**5. Methanogenesis stage**

Methanogenesis is the microbial process, whereby CO2, acetate, or methyl compounds are converted to CH4 in order to generate ATP through the buildup of a sodium ion or proton gradient [31]. Methanogenesis is one of the most metal-rich enzymatic pathways in biology [93]. The contents of Cu, Fe, Ni, and Zn in methanogens (including 10 species of *Methanosarcina*, *Methanococcus*, *Methanobacterium*, *Methanobrevibacter*, etc.) were determined as <10–160 ppm, 0.07–0.28%, 65–180 ppm, and 50–630 ppm, respectively [36].The key enzyme complex in producing biogas from acetate is CODH [15, 94]. CODH cleaves the C-C and C-S bonds in the acetyl moiety of acetyl-CoA, oxidizes the carbonyl group to CO2, and transfers the methyl group to coenzyme M. MCR catalyzes the enzymatic reduction of methyl-coenzyme M to CH4 in methanogenesis, which includes a Ni-containing cofactor called F430 [15, 31, 40]. Besides, the CODH complex is also involved in the formation of acetate by acetogens from H2/CO2 and methanol [95]. MCR is found exclusively in methanogenic archaea [96].

### **5.1 Biogas yields**

The stimulatory effect of trace metal supplementation in certain ranges on anaerobic digestion has been widely reported [10, 21, 46]. 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/orW) and Zn [31].

Low concentrations of Fe have been shown to markedly increase the conversion of acetic acid to CH4 [97]. Fe2+ in concentrations of up to 20 mM has been shown to increase the conversion of acetate to CH4 [98]. It was found that Fe2+ marginally stimulated biogas yield and CH4 content at 37°C and the addition of Fe2+ increased VFA utilization but enhanced H2 utilization considerably [99]. The addition of Fe resulted in a stable process, and its combination with Co contributed to higher biogas production (+9%), biogas production rates (+35%), and reduced VFA concentration while simultaneously degrading the organic fraction of municipal solid waste and slaughterhouse waste [34]. The promoting effect of Fe2+ addition on biogas yields of mixed *Phragmites* straw and cow dung was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities [46].

The optimum or stimulatory concentrations of Ni for batch cultures of methanogens were reported to range between 12 mg/m3 and 5 g/m3 [92]. Pobeheim et al. observed an increase in CH4 production of 25% at day 25 of operation following addition of 10.6 μM Ni [100]. Ni addition of 1–200 μM enhanced the methane production from anaerobic conversion of acetate by 6.30–44.6% compared with the control, respectively [101]. Furthermore, the limitation of Ni in the fermenters led to process instability and was proven to reduce biogas generation [102]. On the other hand, the addition of Ni was found to be beneficial to the methanation process. Ni had increased the ratio of CH4:CO2 [103].

Besides Fe, Ni, and Co, other trace metals like Cu, Cr, and Cd were shown to promote biogas production. Lower concentrations of Cu (1.82 ± 0.01 μg/g dry wt.) and Cr (0.89 ± 0.04 μg/g dry wt.) better served as micronutrients for methanogenic bacteria and might have enhanced the process of methanogenesis and thus CH4 content in the product biogas [9]. Cao et al. harvested five types of plant from Cu-contaminated land, including *Phytolacca americana* L., *Zea mays* L., *Brassica* 

**141**

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

*napus* L., *Elsholtzia splendens*, and *Oenothera biennis* L. and investigated the effects of Cu on anaerobic digestion of these plants. Compared to normal plants with low Cu content, the plants used in remediation with increased Cu levels (100 mg/kg) not only required a shorter anaerobic digestion time but also increased the CH4 content in biogas [25]. 30 and 100 mg/L Cu2+ addition increased the cumulative biogas yields by up to 43.62 and 20.77%, respectively [22]. In another study, 30, 100, and 500 mg/L Cr6+ addition increased the cumulative biogas yields by up to 19.00, 14.85, and 7.68%, respectively, while bringing forward the daily biogas peak yield [21]. Investigations on the anaerobic fermentation of five contaminated crops showed that less than 1 mg/L of Cd in plants promoted or at least had no inhibitory effect on cumulative biogas yields [11]. Jain et al. noted that at low concentrations, Cd and Ni had a favorable effect on the rate of biogas production and its CH4 content, but with increase in concentrations, the rate of biogas production and CH4

Biogas is composed of CH4, CO2, and other trace compositions like H2. Methanogens using H2/CO2 as the matrix usually contain two hydrogenases: one is a hydrogenase that uses coenzyme F420 as the electron acceptor called coenzyme F420-reducing hydrogenase, and the other is coenzyme F420-nonreducing hydrogenase [105]. Hence, the presence of metals in the fermenters influences the biogas composition by impacting the pathways. For example, Cu2+ and Cr6+ addition stimulated biogas production and the generation of CH4 by enhancing the activities

Previously, Fe was found in acetyl-CoA synthase, CH4 monooxygenase, NO-reductase, and nitrite reductase [15]. Fe, together with Ni, was found in hydrogenases of *Methanosarcina barkeri*, which consumes H2 to provide electrons

