The Impact And Mechanism Of Typical Heavy Metals On Dark Fermentative Hydrogen Production

Dark fermentation technology has received widespread attention because of its low operating cost,fast reaction rate,low energy demand,and simple operating conditions,and the production of hydrogen（H₂）using dark fermentation technology can achieve the dual effectiveness of waste treatment and energy production.However,the low efficiency of system H₂production is a bottleneck problem for scaling up dark fermentation technology to industrial scale.Therefore,the reasons for suppressing system H₂production have been widely studied in order to improve the system’s H₂production.Among them,the presence of heavy metals and other factors in actual organic wastes as fermentation hydrogen production substrates may have an impact on microbial activity and thus on the system H₂production efficiency.Mercury,copper and arsenic,as three typical heavy metals,are present in high levels in organic wastes such as sewage sludge and municipal solid waste,and can cause impaired cell function or death.In order to investigate the effects of these three typical heavy metals on anaerobic fermentation hydrogen production,this paper firstly explored the process effects of these three typical heavy metals on the hydrogen production system using glucose as the model substrate,and then explored the mechanism of action affecting H₂production at the microbial community structure level.The main conclusions of the study are as follows:（1）As has an inhibitory effect on the hydrogen production of the system,with the concentration of As³⁺increasing to 1 mg/L,the hydrogen production hardly changed,and when the concentration was≥5 mg/L,the hydrogen production showed a significant decreasing trend.In contrast,the concentration of As³⁺>50 mg/L significantly reduced the efficiency of glucose utilization,inhibited the formation of liquid metabolites,and caused a change in the hydrogen production metabolic pathway of microorganisms from butyric acid type fermentation to other fermentation types.Meanwhile,exposure to high As³⁺concentrations（≥50 mg/L）in the fermentation system resulted in a decrease in bacterial community richness and a decrease in the relative abundance of efficient hydrogen-producing microorganisms,which may have contributed to the significant decrease in hydrogen production in the system.（2）Hg could inhibit hydrogen production in the fermentation system,and the hydrogen production showed a significant decrease with the Hg²⁺concentration greater than 50 mg/L.Meanwhile,Hg²⁺reduced the substrate utilization efficiency and inhibited the formation of liquid metabolites.The distribution of soluble metabolites indicated that the addition of Hg²⁺changed the hydrogen production metabolic pathway of microorganisms from butyric acid type fermentation to mixed acid type fermentation.Comparing the bacterial community structure of the control and Hg-1000(high concentration of Hg²⁺addition with significant inhibition of hydrogen production),the results showed that the exposure of the mixed flora to Hg²⁺resulted in a change in the dominant phylum of the bacterial community and that the presence of Hg²⁺led to a change in the dominant bacteria of the fermentation system from Clostridium sensu stricto（high hydrogen production efficiency）to Escherichia-Shigella and Klebsiella,which reduced the hydrogen production of the system.（3）When Cu²⁺in the system was less than 100 mg/L,the inhibition effect on hydrogen production was not significant,and when Cu²⁺concentration was higher than 500 mg/L,the inhibition on hydrogen production was up to96.80%.Meanwhile,Cu²⁺prolonged the lag period of hydrogen production,reduced the substrate utilization efficiency and inhibited the production of liquid metabolites.The distribution of soluble metabolites indicated that the presence of Cu²⁺changed the hydrogen production metabolic pathway of microorganisms.Comparing the bacterial community structure of the control group and the Cu-500 and Cu-1000 treatment groups,where the inhibition of hydrogen production was evident,the results showed that the species diversity and richness of the mixed flora decreased significantly when exposed to high concentrations of Cu²⁺（≥500 mg/L）,and the presence of Cu²⁺led to a decrease in the relative abundance of efficient hydrogen-producing microorganisms.Analysis of the three major hydrogen production pathways showed that the glycolytic and pyruvate metabolic pathways were inhibited by Cu²⁺and that Cu²⁺significantly down-regulated the abundance of hydrogen production-related genes（e.g.,pfl A,fdo G,por,and E1.12.7.2）,fundamentally inhibiting the efficiency of fermentation hydrogen production.