## 3 Results
### Spatial Structuration of Synthetic Communities Based on Biofilm Ccultivation. #### 3.2.1 Impact of Submerged and Biofilm Cultivation on Colonization Efficiency  
Most oenologists are interested in new fermentation technologies for optimizing the wine production process, either for quality or displaying particular flavor profiles [28]. However, this kind of study on rice wine production technology is very limited. In the context of mixed cultivation, especially with the presence of filamentous fungi involved in the process, biofilm cultivation design with metal structured packing is interesting in order to understand its impact on biofilm formation and microbial interactions for target flavor compounds contributing to rice wine quality. Figure 3 represents the biomass and microbial evolution on the packing and in planktonic phase during single- and co-cultures. Based on observation, the maximal biofilm development always occurred at the level of the air/liquid interface. According to Figure 3A, _S. cerevisiae_ and _L. plantarum_ could grow on artificial rice media containing soluble starch as the carbon source. It was also found that in liquid phase, _S. cerevisiae_ and _L. plantarum_ promoted the growth of each other in a mutualistic way (Figure 3A,C-E) by comparison with single culture. The presence of _L. plantarum_ in all cases did not affect either ethanol production or biomass attachment on packing. Ethanol production was observed in significant amounts for the cultures made with _S. cerevisiae_, single-, or co-culture (Figure 3B). When _S. cerevisiae_ is absent, only very low amount of ethanol is observed. In the same way, the presence of _S. cerevisiae_ in co-culture contributed to higher amount of biomass when a biofilm cultivation device was used (Figure 3C). On the other hand, there was no evolution for either _S. cerevisiae_ or _L. plantarum_ on the metal packing when they were grown without _R. oryzae_ (data not shown), showing the role of _R. oryzae_ in initial colonization. Figure 2: PCA) plot displaying the different aromatic profiles of rice wine by natural and synthetic communities. (**A**) plot of line distribution of 39 volatile compounds. (**B**) PCA plot discriminating each sample. In all cases, _R. oryzae_ was inoculated at an initial concentration of \(10^{6}\) spores/ml. For the cultures involving either _S. cerevisiae_ or _L. plantarum_, their initial concentration is indicated by 0.1, 1, or 10, corresponding to \(10^{5}\), \(10^{6}\) and \(10^{7}\) CFU/mL, respectively. The same symbols represent the samples from biological replications. 2.2 Impact of Biofilm Mode of Cultivation on Flavor Compound Using Natural and Synthetic Communities  
Natural and synthetic communities were cultivated in biofilm mode in order to investigate the impact of species structuration on flavor production. As a result, using biofilm cultivation mode led to a significant improvement of flavor compound production in comparison with submerged culture, for both for natural and synthetic communities (Table 3). As expected, the metallic support of the biofilm cultivation device sustained the selective development of _R. oryzae_, promoting the structuration of the whole community. This mode of fungal development is also recognized as increasing secretion abilities, probably leading in our case to a higher amyloid activity. Additionally, some unpleasant compounds, i.e., pentanoic acid, isovaleric acid, and acetic acid, were produced in high amounts during classical submerged cultivation and were absent in the biofilm mode of cultivation. This mode of cultivation also led to an increase of some flavor compounds, which are important for the organoleptic properties of fermented beverages, i.e., phenylethyl alcohol and isoamyl alcohol. Figure 3: Biofilm and microbial evolution during single and co-culture in biofilm reactor. **(A)** Optical density OD of the planktonic phase. **(B)** Ethanol production.