The anammox process is the latest biological process for removing nitrogenous compounds from wastewater. Anammox has been widely studied for the removal of nitrogen from various wastewaters. Despite intensive research for the past two decades to fully understand these processes, there is still some doubt related to the implementation of full-scale systems. Various characteristics of the anammox process, such as the reaction stoichiometry and the kinetic characteristics are still the predominant subjects. Slow bacterial growth and inhibition are the major causes of unsuccessful trials in the lab scale systems, and further prevent up scaling to pilot scale and the implementation of full-scale. Kinetic parameters of interest, such as the substrate affinity constant and maximum specific growth rate, which have been reported in literature, vary widely. Most studies on the anammox processes have been focused on granule-based cell cultures. Parameter values reported in literature are often hindered by mass transfer resistance associated with larger granules of microbial culture. In this study, the enrichment of free cells suspension culture is described. The free-cells suspension of highly active anammox bacteria was further used for detailed kinetic analysis of the anammox process.
Firstly, the existence and diversity of anammox bacteria from various local habitats was investigated. Batch systems were used to enrich anammox biomass from sludge collected from three municipal wastewater treatment works in Pretoria. Anammox activity was tested and detected in two of the three wastewater treatment works after 90 days of primary enrichment. The activity was confirmed by the consumption of both NH4+ and NO2- in the system. The presence of anammox bacteria was also confirmed by PCR amplification of the 16S rRNA of the anammox using the anammox specific primers. All clones retrieved were closely related to the Brocadia species and were abundant in all habitats tested. The maximum growth rate of anammox for batch experiments was also estimated using a relatively new model.
The denitrifying capability of pre-enriched suspended anammox culture was evaluated using a Sequencing Batch Reactor (SBR) and operated for a period of 120 days. The anammox process achieved high substrate removal with an average total nitrogen removal rate of 2.6 gNL-1 d-1, and reaching a maximum TN removal efficiency of 93%. For ammonium and nitrite, maximum removal efficiencies of 93% and 98%, respectively, were obtained. Stable performance was observed after 10 days of operation. Phylogenetic analysis confirmed the presence of anammox bacteria that are closely related to the Candidatus Brocadia species with 16S rRNA sequence similarity of 98%. In addition, a substrate removal model was also employed to simulate and predict the performance of the anammox reactor. According to the model predictions, the maximum substrate removal rate of the reactor should be 34 g.N.L-1.d-1. According to the model validation, the modified Stover-Kincannon model was suitable for the nitrogen removal description in the anammox SBR with a high correlation coefficient of R2=0.9739.
Further experiments were conducted that focused on improving the anammox process using genetic engineering. In order to improve the anammox nitrogen removal efficiency, E. coli was genetically engineered to express the hydrazine oxidoreductase enzyme, a key enzyme in the anammox process. Batch reactors containing simulated wastewater were inoculated with transgenic E. coli. After six days of incubation, a drastic removal of nitrite and nitrate was observed with maximum removal efficiencies of 93% and 98%, respectively. The start-up was immediate with the transgenic E. coli, as opposed to native anammox, which can take several months. The results obtained from this study have shown genetic engineering technology to be an innovative technology that can speed up the anammox process and improve efficiency and stability. The anammox culture enriched during this study had a biomass specific maximum growth rate of 0.31 d-1 which was slightly higher than most reported granule base cell cultures in literature. Additionally, the intrinsic half saturation constant for ammonium and nitrite were detected to be 0.054 and 0.024mg-N L-1, respectively.