# Fungal-Lactobacteria Consortia and Enzymatic Catalysis for Polylactic Acid Production
## 2 Materials and Methods
### Growth Conditions  
Firstly, bacterium-fungus combinations were tested in liquid medium: GPP (Glucose-Proline-Phosphate) minimal medium [21], Czapek's Dox [29], CDM (chemical defined medium) [30] and plantarum minimal medium (PMM5) [27], all with glucose as a carbon source. In subsequent experiments, we used MRS broth (Condalab) with glucose as a control medium and MRS broth without dextrose and beef extract (Condalab) with 1% (\(w/v\)) starch as the carbon source. In all cases, 15 mL tubes were filled with one-third of its total volume.  
One representative fungus (_T. amestolkiae_), selected for its amyloidtic capacity in the previous screening, was chosen to determine the optimal growth conditions in co-cultures. A 10:1 fungus-bacteria inoculum ratio was used according to previous results [21]. A preinoculum of the selected fungal strains was grown in GPP medium at 28 \({}^{\circ}\)C and 200 rpm for 4 days and filtered through Miracloth to separate spores from hyphae. The spores were counted in a [PERSON] counting chamber under a light microscope (Zeiss, 40\(\times\) magnification), and a spore solution was prepared to inoculate at a final concentration of 5 \(\times\) 10\({}^{6}\) cells/mL. The bacterial inoculum was 10-fold lower (5 \(\times\) 10\({}^{5}\) cells/mL). Both bacterial and fungal microorganisms were co-inoculated in MRS broth with 2% glucose or 1% (\(w/v\)) pure starch from Sigma Aldrich as a carbon source and incubated at 0, 20 or 200 rpm at 28 \({}^{\circ}\)C. Experiments were performed in duplicate. Microbial growth was observed under the light microscope (Zeiss, 40\(\times\) magnification) after 24 h and 72 h of incubation. LA production was measured using the \"l-Lactate and d-Lactate Assay Kits\" (Sigma-Aldrich), as described below.  
The experimental design to test different QSMs and microbial combinations was conducted at 200 rpm and 28 \({}^{\circ}\)C for 72 h, filling one-third of the volume of the 15 mL tubes, and, when necessary, a QSM (farnesol, dodecanol or 3-oxo-C12-HSL) was added at a final concentration of 100 \(\upmu\)M, based on previous works [18]. MRS broth with glucose was used for control experiments, whereas when starch or industrial residues (1% \(w/v\)) were tested, MRS broth without dextrose or beef extract was used. LA production and starch consumption were analyzed by HPLC, as described in Section 2.5. Experiments were performed in triplicate.  
\begin{table}
\begin{tabular}{l c c c} \hline \hline
**Strain** & **Lightening Halo** & \multicolumn{2}{c}{**Colony Diameter (cm)**} \\  & **(cm)** & **Control** & **Starch 1.5\%** \\ \hline _Rhizopus oryzae_ CBS 111718 & Full plate & 3 & 3.5 \\ _Talanromyes amestolkiae_ IJFM A795 & 1 & 2.3 & 4 \\ _Ophiosomica picace_ CECT 20416 & 1 & 0 & 4 \\ _A Aspergillus oryzae_ CECT 20249 & 1 & 2 & 3 \\ _Apergillus oryzae_ ATCC 96995 & 0.6 & 0 & 1.2 \\ _Pencilillium citrium_ CECT 20822 & 0.5 & 1.5 & 1.5 \\ _Hypocrea jecorina_ CECT 20102 & 0.3 & 5.5 & 2.2 \\ _Apergillus nidulans_ MAD2 & 0 & 0 & 4 \\ _Chatomium globosum_ CECT 2701 & 0 & 2.5 & 2.5 \\ _Pencilillium frequentans_ IJFM A569 & 0 & 2.5 & 1.5 \\ _Yarrowia lipolytica_ W29 CBS 7504 & 0.8 & 1.3 & 1.2 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Screening of starch degradation by selected fungal strains.