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.

Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by using microbial enrichments is a promising but largely unexplored approach to obtain elastomeric biomaterials from secondary resources. In this study, several enrichment strategies were tested to select a community with a high mcl-PHA storage capacity when feeding octanoate. On the basis of analysis of the metabolic pathways, the hypothesis was formulated that mcl-PHA production is more favorable under oxygen-limited conditions than short-chain-length PHA (scl-PHA). This hypothesis was confirmed by bioreactor experiments showing that oxygen limitation during the PHA accumulation experiments resulted in a higher fraction of mcl-PHA over scl-PHA (i.e., a PHA content of 76 wt% with an mcl fraction of 0.79 with oxygen limitation, compared to a PHA content of 72 wt% with an mcl-fraction of 0.62 without oxygen limitation). Physicochemical analysis revealed that the extracted PHA could be separated efficiently into a hydroxybutyrate-rich fraction with a higher M_w and a hydroxyhexanoate/hydroxyoctanoate-rich fraction with a lower M_w. The ratio between the two fractions could be adjusted by changing the environmental conditions, such as oxygen availability and pH. Almost all enrichments were dominated by Sphaerotilus sp. This is the first scientific report that links this genus to mcl-PHA production, demonstrating that microbial enrichments can be a powerful tool to explore mcl-PHA biodiversity and to discover novel industrially relevant strains.

Abstract: Using microbial enrichment cultures for the production of waste-derived polyhydroxyalkanoates (PHAs) is a promising technology to recover secondary resources. Volatile fatty acids (VFAs) form the preferred substrate for PHA production. Isobutyrate is a VFA appearing in multiple waste valorization routes, such as anaerobic fermentation, chain elongation, and microbial electrosynthesis, but has never been assessed individually on its PHA production potential. This research investigates isobutyrate as sole carbon source for a microbial enrichment culture in comparison to its structural isomer butyrate. The results reveal that the enrichment of isobutyrate has a very distinct character regarding microbial community development, PHA productivity, and even PHA composition. Although butyrate is a superior substrate in almost every aspect, this research shows that isobutyrate-rich waste streams have a noteworthy PHA-producing potential. The main finding is that the dominant microorganism, a Comamonas sp., is linked to the production of a unique PHA family member, poly(3-hydroxyisobutyrate) (PHiB), up to 37% of the cell dry weight. This is the first scientific report identifying microbial PHiB production, demonstrating that mixed microbial communities can be a powerful tool for discovery of new metabolic pathways and new types of polymers. Key points: • PHiB production is a successful storage strategy in an isobutyrate-fed SBR • Isomers isobutyrate and butyrate reveal a very distinct PHA production behavior • Enrichments can be a tool for discovery of new metabolic pathways and polymers Graphical abstract: [Figure not available: see fulltext.].

The biotechnological production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from organic waste streams by mixed microbial communities is well established at the pilot-level. However, there is limited research on the recovery of the biopolymer from the microbial biomass, while its impact on product quality and product costs is major. When applying solvent extraction, the choice of solvent has a profound influence on many aspects of the process design. This study provides a framework to perform a systematic solvent screening for PHBV extraction. First, a database was constructed of 35 solvents that were assessed according to six different selection criteria. Then, six solvents were chosen for further experimental analysis, including 1-butanol, 2-butanol, 2-ethyl hexanol (2-EH), dimethyl carbonate (DMC), methyl isobutyl ketone (MIBK), and acetone. The main findings are that the extractions with acetone and DMC obtained the highest yields (91-95%) with reasonably high purities (93-96%), where acetone had a key advantage of the possibility to use water as anti-solvent. Moreover, the results provided new insights in the mechanisms behind PHBV extraction by pointing out that at elevated temperatures the extraction efficiency is less determined by the solvent's solubility parameters and more determined by the solvent size. Although case-specific factors play a role in the final solvent choice, we believe that this study provides a general strategy for the solvent selection process.

Bacteria-based self-healing concrete has the ability to heal cracks due to the bacterial conversion of incorporated organic compounds into calcium carbonate. Precipitates seal the cracks, theoretically increasing the service life of constructions. The aim of this paper is to propose a precursor for bacteria-based self-healing concrete derived from organic waste streams, produced is in line with the circular economy principle and ideally more affordable than other substrates. To verify the applicability of the proposed healing agent, some fundamental requirements of the proposed system are studied, such as its influence on functional properties, crack sealing capacity and evidence of bacterial activity in concrete.

Polyhydroxyalkanoate (PHA) production is a promising opportunity to recover organic carbon from waste streams. However, widespread application of waste-derived PHA as biodegradable plastic is restricted by expensive purification steps, high quality requirements, and a fierce competition with the conventional plastic market. To overcome these challenges, we propose a new application for waste-derived PHA, using it as bacterial substrate in self-healing concrete. Self-healing concrete is an established technology developed to overcome the inevitable problem of crack formation in concrete structures, by incorporating a so-called bacteria-based healing agent. Currently, this technology is hampered by the cost involved in the preparation of this healing agent. This study provides a proof-of-concept for the use of waste-derived PHA as bacterial substrate in healing agent. The results show that a PHA-based healing agent, produced from PHA unsuitable for thermoplastic applications, can induce crack healing in concrete specimens, thereby reducing the water permeability of the cracks significantly compared to specimens without a healing agent. For the first time these two emerging fields of engineering, waste-derived PHA and self-healing concrete, both driven by the need for environmental sustainability, are successfully linked. We foresee that this new application will facilitate the implementation of waste-derived PHA technology, while simultaneously supplying circular and potentially more affordable raw materials for self-healing concrete.