Research PapersAn improved temperature-triggered process for glutamate production with Corynebacterium glutamicum☆
Introduction
In 1957, a glutamic-acid-producing bacteria, Micrococcus glutamicus, later renamed Corynebacterium glutamicum was discovered [1]. Since that time, the production of glutamate, mainly used as a flavoring agent, has become an important product for industrial exploitation of microbiology. The currently annual production of glutamate is approximately 900 000 tons and increases each year. In view of this fermentation’s economic impact, extensive studies have occurred to select new strains of C. glutamicum able to produce ever-increasing amounts of glutamate. Bacteria sensitive to various means of triggering glutamate excretion have been discovered. So far, major strategies have been developed industrially: biotin limitation and surfactants addition.
Biotin limitation, was the first process used for the production of glutamate. Final concentrations of glutamate between 10 to 30 g/l have been attained in batch cultures depending on to the strain used [2], [3], [4]. However, the best glutamic acid production reported so far (up to 100 g/l on beet molasses) has been achieved by addition of surfactants [5], [6]. Furthermore, this surfactant process facilitates the exploitation of low cost molasses substrates that contain biotin concentrations much higher than would be compatible with biotin-limitation. However, the requirement for large amounts of surfactants, the introduction of a potential contaminant during the fermentation (surfactants are difficult to sterilize), and the extreme sensibility of the surfactant/biomass ratio [7] are major drawbacks to this process. Other glutamate production processes can be found in the scientific literature: penicillin [6], [8] or tetracaı̈n [9] additions were shown to induce glutamate secretion. However poor glutamate productions were obtained during these studies and they are not realistically extrapolable to industrial fermentation’s conditions.
In 1978, Momose and Takagi [10] isolated a Brevibacterium lactofermentum mutant that was able to secrete 20 g/l glutamate on beet molasses after a temperature shift from 30°C to 40°C. With this strain, glutamic acid production was performed on a biotin-rich medium by controlling fermentation temperature. In 1989, Sun et al. [11] reported the production of 60 g/l of l-glutamate by using a temperature-sensitive mutant of Corynebacterium crenatum on rice hydrolyzate but as for the previous work, final concentrations were too low to enable industrial applications to be contemplated.
In this paper, a fed-batch process enabling high glutamate production in a biotin-rich medium by increasing the culture temperature from 33°C to 39°C is described. The fermentation has been characterized kinetically both in the initial growth phase undertaken at 33°C and the production phase after increasing the temperature to 39°C. The performance obtained with this temperature-sensitive strain of C. glutamicum (strain 2262) is similar to that obtained in surfactant-induced fermentation processes.
Section snippets
Bacterial strain and medium composition
The strain used throughout this study was C. glutamicum 2262. The composition of the glutamate production medium used was based on MCGC medium [12] although citrate (used as chelating agent) was replaced by deferoxamine. Glucose was used as sole carbon sustrate. This medium consisted of: 60 g/liter glucose, 3 g/l Na2HPO4, 6 g/l KH2PO4, 2 g/l NaCl, 8 g/l (NH4)2SO4, 0.4 g/l MgSO4, 7 H2O, 40 mg/l FeSO4, 7 H2O, 3.9 mg/l FeCl3, 0.9 mg/l ZnSO4, 7 H2O, 0.3 mg/l CuCl2, 2 H2O, 3.9 mg/l MnSO4, H2O, 0.1
Cell growth of C. glutamicum
C. glutamicum cells were grown in MCGC medium with glucose as the sole carbon source. The culture was carried out in fed-batch process for 24 h (Fig. 1). During growth at 33°C, exponential growth with a specific growth rate of 0.45 h was rapidly established. When this growth temperature was maintained, exponential growth continued with oxygen transfer eventually provoking a change in metabolic behavior with activation of fermentation pathways leading principally to lactate synthesis
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This research was sponsored by the CNRS (GdR 1197), by the AMYLUM-ORSAN company and as part of the Cell Factory program of the European Commission (B104-CT96-0145)