Anaerobic sludge granulation at high salinity

Industries, such as leather tanning, agro-food, fisheries, petroleum, petrochemical and textile dyeing produce saline wastewater. As a result approximately 5% of the globally generated wastewater is hypersaline (salinity above 3.5%). Because salts have a negative effect on microbial activity, biological treatment processes are usually not considered for such wastewaters and they are treated with more expensive physical-chemical processes. Hence, there is a need to broaden the application of more sustainable biological treatment methods. In particular, anaerobic biological treatment should be considered due to possibility of converting organics into biogas, low energy requirements for operation, and production of small amounts of bio-solids. Amongst the anaerobic biological treatment technologies those based on formation of granular sludge are of special interest. This is due to superb settling velocities and a high methanogenic activity of the granules, which allows for a compact reactor design capable of handling high volumetric organic loading rates. Before this research, anaerobic granular sludge was reported to be unsuitable for treatment of highly saline wastewater because methanogens are inhibited by the salinity and sludge granules disintegrate/do not form. The main objective of this thesis was to accomplish anaerobic granulation at high salinity from dispersed biomass and to investigate strategies of overcoming salt toxicity to microorganisms. In Chapter 2 we investigated the possibility to form granules from dispersed biomass at low (5 g Na+/L) and high salinity (20 g Na+/L) in upflow anaerobic sludge blanket (UASB) reactors under other conditions known to improve fresh water granulation. The wastewater contained a complex, energy rich and proteinaceous substrate – a mixture of glucose, acetate and tryptone. This allowed a surprisingly fast development of anaerobic granules (within 45 days), even at 20 g Na+/L (~ 50 g/L NaCl). Although the COD (chemical oxygen demand, a measurement for organic pollution) removal efficiency was slightly better at 5 g Na+/L compared to 20 g Na+/L, at both salinities the removal efficiency exceeded 98% at organic loading rates as high as 16 g COD/L/d. To remain viable at high salinity, most prokaryotes synthesize or take up from bulk liquid small organic molecules called osmolytes. Uptake of osmolytes from bulk liquid is energetically more favourable compered to synthesis. Furthermore, it is known that methanogens mainly use nitrogen containing molecules, such as amino acids or their derivatives for osmoprotection. Also, extracellular polymeric substances (EPS) – the structural gluing material of granules - in anaerobic granular sludge consist of a large weight fraction of proteins (up to 90%). Thus, we hypothesized, that proteinaceous substrate in Chapter 2 potentially provided bioenergetically favourable synthesis precursors of osmolytes and EPS. This hypothesis was investigated in Chapters 3-5. In Chapter 3 proteins and amino acids were inspected for their potential to alleviate osmotic shock stress of acetoclastic methanogens in granular sludge. Aspartate, glutamate, gelatine and tryptone could all alleviate the negative effects of high salinity on methanogens. Furthermore, analysis of nitrogen containing osmolytes accumulated by salt adapted granular sludge revealed glutamate and N-acetyl-β-lysine as the major osmolytes. This could in part explain the positive effect of amino acids on methanogenic activity: glutamate could be taken up directly from the bulk liquid, while N-acetyl-β-lysine could be synthesized from aspartate after uptake in the cell. Hydrolysis of a protein (gelatine) and a peptide (tryptone) potentially could also provide both of these amino acids, thereby explaining their positive effect on methanogenic activity. In Chapter 4 the (positive) effect of proteinaceous substrates on the rate of anaerobic sludge granulation was investigated. In UASB reactor experiments, glucose and acetate were present in the wastewater, together with a third co-substrate that was different for each reactor. If proteinaceous compounds (tryptone or gelatine) were added as the third substrate, granulation at 20 g Na+/L already was observed after 40-50 days. With starch as the third substrate well settling granule-alike aggregates formed. However, this was only possible after a much longer period (~180 days) than with the proteinaceous substrate. Still, apparently methanogenic adaptation and sludge granulation can be achieved at high salinity even without addition of proteins implying that a broader spectrum of saline wastewater is amenable for anaerobic granular treatment without the need of protein dosing. In Chapter 5, the possibility to estimate the amount of proteinaceous substrate for enhanced granulation based on osmotic pressure calculations was studied. The experimental results agreed with calculations, which allowed for a nine fold decrease of protein concentration compared to the arbitrary chosen amounts in Chapter 2 and Chapter 4. In Chapter 6 microbial molecular and microscopy analyses revealed that in sludge granules of reactors supplied with proteinaceous substrate the dominant methanogenic archaea at two different salinities (5 and 20 g Na+/L) belonged to Methanosaeta in its filamentous form. Interestingly, also the dominant bacteria were present as filaments (Streptococcus at 5 g Na+/L and bacterium belonging to Defluvitaleaceae at 20 g Na+/L). An experiment was also performed in which the granulation at 20 g Na+/L from dispersed biomass was studied without a proteinaceous substrate, but with amino acids leucine and proline instead. In this reactor, the bacteria belonging to Defluvitaleaceae disappeared and the granulation was not achieved. In Chapter 7, ion exchange membranes were prepared with EPS extracted from high salinity adapted granules and shown to selectively transport cations and partially repel anions. Interestingly, EPS exhibited a higher selectivity for potassium transport compared to sodium, even though potassium and sodium have the same valence and similar physical-chemical properties. As potassium selectivity has commercial relevance, future studies focusing on the reason for this selectivity perhaps can result in the development of commercial potassium selective membranes. For microbial cells such improved transport of ions through EPS seems to have a negative effect. In methanogenic activity assays potassium was much more toxic compared to sodium suggesting that cation toxicity may be influenced by properties of EPS, i.e. the better the ion can diffuse through the EPS, the more toxic it is (Chapter 7). Finally, in Chapter 8 the results of this research are discussed in a broader context and future research directions are proposed

