The Variation of Nitrifying Bacterial Population Sizes in a Sequencing Batch Reactor (SBR) Treating Low/Mid/High Concentrated Wastewater

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ABSTRACT
The purpose of this study was to correlate the population size of ammonia-oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) with nitrification performance under various operational conditions (chemical oxygen demand (COD) concentration, dissolved oxygen (DO), and hydraulic retention time (HRT)) and influent allythiourea (ATU) shock. The AOB (genera Nitrosomonas and Nitrosospira) and NOB (genera Nitrobecter and Nitrspira) communities were analyzed using fluorescence in situ hybridization (FISH). AOB and NOB accounted for 6.2±0.9% and 2.5±0.3% in total biomass, respectively. The population sizes of AOB and NOB varied with different levels of COD, DO and HRT. Nitrosomonas and Nitrospira were dominant nitrifying bacteria under conditions favorable for nitrification, while Nitrosospira outcompeted Nitrosomonas under adverse conditions (low [NH4 +], low DO, short HRT, and ATU shock), and Nitrobecter outcompeted Nitrospira at high substrate concentrations (COD and [NH4 +]). Under ATU shock that inhibited the oxidation of NH4 + to NO2 -, the AOB population was substantially reduced with the stepwise increase of ATU dosage, and led to a corresponding decrease of NOB population. There was a discrepancy between nitrifying bacterial populations and their functions. Although AOB outnumbered NOB in all tests and became more dominant at low DO and short HRT, NH4 + oxidation, instead of NO2 - oxidation, was the rate-limiting reaction for nitrification and susceptible to the adverse conditions. The study demonstrated the importance of elucidating the shifts of nitrifying bacterial population in order to optimize process design and operation at different influent characteristics, aeration intensity, retention time, and potential influent toxic shock.

INTRODUCTION
Biological nitrification and denitrification are key processes to remove nitrogen from wastewater and have become more important due to stringent regulation on discharge. Nitrification process has two steps carried out by distinct groups of bacteria: ammonium (NH4 +) is first oxidized to nitrite (NO2 -) by autotrophic ammonia oxidizing bacteria (AOB), then nitrite is oxidized to nitrate (NO3 -) by autotrophic nitrite-oxidizing bacteria (NOB) (Reaction 1 and 2). In anoxic denitrification, nitrite/nitrate is reduced to nitrogen gas (N2) by heterotrophic denitrifiers with the presence of carbon source (e.g. methanol, acetic acid) as electron donor (Reaction 3).

Nitrification is difficult to maintain in municipal and industrial wastewater treatment plants, since autotrophic nitrifying bacteria have a slower growth rate and lower competitiveness for oxygen than aerobic heterotrophs at mid/high chemical oxygen demand (COD) concentrations (Cartstensen et al., 1995, Lee et al. 2001). In conventional activated sludge processes, long retention time, intense aeration, and low COD concentration are necessary to keep nitrifying bacteria active. Sequencing batch reactors (SBR) convert treatment processes from space-course to time-course, in which aerobic-anoxic phases are operated sequentially in a single reactor. Due to its space-saving and operation flexibility, SBRs have been widely used for nitrogen removal (Henze M. 1991, Demouline and Goronszy 1997, Obaja et al. 2003, Kim et al. 2004).

Since microorganisms are critical in the nitrification process, many studies have investigated nitrifying bacterial species and activity. By using classical microbial screening techniques, Hall and Murphy (1980) and Painter (1986) found that Nitrosomonas europa and Nitrobacter winogradskyi are the main AOB and NOB, respectively. In contrast, studies using molecular biology-based techniques including 16S ribosomal RNA (rRNA)-targeted methods have shown a great diversity of nitrifiers in activated sludge. Nitrosospira and Nitrospira were found as main species of AOB and NOB in both bench scale systems (Burrell et al., 1998; Schramm et al., 1998, 1999, Rittmann et al.1999; Morgenroth et al., 2000, You et al., 2003, Gieseke et al., 2002) and wastewater treatment plants (Juretschko et al., 1998, Coskuner and Curtis 2002), while Nitrosomonas and Nitrobacter were still characterized as dominant nitrifying bacteria in some other studies using bench scale systems (Gieseke et al., 2001, Chen et al., 2003, Tsuneda et al., 2003) and wastewater treatment plants (Wagner et al., 1996, Daims et al., 2001, Dionisi et al., 2002, Coskuner and Curtis 2002, Hallin et al., 2005). In addition, although nitrifying bacterial populations (AOB+NOB) are generally supposed to be greater than 5-8% in biomass for good nitrification (Randall et al., 1992, Koch et al., 2001), a wide variation in the percentage of nitrifying bacteria in microbial community has been reported. It varied from 0.34% in activated sludge (Dionisi et al., 2002), to 6-18% in a combined activated sludge and rotating biological contactor process (You et al., 2003) and a sewage plant (Wagner et al, 1995), and to over 50% in a carbon-limited autotrophic nitrifying biofilm (Kindaichi et al., 2004) and a SBR system (Morgenroth et al., 2000). The discrepancy of these studies may reflect differences in AOB and NOB populations among treatment facilities and raise two questions: Does the dominance of specific nitrifying bacterial species vary with operational conditions and influent qualities? What’s the correlation between microbial population and operational conditions in the treatment systems? Understanding these fundamental mechanisms is essential to reliably establish an optimal condition for nitrifying bacteria and thus improve nitrification efficiency. Until now, most studied for nitrifying bacterial population have been conducted in either bench-scale systems (<20 L) fed with synthetic solution or wastewater treatment plants at stable operational status, there is a lack of information for the variation of AOB and NOB populations in a real SBR system under various operation conditions and influent toxic shock.

With these questions in mind, this study comprehensively investigated the population dynamics of different nitrifying bacterial species under a series of operational conditions (low/mid/high COD, dissolved oxygen concentration, and retention time) in a single-family-size SBR system (total volume: 2.8 m3) using 16S rRNA-targeted fluorescent in situ hybridization (FISH). Moreover, the changes of nitrifying bacterial community was examined under allythiourea (ATU) shock, which inhibits the oxidization of NH4 + by chelating copper on ammonia monooxygenase active sites, but reportedly has no effect on the oxidization of NO2 - (Bedard and Knowles. 1989, Gorska et al 1996, Ginestet et al., 1998). Our goal was to find out the response of nitrifying bacteria to different operational conditions, and to establish a correlation between microbial population and nitrogen removal efficiency.

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