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Functions of bacteria and archaea participating in the

bioconversion of organic waste for methane production

Farrukh Raza Amin, Habiba Khalid, Hamed El-Mashad, Chang

Chen, Guangqing Liu, Ruihong Zhang

PII: S0048-9697(20)36537-2
DOI: https://doi.org/10.1016/j.scitotenv.2020.143007
Reference: STOTEN 143007

To appear in: Science of the Total Environment

Received date: 20 June 2020

Revised date: 8 October 2020
Accepted date: 8 October 2020

Please cite this article as: F.R. Amin, H. Khalid, H. El-Mashad, et al., Functions of bacteria
and archaea participating in the bioconversion of organic waste for methane production,
Science of the Total Environment (2020), https://doi.org/10.1016/j.scitotenv.2020.143007

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© 2020 Published by Elsevier.

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Functions of bacteria and archaea participating in the bioconversion of

organic waste for methane production

Farrukh Raza Amina‡, Habiba Khalida‡, Hamed El-Mashadb,c, Chang Chena*, Guangqing Liua*,

Ruihong Zhangb*

a
College of Chemical Engineering, Beijing University of Chemical Technology (BUCT), Beijing

100029, China.

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b
Department of Biological and Agricultural Engineering, University of California, Davis, CA

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95616, United States.
c

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Department of Agricultural Engineering, Faculty of Agriculture, Mansoura University, Egypt,

35516.
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‡
These authors have contributed equally.
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Correspondence:
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*Chang Chen‟s e-mail: chenchang@mail.buct.edu.cn

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*Guangqing Liu‟s e-mail: gqliu@mail.buct.edu.cn

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*Ruihong Zhang‟s e-mail: rhzhang@ucdavis.edu

Tel: +86-10-64442375, Tel: (530)754-9530

Address: 505A Zonghe Building, Beijing University of Chemical Technology, 15th North 3rd

Ring East Road, Beijing 100029, P. R. China

Address: Department of Biological and Agricultural Engineering, 3046 Bainer Hall, University

of California Davis, CA 95616

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Abstract

Anaerobic digestion (AD) is a widely applied technology for treating organic wastes to

generate renewable energy in the form of biogas. The effectiveness of AD process depends on

many factors, among which the most important is the presence of active and healthy microbial

community in the digester. Therefore, it is necessary to explore the microbial ecology in the

anaerobic digesters. However, the deciphering of microbial populations and their functions

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during the AD process of different materials is still incomplete, which restricts the understanding

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of its long-term performance under different operational conditions. This review describes the

type, morphology, function, specific growth conditions, and ecology of commonly found
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hydrolytic, acidogenic, acetogenic bacteria, and archaea during the AD process. The effects of
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microbes on the performance and stability of the digestion process are also presented.
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Furthermore, the article offers a deep understanding of the AD management strategies for the

enhancement of methane production and improve the efficiency of the energy conversion process
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of various organic wastes.

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Keywords: Organic wastes; hydrolytic bacteria; acidogenic bacteria; acetogenic bacteria;

methanogens; anaerobic digestion

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Contents

1. Introduction ........................................................................................................................ 4

2. Types and roles of bacteria ............................................................................................... 6

2.1. Hydrolytic/Fermentative bacteria ............................................................................................ 6

2.2. Acidogenic bacteria ................................................................................................................ 12

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2.3. Acetogenic bacteria ................................................................................................................ 17

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2.4. Sulfate-reducing bacteria....................................................................................................... 23

3. -p
Types and roles of archaea .............................................................................................. 25
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3.1. Hydrogenotrophic/methylotrophic methanogens.................................................................. 26
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3.2. Acetoclastic methanogens ...................................................................................................... 29

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4. Impact of operational conditions on microbial community in AD reactors ............... 30

4.1. Organic loading rate (OLR) and hydraulic retention time (HRT) ............................................ 31
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4.2. Temperature, pH and alkalinity ................................................................................................. 31

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4.3. Nutrient availability .................................................................................................................... 32

5. Practical implications and future research directions .................................................. 34

6. Conclusions ....................................................................................................................... 36

7. References ......................................................................................................................... 38
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1. Introduction

Anaerobic digestion (AD) is widely applied technology for treating organic wastes to generate

renewable energy in the form of biogas. The AD process comprises of four stages; hydrolysis,

acidogenesis, acetogenesis, and methanogenesis. Each of these stages encompasses numerous

microbes which contribute to methane production (S. Wang et al., 2018; Xia et al., 2016) as

shown in Fig. 1 (P. Wang et al., 2018). In the hydrolysis, complex organic substances are

converted to soluble monomers via hydrolytic enzymes that are excreted by microorganisms

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(Nguyen and Khanal, 2018). Acidogenesis serves as an intermediate step in which acidogenic

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bacteria transform soluble monomers into volatile fatty acids (VFAs) (Hendriks et al., 2018).

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Acetogenesis refers to acetate and hydrogen (H2) formation by organic acids and CO2.
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Methanogenesis is the final step in the AD process during which acetate and H2 and CO2, the
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major products of acetogenesis, are converted into methane by anaerobic methanogens (Li et al.,

2019). Preserving the activity of methanogens is a precondition for enhancing methane yield.
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This is because methanogens play an important role in maintaining a low H2 concentration to

thermodynamically drive unfavorable biochemical fermentation reactions (Mccarty and Smith,

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1986; Morris, 2011). The progressive knowledge of the methanogenic communities could help in
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discovering beneficial methanogens for enhancing the methane production. This might lead to

the production and preservation of a wide range of specifically designed consortia of useful

methanogens for improving the AD process (Ribes et al., 2004). Methanogens are very sensitive

to the pH variation, and therefore, a stable pH condition should be maintained. Moreover,

methanogenic activity and methane production process depend on the organic loading rate (OLR)

and temperature (Xin et al., 2018). Controlling the OLR and temperature is important to sustain

the equilibrium between the different steps of the AD process, especially the acidification and
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methanogenesis (Kouas et al., 2018; Pang et al., 2018; Zou et al., 2018). The microorganisms

play a significant role in CH4 production, therefore, it is important to determine their functions

and the optimal operating parameters to ensure efficiency and stability of the AD process

(Mosbæk et al., 2016; Roopnarain et al., 2017; P. Wang et al., 2018). As high-throughput DNA

sequencing technology has rapidly developed, it has been used for the identification of microbes

in the anaerobic digesters (Dennehy et al., 2017; Mu et al., 2017; Xu et al., 2018). Nonetheless,

there is a lack of critical review on research advancements related to the functions of microbes

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during each of the four stages of AD. There is not enough information as how the four different

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groups of microorganisms such as hydrolytic, acidogenic, and acetogenic bacteria and

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methanogenic archaea cooperate with one another to complete the conversion of various
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substrates to clean energy. Moreover, these microorganisms are strongly dependent on the types

of substrates, operation conditions including temperature, organic loading rate (OLR), hydraulic
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retention time (HRT), and F/I ratio to start the fermentation process. The purpose of this study
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was to determine the microbial groups responsible for the anaerobic digestion of different

substrates under different experimental conditions during each stage of AD process. To our best
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knowledge, no previous study has been reported in which an attempt to showcase the detailed
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mechanisms of functions of these microbes during AD has been made. If the functions of the

microbial communities and their optimal operating conditions are known, it can be helpful in

designing AD digesters with the desired microbial ecology, emulating the AD digesters

environment to achieve the maximum rate of energy recovery.

Therefore, the objectives of this review are: to create a database of major bacteria and archaea

participating in the AD of organic wastes, and identify their functional roles and mechanism

during the four stages of AD. Moreover, the influence of operational parameters on the microbial
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activity is also explored. This review may provide beneficial information regarding the

contribution of the associated microorganisms in the AD process, which will be helpful in

designing and operating anaerobic digesters and therefore, enhancing the conversion of different

organic wastes to clean energy.

Fig. 1. Schematic diagram for the AD process

2. Types and roles of bacteria

2.1. Hydrolytic/Fermentative bacteria

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During hydrolysis step, hydrolytic bacteria break down complex carbohydrates, lipids, and

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proteins into simple and soluble sugars, fatty acids, and amino acids, respectively. Hydrolysis of

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organic wastes is generally carried out by the synchronized activities of phylogenetically diverse
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bacteria, which act both as hydrolytic enzyme producers and hydrolysis product utilizers (Li et
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al., 2019). The role of hydrolytic bacteria in AD system has not been widely studied, particularly

if compared to methanogens. Anaerobic hydrolytic bacteria mostly belong to phyla Firmicutes

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and Bacteroidetes (Nguyen and Khanal, 2018). Several hydrolytic bacteria along with their
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functional performance and optimal operational conditions (pH and temperature) are shown in

Table 1. Some hydrolytic and acidogenic bacteria are jointly termed as fermentative bacteria.
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The complex organic materials broken down into intermediate products by hydrolytic bacteria

serve as substrates for fermentative bacteria, which are either facultative or strictly anaerobic

(Azman et al., 2015; Nguyen and Khanal, 2018; Sun et al., 2013). Examples of some commonly

found fermentative bacteria in AD digester are shown in Table 2.

Table 1: Characteristics and functions of mainstream hydrolytic bacteria involved in AD

Complex organic wastes break down into monomers during hydrolysis, which is then utilized

by fermentative bacteria to produce VFAs. Clostridium is very important hydrolytic bacteria

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which is known for degrading lignocellulosic biomass. Clostridium cellulolyticum, Clostridium

thermocellum, Clostridium saccharolyticum, Clostridium phytofermentans, and Clostridium

leptum secrete multienzyme complexes termed as cellulosomes, which are helpful in efficient

degradation of plant cell wall (Fendri et al., 2009). Clostridium cellulolyticum and Clostridium

thermocellum belong to the family Hungateiclostridiaceae and phylum Firmicutes. Clostridium

cellulolyticum hydrolyzes carbohydrates and cellulose to produce CO2, H2, acetate, lactate,

formate and ethanol. This bacterium grows on hexoses and pentoses (Petitdemange et al., 1984).

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Clostridium thermocellum hydrolyzes crystalline cellulose with cellobiose as major product by

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producing cellulosomes to hydrolyze cellulose. This bacterium only grows on hexoses (Freier et

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al., 1988; Liu et al., 2008). Clostridium thermosaccharolyticum, Clostridium saccharolyticum,
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Clostridium phytofermentans, and Clostridium leptum belong to the family Clostridiaceae and

phylum Firmicutes. Clostridium thermosaccharolyticum utilizes pectin to form acetate, butyrate,

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H2, CO2, methanol and traces of ethanol. The methylesterase and polygalacturonate hydrolase
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activity was observed during the initial breakdown of pectin, whereas, no activity was observed

by polygalacturonase (Rijssel and Hansen, 1989). Clostridium saccharolyticum and Clostridium

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phytofermentans hydrolyze carbohydrates to produce CO2, H2, acetate, and ethanol and both can
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grow on hexoses and pentoses (Martin et al., 2018; Murray et al., 1982; Warnick et al., 2002).

Clostridium leptum is a carbohydrate and protein-fermenting bacterium. It works with other

bacteria such as Xylanibacter, Parabacteroides, Clostridium sensu stricto, and Anaerophaga for

producing acids from numerous sugars, including pentoses, hexoses, and complex

polysaccharide matrix, for example, cellulose and xylan (Guo et al., 2015; Hung et al., 2011;

Liang et al., 2015; Moore et al., 1976). During anaerobic digestion, some hydrolytic bacteria

from Clostridium genera grow at mesophilic temperatures (32-37°C) and others grow at
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thermophilic temperatures (58-61°C). The optimal pH for the growth of most of these bacteria

ranges from 6 to 8. A high relative abundance (RA) of Clostridium could be identified in

digesters treating crop residues, animal manures and other lignocellulosic materials (Li et al.,

2018a; M. Wang et al., 2016).

Anaerobacterium chartisolvens belongs to the family Oscillospiraceae and phylum

Firmicutes. It is an obligately anaerobic, which helps in the degradation of carbohydrates. This

species also follows the acidogenic pathways for the production of CO2, H2, acetate, ethanol,

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lactate, and succinate (Horino et al., 2014). During AD, this bacterium works best at a

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temperature of 35°C and pH of 8.0-8.5. Bellilinea caldifistulae is an obligate anaerobe, which

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belongs to the family Anaerolineaceae, and phylum Chloroflexi. It adopts hydrolytic pathways to
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utilize carbohydrates, proteins, and limited variety of sugars, such as glucose, ribose, arabinose,

galactose, mannose, xylose, raffinose and sucrose for growth. The end products of fermentation
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include acetate, formate, lactate, and H2 along with small amounts of propionate and pyruvate in
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medium containing sucrose (Grégoire et al., 2011). This bacterium is found in digesters

operating under thermophilic conditions (55°C) at the optimal pH of 7.0. It has a relatively
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higher abundance in the digestates of sewage sludge and municipal solid waste (MSW). Its
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growth is further enhanced in the presence of hydrogenotrophic methanogens (Yamada et al.,

2007). Gracilibacter thermotolerans and Turicibacter sanguinis belong to the family

Gracilibacteraceae and Erysipelotrichaceae, respectively, and phylum Firmicutes. They

hydrolyze carbohydrates and utilize a wide range of metabolites such as sorbitol, mannitol,

xylose, mannose, glucose, fructose, arabinose, sucrose, and maltose to acetate, ethanol, and

lactate. These bacteria show positive activity of enzymes, including naphthol-AS-BI-

phosphohydrolase, leucine arylamidase, esterase, acid phosphatase, α- & β-glucosidase, and β-

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galactosidase (Bosshard et al., 2002; Lee et al., 2006). Gracilibacter thermotolerans grows best

in AD digesters operating under thermophilic conditions and has high RA in the digestates of

food waste and MSW (Bernat et al., 2019). Whereas, Turicibacter sanguinis grows at

mesophilic digesters treating sewage sludge and dairy wastewater (Yang and Deng, 2020).

