Development of granules-based technology for partial nitrification

Abstract

Side-stream treatment is much more efficient and effective than returning the side stream to the main stream. Currently, partial nitrification followed by either denitrification or ANAMMOX process are being investigated intensively for nitrogen removal from the side stream to replace conventional nitrification-denitrification due to reduced energy and carbon inputs as well as less sludge production. As a precedent step, partial nitrification plays a critical role for the whole nitrogen removal process. Although there are a lot of research to investigate partial nitrification to improve its performance and stability, most of them focus on suspended sludge. Granular sludge as a kind of self-immobilized biofilm with compact structure and large size has distinct advantages such as high biomass retention, high capability to withstand the unfavourable environmental conditions (e.g. pH, toxic compounds and temperature) and loading shock compared with suspended sludge. Therefore, this study mainly focuses on the development of granule sludge based partial nitrification technology for side stream treatment. In the first part of this study, the rapid granulation and the performance by granular sludge for partial nitrification were evaluated in four 3-L sequencing batch reactors. A rapid granulation strategy by forming heterotrophic granules first followed by converting heterotrophic granules to partial nitrifying granules was proposed and validated to shorten the reactor start-up time. With this newly proposed strategy for rapid partial nitrifying granulation, effects of ambient temperature and temperature at 30-33 °C (the temperature of side stream from anaerobic digestion tank), and COD/N ratio (5 and 20) on heterotrophic granulation were investigated. Heterotrophic granules were formed within 9 days at 18-23 ºC and 11 days at 30-33 ºC, respectively, which is faster than most reported granulation periods i.e. 2-4 weeks. By quickly decreasing COD/N ratio, nitrifying bacteria were enriched in the formed heterotrophic granules within 30 days to treat 100 mg N L-1 influent nitrogen, due to the excellent retention of granular sludge in the reactors. On days 37 and 47, it can be claimed that heterotrophic granules have been fully converted into partial nitrifying granules in four reactors, which is around 35-50 days faster than the formation of nitrifying granules directly with suspended sludge by enriching nitrifying bacteria first. In addition, it was found that at lower temperature and COD/N, granules formed a bit more quickly. This part confirmed that the newly proposed strategy for the formation of partial nitrifying granules is very effective, which could be applied in the real situation to start up new reactors or quickly restart the crashed reactors. After the formation of partial nitrifying granules, 4 reactors were operated to evaluate their long-term nitrogen removal performance. The reactor operated at 18-23 ºC and 30-33 ºC achieved specific ammonium oxidation rates of 0.598 and 0.346 mg N mg VSS d-1 , respectively, with nitrogen loading rates of 1.7 and 2.4 g N L-1 d -1 , respectively, suggesting higher specific ammonium oxidation of nitrifying granules at ambient temperature than 30-33 ºC. At both temperatures, ammonium removal efficiency is higher than 98% and nitrite accumulation efficiency is higher than 97%, indicating that temperature is not critical factor for granules to achieve partial nitrification. Although reactor performance was good, it was found that minerals were accumulated in granules gradually, resulting in mineral content as high as 80% after 70-day reactor operation. XRD analysis showed that Ca-phosphate in the form of hydroxyapatite (HAP) is the main inorganic compound in granules in accompany with small amount of CaCO3 in the form of calcite. The second part of this work thus examined this mineral accumulation in partial nitrifying granules in details. Partial nitrifying granules from reactors were sorted into groups with different granule sizes. For the smallest group with granule size from 150 to 250 µm, two distinct colour granules, i.e. brown and yellow, were observed. For larger group with granule size above 710 µm, only brown granules were observed. For all brown granules with different size ranges, ash content of granules was higher than 70%. Both HAP and calcite were found in brown granules with different sizes, with the former preferentially accumulated in the larger granules. The Ca/P ratio in granules is at around 2, which is close to theoretical molar ratio of Ca/P in HAP. Cycle analysis showed that 15 mg L-1 phosphate in the synthetic wastewater was removed by 60% in both particles range <600 µm and >2800 µm, which is mainly due to the reaction of phosphate with high calcium concentration in water, i.e. 100 mg/L Ca in this case (typical calcium concentration in tap water in Southampton) at alkaline conditions such as >8. Compact granule structure results in iv high mass