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An Overlooked Effect of Glycine Betaine on Fermentation: Prevents Caramelization and Increases the <TEX>$\small{L}$</TEX>-Lysine Production
An Overlooked Effect of Glycine Betaine on Fermentation: Prevents Caramelization and Increases the $\small{L}$-Lysine Production
Journal of Microbiology and Biotechnology. 2014. Oct, 24(10): 1368-1376
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : April 07, 2014
  • Accepted : July 08, 2014
  • Published : October 28, 2014
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About the Authors
Jianzhong Xu
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi 214122, P. R. China
Xiuhua Xia
School of Food Science, Jiangnan University, WuXi 214122, P. R. China
Junlan Zhang
OriGene Biotechnology Co., Ltd., WuXi 214122, P. R. China
Yanfeng Guo
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi 214122, P. R. China
Weiguo Zhang
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, WuXi 214122, P. R. China
xujz126@126.com

Abstract
This article focuses on the effects of glycine betaine on preventing caramelization, and increasing DCW and ʟ-lysine production. The additional glycine betaine not only decreased the browning intensity (decreased 4 times), and the concentrations of 5-hydroxymethylfurfural (decreased 7.8 times) and furfural (decreased 12 times), but also increased the availability of glucose (increased 17.5%) for ʟ-lysine production. The DCW and ʟ-lysine production were increased by adding no more than 20 mM glycine betaine, whereas the DCW and ʟ-lysine production were decreased with the reduction of pH values, although pH had a better response to prevent caramelization than did glycine betaine. For ʟ-lysine production, the highest increase (40%) was observed on the media with 20 mM glycine betaine. The crucial enzymes in glycolysis and ʟ-lysine biosynthesis pathway were investigated. The results indicated that additional glycine betaine increases the activity of enzymes in glycolysis, in contrast to the effect of pH. All the results indicated that glycine betaine can be used to prevent caramelization and increase the ʟ-lysine production. By applying this strategy, glucose would not be have to be separated from the culture media during autoclaving so that factories can save production costs and shorten the fermentation period.
Keywords
Introduction
Industrial culture media for ʟ-lysine fermentation contain high concentrations of carbohydrates ( e.g. , glucose and molasses), which have adverse effects for fermentation, such as being non-enzymatic browning-prone during the autoclave sterilization and having high osmolarity. Nonenzymatic browning is the general cause of sugar products darkening [19] , such as caramelization and Maillard. The former is the common name for a group of reactions that occur when carbohydrates are exposed to high temperatures with no amino groups, and the latter takes place in the presence of amino groups [18] . Caramelization is an important reaction in the food industry [19] , but it is not so good for microbial fermentation. There are two main reasons for this phenomenon. One is that the sugar availability is decreased during the caramelization, because a lot of volatile compounds are produced during caramelization [4] . The other is that furfural and 5-hydroxymethylfurfural (5-HMF) are the major compounds in the caramelization product [8] , which are highly toxic to microorganisms [24] . Modig et al . [15] have found that both aldehyde dehydrogenase (ALDH) and pyruvate dehydrogenase (PDH) were strongly inhibited by furfural and less strongly inhibited by 5-HMF. PDH, one of the most important enzymes in the tricarboxylic acid (TCA) cycle, catalyzes the synthesis of acetyl-CoA. In addition, Banerjee et al . [1] have reported that furfural strongly inhibited the hexokinase (HK), which is an important enzyme in glycolysis. The TCA cycle and glycolysis are important for cell growth and metabolites biosynthesis. In order to relieve the restriction of caramelization products (CPs) to the cell growth and to increase the availability of sugar for ʟ-lysine production, the caramelization should be as small as possible. Although caramelization was affected by pH, normally the optimal pH for Brevibacterium or Corynebacterium cell growth was 7.0-7.5 [21] . Therefore, another way should be found to minimize the caramelization during L-amino acid or other organic acids biosynthesis by Brevibacterium or Corynebacterium .
