Manipulation of B. Megaterium Growth for Efficient 2-KLG Production by K

Process Biochemistry 45 (2010) 602–606

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Process Biochemistry

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Short communication Manipulation of B. megaterium growth for efﬁcient 2-KLG production by K. vulgare

Jing Zhang a,b, Jie Liu c, Zhongping Shi b, Liming Liu a,c, Jian Chen a,b,* a State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China b The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China c Jiangsu Jiangshan Pharmaceutical Co., Ltd., Jingjiang 214500, China

ARTICLE INFO ABSTRACT

Article history: In the two-step Vitamin C fermentative production, its precursor 2-keto-L-gulonic acid (2-KLG) was Received 5 June 2009 synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The rates of K. Received in revised form 20 November 2009 vulgare cell growth and 2-KLG production were closely related with B. megaterium concentration in the Accepted 28 November 2009 co-culture system. To enhance the 2-KLG production efﬁciency, a strategy of manipulating B. megaterium growth in the co-culture system and properly releasing its intracellular components was introduced. Keywords: Lysozyme was used speciﬁcally to damage B. megaterium cell wall structure and subsequently inhibit its Bacillus megaterium cell growth. When 10,000 U mLÀ1 lysozyme was fed to the co-culture system at 12 h, the growth rate of Ketogulonicigenium vulgare K. vulgare, sorbose consumption rate, and 2-KLG productivity could increase 27.4%, 37.1%, and 28.2%, 2-Keto-L-gulonic acid Lysozyme respectively. Productivity ß 2009 Elsevier Ltd. All rights reserved.

1. Introduction protein excreted from B. megaterium BM302 with sorbose dehydrogenase activity has been identified [15]. However, the Vitamin C or L-ascorbic acid, an essential and exogenous name and specific function of this protein, particularly its role in nutrient for humans and other primates, plays crucial roles in promoting the metabolites secretion remains unclear. As a result, it biochemical and regulatory reactions, such as scavenging reactive is difficult to achieve the target of 2-KLG production enhancement oxygen [1] and acting as a cofactor for metalloenzymes of living in the co-culture system because the rational or targeted organisms [2,3]. Numerous biotechnological methods have been operational way is not available. developed and exploited to increase its yield and productivity [4– Disrupting the cell wall of B. megaterium could be considered as 8]. The synthesized vitamin C production world-wide (approxi- one of the effective ways to stimulate the secretion. Lysozyme, also mately 110,000 tons annually) mostly originates from glucose via known as muramidase or N-acetylmuramide glycanhydrolase (EC the Reichstein process or the two-step fermentation process [9,10]. 3.2.1.17), is one of the candidates for the cell wall disruption. The modern two-step fermentation process, originally developed Although the function and structure of lysozyme have been in China during 1960s [11], uses Ketogulonicigenium vulgare [12] extensively explored [17–19], application of lysozyme as the cell and Bacillus megaterium [13] as the industrial strains to synthesize walls disrupting agent for industrial strains in order to improve 2-keto-L-gulonic acid (2-KLG), the precursor of vitamin C. Among titer, yield, and productivity of fermentation products has not been the two strains, B. megaterium acts as a companion that secretes previously reported. In this study, lysozyme was employed to some metabolites to stimulate the growth of K. vulgare and thus disrupt the B. megaterium cell wall, and the consequent effect on enhances 2-KLG production [14]. In the past few decades, much the co-culture system performance, namely, the K. vulgare growth effort has been devoted to identify those internal and external and 2-KLG production were carefully investigated. metabolites from B. megaterium through biochemical and molecu- lar methods [14–16], and certain proteins or amino acids were 2. Materials and methods suggested to play a role in this process. Two different proteins 2.1. Microorganisms isolated from B. megaterium capable of improving 2-KLG produc- The strains of K. vulgare and B. megaterium used in this study were kindly tivity by K. vulgare have been identified. Recently, a novel 58-kDa provided by Jiangsu Jiangshan Pharmaceutical Co., Ltd., and stored in the Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University.

