Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge

Polyhydroxyalkanoates (PHAs) can be produced with municipalwasteactivated sludge from biological wastewater treatment processes. Methodsof selective fluorescent staining with confocal laser scanning microscopy(CLSM) were developed and optimized to evaluate the distribution ofPHA storage activity in this mixed culture activated sludge microbialcommunities. Selective staining methods were applied to a municipalactivated sludge during pilot scale PHA accumulation in replicateexperiments. Visualization of stained flocs revealed that a significantbut limited fraction of the biomass was engaged with PHA accumulation.Accumulated PHA granules were furthermore heterogeneously distributedwithin and between flocs. These observations suggested that the PHAcontent for the bacteria storing PHAs was significantly higher thanthe average PHA content measured for the biomass as a whole. Optimizedstaining methods provided high acuity for imaging of PHA distributionwhen compared to other methods reported in the literature. Selectivestaining methods were sufficient to resolve and distinguish betweendistinctly different morphotypes in the biomass, and these observationsof distinctions have interpreted implications for PHA recovery methods.Visualization tools facilitate meaningful insights for advancementsof activated sludge processes where systematic observations, as appliedin the present work, can reveal underlying details of structure-functionrelationships. Municipal activated sludgecan accumulate PHA, but differentbiomass PHA content limits are often found. In this work, stainingmethods to visualize PHA granule distribution and specific biomasscomponents (DNA, RNA, and protein) were developed, optimized, andapplied for a case study.