**Table 2.**

*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) addition. Mean ± standard error.*

*New Advances on Fermentation Processes*

**Lignin (%TS)**

**Metals concentrations (mg/L)**

of inquiry requires further study.

*Mean ± standard error. n = 10.*

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

**Table 1.**

greater biogas generation [45].

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

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

*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.*

**Hemicellulose (%TS)**

Cd(1.0) 19.84 ± 0.94 13.14 ± 0.75 19.27 ± 1.38 52.25 ± 3.07 Cd(1.0) + Fe(10.0) 18.20 ± 0.63 11.96 ± 0.61 15.97 ± 0.90\* 46.13 ± 2.14\* Cd(1.0) + Ni(2.0) 20.82 ± 1.10 15.02 ± 0.75 16.36 ± 0.60 52.20 ± 2.45 Cd(1.0) + Zn(2.0) 12.83 ± 1.07\*\* 12.98 ± 0.64 13.55 ± 1.13\*\* 39.36 ± 2.84\*\* Cu(1.0) 19.63 ± 0.85 13.23 ± 0.75 19.34 ± 1.46 52.21 ± 3.06 Cu(1.0) + Fe(10.0) 16.92 ± 0.90 11.44 ± 0.61 16.46 ± 0.83 44.83 ± 2.34\*\* Cu(1.0) + Ni(2.0) 19.95 ± 1.15 12.05 ± 0.69 20.21 ± 0.74 52.21 ± 2.58 Cu(1.0) + Zn(2.0) 14.56 ± 1.03\*\* 12.34 ± 0.61 14.43 ± 1.18\*\* 41.34 ± 2.82\*\*

**Cellulose (%TS)**

**Total lignocellulose (%TS)**

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

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].

**134**

Lower COD concentrations and higher biogas yields were obtained using higher Ni concentrations. The results demonstrated the balance of different fermentation steps (from hydrolysis to methanogenic phase). Moreover, as the substrates in the Ni-added groups were better degraded in the former three stages (from 4th to 13th day), the left substrates were few, and hence the COD concentrations of Ni-added groups were not increased at the end of the experiments.

The variation of COD in the presence of metals should also be considered under different fermentation temperatures. The required amounts for Ni, Co, Zn, and Fe and in thermophilic glucose fermentation were 10 times more than those required for mesophilic acetate fermentation [49, 50]. In our anaerobic fermentation experiment with acid-pretreated corn stover mixed with fresh cow dung as feedstocks, the COD at 55, 45, 35, and 25 ± 1.0°C were analyzed with and without Cd (1.0 mg/L) addition. The cumulative biogas yields and average COD during the entire fermentation process in Cd-added and no Cd-added group are shown in **Table 2**. It was found that Cd addition resulted in higher biogas yields with the increase of temperature together with the lower COD. Overall, the biogas production should be explained by the variation and/or consumption of the COD concentrations along with VFAs during the fermentation process, rather than the values of COD concentrations.

### **3. Acidogenesis stage**

Acidification is affected by a very diverse group of bacteria, the majority of which are strictly anaerobic. As the acidogenesis stage progresses, the acidic components, including long-chain fatty acids (LCFAs), volatile fatty acids (VFAs), etc., are generated, and they cause a change in the pH. Furthermore, during acidogenesis, organic nitrogen is converted into ammonia [51].

### **3.1 Variation of pH values**

The optimal pH range for efficient methanogenesis ranges from 6.7 to 7.4 [52]. However, the acidogenic bacteria can metabolize organic material down to a pH of around 4. At the beginning of anaerobic fermentation, the pH values are likely to decrease due to the generation of acid components. Thus, buffer solution is suggested for preventing the dramatic pH reduction.

On the one hand, the effect of metal toxicity depends on pH [53]. In general, at high pHs metals have a tendency to form insoluble metal phosphates and carbonates [54], whereas at low pHs the initial leaching of metals from the sludge occurs and hence their solubility increases [55, 56]. Soluble levels of Ni ion were found the highest, while those of Pb ions were found the least as compared to other four heavy metals from pH 4 to 12. At extreme pH of 1, Zn, Pb, and Cd ions showed higher levels than those of Ni, Cu, and Cr. However, Cu, Ni, and Zn ion levels were found higher than those of Pb, Cd, and Cr at an extreme pH of 13. Metal ion levels showed the order of Ni > Cu > Cr > Zn > Cd > Pb between pH 8 and 12. In other pH ranges, metal ions varied with pH [57].

