Diazotrophic behaviour in a non-sterile bioreactor: the effect of O2-availability

Abstract

Current agricultural practices have proven unsustainable due to high reliance on chemical fertilizers. Several environmental problems such as leaching of nitrogen into water bodies and the release of NOx gasses emerge from conventional agriculture. This poses a threat to human health and the environment. Thus, there is a need to develop alternative technologies to safeguard food production in the future. The use of diazotrophic bacteria was identified as a promising route as these bacteria could aid in the nitrogen supply of crops through biological nitrogen fixation. Therefore, the behaviour of a locally isolated diazotrophic consortium was investigated with the prospect of agricultural applications in a non-sterile environment. The behaviour of the consortium was mapped through batch experiments. Concentration profiles of the suspended biomass were obtained through spectrometry, and carbon-compound and nitrogen-compound analyses were employed. The oxygen supply to the reactor was varied to investigate the energy effect of oxygen availability. Mass-transfer limited growth was attained under all aeration conditions. In addition, metagenomic analysis was completed through next-generation sequencing to identify the dominant species in the consortium. Lastly, mass and energy balances were performed to explore mechanisms that could explain the observed behaviour. A repeatable culture, from a process point-of-view, was obtained in a non-sterile bioreactor. Metagenomic analysis indicated Chryseobacterium ssp. and Flavobacterium ssp. were the dominant species, making up approximately 50 % of the microbial community. For each aeration condition, negligible amounts of aqueous metabolites were formed indicating a high selectivity towards biomass production. High oxygen availability resulted in decreased growth efficiencies i.e. the specific energy requirements for biomass synthesis. This was attributed to reduced electron transport chain efficiencies and nitrogenase protection mechanisms. The most efficient growth was measured at an aeration feed composition of 21 % oxygen and 79 % nitrogen. This is consistent with atmospheric conditions. An average yield of 0.20 g/g for biomass synthesis was obtained at this condition with a productivity of 6.03 mg/L.h. For all conditions, the mass and energy balances indicated that sessile biomass with a high C:N served as a carbon sink. The study presents one of the only known investigations of operational conditions on diazotrophic growth in a non-sterile bioreactor. In addition, it provides a strong foundation for the development of a Biological Nitrogen Fixation process with scaling potential.