Quantification of polyhydroxyalkanoate accumulated in waste activated sludge

Exploring the Limits of Polyhydroxyalkanoate Production by Municipal Activated Sludge

“…These concurrent trends of average biomass PHA content and degree of enrichment from six municipal activated sludge sources replicated the experience previously observed by Pei et al , with activated sludge from Bath WWTP. A degree of enrichment of 1 would be indicative of a biomass with 100% selection of the PHA-storing phenotype . In the present work, with levels of less than 0.51 for degree of enrichment, not more than about half of the biomass was active in PHA storage during the assays.…”

“…A degree of enrichment of 1 would be indicative of a biomass with 100% selection of the PHA-storing phenotype. 18 In the present work, with levels of less than 0.51 for degree of enrichment, not more than about half of the biomass was active in PHA storage during the assays. The estimated levels of degree of enrichment could not be readily coupled to be systematically higher or lower for either biological phosphorus removal or nitrification and denitrification WWTP process configurations.…”

“…Samples were fixed with formaldehyde to a final concentration of 3.7% and preserved in a 1:1 ratio mixed with 1X phosphate-buffered saline and pure ethanol before storing at −20 °C. The staining of PHA and non-PHA biomass was performed with BODIPY 493/503 (BODIPY) (Thermo Fisher Scientific, MA, United States) in combination with Sypro Red (Thermo Fisher Scientific, MA, United States), according to Pei et al 17 Fixed sample aliquots of 5 μL were loaded in reaction wells (10 per glass slide). Reaction wells were further provided with 0.5 μL of BODIPY at 2 ng/μL and 0.5 μL of 100 times diluted Sypro Red.…”

“…The fixed and stained samples were evaluated by a Confocal Laser Scanning Microscope LSM 880 (Carl Zeiss, Germany) with Plan-Apochromat 63×/1.4 Oil DIC objectives (Carl Zeiss, Germany). Methods of image capture were as described in Pei et al 17 Each reaction well was first surveyed to get an overall impression. Then, images from 10 randomly selected fields of view containing floc structures were acquired.…”

“…With this metric, it is not possible to distinguish the contribution of individual populations to the total PHA production. In a recent work, a new staining method with microscopy and image analysis was developed and applied to differentiate and quantify between the PHA-accumulating and non-PHA-accumulating biomass fractions in activated sludge. , With this tool, the degree of enrichment for the PHA-accumulating biomass fraction was estimated and directed toward understanding for variations in PHA production processes due to combined factors of amount of polymer stored and fraction of the biomass actively storing polymers. This created an opportunity to explore intrinsic characteristics of microbial cultures to be used for PHA production.…”