On the other hand, the presence of metals in the reactor during the fermentation process can modify pH values. Previous studies found that adding Cu and Cr resulted in a decrease in pH at the beginning of the experiment, but the pH later recovered [21, 22]. The average pH following addition of Cu have been shown to be generally higher than control groups as well as groups in which Cr is added. An investigation on anaerobic digestion of sewage sludge found that pH negatively related with the exchangeable (−0.838, p < 0.01) and residual fractions (−0.753,

**137**

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

**Table 3.** *The NH4 +*

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

p < 0.01) of Cu while positively related to Fe-Mn oxide-bound (0.895, p < 0.01) and organic-bound (0.698, p < 0.05) fractions of Cu [58]. In contrast, pH positively related to carbonate-bound Cr (0.768, p < 0.01) and organic-bound Cr (0.908, p < 0.01) while negatively related to Fe-Mn oxide-bound Cr (−0.899, p < 0.01) [58]. The results suggest the decrease of pH at the beginning of fermentation was probably beneficial for generating both the exchangeable and residual fractions of Cu. The increase of pH after the start-up of the fermentation is probably helpful for partitioning Cr to yield carbonate-bound and organic-bound fractions, thus reduc-

Addition of Fe and Ni has been shown promote an alkalescent environment for anaerobic fermentation. Previous studies found that adding 10.0 mg/L Fe into the fermenter resulted in lower pH values (p < 0.05) when fermentation is around its peak stage [46]. However, following fermentation peak stage, no significant change in pH has been reported even after increasing the Fe concentration from 0.5 to

+


+

+

**+**

) and free ammonia

+ -N con-

+



**-N Total VFAs**


+ -N

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

ing the bioavailability and toxicity of Cr [21].

**+**

+

fermentation; yet in Cu- and Cr-added groups, the NH4

addition significantly enhanced the generation of NH4

**Metals concentrations (mg/L) NH4**

*(55.0 ± 1.0°C) in the presence of different compound metals.*

ing on different experimental conditions [62].

**3.2 Variation of NH4**

in NH4 +

nucleic acids [59]. At NH4

of the fermentation system.

*Mean ± standard error. n = 10.*

5.0 mg/L or Ni concentrations from 0.2 to 2.0 mg/L [46, 48].

**-N concentrations**

Total ammonia (TAN), consisting of ammonium ions (NH4

(FAN, NH3), is produced during anaerobic degradation of proteins, urea, and

et al. [61] and confirmed in a critical review by Chen et al. [42] that NH4

nutrient for microorganism growth [60]. However, it was reported by Math-Alvarez

centrations ranging from 0.6 to 14 g/L inhibited the methanogenic activity depend-


stable with concentrations ranging from 55.98 to 113.82 mg/L [22] and 39.85 to 105.87 mg/L [21], respectively. Thus, Cu and Cr addition contributed to the stability

**Table 3** shows that further addition of other metals may increase the NH4

concentrations in the metal-stressed fermenters. According to our research, Zn

Cd(1.0) 558.39 ± 39.25 910.57 ± 273.75 Cd(1.0) + Fe(10.0) 604.50 ± 37.34 1865.18 ± 684.94 Cd(1.0) + Ni(2.0) 747.13 ± 38.21\*\* 1003.57 ± 219.79 Cd(1.0) + Zn(2.0) 675.34 ± 36.22\* 513.86 ± 195.52 Cu(1.0) 476.63 ± 37.36 369.77 ± 73.28 Cu(1.0) + Fe(10.0) 652.83 ± 61.88\* 1562.24 ± 577.63\* Cu(1.0) + Ni(2.0) 569.31 ± 23.62 1029.20 ± 298.17 Cu(1.0) + Zn(2.0) 671.82 ± 43.40\*\* 804.66 ± 286.14

