Accepted Manuscript

Review

A critical review on the interaction of substrate nutrient balance and microbial

community structure and function in anaerobic co-digestion

Rong Xu, Kai Zhang, Pu Liu, Aman Khan, Jian Xiong, Fake Tian, Xiangkai Li

PII: S0960-8524(17)31660-7
DOI: http://dx.doi.org/10.1016/j.biortech.2017.09.095
Reference: BITE 18923

To appear in: Bioresource Technology

Received Date: 30 June 2017

Revised Date: 13 September 2017
Accepted Date: 15 September 2017

Please cite this article as: Xu, R., Zhang, K., Liu, P., Khan, A., Xiong, J., Tian, F., Li, X., A critical review on the
interaction of substrate nutrient balance and microbial community structure and function in anaerobic co-digestion,
Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.09.095

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A critical review on the interaction of substrate nutrient
balance and microbial community structure and function in
anaerobic co-digestion

Rong Xua, Kai Zhanga, Pu Liub, Aman Khana, Jian Xiongc, Fake Tianc, Xiangkai Lia,*

a
Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations,

School of Life Science, Lanzhou University, Tianshui South Road #222, Lanzhou,

Gansu 730000, People’s republic of China

b
Department of Development Biology Sciences, School of Life Science, Lanzhou

University, Tianshui South Road #222, Lanzhou, Gansu 730000, People’s republic of

China

c
Wuhan Optics Valley Bluefire New Energy Co., Ltd, Fozulingsanlu Wuhan East Lake

Development Zone #29, Wuhan, Hubei 430205, People’s republic of China

E-mail address: xkli@lzu.edu.cn Tel. / fax: +86 931 8912562

1
Abstract: Anaerobic co-digestion generally results in a higher yield of biogas than

mono-digestion, hence co-digestion has become a topic of general interest in recent

studies of anaerobic digestion. Compared with mono-digestion, co-digestion utilizes

multiple substrates. The balance of substrate nutrient in co-digestion comprises better

adjustments of C/N ratio, pH, moisture, trace elements, and dilution of toxic

substances. All of these changes could result in positive shifts in microbial community

structure and function in the digestion processes and consequent augmentation of

biogas production. Nevertheless, there have been few reviews on the interaction of

nutrient and microbial community in co-digestions. The objective of this review is to

investigate recent achievements and perspectives on the interaction of substrate

nutrient balance and microbial community structure and function. This may provide

valuable information on the optimization of combinations of substrates and prediction

of bioreactor performance.

Keywords: Co-digestion; Nutrient balance; Microbial community; Biogas production.

2
Graphical abstract

3
1. Introduction

Nowadays, environmental pollution is a global problem (Liew et al., 2011).

Anaerobic digestion (AD) can convert waste into energy which is ideal for

environmental pollution control (Ahn et al., 2010; Chen et al., 2010). For waste

remediation and resource recovery, AD has been applied for centuries (Weiland, 2010).

The nutrient of substrates is an important aspect of AD. A successful AD process

needs balanced C/N ratios (20–30), suitable pH (6.0–7.5) and other

parameters (Prochazka et al., 2012; Tanimu et al., 2015; Wang et al., 2012). However,

in mono-digestion, a single material is used as a substrate; it is always nutrient

imbalanced and has low biogas production. For example, (i) with animal manure as

the sole substrate, there is a low organic load and high nitrogen concentration, which

inhibits methanogens. (ii) Municipal solid waste has inappropriate materials and high

concentrations of heavy metals. (iii) Agro-industrial wastes lack N, and (iv)

slaughterhouse wastes have high concentrations of N and long chain fatty

acids (Appels et al., 2008; Kanchinadham et al., 2014; Rodriguez-Verde et al., 2014).

To overcome these limitations of mono-digestion, co-digestion was put forward and

applied.

Anaerobic co-digestion (AcoD) is the simultaneous digestion of multiple

substrates with complementary characteristics (Bouallagui et al., 2009; El-Mashad &

Zhang, 2010) With co-digestion, agricultural residues, animal manures, municipal

wastes, and industrial wastes can be maximally utilized in comparison to

mono-digestion (Murto et al., 2004; Wang et al., 2012). Moreover, the combination of
4
co-substrates results in positive effects to the system, such as a better balance (C/N

ratio, pH, moisture), dilution of potentially toxic compounds, and supplement of trace

elements (Davidsson et al., 2008; Esposito et al., 2012; Fernández et al., 2005). Thus,

AcoD is possible way to solve the limitations of mono-digestion, increase biogas

production, improve waste recycling and contribute to environmental

protection (Mata-Alvarez et al., 2014). The studies on this subject has grown quickly

in recent years.

AD is a biochemical process including hydrolysis, acidogenesis, acetogenesis

and methanogenesis, which involves a great variety of microbial groups (Wang et al.,

2017). Microorganisms are the core of the digesters and the efficiency of AD mostly

depends on the active microbial community (Riviere et al., 2009). Generally,

microbial community was correlated with the nutrient of the environment. For

example, in the soil, changes in microbial community composition are often observed

after addition of different types of amendments (Hogberg et al., 2007; Zhong et al.,

2009). Fertilizer amendments are used primarily to increase nutrient to plants, but

they can also affect soil microorganisms. Changes in microbial activity and

composition can in turn influence plant growth (Marschner, 2003). Like soil, the

reactor of AcoD is also a complex ecosystem containing various microorganisms.

