Volatile fatty acids (VFA) may serve as building blocks for Polyhydroxyalkanoates (PHA) production and can be derived from waste streams. Ideal streams for PHA production contain a high Chemical Oxygen Demand (COD) to nutrient ratio, such as (waste)water from a paper-mill factory or candy-bar factory. The (waste)water generated by these companies are usually treated anaerobically with the final product being methane containing biogas. Usually, the methane is burned to produce either heat or electricity. Potentially, more value can be added to these streams by producing VFA and/or PHA. PHA can be produced using microbial enrichment cultures that can be established by cultivation in a selective environment that favours the growth of PHA producing microorganisms. Some advantages of using open cultures are that no sterilization and expensive equipment is required compared to pure culture biotechnology. Open culture biotechnology can be effectively applied when the right selection pressure for a specific microbial trait is identified. The microorganism that is most effective in the given conditions will win the competition, i.e. the strongest will survive. A selection criteria for PHA productis is consuming substrate very fast by first making a storage polymer (in this case PHA) from the supplied substrate. The PHA producers prefer VFA as substrate, hence it is important to maximize the VFA content in the substrate stream. For the production of VFA in the product chain towards PHA it is important to minimize the solid content in the feedstock for PHA production. The objective of the research described in this thesis was to gain more insight in the two-step upstream process for PHA production from agricultural waste streams. The first step concerns the maximization of the VFA concentration in the feedstock. Optimization of VFA production was investigated using the granular sludge process in order to maximize the volumetric VFA production capacity and to minimize the solids concentration in the effluent. Two process variables were investigated regarding the PHA production process. Firstly, the influence of the presence of nutrients on PHA production was investigated using PHA producing enrichment cultures. Secondly, the production of PHA was investigated using the leachate of the organic fraction of municipal solid waste (OFMSW) at pilot scale. @en

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.

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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.

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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.

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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.

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