Insights into microbial acclimation to salinity in high-rate anaerobic digestion

Abstract

In recent years, anaerobic digestion (AD) has been investigated as a suitable biotechnology to treat wastewater at elevated salinities. Nevertheless, when starting up AD reactors with inocula that are not adapted to salinity, low concentrations of sodium (Na+) (i.e., 3.9 g/L or 10 g/L as NaCl) can already cause disintegration of microbial aggregates and wash-out. One of the strategies to overcome this issue is the acclimation of non-saline biomass to saline conditions, which, however, can take over 200 days. Several acclimation strategies have been proposed to shorten this time, including amendments with osmolytes or other cations like potassium (K+) or calcium (Ca2+), a stepwise salinity increase, or using different reactor configurations. Still, no defined approach ensures a stable process performance in the long term, which is crucial for full-scale applications. Therefore, the main objective of this PhD thesis was to study the microbial dynamics and the salinity tolerance of the microbiome during the acclimation of non-saline anaerobic granular sludges fed with saline synthetic media and to develop an acclimation strategy to ensure a reliable and resilient AD reactor operation with a real saline industrial effluent. The first experimental chapter (Chapter 3) studied the acclimation of two non-saline granular sludges in a hybrid expanded granular sludge bed (EGSB) reactor equipped with a pumice stone filter in the upper section, fed with saline synthetic media. After initial granule disintegration due to a sudden salinity increase to 5 g Na+/L, re-aggregation occurred relatively fast (i.e., after 95 days of operation). Na+ replaced Ca2+ as the main cation in the sludge’s matrix, but this did not hamper biomass retention, and the process performance remained stable with average soluble chemical oxygen demand (sCOD) removal of 95%, average methane content of 67%, and relatively low but stable methane yields (average <0.23 NL CH4/g COD removed). The pumice stone filter at the upper section and the low up-flow velocities applied (1 m/hr) were key features for retaining active biomass within the systems during the acclimation process. Then, transferring the acclimated biomass to an up-flow anaerobic sludge blanket (UASB)-type reactor further promoted granulation, achieving comparable methane yields to a second reactor seeded with salinity-acclimated granules from a full-scale plant. Regardless of the origin of the inoculum, 16S rRNA gene sequencing analysis revealed a defined core microbiome of Bacteria (Thermovirga, Bacteroidetes vadinHA17, Blvii28 wastewater-sludge group, Mesotoga, and Synergistaceae) and Archaea (Methanothrix and Methanobacterium), highlighting the importance of these microbial groups in developing halotolerance and for maintaining process stability. The second experimental chapter (Chapter 4) assessed the robustness of two hybrid UASB reactors seeded with previously acclimated biomasses against one seeded with non-saline inoculum, treating a real saline effluent from a dairy processing industry. Such wastewater (ww) had a highly variable organic content (7.6 ± 2.6 g CODtotal/L) and a particular ionic composition with high concentrations of Na+ (5.3 ± 1.3 g/L), chlorides (Cl-, 2.7 ± 1.6 g/L) and phosphates (PO43-, 1.9 ± 0.60 g/L). A stable process performance with CODtotal removal efficiencies >80% and an average methane content of 65% was achieved at an organic loading rate (OLR) < 4 g COD/L d and low ionic strengths (<0.15M), regardless of the inocula origin. A sudden increase in the OLR to 6 g COD/L d and ionic strength to 0.30 M (triggered by a rise in the Cl- levels to 8 g/L) led to process deterioration in all the systems. When fed with a synthetic saline media, as in Chapter 3, the reactors with the previously acclimated biomass recovered while the one with non-acclimated granules remained inhibited. The results of the ionic composition of the biomasses and scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) images revealed that calcium phosphate precipitates formed on the surface of the non-acclimated granules, likely due to the release of Ca2+ ions from their matrix during the osmotic up shock, causing severe mass-transfer limitations and thus, process failure. On the other hand, with the previously acclimated biomasses, calcium phosphate precipitation on the surface of the granules did not occur due to their ionic composition, with Na+ as the main cation instead of Ca2+. However, the process gradually deteriorated in those systems due to a volatile fatty acid (VFA) accumulation caused by increased OLR and presumably the limited bioavailability of trace elements in the wastewater due to the high PO43- concentrations. These results provide practical insights into treating saline industrial effluents since they may contain toxic/inhibitory compounds other than Na+, which can negatively affect the process performance. Finally, Chapter 5 investigated the emergence of filamentous fungi within two of the high rates AD systems used in the previous trials. In both cases, the fungi emergence did not have a negative effect on the process performance. Batch tests revealed that the fungal-prokaryotic consortia had a higher salinity tolerance (up to 20 g Na+/L) than the conventional acclimated bacteria-archaea consortia (12 g Na+/L). Key correlations between the different domains were identified by applying a novel multi-omic method, in which the 16S rRNA gene sequences (the prokaryotic dataset) were integrated with the ITS1 gene sequences (the fungi dataset). The results revealed a syntrophic interaction between acetoclastic methanogenic Methanothrix and ascomycetes from the Scedosporium complex, which was proposed as the main driver of the filamentous emergence in the systems. Overall, this study showed the potential of filamentous fungi for degrading soluble organic pollutants under saline conditions, which can be coupled with methane production. In summary, this PhD thesis provides practical insights to facilitate start-ups and enhance and stabilize methane productivity of AD reactors treating saline effluents.