Microbes can be indicators to AcoD process. Therefore, the qualitative and

quantitative relationship among nutrition of co-substrates, microbial community

structure and biogas production needs to be clarified.

5
 With the advancement of technology, such as high-throughput DNA sequencing,

a great amount of research has investigated the microbial community structure in the

AcoD process (Dennehy et al., 2017; Mu et al., 2017; Shen et al., 2014). However,

insightful analysis of the advantages of co-digestion and relationships between

co-substrates nutrition and microbial community is lacking. Moreover, few reviews

on the interaction of co-substrates nutrition, microbial community and biogas

production have been published. The characterizations of the nutrition and microbial

community in AcoD play a significant role in assessing and enhancing digestion

efficiency (Möller & Müller, 2012; Xu et al., 2014). The objective of this review is to

present the recent achievements and perspectives on the interaction of co-substrates

nutrient balance, microbial community structure and biogas production in AcoD. It

may provide valuable information on the optimization of combinations of substrates

and prediction of bioreactor performance in AcoD.

2. Interaction of co-substrates nutrient, microbial community and

biogas production

2.1. Co-substrates modulate C/N ratio and influence microbial community

In the process of fermentation, ratios of co-substrates are critical, especially the

C/N ratio. The establishment and maintenance of an appropriate C/N ratio were one of

the key factors in a successful AcoD (Zhang et al., 2013; Zhu et al., 2014). Generally,

a C/N ratio of 20 – 30 is considered optimal for AD (Zheng et al., 2015). In

mono-digestion, a single substrate is either carbon-rich or nitrogen-rich, such that it is

difficult to keep a balanced C/N ratio (Table 1). An imbalanced C/N ratio had negative
6
effects on the activity of microbes, which usually denotes an impairment of a

particular microbial function and results in deteriorative phases (Riya et al., 2016; Shi

et al., 2013; Wan et al., 2013). Therefore, in order to obtain a well-organized

microbial community, utilizing complementary co-substrates to modulate C/N ratio is

necessary.

The wastes rich in proteins have a high nitrogen content and most biomass is

carbon-rich material (Palatsi et al., 2011; Wang et al., 2010). The inhibition by a low

C/N ratio can be potentially alleviated by increasing dose of straw as a carbon source.

Many studies have reported that co-digestion of various biomasses with substrate rich

in proteins increased the C/N ratio and biogas production (Cuetos et al., 2011;

Lehtomäki et al., 2007; Li et al., 2013). Co-digestion of swine manure with rice straw

in semi-batch digesters at C/N ratios of 8 (manure only), 20, or 30 were

studied (Riya et al., 2016) In the process of fermentation, methane production of C/N

8 and C/N 20 decreased, while C/N 30 showed stable CH4 production (Figure 1A).

Meanwhile, there has been a few report about the influence of C/N ratio on microbial

community in AcoD. In dry-thermophilic anaerobic digestion of swine manure, with

or without rice straw for 120 days, there was a large increase in Halothermothrix,

Selenomonas, and Halocella affiliated to Firmicutes without large differences among

C/N ratios. During 120–180 days at C/N ratios of 20 and 30, however,

Halothermothrix and Halocella were reduced, while Moorella, Clostridium, and

Selenomonas were increased. Furthermore, archaeal communities were drastically

altered at different C/N ratios. Methanothermobacter and Methanoculleus were

7
dominant in the inoculum. However, in the fermentation process, Methanosarcina

predominated at C/N 20 for 180 days and at C/N 30 at both 120 and 180 days (Figure

1B). During incubation, the predominant archaea were shifted from

Methanothermobacter and Methanoculleus to Methanosarcina.

According to the analysis of the shifted microbes, Halocella is known to degrade

cellulose and produce acid. Increases in this genus after the beginning of incubation

could be due to feeding with rice straw, which contains cellulose. Halothermothrix is

a halophilic bacterium and is capable of producing hydrogen from glucose (Town et

al., 2014). Moreover, Halothermothrix and Halocella were decreased after 180 days at

both C/N 20 and C/N 30, while Clostridium and Selenomonas were increased. Thus,

we can conclude that bacterial communities would be altered from halophilic

communities to non-halophilic ones at C/N 20 and C/N 30 during the fermentation

process. Archaeal communities would be influenced by changes in bacterial

populations (Ros et al., 2013; Turker et al., 2016). Methanothermobacter and

Methanoculleus are known hydrogenotrophic methanogens, whereas Methanosarcina

is an acetoclastic methanogen. A large decrease in Halothermothrix would partially

explain the decrease in Methanothermobacter because the latter produces hydrogen. It

also effects decreases in Thermotogae, which degrade acetate syntrophically with

hydrogenotrophic methanogens. In contrast, increases in Methanosarcina could be

related to increases in bacteria capable of producing acetate, such as Moorella, which

produces acetate from hydrogen and carbon dioxide.