Glycine betaine is one of the major compatible solutes in plants. It is also commonly present in media for microbial growth. In Corynebacterium glutamicum and Brevibacterium flavum , glycine betaine is the preferred osmoregulator [22] . These organisms have been widely used to produce ʟ-lysine in industry [25 - 27] . In order to resist the hyperosmotic stress, glycine betaine must be added into the culture broth during fermentation with C. glutamicum or B. flavum [16] . There are a lot of advantages of the additional glycine betaine: it (i) stimulates the growth rate of the bacterium in hyperosmotic stress condition [10] ; (ii) stabilizes enzymes and resists the decrease of the cytoplasmic volume in cells exposed to hyperosmotic stress condition [22] ; (iii) restores growth and invertase activity at high temperature in vitro [11] ; and (iv) stimulates the respiration recovery rate [22] . In addition, B. flavum is unable to utilize glycine betaine as a sole carbon or nitrogen source, or degrade it [10] . Therefore, glycine betaine is the preferred osmoregulator for B. flavum . However, few people know that glycine betaine is able to prevent caramelization and increase the ʟ-lysine production.
In this paper, to investigate the effects of the additional glycine betaine on preventing caramelization and increasing the ʟ-lysine production, different concentrations of glycine betaine were added to the media before autoclaving and the residual glucose after autoclaving was detected. In addition, the effects of glycine betaine on caramelization, cell growth, and ʟ-lysine production were compared with the effects of pH. This study shows for the first time that glycine betaine is able to prevent caramelization and increase the ʟ-lysine production. Although the increase of the residual glucose and ʟ-lysine production are not large, it is important to note that even the smallest effect has a great impact on the economy of the industrial manufacture for the mass-produced amino acid.
Materials and Methods
- Strains, Growth Media, and Culturing Conditions
Brevibacterium flavum XQ-8 ( B. flavum XQ-8) was screened by repeating random mutagenesis and selected from wild-type strain B. flavum ATCC 14067. B. flavum XQ-8 was resistant to s-2-aminoethyl-ʟ-cysteine (AEC) and Sulfaguanidine (SG), and was a ʟ-threonine auxotroph. This strain was used as the working strain.
For ʟ-lysine fermentation, a preculture was inoculated from a fresh LBG-plate (LB supplemented with 5 g/l glucose) and cultivated overnight. After washing the cells with CgXII-media without carbon source (20g/l (NH 4 ) 2 SO 4 , 5 g/l urea, 1 g/l KH 2 PO 4 , 1 g/l K 2 HPO 4 , 0.25 g/l MgSO 4 ·H 2 O, 42 g/l 3-morpholinopronesulfonic acid, 10mg/l CaCl 2 , 10mg /l FeSO 4 ·7H 2 O, 10mg/l MnSO 4 ·H 2 O, 10mg/l ZnSO 4 ·7H 2 O, 0.2 mg/l CuSO 4 , 0.02 mg/l NiCl 2 ·6H 2 O, 0.2 mg/l biotin, and 0.03 mg/l protocatechuic acid) [9] , the main culture with minimal media was inoculated with OD 600 of 1 in 60ml CgXIIG-media (CgXII supplemented with 40 g/l glucose) in 500 ml Erlenmeyer flasks. To investigate the effects of glycine betaine, glycine betaine was added into the CgXIIG-media to different final concentrations ( i.e. , 5, 10, 15, 20, 25, and 30 mM). These media were adjusted to pH 7.0. To investigate the effects of pH value, the CgXIIG-media were adjusted to different gradient pH values ( i.e. , pH 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0) with HCl or NaOH. The LBG-media and CgXIIG-media were sterilized at 121℃, 0.1 MPa for 20 min. In addition, 0.4 g/l of sterile Lthreonine was added into the culture media. The cultures were cultivated at 30℃ and 120 rpm agitation. In order to eliminate the effect of osmotic pressure, 1.0g/l of sterile ʟ-proline as a compatible solute was added into the culture media after 12 h of incubation [28] .
- Detection of the Browning Intensity at OD420and the Concentrations of 5-HMF and Furfural
The browning intensity of culture media after autoclaving was measured at room temperature at 420 nm (OD 420 ) [17] . 5-HMF and furfural were considered to be a precursor of polymers that lead to the development of brown color during caramelization [1] . Therefore, the concentrations of 5-HMF and furfural were detected after autoclaving. Samples, and 5-HMF and furfural standard solution (Sigma-Aldrich, USA) were filtered (0.45 μm) and analyzed by high pressure liquid chromatography (HPLC; Dionex, USA) with a Kromasil C-18 4.6 × 250mm column (AkzoNobel, Amsterdam, The Netherlands) according to the previous methods reported by Quintas et al . [18] and Mao et al . [13] , respectively.
