Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge

Abstract

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.

Keywords: activated sludge; bioplastic; polyhydroxyalkanoate (PHA); staining; visualization.

Figure 1

Visualization of PHA accumulation in activated sludge by staining PHA with BODIPY (green) and protein SYPRO Red (red). Samples at the start of accumulation (a–c) and after 24 h of accumulation (d–f). Scale bars represent 10 μm.

Figure 2

Direct accumulation replicate experiments with developments over 48 h revealed by combined PHA (green) and protein (red) staining. Yellow represents the overlapping signal between PHA and protein staining. Samples from replicate accumulations ACC 1 (a–d), ACC 2 (e–h), and ACC 3 (i–l). Scale bars represent 10 μm.

Figure 3

Direct accumulation replicate experiments with developments over 48 h revealed by combined PHA (green), DNA (blue), and FISH (red). Magenta represents the overlapping signal between RNA and DNA staining. Yellow represents the overlapping signal between RNA and PHA staining. Samples from replicate accumulation ACC 1 (a–d), ACC 2 (e–h), and ACC 3 (i–l). Scale bars represent 10 μm.

Figure 4

Bright-field images of typically observed floc structures after 0 h (a and b) and 48 h (c and d) direct accumulation. Scale bars represent 50 μm.

Figure 5

Morphology of PHA-accumulating microorganisms after 27 h direct accumulation with PHA biomass content at the saturation level of about 0.48 ± 0.02 gPHA/gVSS. Scale bars represent 10 μm.

Figure 6

Distributions of PHA (green), RNA (red), DNA (blue), and the composites initially (a–f) and after 48 h (g–l) direct accumulation experiments. Scale bars represent 10 μm.

Figure 7

Morphology of PHA-accumulating (green overlay with red) and non-PHA-accumulating microorganisms (only red) from direct accumulation experiments. Scale bars represent 10 μm.

Figure 8

Morphology differences for PHA granules stored in the waste activated sludge from replicate direct accumulation experiments. Scale bars represent 10 μm.