Resumen de Polyhydroxtalkanoates production alongside wastewater treatment by mixed microbial cultures
he idea of implementing a circular economy model has enabled the development of new biotechnological processes that maximize the valorisation of wastes using them as raw materials to obtain value-added products. The polyhydroxyalkanoates (PHA) are a group of biodegradable polymers that are biologically synthesized by a wide range of bacteria. There is increasing attention on PHA production because they are fully biodegradable materials and may be produced by using wastes. These biopolymers may be used in different industrial fields because their mechanical and physical properties are very similar to that of petroleum-based plastics. Nevertheless, the industrial PHA production is mainly limited due to the high production costs associated with the use of pure cultures and specific feedstocks and also because of the high downstream process costs.
The scope of this thesis is to demonstrate the feasibility of biological wastewater treatment alongside PHA production as value-added product. For that purpose, the performance and effectiveness of a three-stage process was investigated including: (1) the acidogenic fermentation of different industrial wastes; (2) the enrichment of a mixed microbial culture with PHA-accumulating organisms; and (3) the accumulation step to improve the PHA content of the enriched-biomass. Finally, the PHA synthetized was extracted and characterized.
The use of inexpensive wastes as substrates for PHA production will help to diminish the overall process costs by ca. 50%. In this sense, the acidogenic fermentation was used for the bio-conversion of different wastes into volatile fatty acids (VFA)-rich streams that can be potentially used as precursors for PHA synthesis. In this thesis, different waste activated sludge with variable sludge residence times (SRT), an olive mill wastewater, glycerol, a winterization oil cake and apple pomace were evaluated under batch acidogenic fermentation experiments to determine their degree of acidification (DA). The higher DA was observed for the waste activated sludge with the lower SRT (69% ±1) followed by the olive mill wastewater (48% ±1). The fermented liquid of each waste was also analysed in terms of total VFA content. H2 production was also assessed. The composition of the produced VFA rich stream was analysed in regard to the odd-to-even ration as it is a crucial parameter to determine their suitability for the production of biopolymers with different properties.A higher odd-to-even ratio is obtained when high amount of propionic or valeric acids (odd-chain carboxylic acids) are produced. The higher odd-to-even ratio was obtained for the winterization oil cake (0.86) with propionic acid as the majoritarian VFA followed by acetic acid. The rest of wastes fermented liquor were mainly composed by acetic and butyric acids.
The research work presented herein also includes the start-up of a 16L sequencing batch reactor for the enrichment of a mixed microbial culture able to accumulate PHA. The process was operated under full aerobic conditions using a feast/famine regimen that alternates periods of presence and absence of carbon source. The enrichment step was evaluated at controlled pH (7.5) and without pH control (oscillating in a range of 8.8 to 9.2). The process performance, the biopolymer content and the depletion of the organic fraction was monitored over 120 days. In this case, acetic acid was used as sole carbon source to attain a better understanding of the PHA accumulation mechanisms. The enriched culture obtained without pH control, exhibited a higher PHA content (36%, gPHA g-1VSS) and thus, it was further used in the fed-batch experiments for the PHA accumulation step. The effect of different pHs and nutrients (nitrogen and phosphorus) concentrations on the PHA maximum content was evaluated. The PHA accumulation was found to be higher without using pH control achieving a PHA content up to 44 % gPHA g-1VSS. The effect of nutrients concentrations was evaluated without using a pH control and a maximum PHA content of 51% gPHA g-1VSS was attained under nitrogen limitation.
Following, a synthetic acidified olive-mill-wastewater (OMW) was used as carbon source for the enrichment of a mixed microbial culture to establish its feasibility to produce PHA. The OMW was selected as low-cost substrate that currently represents an environmental concern. The presence of a phenolic fraction in the OMW represent a problem as it may inhibit the PHA accumulation process. In the present study a strategy based on the feeding the OMW into pulses was implemented to achieve both, the wastewater treatment and the enrichment of the mixed microbial culture in PHA-accumulating microorganisms. Around a 90% of organic matter depletion was achieved and the maximum PHA content in the enriched biomass was 33% gPHA g-1VSS. The PHA accumulation capacity of the enriched biomass was evaluated under different split-fed strategies based on the dissolved oxygen (DO) profile. The PHA content and biopolymer composition were also investigated using different carbon sources and a maximum PHA content of 73 % gPHA g-1VSS was achieved using a mixture of 75:25 acetic to propionic acid.
Finally, the PHA extraction was performed using dimethyl carbonate (DMC) as a green solvent and the recovery efficiency was compared with that obtained using chloroform as solvent. The impact of using a NaOCl pre-treatment or an ethanol precipitation post-treatment on the recovery was also explored. A recovery of 25% ±4 of the copolymer P(HB-co-HV) was attained using DMC and NaOCl which was very similar to the result obtained for chloroform extraction (23% ±2). The structure, thermal properties and molecular weights of the extracted biopolymers was determined. PHB exhibited a higher degree of crystallinity in comparison with the copolymer (35.8% and 14.7%, respectively). The decomposition temperature of both biopolymers were around 240°C.
