**Abstract**

In the past decades, biotechnologies for reutilizing the biomass harvested from the metal-contaminated land draw attention to many scientists. Among those technologies, anaerobic fermentation is proven as an efficient conversion process for biowaste reduction with simultaneous recovery of biogas as an energy source. During the process of anaerobic fermentation, the release of metals from the biomass will impact the growth and performance of microorganisms in reactors, which then results the variation of substrate degradation. In this chapter, the impact of metals on the degradation of substrate at different stages of fermentation process, as indicated by variations of lignocelluloses, chemical oxygen demands (COD), volatile fatty acids (VFAs), etc., will be summarized. The objective is to rationalize the relationship between metal presence and substrate degradability and give suggestions for future research on metal-contaminated biomass reutilization.

**Keywords:** anaerobic fermentation, heavy metal, biodegradation, lignocelluloses, chemical oxygen demands, volatile fatty acid

## **1. Introduction**

The rapid development of industries such as electronic, mining, agrochemical, tannery, and battery industries has led to an increase in the direct and indirect discharge of metals into the environment. Some metals are potentially toxic, and unlike some organic contaminants, they are not biodegradable; thus, they may accumulate in terrestrial and aquatic organisms [1]. Hence, removal of potentially toxic metals (PTM) from contaminated environments has become an issue of urgent concern. In recent years, phytoremediation, which is defined as the use of plants to remove contaminants from contaminated environment, has drawn great attention probably because it is cost-effective and sustainable [2, 3]. However, disposing the biomass residues following phytoremediation is challenging [4–6]. Therefore, there has been a growing interest on the development of inexpensive disposal techniques and improvements in bio-resource utilization to foster sustainability in remediation systems [7].

Anaerobic fermentation is a relatively efficient conversion process for biomass waste reduction with simultaneous recovery of biogas as an energy source [8–11]. In an anaerobic reactor, there are four processes that occur simultaneously, i.e., hydrolysis, acidogenesis, acetogenesis, and methanogenesis [12]. Hydrolysis

process involves the conversion of macromolecules such as proteins, polysaccharides, and fats that compose the cellular mass of the excess sludge into watersoluble molecules with a relatively small molecule (e.g., peptides, saccharides, and fatty acids) [12]. Simple molecules with a low molecular weight such as volatile fatty acids (e.g., acetic, propionic, and butyric acid), alcohols, aldehydes, and gases like CO2, H2, and NH3 are produced via acidification of the hydrolyzed products (acidogenesis) [12]. The acidification products are converted into acetic acids, H2 and CO2, by acetogenic bacteria in a process called acetogenesis. These first three steps of anaerobic digestion are often called acid fermentation, and they help transform the waste biomass into substrates for methanogenesis [12]. In the methanogenesis process, the products of the acid fermentation (mainly acetic acid) are converted into CO2 and CH4.

Microorganisms responsible for anaerobic fermentation require a trace amount of metals (e.g., Ni, Co, Cu, Fe, Zn, etc.) for their optimum growth and performance [13]. Various enzymes involved in anaerobic metabolism use trace metals as their cofactors. For example, methanogenic enzymes such as CO dehydrogenase (CODH) and methyl-H4MPT:HS-CoM methyltransferase use cobalt acts as their cofactor [14].

However, waste biomass often contains varying amounts of metals depending on the source of the biomass. During the process of anaerobic fermentation, the release of metals from the biomass will influence the efficiency of fermentation by affecting the enzyme activity, microorganism community, and even degradation and metabolic pathways [15]. In this chapter, the impact of metals on the degradation of substrate, as indicated by variations of lignocelluloses, chemical oxygen demands, volatile fatty acids, etc., will be summarized. The objective is to rationalize the relationship between metal presence and substrate degradability during different fermentation stages and give suggestions for future research on metalcontaminated biomass reutilization.