- Assay of Residual Glucose
The 100 μl samples were diluted 100 times and used later for assaying the residual glucose by an SBA-40E immobilized enzyme biosensor (Shandong, China).
- Assay of Cell Growth and ʟ-Lysine Production
First, 200 μl samples were taken from the Erlenmeyer flasks every 4 h. Then 100 μl of cultures was used to measure the biomass concentration by photometry (Unic, Shanghai, China) at 600 nm after an appropriate dilution or by gravimetric analysis as described previously [6] . Under these experimental conditions, the correlation factor (g of DCW to OD 600 ) between dry cell weight (DCW) and OD 600 was determined as 0 .327 g DCW l -1 OD -1 . The other 100 μl samples were diluted 100 times and used later for determining the ʟ-lysine concentration by an SBA-40E immobilized enzyme biosensor (Shandong, China). The conversion rate of glucose was calculated according to the formula the total of ʟ-lysine production / the total of glucose.
- Enzyme Activity Assay
The crude enzyme was prepared according to the previous methods reported by Hou et al . [6] . After centrifugation at 4℃ for 30 min at 10,000 × g , the cell-free supernatants were immediately used to determine the enzyme activities. Protein concentrations were determined using the Bradford Protein Quantification Kit (Tiangen, Beijing, China) with bovine serum albumin as the standard. The analyses of enzyme activities and protein concentrations were done in triplicate. Specific activity was given as the number of mU/mg of protein. The activities of PDH [2] , HK [23] , AK [7] , and DDH [3] were determined according to the previous reports.
Results
- The Browning Intensity of Culture Media and the Concentration of 5-HMF in Culture Media
The color changes were different among the culture media with different concentrations of glycine betaine or different gradient pH values after autoclave sterilization (data not shown). These results were consistent with the detected results via spectrophotometry at 420 nm ( Fig. 1 A). The browning intensity of culture media without glycine betaine and pH = 7.0 was OD 420 = 0.185 ± 0.0106 - almost 4 times as many as the media containing 30 mM glycine betaine (OD 420 = 0.047 ± 0.0024) and 6.6 times as many as pH = 2.0 (OD 420 = 0.028 ± 0.0032). It is worth pointing out that the effect of glycine betaine on the browning intensity was worse than that of pH ( Fig. 1 A). It is generally known that 5-HMF and furfural are the precursors of colored compounds in the caramelization [18] . Therefore, we detected the concentrations of 5-HMF and furfural in culture media with different concentrations of glycine betaine or different gradient pH values using HPLC ( Fig. 1 B). The 5-HMF concentration of media without glycine betaine and pH = 7.0 was 0.638 ± 0.0511 g/l - almost 7.8 times as many as the media containing 30 mM of glycine betaine (0.081 ± 0.0058 g/l) and 12.8 times as many as pH = 2.0 (0.050 ± 0.0051 g/l). In addition, the 5-HMF concentration of media was decreased with the increase of glycine betaine concentration or the decrease of pH value ( Fig. 1 B). Similar to the effect on 5-HMF, the furfural concentration of media was decreased with the increase of glycine betaine concentration or the decrease of pH value ( Fig. 1 C). The furfural concentration of media without glycine betaine and pH = 7.0 was 0.445 ± 0.0319 g/l - almost 12 times as many as the media containing 30 mM of glycine betaine (0.037 ± 0.0021 g/l) and 23.4 times as many as pH = 2.0 (0.019 ± 0.0026 g/l).
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Effects of glycine betaine (white column) and pH (gray column) on the browning intensity OD420 (A), the concentration of 5-HMF (B), the concentration of furfural (C), and the concentration of residual glucose (D) after autoclaving. I: 5 mM glycine betaine or pH = 2.0; II: 10 mM glycine betaine or pH = 3.0; III: 15 mM glycine betaine or pH = 4.0; IV: 20 mM glycine betaine or pH = 5.0; V: 25 mM glycine betaine or pH = 6.0; VI: 30 mM glycine betaine or pH = 7.0. The standard errors are shown as bars.
