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

Thiemo Zambanini,Lars M Blank,Guido Meurer,Nick Wierckx,Joerg M Buescher,Wiebke Kleineberg,Eda Sarikaya

doi:10.1186/s13068-016-0553-7

Thiemo Zambanini, Lars M Blank + Show 5 more

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https://doi.org/10.1186/s13068-016-0553-7

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Abstract

BackgroundIn 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.ResultsHere 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 °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.ConclusionsCombining 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.

Highlights

In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative
The strain U. trichophora TZ1, which recently has been reported as promising production organism for malate from glycerol, is capable of producing 200 g L−1 malic acid at an overall rate of 0.74 g L−1 h−1 reaching a maximal production rate of 1.94 g L−1 h−1 and a yield of 0.31 mol mol−1 (31 % of the theoretical maximum assuming CO2 co-fixation or 41 % assuming no CO2 co-fixation)
Malate concentration, glycerol concentration and OD600 in modified Tabuchi medium (MTM) containing 200 g L−1 crude glycerol, 3.2 g L−1 NH4Cl at 28 °C (37 °C during production phase, 48 h) with DO kept at 30 %. pH was kept at 6.5 by automatic addition of NaOH

Summary

Introduction

In order to establish a cost-efficient biodiesel biorefinery, valorization of its main by-product, crude glycerol, is imperative. The fermentation process was improved by medium optimization resulting in a final titer of 113 from 120 g L−1 glucose as substrate [8] This organism is not applicable for industrial malic acid production, especially for food applications, due to the production of aflatoxins [12]. Besides Escherichia coli [13, 14] and Saccharomyces cerevisiae [15], an Aspergillus oryzae strain has been investigated as production organism This strain, overexpressing a C4-dicarboxylate transporter, pyruvate carboxylase, and malate dehydrogenase produced a final titer of 154 g L−1 malic acid from glucose at a rate of 0.94 g L−1 h−1 [16]

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