### **2. Hydrolysis stage**

Hydrolysis is oftentimes the rate-limiting step in the anaerobic digestion process probably because fermentative bacteria require an additional step of excreting extracellular enzymes, such as cellulases and lipases, to carry out the hydrolysis or solubilization process [16, 17]. It can be accelerated by enhancing the accessibility of anaerobic microorganisms to intracellular matter or cellulose using thermal, chemical, biological, and mechanical processes, as well as their combinations [18].

#### **2.1 Lignocellulose degradation**

Lignocelluloses are mainly composed of cellulose, hemicellulose, and lignin [19]. The cellulose and hemicellulose themselves are relatively easy to be broken down by microorganisms; however, their biodegradability decreases when they occur in lignocellulose complexes [20]. The impacts of metals on lignocellulose degradation vary with the metal species, concentrations, and fermentation conditions.

Previous studies showed that the presence of metals at certain concentrations may enhance the degradation of lignocelluloses [21, 22]. In one study, an average lignocellulose content of 87.49 ± 3.19%TS was obtained in a control group but decreased to 80.44 ± 3.41%TS, 77.94 ± 3.50%TS, and 79.45 ± 2.88%TS following the addition of 30, 100, and 500 mg/L Cu and 79.36 ± 3.72%TS, 79.10 ± 2.80%TS, and 76.60 ± 2.97%TS following the addition of 30, 100, and 500 mg/L Cr, respectively. Thus, Cu and Cr addition significantly enhanced the degradation of lignocellulose [21, 22].

**133**

tion states of Ni are Ni+

activities [21].

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

Several studies on methanogenic bacteria found Cu, suggesting it could be a critical component for the enzymes super dismutase and hydrogenase [23]. However, at relatively high concentrations, Cu can inhibit anaerobic fermentation, which results in reduction of degradation efficiency. Cu changes the physiological steady state of the fermentation process by inhibiting the degradation of the substrate and the growth of the microbes [8, 24]. In contrast, Cu has been shown to enhance the biogas production via fermentation [9, 25]. Despite the inhibitory effects of Cu, biogas production was probably enhanced by the addition of sulfide to the digester in stoichiometrically equivalent amounts [26]. Our research suggested that the promoting effect of Cu addition on biogas yields was mainly attributable to better process stability, the enhanced degradation of lignin and hemicellulose, the transformation of intermediates into VFA, and the generation of CH4 from VFA [22]. Cr is one of the heavy metals that have often been blamed for unsatisfactory operation or failure of anaerobic digesters [27]. Contradictory toxicity levels of Cr on anaerobic fermentation have been cited in literatures [28, 29]. This is probably because of differences in availability of Cr in fermenters (which is influenced by the precipitation and adsorption of soluble metals), differences in materials used in the studies [30], and dissimilar operational conditions (e.g., temperature, pH, hydraulic retention time, solid retention time, and mixed liquor volatile suspended solids) [27]. Cr in certain concentrations was found to promote the efficient generation of CH4 by inducing better process stability, enhancing degradation of lignin and hemicellulose, transforming intermediates into VFA, and increasing coenzyme F420

According to our recent study, when compound metals were added into the fermentation reactors, the degradation of lignocelluloses performed differently (**Table 1**). It was found that the addition of Zn into the Cd- or Cu-containing reactors enhanced the degradation of lignin and cellulose significantly which resulted in a significant decrease in the total lignocellulose contents. The addition of Fe together with Cd reduced the cellulose contents and the total lignocellulose contents. In contrast, the addition of Ni into either Cd- or Cu-containing reactors did

Depending on the methanogenic pathway, the general trends of metal requirements are as follows: Fe is the most abundant metal, followed by Ni and Co and smaller amounts of Mo (and/or W) and Zn [31]. Almost all metalloenzymes involved in the pathway of biogas production contain multiple Fe2S2, Fe3S4, or Fe4S4 clusters [17, 31, 32]. Fe is primarily present as Fe-S clusters used for electron transport and/or catalysis, as well as attenuating disturbances associated with the presence of sulfide which often results in a more stable process [31, 33–35]. Zn, like Cu, is present in relatively large concentrations in many methanogens. Zn is important in anaerobic fermentation because it is required by enzymes involved in methanogenesis such as coenzyme M methyltransferase [36]. At certain concentrations, Zn can promote biogas production [37, 38]. For example, during the swine manure anaerobic digestion, Zn concentrations in the range of 125–1250 mg/L

