Improving the stability of glutamate fermentation by Corynebacterium glutamicum via supplementing sorbitol or glycerol
© Cao et al.; licensee Springer. 2015
Received: 26 August 2014
Accepted: 22 December 2014
Published: 4 March 2015
Corynebacterium glutamicum is widely used in glutamate fermentation. The fermentation feature of the strain varies sometimes. These variations may lead to the reduction in the ability of the strain to resist environmental changes and to synthesize glutamate, resulting in abnormal glutamate fermentations.
In the abnormal glutamate fermentations, glutamate accumulation stopped after glucose feeding and the final glutamate concentration was at a lower level (50 to 60 g/L). The r NAD +/r NADH ratio was lower than that in normal batch which was reflected by lower oxidation-reduction potential (ORP) value. The abnormal fermentation performance was improved when glucose was co-fed with sorbitol/glycerol at a weight ratio of 5:1 or adding 10 to 15 g/L of sorbitol/glycerol in the initial medium. Under these conditions, glutamate synthesis continued after substrate(s) feeding and final glutamate concentration was restored to normal levels (≥72 g/L). r NAD +/rNADH ratio, ORP, and pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and cytochrome c oxidase (CcO) activities were maintained at higher levels.
Sorbitol and glycerol were not used as carbon sources for the fermentation. They were considered as effective protective agents to increase cells' resistance ability against environmental changes and maintain key enzymes activities.
KeywordsEnzyme activity Fermentative stability Glutamate fermentation Oxidation-reduction potential Protective substance r NAD +/r NADH
NAD+/NADH ratio is indirectly reflected by oxidation-reduction potential (ORP) [5,6], which represented the redox state of the cells. The optimal ORP range corresponding to different fermentation processes is different. For example, maximum lysine yield was obtained when ORP was controlled between the range of −230 and −210 mV, while the preferable ORP range was −275 to −225 mV in homoserine and valine fermentations [6,7]. The redox state of cells is changed if certain auxiliary substances (such as sorbitol and glycerol) are supplemented and intracellular NAD+/NADH ratio can be varied correspondingly . These auxiliary substrates are usually non-repressive carbon sources. They can protect cells against stress in their living environment, enhance cell viability, and reduce the metabolic burden [9–12]. It was reported that the production of alkaline polygalacturonate lyase and lipase increased by 1.85-fold and 8.7-fold, respectively, when the strategy of methanol/sorbitol co-feeding was adopted [10,13]. Arruda and Felipe found that xylitol productivity could be increased by 35% when glycerol was added in the medium . Therefore, fermentation with mixed carbon sources was considered as an effective way to enhance the targeted metabolite productions.
C. glutamicum used in industry is usually stored at 4°C for a short time and replaced regularly. In this way, production fluctuation resulting from strain change can be avoided. However, sometimes the fermentation characteristics of the strain vary, resulting in decreased glutamate synthesis ability and resistance to environmental changes. In such a case, fermentation performance becomes abnormal and glutamate production also ends at a very low level. Glutamate production fluctuated in a large range, and fermentation stability decreased greatly. Frequent rejuvenation is a common solution to this problem. But it is a costly, time-consuming, and troublesome procedure. Furthermore, glutamate is a low value-added product. It is more economical to adopt a simple way with low operation cost to maintain the fermentative stability. The strategy of feeding mixed substrates has been applied in other fermentations. This was an effective method to decrease cell mortality, maintain the enzyme activity, and promote targeted metabolite production [9–11]. Similar studies with regard to glutamate fermentation are also important, but few have been reported. In this study, sorbitol or glycerol was either co-fed with glucose or added in the initial medium, aiming at protecting the cells against environmental change/stress and stabilizing glutamate productions. Meanwhile, the theoretical mechanism was interpreted. The results gained in this study will provide some useful information and reference to the glutamate fermentation industry in terms of stabilizing the glutamate production.
Strain and culture condition
C. glutamicum ATCC13032 was used in this study. The seed microorganism was grown in a shaker at 32°C and 200 r/min for 8 to 10 h in liquid medium containing (in g/L) glucose 25, K2HPO4 1.5, MgSO4 0.6, MnSO4 0.005, FeSO4 0.005, corn slurry 25, and urea 2.5 (separately sterilized). Initial pH was adjusted to 7.0 to 7.2. The medium for jar fermentation contained (in g/L) glucose 140, K2HPO4 1.0, MgSO4 0.6, MnSO4 0.002, FeSO4 0.002, thiamine 5.0 × 10−5, corn slurry 15, and urea 3.0 (separately sterilized).
Method #1: 50% (w/v) sorbitol or glycerol solution was co-fed with the addition of concentrated glucose solution. The feeding ratio of 1:5 (w/w) sorbitol/glycerol versus glucose was applied.
Method #2: 5 to 15 g /L sorbitol or glycerol was added into the initial medium before inoculation. Only glucose was fed when glucose concentration was lower than 20 g/L.
Analytical and measurement methods
Cell concentration was assayed by spectrophotometer at 620 nm (OD620). Glucose and glutamate concentrations were measured by a biosensor (SBA-40C, Shandong Science Academy, Jinan, China). The concentrations of sorbitol and glycerol were analyzed by HPLC (Hitachi Chromaster Organizer, Hitachi, Ltd, Chiyoda-ku, Japan) equipped with an ion exclusion column (Aminex HPX-87H, 300 mm × 7.8 mm, Bio-Rad, Hercules, CA, USA) and a differential refractive index detector at 30°C. The mobile phase was 0.005 mol/L H2SO4 at a flow rate of 0.6 mL/min . Two electronic balances (JA1102, Haikang Co., Shanghai, China) were connected to the computer and used to monitor the feeding amount of glucose and sorbitol or glycerol solution. O2 and CO2 partial pressure in exhaust gas were on-line measured by a gas analyzer (LKM2000, Lokas Co., Daejeon, Korea). CO2 evolution rate (CER), O2 uptake rate (OUR), and respiratory quotient (RQ) were then calculated by standard formula.
