Characterization of the In Situ Ecophysiology of Novel Phylotypes in Nutrient Removal Activated Sludge Treatment Plants

An in depth understanding of the ecology of activated sludge nutrient removal wastewater treatment systems requires detailed knowledge of the community composition and metabolic activities of individual members. Recent 16S rRNA gene amplicon surveys of activated sludge wastewater treatment plants with nutrient removal indicate the presence of a core set of bacterial genera. These organisms are likely responsible for the bulk of nutrient transformations underpinning the functions of these plants. While the basic activities of some of these genera in situ are known, there is little to no information for the majority. This study applied microautoradiography coupled with fluorescence in situ hybridization (MAR-FISH) for the in situ characterization of selected genus-level-phylotypes for which limited physiological information is available. These included Sulfuritalea and A21b, both within the class Betaproteobacteria, as well as Kaga01, within sub-group 10 of the phylum Acidobacteria. While the Sulfuritalea spp. were observed to be metabolically versatile, the A21b and Kaga01 phylotypes appeared to be highly specialized.


Labeled 14 CO 2 -MAR for the detection of heterotrophic and autotrophic activities
MAR uptake profiles for the A21b and ABS-19 phylotypes revealed that, of the substrates assessed, the former was only able to assimilate pyruvate, and the latter was unable to assimilate detectable amounts of any. HetCO 2 MAR-FISH [1] was therefore applied with a complex carbon source to assess heterotrophic activity of these phylotypes. Labeled 14 CO 2 -MAR was also used to assess various autotrophic activities. Pure culture studies of the only isolate of the Sulfuritalea, S. hydrogenivorans T , demonstrates an ability of the species to oxidize both molecular hydrogen and thiosulphate under anaerobic conditions with nitrate as electron acceptor. Additional MAR-FISH experiments were designed to assess if the related members of the genus present in full-scale sludge performed such activities.

Materials and Methods
Utilization of unlabeled electron acceptors/donors was assessed with the inclusion of NaH[ 14 CO 2 ] to detect CO 2 incorporation during heterotrophic or autotrophic growth [1,2]. In order to remove CO 2 species, mixed liquor was acidified to pH 5 using HCl, allowed to stand for 20 min, sparged with N 2 for 1 h and then adjusted to pH 7 with NaOH [2]. NaH[ 14 CO 2 ] (American Radiolabeled Chemicals Inc., Saint Louis MO, USA) was added to a final concentration of 20 µCi/ml. Unlabeled electron donors/acceptors utilized included: ammonia (1 mM), nitrite (0.5 mM), nitrate (2 mM), thiosulfate (2 mM), formate (2 mM), H 2 (1% [v/v]) and a complex carbon mix consisting of 0.16 g/L yeast extract, 0.16 g/L meat extract and 0.16 g/L casein hydrolysate. A summary of the electron acceptor and donor combinations is given in Table A. Incubation length was 5 h. The first washing step was performed with 0.1 M citrate buffer (pH 3). All other conditions and steps were the same as detailed in the main text.
Quantitative MAR was performed for selected conditions with a custom-made macro (MARQuant) for the Image J software [3]. The script calculated the average number of silver grains per cell, correcting for background MAR signal, for at least 50 cells. Statistical analysis was performed for the Sulf-842 probe-defined population to assess if selected electron donor and acceptor combinations gave a significantly higher level of activity than anaerobic incubation with 14 CO 2 alone (two sample t-test, P < 0.05).

Results and Discussion
MAR signals were not observed to be above background levels (anaerobic incubation with 14 CO 2 alone) for the A21b and ABS-19 phylotypes, for any of the conditions tested (e.g. Figure Ai. and ii.). Although many MAR-positive non-target cells were observed for all conditions (see Figure A), without any positive signal for these target phylotypes it is difficult to conclude anything from these negative results; especially given signal for aerobic HetCO 2 assimilation for the Sulfuritalea spp. was assessed to be negative, despite observed positive aerobic uptake of amino acids for the genus. Increasing the emulsion exposure time from 10 to 15 days only served to increase the background.
Visual assessment of the Sulfuritalea spp. also indicated that most conditions gave negative 14 CO 2 assimilation. Low activity was observed under thiosulphate oxidising conditions (Figure Aiii.), so semi-quantitative MAR (MARQuant) was applied to assess the significance. However, the results were inconsistent due to the low range of activity (data not shown).
The results of this work indicate that the sensitivity of 14 CO 2 -based MAR, as applied here, was not high enough for all species. The lower signal likely relates to the size and activity of the cells. The phylotypes analysed in this study were all relatively small single cells, where the technique has predominantly been applied to assess filamentous [1] and microcolony-producing organisms [4] where positive signal would be easier to visualize, acknowledging that positive single cells were detected in this study (see Figure A). Activity levels of the cells may be reduced by the preparation steps, such as lowering the pH to 5, or a failure to replicate the optimal conditions experienced by the species in the full-scale sample. Therefore, the technique needs to be optimized to achieve the sensitivity required for these, and similar, phylotypes.