Ni is an important trace element for many prokaryotic microorganisms that are in the *Bacteria* and Archaea domains [40]. It is required in the prosthetic groups of a total of eight enzymes that are found in prokaryotic microorganisms, including CODH, acetyl-CoA synthase/decarbonylase, methyl-coenzyme M reductase (MCR), [NiFe]-hydrogenases, superoxide dismutase (Ni-SOD), glyoxylase I, urease, and acireductone dioxygenase [41]. Generally, the biologically relevant oxida-

protein. Ni usually functions either as a redox catalyst, for example, as in the case of hydrogenase or CODH where Ni is liganded by cysteinyl sulfurs [40]. However, Ni

, Ni2+, and Ni3+, and these depend on how Ni is ligated to the

not improve the degradability of the feedstocks.

improved significantly microbial activity [39].

*DOI: http://dx.doi.org/10.5772/intechopen.87161*

#### *Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals DOI: http://dx.doi.org/10.5772/intechopen.87161*

Several studies on methanogenic bacteria found Cu, suggesting it could be a critical component for the enzymes super dismutase and hydrogenase [23]. However, at relatively high concentrations, Cu can inhibit anaerobic fermentation, which results in reduction of degradation efficiency. Cu changes the physiological steady state of the fermentation process by inhibiting the degradation of the substrate and the growth of the microbes [8, 24]. In contrast, Cu has been shown to enhance the biogas production via fermentation [9, 25]. Despite the inhibitory effects of Cu, biogas production was probably enhanced by the addition of sulfide to the digester in stoichiometrically equivalent amounts [26]. Our research suggested that the promoting effect of Cu addition on biogas yields was mainly attributable to better process stability, the enhanced degradation of lignin and hemicellulose, the transformation of intermediates into VFA, and the generation of CH4 from VFA [22].

Cr is one of the heavy metals that have often been blamed for unsatisfactory operation or failure of anaerobic digesters [27]. Contradictory toxicity levels of Cr on anaerobic fermentation have been cited in literatures [28, 29]. This is probably because of differences in availability of Cr in fermenters (which is influenced by the precipitation and adsorption of soluble metals), differences in materials used in the studies [30], and dissimilar operational conditions (e.g., temperature, pH, hydraulic retention time, solid retention time, and mixed liquor volatile suspended solids) [27]. Cr in certain concentrations was found to promote the efficient generation of CH4 by inducing better process stability, enhancing degradation of lignin and hemicellulose, transforming intermediates into VFA, and increasing coenzyme F420 activities [21].

According to our recent study, when compound metals were added into the fermentation reactors, the degradation of lignocelluloses performed differently (**Table 1**). It was found that the addition of Zn into the Cd- or Cu-containing reactors enhanced the degradation of lignin and cellulose significantly which resulted in a significant decrease in the total lignocellulose contents. The addition of Fe together with Cd reduced the cellulose contents and the total lignocellulose contents. In contrast, the addition of Ni into either Cd- or Cu-containing reactors did not improve the degradability of the feedstocks.

Depending on the methanogenic pathway, the general trends of metal requirements are as follows: Fe is the most abundant metal, followed by Ni and Co and smaller amounts of Mo (and/or W) and Zn [31]. Almost all metalloenzymes involved in the pathway of biogas production contain multiple Fe2S2, Fe3S4, or Fe4S4 clusters [17, 31, 32]. Fe is primarily present as Fe-S clusters used for electron transport and/or catalysis, as well as attenuating disturbances associated with the presence of sulfide which often results in a more stable process [31, 33–35]. Zn, like Cu, is present in relatively large concentrations in many methanogens. Zn is important in anaerobic fermentation because it is required by enzymes involved in methanogenesis such as coenzyme M methyltransferase [36]. At certain concentrations, Zn can promote biogas production [37, 38]. For example, during the swine manure anaerobic digestion, Zn concentrations in the range of 125–1250 mg/L improved significantly microbial activity [39].