As we studied, the compositions of biogas varied with temperatures in the presence of heavy metals. The impacts of Cd addition on biogas compositions of anaerobic co-digestion of acid-pretreated corn and fresh cow dung under different fermentation temperatures are shown in **Figure 1**. When temperature increased, the CH4, CO2, and H2 contents also increased, but N2 contents decreased in both Cd-added and control groups. The CH4 contents reached plateau after the fourth day in 55°C group and seventh day in 45°C group (**Figure 1A**). The increase of CO2 contents slowed down after the fourth day in both 55 and 45°C groups (**Figure 1B**). The tendency of CH4 and CO2 contents in 35 and 25°C groups was not detected fully as the biogas yields were too low to be collected by gas bag. Similar contents of H2 were observed in 45, 35, and 25°C groups while lower in 55°C group (**Figure 1C**). The N2 contents decreased more rapidly when fermentation temperature was increased (**Figure 1D**). The results indicated that elevated temperatures accelerated

Cd addition improved the CH4 contents by approximately 6% after the fourth day in 55°C group. Taking the other biogas compositions into account, it was found that Cd addition decreased the CO2 contents in the biogas while having little influences on H2 and N2 contents. However, the impact of Cd on biogas compositions in other three temperature groups was not significant. Therefore, thermophilic fermentation (55°C) promoted the CH4 generation in the presence of Cd in the present study. Low temperature hindered the production of CH4 which agreed with

a previous study that used swine manure as substrate [108].

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

content decreased [104].

**5.2 Biogas compositions**

of coenzyme F420 and methanogenesis [21, 22].

for the reduction of CO2 to CH4 [106, 107].

the start-up of fermentation.

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

*napus* L., *Elsholtzia splendens*, and *Oenothera biennis* L. and investigated the effects of Cu on anaerobic digestion of these plants. Compared to normal plants with low Cu content, the plants used in remediation with increased Cu levels (100 mg/kg) not only required a shorter anaerobic digestion time but also increased the CH4 content in biogas [25]. 30 and 100 mg/L Cu2+ addition increased the cumulative biogas yields by up to 43.62 and 20.77%, respectively [22]. In another study, 30, 100, and 500 mg/L Cr6+ addition increased the cumulative biogas yields by up to 19.00, 14.85, and 7.68%, respectively, while bringing forward the daily biogas peak yield [21]. Investigations on the anaerobic fermentation of five contaminated crops showed that less than 1 mg/L of Cd in plants promoted or at least had no inhibitory effect on cumulative biogas yields [11]. Jain et al. noted that at low concentrations, Cd and Ni had a favorable effect on the rate of biogas production and its CH4 content, but with increase in concentrations, the rate of biogas production and CH4 content decreased [104].

#### **5.2 Biogas compositions**

*New Advances on Fermentation Processes*

exclusively in methanogenic archaea [96].

nogens were reported to range between 12 mg/m3

process. Ni had increased the ratio of CH4:CO2 [103].

**5.1 Biogas yields**

activities [46].

(and/orW) and Zn [31].

Methanogenesis is the microbial process, whereby CO2, acetate, or methyl compounds are converted to CH4 in order to generate ATP through the buildup of a sodium ion or proton gradient [31]. Methanogenesis is one of the most metal-rich enzymatic pathways in biology [93]. The contents of Cu, Fe, Ni, and Zn in methanogens (including 10 species of *Methanosarcina*, *Methanococcus*, *Methanobacterium*,

The stimulatory effect of trace metal supplementation in certain ranges on anaerobic digestion has been widely reported [10, 21, 46]. 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

Low concentrations of Fe have been shown to markedly increase the conversion of acetic acid to CH4 [97]. Fe2+ in concentrations of up to 20 mM has been shown to increase the conversion of acetate to CH4 [98]. It was found that Fe2+ marginally stimulated biogas yield and CH4 content at 37°C and the addition of Fe2+ increased VFA utilization but enhanced H2 utilization considerably [99]. The addition of Fe resulted in a stable process, and its combination with Co contributed to higher biogas production (+9%), biogas production rates (+35%), and reduced VFA concentration while simultaneously degrading the organic fraction of municipal solid waste and slaughterhouse waste [34]. The promoting effect of Fe2+ addition on biogas yields of mixed *Phragmites* straw and cow dung was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase

The optimum or stimulatory concentrations of Ni for batch cultures of metha-

observed an increase in CH4 production of 25% at day 25 of operation following addition of 10.6 μM Ni [100]. Ni addition of 1–200 μM enhanced the methane production from anaerobic conversion of acetate by 6.30–44.6% compared with the control, respectively [101]. Furthermore, the limitation of Ni in the fermenters led to process instability and was proven to reduce biogas generation [102]. On the other hand, the addition of Ni was found to be beneficial to the methanation

Besides Fe, Ni, and Co, other trace metals like Cu, Cr, and Cd were shown to promote biogas production. Lower concentrations of Cu (1.82 ± 0.01 μg/g dry wt.) and Cr (0.89 ± 0.04 μg/g dry wt.) better served as micronutrients for methanogenic bacteria and might have enhanced the process of methanogenesis and thus CH4 content in the product biogas [9]. Cao et al. harvested five types of plant from Cu-contaminated land, including *Phytolacca americana* L., *Zea mays* L., *Brassica* 

and 5 g/m3

[92]. Pobeheim et al.

