The depletion of fresh water sources forces to design innovative integral solutions for the urban water cycle. Usual practice in most cities is to use drinking water to transport waste outside the city via sewer system. For toilet flushing the water quality is less important and seawater could be used as alternative to use of drinking water. Due to high sulphate content in seawater it usage for toilet flushing will increase the sulphate content of wastewater. Sulphate enrichment of wastewater may also origin from industrial wastewater discharges, seawater intrusion in the sewer network or from sulphate presence in the groundwater used for water supply. Sulphate-rich wastewater allows for alternative wastewater treatment solutions such as the novel Sulphate reduction Autotrophic denitrification and Nitrification Integrated (SANI) process, a sulphur-cycle based wastewater treatment process (Wang et al. 2009). The SANI process is a novel treatment concept developed by the Hong Kong University of Science and Technology and TU Delft. Previously, autotrophic (sulphide-based) denitrification has been studied intensively (Shao et al. 2010), also at relatively low temperatures (<30°C) (Kleerebezem & Mendez 2002, Shao et al. 2011). Research on sulphate reduction however, was mainly performed for industrial purposes at higher temperatures (>30°C) (Dries et al. 1998, Vallero et al. 2004). Therefore, the focus of this research was on sulphate reduction processes at lower temperatures and in the context of municipal wastewater treatment. Sulphate reducing bacteria (SRB) play a key role in the sulphur-cycle based wastewater treatment process (e.g. SANI), however SRB can also play a role in a conventional wastewater treatment process. The application of SRB is beneficial due to minimal sludge production, significant coliforms removal by their exposure to in the process produced sulphide, its applicability for selective heavy metal removal, and their ability to form granular sludge. The primary objective of this research was to study the kinetics of SRB found in domestic wastewater treatment plants (WWTPs) in moderate climate (e.g. the Netherlands), and in the temperature range of 10-20°C. A literature review revealed that temperature, carbon source, sulphide toxicity, sulphate and salt level are important parameters to understand the occurrence and performance of SRB in treating domestic wastewater. Consequently, these parameters were studied to evaluate the applicability of SRB to domestic wastewater treatment. The present study comprises both analysis of kinetics of SRB (long and short term effects) and of microbial population (by application of Transcript Restriction Fragment Length Polymorphism (T-RFLP) and clone-sequencing) of SRB. The main conclusions of each chapter will be addressed below. Experimental methods Throughout this study different types of tests were performed, of which the main tests comprised long-term operation of reactors (months), execution of short-term batch experiments (6 hours) and microbiological population analyses. The reactors were operated for a long time at the following standard conditions: temperature of 20°C, hydraulic retention (HRT) of 10 hours, solid retention time (SRT) of 15 days, pH 7.6 and under non-aerated conditions (anaerobic). The influent of reactors contained: acetate and propionate as organic substrates (300 mgCODVFA/L), ammonium (100 mgN/L), phosphate (10 mgP/L), salinity (0.7%) and sulphate (500 mg/L). The effect of change of various operational parameters on SRB were compared to SRB in this standard reactor. In each reactor biomass was evaluated for the growth, conversion rates, morphology and microbial population present. Short-term experiments were conducted to study the separately effect of nutrients (nitrogen, phosphate, sulphide ions, different substrates, et cetera). Microbiological population analyses were performed by T-RFLP and clone sequencing to identify specific microorganisms present in the sludge. SRB in aerobic WWTP In chapter 3 the presence and activity of SRB in aerobic municipal WWTPs were studied, in order to investigate what type of SRB are naturally occurring in the conventional WWTPs. As SRB are strictly anaerobic microorganisms also the ability of SRB to cope with oxygen exposure was studied. Nine WWTPs were subject to sampling to compare the SRB population in the samples taken from biological tanks and influent by T-RFLP and sequencing techniques. The T-RFLP results revealed that the SRB populations were very similar in these nine WWTPs. Also the similarity between the activated sludge of the tanks and influent was high (>76%). Desulfobacter postgatei, Desulfobulbus propionicus, and Desulfovibrio intestinalis seems to be the most common detected SRB species among the nine selected WWTP in the Netherlands. Batch-activity tests (6 hours) using sludge from the WWTP, did not show any SRB activity. This indicated that likely the SRB where derived from the influent with hardly any enhanced cultivation occurring in the treatment plant itself. Furthermore, 2 long-term (>3 SRT) sequencing batch reactors were operated in presence and absence of oxygen, investigating whether SRB can be active in