Medium chain length polyhydroxyalkanoate (mcl-PHA) is a form of polyhydroxyalkanoate (PHA) which has properties similar to rubber. However, most of the research on mcl-PHA has been focused on pure cultures with artificial substrates, both of which increase the cost of the production to economically unviable levels. This is especially the case since the biological and the chemical production are very cheap. To reduce the costs, a mixed culture could be used. By investigating which conditions lead to high mcl-PHA production with a mixed culture, a step can be taken towards an economically viable biodegradable rubber replacement. To develop a culture which was able to produce mcl-PHA, an enrichment was first performed in a sequential batch reactor with a feast-famine regime to select for PHA producers. Additional selection pressures like substrate selection, pH, oxygen flow rate and time between carbon source addition and nitrogen source addition were imposed on the culture. After the culture reached pseudo-steady state, an accumulation was done. The substrate was one of the most important selection pressures. Using octanoate as sole carbon and energy source, the microbial community was successfully enriched in a feast-famine regime with the capacity to produce mcl-PHA. The use of octanoate with a pH of 7 and no reduced oxygen flow resulted in mcl-PHA weight percentages of 27 wt% polyhydroxyoctanoate (PHO) and 5 wt% polyhydroxyhexanoate (PHH) during the cycle. This was raised to 30 wt% PHO and 10 wt% PHH during the accumulation. During the second enrichment, the carbon source, and the oxygen flow of the first enrichment were maintained, but the pH was increased to 8. This led to an increase in mcl-PHA production, to 33 wt% PHO and 7 wt% PHH during the cycle and 44 wt% PHO and 9 wt% PHH during the accumulation. Two other condition changes were also investigated: lowered oxygen flow and uncoupling the system. For the lowered oxygen flow, the oxygen flow rate was reduced to 5% of the original flow rate and the increased pH of 8 of the second enrichment was kept. This resulted in similar values as the enrichment with only increased pH. It is therefore unclear whether the lowered oxygen flow rate could be attributed to the high mcl-PHA production or that the culture performed well despite the lowered oxygen flow. The uncoupled system was a system where the nitrogen source was added 2 hours after addition of the carbon source. Based on the oxygen profile and preliminary results of the enrichment, the culture mainly grew after the addition of carbon and little mcl-PHA was produced. However, scl-PHA production was quite high, with a total of 51 wt% PHB. This work proves that high levels of mcl-PHA could be produced from mixed cultures. This is a good step towards producing an economically viable, biodegradable rubber replacement.

The goal of this experiment has been to characterize storage polymers produced during anaerobic digestion under haloalkaline conditions. The project focused on three research questions: the type of storage polymers produced, which conditions selected for the production of storage polymers and which organisms are responsible for the production of the storage polymers. Based on BODIPY® staining of various batch tests with different conditions, it can be concluded that the produced storage polymer is PHA. The innate fluorescence of the methanogens was used to determine that the methanogens are not the micro-organisms producing and storing the PHA. It is unclear which organisms are
responsible for this. The characterisation and the staining of different digestions and the setup of a new batch test which focused on the influence of trace metals, acetate and toxic compounds were unable to determine the exact conditions which selected for storage polymers. Follow-up research is therefore recommended, for example new digestion series with a new inoculum from the hypersaline soda lakes in Russia instead of the mixture of the sediment from the hypersaline soda lakes and biomass from a previous digestion series. It is also recommended to perform a genomics analysis of the microbial community to find a possible PHA producer.