Pseudobacteroides cellulosolvens and Ruminococcus albus belong to the family

Ruminococcaceae, and phylum Firmicutes. Pseudobacteroides cellulosolvens hydrolyzes

carbohydrates for producing ethanol and lactate as key products and it grows on D-and L-

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arabinose, L-rhamnose, cellobiose, D-fructose, D-glucose, D-galactose, D-mannose, D-xylose,

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micro crystalline cellulose, aesculin, and xylan. The main fermentation products from these

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substrates are acetate, succinate, ethanol, lactate, CO2 and H2 (Horino et al., 2014).
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Ruminococcus albus is known as a plant cell wall degrading ruminal bacterium (Iakiviak et al.,

2016). It produces that cellulosomes, a multi enzymes complex, which enhance the degradation
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process of complex polysaccharides of plant cell wall, such as cellulose and hemicellulose. The
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end products of fermentation include acetate, ethanol, formate, H2, and CO2 (Chen and Dong,

2004). This species is typically found in anaerobic digesters operating at 37°C with an optimal
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pH range of 7.0-7.5, and treating high cellulose content substrates such as paper waste and
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animal manures (Li et al., 2018a). Another most proficient cellulose degrading bacterium is

Fibrobacter intestinalis, which belongs to the family Fibrobacteraceae and phylum

Fibrobacteres. It also digests other plant fiber components such as hemicellulose to formate

(ATCC, 2018; Montgomery et al., 1988; Suen et al., 2011). Fibrobacter intestinalis is known as

a degrader of lignocellulosic materials in the digestive tract of herbivores. It is abundant in

anaerobic digesters treating crop residues and animal manures at an optimal temperature and pH

of 37°C and 6.6 , respectively (M. Wang et al., 2016). Lutaonella thermophila which belongs to
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the family Flavobacteriaceae and phylum Bacteroidetes is hydrolytic bacterium, which

particularly hydrolyzes aesculin, casein, and gelatin. This bacterium is negative for carbohydrate

hydrolysis, but positive for utilizing proteins, amino acids and organic acids (X. Wang et al.,

2016). The metabolites utilized by this bacterium as a carbon source are 2-oxovaleric acid, 4-

hydroxy-L-proline, alanine, cellobiose, glucose, glutamic acid, glycogen, L-alaninamide,

mannose, methyl pyruvate, proline, succinic acid, and leucine. Moreover, it has positive

enzymatic activities for acid phosphatase, alkaline phosphatase, -galactosidase, and gelatinase,

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and works best in the temperature range of 40-45°C at the pH of 8.0. It is mainly found in

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digesters treating cellulose (Arun et al., 2009; Li et al., 2018b).

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Table 2: Characteristics and functions of mainstream fermentative bacterial species involve in AD
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Lactobacillus thermotolerans belongs to family Lactobacillaceae and phylum Firmicutes, it
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hydrolyses aesculin, D-fructose, D-xylose, L-arabinose, ribose, and glucose, D-raffinose, and

melibiose to produce VFAs, CO2 as well as D and L-lactic acid. This bacterium does not utilize
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5-ketogluconate, 2-ketogluconate, gluconate, L-arabitol, D-arabitol, D-fucose, D-tagatose, D-

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lyxose, D-turanose, lactose, adonitol, L-xylose, and D-arabinose (Boonkumklao et al., 2006;

Niamsup et al., 2003). This bacterium grows best at a temperature of 42°C under acidic pH (3.0-
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6.5). It mainly degrades cellulose in lignocellulosic substrates such as crop residues (Pasha and

Rao, 2009). Acetanaerobacterium elongatum belongs to the family Ruminococcaceae, phylum

Firmicutes. It hydrolyzes aesculin, gelatin, and different types of monosaccharides,

disaccharides, and oligosaccharides. The fermentation products include H2, CO2, ethanol, acetate

and glucose. Furthermore, sugars such as raffinose, sucrose, D-fructose, D-galactose, D-glucose,

D-maltose, D-xylose, L-arabinose, cellobiose, starch, and salicin are hydrolyzed to produce

VFAs. During anaerobic digestion, this bacterium shows optimal growth at mesophilic
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temperature (37°C) at a pH of 6.5-7.0. It mainly degrades lignocellulosic materials such as paper

waste and waste water sludge from paper mill (Chen and Dong, 2004). Petrimonas sulfuriphila

belongs to the family Porphyromonadaceae and phylum Bacteroidetes. It is an anaerobic,

fermentative bacterium which ferments glycerol, mannitol, lactate, rhamnose, mannose, maltose,

galactose, arabinose, and glucose to produce acetate, CO2, and H2. It uses elemental nitrate and

sulfur as electron acceptors and reduces them to ammonium and sulfide, respectively. It also

ferments carbohydrates, proteins, and some organic acids to produce acetate, CO2, and H2 and

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grows at the optimum temperature ranging from 37-40°C and pH of 7.2. (Grabowski et al., 2005;

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Huang et al., 2016; Maspolim et al., 2015). Another fermentative bacterium Thermovirga lienii

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belongs to the family Synergistaceae and phylum Synergistetes. It ferments proteins, organic
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acids, and some amino acids (arginine, alanine, glutamic acid, leucine, cysteine), and reduces

cysteine and elemental sulfur to H2S. This bacterium can be found in digesters treating
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proteinaceous substrates such as high protein food waste and wastewater sludge at thermophilic
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temperature (58°C) and pH of 6.5-7.0 (Dahle and Birkeland, 2006; Kim et al., 2019).

Aneurinibacillus tyrosinisolvens is heterotrophic bacterium, which belongs to phylum Firmicutes

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and family Paenibacillaceae helps in hydrolyzing aesculin and degrading L-tyrosine. It also
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generates acid from fermentation of D-fucose, D-glucose, potassium gluconate, N-acetyl-D-

glucosamine, and malic acid, and weakly ferments L-arabinose. However, this bacterium does

not utilize starch, carboxymethyl-cellulose, xanthine, hypoxanthine, n-Capric acid, maltose, D-

mannitol, D-mannose, sodium citrate, and phenylacetate H2, and grows at the optimum

temperature in the range of 10-30°C and pH in the range of 6.0-6.5 (Tsubouchi et al., 2015).

Due to the complexity of organic wastes used in the AD process, researchers have been

utilizing mixed communities of hydrolytic and acidogenic bacteria rather than pure cultures
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(Atasoy et al., 2018; Nguyen et al., 2019; Reddy et al., 2018). The simultaneous presence of

hydrolytic as well as acidogenic bacteria, promotes faster hydrolysis of organic wastes and

formation of VFAs that can be further degraded by functional microbes during AD process.

2.2. Acidogenic bacteria

There are numerous kinds of acidogenic bacteria that are involved in the AD as shown in

Table 3. Acidogenic bacteria have 30 to 40 times greater growth rate than methanogens and

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generally endure a wider range of operational conditions such as temperature and pH (Li et al.,

2019). If the functions and roles of acidogenic bacteria are known, it can be helpful in

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overcoming the factors which may impede the methane production performance. For example,

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inhibition of hydrogen production in the AD reactor operated at higher organic loading can be
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overcome by bio-augmenting the native acidogenic microflora to improve the process efficiency
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in a short time (Mohan et al., 2019). However, several factors, including operational conditions,

survivability, diversity, and functions of the acidogenic bacteria in the AD system need to be
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explored. So far, not enough consideration was given to investigating the functions of acidogenic
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bacteria in both small and large scale AD plants to enhance the biomethane production from

organic wastes (Shrestha et al., 2017).

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Table 3: Characteristics and functions of typical acidogenic bacterial species involve in AD

Advenella faeciporci belongs to the family Alcaligenaceae and phylum Proteobacteria. It is

involved in glucose fermentation and denitrifies nitrite. It uses many compounds as carbon

source such as monomethyl ester, pyruvic acid, methyl ester, citric acid, -and -hydroxy-

butyric acid, itaconic acid, -ketobutyric acid, -ketoglutaric acid, -ketovaleric acid, L-

threonine, L-proline, L-ornithine, L-leucine, L-glutamic acid, D- and L-alanine, alaninamide,

glucuronamide, succinamic acid, bromo succinic acid, succinic acid, sebacic acid, and propionic
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acid. It has positive enzymatic activity for acid phosphatase, 2-ketogluconate esterase (C4) and

alkaline phosphatase. This bacterium was found in the digestate of activated sludge and piggery

wastewater. It grows at optimal temperatures in the range of 25-35°C and pH of 7.0-9.0 (Coenye

et al., 2005; Gibello et al., 2009; Matsuoka et al., 2012; Xenofontos et al., 2016). Alkalitalea

saponilacus belongs to the family Marinilabiliaceae and phylum Bacteroidetes. Its fermentation

products mainly include acetate and propionate, while its metabolites include trehalose, L-

arabinose, D-galactose, D-mannose, D-xylose, sorbitol, sucrose, maltose, melate, and xylan as

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carbon and energy sources. However, it does not use agarose, dextran, dextrin, ethanol, glycerol,

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glycogen, lactose, methanol, raffinose, starch agar, D-glucose, D-mannitol, L-rhamnose, myo-

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inositol, and fumarate. This bacterium grows in digesters digesting manures at an optimal
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temperature range of 35-37°C and pH of 9.7 (Li et al., 2018c; Zhao and Chen, 2012).

Bacteroides caccae belongs to family Bacteroidaceae and phylum Bacteroidetes. It is a

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fermentative bacterium that feeds on sugars to produce succinate, acetate, propionate and
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isovalerate. It does not utilize lactate and threonine (Johnson et al., 1986). This bacterium has

relatively high abundance in the digestate of municipal and piggery wastewater (Hong et al.,
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2008). It was originally isolated from human feces and has been reported to anaerobically digest
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sheep at an optimal temperature and pH of 30.0-37°C and 5.0-5.2, respectively (Li et al., 2018c).

Bifidobacterium animalis belongs to family Bifidobacteriaceae and phylum Actinobacteria and

ferments pentoses but not gluconate. It metabolizes oligosaccharides to produce lactic and acetic

acids. This bacterium was originally found as a symbiont in human intestine and grows at an

optimal temperature and pH of 39.0-41°C and 6.4-7.0, respectively (Bruno et al., 2002; Scardovi

and Trovatelli, 1974; Wirth et al., 2012). Christensenella minuta is a strict anaerobe, which

belongs to the family Christensenellaceae and phylum Firmicutes. It uses numerous sugars,
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including glucose, salicin, D-xylose, D-mannose, L-rhamnose, and L-arabinose to produce

VFAs. However, it does not utilize maltose, lactose, sucrose, trehalose, D-sorbitol, raffinose,

melezitose, D-mannitol, and cellobiose to produce acid. This bacterium has positive enzymatic

activity for β-galactosidase, naphthol-AS-BI-phosphohydrolase, α-arabinosidase, β-glucosidase

and glutamic acid decarboxylase (Morotomi et al., 2012). According to previous study it was

reported that Christensenella minuta produces key enzymes responsible for degrading chitin

derivatives and higher VFA production. There was a strong correlation between RA of this

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bacterium and high production of propionate, butyrate and total VFAs (Gao et al., 2019). It

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grows at an optimal temperature and pH of 37.0-40°C and 7.5, respectively (Morotomi et al.,

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2012). Cloacibacillus porcorum belongs to the family Synergystaceae phylum Synergistetes. It is
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known as amino acids fermenting bacteria, which mainly utilizes arginine, D-tryptophan,

histidine, proline, serine, threonine to produce acetate, propionate, and formate, however,
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butyrate is produced only in the fermentation of serine (Li et al., 2018c; Looft et al., 2013). This
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bacterium was originally isolated from intestinal tract of pig and has been reported in municipal

wastewater and activated sludge digesters. It grows at an optimal temperature of 39°C and pH of
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6.5 (Deng et al., 2016). Corynebacterium humireducens belongs to the family

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Corynebacteriaceae and phylum Actinobacteria, which produces acid from ribose and glucose

but not from arabinose, fructose, galactose, lactose, maltose, mannitol, mannose, salicin, and

xylose. This bacterium has a positive enzymatic activity for alkaline esterase lipase, phosphatase,

esterase, lipase, pyrazinamidase and pyrrolidonyl arylamidase, and grows at an optimal

temperature of 37°C and pH of 9 (Wu et al., 2011). Dechloromonas denitrifican is anaerobic

bacterium, which belongs to the family Azonexaceae and phylum Proteobacteria. It utilizes

acetate, butyrate, isobutyrate, propionate, lactate, isovalerate, succinate, pyruvate, glutamate,

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malate, and Casamino acids as electron donors. However, this bacterium does not utilize H2,

formate, propanol, ethanol, methanol and sugars like glucose, fructose, lactose, xylose,

cellobiose, mannose, and arabinose (Chakraborty and Picardal, 2013; Horn et al., 2005). This

bacterium is responsible for carrying out denitrification and oxidation of organic acids. It is

generally found in the digestates of activated sludge and has been reported to stay in the reactor

even after the acidogenesis phase. It grows at an optimal temperature of 30°C and pH of 7.0 (Lee

et al., 2009). Ferroplasma acidiphilum belongs to the family Ferroplasmaceae phylum

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Euryarchaeota. It is capable of oxidizing ferrous-iron, and helps in biogeochemical cycling of

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sulfur and sulfide metals (Baumler et al., 2007; Golyshina and Timmis, 2005). It is an important

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microorganism that is naturally found in acid mine drainage and is extremely acidophilic
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microbe known so far, which grows at an optimal temperature of 37°C and pH of 1.7-2.2

(Castelle et al., 2015). Geofilum rubicundum belongs to the family Marinilabiliaceae and phylum
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Bacteroidetes. It can assist in reducing nitrate and nitrite. This bacterium has a positive
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enzymatic activity for amylase and gelatinase, and hydrolyze aesculin D-fructose, D-galactose,

D-mannose, L-rhamnose, cellobiose, trehalose, xylose and lactose, and grows at an optimal
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temperature of 33°C and pH of 7.3-8.3 (Miyazaki et al., 2012). Lutispora thermophila belong to
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the family Clostridiaceae and phylum Firmicutes. It is anaerobic, spore-forming and moderately

thermophilic bacterium, which utilizes some amino acids (lysine, methionine, serine, threonine,

cysteine, and tryptophan), pyruvate, hydrolysate, casein, Casamino acids, tryptone, and peptone.