relatively low DO concentration. Firstly a sequencing batch reactor was operated in the absence of oxygen (anaerobically), resulting in a dominant SRB population, then the conditions were altered to low DO conditions achieved by oxygen transfer between the mixed liquor and the oxygen present in the headspace of the reactor. In both reactors biodegradable organic carbon was removed, partly based on SRB activity. Sulphate reducing activity was also obtained under aerobic condition due to the formation of granular sludge, a protective strategy of the bacteria protect against oxygen exposure. In conclusion, SRB are naturally occurring in conventional WWTP, however are not very active. SRB however, can be active under low DO conditions if growing into sludge granules or as biofilms. SRB at low temperatures The SANI process was developed in Hong Kong at relatively high sewage temperature (30°C). The question whether SRB, as part of the SANI-process, could also be applied successfully in moderate climates, was central in chapter 4. Since temperature is a key-parameter in many biological processes, its kinetic effect on SRB performance, as well as the effect on SRB population was studied. Two sequencing batch reactors were operated, for more than 3 SRTs under sufficiently stable conditions, at 10°C and 20°C, to simulate winter and summer conditions of moderate climate, respectively. The study revealed that at 20°C complete readily biodegradable organic substrate (volatile fatty acids: VFA) removal was achieved, while at 10°C only 2/3 of the CODVFA content was removed. A decrease in rate of approximately a factor 2, caused the incomplete CODVFA removal at 10°C. Despite acetate was the only substrate in the effluent, batch experiments indicated that the acetate and propionate consumption rate were equally affected by a temperature decrease from 20°C to 10°C. Increasing the HRT to 13.3 hours assured a complete CODVFA removal also for operations at 10°C. Microbial population analyses (T RFLP and sequencing) revealed that barely any alteration in SRB population occurred, as response on a temperature decrease from 20 to 10°C, in both laboratory reactors (chapter 4). Also in a full-scale WWTP the SRB population hardly altered due to temperature changes in the range of 10-20°C (chapter 3). Temperature in the range of 10-20°C seems therefore not favouring proliferation of other SRB species. The marginal effect of temperature on SRB population and the opportunity to prolong the HRT for temperatures of 10°C in order to achieve complete VFA COD removal, indicate that SRB may be applied in moderate climate successfully. As for normal wastewater treatment process design, the design should be based on the conversion at the lowest temperature. Acetate and propionate feeding The competition between SRB and methanogens is a point of concern for stable process design; both can convert the organic carbon in absence of oxygen or nitrate. Methanogens, however produce methane, which would not be easy to use in the subsequent denitrification step of the SANI process. Along many other factors, the organic substrate type is suggested to play a key-role for this competition. Hence, the effect of acetate, propionate and a mixture of both substrates on the proliferation and activity of both microbial groups were evaluated in chapter 5. Three sequencing batch reactors were operated to investigate the effect of these feed procedures. In the acetate fed reactor, methanogens became dominant, while in the propionate reactor SRB were the dominant population. In the mixed substrate fed reactor both substrates were fully converted by SRB. All operational characteristics such as the substrate consumption rate, yield and growth rate were similar for SRB from the propionate fed and mixed substrate-fed reactors. Nonetheless, low similarity (<35%) between the sludge from propionate- and mixed substrate-feed reactors was found. The SRB population adapted to propionate feeding, could easily switch and consume acetate in similar rates, which suggest that these species can consume both acetate and propionate. These results indicate that under municipal wastewater conditions (20°C) with fluctuations of acetate and propionate in the influent, the SRB are likely to outcompete methanogens more easily as inferred from pure substrate studies on acetate solely. Increased seawater level In general the saline black water (derived from toilet flushing) is mixed with the grey water (from fresh/drinking water usage) before treatment. As a result, it becomes harder to reuse the water than when grey-water would be treated separately. In order to treat these flows separately, the SANI process should be able to treat wastewater with a higher salt and sulphate content. The purpose of chapter 6 was to investigate the effect of higher salt and sulphate concentration on the performance of SRB. Also for industrial effluent with high salt and sulphate levels, these results are of interest. For that reason, three sequencing batch reactors were operated in which three different seawater portions in the sewage were applied. The three feed portions were 20, 60 and 100% seawater, referring to respectively 0.7, 2.1 and 