acid in the catalytic activity of the enzyme invertase (Figure 8) [14,15]. The carboxylate group of the side

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chain of aspartate acts as nucleophile, while the carboxylic group of the glutamate amino acid acts as an acid (donor of the proton) (Figure 8, reaction 2). The conjugate base accepts the proton of the water (Figure 8, reaction 3). The latter acts also as a

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nucleophile (Figure 8, reaction 3) [14,15].

Lipids are triglycerides or esters of fatty acids and glycerol containing long (R)

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chains which are insoluble in water (Figure 9). From an environmental point of view, lipids are important organic constituents in wastewater, which contribute enormously

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to environmental pollution. If they are not removed, the lipids form a layer on the

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surface of the water and thus prevent diffusion of oxygen from the air into the water. This result in the death of aquatic living beings. In this regard, it has been shown that lipases are potential catalysts that can be used to hydrolyze oils from industrial

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effluents, slaughterhouses and frying oils [16]. Experimental observations have shown that the lipases catalyze the hydrolysis of the ester bonds to produce fatty acids and glycerol (Figure 9, reaction 1) [17]. These enzymes contain serine,

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histidine and aspartate amino acid in the active center. It has been found that the aspartate amino acid can be replaced by the glutamate amino acid in the case of Geotrichum candidum lipase [17]. This enzyme uses the glutamate residue to specifically hydrolyze the fatty acids having cis double bonds in the hydrocarbon side

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chains [17]. The reaction mechanism of the lipases is thus similar to that of serine protease (Figure 9, reaction 2).

The catalytic activity of the lipases is influenced by the water molecules present in the structure of these enzymes. From this point of view, it has been observed that

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when the lipases are dehydrated, they lose the ability to catalyze esterification

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reactions involving congested alcohols. This could be explained by the fact that the absence of water affects the mobility necessary to bind the substrates in the active

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center [18,19]. Hence, lipases are less active in solvents which are miscible with

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water because they extract water integrated into the structure of the enzyme [20,21].

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3.2 Acidification

The products of hydrolysis are used by microorganisms as substrates to

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produce acids, alcohols and carbon dioxide (Figure 10). At this level of biomethanation, amino acids could be used as a source of energy for microorganisms. Rupasinghe and his colleagues reported on an enzymatic method for producing glucose, ethanol and acetic acid from apple residues (Figure 10)

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[18,19]. The cellulose was pretreated with acidified aqueous conditions and the hydrolysis was optimized by using commercial enzymes (beta-glucosidase and pectinase). The glucose was fermented using Saccharomyces cerevisiae to produce ethanol. The bioconversion of ethanol to acetic acid was carried out using acetobacter aceti [18,19].

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The enzymatic oxidation of ethanol to acetaldehyde is catalyzed by alcohol dehydrogenase (Figure 10, reaction 2) [22,23]. On the other hand, the oxidation of acetaldehyde to acetic acid is catalyzed by the aldehyde dehydrogenase (Figure 10, reaction 3). These enzymes use nicotinamide adenine dinucleotide (NAD) as coenzyme and glutamate plays the role of a base in the active center of the enzyme

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(dehydrogenase) [22,23]. Regarding the dehydrogenation of ethanol, the mechanism involves the transfer of the hydride ion to NAD+ (oxidized form of NAD)

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and this results in the formation of NADH (reduced form of NAD) and the desired

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product (acetaldehyde) (Figure 10, reaction 2). Dehydrogenation of acetaldehyde involves the nucleophilic attack by a water molecule upon the electrophilic carbon of

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acetaldehyde. This step is followed by the removal of the proton by glutamate and

reaction 3) [22,23]. 3.3 Synthesis of acetate

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the transfer of the hydride ion at the level of the oxidized form of NAD (Figure 10,

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It has previously been observed that the majority of methane is produced from acetate (Figure 1) [24]. In this context, it has been demonstrated that lipase catalyzes the reaction between acetic acid and ethanol to produce volatile natural ethyl acetate

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and water as by-product [25,26]. Because of the reversibility of the reaction, it is important to remove water to optimize the formation of the product. Concerning the mechanism, the lipase utilizes serine, histidine and aspartate amino acid to catalyze the production of acetate from acetic acid and alcohol (ROH). Aspartate amino acid donates a proton to the hydroxyl group of acetic acid (Figure 11, reaction 2). This

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step is followed by serine nucleophilic attack, resulting in the loss of one molecule of water and formation of an intermediate adduct, which in turn reacts with the alcohol (ethanol or methanol) to produce the corresponding acetate (Figure 11, reaction 3) [27].