2.2. Culture conditions * Corresponding authors at: School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China. Tel.: +86 510 85918307; fax: +86 510 85918309. Medium composition for seed culture (medium A) and fermentation (medium B) E-mail addresses: [email protected] (L. Liu), [email protected] (J. Chen). were same as that reported by Yan et al. [20]. The seed culture inoculated from a

1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.016 J. Zhang et al. / Process Biochemistry 45 (2010) 602–606 603 slant was cultivated at 30 8C, 200 rpm, in a 750-mL flask containing 75 mL medium A on a reciprocal shaker for 32 h (K. vulgare), 9 h (B. megaterium), respectively. The seeds were mixed and cultured for another 18 h, and then the mixture of K. vulgare and B. megaterium was transferred into the fermentation medium. The fermenta- tions were carried out in 750-mL flasks containing 75 mL medium B, buffered by À1 5gL CaCO3, or in a 7-L jar fermentor (KF-7 L, Korea Fermentor Co., Inchon, Korea) with 4 L medium B. The inoculums amount was 10% (v/v). In the fermentation with jar fermentor, pH was automatically controlled at 7.0 using 8 M NaOH solution, and the agitation speed was controlled at 400 rpm. The air flow rate was 1.5 L minÀ1. All experiments were done in biological triplicate. All cultivations were carried out at 30 8C.

2.3. Preparation of culture supernatant and cytosolic matrix of B. megaterium

10 mL B. megaterium cell samples were collected at 40 h, and then centrifuged at 4 8C, 7000 Â g for 10 min to obtain culture supernatants. The supernatants were sterilized with 0.22 mm ﬁlters. To obtain cytosolic matrix of B. megaterium,75mL culture medium were collected at 12 h and then centrifuged to separate the cell pellets from the culture. The cell pellets were washed twice with sterile water and disrupted by ultrasonication at 0 8C for 20 min with 30 s on–off intervals. Cell debris was removed by centrifugation at 4 8C, 13,000 Â g for 30 min. The cytosolic matrix was also sterilized with 0.22 mm ﬁlters. At last, the content of proteins in the supernatants and cytosolic matrix of B. megaterium was examined by Bradford assay [21].