The end products of fermentation from tryptone are propionate, acetate, iso-valerate and iso-

butyrate. It also forms H2S by fermenting cysteine. However, weak growth of this bacterium

occurs in the presence of aesculin and gelatin. It has been isolated from a thermophilic bioreactor

digesting municipal solid wastes and grows at an optimal temperature and pH of 55-58°C and
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7.5-8.0, respectively (Shiratori et al., 2008b). Mariniphaga anaerophila belongs to the family

Prolixibacteraceae is a mesophilic, facultative anaerobic chemoheterotrophic bacterium, which

uses O2 and L-cysteine as a substitute electron acceptor and donor, respectively. It ferments

sugars (pentoses, hexoses), soluble starch, and disaccharides and reduces nitrate, and grows at an

optimal temperature of 33-37°C and pH of 7.0-7.5 (Iino et al., 2014; Wang et al., 2015).

Sedimentibacter acidaminivorans belongs to the family Clostridia and phylum Firmicutes. It is

anaerobic bacterium, which grows on amino acids such as arginine, lysine, tryptophan,

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phenylalanine, glycine, methionine, isoleucine, leucine, and valine. However, this bacterium

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does not utilize glutamate, serine, aspartate, threonine, alanine, proline, histidine, cysteine,

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glucose, xylose, mannose, arabinose, lactose, raffinose, trelahose, maltose, sucrose, cellobiose,
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lactate, propionate, crotonate, salicin, benzoate, ethanol, H2/CO2 + acetate and formate + acetate,

sulfate, sulfide, nitrite, elemental sulfur, and nitrate. It grows at an optimal temperature of 30-
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37°C and pH of 6.0-8.5. (Imachi et al., 2016). Thauera aromatica AR-1 belongs to Family
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Zoogloeaceae and phylum Proteobacteria. It is a facultative anaerobe, which ferments 3,5-

dihydroxybenzoate to 1,2,4-trihydroxybenzene via water-dependent hydroxylation of the

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aromatic ring following decarboxylation, and grows at an optimal temperature of 30°C and pH of
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8.0 (Anders et al., 1995; Gallus and Schink, 1998; Molina-Fuentes et al., 2015; Pacheco-Sánchez

et al., 2018).

The above-mentioned information on the substrates utilized by different acidogenic bacteria

provides a fundamental understanding into the key roles of the bacterial populations, which

participate in the acidogenesis step. In general, the predominant phyla that contains most

acidogenic bacteria are Bacteriodetes, Chloroflexi, Firmicutes, and Proteobacteria. Acidogenic

bacteria help in the fermentation of the hydrolysate monomers to acetate, alcohol, butyrate,
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propionate, CO2, H2, and other solvents. They greatly influence concentration as well as

distribution of VFAs, which are also affected by the operating conditions of the AD digester.

Therefore, the functions of acidogenic bacteria should be clearly elucidated to optimize the

process of acidogenesis for efficient product recovery.

2.3. Acetogenic bacteria

Acetogenic bacteria break down alcohols and VFAs into acetate, formate, H2, and CO2 that is

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later utilized by the methanogens. Considering microbial growth, usually, acetogenic bacteria

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exhibit a faster growth rate than methanogens (Amani et al., 2010; P. Wang et al., 2018). The

typical acetogenic bacteria along with their substrates, fermentation products and doubling time

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have been displayed in Table 4. The syntrophic interactions among acetogens and methanogens
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contribute to the smooth operation of AD digester. The acetogenic bacteria can be divided into
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two sub-categories; those producing H2 and acetate and others consuming H2/CO2 to produce

acetate.
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Table 4: Characteristics and functions of typical acetogenic bacterial species involve in AD

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Acetogenic bacteria that produce H2 and acetate include Anaerovorax odorimutans,

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Macellibacteroides fermentans, Saccharofermentans acetigenes, Proteiniphilum acetatigenes,

Levilinea saccharolytica, Hydrogenispora ethanolica, and Hydrogenophaga carboriunda.

Anaerovorax odorimutans belongs to the family Eubacteriaceae and phylum Firmicutes. This

bacterium follows both acidogenic and acetogenic pathways and converts putrescine to acetate,

butyrate, molecular H2 and ammonia. It uses 4-aminobutyrate and 4-hydroxybutyrate for growth

(Matthies et al., 2000). It degrades many anionic surfactants such as alcohol sulfates that could

cause severe inhibition in anaerobic digesters (Feitkenhauer and Meyer, 2002). It also ferments

glucose to form lactate, acetate and ethanol as by-products and helps in sulfate reduction, and
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grows at an optimal temperature of 37°C and pH of 7.2-7.6 (Dhouib et al., 2003; Martins et al.,

2011). Macellibacteroides fermentans belongs to the family Porphyromonadaceae and phylum

Bacteroidetes, metabolizes monosaccharides and disaccharides to produce lactate, acetate,

butyrate, and iso-butyrate. It also utilizes aesculin and gelatin through hydrolysis, and grows at

an optimal temperature of 35-40°C and pH of 6.5-7.5 (Hao et al., 2015; Jabari et al., 2012).

Saccharofermentans acetigenes also belongs to the phylum Firmicutes. This bacterium is

obligatory anaerobe and does not work in a microaerophilic and aerobic environment. It does not

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hydrolyze gelatin. The final fermentation products from glucose include acetate, fumarate, and

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lactate. It produces acids from D-glucose, D-fructose, aesculin, starch, sucrose, adonitol,

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mannitol, dulcitol and inositol, and grows at an optimal temperature of 37°C and pH of 6.5.
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However, weak growth occurs with sugars such as cellobiose, trehalose, melibiose, lactose,

erythritol, and amygdalin (Chen et al., 2010; Hahnke et al., 2016). Proteiniphilum acetatigenes
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belongs to family Dysgonomonadaceae and phylum Bacteroidetes, utilizes peptone, pyruvate,

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glycine and L-arginine as sole carbon and energy sources. This bacterium has weak growth with

tryptone, L-serine, L-threonine and L-alanine. It does not utilize carbohydrates, alcohols and
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fatty acids except pyruvate. The end products of the fermentation include acetic acid and CO2,
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and grows at an optimal temperature of 37°C and pH of 7.5-8.0 (Chen and Dong, 2005).

Levilinea saccharolytica belongs to the family Anaerolineaceae and phylum Chloroflexi. It is a

strict anaerobe that utilizes sugars such as pectin, pyruvate, tryptone, sucrose, ribose, raffinose,

xylose, fructose and glucose and converts amino acids into H2, acetic, and lactic acids. However,

this bacterium weakly ferments Casamino acids, peptone, betaine, xylan, galacatose and

mannose, and grows at an optimal temperature and pH of 37°C and 6.0-7.2, respectively (Guo et

al., 2015; P. Wang et al., 2018; Yamada et al., 2006). Rhizobium cellulosilyticum belongs to the
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family Rhizobiaceae, and phylum Proteobacteria, which utilizes metabolites such as arabinose,

glucose, glutamic acid, malic acid, maltose, mannitol, mannose, and N-acetyl glucosamine as

carbon sources in the degradation of wood. It grows at an optimal temperature and pH of 28°C

and 7.0-7.5, respectively (Garcı´a-Fraile et al., 2007). Hydrogenispora ethanolica belongs to the

phylum Firmicutes. It is spore-forming anaerobic bacterium that utilizes various sugars such as

tryptone, fumarate, glycerol, starch, pectin, raffinose, mannose, galactose, sucrose, ribose,

xylose, fructose, arabinose, maltose, and glucose. The main fermentation products are acetate,

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ethanol and H2, and grows at an optimal temperature of 37-45 °C and pH of 6.0-7.7 (Liu et al.,

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2014). Hydrogenophaga carboriunda belongs to the family Comamonadaceae, and phylum

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Proteobacteria. This bacterium utilizes amino acids other than L-histidine and facilitates the
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degradation of 4-amino benzene sulfonate. It also produces H2 as a fermentation product and

utilizes metabolites, including arbutin, adonitol, arabitol, L-arabinose, citrate, D-glucose, D-

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cellobiose, glucuronic acid, fumaric acid, inosine, glycogen, glucosamine, D-xylose, trehalose,
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succinic acid, salicin, D-sorbose, D-ribose, lactose, inositol, D-mannose, D-rhamnose, melibiose,

pectin, D-maltose, Na-malonate, and raffinose. It grows at an optimal temperature of 25-30 °C

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and pH of 6.5-9.5. However, it does not ferment dulcitol, ornithine, mannitol, melezitose,
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glycerol, D-fructose, D-galactose, Na-gluconate, gluconolactone, sucrose, and sorbitol (Gan et

al., 2011; Mantri et al., 2016; Reinauer et al., 2014).

Acetogenic bacteria that consume H2/CO2 to produce acetate include Acetobacterium

wieringae, Acetobacterium woodii, Acetogenium kivui, Clostridium aceticum, Clostridium

thermoautotrophicum, and Sulfurovum riftiae. Acetobacterium wieringae belongs to family

Eubacteriaceae and phylum Firmicutes, is anaerobic, chemolithotrophic, acetogenic bacterium.

It grows on H2 and CO2 forming acetic acid as the sole end product, and grows at an optimal
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temperature of 30°C and pH of 7.2-7.8 (Braun and Gottschalk, 1982). Acetobacterium woodii

belongs to family Eubacteriaceae and phylum Firmicutes, is obligately anaerobic bacterium that

ferments fructose to acetate. It also utilizes glycerate, glucose, and lactate and can be adapted to

ferment formate. It oxidizes H2 and reduces CO2 and produces succinate from the fermentation

of organic substrates, and grows at an optimal temperature and pH of 30-35°C and 7.3-7.6,

respectively (Bache and Pfennig, 1981; Balch et al., 1977). Acetogenium kivui belongs to the

family Thermoanaerobacteraceae and phylum Firmicutes, uses CO2 and H2 as sole energy

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source to produce acetate, and grows at an optimal temperature and pH of 66°C and 6.4,

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respectively (Leigh et al., 1981). Clostridium aceticum belongs to family Clostridiaceae and

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phylum Firmicutes, is obligately anaerobic bacterium and grows either chemolithotrophically
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with H2 and CO2 or chemoorganotrophically with pyruvate, L-malate, L-glutamate, and fructose.

The product of fermentation is acetic acid, and grows at an optimal temperature of 37°C and pH
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of 8.3-8.5 (Braun et al., 1981). Clostridium thermoautotrophicum belongs to family

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Thermoanaerobacteraceae and phylum Firmicutes, is obligate anaerobe and grows

chemolithotrophically with H2 and CO2 as well as chemoorganotrophically with methanol,

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glycerate, glucose and fructose. The only fermentation product is acetate, and grows at an
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optimal temperature of 56-60°C and pH of 5.6-5.9 (Wiegel et al., 1981). Sulfurovum riftiae

belongs to the family Epsilonproteobacteria and phylum Proteobacteria is a strictly anaerobic

homoacetogenic bacterium, which uses H2 and CO2 as the sole energy source to produce acetate.

It also uses sulfur and thiosulfate as electron donor and nitrate as electron acceptor, and grows at

an optimal temperature of 35°C and pH of 6.0 (Giovannelli et al., 2016; Nabweteme et al., 2016;

Nguyen and Khanal, 2018).

