Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.@en

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.@en