- Concentration of Residual Glucose After Autoclaving
A lot of volatile compounds were synthesized during caramelization, such as hydrocarbons, aliphatic aldehydes, ketones etc . [4] , which led to the decrease of the glucose availability. To investigate the effect of glycine betaine or pH on preventing glucose degradation, the residual glucose of culture media with different concentrations of glycine betaine or different gradient pH values after autoclaving was detected by an SBA-40E immobilized enzyme biosensor (see Material and Methods). The glycine betaine or pH strongly affected the concentration of residual glucose after autoclaving ( Fig. 1 D). The residual glucose concentration of culture media without glycine betaine and pH = 7.0 was 30 ± 1.73 g/l, whereas it was increased with the increase of glycine betaine concentration or the decrease of the pH value ( Fig. 1 D). As compared with the media without glycine betaine and pH = 7.0, the residual glucose concentration was increased to 37 ± 1.45 g/l by adding 30 mM glycine betaine into the media or increased to 39 ± 2.14 g/l at pH = 2.0.
- Growth Performance of B. flavum XQ-8 Under Different Culture Conditions
To investigate the influences of the glycine betaine and pH on cell growth during ʟ-lysine fermentation, we analyzed the DCW and growth curve of B. flavum XQ-8 during growth on culture media with different concentrations of glycine betaine or different gradient pH values. There was a small difference in DCW and maximal specific growth rate (u) among the culture media containing different concentrations of glycine betaine, whereas there was a big difference in DCW and maximal specific growth rate among the different gradient pH values ( Figs. 2 A, 2 B, and 2 C). The DCW of B. flavum XQ-8 remained approximately 12.50 g/l on culture media with different concentrations of glycine betaine ( Fig. 2 A and Table 1 ). However, B. flavum XQ-8 could not grow on media with pH = 2.0, and the growth of B. flavum XQ-8 was strongly retarded during pH ≤ 5.0 ( Fig. 2 B). It should be noted that the growth of B. flavum XQ-8 was also retarded on culture media with 25 or 30 mM glycine betaine ( Fig. 2 C).
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The growth performance and ʟ-lysine production of B. flavum XQ-8 in shake flask during growth on CgXIIG medium with different concentrations of glycine betaine or at different pHs. (A) The DCW on media with 5 mM (■), 10 mM (▲), 15 mM (◆), 20 mM (●), 25 mM (*), and 30 mM (×) glycine betaine; (B) the DCW on media at pH = 2.0 (×), pH = 3.0 (●), pH = 4.0 (■), pH = 5.0 (▲), pH = 6.0 (*), and pH = 7.0 (◆); (C) the specific growth rate (u) of B. flavum XQ-8 on media at pH 7.0 with 0 mM (line 1), 5 mM (line 2), 10 mM (line 3), 15 mM (line 4), 20 mM (line 5), 25 mM (line 6), and 30 mM (line 7) glycine betaine. (D) The ʟ-lysine production on media with 5 mM (■), 10 mM (▲), 15 mM (◆), 20 mM (●), 25 mM (*), and 30 mM (×) glycine betaine; (E) the ʟ-lysine production on media at pH = 2.0 (×), pH = 3.0 (●), pH = 4.0 (■), pH = 5.0 (▲), pH = 6.0 (*), and pH = 7.0 (◆). (F) The specific ʟ-lysine production rate (qlys) of B. flavum XQ-8 on media at pH 7.0 with 0 mM (line 1), 5 mM (line 2), 10 mM (line 3), 15 mM (line 4), 20 mM (line 5), 25 mM (line 6), and 30 mM (line 7) glycine betaine. The histogram in Figs. 2A, 2B, 2D, and 2E represents the total DCW and ʟ-lysine production at the end of fermentation. The standard errors are shown as bars.
The DCW, ʟ-lysine yield, biomass-specific ʟ-lysine yield (YP/X), and substrate-specific ʟ-lysine yield (YP/S) ofB. flavumQX-8 in shake flask during growth on CgXIIG medium containing different concentrations of glycine betaine or at different pHs.
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All data are mean values of three determinations of three independent experiments with errors ≤7%.