8
 Animal manure is a suitable co-substrate with organic wastes are rich in carbon

to improve the C/N ratio and to increase methane yield (Shi et al., 2014; Wang et al.,

2012; Xavier et al., 2015). Methane production can be efficiently increased by using

crop residues and pig manure (Zhou et al., 2016). In C/N ratios of 20 and 30,

respectively, methane production was more high and stable, while in C/N ratio of 10

varied significantly. The C/N ratio had a main influence on the bacterial community

structure (Ren et al., 2014; Resende et al., 2016). From the result of the sequences, the

main bacteria were all affiliated to the phylum Firmicutes in the reactors fed with pig

manure alone and in the inoculum. Members of the Firmicutes have been reported as

the dominant community in digesters. Moreover, most of the data in Firmicutes were

attributed to the class Clostridia and only a few of them were affiliated with the class

Bacilli. In the co-digestion of pig manure and rice straw, the diversity of microbes at

the taxonomical level was greater. Members of the phyla Proteobacteria and

Thermotogae were also found. Species of this class Clostridia were still predominant.

In addition, among the samples based on the mcrA/mrtA-based methanogenic

community structures, the predominance of M.wolfeii existed in all samples. In high

C/N ratio sample, there are another two groups, including M.thermophile and

M.tenebrarum, demonstrated that less inhibition by ammonia would enhance the

diversity of methanogens. Both the bacterial and archaeal community structure were

affected by the co-digestion of substrates as a response to nutritional balance (Jimenez

et al., 2016). The addition of clay can enhance methane yield because it counteracts

the high ammonium and nitrogen concentrations present in swine manure. The

9
phylum Firmicutes and family Methanosarcinaceae, specifically Methanosarcina

species, dominate swine manure anaerobic digestion. However, when rice straw is

used as a co-substrate in co-digestion, the Bacteroidetes phylum becomes dominant.

This allows particular physicochemical conditions that trigger a switch in the

Methanosarcinaceae family, where Methanosaeta species become dominant.

Switchgrass is a common energy source. It has lower nitrogen content and higher

carbon content (Zheng et al., 2015). However, being nitrogen-deficient, it has inherent

limitations to using switchgrass as a substrate in AD (Wan et al., 2013). In order to

keep nutritional balance, a mixture of switchgrass and dairy manure will be a better

feedstock for AD than either single substrate (Astals et al., 2015; Fitamo et al., 2017).

Manures are nitrogen-rich materials. Comparing to the digestion of manure alone, the

addition of a 20% wet mass ratio of switchgrass to the digester of manure resulted in

an 86% increase in biogas production (Frigon et al., 2012). In the study of Zheng et al.,

the highest cumulative biogas production was obtained with a 2:2 co-substrates

ratio (switchgrass to manure) and it was higher than that produced with switchgrass or

manure alone (Zheng et al., 2015). All of the mixtures in co-digestion presented a

higher methane production than the calculated yield, which indicated a positive effect

of co-digesting of dairy manure and switchgrass. A 20% increase by co-digesting

combined dairy manure and wheat straw at 34:1 of VS ratio, similarly by combining

airy manure, chicken manure with wheat straw at a VS ratio of 2.7: 2.7:1 to increase

59% of co-digestion (Wang et al., 2012). In molasses alcohol wastewater (MAW),

C/N ratio is as high as approximately 120:1. A disproportionate ratio of carbon,

10
including some non-usable carbon to nitrogen can cause acidification during

fermentation. The C/N ratio in pig manure (PW) is about 13:1. This relatively low

content of carbon hinders continuous biogas production in the fermentation of pure

PM (Shen et al., 2014). Co-digestion of PM and MAW was performed so that

imbalances in the C/N ratio of either PM or MAW alone during the fermentation

could be modulated. The duration of biogas was comparably longer in the reactors

that contained both PM and MAW.

There are about 50 kinds of bacteria, such as Clostridium, Bacteroides,

Bifidobacterium, Butyrivibrio, Proteobacteria, Pseudomonas, Bacillus, Streptococcus,

Eubacterium and so on in the hydrolysis and acidogenesis process. In the start-up

stage, Bacteroidetes and Firmicutes, include most of the species. In acidogenesis,

Bacteroidetes, Chloroflexi, Firmicutes, and Proteobacteria are the predominant

phyla (Wang et al., 2017). It was indicated that the substrate and mixing ratio resulted

in different C/N ratios. The balance of the C/N ratio had a main influence on the

bacterial composition. Sources of the methanogenic pathway would be different

between digestion of manure alone and co-digestion of manure and rice straw.

Therefore, the C/N ratio affects not only the population structures of bacteria and

archaea but also the microbial community functions and biogas production (Zhang et

al., 2016). Better reactor performance could be due to the predominance of Firmicutes

and greater bacterial diversity. Firmicutes and Chloroflexi have been reported as being

able to degrade a large number of organic compounds under a variety of conditions.

These two groups have been found in a wide range of AcoD. These bacterial phyla

11
had a tolerance on high organic loading. Bacteria are responsible for the degradation

for co-substrates to intermediate metabolites which can be later utilized by

methanogens. Acid-producing bacteria, such as Actinobacter, increased dramatically

at deteriorative phase. A sharp increase of Tenericutes may be the failure indicator in

AD. Clostridia, known as hydrogen producers, the dramatic proliferation of

Clostridia suggested that excessive hydrogen was generated.

