Significant progress has been made over the past decade with pilot scale polyhydroxyalkanoate (PHA) production by direct accumulation using municipal waste activated sludge (WAS). However, industrial upscaling experiences are still lacking in the research literature. In this study, a demonstration scale (4 m³) PHA production process was operated using industrially relevant equipment and compared favourably to those from parallel pilot scale (200 L) production runs. WAS grab samples from a Dutch full scale municipal wastewater treatment plant (WWTP) was used as the biomass source. Final biomass PHA contents and production yields, that are critical for technology viability, were statistically the same between the experiments conducted at pilot scale (0.41 ± 0.02 gPHA/gVSS and 0.42 ± 0.02 gCOD/gCOD) and demonstration scale (0.45 ± 0.05 gPHA/gVSS and 0.39 ± 0.07 gCOD/gCOD). The results furthermore aligned with previous 1 m³ piloting experiences and five year old historical data that similarly used WAS sourced from the same WWTP. Scalability for the technology and a robustness of the applied PHA production methods using WAS were demonstrated. Temperature and foaming control were identified to be critical to upscaled process engineering and design towards successful industrial implementations. The results of the present study, combined with previously produced PHAs and those historical data, support that feedstock quality predictably determines both the average PHA co-monomer content, as well as the blend distribution. PHA solvent extraction from WAS is inherently a blending process. Extraction homogeneously mixes polymer contributions from collectively stored granules from all species of microorganisms in the biomass. Dried PHA-rich biomass batches can be stockpiled and batches can be blended in extraction processes for both recovery and formulation to reach consistent polymer qualities across production batches. More centralized extraction facilities are therefore anticipated to offer economic benefits due to scale and greater opportunities for product quality specification and control. Research findings are presented herein of the production scale comparative study along with practical perspectives of technological readiness for realizing WAS based industrial scale PHA production, quality control, and the supply chains that will be necessary for successful commercial implementation.

Poly(3-hydroxybutyrate) (PHB) was accumulated in full-scale municipal waste activated sludge at pilot scale. After accumulation, the fate of the PHB-rich biomass was evaluated over two weeks as a function of initial pH (5.5, 7.0 and 10), and incubation temperature (25, 37 and 55 °C), with or without aeration. PHB became consumed under aerobic conditions as expected with first order rate constants in the range of 0.19 to 0.55 d⁻¹. Under anaerobic conditions, up to 63 percent of the PHB became consumed within the first day (initial pH 7, 55 °C). Subsequently, with continued anaerobic conditions, the polymer content remained stable in the biomass. Degradation rates were lower for acidic anaerobic incubation conditions at a lower temperature (25 °C). Polymer thermal properties were measured in the dried PHB-rich biomass and for the polymer recovered by solvent extraction using dimethyl carbonate. PHB quality changes in dried biomass, indicated by differences in polymer melt enthalpy, correlated to differences in the extent of PHB extractability. Differences in the expressed PHB-in-biomass melt enthalpy that correlated to the polymer extractability suggested that yields of polymer recovery by extraction can be influenced by the state or quality of the polymer generated during downstream processing. Different post-accumulation process biomass management environments were found to influence the polymer quality and can also influence the extraction of non-polymer biomass. An acidic post-accumulation environment resulted in higher melt enthalpies in the biomass and, consequently, higher extraction efficiencies. Overall, acidic environmental conditions were found to be favourable for preserving both quantity and quality after PHB accumulation in activated sludge.

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.