*-N and total VFA concentrations during the anaerobic co-digestion of corn stover and cow dung* 

in the control group fluctuated in the range 9.70–157.34 mg/L before the 21st day of

Previous studies showed that Cu or Cr addition induced remarkable differences

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

p < 0.01) of Cu while positively related to Fe-Mn oxide-bound (0.895, p < 0.01) and organic-bound (0.698, p < 0.05) fractions of Cu [58]. In contrast, pH positively related to carbonate-bound Cr (0.768, p < 0.01) and organic-bound Cr (0.908, p < 0.01) while negatively related to Fe-Mn oxide-bound Cr (−0.899, p < 0.01) [58]. The results suggest the decrease of pH at the beginning of fermentation was probably beneficial for generating both the exchangeable and residual fractions of Cu. The increase of pH after the start-up of the fermentation is probably helpful for partitioning Cr to yield carbonate-bound and organic-bound fractions, thus reducing the bioavailability and toxicity of Cr [21].

Addition of Fe and Ni has been shown promote an alkalescent environment for anaerobic fermentation. Previous studies found that adding 10.0 mg/L Fe into the fermenter resulted in lower pH values (p < 0.05) when fermentation is around its peak stage [46]. However, following fermentation peak stage, no significant change in pH has been reported even after increasing the Fe concentration from 0.5 to 5.0 mg/L or Ni concentrations from 0.2 to 2.0 mg/L [46, 48].

#### **3.2 Variation of NH4 + -N concentrations**

*New Advances on Fermentation Processes*

concentrations.

**3. Acidogenesis stage**

**3.1 Variation of pH values**

metal ions varied with pH [57].

Lower COD concentrations and higher biogas yields were obtained using higher Ni concentrations. The results demonstrated the balance of different fermentation steps (from hydrolysis to methanogenic phase). Moreover, as the substrates in the Ni-added groups were better degraded in the former three stages (from 4th to 13th day), the left substrates were few, and hence the COD concentrations of Ni-added

The variation of COD in the presence of metals should also be considered under different fermentation temperatures. The required amounts for Ni, Co, Zn, and Fe and in thermophilic glucose fermentation were 10 times more than those required for mesophilic acetate fermentation [49, 50]. In our anaerobic fermentation experiment with acid-pretreated corn stover mixed with fresh cow dung as feedstocks, the COD at 55, 45, 35, and 25 ± 1.0°C were analyzed with and without Cd (1.0 mg/L) addition. The cumulative biogas yields and average COD during the entire fermentation process in Cd-added and no Cd-added group are shown in **Table 2**. It was found that Cd addition resulted in higher biogas yields with the increase of temperature together with the lower COD. Overall, the biogas production should be explained by the variation and/or consumption of the COD concentrations along with VFAs during the fermentation process, rather than the values of COD

Acidification is affected by a very diverse group of bacteria, the majority of which are strictly anaerobic. As the acidogenesis stage progresses, the acidic components, including long-chain fatty acids (LCFAs), volatile fatty acids (VFAs), etc., are generated, and they cause a change in the pH. Furthermore, during acido-

The optimal pH range for efficient methanogenesis ranges from 6.7 to 7.4 [52]. However, the acidogenic bacteria can metabolize organic material down to a pH of around 4. At the beginning of anaerobic fermentation, the pH values are likely to decrease due to the generation of acid components. Thus, buffer solution is sug-

On the one hand, the effect of metal toxicity depends on pH [53]. In general, at high pHs metals have a tendency to form insoluble metal phosphates and carbonates [54], whereas at low pHs the initial leaching of metals from the sludge occurs and hence their solubility increases [55, 56]. Soluble levels of Ni ion were found the highest, while those of Pb ions were found the least as compared to other four heavy metals from pH 4 to 12. At extreme pH of 1, Zn, Pb, and Cd ions showed higher levels than those of Ni, Cu, and Cr. However, Cu, Ni, and Zn ion levels were found higher than those of Pb, Cd, and Cr at an extreme pH of 13. Metal ion levels showed the order of Ni > Cu > Cr > Zn > Cd > Pb between pH 8 and 12. In other pH ranges,

On the other hand, the presence of metals in the reactor during the fermentation process can modify pH values. Previous studies found that adding Cu and Cr resulted in a decrease in pH at the beginning of the experiment, but the pH later recovered [21, 22]. The average pH following addition of Cu have been shown to be generally higher than control groups as well as groups in which Cr is added. An investigation on anaerobic digestion of sewage sludge found that pH negatively related with the exchangeable (−0.838, p < 0.01) and residual fractions (−0.753,

groups were not increased at the end of the experiments.

genesis, organic nitrogen is converted into ammonia [51].

gested for preventing the dramatic pH reduction.