2.4. Experimental design

2.4.1. Enhancement of K. vulgare growth and 2-KLG production by B. megaterium In order to determine the K. vulgare growth stimulating components, the following five experiments were designed: (1) K. vulgare were grown alone; (2) K. vulgare were grown with B. megaterium supernatants (0.05 mg mLÀ1 protein); (3) K. vulgare were grown with B. megaterium cytosolic matrix (0.05 mg mLÀ1 protein); Fig. 1. Effect of B. megaterium addition volume (A) and time (B) on K. vulgare growth. (4) K. vulgare were grown with B. megaterium cytosolic matrix and supernatants 5%, 10% and 15% (v/v) B. megaterium broth were added to the culture broth at the (0.05 mg mLÀ1 protein); (5) B. megaterium (10% v/v) and K. vulgare were in mixed beginning of the fermentation (A), and B. megaterium (10%, v/v) added at 0, 2, 4, 6, and cultured. 8 h and harvested at 68 h (B). Initial sorbose concentration was 80 g LÀ1. Error bars indicate standard deviations (n = 3). Statistically significant differences (P < 0.05) 2.4.2. Effect of lysozyme concentration on K. vulgare and B. megaterium cell viability were determined by Student’s t-test and are indicated with an asterisk. ^, Without & ~ and 2-KLG production B. megaterium; , 5% (v/v) B. megaterium; , 10% (v/v) B. megaterium; Â, 15% (v/v) B. Resting cells were prepared as follows: B. megaterium (12 h) and K. vulgare (30 h) megaterium. were collected separately and washed twice with water, then re-suspended and mixed in 750-mL flask with medium B. Lysozyme dissolved in sterile water was added to B. megaterium and K. vulgare resting cells in amount of 1000, 5000 and K. vulgare growth was also investigated in detail (Fig. 1B). It was À1 10,000 U mL , respectively. found that adding B. megaterium at different time could all significantly enhance K. vulgare growth. The highest final cell 2.5. Assay methods density of K. vulgare was achieved when B. megaterium was added 2-KLG in the fermentation broth was determined by HPLC using an Aminex HPX- at the beginning of the fermentation. Delaying addition time, even À1 87H column (Bio-Rad) at 35 8C with a flow rate of 0.5 mL min and 0.4 mM H2SO4 though the promotion effect of K. vulgare growth is continuously as the eluent [22]. Sorbose concentration was determined via the sulfuric anthrone decreased, the final amount of K. vulgare in presence of B. reaction and detected at 620 nm [23]. K. vulgare and B. megaterium were enumerated by hemacytometer count and confirmed by standard plate count. megaterium is still larger than that of the control (without B. megaterium addition). This result indicated that B. megaterium may 2.6. Statistical analysis secrete certain bioactive component to promote K. vulgare growth. This result also strongly suggested that B. megaterium itself or the Student’s t-test was employed to investigate statistical differences of K. vulgare metabolites produced is vital for obtaining high level or amount of growth and 2-KLG production with or without B. megaterium addition. Samples with P values of <0.05 were considered statistically different. K. vulgare. A question remaining unanswered is that, whether the intracellular or extracellular metabolites, or both of them 3. Results and discussion stimulated the growth of K. vulgare. To answer the question, five experiments in the 7-L jar fermentor were designed and conducted 3.1. Enhancement of K. vulgare growth by B. megaterium (see Section 2.4.1) and the results were summarized in Table 1.No enhanced K. vulgare growth was detected only when the Although K. vulgare can self-survive in the fermentation centrifugal supernatant of B. megaterium was supplemented to medium, but the growth is very poor (cfu 7.85 Â 108,36h) the culture. However, the concentration of K. vulgare reached (Fig. 1A). Interestingly, the growth of K. vulgare can be promoted 1.19 Â 109 cfu when adding the sonicated extracts of B. mega- by the presence of B. megaterium in the K. vulgare culture broth, and terium, and similar cell levels could also be achieved for the cases a positive relationship between K. vulgare growth and the number #4 and #5 (refer to Table 1). This result strongly suggested that of B. megaterium bacteria in the mixed culture could be observed as both the cytosolic matrix of B. megaterium and B. megaterium cells shown in Fig. 1A. When 5% and 10% (v/v) B. megaterium broth were stimulated the growth of K. vulgare, and the highest 2-KLG added to the culture broth, the K. vulgare amount increased to production were achieved in mixed culture. Among the different 1.23 Â 109 and 1.25 Â 109 (cfu), respectively, which was 56.6% and culture strategies, the cytosolic matrix of B. megaterium played a 59.2% higher than that of the control. More importantly, the central role in achieving high growth of K. vulgare. The major presence of B. megaterium can efficiently reduce the lag time (6 h, results could be summarized and interpreted as follows: (1) the red line in Fig. 1A) and the time to achieve the highest cfu (9 h, blue highest K. vulgare concentration was detected when adding B. line in Fig. 1A). The effect of the addition time of B. megaterium on megaterium at beginning of the fermentation; (2) K. vulgare growth 604 J. Zhang et al. / Process Biochemistry 45 (2010) 602–606

Table 1 Effect of different B. megaterium components on cell growth and 2-KLG production of K. vulgare. The fermentation time was 68 h. The values are the mean Æ standard deviation for experiments of triplicate bioreactors.