- ʟ-Lysine Production of B. flavum XQ-8 Under Different Culture Conditions
As compared with growth on culture media without glycine betaine, B. flavum QX-8 showed a higher ʟ-lysine production during growth on media with 5, 10, 15, 20, 25, and 30 mM glycine betaine, at 15 ± 0.76, 16 ± 0.67, 19 ± 1.03, 19 ± 0.68, 21 ± 0.76, 20 ± 0.73, and 17 ± 0.87 g/l, respectively ( Fig. 2 D, Table 1 ). However, the ʟ-lysine production was decreased with the decrease of pH values ( Fig. 2 E). The highest increase (40%) was observed for media with 20 mM glycine betaine, but the increase was also significant for media with 25 mM (33.3%) and 10 or 15 mM (26.7%) glycine betaine. Although the ʟ-lysine production of B. flavum QX-8 was higher, the time of reaching the maximum on media with 15, 20, and 25 mM glycine betaine was longer (peak reached at 32 h) than on media without glycine betaine or pH = 7.0 (peak reached at 28 h, Fig. 2 F). As shown in Fig. 2 E, the ʟ-lysine production was rising constantly during growth on media with pH ≤ 5.0 except on media with pH = 2.0. In addition, the maximal specific ʟ-lysine production rate (q lys ) of B. flavum QX-8 on media with different concentrations of glycine betaine was higher than on media without glycine betaine ( Fig. 2 F). Moreover, the biomass-specific ʟ-lysine yield (Y P/X ) and substrate-specific ʟ-lysine yield (Y P/S ) of B. flavum QX-8 on medium with different concentration of glycine betaine were better than on media with different gradient pH values ( Table 1 ). It is worth pointing out that the highest Y P/X (Y P/X = 1 .661 g [ g DCW] -1 ) and Y P/S (Y P/S = 0.525 g [g glucose] -1 ) were observed during growth on media with 20 mM glycine betaine.
- Activity of Crucial Enzymes in Glycolysis and ʟ-Lysine Biosynthesis Pathway in B. flavum QX-8 During Cultivation on Different Conditions
Previous research has reported that 5-HMF and furfural strongly inhibit the activities of HK and PDH of glycolysis [1 , 15] . To investigate the effects of glycine betaine and pH on the crucial enzymes in glycolysis and ʟ-lysine biosynthesis pathway, we detected the specific activities of HK and PDH (belonging to glycolysis), and AK and DDH (belonging to the ʟ-lysine biosynthesis pathway). As shown in Table 2 , the activity of HK and PDH were increased by adding glycine betaine, whereas the activity of AK and DDH changed very little under different concentrations of glycine betaine. Interestingly, the activities of HK, PDH, AK, and DDH were strongly decreased with the decrease of pH value. It is important to note that the activities of HK and PDH were slightly decreased when adding more than 20 mM glycine betaine.
Specific activity of enzymes ofB. flavumQX-8 in shake flask during growth on CgXIIG medium containing different concentrations of glycine betaine or at different pHs.
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PDH: Pyruvate dehydrogenase; HK: Hexokinase; AK: Aspartokinase; DDH: Diaminopimelate dehydrogenase. Symbol “-”: No detection; All data are mean values of three determinations of three independent experiments with errors≤10%.
Discussion
Industrial culture media for ʟ-lysine fermentation contain high concentrations of carbohydrates, such as glucose and molasses, which will inevitably lead to a high-intensity caramelization during autoclaving. In the present study, we focussed on the overlooked effect of glycine betaine on ʟ-lysine fermentation. Although glycine betaine is wildely used as osmoregulators in ʟ-lysine fermentation by Corynebacterium or Brevibacterium [16] , the viewpoint that the glycine betaine is able to prevent caramelization and increase the ʟ-lysine production is presented for the first time.
There were big differences of color change in CgXIIG medium containing different concentrations of glycine betaine or at different pHs ( Fig. 1 A). The variation of color change is related to the intensity of caramelization, which leads to the color changes [18] . The typical brown color developed during caramelization is attributed to the production of polymeric products [19] , and it is well known that 5-HMF and furfural are the precursors of polymeric products [18] . Thus, the intensity of browning and the concentration of 5-HMF and furfural can be used as measures to determine the intensity of caramelization of culture media. The intensity of browning (OD 420 ) and the concentrations of 5-HMF and furfural were decreased by adding glycine betaine or decreasing pH values. With the addition of more glycine betaine or the lower pH, the intensity of browning and the 5-HMF and furfural concentrations decreased even more ( Figs. 1 A, 1 B, and 1 C). The above experimental results show that glycine betaine is able to prevent caramelization. Interestingly, in the case of the effect on browning intensity and the concentration of 5-HMF concentration of furfural, pH appeared to work better than glycine betaine ( Figs. 1 A, 1 B, and 1 C).