The change of C/N ratio of co-substrates led to a shift in bacterial community

and the archaeal community can be influenced by changes in bacterial community.

The interaction between bacteria and archaea is influenced by C/N ratio in

co-substrates. Therefore, source of the methanogenic pathway are different between

mono-digestion and co-digestion. There are mainly three types of methanogens,

namely acetoclastic, hydrogenotrophic, and methylotrophic. Most of the CH 4 is

produced by the first two types (Shin et al., 2010). Methanobacterium,

Methanothermobacter, Methanospirillum, Methanobrevibacter and Methanoculleus

are the most commonly identified hydrogenotrophic methanogens (Collins et al., 2003;

Ziganshin et al., 2013). Acetoclastic methanogens are belonging to Methanosaeta and

Methanosarcina. Between mono-digestion and co-digestion, the source of the

methanogenic pathway would be different (Wang et al., 2017). Methanogens

compositions have no notable changes between stable and deteriorative phases. But

the distinct difference was observed in the relative abundance of each genus.

12
 The modulation of the C/N ratio in co-digestion influence the microbial

community and improve the biogas yield. Meanwhile, the shifts in microbial

community enhance various waste utilization and recycling.

2.2. Co-substrates adjust pH and influence microbial community

The original pH of different substrates has a wide range (Table 2), but the

optimal pH range for AD is 6.0 - 8.0 (Shi et al., 2014). Whether the pH is too high or

low, the growth and activities of microbes are inhibited (Franke-Whittle et al., 2014;

Gomez-Romero et al., 2014). The natural pH for methanogens is about 7.0 and

5.5-6.5 for acidogens (Sawatdeenarunat et al., 2015). Co-digestion of straw and

manures not only keep the balance of nutrition for microorganisms, but also adjust pH

in the digestion by improving the buffer capacity (Leitao et al., 2006). The pH

variation was only affected by an OTU assigned to Clostridium, whereby an increase

in abundance related to an increase in pH, and by some other non-abundant

Firmicutes OTUs (Zhang et al., 2016).

The failure of fermentation can be caused by acidification and methanation

imbalance (Prochazka et al., 2012; Zhang et al., 2011). An insufficient buffering

capacity leads to a dramatic drop in pH. The change of pH had inhibition to the

functions of microorganisms, especially methanogenic archaea, which finally disrupts

the production of biogas, causing a decrease in biogas production. Therefore, pH and

buffering capacity are common indicators to control the process of digestion (Murto et

al., 2004; Wan et al., 2013; Zhang et al., 2013). In AcoD of pig manure and food

waste, the methane yield did not drop extensively despite elevated VFA
13
concentrations; this is due to the high pH and extensive buffering capacity (Dennehy

et al., 2017). Fermentation with manure and switchgrass (2:2 ratio) obtained a higher

methane than others (Figure 2A) (Zheng et al., 2015). 2:2 ratio digesters had a higher

resistance to pH fluctuations (Figure 2B). Chloroflexi clones were not detected in

digesters with ratios of 1:3 and 0:4, because of varied pH conditions (Figure 2C).

In most digestion processes, hydrolysis is considered to be the rate limiting phase.

However, in the AcoD of biodegradable substances, the rate is limited by

metanogenesis, rather than by hydrolysis. This is due to the rapid acidification of

these wastes to volatile fatty acids (VFAs) resulting in a rapid decrease in process pH

and subsequent inhibition of necessary methanogens activity (Ren et al., 2014). It was

mainly due to the acid resistance of some Firmicutes species, which can grow at low

pH. Firmicutes continued to be active in digestion when the pH restored.

Anaerophaera and Aminomonas played an important role in amino-acid-utilizing with

ammonia, acetate and propionate as end-products, which maintained a neutral pH of

AcoD. Chloroflexi plays an important role in AD and can degrade both

monosaccharaides and polysaccharides, as well as generate acetic acid (Riviere et al.,

2009). Some species belonging to Chloroflexi are associated with hydrogenotrophilic

methanogens (Yamada et al., 2005). However, Chloroflexi abundance was sensitive to

pH fluctuation. The low pH results in a decrease of the relative abundance of

Chloroflexi. Consequently, a stable pH is important for retaining original Chloroflexi

and enriching of Chloroflexi.

In digestion process, the fermentation bacteria converted the substrates into

14
various acids. In mono-digestion, the buffer capacity was insufficient to prevent the

decline in pH, so the functioning of methanogens was inhibited by low pH conditions.

Consequently, methane production in these digesters was suspended due to an

imbalance of acidification and methanation. Co-digestion with buffering materials can

adjust pH and reduce the inhibition of acidification to methanogens. Thus, the

methane productivity and waste recycling efficiency were improved.