Parameters Cell concentration (cfu mLÀ1) 2-KLG concentration (g LÀ1)

K. vulgare + – 7.85E+08 Æ 7.5E+06 39.8 Æ 0.3 Supernatant of B. megaterium (A) 7.93E+08 Æ 1.1E+07 48.6 Æ 1.1 Cytosolic matrix of B. megaterium (B) 1.19E+09 Æ 3.2E+06 60.7 Æ 1.4 Mixture of A and B 1.22E+09 Æ 5.8E+06 68.2 Æ 1.2 B. megaterium 1.25E+09 Æ 8.2E+06 72.5 Æ 2.6

promotion was effective when B. megaterium was fed in the initial 3.3. Effect of lysozyme on 2-KLG production in K. vulgare and B. phase of the fermentation (before 18 h); (3) the regermination of B. megaterium megaterium spores is thought to utilize a part of carbon source and lead to 2-KLG yield decrease, but do not further facilitate K. vulgare The effect of lysozyme concentration on 2-KLG production was growth. As a result, a promise strategy is to disrupt B. megaterium studied in batch co-cultivation using K. vulgare and B. megaterium cell wall and leak its intracellular components to the culture broth in shake flaks (Table 2). With the increase of lysozyme by lysozyme. concentration, the final cell density of K. vulgare continuously improved, suggesting that the release of B. megaterium cytosolic 3.2. Effect of lysozyme concentration on cell viability in K. vulgare and matrix enhances K. vulgare growth. The improved K. vulgare B. megaterium growth achieved by adding 10,000 U mLÀ1 lysozyme into the culture broth led to a higher sorbose consumption rate (1.38 g As mentioned above, the release of B. megaterium cytosolic (L h)À1) and 2-KLG productivity (1.29 g (L h)À1), which was 23.0% matrix through destruction of its wall structure seems to be an and 24.7% higher than those of the control (no lysozyme addition), efficient and ideal strategy for increasing the titer, yield, and respectively. The addition of lysozyme did not alter the titer and productivity of 2-KLG. As described in several reports, B. yield of 2-KLG (Table 2). Therefore, the specific productivity (g- megaterium and K. vulgare co-existence and co-culture are the KLG (g-cell h)À1) with 10,000 U mLÀ1 lysozyme addition was integrated industrial process of vitamin C production. K. vulgare, decreased 22.8% compared with the corresponding values of the a Gram-negative bacterium, can accumulate a high titer of 2-KLG, control (no lysozyme addition), and this result was identical with the precursor of vitamin C, in the culture broth with the help of B. that reported by Lu et al. [13], in which the specific productivity megaterium, a Gram-positive bacteria. Lysozyme, a 1,4-b-N- was decreased 26.3% when B. megaterium culture supernatant acetylmuramidase could cleave the glycosidic bond between the addition amount increased from 3 to 9 mL. These results suggested C-1 of N-acetylmuramic acid (Mur NAc) and the C-4 of N- that 2-KLG productivity does not depend on the biomass amount of acetylglucosamine (Glc NAc) in the bacterial peptidoglycan. The addition of lysozyme to the culture of B. megaterium and K. vulgare resulted in a pronounced decrease of B. megaterium cell survival rate and colony number. However, the growth of K. vulgare was not affected even when the lysozyme concentration in the broth reached at 10,000 U mLÀ1 (Fig. 2). From the cell wall structure of K. vulgare and B. megaterium, it could speculate that the peptidoglycan layer of the K. vulgare outer membrane can prevent its destruction by lysozyme [18], so the growth of K. vulgare was not affected. On the other hand, the peptidoglycan layer, main components of B. megaterium cell wall, was destroyed and then B. megaterium cell lysed by lysozyme. This result demonstrated that the B. megaterium cytosolic matrix can be released by lysozyme disruption while K. vulgare growth unaffected.