In order to enhance the ʟ-lysine production, it is necessary to increase the availability of sugar and to maintain a certain mount of biomass concentration. As shown in Fig. 1 D, the glycine betaine and pH affected strongly the concentration of glucose after autoclaving. The residual glucose concentration of culture media was increased by adding different concentrations of glycine betaine or decreasing the pH values ( Fig. 1 D). On the one hand, some non-volatile polymeric products were produced [19] , which were not utilized by the microorganism. On the other hand, a lot of volatile compounds were synthesized during caramelization, such as hydrocarbons, aliphatic aldehydes, ketones, etc . [4] , which will dissipate into the ambient air. The above-mentioned results directly affect the availability of glucose for ʟ-lysine production. In addition to the availability of glucose, biomass is an important factor for ʟ-lysine production. There were small differences in the total DCW and specific growth rate among the culture media containing no more than 20 mM glycine betaine ( Figs. 2 A, 2 C). This does not mean that additional glycine betaine does not affect the cell growth. Lee et al . [12] have reported that fermentation in media containing 5-HMF or furfural showed decreased cell growth and longer times to reach the maximum production. As showed in Figs. 1 B, and 1 C, the concentrations of 5-HMF and furfural are decreased by adding glycine betaine. In addition, the availability of glucose is increased by adding glycine betaine ( Fig. 1 D). Based on the above results, the DCW should be gradually increased with increasing the addition of glycine betaine. However, the DCW is regulated by at least three factors: (i) the availability of glucose [25] ; (ii) the concentration of inhibitory compounds, such as 5-HMF and furfural [12] ; and (iii) the osmotic pressure of the culture media [16] . The osmotic pressure will lead to cell lysis or dehydration, thereby preventing cell growth [16] . Additional glycine betaine will change the concentration of substrate and then increase the osmotic pressure, because glycine betaine is not metabolized by C. glutamicum [5] . In addition, Meury [14] has found that the cell growth was decreased early in the addition of glycine betaine, indicating that exogenous glycine betaine may harm cell growth. Although additional glycine betaine leads to an increase in the availability of glucose and to a decrease in the concentrations of 5-HMF and furfural, it is also harmful for cell growth and increases the osmotic pressure. Therefore, the difference of DCW among the different concentrations of glycine betaine is small. Interestingly, there was a big difference in DCW and maximal specific growth rate among the different gradient pH values. The optimal pH for Brevibacterium or Corynebacterium cell growth was 7.0-7.5; this may be a reason why the DCW and maximal specific growth rate had a big difference among the different gradient pH values [21] . It is strange that the DCW of B. flavum XQ-8 on media with 25 and 30 mM glycine betaine was lower than on media without glycine betaine or at pH = 7.0 ( Fig. 2 A). Although the reason for the DCW decrease and growth retardation is obscure, the initial growth retardation phenomenon is similar to previous reports [14] .
As compared with growth on culture media without glycine betaine, B. flavum QX-8 showed a higher ʟ-lysine production during growth on media with different concentrations of glycine betaine ( Fig. 2 D and Table 1 ). This is because the additional glycine betaine increases the availability of glucose but decreases the concentrations of 5-HMF and furfural ( Figs. 1 B, 1 C, and 1 D). However, interestingly, the increase of ʟ-lysine production is not consistent with the increase in concentration of glycine betaine. We can find that the DCW of B. flavum XQ-8 was decreased by adding 25 or 30 mM glycine betaine, which may be the reason for the decrease of ʟ-lysine production during cultivation on culture media with 25 or 30 mM glycine betaine. Moreover, the glucose was not completely consumed during growth on CgXIIG medium containing 25 or 30 mM glycine betaine (data not shown). It should be noted that the time of reaching the maximal ʟ-lysine production on media with more than 5 mM glycine betaine was longer (reached peak at ≥32 h) than on media without glycine betaine or with 5 mM glycine betaine (peak reached at 28 h, Fig. 2 F) for reasons of DCW. However, interestingly, the ʟ-lysine production was decreased with the decrease of pH value, although pH usually does better work than glycine betaine on preventing caramelization ( Fig. 2 E). The decrease of DCW may be at least partly to blame. Because of the increase of the glucose availability, the biomass specific ʟ-lysine yield (Y P/X ) and the substrate-specific ʟ-lysine yield (Y P/S ) were increased by adding glycine betaine ( Table 1 ).The highest Y P/X and Y P/S were observed when adding 20 mM glycine betaine into culture media. This indicates that 20 mM glycine betaine is helpful to increase the ʟ-lysine production during cultivation on media with 40 g/l glucose. Interestingly, although the availability of glucose for ʟ-lysine production was increased, the Y P/X and Y P/S decreased with the decrease of pH value.
