Anaerobic microbial processes for energy conservation and biotransformation of pollutants

Anaerobic microbial processes are commonly applied in the treatment of domestic and industrial wastewaters. Anaerobic digestion (AD) of wastewater has received a great deal of attention, but many aspects related to the complex interactions between microorganism, and how that is affected by the presence of certain toxic, are not yet fully understood. A particular case of this is the effect of heavy metals or chlorinated compounds. These compounds are known to have a strong impact in methanogens, a phylogenetic diverse group responsible for the last step of the AD process. The negative effect of sulphate towards methanogenesis is mainly related to outcompetition of methanogens by sulphate-reducing bacteria (SRB), or to toxicity caused by the sulphide generated from sulphate reduction. Heavy metals are part of many enzymes and cofactors and, in low concentrations, may beneficiate microbial activity. However, high concentrations of metals may disrupt enzyme function and structure. In cases where metal concentration is high, the presence of sulphate or sulphide might be favourable because sulphide precipitate with metals and detoxify the environment. In <strong>Chapter 2 </strong>we provide a review on the current knowledge on the effects of heavy metals and sulphate on AD, with special focus on methanogenesis. From this literature study, it came out that the influence of some metals, such as Co, is not extensively studied and that the potential of biologically produced sulphide as metal detoxification method in AD is still quite unexplored. In <strong>Chapter 3</strong> we explored different strategies to improve methane production. Low concentrations of Ni and Co were supplemented to anaerobic sludge and the impact on methane production was evaluated. Although in contrast with other studies, no beneficial effect of metal supplementation was observed. Further on, the impact of high concentrations of Ni and Co added to anaerobic sludge was evaluated, as well as the use of sulphide as a detoxification strategy. This was evaluated in terms of impact on methane production and in changes in the microbial communities. The results showed that sulphide can be used as a method for metal detoxification, but in the case of biological produced sulphide, the competition between SRB and methanogens needs to be considered. Chlorinated compounds are widely used and commonly found in wastewaters. Several methanogenic metal-containing cofactors are reported to be involved in reductive dechlorination. Therefore, in <strong>Chapters 4 </strong>and<strong> 5</strong> the potential of metal supplementation to enhance the dechlorination process was studied. In <strong>Chapter 4</strong>, the enrichment of methanogenic cultures able to perform reductive dechlorination of 1,2-dichloroethene (DCE) and tetrachlorethene (TCE) using different inoculum sources and substrates is described. Differences in physiological performance and in the microbial communities were evaluated. The results showed that the microbial community can be influenced by inoculum and substrate as well as by the chlorinated compound used. The enriched cultures presenting the best dechlorination performance were selected and used for metal supplementation studies with Ni, Co, and Fe. The results showed a clear positive impact of metal addition, both on methane production and reductive dechlorination. Further research on metal supplementation to enhance dechlorination was performed in pure cultures of <em>Methanosarcina barkeri</em>, a methanogen known to be able to reduce DCE (<strong>Chapter 5</strong>). In this case, it was observed that metal supplementation could improve methane production and reductive dechlorination, but the effect is dependent on the metal and concentration used. It was found that methanogenesis and reductive dechlorination can be affected in a different way by the same metal. Finally, in <strong>Chapter 6</strong> the impact of sulphate on a methane-producing bioelectrochemical system (BES), an emerging technology that can be applied to wastewater treatment, was studied. The results showed an unexpected fast sulphate removal in the system and a limited impact caused by sulphate addition on methane production. The sulphate removal could only partially be explained by microbial activity, but the results demonstrated the ability of microbial communities to evolve and adapt to new operational conditions. In conclusion, the work presented in this thesis gave insights on the impact of heavy metals and sulphate in methanogenic systems. Furthermore, different approaches to maximise methane production were evaluated. In particular, it was shown that metal supplementation can be a promising strategy to improve anaerobic microbial processes, such as methanogenesis and reductive dechlorination.