Enhanced malic acid production from glycerol with high-cell density Ustilago trichophora TZ1 cultivations

Background: In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative. Recently, Ustilago trichophora TZ1 was found to efficiently produce malic acid from glycerol. By adaptive laboratory evolution and medium optimization, titer and rate could be improved significantly. Results: Here we report on the investigation of this strain in fed-batch bioreactors. With pH controlled at 6.5 (automatic NaOH addition), a titer of 142 +/- 1 g L-1 produced at an overall rate of 0.54 +/- 0.00 g L-1 h(-1) was reached by optimizing the initial concentrations of ammonium and glycerol. Combining the potential of bioreactors and CaCO3 as buffer system, we were able to increase the overall production rate to 0.74 +/- 0.06 g L-1 h(-1) with a maximum production rate of 1.94 +/- 0.32 g L-1 reaching a titer of 195 +/- 15 g L-1. The initial purification strategy resulted in 90 % pure calcium malate as solid component. Notably, the fermentation is not influenced by an increased temperature of up to 37 degrees C, which reduces the energy required for cooling. However, direct acid production is not favored as at a lowered pH value of pH 4.5 the malic acid titer decreased to only 9 +/- 1 g L-1. When using crude glycerol as substrate, only the product to substrate yield is decreased. The results are discussed in the context of valorizing glycerol with Ustilaginaceae. Conclusions: Combining these results reveals the potential of U. trichophora TZ1 to become an industrially applicable production host for malic acid from biodiesel-derived glycerol, thus making the overall biodiesel production process economically and ecologically more feasible.