Fig. 3. Time course of the batch fermentation process for K. vulgare and B. megaterium. Initial sorbose concentration was 80 g LÀ1. Error bars indicate standard deviations (n = 3). &, K. vulgare without lysozyme; &, K. vulgare with 10,000 U mLÀ1 lysozyme; ~, B. megaterium without lysozyme; ~, B. megaterium with 10,000 U mLÀ1 lysozyme; *, 2-KLG production without lysozyme; *, 2-KLG Fig. 2. Effect of hen egg white lysozyme on resting cells of K. vulgare and B. production with 10,000 U mLÀ1 lysozyme; ^, sorbose consumption without megaterium. lysozyme; ^, sorbose consumption with 10,000 U mLÀ1 lysozyme. J. Zhang et al. / Process Biochemistry 45 (2010) 602–606 605

K. vulgare only. When the biomass of B. megaterium achieved a Æ 1.9 2.2 0.05 0.01 0.04 maximum value at 12 h, the biomass of K. vulgare and the 2-KLG Æ Æ Æ Æ Æ titer arrived at 2.1 Â 109 cfu mLÀ1 and 72.9 g LÀ1, respectively 75.4 1.4E+09 58.3 0.77 0.91 1.18 1.1E+08 (Table 2). If lysozyme was added at early fermentation stage (before 12 h), the amount of B. megaterium was not large enough to keep K. vulgare optimal growth and 2-KLG titer reach the expected Æ 1.7 1.5 0.04 0.06 0.02 values. When lysozyme was added at later stage (after 12 h), both Æ Æ Æ Æ Æ the sorbose consumption and 2-KLG production were signiﬁcantly 76.3 1.5E+09 66.3 0.87 1.11 1.27 1.2E+08 lower than those of the early addition case (12 h, Table 2). It is predicted that the effect of B. megaterium will reach maximum when lysozyme was added at 12 h since lysozyme will release

Æ most intracellular metabolites. As a result, adding lysozyme 1.2 2.1 0.08 0.05 0.06 Æ Æ Æ Æ Æ around 12 h is the best choice for the fermentation performance enhancement. 77.2 2.1E+09 72.9 0.94 1.40 1.48 1.1E+08

3.4. Batch culture process with lysozyme addition in a jar fermentor Æ 1.1 2.3 0.03 0.04 0.04 Batch fermentation with 10,000 U mLÀ1 lysozyme added at Æ Æ Æ Æ Æ 12 h was conducted in 7-L jar fermentor. K. vulgare grew in same 76.6 8.8E+08 44.3 0.58 0.55 0.96 1.5E+07 pattern as that of control (no lysozyme addition) during 0–12 h. However, after 12 h when lysozyme was added, the growth of K. vulgare, sorbose consumption rate, and 2-KLG productivity Æ 1.5 1.2 0.02 0.01 0.03 increased sharply. Compared with the corresponding values of Æ Æ Æ Æ Æ the control (no lysozyme addition), those rates increased 27.4%, 75.1 7.9E+08 38.5 0.51 0.42 0.82 1.2E+07 37.1%, and 28.2%, respectively (Fig. 3). As a result, the fermentation with lysozyme addition at 12 h could end at 56 h, the time shortened 20.6% as compared with that of control. Æ 1.7 2.8 0.05 0.03 0.01 Æ Æ Æ Æ Æ Acknowledgements 77.4 2.1E+09 72.5 0.94 1.29 1.38 2.2E+08 This work was supported by a grant from the National Outstanding Youth Foundation of China (Grant No.: 20625619);

Æ the National Basic Research Program of China (973) (Grant No.: 1.8 1.9 0.04 0.03 0.01 Æ Æ Æ Æ Æ 2007CB714303); the Key National High-Tech Research and Devel- . 77.3 2.1E+09 72.5 0.94 1.29 1.38 1.9E+08 opment Program of China (863) (Grant No.: 2006AA020303) and the National Key Technology R&D Program of China during the 11th Five-Year Plan Period (Grant No.: 2007BAI46B02 & 2007BAI26B0). Æ B. megaterium 1.1 0.06 0.02 3.5 0.03 Æ Æ Æ Æ Æ References and 76.6 1.9E+09 70.9 0.93 1.27 1.37 2.1E+08 [1] Kayali HA, Tarhan L. The impact of Vitamins C B-1 and B-6 supplementation on antioxidant enzyme activities, membrane total sialic acid and lipid peroxida- tion levels in Fusarium species. Process Biochem 2006;41:1608–13. K. vulgare

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