Ni is an important trace element for many prokaryotic microorganisms that are in the *Bacteria* and Archaea domains [40]. It is required in the prosthetic groups of a total of eight enzymes that are found in prokaryotic microorganisms, including CODH, acetyl-CoA synthase/decarbonylase, methyl-coenzyme M reductase (MCR), [NiFe]-hydrogenases, superoxide dismutase (Ni-SOD), glyoxylase I, urease, and acireductone dioxygenase [41]. Generally, the biologically relevant oxidation states of Ni are Ni+ , Ni2+, and Ni3+, and these depend on how Ni is ligated to the protein. Ni usually functions either as a redox catalyst, for example, as in the case of hydrogenase or CODH where Ni is liganded by cysteinyl sulfurs [40]. However, Ni

*New Advances on Fermentation Processes*

acid) are converted into CO2 and CH4.

contaminated biomass reutilization.

**2.1 Lignocellulose degradation**

**2. Hydrolysis stage**

cofactor [14].

process involves the conversion of macromolecules such as proteins, polysaccharides, and fats that compose the cellular mass of the excess sludge into watersoluble molecules with a relatively small molecule (e.g., peptides, saccharides, and fatty acids) [12]. Simple molecules with a low molecular weight such as volatile fatty acids (e.g., acetic, propionic, and butyric acid), alcohols, aldehydes, and gases like CO2, H2, and NH3 are produced via acidification of the hydrolyzed products (acidogenesis) [12]. The acidification products are converted into acetic acids, H2 and CO2, by acetogenic bacteria in a process called acetogenesis. These first three steps of anaerobic digestion are often called acid fermentation, and they help transform the waste biomass into substrates for methanogenesis [12]. In the methanogenesis process, the products of the acid fermentation (mainly acetic

Microorganisms responsible for anaerobic fermentation require a trace amount

However, waste biomass often contains varying amounts of metals depending on the source of the biomass. During the process of anaerobic fermentation, the release of metals from the biomass will influence the efficiency of fermentation by affecting the enzyme activity, microorganism community, and even degradation and metabolic pathways [15]. In this chapter, the impact of metals on the degradation of substrate, as indicated by variations of lignocelluloses, chemical oxygen demands, volatile fatty acids, etc., will be summarized. The objective is to rationalize the relationship between metal presence and substrate degradability during different fermentation stages and give suggestions for future research on metal-

Hydrolysis is oftentimes the rate-limiting step in the anaerobic digestion process

Lignocelluloses are mainly composed of cellulose, hemicellulose, and lignin [19]. The cellulose and hemicellulose themselves are relatively easy to be broken down by microorganisms; however, their biodegradability decreases when they occur in lignocellulose complexes [20]. The impacts of metals on lignocellulose degradation

Previous studies showed that the presence of metals at certain concentrations may enhance the degradation of lignocelluloses [21, 22]. In one study, an average lignocellulose content of 87.49 ± 3.19%TS was obtained in a control group but decreased to 80.44 ± 3.41%TS, 77.94 ± 3.50%TS, and 79.45 ± 2.88%TS following the addition of 30, 100, and 500 mg/L Cu and 79.36 ± 3.72%TS, 79.10 ± 2.80%TS, and 76.60 ± 2.97%TS following the addition of 30, 100, and 500 mg/L Cr, respectively. Thus, Cu and Cr

probably because fermentative bacteria require an additional step of excreting extracellular enzymes, such as cellulases and lipases, to carry out the hydrolysis or solubilization process [16, 17]. It can be accelerated by enhancing the accessibility of anaerobic microorganisms to intracellular matter or cellulose using thermal, chemical, biological, and mechanical processes, as well as their combinations [18].

vary with the metal species, concentrations, and fermentation conditions.