**136**

Total ammonia (TAN), consisting of ammonium ions (NH4 + ) and free ammonia (FAN, NH3), is produced during anaerobic degradation of proteins, urea, and nucleic acids [59]. At NH4 + -N concentrations below 200 mg/L, TAN is an important nutrient for microorganism growth [60]. However, it was reported by Math-Alvarez et al. [61] and confirmed in a critical review by Chen et al. [42] that NH4 + -N concentrations ranging from 0.6 to 14 g/L inhibited the methanogenic activity depending on different experimental conditions [62].

Previous studies showed that Cu or Cr addition induced remarkable differences in NH4 + -N concentrations compared to a control group [21, 22]. The NH4 + -N values in the control group fluctuated in the range 9.70–157.34 mg/L before the 21st day of fermentation; yet in Cu- and Cr-added groups, the NH4 + -N values were relatively stable with concentrations ranging from 55.98 to 113.82 mg/L [22] and 39.85 to 105.87 mg/L [21], respectively. Thus, Cu and Cr addition contributed to the stability of the fermentation system.

**Table 3** shows that further addition of other metals may increase the NH4 + -N concentrations in the metal-stressed fermenters. According to our research, Zn addition significantly enhanced the generation of NH4 + -N in both Cd and Cu


#### **Table 3.**

*The NH4 + -N and total VFA concentrations during the anaerobic co-digestion of corn stover and cow dung (55.0 ± 1.0°C) in the presence of different compound metals.*

contained fermenters. Addition of Ni induced higher NH4 + -N concentrations in Cd-stressed anaerobic fermentation process, while Fe had a similar response in Cu-stressed fermentation processes. The results suggest that metal mixtures benefited from the degradation of substrate containing nitrogen, such as proteins (**Table 3**) together with the degradation of lignocelluloses (**Table 1**).

#### **3.3 Variation of long-chain fatty acids (LCFAs)**

Long-chain fatty acids (LCFAs) are the intermediate products of lipids' hydrolysis and thus are abundant in lipid-rich substrates such as slaughterhouse wastewater and dairy industrial sludge [63, 64]. LCFAs (e.g., oleic acid) are often degraded through β-oxidation [65] to form acetate, hydrogen, and short-chain fatty acids (SCFAs). Short-chain fatty acids are further catabolized to acetate and hydrogen following cycles of β-oxidation [66]. LCFAs can inhibit the activities of the microorganisms involved in all the AD steps [67] by attaching to bacterial cell membrane, thus limiting mass transfer [68]. It was reported that LCFAs concentration of 0.2 g/L oleate had a profound inhibitory effect, while biogas production ceased when the concentration was increased to 0.5 g/ L [69]. Hwu et al. [70] reported 50% inhibition of methanogenesis in batch reactors at 0.1–0.9 g L<sup>−</sup><sup>1</sup> oleate, depending on the origin of the bacterial inoculum. It has been previously documented that LCFAs could inhibit the activity of hydrolytic, acidogenic, and acetogenic bacteria and methanogenic archaea [68, 69, 71]. However, many studies have reported an adaptation of the microbial communities during the degradation of LCFAs [70, 72]. Moreover, the archaeal community was found to be more tolerant to increased LCFA concentration levels compared to the bacterial community [73].

Metals play a major role in several metabolic pathways and thus will impact of transformation of LCFAs during fermentation process. In general, adding adequate concentrations of microelements may accelerate the degradation of short-chain fatty acids (SCFAs) and LCFAs and would be beneficial for the anaerobic monodigestion of food waste [74]. However, there is lack of studies on the responses of LCFAs to metal stress. Further studies are necessary for revealing the underlying mechanisms.