2.3. Co-substrates dilute toxic substances and influence microbial community

2.3.1. Dilution of Ammonia

Protein-rich substrates contribute to nearly most of the nitrogen, which enters the

solution as NH3 or the NH4+ ions (Fountoulakis et al., 2010; Hagen et al., 2014). The

appropriate concentration of ammonia ensures sufficient buffer capacity and improve

the stability of AD. However, high ammonia causes digester failure. Degradation of

chicken manure observed threshold level of unionized NH3 that is toxic to

methanogenesis ranged above 250 mg/L (Bujoczek et al., 2000). The reactor fed with

cattle and chicken manures showed the highest yield of ammonia (up to 5.93 g/L

ammonia), which come from chicken manure. The diffusion of NH 3 across cell

membranes inhibit biogas production by the loss of cellular potassium,

de-potentiation of the cell membrane, and accumulation in the cytoplasm (FitzGerald

et al., 2015). Increasing the organic loading rate resulted in an increase of VFA and a

drop in CH4 yield. This indicated strong inhibition of acetogenesis and

methanogenesis (Ziganshin et al., 2013).

15
 VFA/TA ratio lower than 0.35 showed the stable operation of AD. Total ammonia

concentration (TAN) and the free ammonia concentration (FAN), are the end-products

of digestion of nucleic acids, proteins and urea. The levels of TAN and FAN increased

significantly during the AD process, reaching values lower than 5000 mg/L and 280

mg/L, respectively. They are potential inhibitory chemicals to AD, especially to the

activity of microorganisms (Ros et al., 2017). FAN concentration higher than 600 mg

L-1 results in inhibition to the activity of methanogens (Dai et al., 2013; Rajagopal et

al., 2013). According to the Kayhan et al., FAN inhibited the propionate degrading

acetogenic bacteria and caused increasing of propionate concentration in these

digesters (Kayhanian, 1999). For TAN, values ranging from 4100 to 5700 mg L -1

cause toxicity or complete inhibition to methanogenic activity (Prochazka et al.,

2012).

Similar results were reported in a pig manure anaerobic digester. When PS was at

the highest percentage, Firmicutes increased principally due to Clostridium and

Turicibacter enrichment. Clostridia are efficient hydrogen producers, e.g. Clostridium

degrades organic acids in a syntrophic association with hydrogenotrophic

methanogens (Kim et al., 2014). Turicibacter is able to degrade carbohydrates in gut

microbiota. Accumulations of ammonia had inhibition to acetoclastic methanogens.

However, in the early stages a notable decrease in Methanomicrobiales and

Methanosarcinales was observed, while Methanobacteriales was enriched. This

indicated that the digester trended from a common activity of hydrogenotrophic and

aceticlastic methanogens to principally hydrogenotrophic methanogens. With PS

16
increasing, Methanosaeta reduced; this genus uses acetate as a sole substrate for

energy consumption at low levels. Because of 5–10 times higher affinity for substrate,

Methanosaeta holds a competitive advantage over Methanosarcina spp. When acetate

is limiting, Methanosaeta might be expected to out-compete Methanosarcina.

Comparing to Methanosaeta spp., Methanosarcina spp. are able to generate

methane by both acetoclastic and the hydrogenotroph methanogenesis pathways. It

makes them more tolerant to inhibitors of the acetoclastic pathway (Yi et al., 2014).

Therefore, co-digester dominantly based on Methanosarcina spp. could achieve stable

methanogenesis, as their special morphological characteristics and flexibility in

methabolism. In addition, higher concentrations of VFA and higher OLR induced the

selective proliferation of Methanosarcina (Li et al., 2014). According to previous

studies, ammonia may affect archaea of methanogenesis in two ways. Ammonium ion

may inhibit enzymes mediated methane producing directly. In addition, ammonia

molecule may diffuse into cells and cause proton imbalance or potassium

deficiency (Rajagopal et al., 2013). Therefore, among many ammonia inhibition

control strategies, the optimization of the feedstock for co-digestion is possibly the

easiest and cost-effective technique to reduce the ammonia toxicity.

2.3.2. Dilution of VFAs, FAs, and FVFAs

When FW is used as alone substrate, especially at high OLR rate of

hydrolysis/acidogenesis, the high concentration of VFA accumulated from

hydrolysis/acidogenesis can eventually lead to an irreversible acidification.

Co-digestion food waste with activated sludge may be an alternative. In the

17
mono-digestion of chicken manure, AD systems are easy to suffer from ammonia

toxicity. However, co-digestion of chicken manure and corn stover was operated with

stable performance without VFA accumulation (Li et al., 2014). In the study by

Molinuevo-Salces et al., the addition of vegetable processing waste as co-substrate in

swine manure digestion increase the volatile removal (Molinuevo-Salces et al., 2010).

Considering VFAs, a significant decrease in the abundance of these acids,

particularly propionate, was related to an increase in Syntrophomonas.

Syntrophomonas is known to beta-oxidized saturated fatty acids to acetate or acetate

and propionate (Park & Li, 2012; Riggio et al., 2015). Propionate significantly

decreased following an increase in OTUs belonging to the order of the class

Clostridia, which are known to be involved in syntrophic acetate oxidation activities.

Acetate concentration has a crucial impact on acetotrophic methanogens.