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Other examples of acetogenic bacteria that work in syntrophic association with

hydrogenotrophic methanogens to produce acetate are Syntrophaceticus schinkii,

Syntrophomonas wolfei, Syntrophus aciditrophicus, Syntrophobacter wolinii, and

Syntrophorhabdus aromaticivoran. Syntrophaceticus schinkii belongs to family (not assigned)

and phylum Firmicutes, is a syntrophic acetate-oxidizing bacterium which works in co-culture

with hydrogen-consuming methanogen. It utilizes ethanol, betaine and lactate as carbon and

electron sources, and grows at an optimal temperature ranging from 37-60°C and pH from 6.0-

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8.0 (Schnürer et al., 2018; Westerholm et al., 2016, 2010). Syntrophomonas wolfei belongs to the

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family Syntrophomonadaceae and phylum Firmicutes. It converts butyrate to CH4 and CO2 in

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syntrophic association with methanogenic partner such as Methanospirillum Hungatei as shown
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in Fig. 2. It grows at an optimal temperature and pH of 37°C and 5.4-7.4, respectively (Schmidt

et al., 2013). Syntrophus aciditrophicus belongs to the family Syntrophaceae and phylum
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Proteobacteria. It is a metabolic specialist, which degrades short-chain fatty acids and aromatic
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acids (i.e., cyclohexane-1-carboxylate, benzoate or butyrate) to acetate, CO2, formate, and H2.

This mechanism requires a hydrogenotrophic partner for removing H2 and formate such that
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catabolic reactions remain thermodynamically favorable. This bacterial strain helps in oxidation
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of crotonate to acetate and also in its reduction to benzoate and cyclohexane carboxylate, and

grows at an optimal temperature and pH of 28-37°C and 7.0-7.8, respectively (Elshahed et al.,

2001; Jackson et al., 1999; James et al., 2016; Mouttaki et al., 2009). Syntrophobacter wolinii

belongs to family Syntrophobacteraceae and phylum Proteobacteria, degrades propionate in

coculture with an H2 using organism and in the absence of light or exogenous electron acceptors

such as O2, sulfate, or nitrate. It produces acetate and, presumably, CO2 and H2 (or formate) from

propionate, and grows at an optimal temperature ranging from 30-35 °C and pH of 6.1 (Boone
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and Bryant, 1980; Liu et al., 1999). Syntrophorhabdus aromaticivoran belongs to the family

Syntrophorhabdaceae and phylum Proteobacteria. It is an obligatory anaerobic mesophilic

bacterium, which is capable of degrading p-cresol, phenol, 4-hydroxybenzoate, benzoate, and

isophthalate in association with an H2-scavenging methanogen partner, which produces acetate

and CH4 as final products, and grows at an optimal temperature ranging from 35-37°C and pH of

7.0 (Qiu et al., 2008). The reactions involved in the acetate and hydrogen metabolism are shown

below:

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Aceticlastic methanogenesis:

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Syntrophic acetate oxidation:
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Hydrogen consuming acetogenesis:

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Hydrogen consuming methanogenesis:

Hence, all through this step, it is found that the unifying characteristics of acetogenic bacteria

are producing acetate as a main product formed by CO2 to acetate. These acetogens use versatile

pathways for acetate production from organic acids and sugars (pentoses and hexoses). Many

acetogens, on the other hand, are chemolithotrophic and convert H2 and CO2 to acetate. Some
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acetogens work in syntrophic association with methanogens to convert butyrate into CH4 and

CO2. The predominant acetogens Syntrophomonas wolfei, Syntrophus aciditrophicus,

Syntrophorhabdus aromaticivoran, Syntrophaceticus schinkii, Thermacetogenium phaeum, and

Thermotoga lettingae are commonly found in digesters treating complex feedstocks. These

acetogens significantly influence acetogenesis for maintaining the stability of AD operation,

particularly by utilizing those fatty acids, whose high concentration leads to inhibition.

Fig. 2. Conceptual illustration of the effect of conductive materials on the stimulation of syntrophic methanogenesis

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a) ethanol, b) acetate, c) propionate, d) butyrate, and e) complex organic substrates. DIET pathway is represented by
red arrows (Yin and Wu, 2019)

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2.4. Sulfate-reducing bacteria

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Sulfate reducing bacteria (SRBs) include a metabolically diverse group of obligatory
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anaerobic microorganisms, which belong to different families and genera. SRBs are commonly

detected in digesters treating wastewaters. SRBs utilize sulfate as an electron acceptor to oxidize
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organic substances while producing H2S (Liu et al., 2018). They either function independently or
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work with hydrogen-scavenging methanogens in syntrophic association to degrade propionate

and butyrate, which serve as carbon sources and electron donors/acceptors to produce acetate
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(Table 4). Desulfobulbus propionicus belongs to family Desulfobulbaceae and phylum

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Proteobacteria, is a sulfate-reducing bacterium. It uses alcohols, lactate and propionate, as

carbon sources and electron donors to produce acetate, and grows at an optimal temperature

ranging from 30-39°C and pH of 7.2 (Widdel and Pfennig, 1982). Desulfotomaculum peckii

belongs to family Peptococcaceae and phylum Firmicutes, is anaerobic thermophilic sulfate-

reducing bacterium and utilizes H2/CO2, propanol, butanol and ethanol as carbon and energy

sources in the presence of sulfate as terminal electron acceptor. This bacterium does not use

fumarate, formate, lactate and pyruvate. The end product of fermentation from butanol is
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butyrate, while propanol and ethanol are oxidized to propionate and acetate, respectively. It

grows at an optimal temperature of 55-60°C and pH of 6.0-6.8. This bacterium utilizes, sulfate,

sulfite and thiosulfate as terminal electron acceptors but does not utilize elemental sulfur, iron

(III), fumarate, nitrate and nitrite (Jabari et al., 2013). Desulfovibrio desulfuricans belongs to

family Desulfovibrionaceae and phylum Proteobacteria, is a sulfate reducing bacteria that grows

in chemostat culture with H2 along with limiting concentrations of nitrate, nitrite or sulfate as

sole energy source to produce organic acids, and grows at an optimal temperature of 32°C and

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pH of 6.5-7.0 (Boon et al., 1977; Herrera et al., 1993; Seitz and Cypionka, 1986). Desulfococcus

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multivorans belongs to family Desulfobulbaceae and phylum Proteobacteria, is a sulfate-

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reducing bacterium that works with hydrogen-scavenging methanogens in syntrophic iso-
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butyrate oxidation. The fermentation product is acetate after initial conversion of iso-butyrate to

butyrate, and grows at an optimal temperature of 30-35°C and pH of 7.0-7.3 (Peters et al., 2004;
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Schauder et al., 1986; Stieb and Sehink, 1989). Pelotomaculum thermopropionicum belongs to
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family Peptococcaceae and phylum Firmicutes, is anaerobic, syntrophic propionate-oxidizing

bacterium, which grows fermentatively on fumarate and pyruvate. When this bacterium is grown
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in co-culture with hydrogenotrophic methanogen such as Methanothermobacter

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thermautotrophicus, it utilizes ethylene glycol, 1-propanol, 1,3-propanediol, 1-pentanol, 1-

butanol, lactate, ethanol and propionate, and grows at an optimal temperature of 55°C and pH of

7.0 (Imachi et al., 2002). Sporobacter termitidis is obligatory homoacetogenic anaerobe that

grows on methylated sulfides from methylated aromatic compounds, if cysteine or sulfide exists

in the medium. Other metabolites used as a sole energy source by this bacterium include, 3,4,5-

trimethoxycinnamate (TMC) sinapate (33-dime thoxy-4-hydroxycinnama), 3,4-dimethyl-

hoxycinnama, 3,5-dimethoxy-4-hydroxybenzoate, ferulate, nillate (4-hydroxy-3-

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methoxybenzoate) to produce acetate and methanethiol, and grows at an optimal temperature of

32-35°C and pH of 6.7-7.2 (Grech-Mora et al., 1996). Thermacetogenium phaeum belongs to the

family Thermoanaerobacteraceae and phylum Firmicutes, which oxidizes acetate to CO2 in co

culture with hydrogenotrophic methanogens to produce methane. It also oxidizes acetate to CO2

in pure culture with reduction of sulfate and thiosulfate as the electron acceptor, and grows at an

optimal temperature and pH of 28-58°C and 6.8, respectively (Hattori et al., 2000). Thermotoga

lettingae belongs to the family Thermotogaceae and phylum Thermotogae, which degrades

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methanol to CO2 and H2 in syntrophic association with Methanothermobacter

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thermautotrophicus or Thermodesulfovibrio yellowstonii. It grows at an optimal temperature and

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pH of 65°C and 7.0, respectively. However, in the presence of elemental sulfur or thiosulfate,
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methanol degrades to CO2 and alanine, whereas in pure culture, it ferments methanol to acetate,

CO2, and H2 (Balk et al., 2002).

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3. Types and roles of archaea

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Methanogenesis, is carried out by specialized group of microbes called methanogens, which

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are categorized as: hydrogenotrophic, acetoclastic, and methylotrophic. Hydrogenotrophic

methanogens utilize H2 and formate to produce CH4. Acetoclastic methanogens are helpful in
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converting acetate to CH4 and CO2. Methylotrophic utilize methyl compounds including

methylsulfides, methylamines, and methanol to produce CH4. In typical AD digesters,

approximately 70% of CH4 is generated from acetate and the remaining from CO2 and H2. A

very small amount of methane is generated by methylotrophic methanogenesis (Venkiteshwaran

et al., 2015). The predominant methanogens that play vital role in methane production process

are discussed below.

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3.1. Hydrogenotrophic/methylotrophic methanogens

The role of hydrogenotrophic methanogens is critical for ensuring an efficient methane

production process. The growth rate of hydrogenotrophic methanogens (4-12 h) is much faster

than acetoclastic methanogens (4-9d). The low hydrogen partial pressure (<10-4) is a vital factor,

which describes process stability or setbacks. Table 5 shows different hydrogenotrophic species

present in the anaerobic conversion of selected soluble substrates. The description of shape, size,

and optimal conditions needed for achieving high conversion of these substrates is also shown in

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the Table 5.

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Table 5: Characteristics and functions of typical hydrogenotrophic and methylotrophic archaeal species involve in AD

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Many isolated mesophilic and thermophilic archaea belong to the family Methanobacteriaceae
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and phylum Euryarchaeota as shown in Fig. 3. They mainly utilize formate, H2/CO2 as
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substrates (Demirel and Scherer, 2008). Methanospirillum lacunae, Methanospirillum hungatei,

Methanobrevibacter gottschalkii, Methanobrevibacter olleyae, Methanobrevibacter millerae,

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Methanobacterium beijingense, Methanobrevibacter boviskoreani, Methanobrevibacter

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ruminantium, Methanobacterium formicicum, Methanobacterium oryzae, Methanobacterium

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subterraneum, Methanobacterium movens, Methanobacterium flexile, Methanoculleus

bourgensis, Methanoculleus thermophilicum, Methanoculleus horonobensis, Methanoculleus

receptaculi, Methanoculleus submarinus, Methanoculleus sediminis, and Methanoculleus

taiwanensis, are strictly anaerobic hydrogenotrophic archaea, which utilize formate and H2/CO2

as substrates for CH4 production (Asakawa and Nagaoka, 2003; Battumur et al., 2016; Chen et

al., 2015; Iino et al., 2010; Joulian et al., 2000; Kotelnikova et al., 1998; Lee et al., 2013; Ma et

al., 2005; Maestrojuan et al., 1990; Mikucki et al., 2003; Rea et al., 2007; Rivard and Smith,

1982; Shcherbakova et al., 2011; Shimizu et al., 2013; Smith and Hungate, 1958; Tian et al.,
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2010a; Weng et al., 2015; Zhu et al., 2011a). Methanospirillum stamsii also uses H2/CO2 and

formate for CH4 production but have a very low growth rate with formate (10-20 mmol-1), even if

the medium is supplemented with tungsten. Acetate and yeast extract promote the growth of this

archaeon, but are not required (Parshina et al., 2014). Other common methanogens found in AD

digesters are Methanobacterium aggregans, Methanobacterium congolense, Methanobacterium

paludis, Methanobacterium aarhusense, Methanobacterium espanolae, Methanobacterium

uliginosum, Methanobacterium ivanovii, Methanobacterium alcaliphilum, Methanobacterium

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petrolearium, Methanobacterium kanagiense, Methanobacterium ferruginis, Methanoculleus

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hydrogenitrophicus, and Methanospirillum psychrodurum, are strictly anaerobic

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hydrogenotrophic archaea, which utilize only H2/CO2 as substrates for CH4 production (Brauer et
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al., 2014; Cadillo-quiroz et al., 2014; Cuzin et al., 2001; Fabbri et al., 2012; Kern et al., 2015;

Kitamura et al., 2011b; Konig, 1984; Mori and Harayama, 2011; Patel et al., 1990; Shlimon et
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al., 2004; Tian et al., 2010b; Zhou et al., 2014). Methanobrevibacter acididurans is acid tolerant
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hydrogenotrophic methanogen, which requires acetate and rumen fluid to grow on H2/CO2 as

substrate to produce CH4 and does not require coenzyme in the presence of rumen fluid (Savant
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et al., 2002). Methanobrevibacter smithii uses formate only instead of H2/CO2 as a substrate for
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CH4 production. Compared with other methanogens, Methanobrevibacter smithii is rich in genes,

which are involved in utilizing formate during methanogenesis. These genes play a vital role in

the vitamin cofactor synthesis used by enzymes during methanogenesis including riboflavin,

coenzyme M synthase, and methyl group carriers (Khelaifia et al., 2013).