#### **3.4 Variation of total volatile fatty acids**

VFAs are the intermediary products of the anaerobic fermentation and a precursor for methanogenesis. The concentration of VFA is an important index to evaluate the efficiency of hydrolysis, acidification, and methanogenesis [75]. Trace metal supplementation is one method to increase VFA utilization [76].

Many studies worked on the impacts of heavy metals on the degradation of VFAs [77–79]. At the beginning of the fermentation, the total VFAs often increase due to hydrolysis of substrate and the accumulation of acidic hydrolytic products [21, 22], together with the decrease of pH values. It was reported that a pH range of 5.7–6.0 was recommended as optimal to produce VFAs [80]. During this period, high concentration of Cu was found to inhibit the acidification process [22], while high concentration of Cr inhibited the methanogenesis [21], resulting in low biogas yields. Later on, the VFAs were shown to be consumed during the biogas production, and supplementing metals greatly benefited the process [21, 22].

Supplementing metals may promote the degradation of VFAs, while a metal deficiency may result in the accumulation of VFAs, which often inhibits the anaerobic processes. For example, a previous study found Fe and Ni deficiency during anaerobic digestion of wheat stillage resulted in a rapid accumulation of VFAs [81]. In another study, excluding Co, Zn, and Ni from the methanol-based feed of an

**139**

*Biodegradability during Anaerobic Fermentation Process Impacted by Heavy Metals*

UASB reactor induced lower specific methanogenic activity (SMA) and the accu-

A strong relationship has been previously reported between VFAs and different forms of metals [58]. For example, there was a strong correlation between the organic-bound Cr and VFAs (*r* = −0.846, p < 0.01), indicating that decrease in VFA enhanced the transformation of Cr from unstable species to organic-bound fractions, thus reducing Cr bioavailability and toxicity [58]. As a result, there was an improvement in the CH4 yield. However, the relationships between VFA and metals have been shown to depend on the form and species of the metal. For combining metals, the addition of Fe into Cu-contained fermenters significantly increased the total VFA concentrations (**Table 3**) and resulted in higher biogas yields (data not

Many factors, including substrate concentration, hydraulic retention time, temperature, pH, and process configuration, affect the performance of the acidogenesis phase [31, 85]. However, these factors are particularly susceptible to the presence

The C2-C7 organic acids are predominant intermediates in the anaerobic digestion of organic matter. The anaerobic oxidation of the C3-C7 substrates is coupled to a reduction of protons (H2 formation), and the oxidation of C3 and C4 organic acids is thermodynamically unfavorable (endergonic process) under standard

Lin studied the effects of Cr, Cd, Pb, Cu, Zn, and Ni on VFA degradation in anaerobic digestion by using serum bottle assays with acetic acid acclimated seed sludge (AASS) and mixed acid acclimated seed sludge (MASS) [87]. The relative toxicity of heavy metals to degradation of acetic acid (HAc), propionic acid (HPr), and n-butyric acid (n-HBu) was Cd > Cu > Cr > Zn > Pb > Ni, Cd > Cu > ≒ Zn ≒ Cr > Pb > Ni, and Cd > Cu > Cr > Zn > Pb > Ni, respectively [87]. Cd and Cu were the most, and Pb and Ni were the least toxic heavy metals to VFA-

degrading organisms. To some heavy metals, VFA-degrading acetogens were more sensitive than HAc-utilizing methanogens. The order of sensitivity of the VFA degradation to the metallic inhibition was HPr > HAc ≒ HBu for Cr, HAc > HPr ≒ HBu for Cd and Pb, HPr > HAc > HBu for Zn, HAc ≒ HPc ≒ HBu for Cu, and HAc > HPR > HBu for Ni. Mixtures of the heavy metals caused synergistic inhibi-

Lin et al. carried out a systematic study on the effect of trace metal supplementation on anaerobic degradation of butyric acid [88]. The results showed that the stimulatory effects were in the following order: Cu2+ < Fe3+ < Zn2+ < Ni 2+ < Mn2+ and the normal and isoHBu degradation activities of the methanogens increased by 14–25% and 17–43%, respectively [88]. Kim et al. reported that the supplementation of Ca, Fe, Co, and Ni to a thermophilic non-mixed reactor was required in order to achieve a high conversion of propionate at high concentrations of VFAs [89].