Methanosaeta can only use acetate for CH4 production, but its growth is favored by

low concentrations of acetate. Acetate also significantly increased when

Methanosarcina increased and seemed also to be significantly associated with the

phylum Firmicutes and with Methanosarcina, OTUs positively correlated with

acetoclastic methanogens, because acetate is a substrate for Methanosarcina

metabolism. On the other hand, acetate variation was not related to variation in the

hydrogenotrophic methanogen Methanothermobacter thermautotrophicus, since

methane is mainly produced via syntrophic acetate oxidation association followed by

hydrogenotrophic methanogenesis. Butyrate resulted in significant changes in the

abundance of Syntrophomonas and Anaerobaculum, a genus able to reduce substrates

18
to butyrate with glucose as an electron donor. Sporanaerobacter and Proteiniphilum

were capable of acetogenesis, which accelerated the transfer of VFA to acetate.

Methanosarcina, another acetoclastic methanogenesis, is dominant genus instead of

Methanosaeta at high VFA concentrations.

Due to rapid accumulation of VFAs, AD of food waste usually results in process

inhibition. However, the inhibition can be alleviated by co-digestion with sewage

sludge. Microbial communities played an important role in this process. Firmicutes,

Proteobacteria, Bacteroidetes and Actinobacteria were found as predominant phyla in

the bacterial community. In acidification phase, Firmicutes increased significantly.

After that, the abundance of Firmicutes increased much further on day 12 for

MW-FW and changed little to the end. Bacteroidetes also increased for MW-SS and

MW-FW. Proteobacteria and Actinobacteria decreased notably for MW-SS and

MW-FW. The great changes of these four dominant phyla all appeared on day 5,

which could be an indicator at acidification phase corresponding to VFAs

accumulation. According to the genus analysis, Proteinniborus and Parabacteroides

both belonging to phylum Bacteroidetes were dominant genera for the VFAs

production. Proteiniborus is a kind of protein-specific utilizing bacteria. Its

metabolites mainly include hydrogen, acetic acid, ethanol, carbon dioxide and a trace

amount of propionic acid. Aminobacterium is also a kind of protein-degrading

bacteria producing acetate, propionate and succinate. Its abundance is higher in the

digesters rich in proteins. Parabacteroides plays important roles in the degradation of

polysaccharides. Acetate and succinate are its primary fermentation end products.

19
Petrimonas and Bacteroides are acidogens which abundance could indicate the VFAs

accumulation. In days 0-5 (acidification phase), Methanosaeta, acetoclastic

methanogen, is the dominant genus. However, its abundance decreased significantly

in days 5-33 (active methane production phase) (Wang et al., 2017). Methanosarcina

increased significantly in the methanogenesis. Many studies indicated that

Methanosaeta and Methanosarcina were competitive genera in AD. Methanosarcina

is more tolerant to the inhibitors than Methanosaeta. Methanoculleus,

hydrogenotrophic methanogens, became dominant at the end of digesting process due

to the exhaust of acetic acid.

FA and FVFA are also inhibitors, which can freely diffuse into the cell and cause

potassium deficiency and proton imbalance (Alvarez et al., 2010; Koch et al., 2015).

In AD, acidogens and methanogens can be inhibited by both FA and FVFA;

methanogens are more sensitive than acidogens. The resistance of hydrogen

methanogens to ammonia inhibition is higher than acetoclastic methanogenesis. In

fact, the low methane yield of digesters was due to the slow increase in methanogens.

The FA in manure digesters was the highest, while the FVFA in straw digesters was

the highest. Thus, FA can easily inhibit the digestion of manure and the FVFA may

easily inhibit the digestion of straw.

2.3.3. Dilution of Long chain fatty acids (LCFAs)

LCFAs are the primary component of fat, oil, and grease (FOG). LCFAs are

degraded to acetate and H2 via a beta-oxidation pathway, which is subsequently

converted to methane. The methanogens can be suppressed by high LCFA levels. A

20
key inhibitory mechanism of LCFAs is attributed to negative effects of microbial cell

membranes, including enzyme activity inhibition, electron transport chain disruption

and cell lysis. The other inhibition mechanism is due to LCFA adsorption on the

surface of microbial cell membranes and leads to mass transfer limitation. Previous

studies revealed that the acetoclastic population is more sensitive to the LCFA

inhibition than the acidogenic population (Park & Li, 2012). Likewise, the detrimental

effect of LCFA on the archaea of methanogenesis may have four reasons: sludge

flotation and washout, transport limitation from bacteria being coated in a layer of

LCFAs, releasing biogas by LCFA, or LCFA toxicity on methanogens.

2.3.4. Dilution of Others: phenol, sulfate, heavy metals

Besides ammonia and VFAs, other toxic substances exist in AD, such as phenol,

sulfate and heavy metals. Co-digestion can dilute these toxic effects on microbes. The

high toxicity of olive mill waste-water (OMW) is entirely ascribable to

phenols (Gannoun et al., 2016). Abattoir wastewater (AW) contains a high

concentration of biodegradable organics and were mostly in the form of fats and

proteins, adequate nitrogen, sufficient alkalinity, and micronutrients for bacterial

growth. Due to the accumulation of recalcitrant products such as phenols, major

species disappeared or decreased and inhibit the activities of microorganisms,

particularly methanogens. Co-digestion of OMW with AW can dilute phenols. The

AcoD of OMW/AW (40%/60%) enhanced the biogas production and it could be

attributed to the decrease of polyphenol toxicity under thermophilic conditions.

