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.

Bitumen is a key constitutive material in asphalt pavements. It binds together the rock scaffolding of a pavement. Bitumen provides asphalt pavement with flexibility and enables it to respond to traffic loading and return to its original condition after the loading, i.e. bitumen restores/repairs the damage. In Porous Asphalt (PA) or Stonemastic Asphalt Mix (SMA) asphalt mixtures, due its open graded structure, bitumen modifiers are used to improve or restore bitumen physical and mechanical performance. Traditionally bitumen modifiers are made with products of crude oil, such as: ethylene vinyl acetate (EVA) copolymers and styrene–butadiene–styrene (SBS). As crude oil production declines and the environmental and financial costs of crude oil extraction increase, there is a need to identify environmentally sustainable alternatives to use in bitumen. Bitumen modifiers generated from biological sources offer an environmentally friendly and economically viable alternative to crude oil based bitumen modifiers. This paper describes how PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate), a bio-based co-polymer, might be used as an alternative bitumen modifier. The effect of PHBV on 70/100pen was investigated for this paper. The chemical and physical effect of the PHBV on the bitumen performance was investigated using Gel Permeation Chromatography, Fourier Transformed Infrared Spectroscopy, Differential Scanning Calorimetry, microscopic imaging and Dynamic Shear Rheometer tests. The results indicate that PHBV has significant potential as a bitumen bio-polymer modifier.

(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) obtained from waste/wastewater using a mixed microbial culture (MMC) usually varies in its properties due to daily variation in the waste/wastewater composition applied as feedstock. In the current study, the average molecular weight (MW) of PHBV was purposely reduced from about 1 MDa to about 200 kDa by drying the PHBV-rich biomass at elevated temperature of 120 °C for 18 h to ease extraction and handling. Furthermore, conversion into value-added chemicals such as trans-crotonic acid (trans-CA) and trans-2-pentenoic acid (2-PA) by thermal decomposition (pyrolysis) benefits from the lower MW. For the extraction of low MW PHBV, the use of the bio-based solvents 2-methyltetrahydroxyfuran (2-MTHF) and dihydrolevoglucosenone (cyrene) was studied. The maximum extraction yield of 62 ± 3 % with purity of > 99 % was achieved with 2-MTHF at 80 °C for an hour with high biomass to solvent ratio of 5 % (g/mL). Cyrene-based extractions resulted in the highest yield of 57 ± 2 % with purity of > 99 % at 120 °C in 2 h with 5 % (g/mL) biomass to solvent ratio. The mass balance closure over the extraction process indicated that about 15 % and 10 % of polymer has remained in the residual biomass after extraction by 2-MTHF and cyrene, respectively. The performance of these new solvents to extract polymers with various average MW was compared to the benchmark extractions using chloroform and dimethyl carbonate (DMC). It was found that for the polymers with low average MW the extraction efficiency of the proposed solvents exceeds the benchmark solvents.

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.

In this study, we investigated the operational performance and product spectrum of glucose-fermenting anaerobic granular sludge reactor at pH 4. A selective environment for the growth of granules was implemented by the introduction of a 2 min settling phase, a hydraulic retention time of 6 h and a solid retention time of 12 ± 3 days. The fermentation products were ethanol, lactate, and volatile fatty acids (VFA) with yields of 0.55 ± 0.03, 0.15 ± 0.02, and 0.20 ± 0.04 gram chemical oxygen demand (gCOD)/gCOD glucose, respectively. The obtained product spectrum was remarkably different from the VFA-dominated product spectrum reported in a previous study when the same system was operated at higher pH (4.5–5.5). The shift in product spectrum coincided with a shift in the microbial community structure with the dominance of eukaryotic Candida tropicalis, Pichia jaroonii, and prokaryotic Lactobacillus species instead of the Clostridia species obtained at higher pH-values. The control of the microbiomes and the associated product spectra provides bioprocess engineers with the option to tailor a suitable precursor compound mixture for subsequent chain elongation fermentation or PHA biopolymer production.

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.

Polyhydroxyalkanoate (PHA) accumulating microbial enrichment was established on volatile fatty acids (VFAs) containing leachate derived from the organic fraction of municipal solid waste (OFMSW). The enrichment was based on a 12-h feast-famine batch cycle and an exchange ratio of 50% in which VFAs were completely consumed in less than 50 min during stable periods of operation. No pH control was applied in the system, and the pH went as high as 9 due to the presence of amongst others, ammonia [500 mg·L-1 total ammonia nitrogen (TAN) on average]. The degree of enrichment was evaluated with fluorescence in situ hybridization (FISH), and a yet unknown genus of large (3-5 μm diameter) beta-proteobacteria appeared dominant in the culture. A method for estimating the fraction of PHA accumulating active biomass in the total volatile suspended solids was established, and the results indicated an increase of this fraction from 25% to 56% after implementing two modifications in the operational protocol: (1) a pretreatment of the substrate removing virtually all settleable solids; and (2) a settling phase in the enrichment reactor after the feast phase, selectively removing nonsettleable solids and slowly degradable substrates. The PHA accumulation potential of the culture was 77±18 wt% PHA (n=3) after 3 h in batch accumulation experiments. The results suggest the potential feasibility of PHA production under conditions that were previously considered economically favorable but technically difficult.

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.

The wide variety of organic carbon to nitrogen and phosphorous ratios that are encountered in different wastewaters has a major impact on the poly(3-hydroxybutyrate) (PHB) accumulation potential of microbial communities. In this study we investigated the influence of the substrate composition in terms of the carbon to nitrogen (C/N) or phosphorus (C/P) ratio on the PHB accumulation performance. A multi-reactor set-up was used, enabling parallel experiments using identical inoculum of an enrichment culture dominated by Plasticicumulans acidivorans. In all experiments simultaneous PHB production and growth was observed. Generally, when trace amounts of growth nutrients were present the PHB production yield on substrate remained high for at least 12 h. Interestingly, from the carbon to nutrient ratio in the substrate, the PHB wt% could be accurately predicted in the accumulations. This study demonstrates that strict uncoupling of microbial growth and PHA accumulation is not required for achieving high cellular PHA-contents. Herewith the range of wastewaters that enable a cellular PHA content of 80 % or higher for at least 12 h is expanded to C:N and C:P-ratios exceeding COD:N of 26 gCOD:gNH₄-N and COD:P of 511 gCOD:gPO₄-P respectively.

In this study, the suitability of paper industry wastewater for production of polyhydroxyalkanoate (PHA) was investigated in a pilot reactor in an industrial setting. The pilot plant was designed as a three-step process comprising (1) anaerobic fermentation for maximization of the volatile fatty acid (VFA) concentration, (2) enrichment of PHA-producing biomass, and (3) accumulation for maximization of the PHA content of the biomass. After fermentation, the paper mill process water contained a VFA fraction of 78% on a chemical oxygen demand (COD) basis. The length of the feast phase in the enrichment process stabilized at 45 min±4 min after 18 days of operation. At the end of the feast phase all VFA was consumed and the PHA content of the volatile suspended solids (VSS) was 0.50 g PHA/g VSS±0.05 g PHA/g VSS. The acquired microbial community was dominated by Plasticicumulans acidivorans, a PHA-producing microorganism previously found to dominate VFA-fed laboratory reactors. The maximum PHA content achieved after accumulation was 0.70 to 0.80 g PHA/g VSS. An overall PHA yield of 34% on a COD basis was achieved. Improving the VFA fraction in the product spectrum of the fermentation and minimization of acid and base consumption for pH control were identified as major bottlenecks.