addition significantly enhanced the degradation of lignocellulose [21, 22].

of metals (e.g., Ni, Co, Cu, Fe, Zn, etc.) for their optimum growth and performance [13]. Various enzymes involved in anaerobic metabolism use trace metals as their cofactors. For example, methanogenic enzymes such as CO dehydrogenase (CODH) and methyl-H4MPT:HS-CoM methyltransferase use cobalt acts as their

**132**


*\*p < 0.05. \*\*p < 0.01.*

#### **Table 1.**

*The average contents of cellulose, hemicellulose, and lignin and total lignocellulose during the anaerobic co-digestion of corn stover and cow dung (55.0 ± 1.0°C) in the presence of different compound metals.*

can act synergistically in Ni–Cu, Ni–Mo–Co, and Ni–Hg systems or antagonistically in Ni-Cd and Ni-Zn systems [42]. Ni was also found to decrease the toxicity of Cd and Cu [43]. However, the combination of Ni and Cd or Cu has been shown that they do not promote the degradation of lignocelluloses (**Table 1**). However, this line of inquiry requires further study.

#### **2.2 Variation of chemical oxygen demands (COD)**

The soluble organic components in the fermenter, shown as COD, originate from the hydrolysis process that liquefies large molecules; long-chain natural polymers of the substrate-like cellulose, hemicellulose, lignin, and polysaccharides; and proteins by extracellular enzymes [17, 44]. Previous studies on anaerobic fermentation of crops and manure showed that the COD in the reactor increased and then decreased because organic matter in the liquid was generated first and then consumed to produce the biogas. Therefore, greater COD did not cause greater biogas generation [45].

Previous studies demonstrated that COD initially increased and then decreased in the presence of Cu [22]. During the initial stage of the fermentation, the substrate was rapidly hydrolyzed into small organic molecules, bringing about an increase of COD in the first 5 days. Later, the COD of the Cu-added groups decreased. The COD of the control group decreased more slowly than those of the Cu-added groups, and the discrepancy between them increased during the fermentation. Taking the whole fermentation process into account, the COD in the Cu addition groups were relatively lower than the control group. It was suggested that Cu addition enhanced the utilization of organic molecules in the fermentation (as indicated by the decrease of COD) and the biogas production [22]. A similar promoting effect was found in the Cr-stressed anaerobic fermentation process [21]. However, Cr addition did not yield lower COD than the control group.

The COD were generally lower in Fe-added groups than in the control group [46]. Fe addition induced a stable and excellent COD conversion rate suggesting a more efficient utilization of soluble organic components in the fermenter that consequently improves biogas yields [47]. Likewise, Ni addition influenced the biogas production, and this can also be partly explained by the Ni effect on COD [48].

**135**

**Treatments** No Cd added

Cd added

**Table 2.**

*addition. Mean ± standard error.*

**Parameters** COD (mg/L) Biogas yield (mL/g TS)

COD(mg/L) Biogas yield (mL/g TS)

**55.0 ± 1.0°C** 10172.01 ± 1246.81

67.65 ± 1.16 9651.83 ± 1505.46

341.62 ± 5.88 *The average COD and cumulative biogas yields during the anaerobic co-digestion of acid-pretreated corn stover and cow dung (55.0, 45.0, 35.0, and 25.0 ± 1.0°C) with and without Cd (1.0 mg/L)* 

**45.0 ± 1.0°C** 11219.30 ± 1596.13

58.45 ± 1.01 11052.13 ± 1612.40

68.35 ± 1.18

**35.0 ± 1.0°C** 12116.95 ± 2317.61

19.42 ± 0.32 13192.66 ± 2518.87

20.51 ± 0.34

**25.0 ± 1.0°C** 9517.14 ± 1199.70

15.96 ± 0.27 9829.71 ± 398.46

15.84 ± 0.27

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

*DOI: http://dx.doi.org/10.5772/intechopen.87161*


#### *Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals DOI: http://dx.doi.org/10.5772/intechopen.87161*
