Fatihah Suja’

Universiti Kebangsaan Malaysia Malaysia

1. Introduction

Nitrification is a two step process namely ammoniacal oxidation and nitrite oxidation. Oxidation of ammonium to nitrite is carried out by autotrophic bacterium mainly Nitrosomonas (e. g. N. europaea, N. oligocarbogenes) and Nitrosospira while conversion of nitrite to nitrate is performed by Nitrobacter (e. g. N. agilis, N. winogradski) and Nitrospira. However, ammoniacal oxidation is considered as the limiting or critical process in nitrification since the ammonia-oxidizing bacteria (AOB) has very low growth rate (Metcalf and Eddy 1991). Various approaches, both culture dependent and independent have been applied to analyze and compare the microbial structure of biomass. However, culture dependent methods are biased by the selection of species which obviously do not represent the real dominant structure (Wagner et al 1995; Lipponen et al 2002). Recently, the development of culture independent molecular techniques, like fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR) or denaturing gradient gel electrophoresis (DGGE) improved the analysis of environmental samples.

Whole cell fluorescene in situ hybridization (FISH) is a technique that uses fluorescently labelled phylogenetic oligonucleotide probes to detect specific whole cells/organisms in biological samples. It can be a valuable tool for the study of microbial dynamics in natural environments (Li et al 1999; Liu et al 2002, Eschenhagen et al 2003). These probes could be designed using the wealth of 16S and 23S rDNA sequence data available to target species, genera subdivisions or divisions in-situ and could be labelled with fluorescent groups, radioactive groups or antigens for immunological detection (Amann 1995).

A combination of the FISH approach with the application of scanning confocal laser microscopy (SCLM) allows non-destructive studies of the three dimensional arrangements of bacterial population identified and out-of-focus fluorescence (Wagner et al 1995). Biological Aerated Filters (BAFs) also have a long history of successfully removing nitrogen in wastewater treatment plants (Chen et al 2000; Quyang et al 2000; Chui et al 2001). Biofilm in the reactors bears great potential for simultaneous and efficient removal of nitrogen (Fdz — Polanco et al 2000). Therefore, an assessment of nitrogen removal efficiency has been made to detect any deterioration to the performance. A possible adverse effect of reduced mass of biofilm in the partial-bed reactor was foreseen for the reason that the slow-growing nitrifiers

will be more easily washed out at lower mean solids retention times (SRT) (Gieseke et al 2002). The denitrification process may also be disrupted because the biofilm provides potential anaerobic conditions in which denitrification flourishes.

Fdz-Polanco et al (2000) pointed out the importance of understanding the spatial distribution of the microbial population, and its activity, for the optimisation of nitrogen removal performance in reactors treating wastewater. The performance of the full and partial-bed reactors for nitrogen removal has been examined (Fatihah 2004). It was verified that the full — and partial-bed reactors have the capacity to remove 79.3 ±7.7 % and 79.4 ±3.6 % nitrogen at carbon organic loadings of 5.71 ±0.16 kg COD/m3.d, corresponding to nitrogen loadings of 0.24 ± 0.02 kg N/m3.d. At this condition, the organic carbon removal efficiency was 5.34 kg COD/m3.d for the full-bed and 5.22 kg COD/m3.d for the partial-bed. The successful removal of nitrogen indicates the existence of ammonia-oxidizing bacteria (AOB) in both reactors.

From the perspective of engineering design, it is important to be able to predict the functional groups of bacteria that are most favoured by various applied reactor conditions. In this respect, knowledge of their activities is more important than that of the detailed microbial population (Beer and Muyzer 1995). The nitrogen removal process in such systems is typically initiated by chemoliautotrophic ammonia-oxidizing bacteria converting ammonia to nitrite and traces of oxidized nitrogen gases. Subsequently nitrite-oxidizing bacteria catalyse the oxidation of nitrite to nitrate, and the process is then completed by denitrification (Metcalf and Eddy 1991). Clearly the oxidation processes of nitrification are an essential prerequisite for the whole removal process. In addition, retaining a large amount of nitrifying bacteria within the reactor can be difficult to achieve, due to their relatively low rates of respiration, and their subsequent sensitivity to DO and temperature, thereby making nitrification the rate-determining microbial system in the entire nitrogen removal process (Tsuneda et al 2003).

Since the number and the physiological activity of the ammonia oxidizers are generally the rate-limiting parameters, the rapid and reliable identification of this autotrophy is an important task. The aerobic ammonia oxidizers belong to a very restricted group of autotrophs with Nitrosomonas and Nitrosospira being the best-known oxidizers (Sliekers et al 2002), dominated by P-Proteobacteria (Wagner et al 1995; Eschenhagen et al 2003). Rowan et al (2003) found that detection of ammonia-oxidizing bacteria using PCR amplified 16S rRNA gene in a laboratory-scale BAF reflects the dominant AOB within a full-scale plant.

If the partial-bed reactor exhibited comparable nitrogen removal performance, intriguing questions would arise: would the slow-growing nitrifying bacteria’s preference for attachment on biofilm thereby enhancing sludge retention time (SRT), be challenged by bacterial growth in suspension: or would there be other factors related to reactor configuration that satisfied the need for nitrifying bacteria to grow in the partial-bed reactor. Since, for any high rate system, the AOBs need to reside within the biofilm that has a longer SRT than the suspended growth, it is interesting to locate the microorganisms along the height of both the full- and partial-bed reactors. The detailed aspects to be evaluated in this part include:

• to detect and enumerate the presence of AOBs in the biofilm and suspended growth biomass using fluorescence in situ hybridization (FISH) technique in combination with confocal laser scanning microscopy (CLSM)

• to correlate changes in the proportion of AOBs to all bacteria along the reactor heights in relation to the reactor configuration

• to associate factors that contribute to the changes in the AOB proportion