As described previously, the activities of HK and PDH were increased by adding no more than 20 mM glycine betaine because of the decrease of the 5-HMF and furfural concentrations [1 , 15] . It is strange that the activities of HK and PDH were slightly decreased when adding more than 20 mM glycine betaine, but the reasons are not clear. We speculate this may be the effect of excessive glycine betaine. Interestingly, the activities of AK and DDH changed very little under different concentrations of glycine betaine, indicating that 5-HMF or furfural does not affect the activities of AK and DDH. It is strange that the activities of HK, PDH, AK, and DDH were decreased with the decrease of pH value ( Table 2 ). The reason could be that the transcription and translation of the enzymes’ genes are interrupted at a low pH value [20] .
In conclusion, we have studied the effect of glycine betaine on the ʟ-lysine production, and demonstrated that glycine betaine is able to prevent caramelization and increase the ʟ-lysine production. These results provide a new strategy that adding glycine betaine into culture media can avoid caramelization during autoclaving. By applying the principle of this strategy, glucose need no longer be separated from the culture media during autoclaving and the wastage of glucose via caramelization is decreased, so that factories can save production costs and shorten the fermentation period.
Acknowledgements
This work was financially supported by the Program of Chinese 863 National High-Tech Research and Development Plan Project (No. 2008AA02Z212) and by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
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Banerjee N , Bhatnagar R , Viswanathan L 1981 Inhibition of glycolysis by furfural in Saccharomyces cerevisiae. Eur. J. Appl. Microbiol. Biotechnol. 11 226 - 228    DOI : 10.1007/BF00505872
Blombach B , Schreiner ME , Moch M , Oldiges M , Eikmanns BJ 2007 Effect of pyruvate dehydrogenase complex deficiency on L-lysine production with Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 76 615 - 623    DOI : 10.1007/s00253-007-0904-1
Correia A , Martin JF , Castro JM 1996 Targeted integration of foreign genes into repetitive sequences of the Brevibacterium lactofermentum chromosome. FEMS Microbiol. Lett. 142 259 - 264    DOI : 10.1111/j.1574-6968.1996.tb08440.x
Fadel HHM , Farouk A 2002 Caramelization of maltose solution in presence of alanine. Amino Acids 22 199 - 213    DOI : 10.1007/s007260200008
Farwick M , Siewe KM , Krämer R 1995 Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum. J. Bacteriol. 177 4690 - 4695
Hou XH , Ge XY , Wu D , Qian H , Zhang WG 2012 Improvement of ʟ-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes. J. Ind. Microbiol. Biotechnol. 39 63 - 72    DOI : 10.1007/s10295-011-1000-1
Jakobsen ØM , Brautaset T , Degnes KF , Heggeset TMB , Balzer S , Flickinger MC 2009 Overexpression of wildtype aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus. Appl. Environ. Microbiol. 75 652 - 661    DOI : 10.1128/AEM.01176-08
Kroh LW 1994 Caramelization in food and beverages. Food Chem. 51 373 - 379    DOI : 10.1016/0308-8146(94)90188-0
Keilhauer C , Eggeling L , Aahm H 1993 Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvBilvN-ilvC operon. J. Bacteriol. 175 5595 - 5603
Kawahara Y , Nakamura T , Yoshihara Y , Ikeda S , Yoshii H 1990 Effect of glycine betaine on the sucrose catabolism of an L-lysine producing mutant of Brevibacterium lactofermentum. Appl. Microbiol. Biotechnol. 34 340 - 343