21
 Regarding heavy metals, there are high concentrations of Cu, Ni and Zn in pig

manure, cattle manure and poultry manure. Moreover, cations (e.g. Na+, K+, Ca2+,

Mg2+) influence methane production in a charge-dependent manner. These cations

were possibly inhibiting a Na+ export channel, which is necessary for the final

methane production (FitzGerald et al., 2015).

In addition, alcohol wastewater (MAW) is rich in sulfate, which can reduce

bacteria competing with methanogens during AD (Shen et al., 2014). Compared to

fermentation of MAW alone, by adding PM to MAW, the increased nitrogen content

increased the buffering capacity of the system against H2S toxicity.

2.4. Co-substrates adjust moisture and microbial community

Moisture is an important parameter in AD (Abbassi-Guendouz et al., 2012).

Using straw as a single substrate showed only 77.6 mL methane yield. However,

when added cabbage waste (CW), it showed 120 mL methane yield. CW consists of

approximately 90% water and co-digestion with CW increase the water content of the

digester. According to the analysis of microbial community, Bacteroidetes and

Firmicutes were enriched after CW addition, whereas the richness of Proteobacteria

decreased (Wu et al., 2016). The bacteria from Firmicutes phylum are able to

metabolize various substrates including sugars and lignocellulose, which is the main

components of CW. Therefore, the addition of CW increase the moisture content in

the digester, enrichment of Firmicutes, increase biogas yield and waste utilization

efficiency.

22
 Furthermore, Yao et al. reported that the benefits of water-free co-digestion of

vegetable processing wastes with cattle slurry was obvious in the high capacity of

VPW treatment and biogas yield (Yao et al., 2014). The 42.9% treatment capacity or

production efficiency could be achieved. This experiment was conducted without

water addition and it was superior to solid-state AD from the view of water

conservation. Hence, the economic benefit was significant. However, no microbial

community analysis was conducted in the study.

2.5. Co-substrates supplement trace elements and influence microbial community

Besides micronutrients such as carbon, nitrogen, phosphorus and sulphur,

microbes in AD also require the growth factor of trace elements at relatively lower

concentration. Many researchers have studied the importance of trace elements in

AD (Choong et al., 2016; Sawatdeenarunat et al., 2015). Most of the previous studies

reported that trace elements addition had positive impacts on AD process, including

mainly longer term on digester stability, greater organic matter degradation, low VFA

generation and higher biogas yield (Choong et al., 2016; Rajagopal et al., 2013). The

trace elements must be adequate for supporting the metabolism of microorganisms in

order to maintain the effective digestion process. Otherwise, the performance of

anaerobic digestion will significantly deteriorate.

Instead of dosing the micronutrients into the reactor, the trace elements existing

in the substrates should be well utilized (Demirel & Scherer, 2011). The trace

elements in the co-substrate (swine wastewater) had improved the digesting efficiency

of food waste, without VFA accumulation and with a higher biogas yield and (Agler et
23
al., 2008; Zitomer et al., 2008). The concentrations of Fe, Ni, and Mo in food waste

were only about one-tenth of those in the piggery wastewater. AcoD of food waste

with the trace element-rich piggery wastewater resulted in significantly improved

methane production rates without VFA accumulation and process stability, while the

food waste alone in a single stage reactor was not feasible under the conditions

examined, even with pH control (Zhang et al., 2011). It was suggested that trace

element supplemented from co-substrates was a key factor in enhancing co-digestion

performance.

Many studies had observed trace elements are essential to methanogens. Banks et

al. (2012) studied the concentrations of Se and Co (0.16 and 0.22 mg/kg of food waste)

for the stable AD at 5000-6100 mg/L of TAN concentrations was 5000 mg/L, but in

control reactors without these elements, the concentrations of TAN was increased up

to 6100 mg/L. Particularly selenium (Se), molybdenum (Mo) and tungsten (W) have

been reported as important trace elements required by methanogens for the oxidation

of formate by enzyme formate dehydrogenase (Banks et al., 2012). In addition, trace

elements can improve the growth and metabolic activities of microbes (Appels et al.,

2008; Zhang et al., 2011). Addition of night soil waste into a mixture of MSW and

FVW enhanced the numbers of Anaerovorax (Clostridiales order), diversifying the

microbial consortia. The content of these elements in methanogens can be

summarized in the order of Fe >> Zn > Ni > Cu ≈ Co ≈ Mo > Mn (Demirel & Scherer,

2011). It clearly indicated that trace elements indeed play essential roles as the

methanogens building elements and growth factor. Besides the role as a growth factor,

24
Fe was discovered to be also important in stimulating the formation of cytochromes

and ferroxins (Fd) which are vital for cellular energy metabolism.

