Abstract:
Heterotrophic denitrification is a common biological process for nitrogen removal in wastewater treatment plants. Heterotrophic denitrifying microbes reduce nitrates/nitrites by consuming organic carbon and produce nitrogen gas and carbon dioxide as by-product. Carbon dioxide, however, is a major contributor to greenhouse gasses, furthermore, through incomplete denitrification, denitrifiers can also produce nitrous oxide, another potent greenhouse gas. Due to environmental concerns, New Zealand has recently set a goal of being net carbon neutral by 2050. Therefore, novel denitrification methods need to be developed and implemented. Hydrogenotrophic denitrification is an alternative method for denitrification, that consumes inorganic carbon (i.e., carbon dioxide) with hydrogen gas as an electron donor, to reduce nitrates/nitrites. This process has the advantages of being low in cost, has high nitrogen removal rates, and is over all environmentally friendly.
New Zealand laws for biological imports makes it difficult to obtain hydrogenotrophic microbes. Therefore, it was deemed necessary to obtain a hydrogenotrophic community using indigenous species. This method was investigated to help New Zealand maintain their wastewater effluent quality standards while reducing greenhouse gas emissions. Seed inoculum for hydrogenotrophic communities were chosen from two sources 1) Heterotrophic waste activated sludge, from a wastewater treatment plant and 2) Beach sediment, located near a wastewater pipe outfall, to consider treatment of nitrogen for industrial saline wastewater. These two sources allowed to investigate hydrogenotrophic communities in (1) non-saline and (2) saline environments (seawater salinity ~4%). If successful, nitrogen can be removed through hydrogenotrophic denitrification for conventional domestic wastewater or industrial wastewater sources (i.e seafood processing industry that has high salinity (3%-6%)).
This thesis elucidated key species that could support the process of hydrogenotrophic denitrification. Overall, the study recognized Thauera sp. str. UOA1 as a key hydrogenotrophic denitrifier in the non-saline environment. Paracoccus sp. str. UOA2 was also identified as a key hydrogenotrophic denitrifier and was isolated as a pure culture from the mixed community. Paracoccus sp. str. UOA2 was observed to accumulate high concentrations of nitrite. This was shown to directly support the growth of Thauera sp. str. UOA1 that requires the accumulation of nitrite for respiration, due to the absence of a vital nitrate transport enzyme (NarK1).
Alternatively, in the saline environment, Zobellella sp. str SUOA1 and Thauera sp. str. SUOA2 were identified as the key denitrifiers. However, compared to the non-saline community, the denitrification performance was considerably inhibited (28 mg NO3--N L-1d-1 vs 75 mg NO3--N L-1d-1). This was attributed to the osmotic pressures of the species created by the harsh saline environment. This meant, the species are required to produce compatible solutes within its cells to balance the osmotic pressure. This is a problem because synthesis of compatible solutes is an energy intensive process (55 ATP), and severely inhibits the denitrification capabilities. This energy requirement creates an uncertainty when developing autotrophic denitrifying reactors for saline waters. However, genomic analysis determined that both Zobellella sp. str SUOA1 and Thauera sp. str. SUOA2 could transport a range of compatible solutes, which requires less energy (2 ATP). Therefore, by providing betaine (a commercially cheap compatible solute), it was shown to provide a greater stability for denitrification.
To complete the nitrogen removal process in saline environments, it was also deemed necessary to understand the impact of salinity for nitrifying communities. Nitrifying communities include two distinct groups of microbes known as ammonia oxidizers (AOM) and nitrite oxidizing bacteria (NOB). In this case seed inoculum was chosen from carriers at a local aquarium. After having enriched the nitrifiers in the saline environment (4%), it was found that the salinity
inhibits NOB, more than AOM. Therefore, the nitrification resulted in high nitrite accumulation instead of nitrate. Although high concentrations of nitrite are toxic to microbes, the hydrogenotrophic denitrifiers in the saline study were found to be efficient at nitrite reduction (40 mg NO2--N L-1d-1). Therefore, it was proposed to expand the saline study to develop a pilot scale reactor, by utilizing the advantages of the shortcut nitrogen removal process, nitrite shunt denitrification. In this way not only do we save the organic carbon costs from using hydrogenotrophic denitrification, but the process also saves an additional 25% in aeration costs, further improving the carbon footprint of the process. The results support; 1) the development of a pilot scale reactor for hydrogenotrophic denitrification using the enriched community as a seed inoculum, 2) the development of a pilot scale reactor for nitrite shunt hydrogenotrophic denitrification in saline environments. 3) the possibility for partial denitrification supported anammox nitrogen removal.