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.

The developments of mixed culture polyhydroxyalkanoate production has been directed to maximize the biomass PHA content with limited attention to polymer quality. Direct comparison of PHA accumulation literature is challenging, and even regularly contradicting in reported results, due to underlying differences that are not well expressed. A study was undertaken to systematically compare the commonly reported process conditions for PHA accumulation by full-scale municipal activated sludge. A biomass acclimation step combined with a pulse-wise feeding strategy resulted in maximum average PHA contents and product yields. pH control and active nitrification did not result in observable effects on the PHA productivity. Under these conditions a high molecular weight polymer (1536 ± 221 kDa) can be produced. Polymer extraction recoveries were influenced by the PHA molecular weight. A standard protocol for an activated sludge PHA accumulation test including downstream processing and standardized extraction has been developed and is available as supplementary material.

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.

Microbial community-based polyhydroxyalkanoate (PHA) production has been demonstrated repeatedly at pilot scale. Ammonium, normally present in waste streams, might be oxidized by nitrifying bacteria resulting in additional aeration energy demand. The use of low dissolved oxygen (DO) concentrations allowed to reduce nitrifying rates by up to 70% compared to non-oxygen limiting conditions. At lower DO concentrations nitrate was used as alternative electron acceptor for PHA production and therefore, a constant PHA production rate could only be maintained if nitrate was sufficiently available. An optimum DO concentration (0.9 mgO₂/L) was found for which nitrification was mitigated but also exploited to supply requisite heterotrophic nitrate requirements that maintained maximum PHA production rates. PHA accumulations with such DO control was estimated to reduce oxygen demand by about 18%. This work contributes to establish fundamental insight towards viable industrial practice with the control and exploitation of nitrifying bacteria in microbial community-based PHA production.

Volatile fatty acids (VFA) may serve as building blocks for the production of chemicals and polymers. A technology enabling high-rate VFA production from carbohydrate-rich wastewater is the anaerobic granular sludge process. In this study, the characteristics of an anaerobic granular sludge process fermenting glucose was evaluated at different solid retention times (SRT). A lab-scale anaerobic sequencing batch reactor fed with 6 g·L-1 glucose was operated at a pH of 5.5 and at SRT values of 1-2, 10-20, and 40-50 days and operated in total for 215 days. A low sludge volume index (SVI) of 1,144 mL·gTSS-1 allowed for the high SRT and high volatile suspended solid (VSS) concentration that reached 59 gVSS·L-1. This high VSS concentration enabled a glucose consumption rate of 1,100 gCOD·L-1·day-1 at an SRT of 40-50 days. Two product spectra were obtained: (1) a propionate:acetate mixture with a ratio of 2.05∶1 (molpropionate:molacetate) produced at an SRT of 40-50 days; and (2) an acetate dominated product spectrum was obtained at 1-2 days and 10-20 days SRT (0.71-0.75 molacetate·molVFA-1). Overall, a high VFA yield between 0.77 and 0.79 was obtained throughout all enrichments. This work demonstrates that high-rate VFA production combining high yields and low solid concentrations in the effluent technologically can be achieved. This work contributes to the implementation of waste-based production of VFA using anaerobic granular sludge.