Fig. 3. Phylogenetic tree of acetoclastic, hydrogenotrophic, and methylotrophic methanogens at phylum, class, order,
family, and genus level.

Methanomassiliicoccus luminyensis is a methylotrophic archaeon with H2 as the electron

donor and produces CH4 by reducing methanol with a growth yield of 2.4 g cells/mol CH4. It
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was further demonstrated that most of the methanogens especially Methanosarcina barkeri and

Methanosarcina mazei species transfer methyl moieties from methanol or methylamines to HS-

CoM forming methyl-coenzyme M (methyl-CoM) as an important intermediate for

methanogenesis. Methanomassiliicoccus luminyensis cannot produce CH4, if H2 or methanol is

used as the only energy source (Kern et al., 2016; Kröninger et al., 2017). Methanosphaera

stadtmanae, produces CH4 from methanol and H2 only, and 95% of the cell carbon comes from

acetate and CO2 (Miller and Wolin, 1985; Weiss et al., 2008). The growth of this archaeon is

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completely inhibited by nitrate. Methanolobus chelungpuianus uses Methanol + trimethylamine

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as substrates. However, it does not utilize formate, acetate, dimethylamine, isobutanol, 2-butanol,

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2-propanol, ethanol, methyl sulfide and methylamine (Wu and Lai, 2011). Methanimicrococcus
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baltticola produces CH4 by reducing methylated amines and methanol with H2. It requires

acetate, coenzyme M, tryptic soy broth, vitamins, and yeast extract for growth (Sprenger et al.,
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2000; Weiss et al., 2008). Methanobacterium lacus uses catabolic substrates such as methanol +
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H2 and H2 + CO2 for growth, but does not utilize acetate, formate, iso-butanol, 2-propanol or

methylamine + H2. This archaeon does not require rumen fluids, vitamins, acetate and yeast
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extract for growth, however, their presence stimulates growth (Borrel et al., 2012).
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Methanobacterium veterum, Methanobacterium bryantii utilizes H2 + CO2, Methanol + H2,

CH3NH2 + H2, and their growth are stimulated by acetate. It also utilizes isobutanol, isopropanol

and H2/CO2 for CH4 production and both methanogens have a very close affinity with each other

(Krivushin et al., 2010). Methanoculleus chikugoensis, Methanobacterium movilense,

Methanoculleus marisnigri, and Methanoculleus palmolei use a wide variety of substrates

including formate, cyclopentanol/CO2, 2-butanol/CO2, H2/CO2, and 2-propanol/CO2 for methane

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production (Asakawa and Nagaoka, 2003; Chen et al., 2015; Dianou et al., 2001; Schirmack et

al., 2014b; Shimizu et al., 2013; Weiss et al., 2008).

3.2. Acetoclastic methanogens

The acetoclastic archaea are strictly anaerobic, and utilize acetate to produce CH4 and CO2 (P.

Wang et al., 2018). Acetoclastic methanogens are sensitive to lower pH as compared to

hydrogenotrophic methanogens, which can even work at pH < 5. At low pH values acetate

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oxidizing syntrophs along with hydrogenotrophic methanogens can overcome the acetoclastic

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methanogens (Kim et al., 2004). Table 6 shows the shape size, and the optimal conditions for the

growth of acetoclastic, methylotrophic and neutrophilic archaeal species.

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Table 6: Characteristics and functions of typical acetoclastic archaeal species involve in AD
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Methanothrix concilii, Methanothrix harundinacea, Methanothrix soehngenii, and
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Methanothrix thermophilla, belongs to the family Methanosaetaceae and phylum

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Euryarchaeota. They are strictly anaerobic and acetoclastic methanogens, using acetate for

producing CH4 and CO2 without the ability to utilize H2, CO2, formate, methanol, and
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methylamines (Huser et al., 1982; Kamagata et al., 1992; Ma et al., 2006; Patel and Sprott,
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1990). Some methanogens adopt both acetoclastic and methylotrophic pathways such as

Methanosarcina flavescens, Methanosarcina barkeri, and Methanosarcina mazei (Kern et al.,

2016). They are strictly anaerobic methanogens and belong to the family Methanosarcinaceae

and phylum Euryarchaeota. Methanogen communities have higher diversity in the AD digester

operating at 37 °C as compared to the digester operating at 55 °C (Li et al., 2019; Nguyen and

Khanal, 2018). If the temperature is further lowered to 25 °C, the microbial community may shift

from acetoclastic to hydrogenotrophic methanogens, but the underlying mechanism is still not

clear and needs additional research as stated by Venkiteshwaran (Venkiteshwaran et al., 2015).
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They can produce CH4 from CO2/H2, CO, acetate, monomethylamine, dimethylamine,

trimethylamine and methanol (Archer and King, 1983; P. Wang et al., 2018).

In conclusion, it is important to identify key methanogens and their metabolic pathways

involved in the methanogenesis phase of anaerobic digestion is important. Hydrogenotrophic

methanogens utilize only H2 and CO2. Since the partial pressure of hydrogen is a significant

factor in defining the stability in the AD process, these methanogens play a key role in the

efficiency of the AD process. The hydrogenotrophic microorganisms in the AD of fruit and

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vegetable wastes are predominantly Methanosphaera and Methanobrevibacter. Acetoclastic

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methanogens are obligatory anaerobes that reduce methyl groups and utilize acetate for their

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growth. Methanothrix species have been found to be dominant acetoclastic methanogens playing
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a significant role in the AD of sewage sludge and municipal waste, whereas, Methanoculleus and

Methanosarcina are generally dominant in the AD of manures (Bouallagui et al., 2004;

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McMahon et al., 2004). The information provided here gives deep insights into the ways in
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which environmental and operational conditions influence the microbial populations for

optimizing and maintaining a favorable ambience for their activity and growth, thereby
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improving efficiency of AD process.

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4. Impact of operational conditions on microbial community in AD reactors

Lately, some researchers have reported the impact of operational conditions on the structure

and changes in the microbial populations during AD, mainly concentrating on the methanogenic

pathways. Archaea have a lower growth rate than bacteria and can be easily affected by

fluctuations in operational parameters including pH, VFAs, and ammonia concentrations. Other

factors, including temperature, organic loading rate (OLR), occurrence of toxic compounds,

substrate composition and concentration, might lead to a shift in the archaeal community and
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influence the whole AD process as shown in Fig. 4 (Li et al., 2019; Regueiro et al., 2016;

Rodriguez-verde et al., 2014; Vanwonterghem et al., 2015).

Fig. 4. Operational parameters for microbial community in AD process

4.1. Organic loading rate (OLR) and hydraulic retention time (HRT)

The OLR is an essential operational parameter of AD that needs to be controlled for avoiding

disruptions in the process (Abendroth et al., 2015; Braz et al., 2019). OLR indicates the balance

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among microorganisms and substrate, which must be controlled for maintaining the equilibrium

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between methanogenesis and acidification (Kouas et al., 2018). An OLR shock generally

imbalances hydrolysis, acidogenesis, acetogenesis, and methanogenesis steps. Accordingly,

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accumulation of VFAs occurs, which drops pH and further leads to the failure of methanogenesis
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(Braz et al., 2019; He et al., 2017). However, while reducing HRT and increasing OLR, there is a
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shift in microbial community, where slow-growing methanogens could be washed out

(Chojnacka et al., 2015; Suryawanshi et al., 2010; Vanwonterghem et al., 2015). Moreover,
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when the feedstock is overloaded, the rate of VFAs intermediate formation is higher, which
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deteriorates AD performance by significantly disturbing the microbial community through

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additional accumulation of intermediate products, which negatively affect the activity of

methanogens (Croce et al., 2016).

4.2. Temperature, pH and alkalinity

Several microbial communities showed a high growth and became abundant with the change

in temperature, while others were increasingly washed out from the AD digesters (Peces et al.,

2018). The appropriate temperature range is wide as methanogens can effectively work with

different temperature ranges, thus, classified as mesophilic (37 °C) and thermophilic (55 °C)

(Pang et al., 2018; Wan et al., 2018; Zou et al., 2018). Hydrolytic and acidogenic bacteria
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generally operate at the optimal pH value ranging between 5-6 (Demirer and Chen, 2004).

However, pH value less than 5 decreases the VFAs yield and efficiency of acidogenesis, whereas,

at pH value of 4.5, ethanol-type fermentation has been reported instead of VFAs production (Ren

et al., 1997). High VFAs accumulation in anaerobic digesters causes pH drops to levels that can

inhibit microorganisms (Cater et al., 2013). Besides decreasing the pH, high concentrations of

VFAs inhibit the process of methanogenesis. However, the inhibition is considerably greater at

lower pH values (Deublein and Steinhauser, 2008). The pH affects the ratio of undissociated to

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dissociated forms of VFAs, where the undissociated is lethal to microbes as it easily diffuses

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through their cell membrane causing damage by lowering the intracellular pH (Kadam and

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Boone, 1996). Archaea are tremendously influenced by fluctuations in pH, whereas, fermentative
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microbes are less affected by pH variations and grow well in a wide range of pH (Hwang et al.,

2004; Li et al., 2019). The effect of pH on the growth of microorganisms is described in the next
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section. The optimal growth of methanogens occurs at ≥30 °C, but thermophilic methanogens
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have temperature optimal near 55°C. Generally, thermophilic AD process allows high OLR,

reduced HRT, higher conversion efficiency, and pathogen disinfection. However, the digestion
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under mesophilic temperature is more stable and energy efficient with a lower risk of ammonia
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nitrogen toxicity (Enright et al., 2009; Gonzalez et al., 2018; Zhang et al., 2012). It is found that

most species had the optimal growth at pH ranging from 6 to 8, whereas, pH less than 5.6

negatively influences the microbial growth (Garcia et al., 2000).

4.3. Nutrient availability

Nutrients are essential for the growth of microbial community participating during the

methane production process. Apart from phosphate or nitrogen, the lack of inorganic nutrients

hinders the methanogens metabolism and therefore, negatively affects the AD process. Brulé et
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al. (2013) reported that lower CH4 production along with high VFAs concentrations in the AD

system was attributed to the lack of trace elements. The increase in the concentration of trace

elements in the appropriate range corresponds to a high growth rate of archaea. Even though

trace elements influence bacterial metabolism in many ways, archaea participating in the final

stages of AD require a higher concentration of Cobalt (Co), Nickel (Ni), Copper (Cu) and Iron

(Fe), as compared to fermentative bacteria (Brulé et al., 2013; Gerardi, 2003; Hendriks et al.,

2018). Fe is an important nutrient and is needed for stimulating the growth of hydrogenotrophic

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methanogens and is also useful in promoting the availability of other metals (Feng et al., 2014),

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whereas, high Ca and Mg concentration, enhance the AD digester alkalinity as well as provide

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vital elements for the growth of some useful methanogens (Chen et al., 2008; Thanh et al., 2016).
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The amount of essential trace metals needed for stable AD process might differ for microbial

species depending on their methanogenic pathway. Fe is abundantly utilized after Ni, Co, Mo,
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and Zn in catalysis and electron transport. According to a study, the addition of 10 µM Fe had
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doubled the methanogenic activity of the sludge in a UASB reactor (Zandvoort et al., 2003).

Generally, Fe and Ni occur in the form of Ni-Fe-S cluster and Fe-S cluster, forming enzyme
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subunits, including hydrogenase and acetyl-CoA synthase (Myszograj et al., 2019). Many
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anaerobic bacteria depend on Ni if CO2 and H2 are the sole energy source. The nickel

tetrapyrrole, coenzyme F430, binds to methyl-S-CoM reductase, which catalyses CH4 formation

from methyl-S-CoM by acetoclastic and hydrogenotrophic methanogens. Co occurs in the form

of cobamides participating in the methyl group transfer. Mo and Tungsten (W) are noncovalent

bound to co-factors molybdopterin and tungstopterin, which reduce CO2 to formate by formate

dehydrogenase (Deublein and Steinhauser, 2008). Mo also has a significant role in the syntrophic
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propionate oxidation. The function of other trace elements during the AD process is displayed in

Table 7.

Table 7: Role of trace elements in the process of AD (Schattauer et al., 2011)

In general, due to the diversity and variability of feedstocks, unexpected fluctuations in the

operation of anaerobic digesters have been reported in the literature. This causes reduction in

biogas production, leading to high operational cost and inferior digestion performance. Therefore,

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it is vital to optimize the operational conditions in order to guarantee sound anaerobic digestion

performance.