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3.4 Synthesis of methane Bacteria degrade ethyl acetate to produce ethanol and acetic acid. The latter

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is then metabolized to produce methane and carbon dioxide with the assistance of

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decarboxylase as a catalyst. It has been reported that decarboxylation involves the formation of negatively charged intermediate compounds. In this perspective, the

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electrophilic substitution of carbon dioxide by a proton involves the loss of carbon

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dioxide to form a carbanion (methyl anion), which accepts a proton from the catalytic amino acid residue, in this case, lysine, to generate the desired product (methane) (Figure 12) [25,28]. It has been observed that lysine and cysteine are at the center

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of catalytic activity of decarboxylase (Figure 12) [26].

4. Current industrial applications to optimize biomethanation efficiency

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Cellulosic materials have relatively low cost and plentiful supply. Organisms that are producing cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products are considered for “consolidated bioprocessing” (CBP). Biological hydrogen and methane production biomass able to convert lignocellulose into biofuels show great potential as a promising alternative to

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conventional hydrogen and methane production methods, but utilization of these materials is limited due to their low degradability. As hydrolysis is the first step of anaerobic digestion, preliminary degradation of lignocellulose is crucial for efficient bioconversion of biomass to biofuels [29,30]. The low rate of hydrolysis of lignocellulose results in the slowdown of the entire process. Therefore, the

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lignocellulosic material requires special preliminary treatment before further processing [31], which may be carried out by physical, chemical and biological

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methods including, but not limited to, e.g., comminution, acid hydrolysis, ammonia

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fiber expansion, steam explosion [32].

Increase in the biogas yield production from hemicelluloses using anaerobic

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digestion and decrease in the time of this process can be achieved by several

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pretreatment technologies [31,33,34,35,36]. Pretreatments with enzymes that will convert lignocellulosic biomass into biogas are necessary also for elimination of structural barriers like crystalline cellulose structure [37,38,39,40]. In the literature,

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two enzymatic pretreatment strategies for lignocelluloses have been reported: 1) helping to degrade cellulose by addition of laccases and peroxidises, 2) reinforcing cellulose and other polysaccharides metabolism by addition of cellulosic enzymes.

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The first strategy utilizes enzymes from various fungi. Anaerobic fungi, belonging to the phylum Neocallimastigomycota, key players in the digestive system of various animals, produce a plethora of plant carbohydrate hydrolysing enzymes that can degrade both amorphous and crystalline cellulose [41].

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Combined with the invasive growth of their rhizoid system their contribution to cell wall polysaccharide decomposition may greatly exceed that of bacteria [42,43]. The yield of end-products of enzymes used as pretreatment depend on species, sources and substrates of fungi [44,45]. Anaerobic fungi have widely been isolated from wild and domestic ruminants, such as Holstein steers rumen, ovine rumen,

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cattle rumen, cattle faeces, buffalo rumen or faeces, goats, wild bluebull, elephants, deer, zebras, grazing sheep [46,47,48,49,51]. Sijtsma and Tan (1993) reported that

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Neocallimastix isolate from the yaks faeces could secrete enzymes that degrade the

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cell wall, including exoglucanase [52].

Some studies suggest that anaerobic fungi and methanogens, e.g.

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methanogenic Archaea, a diverse group of strictly anaerobic Euryarchaeota, can

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form stable co-cultures that have higher ability to degrade lignocellulose than pure anaerobic fungi [53,54]. Such co-culture changes metabolism pathways of fungi, resulting in the production of methane and more acetate and ATP, but less lactate,

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ethanol and succinate, since it can provide more energy for fungi by enhancing cellulose fermentation kinetics [54]. The strength of such cooperation was confirmed in the study of Wei et al. (2016) showing that the anaerobic fungi and associated

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indigenous methanogens in the rumen of grazing yaks have a strong capability to degrade lignocellulose [55]. Another practical example are extremophile-derived enzymes (enzymes

present

in

Extremophilic

microorganisms

called

thermophiles

and

hyperthermophiles). Due to their optimal activity and stability under extreme

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conditions, these enzymes are able to catalyze chemical reactions under harsh conditions similar to those found in industrial processes. Those organisms are found in environments where temperatures are above 100°C such as hot springs and deep sea vents, geysers and highly geothermal volcanic areas [56,57] Hyperthermophiles, e.g., Methanocaldococcus jannaschii, posses the anaerobic metabolic pathways