About 70% of CH4 is generated from acetic acid [86]. The acetate utilization rates required per gram of VSS are used to estimate the nutrient supplementation required to prevent limitations in methanogenic activity [90]. Addition of Fe was found to have a stimulatory effect on acetate utilization by methanogens [76]. Bhattacharya et al. [91] found adding 20 mg/L Zn2+ resulted in a complete inhibition of acetate degradation due to Zn toxicity to methanogenesis. Ni sites in the acetyl-CoA decarboxylase/synthase enzyme complex have been identified. This enzyme seemed to have an important role in the conversion of acetate to CH4 [92].

and subsequent interactions with heavy metal ions [86].

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

mulation of VFAs [82–84].

**4. Acetogenesis stage**

conditions [77].

tion on HAc degradation [87].

shown).

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

UASB reactor induced lower specific methanogenic activity (SMA) and the accumulation of VFAs [82–84].

A strong relationship has been previously reported between VFAs and different forms of metals [58]. For example, there was a strong correlation between the organic-bound Cr and VFAs (*r* = −0.846, p < 0.01), indicating that decrease in VFA enhanced the transformation of Cr from unstable species to organic-bound fractions, thus reducing Cr bioavailability and toxicity [58]. As a result, there was an improvement in the CH4 yield. However, the relationships between VFA and metals have been shown to depend on the form and species of the metal. For combining metals, the addition of Fe into Cu-contained fermenters significantly increased the total VFA concentrations (**Table 3**) and resulted in higher biogas yields (data not shown).

### **4. Acetogenesis stage**

*New Advances on Fermentation Processes*

contained fermenters. Addition of Ni induced higher NH4

**3.3 Variation of long-chain fatty acids (LCFAs)**

in Cd-stressed anaerobic fermentation process, while Fe had a similar response in Cu-stressed fermentation processes. The results suggest that metal mixtures benefited from the degradation of substrate containing nitrogen, such as proteins

Long-chain fatty acids (LCFAs) are the intermediate products of lipids' hydrolysis and thus are abundant in lipid-rich substrates such as slaughterhouse wastewater and dairy industrial sludge [63, 64]. LCFAs (e.g., oleic acid) are often degraded through β-oxidation [65] to form acetate, hydrogen, and short-chain fatty acids (SCFAs). Short-chain fatty acids are further catabolized to acetate and hydrogen following cycles of β-oxidation [66]. LCFAs can inhibit the activities of the microorganisms involved in all the AD steps [67] by attaching to bacterial cell membrane, thus limiting mass transfer [68]. It was reported that LCFAs concentration of 0.2 g/L oleate had a profound inhibitory effect, while biogas production ceased when the concentration was increased to 0.5 g/ L [69]. Hwu et al. [70] reported 50%

(**Table 3**) together with the degradation of lignocelluloses (**Table 1**).

inhibition of methanogenesis in batch reactors at 0.1–0.9 g L<sup>−</sup><sup>1</sup>

concentration levels compared to the bacterial community [73].

supplementation is one method to increase VFA utilization [76].

tion, and supplementing metals greatly benefited the process [21, 22].

on the origin of the bacterial inoculum. It has been previously documented that LCFAs could inhibit the activity of hydrolytic, acidogenic, and acetogenic bacteria and methanogenic archaea [68, 69, 71]. However, many studies have reported an adaptation of the microbial communities during the degradation of LCFAs [70, 72]. Moreover, the archaeal community was found to be more tolerant to increased LCFA

Metals play a major role in several metabolic pathways and thus will impact of transformation of LCFAs during fermentation process. In general, adding adequate concentrations of microelements may accelerate the degradation of short-chain fatty acids (SCFAs) and LCFAs and would be beneficial for the anaerobic monodigestion of food waste [74]. However, there is lack of studies on the responses of LCFAs to metal stress. Further studies are necessary for revealing the underlying