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It is reported that the microorganisms such as bacteria, protozoa, and fungi are together play a

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role in the anaerobic digestion process. Despite the complex interrelationships between bacteria,
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protozoa, and fungi, bacteria are believed to play a significant role in the AD process due to
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metabolic diversity and numerical predominance, nonetheless protozoa have been reported to

digest 25-30% of the total fiber (Lee et al., 2000). The extent of fungi involvement in AD is yet
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to be understood in depth and needs further research. This will enable the readers to better
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understand the effects of interaction among microorganisms which range from synergism to

antagonism depending both on the participation of different microbial and the type of feedstock.
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5. Practical implications and future research directions

The anaerobic digestion of organic feedstocks proceeds effectively via the interactions among

different microorganisms in the digester to produce methane. The AD process is feasible if

different types of perturbations which impede the smooth operation of anaerobic digesters are

managed. Although the functions of different microorganisms and operational parameters were

highlighted in this work, there should be strategies to alleviate the stresses in AD digesters and
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reactivation of the bioreactors. This section discusses the practical implications and future

research directions for successful AD operations.

One of the important implications of this work is that it can be helpful in the modification of

bioreactors to maximize the retention of useful microbes to improve the AD of organic wastes to

produce renewable energy. Based on their growth patterns and characteristics, some of the

microbes could be suggested as rarely active and others as core anaerobic microbes in AD. The

findings of this review can be useful in assessing which microbes are preferred in the dynamic

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environment of AD reactors. On the other hand, to improve the methane production, a

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pretreatment unit could be designed to disintegrate the former microbes, and the anaerobic

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digester can be kept at conditions favorable for the latter and effective strategies can be brought
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into practice to improve the activities of microbial populations in the AD process.

It is important to note that previously published work on the roles of microbes focused only on
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the laboratory scale experiments. At present there is no systematic study on the application and
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function of useful microbes in full-scale anaerobic systems due to the AD process complexity

and large volume of digesters. Hence, it is imperative to study the performance of AD process by
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introducing microbial consortium with desired characteristics. Another important impeding

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factor to its real applications is that most feedstocks for AD are complex and designing microbial

cultures for the effective degradation of these substrates is quite challenging. Therefore, future

research should focus on addressing these challenges along with the modification of the AD

reactors configuration to eliminate the operational challenges influencing the performance of

microbes during AD process.

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Conclusively, future research may include adoption of desired operational conditions and

modification of bioreactor configurations to efficiently operate AD and improve the prospect of

renewable energy production from various feedstocks.

6. Conclusions

The bioconversion of organic matter via anaerobic digestion is effectively carried out with

active participation of many microbes through all the four stages of anaerobic digestion. Most of

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the hydrolytic and fermentative bacteria participating in the hydrolysis step belong to the phylum

Firmicutes and Bacteroidetes. They mainly hydrolyze carbohydrates, proteins, and lipids to

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produce simple and soluble sugars, fatty acids, and amino acids, respectively. Acidogenic

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bacteria participate in the acidogenesis step generally belong to the phylum Chloroflexi,
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Firmicutes and Proteobacteria. They mainly degrade amino acids and sugars to produce VFAs.
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Acetogenic bacteria belong to phylum Proteobacteria and Firmicutes and have two

subcategories namely homoacetogenic bacteria and syntrophic acetate oxidizing bacteria. The
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homoacetogenic bacteria utilize H2 and CO2 as sole energy source to produce acetate, whereas,
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syntrophic acetate oxidizing bacteria oxidize acetate into H2 and work in association with

hydrogen utilizing methanogens to produce CH4. Most methanogens belong to the family
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Methanobacteriaceae and phylum Euryarchaeota. They utilize simple soluble substrates

including acetate, H2, CO2 and formate to produce methane. Various approaches have been taken

in order to expand AD such as lowering the cost and operational barriers and enhancing the

activity of microbes for bringing engineering improvements. The major opportunity to enhance

the CH4 production is the adequate exploitation of microbial communities that are usually

complex and have often been considered as a black box in the current engineering and

management tools for designing AD operations. These findings provide an improved

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understanding of functions of microbial community participating in a complex AD process of

organic wastes for renewable energy production.

Acknowledgement

This study was funded by the National Key Research and Development Program of China

(2018YFD0800103), China Association for International Education (CAFSA), and the Teaching

Reform Program in Graduate Education at the Beijing University of Chemical Technology (G-

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JG-PT201914).

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Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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Fig. 3. Schematic diagram for the AD process

Fig. 4. Conceptual illustration of the effect of conductive materials on the stimulation of

syntrophic methanogenesis a) ethanol, b) acetate, c) propionate, d) butyrate, and e) complex
organic substrates. DIET pathway is represented by red arrows (Yin and Wu, 2019)

Fig. 3. Phylogenetic tree of acetoclastic, hydrogenotrophic, and methylotrophic methanogens at

phylum, class, order, family, and genus level.

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Fig. 4. Operational parameters for microbial community in AD process

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Table 2
Characteristics and functions of mainstream hydrolytic bacteria involved in AD
Bacterial Species DT/GR (hr) Shape/Size Optimal conditions Functions References
pH Temperature
Anaerobacterium NG Curved rod/0.8-1µm 8.0-8.5 35 °C Mainly degrades carbohydrates (Horino et al., 2014)
chartisolvens width, 3.5-15µm
length.
f
to produce acetate, ethanol,

o
lactate and succinate.

Bellilinea caldifistulae 45 Multifilamentous/0.2-

0.4µm
7.0 55 °C
o
Utilizes carbohydrates, proteins

r
and a limited variety of sugars to
(Grégoire et al., 2011;
Yamada et al., 2007)
width, >100µm
length.

- p acetate, formate, lactate and H2.

e
Clostridium With Slightly curved 5.2-5.5 32-35 °C Hydrolyzes varieties of (Petitdemange et al., 1984)
cellulolyticum cellobiose (7),
cellulose (15),
and glucose
rod/0.6-1µm width, 3-
6µm length.

P r carbohydrates and major

fermentation products from
cellulose are CO2, H2, acetate,

Clostridium
thermocellum
(10)
With cellulose
(6.5),
Lens and rod
shaped/0.4-0.6µm
a l
6.7-7.0 58-61 °C
lactate, formate and ethanol.
Hydrolyzes crystalline cellulose
with cellobiose as major product.
(Freier et al., 1988; Liu et
al., 2008)
cellobiose
(2.5)
n
width, 2-5µm length.

r
Clostridium
thermosaccharolyticum
GR=0.58 h-1 Rod.

u 6.0 58 °C Hydrolyzes polysaccrides and

major fermentation products are
(Aduse-opoku and
Mitchell, 1988; Rijssel and

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acetate, butyrate, H2, CO2, Hansen, 1989)
methanol and ethanol.
Clostridium leptum 24 Straight rod/0.6- 5.3-5.8 35-37 °C Hydrolyzes carbohydrate and (Guo et al., 2015; Hung et
0.8µm width, 1.3- protein to produce VFAs. al., 2011; Liang et al.,
2.8µm length. 2015; Moore et al., 1976)
Clostridium 2.3 Curved rod/0.5- 6.0-9.0 35-37 °C Cellulolytic bacterium. Major (Martin et al., 2018;
phytofermentans 0.8µm width, 3-15µm end products are acetate, ethanol, Warnick et al., 2002)
length. H2, & CO2, and minor end
products are lactate & formate.
Clostridium 48 Spindle shaped 7.4 37 °C Hydrolyzes wide range of (Murray et al., 1982)
saccharolyticum rod/0.5-0.7µm width, carbohydrates. Its fermentation
3µm length. products are CO2, H2, acetate,
and ethanol.
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Fibrobacter intestinalis NG Rod/ 0.3-0.4µm 6.6 37 °C Degrades cellulose and (ATCC, 2018;
width, 0.8-2µm hemicellulose. Major Montgomery et al., 1988;
length. fermentation end products Suen et al., 2011)
include acetate, formate, formic
acid, and succinic acid.
Gracilibacter 3.1 Curved rod/0.2- 6.8-7.7 42.5-46.5 °C Ferment carbohydrates and (Lee et al., 2006)
thermotolerans 0.4µm width, 2-7µm sugars to acetate, ethanol and
length. lactate.
Lutaonella thermophila 24 Rod/0.4-0.5µm width, 8.0 40-45 °C Hydrolyzes proteins and sugars (Arun et al., 2009)
1-2µm length.
f
to produce VFAs and CO2, H2,

o
formate, acetate and butyrate.

o
Pseudobacteroides NG Straight and slightly 8.0-8.5 35 °C Hydrolyzes carbohydrates and (Horino et al., 2014)

r
cellulosolvens curved rods. produce ethanol and lactate.

p
Ruminococcus albus 2 Cocci. 7.0-7.5 37 °C Degrades plant cell wall, (Chen and Dong, 2004;

-
cellulose and hemicellulose. Its Iakiviak et al., 2016)
main final fermentation products

r e include acetate, formate, ethanol,

H2 and CO2.

P
Turicibacter sanguinis NG Rod/0.5-2µm width, 7.5 37 °C Carbohydrates degrading (Bosshard et al., 2002)
0.7-7µm length. bacteria with lactate as the major

DT; doubling time, GR; growth rate, NG; not given

a l fermentation product.

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Table 2
Characteristics and functions of mainstream fermentative bacterial species involve in AD

Bacterial Species DT/GR (hr) Shape/Size Optimal conditions Functions References

pH Temperature
Acetanaerobacterium 72 Thin Rod/0.2-0.4µm 6.5-7.0 37 °C Hydrolyzes proteins and sugars to (Chen and Dong, 2004)
elongatum width, 4-8µm length. acetate, ethanol, hydrogen and CO2.

f
Aneurinibacillus NG 0.5-1µm width, 2- 6.0-6.5 10-30 °C Helps in hydrolysis of aesculin and (Tsubouchi et al., 2015)
tyrosinisolvens 6.3µm length. degradation of L-tyrosine. It also

o
ferments some organic acids and

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sugars.
-1
Lactobacillus GR=0.20 h Rod/1µm width, 2- 3.0-6.5 42 °C Hydrolyzes carbohydrates and (Boonkumklao et al., 2006;

-p
thermotolerans 3µm length. produces VFAs and CO2 as well as D Niamsup et al., 2003)
and L-lactic acid.
Petrimonas 2 Rod/0.7-1µm width, 7.2 37-40 °C Hydrolyzes carbohydrates, proteins (Grabowski et al., 2005;
sulfuriphila length 1.5-2µm.

r e and sugars to produce acetate, H2 and

CO2.
Huang et al., 2016;
Maspolim et al., 2015)

P
Thermovirga lienii NG Rod/0.4-0.8µm 6.5-7.0 58 °C Able to hydrolyzes protein-rich (Dahle and Birkeland,
width, 2-3µm length. substrates, few amino acids and 2006)

DT; doubling time, GR; growth rate, NG; not given

a l restricted number of organic acids.

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Table 3
Characteristics and functions of typical acidogenic bacterial species involve in AD

Bacterial Species DT/GR (hr) Shape/Size Optimal conditions Functions References

pH Temperature
Advenella faeciporci NG Coccus/0.75µm width, 7.0-9.0 25-35 °C Helps in glucose and hydrocarbons (Coenye et al., 2005;
1.5-2µm length. fermentation and denitrifies nitrite. Gibello et al., 2009;

f
Matsuoka et al., 2012;
Xenofontos et al.,

Alkalitalea NG Rod/0.4µm width, 9.7 35-37 °C

o o
Utilizes amino acids and sugars as
2016)
(Zhao and Chen, 2012)
saponilacus 5.5µm length.

r
energy source to produce propionate

p
and acetate.

