Controlling Biofilm Growth and Lactic Acid Production by Rhizopus oryzae in a Rotating Fibrous Bed Bioreactor: Effects of Dissolved Oxygen, Rotational Speed, and Urea Concentration

Oxygen was found to be the critical factor affecting lactic acid production by Rhizopus oryzae in a rotating fibrous bed bioreactor (RFBB), which was used to immobilize fungal mycelia and control fungal morphology during fermentation. A lack of oxygen resulted in low lactic acid production because the pathway becomes shunted to ethanol production, and it eventually leads to a loss in fungal activity and cell death. However, simply maintaining a high level of dissolved oxygen (DO) in the RFBB did not significantly improve lactic acid production, whereas increasing oxygen transfer rate led to a slight increase in lactic acid productivity. Ethanol production occurred even though a high oxygen transfer rate in the fermentation medium was maintained, indicating an anoxic condition occurring in the biofilm due to diffusion limitation. The effective diffusivities of oxygen and glucose in the biofilm were determined experimentally and then used in a diffusion model to estimate the critical biofilm thickness, which was found to be much smaller than the actual thickness of the biofilm attached on the fibrous matrix, suggesting the occurrence of oxygen starvation in the overgrown biofilm. Controlling the biofilm thickness to prevent oxygen diffusion limitation could be achieved by shaving off the fungal hyphae at the outer region or limiting cell growth by reducing the nitrogen concentration in the medium. Shaving occurs when the shear stress is higher than the hyphae tensile strength, and the shaving rate is proportional to the specific energy dissipation rate, which in turn is affected by the rotational speed of the fibrous matrix. However, increasing the rotational speed did not improve lactic acid production because the shear stress generated from the fluid motion was not high enough to shave off fungal hyphae. The growth and metabolic pathway of R. oryzae immobilized on the fibrous matrix also can be controlled by manipulating the medium composition, particularly the nitrogen source. It was found that biomass production was limited and lactic acid production increased in the absence of urea in the production phase. However, a lack of nitrogen source could reduce cell activity and product synthesis in long-term cultivation. Therefore, periodical addition of urea during the production phase is recommended to prevent biofilm overgrowth, delay sporulation, and maintain high cell viability and fermentation productivity.