Introduction

Coenzyme Q10 (Ubiquinone 10, CoQ10) is an essential lipid-soluble component of membrane-bound electron transport chains. CoQ10 is naturally present in foods and is also synthesized in the body. In humans, CoQ10 boosts energy, enhances the immune system, and acts as an antioxidant (Ernster and Dallner 1995). CoQ10 functions in the transport of electrons from Complex I or II to the cytochrome bc 1 complex found in the inner mitochondrial membrane of eukaryotes and in the plasma membrane of prokaryotes (Trumpowder 1981; Brandt and Trumpower 1994). In addition to its central role in the mitochondrial respiratory chain, CoQ10 is involved in aspects of cellular metabolism and is increasingly being used in therapeutic applications for several diseases such as heart disease, high blood pressure, high cholesterol, immune deficiencies, and Alzheimer’s disease (Choi et al. 2005; Al-Hasso 2001; Singh et al. 1999; Human et al. 1997; Hanioka et al. 1994).

Microorganisms that synthesize CoQ10, including photosynthetic bacteria and yeasts, have been selected and used for the biological synthesis of CoQ10 (Kaplan et al. 1993; Choi et al. 2005). Through fermentation processes, the specific CoQ10 content and CoQ10 production were increased up to 8.7 mg g-dry cell weight (DCW)−1 and 412 mg l−1 of CoQ10 (Yoshida et al. 1998; Lee et al. 2007). A genetically engineered microorganism synthesizing CoQ10 has also been constructed (Lee et al. 2004; Park et al. 2005).

Recently, we searched for CoQ10-producing bacteria and selected one strain of Agrobacterium tumefaciens KCCM 10413 as a potent CoQ10 producer (Lee et al. 2007). Lactate has been reported to influence the content of some metabolites (Matsuoka et al. 1996; Naritomi et al. 1998), is economical and easy to deliver during liquid fermentation. However, its effect on CoQ10 production has not been studied. In the current study, we investigated the effect of lactate on CoQ10 production by A. tumefaciens.

Materials and methods

Microorganism and media

Agrobacterium tumefaciens KCCM 10413, a mutant strain, was used in this study. Cell cultures were maintained in a frozen state at −70°C. The seed medium contained (l−1) 60 g glucose, 15 g yeast extract, 15 g peptone, and 7.5 g sodium chloride. The production medium in batch culture consisted of (l−1) 50 g sucrose, 40 g CSP(Corn steep powder), 10 g (NH4)2SO4, 0.5 g K2HPO4, 0.5 g KH2PO4, 0.25 g MgSO4·7H2O, and 2 g calcium carbonate. To evaluate the effect of the initial concentrations of lactate, the concentrations of lactate (0.5, 1.0, 1.5, 2.0, 2.5 g l−1) were varied in the production media and pH was controlled at 6.5 by ammonium hydroxide (30%).

Culture conditions

A suspension of frozen cells was inoculated into a 250-ml flask containing 50 ml of seed medium and incubated at 32°C and 200 rev min−1 for 12 h. For flask culture, the culture broth of 5 ml was transferred to a 500-ml baffled-flask containing 100 ml of production medium and incubated at 32°C and 200 rev min−1 for times ranging from 72 to 96 h. For fermentor culture, the culture broth of 100 ml, which was obtained from the 500-ml baffled-flask after 12 h incubation, was transferred to a 5-l jar fermentor (KoBiotech Co.) containing a working volume of 2 l production medium. Temperature, agitation speed, and pH during the culture were 32°C, 500–900 rev min−1, and 7.0 respectively. Dissolved oxygen (DO) concentration was measured on a DO-analyser (KoBioTech Co., Ltd.) The pH values of culture broths were monitored using a pH-meter (Mettler Toledo Co., Ltd.) and controlled by ammonium hydroxide (30%).

Fed-batch culture

The working volume in a fed-batch culture increased from 2.0 to 2.8 l by intermittent feeding of 75% sucrose solution. When the sucrose concentration in the culture broth was limited, the feeding solution of 160 ml was added intermittently 5 times. The total added amount of feeding solution was 800 ml (600 g) and total use of sucrose concentration was 250 g l−1 in the fed-batch culture. The other culture conditions of the fed-batch culture were the same as described in fermentor cultures.

Extraction and measurement of CoQ10

For the assay of CoQ10, CelLytic B (Sigma-Aldrich) cell lysis reagent solution of 0.5 ml was added to a cell pellet prepared by centrifugation of 0.5 ml culture broth. After 30 min incubation, a solvent mixture of propanol and hexane (3:5) was added to the solution of cell lysis and mixed vigorously. The solution of solvent phase and that obtained by a second extraction from the aqueous phase were combined and evaporated to dryness using a speed vacuum concentrator (Hanil R&D Co., Ltd.). The dry residue was dissolved in ethanol and applied to a high-pressure liquid chromatography (HPLC) system (Waters 2690) with a Capcell Pak C18 column (Shodex) coupled to a u.v. detector (Waters 486). The column was eluted with a solvent mixture of methanol and ethanol (13:7) at a flow rate of 1.0 ml min−1.