Calcium effect on microbial activity and biomass aggregation during anaerobic digestion at high salinity

The potential effect of different Ca2+ additions (150, 300, 450, 600 and 1000 mg/L) on microbial activity and aggregation, during anaerobic digestion at moderate (8 g/L Na+) and high salinity (20 g/L Na+) has been investigated. Batch tests were carried out in duplicate serum bottles and operated for 30 days at 37 °C. At 8 g/L Na+, methanogenic activity and protein degradation were comparable from 150 to 450 mg/L Ca2+, and a significant inhibition was only observed at a Ca2+concentration of 1000 mg/L. In contrast, at 20 g/L Na+, 150 to 300 mg/L were the only Ca2+ concentrations to maintain chemical oxygen demand (COD) removal, protein hydrolysis and methane production. Overall, increasing Ca2+ concentrations had a larger impact on acetotrophic methanogenesis at 20 g/L than at 8 g/L Na+. Increasing Ca2+ had a negative effect on the aggregation behaviour of the dominant methanogen Methanosaeta when working at 8 g/L Na+. At 20 g/L Na+ the aggregation of Methanosaeta was less affected by addition of Ca2+ than at 8 g/L Na+. The negative effect appeared to be connected with Ca2+ precipitation and its impact on cell-to cell communication. The results highlight the importance of ionic balance for microbial aggregation at high salinity, bringing to the forefront the effect on Methanosaeta cells, known to be important to obtain anaerobic granules.</p

Microbial Community Drivers in Anaerobic Granulation at High Salinity

In the recent years anaerobic sludge granulation at elevated salinities in upflow anaerobic sludge blanket (UASB) reactors has been investigated in few engineering based studies, never addressing the microbial community structural role in driving aggregation and keeping granules stability. In this study, the combination of different techniques was applied in order to follow the microbial community members and their structural dynamics in granules formed at low (5 g/L Na+) and high (20 g/L Na+) salinity conditions. Experiments were carried out in four UASB reactors fed with synthetic wastewater, using two experimental set-ups. By applying 16S rRNA gene analysis, the comparison of granules grown at low and high salinity showed that acetotrophic Methanosaeta harundinacea was the dominant methanogen at both salinities, while the dominant bacteria changed. At 5 g/L Na+, cocci chains of Streptoccoccus were developing, while at 20 g/L Na+ members of the family Defluviitaleaceae formed long filaments. By means of Fluorescence in Situ Hybridization (FISH) and Scanning Electron Microscopy (SEM), it was shown that aggregation of Methanosaeta in compact clusters and the formation of filaments of Streptoccoccus and Defluviitaleaceae during the digestion time were the main drivers for the granulation at low and high salinity. Interestingly, when the complex protein substrate (tryptone) in the synthetic wastewater was substituted with single amino acids (proline, leucine and glutamic acid), granules at high salinity (20 g/L Na+) were not formed. This corresponded to a decrease of Methanosaeta relative abundance and a lack of compact clustering, together with disappearance of Defluviitaleaceae and consequent absence of bacterial filaments within the dispersed biomass. In these conditions, a biofilm was growing on the glass wall of the reactor instead, highlighting that a complex protein substrate such as tryptone can contribute to granules formation at elevated salinity.</p

