Anaerobic wastewater treatment of high salinity wastewaters: impact on bioactivity and biomass retention

Anaerobic sludge bed reactor systems like the upflow anaerobic sludge blanket (UASB) and expended granular sludge bed (EGSB) reactors are currently the mostly applied high-rate reactor systems for anaerobic wastewater treatment. The success of both systems has changed the world conception of wastewater treatment with energy recovery being an intrinsic part of the treatment process, avoidance of excess sludge problems and extremely low space requirement. Nevertheless, while broadening the UASB reactor application to a diverse type of wastewaters, high salinity wastewaters were found to give an adverse effect to the granulation processes. Accumulation of Na+at high concentrations produced weaker and fluffy granules endangering the applicability of the mentioned sludge bed systems. In this thesis, research was conducted to investigate the mechanisms of destabilization of the granules at high Na+concentrations, while trying to improve the granules’ properties. Chapter 1gives an overview of high salinity wastewaters, the application and the bottlenecks of anaerobic wastewater treatment (AnWT) technology under extreme conditions. Focus is given to the granulation process as a key factor in the operation of high rate anaerobic reactors. Indeed, it is a complex process that involves physicochemical as well as biological mechanisms. A short overview of the previous research on anaerobic wastewater treatment processes for high salinity wastewaters is discussed, followed by granulation theories and processes. Referring to the imbalance in the monovalent to divalent cation ratio, the Ca2+augmentation approach is discussed in this chapter as a tool to establish a favourable ratio for the required granulation process. The chapter also discusses the need for extracellular polymeric substances (EPS) production, depending on the types of substrates, as a major factor for a successful granulation process. Specifically for high Na+concentrations, also the importance of K+and more specifically the K+/Na+ratio is discussed as a control regulator to alleviate the negative effects of Na+. Finally, the review discusses microbiological aspects related to the anaerobic treatment of high salinity wastewaters such as the adaptation of sludge to high Na+concentrations and the presence of halophilic/halotolerent microorganisms and their application in anaerobic wastewater treatment. In Chapter 2, the effects of high Na+concentrations on methanogenic sludge bed reactor systems were investigated. In three different UASB reactors a rapid acclimation to 5 or 15 g Na+/L was observed, showing satisfactory results for a period of 100 days, when the reactors were fed with a mixture of acetate, gelatine and ethanol. Loading rates up to at least 18 kg COD/m3.d gave a good COD removal performance and the cultivated sludge showed high specific methanogenic activities (SMA) on acetate, propionate, butyrate and H2compared to the inoculated granules. Remarkably, only the reactor which was operated at a lower COD (5 instead of 50 g/L) exhibited severe washout, probably due to the much shorter HRT that was applied, i.e. 12 hours compared to 120 hours. Interestingly, the SMA of the biomass that washed out from this reactor showed considerably higher SMAs than the washed-out biomass from the other reactors. The performance of the granular sludge systems in this study shows the appropriateness of anaerobic inocula, pre-grown under saline conditions for the anaerobic treatment of high salinity wastewater. In Chapter 3the production of EPS in UASB reactor systems that were operated under high salinity conditions was investigated. Four different UASB reactors were operated at loading rates up to 22 kg COD/m3.d with different acetate:gelatine:starch ratios as the substrate. Reactors were fed with partially acidified and fully acidified substrates, i.e. PAS and FAS respectively, and Na+concentrations of either 10 or 20 g Na+/L. One of the reactors additionally received 1 g of Ca2+/L. All four reactors showed a good performance with COD removal efficiencies exceeding 90 %. Proteins were the dominant EPS and the PAS-fed granules gave much higher EPS concentrations than FAS-fed granules. However, the proteins concentration was found not dependent on the Na+ concentration in the feed. Interestingly, the granules from the reactors which were operated at a Na+/Ca2+ratio of 770 contained more polysaccharides than granules from reactors operated at a high Na+/Ca2+ratio of 1540. SEM images showed that that PAS-fed granule had a smoother granule surfaces than FAS-fed granule. PAS-fed granules also were considerably larger than FAS-fed granules. When the influent contained additional Ca2+, clear cracks or fissures could be observed on the surface of the granules. Na+ concentrations of 10 g/L seemed to increase the granule size, which may have been caused by swelling of the EPS matrix. Shear tests indicated that PAS-fed granules were stronger than the FAS fed granules and that Ca2+ addition had a positive effect on