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.