3.5% salinity and 500, 1,500 and 2,500 mg/L sulphate. In the reactors operated with 20 and 60% seawater portion the same dominant SRB species was present, while the SRB population shifted in the reactor with a 100% seawater portion. The biodegradable organic carbon was in each reactor fully converted. The biomass-specific biological sulphate reduction rate decreased significantly (~45%) when salinity increased from 0.7 to 3.5%. Still, complete acetate and propionate removal occurred even for the 100% seawater feed. In conclusion, the performance of SRB and the effluent quality of SANI process should not be affected when the original SANI process is fed with black water. Effect of nutrients and salinity When saline black water would be separately treated, increased nutrient concentrations can be expected in the influent. For instance, the nitrogen, phosphate, COD (acetate and propionate), salt composition, salt en sulphate level can vary drastically in the influent as a result of separate black water treatment. Further, variation of these nutrients as result of regular influent fluctuation can be expected. The main goal in chapter 7 was to study the effect of the selected nutrients and ions on the sulphate reduction rate, as measure of the SRB performance. For this purpose batch tests, (6 hours) with varying feed composition were conducted. The sludge from the reactors used in chapter 6, was used as inoculum. The average value of each nutrient and ion was applied, as well as a 10 fold higher concentration. These batch tests revealed that increased level of propionate (4,000 mgCODVFA/L) decreased the sulphate reduction rate, while solely acetate and mixture of acetate and propionate (with equal CODVFA concentrations) did not decrease the sulphate reduction rate. Higher concentrations of ammonium and phosphate in the influent did not lead to a change in sulphate reduction rate. Nitrate however, became inhibitory to SRB at levels higher than 500 mgN/L, due to the formation of nitrite (<10 mgN/L). Batch test with separate sulphate or NaCl (salinity) increase in concentration demonstrated that the inhibition of increased salinity (3.5% salinity and 2,500 mgSO42-/L) of the sewage was mainly caused by the increase of salinity. Furthermore also the salt composition had effect on the sulphate reduction rates; the ions Na+ and K+ affected more severely the sulphate reduction rate than Mg2+. In short, assuming the minor effects of CODVFA, N and P in ranges typical for domestic wastewater, and the adaptation opportunities to higher salinity, SRB can be applied successfully for the treatment of saline black water and under usual fluctuation of nutrients concentrations in the influent. Toxicity of sulphide Results of chapter 4 and 6 showed that the sulphate reduction rate within the sequencing batch cycle operation declined, suggesting the toxicity of sulphide on SRB or a limitation due to depletion of volatile fatty acids (CODVFA). In chapter 8 the cause of the sulphate reduction rate decline was studied, as well as the toxicity effect of sulphide and the ability of SRB to adapt to higher sulphide concentration. Batch tests (6 hours) with different initial sulphide levels demonstrated that sulphide presence decreased the sulphate reduction rate substantially. Batch tests with increased CODVFA content neither resulted in higher rates, nor in more sulphide production. Therefore, the decline in rate over time within the reactor was rather caused by sulphide toxicity than CODVFA depletion. The sulphide toxicity affected acetate and propionate consumption equally. To study the adaptability of SRB to higher sulphide levels, two long-term sequencing batch reactors (>3 SRTs) were operated. The reactors were fed with 400 or 800 mgCODVFA/L, resulting in respectively 200 or 400 mg/L sulphide. After changing the feed composition from 400 to 800 mgCODVFA/L, no additional CODVFA was consumed indicating the sludge suffered from sulphide toxicity. However, after a month of operation, all CODVFA was oxidized by SRB. Clone sequencing results revealed that the SRB species differ between the 400 and 800 mgCODVFA/L fed reactors, indicating that a SRB were able to resist higher sulphide level were selected to achieve complete organic carbon removal. The achieved adaption of SRB to sulphide was accomplished by the occurrence of a new dominant species within the reactor. In conclusion, since 400 mg/L CODVFA is usual for domestic wastewater (resulting in 200 mg/L sulphide), no sulphide toxicity issues are expected for the application of SRB in domestic wastewater treatment. Final remarks This research contributed to a better understanding of SRB application in (domestic) wastewater treatment. It revealed that SRB could perform well at moderate climate additions, and no major bottlenecks were identified regarding saline black water treatment. Furthermore, fluctuation of acetate and propionate in the influent does not seem to affect the performance of SRB, and propionate seems even beneficial for SRB in preventing methanogenesis. In the future more attention should be given to the integration of these findings with the SANI process, as well as on the overall seawater toilet-flushing concept.