Pilot-scale Production of Functionalized mcl-PHA from Grape Pomace Supplemented with Fatty Acids

Amongst the polyhydroxyalkanoate (PHA) family, the class of medium-chain-length PHA ( mcl-PHA) that contains C6 to C14 monomers has attracted a lot of attention due to its extraordinary diversity. In particular, the possibility to adjust material properties by controlling the monomeric composition, and functionalize it via introduction of vinyl groups is of great interest1. Whereas short-chain-length PHAs (scl-PHA, C4-C5 monomers) are readily available on the market2,3, largescale commercialization of mcl-PHA is still to be established. Reasons for this are lower PHA yields, more costly substrates (e.g. fatty acids) as well as the difficulty to control precisely the monomeric composition and thus the material properties of the biopolymer produced. In general, the carbon substrates used for the bacterial fermentations contribute to at least 30 % of the production cost4. Therefore, any notable improvement in this respect would have a remarkable impact on the production cost. Mcl-PHAs are produced as intracellular granules by Pseudomonas from either related substrates like fatty acids via the b-oxidation pathway5 or by unrelated sources like carbohydrates via de novo fatty acid synthesis6. The polymer composition is nearly constant when using unrelated sources, since the monomers are always derived from acetyl-CoA. In contrast, when fatty acids are used as mcl-PHA precursors, it is directly related to their composition and thus can be quite easily adjusted provided a well-defined fermentation strategy is applied7. As a result, fatty acids are normally used to produce tailor-made mcl-PHAs in a reproducible, controlled way. Nevertheless, cheaper alternative carbon sources may be used for the growth phase in order to reduce the overall cost of the bioprocess. The strategy then consists of (1) producing large amounts of biomass from a non-costly substrate (= growth phase), and (2) adding the fatty acids that are necessary for the accumulation of tailor-made mcl-PHAs (= mcl-PHA accumulation phase). As examples, Kim and co-workers designed a 2-step fed-batch cultivation with Pseudomonas putida BM01 using glucose for the growth phase and octanoate for the biopolymer accumulation phase8, and Sun and co-workers did a similar bioprocess with P. putida KT2440 growing on glucose and accumulating mcl-PHA from nonanoic acid9. More recently, Le Meur and co-workers genetically engineered P. putida KT2440 so that it could grow on xylose and produce mcl-PHA from octanoate during the accumulaPilot-scale Production of Functionalized mcl-PHA from Grape Pomace Supplemented with Fatty Acids