# Fungal-Lactobacteria Consortia and Enzymatic Catalysis for Polylactic Acid Production
## 3 Results and Discussion
### Enzymatic Synthesis of PLA  
The recovery of LA obtained by microbial fermentation is out of the scope of this work. However, the enzymatic synthesis of PLA bioplastic is an interesting objective, since current manufacturing procedures use toxic solvents and/or high temperatures. In this context, a recombinant form of the sterol esterase/lipase naturally secreted by _O. picene_ and produced in _P. pastoris_ (denominated OPEr) has been extensively studied for its efficiency and versatility [37]. Since this enzyme has shown better properties than several commercial lipases in esterification, transesterification and hydrolysis [49], we have evaluated its efficiency for PLA synthesis, either from lactic acid (by direct polycondensation) or from its cyclic dimer, lactic (by enzymatic ring opening polymerization, eROP). The first reaction involves the release of water as a product of the condensation of LA monomers, which deviates the reaction to hydrolysis once a certain amount of PLA has been formed. As a result, the PLA produced by this procedure usually has a low polymerization degree [50]. The samples were qualitatively analyzed by MALDI-TOF (Figure 7), which allowed detecting patterns characteristic of oligomers of different lengths resulting from addition of 72 Da, which corresponds to the mass of LA residues. The signals observed in the spectrum of the negative control (without enzyme) were attributed to the Na\({}^{+}\) adducts of chains from four (329 Da) to seven LA units (547 Da), as LA naturally forms small chains. However, the spectra of samples treated with OPEr showed bigger LA chains, up to 19-LA units (1386 Da), indicating that the condensation reaction occurred. Figure 6: LA production in _T. amestolkiae–L. plantarum_ M9 BG6-B2 consortia growing with industrial starch waste (**A**) or bagasse (**B**) as carbon sources. On the other hand, enzymatic ROP (eROP) is known to produce PLAs of a higher polymerization degree than direct polycondensation. In this case, the substrate for the reaction is lactide, which is a relatively insoluble compound that requires toxic solvents and/or very high temperatures to be polymerized in bulk [51]. Therefore, apart from testing reactions in bulk, we have also analyzed the suitability of using ethyl lactate as a green solvent for the reaction catalyzed by OPEr. PLA synthesis was followed on this occasion by \({}^{1}\)H-NMR (Figure 8). As no peaks from short chain oligomers were observed by MALDI-TOF in a m/z range from 200 Da to 5 kDa, this suggested that longer polymers had been synthesized. The lactide:PLA ratios were similar regardless of how the substrate was incorporated to the reaction mixture (Figure 8A-C). Thus, fractioned addition of lactide does not translate to an improvement of the reaction's efficiency. This is different to what is observed in other synthetic reactions catalyzed by lipases [52]. From the same plots, it can be deduced that reactions take longer to begin in ethyl lactate than in bulk; however, the final conversion at day 3 is virtually complete in ethyl lactate, whereas there is lactide left in bulk reactions. Thus, the solubility of this substrate in ethyl lactate will probably favor its complete polymerization. In terms of substrate conversion only, these results are good compared with some previously published works. [PERSON] et al. [52] catalyzed the eROP of d-lactide with Novozyme 435 (N435) (12.5%, wt) in toluene at 70 \({}^{\circ}\)C for 3 days, and \({}^{1}\)H-NMR analysis revealed a substrate conversion of 33%, quite lower than the conversion obtained here. [PERSON] et al. [53] attempted the eROP of l-lactide in a green solvent as well, the ionic liquid 1-hexyl-3-methylimidazolium hexafluorophosphate [HMIM][PF6] mediated by N435 (10%, wt). The highest l-PLA yield (63%) was attained after 7 days at 90 \({}^{\circ}\)C. Nevertheless, [PERSON] et al.