VFAs are the intermediary products of the anaerobic fermentation and a precursor for methanogenesis. The concentration of VFA is an important index to evaluate the efficiency of hydrolysis, acidification, and methanogenesis [75]. Trace metal

Many studies worked on the impacts of heavy metals on the degradation of VFAs [77–79]. At the beginning of the fermentation, the total VFAs often increase due to hydrolysis of substrate and the accumulation of acidic hydrolytic products [21, 22], together with the decrease of pH values. It was reported that a pH range of 5.7–6.0 was recommended as optimal to produce VFAs [80]. During this period, high concentration of Cu was found to inhibit the acidification process [22], while high concentration of Cr inhibited the methanogenesis [21], resulting in low biogas yields. Later on, the VFAs were shown to be consumed during the biogas produc-

Supplementing metals may promote the degradation of VFAs, while a metal deficiency may result in the accumulation of VFAs, which often inhibits the anaerobic processes. For example, a previous study found Fe and Ni deficiency during anaerobic digestion of wheat stillage resulted in a rapid accumulation of VFAs [81]. In another study, excluding Co, Zn, and Ni from the methanol-based feed of an

+


oleate, depending

**138**

mechanisms.

**3.4 Variation of total volatile fatty acids**

Many factors, including substrate concentration, hydraulic retention time, temperature, pH, and process configuration, affect the performance of the acidogenesis phase [31, 85]. However, these factors are particularly susceptible to the presence and subsequent interactions with heavy metal ions [86].

The C2-C7 organic acids are predominant intermediates in the anaerobic digestion of organic matter. The anaerobic oxidation of the C3-C7 substrates is coupled to a reduction of protons (H2 formation), and the oxidation of C3 and C4 organic acids is thermodynamically unfavorable (endergonic process) under standard conditions [77].

Lin studied the effects of Cr, Cd, Pb, Cu, Zn, and Ni on VFA degradation in anaerobic digestion by using serum bottle assays with acetic acid acclimated seed sludge (AASS) and mixed acid acclimated seed sludge (MASS) [87]. The relative toxicity of heavy metals to degradation of acetic acid (HAc), propionic acid (HPr), and n-butyric acid (n-HBu) was Cd > Cu > Cr > Zn > Pb > Ni, Cd > Cu > ≒ Zn ≒ Cr > Pb > Ni, and Cd > Cu > Cr > Zn > Pb > Ni, respectively [87]. Cd and Cu were the most, and Pb and Ni were the least toxic heavy metals to VFAdegrading organisms. To some heavy metals, VFA-degrading acetogens were more sensitive than HAc-utilizing methanogens. The order of sensitivity of the VFA degradation to the metallic inhibition was HPr > HAc ≒ HBu for Cr, HAc > HPr ≒ HBu for Cd and Pb, HPr > HAc > HBu for Zn, HAc ≒ HPc ≒ HBu for Cu, and HAc > HPR > HBu for Ni. Mixtures of the heavy metals caused synergistic inhibition on HAc degradation [87].

Lin et al. carried out a systematic study on the effect of trace metal supplementation on anaerobic degradation of butyric acid [88]. The results showed that the stimulatory effects were in the following order: Cu2+ < Fe3+ < Zn2+ < Ni 2+ < Mn2+ and the normal and isoHBu degradation activities of the methanogens increased by 14–25% and 17–43%, respectively [88]. Kim et al. reported that the supplementation of Ca, Fe, Co, and Ni to a thermophilic non-mixed reactor was required in order to achieve a high conversion of propionate at high concentrations of VFAs [89].

About 70% of CH4 is generated from acetic acid [86]. The acetate utilization rates required per gram of VSS are used to estimate the nutrient supplementation required to prevent limitations in methanogenic activity [90]. Addition of Fe was found to have a stimulatory effect on acetate utilization by methanogens [76]. Bhattacharya et al. [91] found adding 20 mg/L Zn2+ resulted in a complete inhibition of acetate degradation due to Zn toxicity to methanogenesis. Ni sites in the acetyl-CoA decarboxylase/synthase enzyme complex have been identified. This enzyme seemed to have an important role in the conversion of acetate to CH4 [92].
