Develop Biological Denitrification Strategies to Reduce Greenhouse Gas Emissions under Multi-extreme Conditions

Abstract

As a prevalent nitrogen removal process, heterotrophic denitrification plays a crucial role in wastewater treatment plants. Through the process of denitrification, heterotrophic microbes utilize organic carbon to reduce nitrates and nitrites, ultimately releasing nitrogen gas (N2). Notably, incomplete denitrification can also yield nitrous oxide (N2O), a potent greenhouse gas, which is of particular concern due to its substantial impact on the environment. Given the environmental implications and New Zealand's commitment to achieving carbon neutrality by 2050, there is a pressing need for innovative denitrification methods. This thesis explores hydrogenotrophic denitrification, which employs inorganic carbon (i.e., carbon dioxide) and hydrogen gas to reduce nitrates, as an alternative to conventional heterotrophic denitrification. Notwithstanding its environmental merits, hydrogenotrophic denitrification generally presents lower nitrate removal efficacy compared to its heterotrophic counterpart. To address this, the research proposes a mixotrophic denitrification strategy that utilizes both inorganic and organic carbon, potentially offering lower nitrous oxide emissions with almost comparable nitrate removal efficiency. Because New Zealand's stringent biosafety laws hinder the utilization of foreign microbial species in large-scale applications, the study necessitated the use of indigenous inoculants to enrich both heterotrophic and autotrophic denitrification communities. These communities demonstrated the efficacy of using New Zealand indigenous microorganisms to maintain wastewater effluent quality standards and reduce greenhouse gas emissions. In addition, this study investigated wastewater denitrification under challenging conditions that resemble industrial wastewater sources. Specifically, high salinity (3.5%) and extreme multi-conditions (1.5% salinity and pH 5.5) that mirror seafood processing wastewater, scenarios induced by seawater intrusion, and the particularities of dairy wastewater in New Zealand. The latter is characterized by its demanding management due to notable pH fluctuations and high BOD loads (Daly, 2016). The former, seawater intrusion, is a pervasive issue that compromises freshwater aquifers, particularly in coastal regions, by introducing saltwater into freshwater zones, which can be exacerbated by factors like groundwater pumping and reduced freshwater flow (Barlow & Reichard, 2010). The seed inoculum, derived from the heterotrophic activated sludge of a wastewater treatment plant, was subjected to these extreme conditions, aiming to replicate and understand the potential implications and management strategies of such environments on wastewater treatment processes. The results demonstrated that the specific nitrate removal rate of heterotrophic denitrification under high salinity was 55 mg NO3-N /g MLSS/d, while under multi-extreme condutions, it was 58 mg NO3-N /g MLSS/d. Concurrently, the rate for hydrogenotrophic denitrification under multi-extreme conditions was observed to be 50 mg NO3-N /g MLSS/d. Furthermore, the data on N₂O concentration revealed a significant decline in hydrogenotrophic denitrification compared to heterotrophic conditions. While the heterotrophic system accumulated 0.98% N₂O in the reactor headspace, no N₂O accumulation was observed in the hydrogenotrophic system. Lastly, mixotrophic denitrification exhibited also reduced nitrous oxide accumulation potentially due to the relative ratio of nitric oxide reductase (Nir) to nitrous oxide reductase (Nos), a hypothesis corroborated by proteomic results. Future research directions include exploring gene regulation mechanisms, expression patterns, shifts between autotrophic and heterotrophic denitrification, and the interplay between hydrogenotrophic and heterotrophic denitrification. To identify the key species of the hydrogenotrophic denitrification enrichmet cultures, a pure culture of a Paracoccus species was isolated from the hydrogenotrophic mixed culture. This particular species exhibited a notable tendency to accumulate nitrite under the imposed multi-extreme conditions in this study. Furthermore, the genomic data supported its capability to synthesize Polyhydroxyalkanoates (PHA) and carboxysomes, enhancing its adaptability to environments characterized by limited carbon availability.