Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by Propionibacterium freudenreichii ET-3 Fed-Batch Culture

Microorganism and media.ET-3 was used throughout this study. This strain was isolated from Swiss cheese and was stocked at the Food Functionality Research Institute of Meiji Dairies, Odawara, Japan. The preculture medium contained 10% (wt/wt) whey powder (Meiji Dairies, Tokyo, Japan) and 0.1% (wt/wt) beer yeast extract (Asahi Food and Health, Tokyo, Japan). The fermentation medium contained 3.0% (wt/wt) skim milk powder (Meiji Dairies, Tokyo, Japan), 3.4% milk protein concentrate (Murray Goulburn, Melbourne, Australia), and 0.5% (wt/wt) beer yeast extract (Asahi Food and Health). The whey powder was dissolved in a volume of deionized water that was 50 to 70% of the volume that was used to dissolve the medium and was digested using 0.07% (wt/wt) protease (Amano A; Amano Pharmaceutical, Tokyo, Japan) at 47°C for 3 h. The skim milk powder and milk protein concentrate were also dissolved after mixing using the procedure described above and were digested using 0.25% protease (Amano A) at 47°C for 6 h. During enzyme digestion, the pH was controlled in the range from 6.6 to 7.0 using K₂CO₃. The media were autoclaved at 121°C for 15 min.

Culture method.One milliliter of a frozen ET-3 culture stored at −80°C was inoculated into 100 ml of the preculture medium. After static incubation for 48 h at 37°C, 20 ml of the culture was inoculated into a 3-liter jar fermentor (BMS 03PI; Able, Tokyo, Japan) containing 2.0 liters of the fermentation medium. The temperature of the culture was controlled at 33°C, and the pH of the medium was adjusted to 6.5 using K₂CO₃ during the culture period. An anaerobic culture was grown using filter-sterilized nitrogen gas. The agitation speed and the flow rate of nitrogen in the anaerobic culture were controlled at 100 rpm and 0.4 liter min⁻¹, respectively. All the fed-batch cultures in this study were grown under anaerobic conditions and were started 72 h after the anaerobic batch culture was started by feeding a lactose solution at a constant rate. The concentration of the lactose solution fed was 1.5 M, and the lactose feeding rate was maintained in the range from 0.62 to 2.54 ml h⁻¹. The lactose in the anaerobic batch culture was almost entirely depleted after 72 h of culture. Samples of the cultures were taken at 24-h intervals. The volume of the samples withdrawn from the fermentor was almost the same as the volume of the lactose solution added for each 24-h period, which maintained the working volume of the fed-batch cultures at approximately 2.0 liters. For the aerobic batch culture, oxygen gas was supplied at a rate of 0.4 liter min⁻¹, and the agitation speed was controlled at 100 rpm. These aerobic conditions provided the minimum oxygen transfer rate (0.38 mg liter⁻¹ h⁻¹) necessary to induce ET-3 to change its metabolism (5).

Quantification of DHNA, MK, organic acids, and biomass.DHNA, MK, and organic acid contents were measured using a high-performance liquid chromatography system. The details of the measurement procedures used for these products have been described previously (4). Biomass was expressed in terms of dry cell weight.

Effect of lactose limitation on DHNA production.To investigate the effect of lactose limitation on DHNA production, ET-3 was cultured by feeding a lactose solution at a constant rate under anaerobic conditions (Fig. 3). The initial lactose concentration in this culture was 0.08 M, and 0.20 mol of lactose was fed during the fed-batch culture experiment. An anaerobic batch culture was also grown as a control (Fig. 3). To the anaerobic batch culture, 0.20 mol of lactose was added before the culture was started. As shown in Fig. 3, no lactose was detected throughout the lactose feeding period. MK production stopped during the lactose feeding period, although it increased with cell growth in the anaerobic batch culture. In contrast, DHNA production increased in the anaerobic fed-batch culture, and the maximum DHNA concentration in the anaerobic fed-batch culture was 1.7 times that in the anaerobic batch culture. There was no substantial difference in propionate or acetate production between these two cultures.

• Open in new tab
• Download powerpoint

FIG. 3.

Profiles of anaerobic batch and anaerobic fed-batch cultures of ET-3. The anaerobic fed-batch culture was started 72 h after the anaerobic batch culture was started. During the fed-batch culture, 0.20 mol of lactose was fed, and the lactose feeding rate was maintained at 1.90 ml h⁻¹. The concentration of the lactose solution fed was 1.5 M. In the anaerobic batch culture, 0.20 mol of lactose was added before the culture was started. The vertical and horizontal arrows indicate the start time and the period of lactose feeding, respectively. ⋄, anaerobic batch culture (control experiment); ⧫, anaerobic fed-batch culture. DCW, dry cell weight.

Effect of lactose feeding rate on DHNA production.In a preliminary experiment, an anaerobic fed-batch culture in which a lactose solution was fed at a rate of 1.90 ml h⁻¹ was grown for 140 h to add 0.40 mol of lactose (data not shown). In this culture, DHNA production stopped after 0.24 mol of lactose had been added, and the maximum DHNA concentration was 0.30 mM. In our previous study (4), propionate accumulation inhibited DHNA production, and the propionate concentration at the time that DHNA production stopped was 0.35 M; this concentration was higher than the concentration at which DHNA production is inhibited. Therefore, 0.24 mol of lactose was used as the total amount of lactose that was added during the anaerobic fed-batch culture in the subsequent anaerobic fed-batch cultures.