granule strength. The calcium content of these granules also was higher with 150 mg/g TSS compared to 60 mg/g TSS in the other reactors. Batch tests at high Na+concentrations confirmed calcium leaching from the granules. When granular sludge was exposed to 20 g Na+/L in batches, leaching of Ca2+from granules took place with a maximum obtained after 10 days. The calcium content of the granules decreased from approximately 85 to 52 mg/g TSS. Therefore, the highest Na+concentrations resulted in the weakest granules. Chapter 4describes the results of batch incubation studies in which anaerobic granular sludge was augmented with 0.3 g Ca2+/L at Na+concentrations of 20 g/L. Experiments followed the previously described results of Chapter 3 showing that high Na+concentrations caused Ca2+leaching from anaerobic granules. Extensive SEM-EDX and SEM-BSE measurements confirm leaching of calcium from the granules when these are exposed to higher Na+ concentrations. Moreover Ca2+additions seemed to indeed maintain the Ca2+content of granular sludge. Initial attachment of microorganism is very important for the development of granules and biofilms. Therefore, biofilm studies were conducted and explained in Chapter 5. Four different biofilm reactors with a non-woven carrier material were exposed to different Na+ concentrations (10 or 20 g/L) and inoculated with crushed granules. Acetate was used as the substrate. One of the reactors received 0.3 g Ca2+/L and another one 0.7 g K+/L. The reactors were operated as sequencing batches. The reactor fed with K+ gave the best performance. Lower salinity (10 compared to 20 g Na+/L) improved the performance and the reactor which received calcium gave the worst results. This finding contradicts with previous experiments in Chapter 3. However, the situation in the biofilm reactors is completely different because the biofilms had to form from scratch. FISH-CLSM images revealed no significant visible differences in microbial coverage (i.e. bacteria and archea) of the non-woven fabric, except for the calcium enriched reactor. After 40 days of operation, it was clearly observed that 20 g Na+/L does not prevent the initial microbial attachment under anaerobic conditions. From 16 sRNA DGGE measurements it was shown that the biofilms had a similar population and that this population did not change very much in time. The Archea were related to Methanosaeta harundinacae (acetoclastic), Methanolinea tarda and Methanobacterium subteraaneum (both hydrogenotrophic). Apparently these species can easily adapt to high salinity. However, the biofilms in the reactors did not show any of the known acetate-oxidizing bacteria that are expectedly needed for the production of H2from acetate as source for the growth of the found hydrogenotrophic Archea. Therefore, it is assumed that H2leakage by acetoclastic methanogen explain their presence in the reactor. Four identical UASB reactors treating concentrated wastewaters (10 – 30 g COD/L) were operated at 20 g Na+/L and in detail described in Chapter 6. PAS and FAS substrates were fed to different reactors to compare the effect of different organic substrates on granule activity, stability and growth. The effect of calcium augmentation on anaerobic granules’ properties was studied by feeding two of the reactors with additional calcium at a concentration of 0.3 g Ca2+/L. A beneficial effect of potassium was demonstrated in Chapter 5 and it therefore was added to all the reactors, at a concentration of 0.7 g K+/L. The treatment performance of the reactors was compared during a period of 120 days at increasing volumetric organic loading rates (OLRs). Physicochemical and microbiological properties of the anaerobic granules were determined and discussed. The results showed that high COD removal efficiencies are possible at 20 g Na+/L, up to an organic loading rate of at least 14 g COD/L.d. At a loading rate of 25 g COD/L.d the performance and stability of all reactors deteriorated. There were indications that calcium augmentation had a positive effect on biomass retention, but this could not be further quantified. The microbial assays gave similar results as in Chapter 5. Compared to the inoculum, bacterial diversity in FAS-fed granules did not change significantly but was changed considerably in PAS-fed granules. Complex organic PAS feed resulted in more complex bacterial populations that were not related to archea. The bacterial presence of a dominant phylotype, belonging to the family of Marinilabiliaceaeand specifically Alkaliflexus imshenetskiiwas evidenced. Members of Marinilabiliaceae are capable of degrading polymeric substances such as starch and gelatine. The dominant archeal species in the reactors was related to Methanosaeta harundinacea. Methanosaetahave been found to play a major role in granulation In Chapter 7the results of this research are discussed in a general context. Directions for further research are presented focussing on the increase in biomass activity and biomass retention in high salinity wastewaters by improving the adaptability of microbes and the anaerobic sludge granulation process.