Past studies have repeatedly demonstrated the technical feasibility to produce polyhydroxyalkanoate (PHA) using bacterial biomass of mixed microbial cultures (MMCs). Commercial quality grades of poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV, can be produced with control of average monomer composition. However, demonstration of PHBV production and recovery with quality control of molecular weight (MW) distribution has been lacking in the research literature. Towards this goal, a workflow has been developed for characterizing molecular weight control by thermal treatment pre-processing of dried PHA-rich biomass before solvent extraction. Dimethyl carbonate (DMC) was a suitable solvent in this workflow in the routine evaluation of extractable PHA. From assessments of DMC extraction using differential scanning calorimetry, 125 °C was selected for nominally 100 percent extraction yield independent of polymer 3-hydroxyvalerate (3HV) content (2 to 41 wt.% 3HV) and molecular weight (100 to 1400 kDa). Intrinsic viscosity measurements of PHBV in DMC at 60 °C was used for molecular weight monitoring. Mark-Houwink constants, α (0.738 ± 0.010) and LogK (-2.016 ± 0.025), were estimated for a PHBV co-polymer blend having 36 wt.% 3HV. A model of random scission supported that weight average molecular weight (M_w) was a more robust metric, compared to number average molecular weight (M_n), for assessing the polymer scission rates. During isothermal heat treatment for a given biomass batch, interpreted scission rate was reproducible and commonly, but not always, constant in time. Scission rates between biomass batches were also variable. Measured properties of the polymer in the biomass (thermal stability, biomass PHA content, PHBV grade, initial moisture content) could not be correlated to this observed batch-to-batch variation of scission rate. Molecular weight loss before extraction did not influence the melting temperatures of the co-polymer blends of PHBV evaluated over a wide sub-eutectic range of average 3HV content. Molecular weight changes for these PHBV co-polymer blends were considered to have likely influenced the nature of blend 3HV distribution, and consequently, crystallization behaviour. Molecular weight loss effects on crystallization behaviour at constant PHBV average 3HV wt.% content could then have contributed to the observed variability for glass transition temperatures and melting enthalpies. However, a reproducible correlation between this variability and MW change was not observed.

Municipal activated sludge can be used for polyhydroxyalkanoate (PHA) production, when supplied with volatile fatty acids. In this work, standardized PHA accumulation assays were performed with different activated sludge to determine (1) the maximum biomass PHA content, (2) the degree of enrichment (or volume-to-volume ratio of PHA-accumulating bacteria with respect to the total biomass), and (3) the average PHA content in the PHA-storing biomass fraction. The maximum attained biomass PHA content with different activated sludge ranged from 0.18 to 0.42 gPHA/gVSS, and the degree of enrichment ranged from 0.16 to 0.51 volume/volume. The average PHA content within the PHA-accumulating biomass fraction was relatively constant and independent of activated sludge source, with an average value of 0.58 ± 0.07 gPHA/gVSS. The degree of enrichment for PHA-accumulating bacteria was identified as the key factor to maximize PHA content when municipal activated sludge is directly used for PHA accumulation. Future optimization should focus on obtaining a higher degree of enrichment of PHA-accumulating biomass, either through selection during wastewater treatment or by selective growth during PHA accumulation. A PHA content in the order of 0.6 g PHA/g VSS is a realistic target to be achieved when using municipal activated sludge for PHA production.

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.

Activated sludge from municipal wastewater treatment processes can be used directly for the production of biodegradable polyesters from the family of polyhydroxyalkanoates (PHAs). However, municipal activated sludge typically cannot accumulate PHAs to very high levels and often low yields of polymer produced on substrate are observed. In the present work, it was found that the presence of calcium promotes selective growth and enrichment of the PHA-storing biomass fraction and significantly improved both PHA contents and yields. Calcium addition resulted in PHA contents of 0.60 ± 0.03 gPHA/gVSS and average PHA yields on substrate of 0.49 ± 0.03 gCOD_PHA/gCOD_HAc compared to 0.35 ± 0.01 gPHA/gVSS and 0.19 ± 0.01 gCOD_PHA/gCOD_HAc without calcium addition. After 48 h, three times more PHA was produced compared to control experiments without calcium addition. Higher PHA content and selective biomass production is proposed to be a consequence of calcium dependent increased levels of passive acetate uptake. Such more efficient substrate uptake could be related to a formation of calcium acetate complexes. Findings lead to bioprocess methods to stimulate a short-term selective growth of PHA-storing microorganisms and this enables improvements to the techno-economic feasibility for municipal waste activated sludge to become a generic resource for industrial scale PHA production.

Conversion of organic waste and wastewater to polyhydroxyalkanoates (PHAs) offers a potential to recover valuable resources from organic waste. Microbial community-based PHA production systems have been successfully applied in the last decade at lab- and pilot-scales, with a total of 19 pilot installations reported in the scientific literature. In this review, research at pilot-scale on microbial community-based PHA production is categorized and subsequently analyzed with focus on feedstocks, enrichment strategies, yields of PHA on substrate, biomass PHA content and polymer characterization. From this assessment, the challenges for further scaling-up of microbial community-based PHA production are identified.