Analysis of cell mass, sucrose, and lactate

The cell mass was determined by using a calibration curve relating optical density at 660 nm and dry cell weight (DCW). The optical density at 660 nm was measured with a spectrophotometer (Beckman DU 530). DCW was determined after the culture broth was centrifuged at 6,000g, washed with distilled water and drying at 121°C for overnight to a constant weight. The concentrations of residual sucrose in the culture broth were determined by the HPLC system (Waters 510) with a high performance carbohydrate column (Waters) coupled to a refractive index detector (Waters 2410). The column was eluted with CH3CN/H2O (80/20, v/v) at a temperature of 30°C and a flow rate of 1.5 ml min−1. The concentrations of residual lactate in the culture broth were determined by the IC system (DX−120, Dionex) with Ionpac ICE-AS6 column (Dionex) and Suppressor device coupled to a conductivity detector (Dionex). The column was eluted with 0.4 mM heptafluorobutyric acid at a temperature of 30°C and a flow rate of 1.0 ml min−1.

Results and discussion

Optimization of nitrogen source for CoQ10 production

To select an optimum nitrogen source for CoQ10 production, various nitrogen sources, such as corn steep powder (CSP), yeast extract, peptone, malt extract, casein, tryptone, and soytone were tested (Fig. 1). The quantity of nitrogen source used corresponded to nitrogen content of 25.0 g of yeast extract l−1. DCW, the specific CoQ10 content, and CoQ10 production were varied widely depending on the nitrogen sources. The highest CoQ10 production was achieved in CSP-containing medium. CSP, a by-product of starch manufacture (Foda et al. 1973) contains 20–25% of lactate and its content depends on the source of corn and processing (Nelson 1994). The effect of lactate on CoQ10 production by A. tumefaciens was further investigated.

Fig. 1
figure 1

Effects of various nitrogen sources (CSP, corn steep powder; YE, yeast extract; ME, meat extract; BE, beef extract; Pep, peptone; Tryp, tryptone; Soy, soytone) on DCW and CoQ10 production by Agrobacterium tumefaciens KCCM 10413 in a 500 ml baffled flask. DCW (), specific CoQ10 content (), and CoQ10 concentration (). The quantity of used nitrogen source corresponds to nitrogen content of 25.0 g l−1 yeast extract. Each value represents the mean of triplicate measurements and varied from the mean by not more than 15%. Each error bar represents the standard deviation of triplicate measurements

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Supplementation of lactate increases DCW and CoQ10 production

In a batch culture, A. tumefaciens produced 48.4 g of DCW l−1 and 320 mg of CoQ10 l−1 in 96 h, corresponding to a specific CoQ10 content of 6.61 mg g-DCW−1. To determine the effect of lactate on DCW and the specific CoQ10 content, various concentrations of lactate (0.5, 1.0, 1.5, 2.0, and 2.5 g l−1) were supplemented (Table 1). DCW increased with the lactate concentration up to 1.5 g l−1 and then decreased significantly above 1.5 g of lactate l−1, but the specific CoQ10 content increased slightly as the lactate concentration increased. The specific CoQ10 content was the highest with 2.5 g of lactate l−1 but DCW was much lower than that with 1.5 g of lactate l−1. Therefore the optimum supplemental lactate concentration for CoQ10 production was found to be 1.5 g l−1, resulting in 55.6 g of DCW l−1, 7.0 mg g-DCW−1 of the specific CoQ10 content, and 389.8 mg l−1 of CoQ10 production, respectively.

Table 1 Effect of supplemental lactate on CoQ10 production by Agrobacterium tumefaciens KCCM 10413 with batch culture. Each value represents the mean of triplicate measurements and varied from the mean by not more than 15%
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Fed-batch culture with lactate supplementation

Lactate supplementation was further tested in a fed-batch culture and the highest CoQ10 production was obtained with 1.5 g of lactate l−1 (Fig. 2). A. tumefaciens produced 62.2 g of DCW l−1 and 562.3 mgof CoQ10 l−1 in 96 h, corresponding to the specific CoQ10 content of 9.1 mg g-DCW−1. In a fed-batch culture, 1.5 g l−1 of supplemental lactate improved DCW and CoQ10 production by 16.0 and 22.8%, respectively.

Fig. 2
figure 2

Effect of lactate supplementation on DCW, CoQ10 production, and the specific CoQ10 content. Cultivations of Agrobacterium tumefaciens KCCM 10413 were performed for 96 h with fed-batch culture. DCW (•), CoQ10 (▼), specific CoQ10 content (▴). Each value represents the mean of triplicate measurements and varied from the mean by not more than 15%. Each error bar represents the standard deviation of triplicate measurements

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It has been reported lactate stimulates cell growth (Roberto and Mayra 2002), and acts as an accelerator driving the tricarboxylic acid cycle (TCA cycle), as well as an energy producer (Matsuoka et al. 1996). According to Naritomi, the ATP content of viable cells growing on the lactate-supplemented medium was maintained at a higher level than that of cells growing on the no-lactate-supplemented medium (Naritomi et al. 1998). In this study, lactate supplementation increased DCW and the specific CoQ10 content in A. tumefaciens culture, probably by accelerating TCA cycle and energy production as reported previously (Roberto et al. 2002; Matsuoka et al. 1996), leading to the increase of CoQ10 production.

Conclusion

The highest CoQ10 production was achieved with CSP as a nitrogen source by A. tumefaciens KCCM 10413. Supplementation with 1.5 g of lactate l−1 further improved CoQ10 production by 22.8% to give 562.3 mg of CoQ10 l−1, which is the highest reported production of CoQ10. Improved CoQ10 production arising from lactate supplementation can be ascribed to the high cell concentration and specific CoQ10 content derived from the acceleration of TCA cycle and energy production. Our results improve the understanding of CoQ10 biosynthesis in A. tumefaciens and should contribute to better industrial production of CoQ10 by biological processes.