Understanding of Nitrifying and Denitrifying Bacterial Population Dynamics in an Activated Sludge Process

Nitrogen removal from wastewater has been an important objective in treatment since the 1960s and is one of the most important biological processes used. The progression of knowledge has evolved in stages moving from simple stoichiometric equations into the modern activated sludge models of today. These models use surrogates such as volatile solids for biomass and outcome parameters such as nitrate and nitrite in the secondary effluent to simulate biological activity. Thus, even the most complex models fail to capture the cyclical nature of bacterial abundance and the operating parameters which drive these cycles in full-scale plants. Better understanding of microbial communities has been attempted through the application of florescent in situ hybridization (FISH), which has determined the presence of specific organisms and the distributions of nitrifying and denitrifying populations within a single grab sample. New techniques such as quantitative polymerase chain reaction (qPCR) allowed the identification and quantification of nitrifying and denitrifying bacterial populations over time in full- scale plants. This has permitted the determination of relationships between organisms and operating parameters, which is missing from the majority of earlier microbial studies of wastewater treatment processes.Intense monitoring of bacterial populations involved in nitrification and denitrification was used in this dissertation to identify and illustrate how application of these molecular tools can be used improve plant performance. The overall findings of this study showed that plant performance should be optimized seasonally for maximum nitrification and to maximize denitrification anoxic dissolved oxygen needs to be carefully monitored during the winter and spring to prevent excess oxygen from inhibiting denitrification activity. Furthermore, this study suggests that consortia of bacterial groups carried out denitrification and no one single group could be identified which represented more than 50% of the population. This latter finding suggests that interactions, of what might otherwise be considered as minor groups, become important in understanding overall influences on the denitrification process. This was shown by the inhibition of the abundance of denitrifying bacteria through the production of nitrite by a bulking organism (Thiothrix eikelboomii). In the first study, we determined the nitrifying populations (ammonia oxidizing bacteria, Nitrobacter spp. and Nitrospira spp.) and the total bacterial population were most affected by five of the major physicochemical parameters. Water temperature, nitrite produced, nitrate produced, solids retention time, and pH were found to be the major physicochemical parameters controlling these bacterial dynamics. Two clusters in Principal Component 1(PC1) reflected a seasonality shift at 26.9oC. Temperature was found to be the parameter most directly affecting all bacterial populations in the warmer seasons (July-December), while nitrite produced and pH showed direct negative impacts on the bacterial populations in the cooler seasons (January-June) in the principal component analysis plot. PC1 and PC2 together accounted for 59.8% of the total variance, and the first six Principal Components accounted for 90.2% of total variance. Nitrifying and total bacterial abundance were strictly dependent on temperature in the summer time and inhibited by pH and nitrite in the winter season. This study found SRT needs to be extended by approximately 3.6 days to achieve optimum nitrification and the reduction of the ammonia-oxidizing bacteria: nitrite-oxidizing bacteria ratio of 9.5:1 to 2:1, because the SRT is too short for the Nitrobacter spp. and Nitrospira spp. growth rates.In the second study, two major denitrifying microbial groups, Thauera-like bacteria and Zoogloea-Methyloversatilis-like bacteria, which accounted for 34% on average of the total bacterial community measured using quantitative PCR (qPCR), were investigated in relation to the denitrification ability in a full scale plant. In this study of 11-months in warm wastewater (23-28.6 oC), dissolved oxygen (DO) in the anoxic zone was the most important parameter that determined denitrification efficiency when the temperature was below 27oC. Zoogloea-Methyloversatilis-like bacteria correlated significantly with denitrification (r= 0.52, p < 0.05) under hypoxic conditions (0.2 mg l-1 < DO < 0.6 mg l-1), while Thauera-like bacteria abundance negatively correlated (r= -0.55, p < 0.05) with DO and significantly correlated with denitrification (r= 0.55, p < 0.05) under strict anoxic condition (DO < 0.2 mg l-1). Methanol dosing only played an important role in the Zoogloea-Methyloversatilis-like bacterial abundance (r=0.45, p < 0.05) when temperature was above 27oC, and led to no correlation with Thauera-like bacteria. Thauera-like bacteria abundance correlated positively with COD removal when temperature was less than 27oC (r= 0.52, p< 0.05). Operating the anoxic zone of the plant in the higher DO range (0.2 mg l-1 < DO <0.6 mg l-1) when the temperature was below 27oC reduced the overall denitrification and corresponded to a reduction in Thauera-like bacteria bacterial abundance. At temperatures above 27oC, the denitrification efficiency was independent of the DO level, with the same number of events of denitrification exceeding 70% for both hypoxic and strict anoxic conditions.In the third study, we found that Burkholderia-like bacterial populations and Paracoccus-like bacterial populations were out grown by Thiothrix Eikelboomii (bulking organisms) during three different time periods (March, May, July). Anoxic DO was the most important factor creating negative impacts on the bacterial abundances of Burkholderia-like bacteria (r = -0.45, p < 0.05) and Paracoccus-like bacteria (r = -0.46, p < 0.05). However, hypoxic DO positively correlated with Thiothrix-like bacteria (r =0.38, p <0.05). Nitrite accumulations also imposed a negative impact on Paracoccus-like bacteria (r = -0.33, p<0.05) but correlated positively with Thiothrix Eikelboomii (r = 0.44, p < 0.05), primarily due to fluctuations in the build-up of nitrite accumulation (up to 0.6 mg l-1). Elevated anoxic dissolved oxygen (DO), nitrite buildup, and increasing temperature have significantly influenced the increasing population of bulking oraganisms and the suppressed population dynamics of denitrifying bacterial populations