*Methanobrevibacter*, etc.) were determined as <10–160 ppm, 0.07–0.28%, 65–180 ppm, and 50–630 ppm, respectively [36].The key enzyme complex in producing biogas from acetate is CODH [15, 94]. CODH cleaves the C-C and C-S bonds in the acetyl moiety of acetyl-CoA, oxidizes the carbonyl group to CO2, and transfers the methyl group to coenzyme M. MCR catalyzes the enzymatic reduction of methyl-coenzyme M to CH4 in methanogenesis, which includes a Ni-containing cofactor called F430 [15, 31, 40]. Besides, the CODH complex is also involved in the formation of acetate by acetogens from H2/CO2 and methanol [95]. MCR is found

**5. Methanogenesis stage**

**140**

Biogas is composed of CH4, CO2, and other trace compositions like H2. Methanogens using H2/CO2 as the matrix usually contain two hydrogenases: one is a hydrogenase that uses coenzyme F420 as the electron acceptor called coenzyme F420-reducing hydrogenase, and the other is coenzyme F420-nonreducing hydrogenase [105]. Hence, the presence of metals in the fermenters influences the biogas composition by impacting the pathways. For example, Cu2+ and Cr6+ addition stimulated biogas production and the generation of CH4 by enhancing the activities of coenzyme F420 and methanogenesis [21, 22].

Previously, Fe was found in acetyl-CoA synthase, CH4 monooxygenase, NO-reductase, and nitrite reductase [15]. Fe, together with Ni, was found in hydrogenases of *Methanosarcina barkeri*, which consumes H2 to provide electrons for the reduction of CO2 to CH4 [106, 107].

As we studied, the compositions of biogas varied with temperatures in the presence of heavy metals. The impacts of Cd addition on biogas compositions of anaerobic co-digestion of acid-pretreated corn and fresh cow dung under different fermentation temperatures are shown in **Figure 1**. When temperature increased, the CH4, CO2, and H2 contents also increased, but N2 contents decreased in both Cd-added and control groups. The CH4 contents reached plateau after the fourth day in 55°C group and seventh day in 45°C group (**Figure 1A**). The increase of CO2 contents slowed down after the fourth day in both 55 and 45°C groups (**Figure 1B**). The tendency of CH4 and CO2 contents in 35 and 25°C groups was not detected fully as the biogas yields were too low to be collected by gas bag. Similar contents of H2 were observed in 45, 35, and 25°C groups while lower in 55°C group (**Figure 1C**). The N2 contents decreased more rapidly when fermentation temperature was increased (**Figure 1D**). The results indicated that elevated temperatures accelerated the start-up of fermentation.

Cd addition improved the CH4 contents by approximately 6% after the fourth day in 55°C group. Taking the other biogas compositions into account, it was found that Cd addition decreased the CO2 contents in the biogas while having little influences on H2 and N2 contents. However, the impact of Cd on biogas compositions in other three temperature groups was not significant. Therefore, thermophilic fermentation (55°C) promoted the CH4 generation in the presence of Cd in the present study. Low temperature hindered the production of CH4 which agreed with a previous study that used swine manure as substrate [108].

**Figure 1.**

*Biogas composition under Cd stress with fermentation temperature of 55, 45, 35, and 25°C (A) CH4 contents, (B) CO2 contents, (C) H2 contents, and (D) N2 contents.*

#### **6. Conclusions**

This book chapter reviewed the past findings in the impacts of metals on different stages of the anaerobic degradation process. The requirements of metals by the enzymes involved in the anaerobic process resulted in the different performances with varied metal species and bioavailability. In general, metals in certain concentrations were able to promote the lignocellulose degradation, the generation and consumption of organic components in the fermenters like LCFAs and VFAs, the biogas production, as well as the CH4 contents. The mechanisms of metals were studied by many scientists, focusing on the enzyme activities, microbial communities, etc.

Although a large amount of research has been carried out on individual stages or the entire anaerobic fermentation process, there are still challenges on controlling metal-stressed anaerobic degradation process for optimal utilization of metalcontaminated biowastes. Further work on bioavailability of metals during anaerobic fermentation process and detailed compositions of intermediary products, the relationships between microbial functions and metal species, etc. are recommended for better understanding of the metal-stressed anaerobic degradation process.

#### **Acknowledgements**

This work was funded by the Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07101-003, 2015ZX07204-007, 2015ZX07203-011), the Fundamental Research Funds for the Central Universities (2018MS051).

**143**

**Author details**

\*, Huayong Zhang1

provided the original work is properly cited.

North China Electric Power University, Beijing, China

\*Address all correspondence to: yonglantian@ncepu.edu.cn

1 Research Center for Engineering Ecology and Nonlinear Science,

2 Marine Biology Institute, Shantou University, Shantou, Guangdong, China

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Edmond Sanganyado2

Yonglan Tian1

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

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

**Conflict of interest**

No conflict of interest.

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