Resource recovery from organic waste streams by microbial enrichment cultures

Polyhydroxyalkanoate (PHA) is a natural product that can potentially replace a part of the chemicals and plastics derived from fossil sources. One of the main barriers for market entry of PHA is its relatively high price compared to conventional (fossil) feedstocks. This high price is related to current industrial production methods which are based on the cultivation of pure microbial cultures of a single species that a.o. has to be protected from contaminations from unwanted microorganisms that invade the systems from the surroundings. These production methods consequently have to rely on expensive substrates and pre-sterilized equipment. It was proposed that the costs of PHA production can be reduced significantly by replacing the existing industrial practices with open cultures that do not require sterile conditions and use organic waste streams as a feedstock. Open culture processes have a free exchange with the surroundings and therefore any organism present in nature can in principle enter these systems. To make an open process for PHA production feasible, a selective environment needs to be applied that enriches for species with high PHA accumulation capacity. PHA is produced by numerous microorganisms in natural ecosystems as a reserve compound to balance metabolic requirements during the absence of external energy and carbon sources. Based on this ecological role of PHA, selective environments can be designed that provide a competitive growth advantage to species with a superior PHA producing capacity. One approach for selective cultivation of PHA producing species is the feast-famine process, in which the substrate is dosed in short pulses followed by relatively long periods (hours) of absence of external substrate. This process is relatively well understood viz. controlled conditions at lab-scale e.g. the enrichment of PHA producing cultures dominated by the specialised genus Plasticicumulans (that can accumulate up to 0.9 gPHA gVSS-1) was reported for sequencing batch reactors that were operated under feast-famine conditions and at short solid retention time (i.e. 24 h) in a relatively long cycle (12 h) (Johnson et al. 2009; Jiang et al. 2011). The objective of this thesis was the development of processes for resource recovery from wastewater with microbial enrichment cultures and to evaluate the industrial relevance of waste based PHA production, with a focus on the upstream part of the product chain: the production of PHA rich biomass. To this end, we investigated several topics related to the the production of PHA from waste water using a three-step process: (1) pre-treatment to maximize the VFA concentrations, (2) enrichment of a microbial culture with high PHA storing capacity and (3) maximization of the PHA content in a fed-batch accumulation step. The first chapter contains a general introduction of the topic and an explanation of the relevance and scope of the research. In the second chapter, the pre-treatment of organic waste streams was investigated. The goal was to develop a process for efficient production of a VFA, the preferred substrate for PHA production. A granular sludge process that produces VFA at high rate, yield and purity while minimizing potential operational costs in an anaerobic sequencing batch reactor (ASBR) at low pH was developed using a model substrate (glucose). The inclusion of a short (2 minute) settling phase before effluent discharge enabled effective granulation and very high volumetric conversion rates of 150-300 kgCOD m-3 d-1. The product spectrum remained similar at the tested pH range with acetate and butyrate as the main products, and a total VFA yield of 60-70% on chemical oxygen demand (COD) basis. The requirement for base addition for pH regulation could be reduced from 1.1 to 0.6 mol OH- (mol glucose)-1 by lowering the pH from 5.5 to 4.5. Moreover, a virtually solid-free VFA stream could be achieved, which is advantageous to achieve high PHA contents in the accumulation step. Wastewater often contains a fraction of lipids, these are not easily converted to volatile fatty acids in a pre-fermentation step. In the third chapter of this thesis, the conversion of lipids in the feast-famine process was investigated. It was found that lipids do not contribute to PHA production in a standard feast-famine SBR. Instead, lipid-accumulating organisms were enriched. Further optimisation could potentially lead to a process for lipids recovery from wastewater, for instance for the production of biodiesel. A modelling approach was used to compare the experimental data from the pilot- and lab-scale experiments. There are many models for feast-famine processes found in literature, and the differences between the models used by different research groups hinders easy comparison experimental data. To enable better comparison of experimental results, a (concept) generalized model was developed in chapter four. Based on experimental data available in literature we have proposed model improvements for (1) modeling mixed substrates uptake, (2) growth in the feast phase, (3) switching between feast and famine phase, (4) PHA degradation and (5) modeling the accumulation phase. Finally, we provide an example of a simple uniform model. In chapter five the industrial relevance of waste-based production is investigated in a pilot experiment at an industrial location. The Mars candy bar factory in Veghel, The Netherlands, was selected because of its favourable waste water properties: high VFA and low nitrogen content. The pilot setup was according the earlier described three-step process: (1) fermentable COD was converted into mainly VFA in an anaerobic pre-treatment step resulting in an average VFA fraction of 0.64 gCOD gCOD-1; (2) selective enrichment in a 200 l SBR led to a microbial culture dominated by P. acidivorans; (3) the PHA content of the biomass was maximized in a fed-batch reactor resulting in an average PHA content of 0.7 gPHA gVSS-1. The dominant presence of P. acidivorans indicated that the selective pressure in the pilot experiment was similar to the lab. The difference in the PHA content achieved in pilot and lab (0.9 gPHA gVSS-1) could be explained by two main factors: the presence of non-VFA COD and solids in the wastewater . In chapter six an outlook for future development is provided. To replace existing chemical and polymer feedstocks with PHA, further optimization of the process is required. Amongst others minimization of acid and base consumption for pH control, production of a clean effluent water, and the recycling of effluent water will still significantly contribute to process efficiency. Nevertheless, in the perspective of these results, we believe the optimization of waste-based PHA production in conceptually not limited by the bioprocesses investigated in this thesis. Instead the most important bottleneck for successful market entry is the development of economic down-stream processing and product utilization routes that enable conversion of the PHA-containing sludge into a marketable product.