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used for production of methane from hydrogen and carbon dioxide [58]. From an industrial viewpoint, hyperthermophilic enzymes possess certain advantages: their

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enzymes are active and efficient under high temperatures (above 100°C), extreme

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pH values (4.0 to 7.5), high substrate concentrations, high pressure and are highly

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resistant to denaturing agents and organic solvents [59, 60, 61] Unfortunately, the costs of using enzymatic processes in industrial applications are

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very high, which increases the cost of waste conversion to biogas. Nevertheless, efforts to improve the cost efficiency are growing. DuPont Org., winner in the

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European Community project DEMETER (Demonstrating More Efficient Enzyme Production To Increase Biogas Yields, January 2016) provides a good best practice example. They developed and applied a fungal expression technology for producing enzymes that break down plant fibers (carbohydrates such as cellulose and

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hemicellulose) and protein-rich materials, resulting in sugars and amino acids more suitable for biogas-producing microorganisms [62].

5. Conclusions and perspectives

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Proteins, carbohydrates, and lipids are essential constituents of domestic, agricultural and industrial organic waste. These organic molecules have functional groups, at which hydrolysis, acidification and decarboxylation reactions catalyzed by specific enzymes take place. This paper explains in an understandable way the different steps of enzymatic reactions involved in the production of methane and

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fertilizer from residual organic substances. This allows guiding future research efforts in terms of biomethanation optimization, e.g., by maximizing biomethane production

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and biofertilizer quality. Knowledge of enzymatic mechanisms involved in

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Conflict of interest statement

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reinforce the process of methane production.

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biomethanation can help to define precisely the pretreatment strategies that will

We declare that we do not have a conflict of interest regarding the publication of this

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paper.

Acknowledgements

Céline Vaneeckhaute is funded by the Natural Science and Engineering

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Research Council of Canada (NSERC) through the award of an NSERC Discovery Grant (RGPIN-2017-04838) and by the Fonds de Recherche du Québec Nature et Technologies (FRQNT) through the award of a Research Support for New Academics Grant (2019-NC-253483).

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Carbohy drates

Lipids

Amino acids

Sugars

Fatty acids

Acidific ation

Acetate synthesis

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Acetate Water

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Hydrol ysis

Polypep tides

Methane synthesis

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Figure 1 – Processes involved in anaerobic digestion and products obtained.

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Figure 2 – Triad of amino acids forming the catalytic center of certain proteases.

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Figure 3 – Examples of reactions with polar amino acids occurring in the catalytic center of an enzyme: 1) Donation of protons in alkaline solution, 2)

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neutral solution.

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Acceptance of protons in acidic solution, 3) Neutrality of the side chains in

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Figure 4 – Serine protease mechanism: 1) Reaction between aspartate,

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histidine and serine, 2) Nucleophilic attack of the polypeptide by the serine.

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Figure 5 – 1) Hydrolysis of starch, 2) Catalytic mechanism involving glutamate and formation of the cyclic oxocarbonium ion, 3) Reaction of the ion with water

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and aspartate to produce glucose.

Figure 6 – Hydrolysis of cellulose with cellulase produced by the fungus Trichoderma viride.

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Figure 7 – Hydrolysis mechanisms of cellulose.

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Figure 8a – Degradation of sucrose with the enzyme invertase.

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Figure 8b – Reaction mechanisms for sucrose hydrolysis: 1) Formation of D(+)-glucose, 2) Formation of D-(-)-fructose.

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Figure 9 – Hydrolysis of lipids with lipase: 1) Formation of fatty acids and

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glycerol, 2) Reaction of the triglyceride with serine.

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Figure 10 – 1) Formation of carbon dioxide and ethanol from glucose, 2)

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Oxidation of ethanol to acetaldehyde, 3) Oxidation of acetaldehyde to acetic

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acid.

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Figure 11 – Formation of acetate from acetic acid and ethanol: 1) Basic reaction, 2) Enzymatic reaction between acetic acid and aspartate amino acids,

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3) Enzymatic reaction of serine to form acetate.

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Figure 12 – Reaction mechanisms for the formation of methane with lysine.