# Anaerobic Digestion of Duckweed Used to Remediate Water Contaminated with Zinc and Ammonium
## 4 Discussion
### Anaerobic Digestion of Duckweed Used for Zn Phytoremediation  
#### 4.2.1 Effect of Zn on Methane Production Step  
The presence of Zn can stimulate or inhibit the AD process, depending on its concentration. Zn is an essential trace element of various enzymes that catalyze anaerobic reactions and transformations in the AD process, and it is also part of enzymes such as formate dehydrogenase, superoxide dismutase (SOD), and hydrogenase [62]. Although high Zn concentrations (50-630 mg/L) can accumulate in methanogenic archaea, Zn has not yet been shown to be an essential element for methanogenesis [63]. However, [PERSON] and [PERSON] found that Zn was a necessary trace element to maintain the cell metabolism of methanogens, whereas more than 5 mg/L Zn inhibited the growth of methanogens [64].  
[PERSON] et al. observed the highest biomethane production from activated sludge at a Zn concentration of 93 mg/L [51]. In another study, 4 mg/L Zn promoted the AD of dairy manure to obtain the highest biomethane production of 331.85 mL/g VS, which was 37.59% higher than that of the control [62]. [PERSON] et al. obtained the highest methane yield using de-oiled grease trap waste at 2.1 mg/L Zn, a short lag time, and a decrease in maximum methane production with an increasing Zn concentration [65]. However, [PERSON] et al. reported a 40% reduction in biogas production from the AD of olive mill waste with respect to the base case, because the addition of Zn delayed the degradation during the acidogenic-anaerobic treatment [66]. [PERSON] et al. reported that the maximum methanogen activity was obtained in a digestive system with 2 mg/L Zn, and the inhibition effect on methanogenic activity increased as Zn concentrations increased to more than 3 mg/L [26].  
The liquid fraction of the acid-pretreated duckweed (Figure 6b) contained more available organic matter in comparison to the solid fractions (Figure 6a), thus giving higher methane production (Figure 5b vs. Figure 5a), as also reported by [PERSON] et al. [51]. However, higher initial Zn concentrations in liquid fractions (Figure 5d) exerted toxic effects and inhibited AD, which reduced the methane yield of the liquid fractions of duckweed cultured with Zn (T2, T3, T5, and T6 versus T1 in Figure 5b). Since the Zn concentrations in the liquid fractions of duckweed cultured with both Zn and NH\({}_{4}\)\({}^{+}\)-N were lower than those of duckweed grown in the presence of solely Zn, a higher methane production was observed in the liquid fractions of duckweed cultured with both Zn and NH\({}_{4}\)\({}^{+}\)-N. The lower biomethane production can be explained by the fact that a higher initial Zn concentration was inhibitory to methanogens in the liquid hydrolysate of duckweed cultured with solely Zn and affected the methane yield. Methanogens were not inhibited in the control incubation (T1), and the liquid fractions of duckweed cultured without Zn had higher methane production in comparison to the liquid fractions of duckweed cultured in the presence of Zn (T2 and T3).  
In the solid fraction of the (acid) pretreated duckweed, the rapid acidification of the cellulose and hemicellulose resulted in a low pH (Figure 6c) and inhibited the hydrolysis process. Thus, the high biomass concentrations prolonged the lag phase of methane production from the solid fraction due to the absence of the enzymatic hydrolysis of non-soluble organic polymers to soluble forms available to microorganisms in the initial experiment, especially for duckweed cultured with Zn at 5.0 mg/L. Indeed, adjusting the pH to 7 did improve the methane production in AD tests with the solid fraction of pretreated duckweed.