Utilization of transient electroporation in intensified bioprocessing : a study for the enhancement of L-Glutamate production by corynebacteria

Abstract

Although L-glutamate production by Corynebacteria fermentation has been well-established in the biotechnology industry since 1960’s, the demand for this building block for human consumption is increasing enormously. Researchers have therefore been trying to improve the yield and productivity of amino acids by developing genetically modified organisms or through the use of metabolic engineering tools. In this study, a new approach by which the utilization of transient electropermeabilization for increasing the membrane permeability of these bacteria to L-glutamate secretion will be investigated, thus potentially enhancing the yield of glutamic acid production.

Micrococcus glutamicus (a strain of Corynebacteria) cultivated in CGXII Minimal Medium was successfully used to produce L-glutamate under different growth conditions i.e., biotin limitation, surfactant (Tween 40) addition and ethambutol addition. This study clearly showed that the production of L-glutamate is influenced by the concentration of these agents and by their time of addition into the fermentation medium. The highest amount of L-glutamate was found to be 57mM at a biotin concentration of 1µg l-1. A simple method based on centrifugation and acid-base addition was developed in which L-glutamate could be separated with a purity of 90% from the fermentation broth. This research confirmed that cell growth, viability, substrate consumption and L-glutamate production by M. glutamicus are notably affected by the increase of medium osmolality (caused by the addition of NaCl). This phenomenon is often observed because of the use of high concentration industrial medium or accumulating amino acids into the extracellular medium during fermentation.

This study confirmed that the conductivity of medium in which cells are suspended represents a major barrier for excreting the intracellular protein or enzyme by membrane electropermeabilization, regardless of the electric field strength and the number of pulses applied. The electroenhancement of L-glutamate secretion by the above-mentioned treatments is limited due to the high conductivity of fermentation broth. However, an increase (about 19%) of total protein excreted from M. glutamicus as compared to the control was obtained in cells suspended in a less conductive medium (dH2O) and electroporated at 12.5kV cm-1, 200 and 25µF with 3 pulses. It was also observed that electroporation factors, especially field strengths, pulse numbers, medium conductivity and the configuration of cells have a major impact on cell viability. The yield of electrosecretion will be limited due to the reduction in cell viability during continuous microbial fermentation and metabolite extraction by the high intensity electric pulses used. This research has established many of the factors requiring consideration when using electroporation for bioprocess intensification.