-
Bacteroides caccae 48 Rod/1.4-1.6µm width, 5.0-5.2 30-37 °C Ferments carbohydrates to produce (Johnson et al., 1986)
2.5-12µm length. succinate and acetate.
Bifidobacterium
animalis
5.5 Short rod/0.3µm width,
1.3-1.5µm length.
6.4-7.0

r
39-41 °C
e Ferments sugar to lactate and acetate. (Bruno et al., 2002;
Scardovi and

Christensenella
minuta
96 Rod/0.4µm width, 1.8-
1.9µm length.
7.5

l P
37-40 °C Utilize various sugars and produce
VFAs as fermentation products.
Trovatelli, 1974)
(Morotomi et al., 2012)

Cloacibacillus
porcorum
8 Curved Rod/1µm

n
width, 4.25µm length. a
6.5 39 °C Ferments amino acids to produce
acetate, formate, and propionate.
(Li et al., 2018c; Looft
et al., 2013)

Corynebacterium 24

u r
Rod/0.5-0.7µm width, 9.0 37 °C
Butyrate is resulted from Serine only.
Utilizes organic acids with lactate, (Wu et al., 2011)

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humireducens 1-2µm length. formate, acetate and ethanol as electron
donors.
Dechloromonas NG Rod/0.5µm width, 7.0 30 °C Denitrifying bacteria and nitrate (Chakraborty and
denitrifican 1.7µm length. reducers. They also use metabolites Picardal, 2013; Horn et
such as acetic, glutamic, lactic, malic, al., 2005)
propionic, pyruvic, and succinic acid as
carbon source.
Ferroplasma NG 0.3-3µm width. 1.7-2.2 37 °C Ferrous-iron oxidizing, biogeochemical (Baumler et al., 2007;
acidiphilum cycling of sulfur and sulfide metals; Golyshina and Timmis,
sulfate-containing salt. 2005)
Geofilum rubicundum 72-96 Filamentous/0.2-0.4µm 7.3-8.3 33 °C Nitrate and nitrite reducing bacteria and (Miyazaki et al., 2012)
width, 4-22µm length. also utilize sugars to produce acetate,
ethanol, and lactate.
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Lutispora NG Rod/0.7µm width, 7.5-8.0 55-58 °C Utilizes amino acids to acetate, iso- (Shiratori et al., 2008a)
thermophila 6.7µm length. butyrate, propionate and iso-valerate
and also hydrolyzes aesculin and
gelatin.
Mariniphaga NG Rod/0.5-0.6µm width, 7.0-7.5 33-37 °C Ferments sugars to produce organic (Iino et al., 2014; Wang
anaerophila length 1.9-6.9µm. acids. et al., 2015)
Sedimentibacter NG Rod/0.4-1.4µm width. 6.0-8.5 30-37 °C Plays a key role in amino acid (Imachi et al., 2016)
acidaminivorans degradation to produce VFAs.
Thauera aromatica
AR-1
21 Rod/0.5-1.5µm width,
1-2.5µm length.
8.0 30 °C

o f
Degrades aromatic compounds in the
presence of nitrate.
(Anders et al., 1995;
Gallus and Schink,
1998; Molina-Fuentes

r o et al., 2015; Pacheco-

Sánchez et al., 2018;

DT; doubling time, GR; growth rate, NG; not given

-p Philipp et al., 2002)

r e
l P
n a
u r
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Table 4
Characteristics and functions of typical acetogenic bacterial species involve in AD

Bacterial Species DT/GR (hr) Shape/Size Optimal conditions Functions References

pH Temperature
Acetobacterium >10 Rod/1µm width, 1- 7.2-7.8 30 °C Growth occurs with H2 and CO2 to (Braun and Gottschalk,
wieringae 2µm length form acetate. 1982)

Acetobacterium With 2,3- Short rod/1µm width, 7.3-7.6 30-35 °C

o f
Oxidizes H2 and reduces CO2 to (Bache and Pfennig,

o
woodii butanediol and 2µm length. produce acetate. 1981; Balch et al.,

r
acetoin (8.3) 1977)

Acetogenium kivui 2 Rod/0.7-0.8µm width,

2-7.5µm length.
6.4 66 °C

- p Oxidizes H2 and reduces CO2 to

produce acetate.
(Leigh et al., 1981)

r e
Anaerovorax
odorimutans
GR=0.044 h-1 Slightly curved
rod/0.7-0.8µm width,
1.9-2.7µm length.
7.2-7.6

l P37 °C Converts putrescine to acetate,

butyrate, molecular H2 and ammonia.
It uses 4-aminobutyrate and 4-
(Matthies et al., 2000)

Clostridium aceticum With H2 and

n
0.8-1µm width, 5µm
a
8.3-8.5 37 °C
hydroxybutyrate for growth.
Converts H2 and CO2 into acetate. (Braun et al., 1981)
CO2 (20-25)
and with
fructose (8) r
length with H2, CO2

u
and 40µm length with
fructose length.

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Clostridium With glycerate Rod/0.8-1µm width, 3- 5.6-5.9 56-60 °C Growth occurs with H2 and CO2 as (Wiegel et al., 1981)
thermoautotrophicum (2) and under 6µm length. well as fructose, glucose, glycerate,
chemo and methanol to produce acetate.
lithotrophic
conditions (8)
Desulfobulbus 10 Lemon or onion 7.2 30-39 °C Utilizes alcohols, lactate and (Widdel and Pfennig,
propionicus shaped/1.0-1.3µm propionate, as carbon sources and 1982)
width, 1.8-2µm length. electron donors to produce acetate.

Desulfococcus 28, Rod shaped. 7.0-7.3 30-35 °C Sulfate reducing bacteria, oxidizes (Peters et al., 2004;
multivorans With sulfate organic acids to CO2. Schauder et al., 1986;
(43.2), and Stieb and Sehink, 1989)
with sodium
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benzoate (35).

Desulfotomaculum NG Rod/1µm width, 2- 6.0-6.8 55-60 °C Oxidizes CO2, H2, propanol, butanol, (Jabari et al., 2013)
peckii 5µm length. and ethanol to propionate, butyrate,
and acetate.

Desulfovibrio
desulfuricans
With sulfate
(5.6), nitrate
(4.6), and
Rod/0.5µm width,
1.8µm length.
6.5-7.0 32 °C

f
Grows in chemostat culture with H2
and reduces sulfates to form organic
acids.
o
(Boon et al., 1977;
Herrera et al., 1993;
Seitz and Cypionka,

Hydrogenispora
nitrite (3.6).
NG Rod/0.3-0.5µm width, 6.0-7.7 37-45 °C
r o
Ferment various sugars into acetate,
1986)
(Liu et al., 2014)
ethanolica 3-18µm length.

-p ethanol and H2.

Hydrogenophaga
carboriunda
NG Rod/0.5µm width,
2µm length.
6.5-9.5

r
25-30 °C
e Ferments sugars and amino acids to
produce H2.
(Gan et al., 2011;
Mantri et al., 2016;

l P Reinauer et al., 2014)

Levilinea
saccharolytica
56 Filamentous/0.4-
0.2µm width,

n a
6.0-7.2 37 °C Ferments amino acids and sugars into
H2, lactic acids, and acetate.
(Guo et al., 2015; P.
Wang et al., 2018;

r
length >100µm. Yamada et al., 2006)

Macellibacteroides 72
u
Rod/0.5-0.1µm width, 6.5-7.5 35-40 °C Metabolizes monosaccharides and (Hao et al., 2015; Jabari

Jo
fermentans 2-3µm length. disaccharides to produce acetate, et al., 2012; Rout et al.,
butyrate and iso-butyrate. 2017)

Pelotomaculum GR=1.65±0.17 Spherical//0.7-0.8µm 7.0 55 °C Ferments pyruvate, fumarate, and (Imachi et al., 2002)
thermopropionicum day-1 with width, 1.7-2.8µm lactate.
pyruvate. length.

Proteiniphilum 11.2 Rod/0.6-0.9 µm width, 7.5-8.0 37 °C Acetic acid is the main fermentation (Chen and Dong, 2005)
acetatigenes 1.9-2.2 µm length. product from yeast extract, peptone,
pyruvate, and L-arginine.
 Journal Pre-proof

Rhizobium NG Rod. 7.0-7.5 28 °C Utilizes sugars as carbon sources to (Garcı´a-Fraile et al.,

cellulosilyticum produce acetate and formate. 2007)

Saccharofermentans 6.2 Oval/ 0.6-0.9 µm 6.5 37 °C Sugar-fermenting bacteria which (Chen et al., 2010;
acetigenes width, 1.2-1.8µm degrade sugars into acetate, lactate, Hahnke et al., 2016)
length. and fumarate.

Sporobacter
termitidis
25 Slightly curved
rod/0.2-0.4µm width,
1-2µm length.
6.7-7.2 32-35 °C

f
Uses methylated sulfides from
methylated aromatic compounds in

o
the presence of cysteine.
(Grech-Mora et al.,
1996)

Sulfurovum riftiae 3 Rod/0.4µm width,

1.05µm length.
6.0 35 °C
ro
Utilizes H2 as the only energy source,

p
CO2 is used as the sole carbon source
(Giovannelli et al.,
2016; Nabweteme et

e -for production of acetate. al., 2016)

Syntrophobacter
wolinii
With
Desulfovibrio
sp. (87±7) and
Rod/0.6-1µm width, 1-
4.5µm length.
6.1

P r
30-35 °C Utilizes propionate to produce acetate,
H2 and CO2.
(Boone and Bryant,
1980; Liu et al., 1999)

Methano-
spirillum
hungatei
a l
rn
(161±18)
Syntrophaceticus NG Curved rod/0.5-0.7µm 6.0-8.0 37-40 °C Oxidizes acetate in cocultivation with (Schnürer et al., 2018;
schinkii

o u
width, 2-5µm length. 42-45 °C
49-60 °C
hydrogen consuming methanogens. Westerholm et al.,
2016, 2010)

Syntrophomonas
wolfei
84
J
Helical rod/0.5-1µm
width, 2-7µm length.
5.4-7.4 37 °C Helps to convert butyrate to H2 and
CO2.
(McInerney et al., 1981;
Schmidt et al., 2013)

Syntrophorhabdus 480, Thin rod/0.4-0.8µm 7.0 35-37 °C Capable of degrading aromatic (Qiu et al., 2008)
aromaticivorans GR=0.025 width, 1.2-2.5µm compounds to acetate and H2.
day-1 length.

Syntrophus GR=0.006h−1 Rod/0.5-0.7µm width, 7.0-7.8 28-37 °C Work in syntrophic relationship with (Elshahed et al., 2001;
aciditrophicus with benzoate 1-16µm length. H2 or formate utilizing microbes to Jackson et al., 1999;
degrade benzoate and certain fatty James et al., 2016;
acids to produce acetate, CO2, Mouttaki et al., 2009)
 Journal Pre-proof

formate, and H2.

Thermacetogenium 22.8 Curved and rod/0.4- 6.8 58 °C Oxidizes acetate by working with (Hattori et al., 2000)
phaeum 0.7 width, 2-12.6µm hydrogenotrophic methanogens.
length.

Thermotoga lettingae 4 Rod/0.5-1 width, 2-

3µm length.
7.0 65 °C

f
Acetate oxidizing syntroph that
degrades glucose to acetate and then
to CO2.
o
(Balk et al., 2002)

DT; doubling time, GR; growth rate, NG; not given

r o
- p
r e
l P
n a
u r
Jo
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Table 5
Characteristics and functions of typical hydrogenotrophic and methylotrophic archaeal species involve in AD

Archaeal Species DT/GR (hr) Shape/Size Optimal Conditions Methane References

Producing
pH Temperature
Substrates
†
Methanimicrococcus baltticola* 3.1 Lens-shaped/width 7.2-7.7 39 °C Methanol and (Sprenger et al.,
0.3µm. methylated amines. 2000; Weiss et al.,

Methanobacterium aarhusense** NG Filamentous/5-18 µm 7.5-8 45 °C

o f
H2/CO2.
2008)
(Shlimon et al.,

o
length, width 0.7µm. 2004)

Methanobacterium aggregans** NG Curved Rod/2-2.5µm 6.5-7.0

p r
40 °C H2/CO2. (Kern et al., 2015)
length, width 0.2-
0.5µm.

e -
Methanobacterium alcaliphilum** NG
width 0.5-0.6µm.

P r
Rod/2-25µm length, 8.1-9.1 37 °C H2/CO2. (Cadillo-quiroz et
al., 2014)

Methanobacterium arcticum** GR= 0.026h−1

with H2/CO2;
0.014h−1 with
a l
Curved rod/3-6µm
length, width 0.45-
6.8-7.2 37 °C Formate, H2/CO2. (Shcherbakova et al.,
2011)

n
0.5µm.

Methanobacterium beijingense**
formate.

grown in
u r
GR=0.049h-1 when Rod/3-5µm length,
width 0.4-0.5µm.
7.2-7.7 37 °C Formate, H2/CO2. (Ma et al., 2005)

Jo
H2/CO2 medium;
GR= 0·030, 0·023
and 0·021 h−1 in
the absence of
acetate, yeast
extract and both.
Methanobacterium bryantii† GR=0.036h−1 with Rod/10-15µm length, 6.9-7.0 37 °C 2-Propanol, 2- (Krivushin et al.,
H2/CO2. width 0.5-1µm. butanol, acetate. 2010; Shcherbakova
et al., 2011)
Methanobacterium congolense NG Rod/2-10µm length, 7.2 37-42 °C H2/CO2 only (Cuzin et al., 2001)
width 0.4-0.5µm. substrates for growth
and CH4 production.
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Methanobacterium espanolae** 10 Rod/6µm length, width 5.6-6.2 35 °C H2/CO2. (Patel et al., 1990)
0.8µm.

Methanobacterium ferruginis** 18.5 Rod/2.3-3.7µm length, 6.0-8.0 40 °C H2/CO2. (Mori and

width 0.3µm. Harayama, 2011)

Methanobacterium flexile** GR = 0.032h-1 Rod/2-5µm length, 7.0-7.5 35-38 °C Formate, H2/CO2. (Zhu et al., 2011a)
width 0.3-0.5µm.