EPS glycoconjugate profiles shift as adaptive response in anaerobic microbial granulation at high salinity

Anaerobic granulation at elevated salinities has been discussed in several analytical and engineering based studies. They report either enhanced or decreased efficiencies in relation to different Na+ levels. To evaluate this discrepancy, we focused on the microbial and structural dynamics of granules formed in two upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewater at low (5 g/L Na+) and high (20 g/L Na+) salinity conditions. Granules were successfully formed in both conditions, but at high salinity, the start-up inoculum quickly formed larger granules having a thicker gel layer in comparison to granules developed at low salinity. Granules retained high concentrations of sodium without any negative effect on biomass activity and structure. 16S rRNA gene analysis and Fluorescence in Situ Hybridization (FISH) identified the acetotrophic Methanosaeta harundinacea as the dominant microorganism at both salinities. Fluorescence lectin bar coding (FLBC) screening highlighted a significant shift in the glycoconjugate pattern between granules grown at 5 and 20 g/L of Na+, and the presence of different extracellular domains. The excretion of a Mannose-rich cloud-like glycoconjugate matrix, which seems to form a protective layer for some methanogenic cells clusters, was found to be the main distinctive feature of the microbial community grown at high salinity conditions.</p

Functional Insights of Salinity Stress-Related Pathways in Metagenome-Resolved Methanothrix Genomes

Recently, methanogenic archaea belonging to the genus Methanothrix were reported to have a fundamental role in maintaining stable ecosystem functioning in anaerobic bioreactors under different configurations/conditions. In this study, we reconstructed three Methanothrix metagenome-assembled genomes (MAGs) from granular sludge collected from saline upflow anaerobic sludge blanket (UASB) reactors, where Methanothrix harundinacea was previously implicated with the formation of compact and stable granules under elevated salinity levels (up to 20 g/L Na+). Genome annotation and pathway analysis of the Methanothrix MAGs revealed a genetic repertoire supporting their growth under high salinity. Specifically, the most dominant Methanothrix (MAG_279), classified as a subspecies of Methanothrix_A harundinacea_D, had the potential to augment its salinity resistance through the production of different glycoconjugates via the N-glycosylation process, and via the production of compatible solutes as Nε-acetyl-β-lysine and ectoine. The stabilization and reinforcement of the cell membrane via the production of isoprenoids was identified as an additional stress-related pathway in this microorganism. The improved understanding of the salinity stress-related mechanisms of M. harundinacea highlights its ecological niche in extreme conditions, opening new perspectives for high-efficiency methanisation of organic waste at high salinities, as well as the possible persistence of this methanogen in highly-saline natural anaerobic environments

Data_Sheet_1_EPS Glycoconjugate Profiles Shift as Adaptive Response in Anaerobic Microbial Granulation at High Salinity.PDF

<p>Anaerobic granulation at elevated salinities has been discussed in several analytical and engineering based studies. They report either enhanced or decreased efficiencies in relation to different Na⁺ levels. To evaluate this discrepancy, we focused on the microbial and structural dynamics of granules formed in two upflow anaerobic sludge blanket (UASB) reactors treating synthetic wastewater at low (5 g/L Na⁺) and high (20 g/L Na⁺) salinity conditions. Granules were successfully formed in both conditions, but at high salinity, the start-up inoculum quickly formed larger granules having a thicker gel layer in comparison to granules developed at low salinity. Granules retained high concentrations of sodium without any negative effect on biomass activity and structure. 16S rRNA gene analysis and Fluorescence in Situ Hybridization (FISH) identified the acetotrophic Methanosaeta harundinacea as the dominant microorganism at both salinities. Fluorescence lectin bar coding (FLBC) screening highlighted a significant shift in the glycoconjugate pattern between granules grown at 5 and 20 g/L of Na⁺, and the presence of different extracellular domains. The excretion of a Mannose-rich cloud-like glycoconjugate matrix, which seems to form a protective layer for some methanogenic cells clusters, was found to be the main distinctive feature of the microbial community grown at high salinity conditions.</p