PHA validation data
An ion chromatographic method for measuring polyhydroxyalkanoate (PHA) in the form of its monomers (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV) in wastewater was optimized and used to evaluate the PHA content of cells sampled from the aeration basin of a municipal wastewater treatment facility. This is a safer alternative to the more popular gas chromatographic (GC) method. This updated method offers a more sensitive calibration range, improved by two orders of magnitude from 1-16 mg/L to 0.05-5.0 mg/L. Relative standard deviation was reduced from 2% for total PHA in the original method to 0.5% for 3HB and 0.7% for 3HV using the updated protocol. Elution time also was reduced by 20% from 31 minutes to 25 minutes. A relationship was established between sample solids content and PHA extraction efficiency in order to avoid error from incomplete PHA extraction from high solids samples, expanding the application of this method to activated sludge processes with high mixed-liquor suspended solids. Application-based method validation was accomplished using three approaches: (1) commercially available standards, (2) basin profiles of PHA dynamics in a full-scale activated sludge basin, and (3) batch enrichment experiments comparing PHA production dynamics. The results of these three approaches were consistent with published literature. Basin profiles yielded data that reflected expected PHA dynamics in an A2O basin, with intracellular PHA highest just after VFA uptake then decreasing through the aerobic zone. PHA enrichment experiments using activated sludge batch reactors fed with acetate, propionate, and 50/50 mixed-feed showed highly variable PHA yield and monomer distribution. In order of relative total PHA yield from lowest to highest, propionate fed batch reactors produced the least total PHA (17% 3HB, 83% 3HV), mixed VFA fed reactors produced more PHA (69% 3HB, 31% 3HV), and acetate fed bioreactors yielded the highest total PHA (78% 3HB, 22% 3HV). Varying doses of acetate resulted in consistent VFA conversion to PHA regardless of concentration. Comparison of PHA production from basins with differing carbon loading showed that 82% of VFA as COD was converted to PHA from sludge from carbon-poor basins, while 45% of VFA as COD was transformed into PHA in sludge from historically carbon-rich basins. These results validate this updated method as a sensitive and accurate method for measuring PHA in wastewater.