Enzyme activity assay
Where E bef(k) and E aft(k) referred to the activities of k-th enzyme (PDH, ICDH, and CcO) before and after glucose/mixed carbon sources feeding.
Modeling and calculation of r NAD +/r NADH ratio
The main products were glutamate and CO2, because the concentrations of other byproducts (lactate, acetate, and other amino acids) were very low.
Pentose phosphate (PP) pathway was ignored because it was not related to generation/consumption of NADH or NAD+.
The intermediate carbon metabolites were in pseudo-steady-state, and the net accumulation of them was 0. But it was not applied for NADH and NAD+. r NAD +/r NADH ratio was closely and positively associated with the NAD+/NADH ratio, while r NAD + actually represented NADH consumption rate (r (C) NADH) and r NADH represented NADH formation rate (r (F) NADH). NADH formation rate (r (F) NADH) could differ with its consumption rate (r (C) NADH), which led the variation in r NAD +/r NADH ratio.
Glutamate fermentation was a non-growth associated process; the cell concentration in production phase basically stayed at a constant level or declined slightly. So, we used the volume reaction rate to replace the specific reaction rate for convenience purpose.
Where r F NAD +(t) is the NAD+ formation rate (mmol/L/h), r F NADH(t) NADH formation rate (mmol/L/h), r U O2(t) O2 uptake rate (mmol/L/h), r F CO2(t) CO2 evolution rate (mmol/L/h), r U CO2(t) CO2 uptake rate (mmol/L/h), r GLC(t) glucose consumption rate (mmol/L/h), and r GLU(t) glutamate production rate (mmol/L/h).
Results and discussion
Fermentation performance of normal and abnormal batches
r NAD + / r NADH ratio at different instants under different operation conditions
Therefore, the abnormal fermentation was due to the decrease of resistance ability in response to the environmental alterations. In addition, the permeability of cell membrane increased to secrete glutamate extensively in the production phase, and the glucose addition easily brought about shock or stress in the living environment. The carbon flux distribution in vivo was adjusted, and consequently, the metabolism of NAD+ and NADH was changed. On the other hand, PDH and ICDH required NAD+ as the coenzyme and less NAD+ amount restricted the enzymes' catalytic actions. As a result, the metabolic flux was redistributed and a series of abnormal effects arose.
Cells might be more tolerant to the environmental change if some osmoregulators, such as trehalose or betaine, were added before or at the same time when the environmental shock/stress occurred. However, trehalose and betaine are expensive. Sorbitol and glycerol were cheaper, and they were also efficient environmental shock/stress protective reagents. Hence, fermentation performance when co-feeding sorbitol/glycerol with glucose or adding sorbitol/glycerol in the initial medium was studied.
Fermentation performance in presence of sorbitol
Fermentation performance in presence of glycerol
From the results above, it could be concluded that abnormal glutamate fermentations could be restored to normal by supplementing the media with sorbitol or glycerol, especially when sorbitol or glycerol was added in the initial medium. It has been reported that sorbitol and glycerol could be assimilated by yeast and Escherichia coli to increase the targeted product yield by effectively providing the required energy [22,23]. Furthermore, both sorbitol and glycerol were used as effective protective agents of cell viability and enzymes due to their hygroscopicity, freezing tolerance, and oxidation resistance. The major role that sorbitol and glycerol played in the restoration of abnormal glutamate fermentation was analyzed subsequently.
Investigation of the role of sorbitol and glycerol during glutamate fermentation
Relative enzymatic activities of PDH, ICDH, and CcO after substrate(s) feeding (26 h) in different batches
0.84 ± 0.057
0.58 ± 0.043
0.72 ± 0.052
0.72 ± 0.036
0.76 ± 0.078
0.72 ± 0.061
1.18 ± 0.051
0.72 ± 0.039
0.97 ± 0.061
0.92 ± 0.032
1.24 ± 0.076
1.27 ± 0.092
0.50 ± 0.063
1.50 ± 0.089
0.79 ± 0.043
0.75 ± 0.057
0.25 ± 0.04
Sorbitol and glycerol served as shield materials during glutamate fermentation. The activity of key enzymes could be properly maintained when supplementing sorbitol or glycerol. The r NAD +/r NADH ratio was increased, and ORP was maintained around the normal range. The stability of glutamate fermentation was improved efficiently by adding sorbitol or glycerol, and the improvement was more obvious when sorbitol/glycerol was added in the initial medium.
Feasibility analysis in industry
The fermentation features of C. glutamicum changed during preservation process and glutamate accumulation stopped after glucose feeding, leading to an abnormal fermentation. This abnormal fermentation performance could be restored to normal by co-feeding sorbitol or glycerol with glucose or adding them in the initial medium. Restoration was more effective when sorbitol or glycerol was added in the initial medium. Glutamate fermentation stability was also improved efficiently. In these cases, sorbitol and glycerol were used as protective agents. When sorbitol or glycerol was added, the adaptive capability of cells to environmental change was promoted and the activities of PDH/ICDH/CcO could be maintained. The usage efficiency of NADH was improved, and r NAD +/r NADH ratio increased to normal level which was reflected by higher ORP value. These results provided theoretical basis and feasibility for stabilizing glutamate fermentation in its industrial production.
The authors thank the financial sponsors from the National High-Tech Program (#2006AA020301) and Major State Basic Research Development Program (#2007CB714303) of China.
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