Kawahara Y , Yoshihara Y , Ikeda S , Hirose Y 1990 Effect of glycine betaine, an osmoprotective compound, on the growth of Brevibacterium lactofermentum. Appl. Microbiol. Biotechnol. 33 574 - 577    DOI : 10.1007/BF00172554
Lee H , Cho DH , Kim YH , Shin SJ , Kim SB , Han SO 2011 Tolerance of Saccharomyces cerevisiae K35 to lignocellulosederived inhibitory compounds. Biotechnol. Bioproc. Eng. 16 755 - 760    DOI : 10.1007/s12257-010-0474-4
Mao LY , Zhang L , Gao NB , Li AM 2012 FeCl3and acetic acid co-catalyzed hydrolysis of corncob for improving furfural production and lignin removal from residue. Bioresour. Technol. 123 324 - 331    DOI : 10.1016/j.biortech.2012.07.058
Meury J 1988 Glycine betaine reverses the effects of osmotic stress on DNA replication and cellular division in Escherichia coli. Arch. Microbiol. 149 232 - 239    DOI : 10.1007/BF00422010
Modig T , Liden G , Taherzadeh MJ 2002 Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem. J. 363 769 - 776    DOI : 10.1042/0264-6021:3630769
Peter H , Bader A , Burkovski A , Lambert C , Krämer R 1997 Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes. Arch. Microbiol. 168 143 - 151    DOI : 10.1007/s002030050480
Phongkanpai V , Benjakul S , Tanaka M 2006 Effect of pH on antioxidative activity and other characteristics of aromatization productions. J. Food Biochem. 30 174 - 186    DOI : 10.1111/j.1745-4514.2006.00053.x
Quintas MAC , Brandão TRS , Silva LM 2007 Modelling colour changes during the caramelisation reaction. J. Food Eng. 83 483 - 491    DOI : 10.1016/j.jfoodeng.2007.03.036
Quintas MAC , Fundo JFC , Silva LM 2010 Sucrose in the concentrated solution or the supercooled “state”: a review of caramelisation reactions and physical behavior. Food Eng. Rev. 2 204 - 215    DOI : 10.1007/s12393-010-9022-4
Qiu RD , Zhu JB , Wang L , Ji XS , Lu CD , Yuan ZY 1999 Expression of recombinant human p53 in methylotrophic yeast Pichia pastoris. Chinese J. Biotechnol. 15 477 - 481
Sheng P 2000 Microbiology. Higher Education Press Beijing (in Chinese).
Skjerdal OT , Sletta H , Flenstad SG , Josefsen KD , Levine DW , Ellingsen TE 1995 Changes in cell volume, growth and respiration rate in response to hyperosmotic stress of NaCl, sucrose and glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum. Appl. Microbiol. Biotechnol. 43 1099 - 1106    DOI : 10.1007/BF00166932
Wierckx N , Koopman F , Ruijssenaars HJ , de Winde JH 2011 Microbial degradation of furanic compounds: biochemistry, genetics, and impact. Appl. Microbiol. Biotechnol. 92 1095 - 1105    DOI : 10.1007/s00253-011-3632-5
Willis AW 1982 Methods in Enzymology: Carbohydrate Metabolism. Academic Press New York.
Xu JZ , Han M , Zhang JL , Guo YF , Qian H , Zhang WG 2013 Improvement of ʟ-lysine production combines with minimization of by-products synthesis in Corynebacterium glutamicum. J. Chem. Technol. Biotechnol.    DOI : 10.1002/jctb.4278.
Xu JZ , Zhang JZ , Guo YF , Jia QD , Zhang WG 2014 Heterologous expression of Escherichia coli fructose-1,6-bisphosphatase in Corynebacterium glutamicum and evaluating the effect on cell growth and ʟ-lysine production. Prep. Biochem. Biotechnol. 44 493 - 509    DOI : 10.1080/10826068.2013.833115
Xu JZ , Zhang JZ , Guo YF , Zai YG , Zhang WG 2013 Improvement of cell growth and production of ʟ-lysine by genetically modified C. glutamicum during growth on molasses. J. Ind. Microbiol. Biotechnol. 40 1423 - 1432    DOI : 10.1007/s10295-013-1329-8
Xu S , Zhou JW , Liu LM , Chen J 2010 Proline enhances Torulopsis glabrata growth during hyperosmotic stress. Biotechnol. Bioproc. Eng. 15 285 - 292    DOI : 10.1007/s12257-009-0131-y