# Fungal-Lactobacteria Consortia and Enzymatic Catalysis for Polylactic Acid Production
## 3 Results and Discussion
### LA Production from Glucose and Starch and the Effect of QSMs  
Once the agitation (200 rpm) and the basal medium (MRS) were selected from the previous experiment, using _T. amestolkiae_ as a representative fungus we evaluated the efficiency of the synthesis of LA in fungus-bacteria consortia formed by one of the three preselected fungi in combination with the two bacterial strains. As control conditions, the production of lactic acid in pure cultures of the bacterial strains and _R. oryzae_ (this fungus was also capable of releasing lactic acid naturally) was also analyzed. To test the effect of adding _quorum sensing_ molecules, cultures with or without farnesol, dodecanol or 3-oxo-C12-HSL were compared. LA production yield was analyzed by HPLC as described in Section 2.5 and calculated with respect to the theoretical maximum releasable from the substrate (100%) in media with glucose (Figure 2) or pure commercial starch (Figure 3) as the carbon source. As shown in Figure 2, the production of LA in a control medium with glucose and without added QSMs varied from 15% in _R. oryzae_ (labeled as R in Figure 2) to 98% in _L. plantarum_ WCFS1 alone (labeled as W) and in its consortium with _T. amestolkiae_ (TW); despite most of these differences being not statistically significant, trends could be observed. The effect of supplementing with one of the QSMs was different in each case, although in most cases it was neutral or positive for LA release. The most relevant enhancements were due to farnesol (in W, TW and TM). A clear decrease in LA was observed in the consortium _R. oryzae_-_L. plantarum_ M9 MG6-B2 (RM) with farnesol, the consortium _R. oryzae_-_L. plantarum_ WCFS1 with dodecanol and _L. plantarum_ WCFS1 alone with dodecanol. _R. oryzae_ was unsensitie to any of the signal molecules. Figure 2: LA production (% of the theoretical maximum) in pure cultures or co-cultures of the microorganisms selected with glucose as carbon source (24 h, 200 rpm, 28 \({}^{\circ}\)C). Microorganisms: _R. oryzae_ (R), _T. amestolkiae_ (T), _O. picaea_ (O), _L. plantarum_ M9 MG6-B2 (M), _L. plantarum_ WCFS1 (W) and their combinations. Farnesol (FaOH), 3-oxo-C12-HSL (HS) or dodecanol (Dodec.) were added to the cultures and the LA produced was compared with that from untreated cultures (control). The low production of lactate by _R. oryzae_ alone is remarkable [40]; however, it is interesting to note that up to 7 mM succinic acid was also detected in the culture medium, which means a metabolic change to secondary products of the TCA cycle. The production of this acid in trace amounts has been previously reported [41, 42], and the higher concentrations detected in the current work might be due to transformation of components of the MRS medium into this by-product. Its presence may indicate a deviation of the metabolic fluxes affecting the synthesis of LA, and its production is not affected by any of the QSMs used, the combinations with bacteria or the C source. As expected, the maximum LA production from starch (65% corresponding to 72 mM, Figure 3) was, in general, much lower than that from glucose (99% corresponding to 200 mM, Figure 2) since the theoretical maximum yield from glucose is twice that from starch. In terms of percentage regarding the initial substrate concentration, production from glucose is much more efficient since starch consumption requires high amylase activity to depolymerize it to monosaccharides assimilable by the LA producer. In this sense, the disappearance of starch was directly correlated to LA concentration. In control cultures without QSMs, _R. oryzae_ (R) produced a moderate amount of LA (around 30% of the maximum theoretical value), confirming the activity of its extracellular amylase and its intracellular L-lactate dehydrogenase. However, both LAB cultures produced low concentrations of LA. Although the value was slightly higher in _L. plantarum_ WCFS1, the poor amylolytic capacity of this strain in these conditions was corroborated.