transfer resistance, which might further create a more alkaline environment in granules due to pH gradient to facilitate the precipitation of CaP. For yellow granules with size ranging from 150 to 250 µm, ash content only was 3% and specific ammonia removal rate was 35 mg N g-1 h -1 , indicating that yellow granules are newly formed fresh granules which play a major role for nitrogen removal in the reactors. Although the structure of partial nitrifying granules was very stable during 150 days operation, the gradual accumulation of minerals in granules actually led to the reduced microbial activity. Therefore, further investigation is needed to control the mineral accumulation and ash content increase. Extracellular polymeric substances (EPS) were reported much higher in granular sludge than in suspended sludge because of the strong hydraulic selection pressure used to form granules, which can stimulate more EPS production to adhere cells to form compact aggregates. Furthermore, the interaction between EPS and metal ions, and the binding of metal ions with EPS provided localized high metal ion concentration, leading to a saturated local environment for inorganic precipitation, which were also widely reported especially in geography studies. The third part of this study thus intended to explore how mineral precipitates affect EPS analysis and the relationship between mineral precipitates and EPS in sludge and how to produce a reliable and accurate analysis of EPS with high mineral precipitates. It was found that high mineral precipitate in sludge mainly interfere protein analysis with the Lowry method. It was found that the highly bound Ca within EPS instead of free calcium ions in the solutions of extracted EPS, from partial nitrifying granules, reacted with Lowry reagents to form precipitates to interfere the optical density readings of spectrophotometer. How bound Ca with EPS can form inorganic precipitates with Lowry reagents was out of the study scope although it is very interesting with regard to the mechanism of the precipitate accumulation in partial nitrifying granules with high EPS content and high influent water hardness levels (in this case, high Ca ion concentration). But an improved protein analysis was found in this research work by simply prolonging the incubation time from 1 hr to 3 hr to allow newly formed precipitates to settle down to the bottom of cuvette to minimize the interference of suspended particles on optical density readings. After 3 hr incubation, the difference of optical readings with/without Calcium dropped from 38.6% to less than 0.1%, suggesting the effectiveness of this incubation prolongation for protein analysis with Lowry method. Although partial nitrifying granules are more resistant to unfavourable conditions compared with suspended sludge, it is still unavoidable in some circumstances that nitrifying bacteria are inhibited such as at high salinity levels or high toxic compound levels. The fourth part of the study examined the reactivation of inhibited nitrifying granules as well as suspended nitrifying sludge by exerting a weak electric field (EF). The experiment studied the inhibition of partial nitrifying sludge with 2-4% salinity and 50-100 mg phenol L-1 , respectively. It was found that the application of 2 Vcm-1 electric field for 90 minutes could increase the activity of nitrifying granules (that were not inhibited) by 30% compared to the control. Suspended sludge showed 164% and 169% more activity than control after EF treatment while intact granules showed 43% and 18% more activity compared with the control, at 2% and 4% salinity, respectively. However, different results were obtained for phenol inhibition tests. After phenol shock, all sludges were not able to recover their activity completely. The EF treated intact granules showed a recovery of 55% and 45%, after shock at 50 and 100 mg phenol L-1 , respectively. The suspended sludge showed only negligible recovery after phenol inhibition, indicating granules are more resistant to toxic compounds than suspended sludge. In addition, it is suggested that EF treatment is effective to reactivate nitrifying activity after unfavourable condition shock. Overall, this study has demonstrated the rapid formation of partial nitrifying granules, which is promising for industrial application to shorten the reactor start-up period. The rapidly formed partial nitrifying granules also can maintain long-term stable operation, indicating its prospect to replace conventional partial nitrification by suspended sludge for side stream treatment. The mineral accumulation in partial nitrifying granules due to high water hardness level, however, needs further investigation to minimize its negative effects on the microbial activity although precipitation of CaP is favourable for phosphorus removal from side stream by partial nitrifying granules.

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ORCID for Yongqiang Liu: orcid.org/0000-0001-9688-1786

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