Methanobacterium formicicum** NG Rod/5-15µm length, 7.0 37-45 °C

o f
Formate, H2/CO2. (Battumur et al.,
width 0.7µm.

r o 2016)

Methanobacterium ivanovii** NG Rod/1-15µm length,

width 0.5-0.8µm.
7-7.4

- p 45 °C H2/CO2. (Cadillo-quiroz et
al., 2014)

Methanobacterium kanagiense** 21
r
Rod/1.6-5µm length,
e
7.5-8.5 40 °C H2/CO2. (Kitamura et al.,

P
width 0.35-0.5µm. 2011a)

Methanobacterium lacus**† 35

a l
Rod/2-15µm length,
width 0.2-0.4µm.
6.5 30 °C H2/CO2 and
methanol+H2.
(Borrel et al., 2012;
Brauer et al., 2014)

Methanobacterium movens* GR=0.027h−1

u rn
Rod/2-5µm length,
width 0.3-0.5µm.
7.2-7.5 35-38 °C Formate, H2/CO2. (Zhu et al., 2011a)

Methanobacterium movilense**

J o
GR=0.027 h−1 Rod/3.5-4µm length,
width 0.6-0.7µm.
7.4 33 °C 2-butanol, 2-
propanol H2/CO2.
(Schirmack et al.,
2014a; Zhu et al.,
2011b)
Methanobacterium oryzae** NG Filamentous rod/3- 7.0 40 °C Formate, H2/CO2. (Joulian et al., 2000)
10µm length, width
0.3-0.4µm.
Methanobacterium paludis** 35 Rod/1.5-2.8µm length, 5.4-5.7 32-37 °C H2/CO2. (Brauer et al., 2014;
width 0.6µm. Cadillo-quiroz et al.,
2014)
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Methanobacterium petrolearium** 39.5 Rod/2.4-4.7µm length, 6.5 35 °C H2/CO2. (Mori and

width 0.3µm. Harayama, 2011)

Methanobacterium subterraneum** 2.5 Rod/width 0.1-0.15µm. 7.8-8.8 36-45 °C Formate, H2/CO2. (Kotelnikova et al.,
1998)

Methanobacterium uliginosum NG Rod/1.9-3.9µm length, 6-8.5 40 °C H2/CO2. (Konig, 1984)

width 0.2-0.6µm.

Methanobacterium veterum**† GR= 0.026h−1, Rod/2-8µm length, 7.2-7.4 28 °C

o f
H2/CO2, (Krivushin et al.,
with H2+ CO2
GR= 0.012h−1
width 0.4-0.45µm.

r o Methanol+H2,
CH3NH2+H2.
2010)

Methanol+ H2+
methylamine.
GR=0.040 h-1
- p
e
Methanobrevibacter acididurans** Cocci/width 0.3- 6.0 35 °C H2. (Savant et al., 2002)

r
0.5µm.

Methanobrevibacter boviskoreani** NG

l
width 0.6µm. P
Rod/1.5-1.8µm length, 6.5-7.0 37-40 °C Formate, H2/CO2. (Lee et al., 2013)

Methanobrevibacter gottschalkii* NG

n a
Coccobacillus/0.9µm 6.5-7.0 37-41 °C Formate, H2/CO2. (Rea et al., 2007)

u r length, width 0.7µm.

Jo
Methanobrevibacter millerae** NG Coocobacilli/width 7.0-8.0 36-42 °C Formate, H2/CO2. (Rea et al., 2007)
0.5-1.2µm.

Methanobrevibacter olleyae** NG Coocobacilli/width 7.5 25-39 °C Formate, H2/CO2. (Rea et al., 2007)
0.3-1µm.

Methanobrevibacter ruminantium** NG Encapsulated Rod. 6.0-7.0 35-40 °C Formate, H2/CO2. (Rea et al., 2007;
Smith and Hungate,
1958)
Methanobrevibacter smithii** 3 Rod. 7.0 37-39 °C Formate. (Amani et al., 2010;
Khelaifia et al.,
2013; Rea et al.,
2007)
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Methanoculleus bourgensis* 18 Width 1-2µm. 6.7 35-40 °C Formate, H2/CO2. (Asakawa and
Nagaoka, 2003;
Dianou et al., 2001;
Wandera et al.,
2019; Weiss et al.,
2008)
Methanoculleus chikugoensis** NG Cocci/width 1-2µm. 6.7-7.2 25-30 °C Formate, H2/CO2, 2- (Dianou et al., 2001)
butanol/CO2, 2-
propanol/CO2, and

o f
cyclopentanol/CO2
as substrates for

o
methanogenesis.

r
Methanoculleus horonobensis** NG Cocci/width 0.7- 6.7-6.8 37-42 °C Formate, H2/CO2. (Shimizu et al.,
1.6µm. 2013)

GR= 0.031h−1
- p
e
Methanoculleus hydrogenitrophicus Cocci/width 0.8-2µm. 6.6 37 °C H2/CO2. (Tian et al., 2010a)

Methanoculleus marisnigri** NG
P r
Cocci/width 1-2µm. 6.2-6.6 20-25 °C Formate, H2, 2- (Dianou et al., 2001;

a l propanol/CO2, 2-
butanol/CO2.
Maestrojuan et al.,
1990)

n
Methanoculleus palmolei** 13.5 Cocci/width 1.25-2µm. 6.9-7.5 40 °C Formate, H2/CO2, 2- (Shimizu et al.,

u
-1r propanol+CO2, 2-
butanol+CO2
2013; Zellner et al.,
1998)

Jo
Methanoculleus receptaculi** GR=0.084h Cocci/0.8-1.7µm. 7.5-7.8 50-55 °C Sodium formate, (Chen B, Zhou D,
H2/CO2. 2008; Shimizu et al.,
2013)
Methanoculleus submarinus* 18.7 Cocci/width 0.8-2µm. 4.8-7.7 45 °C H2+CO2 or Formate, (Mayer et al., 2019;
acetate. Mikucki et al., 2003)

Methanoculleus sediminis** 15.07 Cocci/width 0.5-1µm. 7.1 37 °C Formate, H2/CO2. (Chen et al., 2015)

Methanoculleus taiwanensis** 6.7 Cocci/width 0.6- 8.08 37 °C Formate, H2/CO2. (Chen et al., 2015;
1.5µm. Weng et al., 2015)
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Methanoculleus thermophilicum 20 Cocci/width 1-1.3µm. 7.0 55 °C Formate, H2/CO2. (Rivard and Smith,
1982; Shimizu et al.,
2013)
Methanolobus chelungpuianus**† GR= 0.041h−1 and Cocci/width 0.5-1µm. 7.0 37 °C Methanol and (Wu and Lai, 2011)
0.091h−1 Trimethylamine.

Methanomassiliicoccus luminyensis**† 32 Cocci/width 0.7-1µm. 7.6 37 °C H2 or methanol. (Dridi et al., 2012)

Methanosphaera stadtmanae**† 3 Spherical/width 1µm. 6.5-6.9 36-40 °C

o f
Methanol and H2. (Khelaifia et al.,

r o 2013; Miller and

Wolin, 1985; Weiss

p
et al., 2008)
Methanospirillum hungatei* 20.7 Filamentous/7.4-10µm
length width 0.4-

e -
7.0-9.0 37-45 °C H2, Formate or
H2/CO2 to produce
(Iino et al., 2010)

r
0.5µm. CH4.
Methanospirillum lacunae* 32.3 Curved Rod/11-25µm 7.5 30 °C H2, Formate or (Iino et al., 2010)

0.6µm.

l P
length, width 0.5- H2/CO2 to produce
CH4.
Methanospirillum psychrodurum* GR=0.065 h-1

a
Rod/11-62µm length, 7.0 25 °C H2/CO2. (Zhou et al., 2014)

rn
width 0.4-0.5µm.
Methanospirillum stamsii* 39.8 Rod/7-25µm length, 7.0-7.5 20-30 °C, H2/CO2, very weak (Parshina et al.,

u
width 0.4-0.5µm. growth with 2014)
Formate.

o
*Motile, **Non-motile, †Methylotrophic, DT; doubling time, GR; growth rate, NG; not given

J
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Table 6
Characteristics and functions of typical acetoclastic archaeal species involve in AD
Archaeal Species DT/GR Shape/Size Optimal Conditions Methane Producing Substrates References
(hr)
pH Temperature
Methanosarcina 22 Irregular cocci/1-2µm 7.0 45 °C Acetate, mono-, di-, and (Archer and King, 1983; Kern
flavescens* width. trimethylamine, methanol, H2, et al., 2016; P. Wang et al.,
and CO2. 2018)
Methanosarcina barkeri* 30 Irregular cocci/1.5-2µm
width.
7.0 30-40 °C
f
Acetate, mono-, di-, and

o
trimethylamine, methanol, CO,
H2, and CO2.
(Bock and Schonheit, 1995;
Bryant and Boone, 1987)

Methanosarcina mazei* 51 Cocci/1-2mm width. 6.5-7.2 40-42 °C

r o
Acetate, mono-, di-, and
trimethylamine, methanol, and
(Liu et al., 1985; Maestrojuan
and Boone, 1991)

Methanothrix 65 Rod/0.8µm width, 2.5- 7.1-7.5 35-40 °C

- p
H2, CO2.
Acetate requires for growth and (Patel and Sprott, 1990)
(Methanosaeta) concilii*
Methanothrix
(Methanosaeta)
28
6.0µm length.
Rod/0.8-1µm width, 3-
5µm length.
7.2-7.6

r
34-37 °C
e CH4 production.
Acetate requires for growth and
CH4 production.
(Ma et al., 2006)

harundinacea*
Methanothrix NG Rod/1-1.3µm width.

l
7.0
P
50-60 °C Acetate requires for growth and (Kamagata et al., 1992; Oren,

a
(Methanosaeta) CH4 production. 2014)
thermophilla*
Methanothrix soehngenii* 168

r n
Filamentous rod/width
0.8µm, 2µm length.
7.4-7.8 37 °C Acetate requires for growth and
CH4 production.
(Huser et al., 1982; Jetten et
al., 1992, 1989)

u
*Non-motile, DT; doubling time, GR; growth rate, NG; not given

Jo
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Table 7
Role of trace elements in the process of AD (Schattauer et al., 2011)
Elements General functions (microorganisms) Role in methanogenesis Recommended conc. of trace
elements (mg/l) in AD

Boron Cofactor of enzymes. [0.001-11]4.

Calcium Membrane permeability. [>0.54-40]5.

Improves action of other metals1.

o f
ro
Eliminates toxic impacts by other metals.

Chromium Glucose metabolism. [0.005-52]4.

Cobalt Presents in corrinoids2.

e -p Methyltransferase3. [0.024-10]6.

Activates metallic enzyme1.

Carboxylpeptidase activator1.

P r
Needed for vitamin B12 synthesis (cyanocbalamin)1.

Can inhibit metabolism1.

a l
Carbon monoxide hydrogenase2.
r n
Copper Activates metallic enzyme1.
u [1-10]8

Jo
Can inhibit metabolism chelates and decreases their toxicity1.

Superoxide dismutase and hydrogenase in CH4 producing bacteria, in facultative

anaerobes2.

Pigments.

Iron Electron acceptor in cytochromes (Fe +3) 1,2. Formyl-MF- [>0.28-50.4]5.

dehydrogenase3.
Synthesis of acotinase, catalase, and peroxidase1.
Carbon monoxide,
Essential in ferredoxin, carbon monoxide and formate hydrogenase2. dehydrogenase, acetyl-
CoA synthesis3.
 Journal Pre-proof

Redox property. Hydrogenases3.

Magnesium Activates kinases and phosphotransferase1. [360-4800]5.

Manganese Activates bacterial enzymes such as iso-citric dehydrogenase and malic enzyme1. [0.005-55]4.

Interchanges with magnesium in kinase reactions1.

Stabilizes methyltransferase in bacteria producing CH42.

Redox reaction2.

Cofactor of various enzymes.

o f
Molybdenum Presents in formate dehydrogenase2.
r oFormyl-MF- [0.16-50]6.

Inhibitor of sulphate reducing bacteria2.

- p dehydrogenase3

Formate dehydrogenase3

Nickel
Cofactor of various enzymes.

Carbon monoxide dehydrogenase . 2

r e Carbon monoxide [0.024-0.62]6.

l
Synthesis of Coenzyme A, factor F430, CH3-CoM reductase2.
P dehydrogenase3.

Methyl reductase3.
Stabilizes DNA, RNA2.

n a Hydrogenases3.

r
Cofactor of urease.

Selenium

u
Hydrogenase, formate dehydrogenase in CH4 producing bacteria2. Formyl-MF- [0.079-0.79]5.

Jo
dehydrogenase3.

Formate dehydrogenase3.

Carbon monoxide,
dehydrogenase/ acetyl-
CoA synthesis3.

Sulphur Cofactor and component of many proteins. Carbon monoxide [0.32-13000]5.

dehydrogenase3.

Hydrogenases3.
 Journal Pre-proof

Zinc Activates metallic enzymes1. [15.8-117.1]7.

Responsible for carbonic anhydrase and carboxylpeptidase activity1.

Stimulates cell growth1.

Cofactor of RNA and DNA polymerase.

Can aggravate the toxic effects of other metals and inhibit metabolism1.

Hydrogenase in CH4 producing and sulphate reducing bacteria2.

Formate dehydrogenase2.
o f
Superoxide dismutase2.
r o
- p
1. (Burgess et al., 1999), 2. (Oleszkiewicz and Sharma, 1990), 3. (Somitsch, 2007), 4. (Sahm, 1981), 5. (Takashima et al., 1990), 6. (Pobeheim et al., 2010), 7.
(Zhang et al., 2017), 8. (Gerardi, 2003)

r e
l P
n a
u r
Jo
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Highlights
 Systematic study on roles of bacteria and archaea participating in four stages of

anaerobic digestion is given

 Microbial ecology of digesters is strongly affected by temperature, organic loading

rate and F/I ratio

 The review gives deep understanding of the anaerobic digestion strategies to enhance

of
methane production

ro
-p
re
lP
na
ur
Jo
Figure 1
Figure 2
Figure 3
Figure 4