ABSTRACT Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase ( amoA ) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA gene-specific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.

Production of hydrogen sulphide by sulphate reducing bacteria (SRB) is a serious problem in anaerobic wastewater treatment, because it causes corrosion and reduces the value of methane in the biogas produced. The surface and adhesion characteristics of SRB as measured through the zeta potential are different from those of methanogens. We therefore tested the hypothesis that by choosing a carrier material with the proper surface characteristics (zeta potential) it should be possible to selectively immobilize methanogens while excluding SRB. In a series of batch tests with different support materials complete elimination of SRB was obtained on supports made of nylon, a result in line with our original hypothesis. Maximum preferential immobilization of methanogens was obtained at a temperature of 37 °C with media containing volatile fatty acids (VFA) as the carbon source. Preferential immobilization of methanogens was achieved in an anaerobic fluidized bed reactor (AFBR) with nylon granules as support and in an anaerobic rotating biological contactor (ARBC) with an acrylic disc as support. H2S-free biogas containing 63% and 38% methane were obtained from the AFBR and ARBC, respectively.

A pilot-scale primary maturation pond was spiked with (15)N-labelled ammonia ((15)NH(4)Cl) and (15)N-labelled nitrite (Na(15)NO(2)), in order to improve current understanding of the dynamics of inorganic nitrogen transformations and removal in WSP systems. Stable isotope analysis of delta(15)N showed that nitrification could be considered as an intermediate step in WSP, which is masked by simultaneous denitrification, under conditions of low algal activity. Molecular microbiology analysis showed that denitrification can be considered a feasible mechanism for permanent nitrogen removal in WSP, which may be supported either by ammonia-oxidising bacteria (AOB) or by methanotrophs, in addition to nitrite-oxidising bacteria (NOB). However, the relative supremacy of the denitrification process over other nitrogen removal mechanisms (e.g., biological uptake) depends upon phytoplanktonic activity.