3. Future prospects:

Despite the advantage of the co-digestion process, there is a great challenge in

AcoD. The co-digestion may lead not only to positive synergistic effects, but also to

antagonistic interactions resulting in lower biogas productivity. Therefore, besides

substrate nutrition, the optimization of temperature, OLR, HRT and other factors that

affect the co-digestion performance is necessary. Establishing optimal physical and

chemical conditions in an AcoD may enrich functional microorganisms and construct

the optimal microbial community. Further research, using advanced sequencing

techniques, is needed to classify in detail any unknown microbes and understand their

specific roles in the complex anaerobic degradation process. More studies are needed

to link microbial community descriptors to AcoD models for the simulation, control,

and optimization of digesters.

Moreover, bioaugmentation concepts can be developed aiming at the application

of such pre-adapted consortia to treat more waste which is difficult to degrade or toxic

to the environment. Exploring the community response to the addition of

co-substrates can offer an engineered community structure through altering

co-substrates and their composition for a better waste management and recycling.

Furthermore, in AcoD, numerous studies concerned with the structure and succession

of microbial communities were in laboratory-scale, the researches in full-scale

reactors are scarce. Thus, further studies need more research in full-scale so that the
25
information of the microbial community can be a good indicator in the application of

AcoD.

4. Conclusion:

Because of the better substrate nutrient balance than mono-digestion,

co-digestion has higher ability to modulate C/N ratio, improve buffer capacity, dilute

toxic substances, increase biodegradation, adjust moisture and supplement trace

elements. These nutritional changes result in microbial community structure shifting

and improve the synergistic effects of fermentative bacteria and Methanoarchaea.

Thus, the positive interactions between co-substrates and microbial community

structures and functions promote the biogas production in AD.

Acknowledgements:

Our work was financially supported by MOST international cooperation

grant (2014DFA91340); National Natural Science Foundation of China

grant (31470224);Gansu provincial international cooperation grant 1604WKCA013;

Gansu provincial international cooperation grant 1504WKCA089-2.

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Figure Captions:

Figure 1. The effects of different C/N ratios on (A) methane production and (B)

microbial relative abundance in the digestions of swine manure and rice straw.

Figure 2. The co-digestion of switchgrass and dairy manure improve the (A) methane

yield than mono-digestion of each substrate (B) buffer capacity than mono-digestion

of switchgrass. (C) The shifts of microbial community in co-digestion of mixed

substrates.

35
Fig.1

15×11.42 cm (1200×1200DPI)

36
Fig.2

20×9.33 cm (1200×1200DPI)

37
Table 1 The original C/N ratios of different substrates
Substrates C/N ratio References

dairy manure 2.3 (Zheng et al., 2015)

concentrated effluent 3.0-6.6 (Brown & Li, 2013; Shi et al., 2013)

sewage sludge 6.76-10 (Silvestre et al., 2014; Zhang et al., 2014)

wastewater sludge 11.72 (Alagoz et al., 2015)

chicken manure 10.1 (Li et al., 2014)

pig manure 7.4-12.96 (Ren et al., 2014; Zhou et al., 2016)

cow manure 5.2-16.75 (Wang et al., 2016; Zhang et al., 2013)

soybean processing waste 12.5 (Zhu et al., 2014)

fruit & vegetable waste 18.88 (Ros et al., 2017)

food waste 11.5-34 (Capson-Tojo et al., 2017; Gou et al., 2014)

grease waste 39 (Silvestre et al., 2014)

switchgrass 32.6 (Zheng et al., 2015)

olive pomace 36.61 (Alagoz et al., 2015; Riggio et al., 2015)

Green waste 41.2 (Chen et al., 2014)

yard waste 55.3 (Brown & Li, 2013)

Hay 76 (Zhu et al., 2014)

silver grass 81.67 (Wan et al., 2013)

corn straw 43.3-52.41 (Wang et al., 2016)

corn stover 63.2-79.7 (Li et al., 2014; Yong et al., 2015)

rice straw 125 (Zhou et al., 2016)

cassava dregs 152 (Ren et al., 2014)

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Table 2. The original pH of different substrates

Substrates pH References

food waste 4.1-5.6 (Capson-Tojo et al., 2017; Gou et al., 2014)

apple pulp 4.2 (Riggio et al., 2015)

silver grass 5.57 (Wan et al., 2013)

olive pomace 6.1 (Alagoz et al., 2015)

effluent 6.6-7.8 (Brown & Li, 2013; Shi et al., 2013)

Feedstock 6.6 (Agyeman & Tao, 2014)

Hay 6.8 (Zhu et al., 2014)

Cow slurry 6.6-6.94 (Wang et al., 2016; Zhang et al., 2013)

Cardboard 7.1 (Capson-Tojo et al., 2017)

Cassava dregs 7.35 (Ren et al., 2014)

Sewage sludge 7.46 (Silvestre et al., 2014; Zhang et al., 2014)

Pig manure 7.08-8.41 (Ren et al., 2014; Zhang et al., 2011)

Cattle manure 8.3-9.2 (Zhang et al., 2013; Zheng et al., 2015)

Soybean processing waste 8.8 (Zhu et al., 2014)

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Highlights:

1. Co-digestion with complementary substrates has more balanced nutrient.

2. Balanced nutrient of co-digestion causes positive shifts of microbial

community.

3. The positive shifts of microbial community improve AcoD efficiency.

40