Expanding methanogenic consortia resilience: The role of trace element interactions, Electroactive taxa, and Molecular stress responses

Abstract

In current times, anaerobic digestion is a relevant technology to push the bio economy into its phase of high productivity, and to support a just transition. The sustainable production of biogas is centrally a microbial process. These processes are amenable to instability caused by disturbances and perturbations. For example, trace element imbalance and organic rate overloading. In attempts to improve the methane yields while encountering complex process challenges, stability boosting strategies have been extensively studied. While, trace element dosing is essential for anaerobic digestion, the synergistic and antagonistic effects are not well understood. Additionally, the electroactive taxas part in methanogen resilience and molecular markers underpinning microbial adaptation to stressed conditions remain mostly unexplored. This thesis aims to uncover the functional role of trace element interactions, electroactive microbes and molecular markers in raising methanogen resilience when exposed to stress. Major microbial community members have been identified, yet the main microbes arising to the occasion of process disruptions are syntrophic propionate oxidisers (SPOB). The bipartite syntrophic breakdown of propionate to hydrogen, CO2 and, methane, takes place at the brink of thermodynamic equilibrium, consequently leading to low energy gains. Since, the resilience of this process relies heavily on enhanced interspecies electron transfer (IET) between the partners. The key to strengthening methanogen resilience identified were: improving routes of IET, bio-augmentation of microbes, addition of conductive materials, process adjustments (low organic loading rates, metal supplementation). Of these, three main effectors of methanogenic resilience were selected and individually scrutinised in this thesis. The first experimental chapter examined the exposure of methanogenic granules to a trace element (TE) mixture alongside molybdenum (Mo), tungsten (W) or selenium (Se) would impact (i) extracellular polymeric substances (EPS) protein and carbohydrate content, (ii) microbial composition, and (iii) functional potential. The results showed that, Mo and W increased the concentration of soluble Fe in abiotic controls, enhancing Fe retention. The presence of W, Mo, W+Se, and Se had a positive effect on methane production, with W+Se and W enhancing acetoclastic and hydrogenotrophic methanogenesis. Additionally, Se increased EPS protein and carbohydrate contents in the biomass. Shifts in the microbiome composition were mainly driven by Mo and Se, which typically enriched Capriciproducens, Macelibacteroides and Clostridium sensu stricto 5 taxa. Functional analysis suggested an enrichment of nucleotide metabolism and, importantly, Vitamin (B12, B6 and B9) metabolic potential. The main takeaway was that Fe dynamics dictates the retained concentration of Mo, W, and W+Se, this is essential to optimize methane production through tailored metal supplementation combinations. 16The second experimental chapter assessed the stress mitigation capacity electroactive taxa provides to propionate oxidation and methanogenesis processes after trace element overdoses. Duplicate reactors from propionate enrichment (PE) and electroactive enrichment (EE) group after 62 day trail time, showed that EE reactors improved faster from TE-induced performance decline, achieving higher propionate elimination (75% vs 57%) and superior specific methanogenic activity after 33 days. Notably, EE biomass retained significantly more tungsten (290 vs 45.7 mg W kg-1) than PE. The basis for this was linked to the (i) differential enrichment of Uncultured Geobacteracea, (ii) enhanced mcrA expression (iii) putative cytochrome based respiration and (iv) putative heme and co-factor biosynthesis. This demonstrated that ethanol-fed Geobacteracae can significantly enhance W and Co retention and stabilise propionate oxidation and methanogenesis under severe TE stress. Given longer recovery time, complete rescue of reactor would be possible. The indication that electroactive taxa aided methanogen robustness was critical. However, since the reactor biomass deteriorated identifying responsible modules would prove to be complex. The third experimental chapter attempted to identify the means by which electroactive taxa & direct interspecies electron transfer (DIET) specific genes propel propionate oxidisers & methanogens under pulsed metal dosing conditions. Duplicate reactor set up in groups named; propionate enrichment (PE) and electroactive enrichment (EE). The results showed that after 49 days, 8 pulses of (2 ppm/ week), the test reactors maintained optimal sCOD removal and methane production with increased methyl- dependent methanogenesis. W, Mn, Se dosing increased W retention (52 vs 30 mg W kg-1 , p>0.01). EE reactor biomass produced methane even when exposed to 80X metal dosing concentration, while PE failed to do so. Key metagenome-assemebled genomes identified were Smithellaceae, Syntrophobacter (propionate oxidizer), Methanothrix, Methanospirillum (methanogen), and Geobacteraceae (electroactive).Metagenomes revealed the presence of type 4 secretion system, type 4 pili (two-component system) TCS, CO2 reduction genes and EPS/ Lipopolysacchride (LPS) modification modules. Geobacteraceae facilitated, W-based promotion of methanogenesis, IC50 elevation, through likely mechanisms that retrofit existing methanogens as DIET capable were observed in chapter 3 and 4. Finally, in the fourth experimental chapter, the utility of selected DIET-related genes (pilA and hgtR) was tested against established markers (16SrRNA and mcrA), to determine their responses. To our knowledge, this chapter investigates for the first time the response of a metabolic gene panel to organic loading rate (OLR) stress in propionate- degrading methanogenic consortia in lab-scale upflow anaerobic sludge blanket (UASB) reactors. The experimental phases included stabilisation (1.4¿2.8 g COD/L/day), 17electroactive enrichment, OLR shock (6 g COD/L/day), and early recovery. Quantitative PCR was used to assess the abundance of key functional genes (16SrRNA, mcrA, pilA, and hgtR). During stabilisation, ~200 mL CH4/h was produced, the mcrA/16SrRNA ratio was 0.78¿2.64, and pilA and hgtR abundances were 1.29¿2.27 × 105 and 2.12¿4.37 × 104 copies/gVS. Following the OLR shock, methane production ceased entirely, accompanied by a sharp decline in the mcrA/16S ratio (0.08¿0.24) and significant reductions in pilA (1.43-log) and hgtR (1.34-log) abundance. Partial recovery of pilA and hgtR abundance (1.19 × 105 and 8.57 × 104) was observed in the control reactor after the early recovery phase. The results highlight the utility of mcrA, 16SrRNA, pilA, and associated ratios, as reliable indicators of OLR stress in lab-scale UASB reactors. This chapter advanced the understanding of molecular stress responses in propionate-degrading methanogenic consortia, focusing on DIET in process stability and recovery. In summary, this PhD thesis provided theoretical and practical insights to the potential routes of augmenting methanogenic resilience and stability, thereyby leading to stronger, robust and resilient AD processes.