To investigate the optimal lactose feeding rate for DHNA production, anaerobic fed-batch cultures were grown with various feeding rates (Fig. 4). The feeding rates in these anaerobic fed-batch cultures were maintained at 2.54, 1.90, 1.31, 0.90, and 0.62 ml h⁻¹. As shown in Fig. 4, the lactose concentration during all the fed-batch cultures remained constant at approximately zero. The MK concentration and dry cell weight decreased as the lactose feeding rate decreased. However, the DHNA concentration increased as the lactose feeding rate decreased. The maximum DHNA concentration in these anaerobic fed-batch cultures was observed in the cultures with lactose feeding rates of 0.90 and 0.62 ml liter⁻¹ and was 0.40 mM. Therefore, a lactose feeding rate of 0.90 ml liter⁻¹ was employed for the subsequent anaerobic fed-batch cultures by considering the time needed to reach the maximum DHNA concentration. The rates of production of propionate and acetate decreased in proportion to the lactose feeding rate; however, the final concentrations of these organic acids were practically the same in all the cultures (Fig. 4). The maximum DHNA and MK concentrations in each fed-batch culture are shown in Table 1. Table 1 also shows the sum of the maximum DHNA and MK concentrations in each fed-batch culture. The sums are almost the same, ranging from 0.41 to 0.44 mM, regardless of the lactose feeding rate.

• Open in new tab
• Download powerpoint

FIG. 4.

Effects of lactose feeding rate on the production of organic acids, MK, and DHNA and cell growth. During the fed-batch cultures, the lactose feeding rates were maintained at 2.54, 1.90, 1.31, 0.90, and 0.62 ml h⁻¹, and it took 63, 86, 122, 178, and 260 h to feed 0.24 mol of lactose, respectively. The concentration of the lactose solution fed was 1.5 M. The vertical and horizontal arrows indicate the start time and the periods of lactose feeding, respectively. The symbols between the horizontal arrows correspond to the symbols in the plots, as follows: ⋄, 2.54 ml h⁻¹; ⧫, 1.90 ml h⁻¹; ▵, 1.31 ml h⁻¹; ▴, 0.90 ml h⁻¹; ○, 0.62 ml h⁻¹. DCW, dry cell weight.

Table 2 shows the specific lactose consumption, DHNA production, MK production, and cell growth rates in the anaerobic fed-batch cultures. The specific lactose consumption rate decreased in proportion to the lactose feeding rate. The specific MK production and cell growth rates markedly decreased as the lactose feeding rate decreased. In contrast to these results, the specific DHNA production rate remained constant at approximately 0.18 μmol g⁻¹ h⁻¹ when the lactose feeding rate was maintained at more than 0.90 ml h⁻¹.

DHNA production after a switch from an anaerobic fed-batch culture to an aerobic culture.As described above, DHNA production was inhibited by propionate that accumulated in the anaerobic fed-batch culture. However, in our previous study (4) we showed that ET-3 consumed propionate and produced DHNA continuously even after propionate accumulated by switching from anaerobic conditions to aerobic conditions at the time of lactose depletion. As shown in Fig. 2B, under aerobic conditions, propionibacteria consumed propionate and produced acetate via the reverse methylmalonyl coenzyme A pathway. To decrease the inhibitory effect of propionate on DHNA production, an anaerobic fed-batch culture was switched to an aerobic batch culture at the end of the lactose feeding period (Fig. 5). The propionate concentration started to decrease immediately after the switch to aerobic conditions, and the acetate concentration increased continuously even after the end of lactose feeding. DHNA was produced continuously after the end of lactose feeding, although the level of MK produced was low throughout the cultivation. However, an increase in the dissolved oxygen (DO) concentration was observed from 96 to 120 h in the aerobic batch culture (data not shown). The DHNA concentration decreased sharply during this time because the structure of DHNA is sensitive to oxidation. The maximum DHNA concentration in this culture was 0.51 mM.

• Open in new tab
• Download powerpoint

FIG. 5.

Profiles of cultivation in which an anaerobic fed-batch culture was switched to an aerobic culture. During the fed-batch culture, the lactose feeding rate was maintained at 0.90 ml h⁻¹, and 0.24 mol of lactose was fed. The concentration of the lactose solution fed was 1.5 M. The vertical arrow indicates the start time of lactose feeding. The solid and dotted horizontal arrows indicate the periods of lactose feeding and aerobic culture, respectively. DCW, dry cell weight.

DHNA production by repeated alternation between anaerobic fed-batch and aerobic cultures.Propionibacteria are sensitive to oxygen (2, 21, 25). In our previous study (3, 4) we also showed that in aerobic cultures grown for a long time there was an increase in the DO concentration, resulting in a marked decrease in the DHNA concentration; this was despite the fact that the concentration of propionate as a carbon source at the time was sufficiently high. To protect ET-3 from exposure to oxygen for a long period, a cultivation method involving repeated alternation between anaerobic and aerobic batch cultures was developed in our previous study (3). This cultivation method increased the DHNA concentration continuously by maintaining the DO concentration at approximately zero. In this study, we applied this method to a cultivation in which an anaerobic fed-batch culture was switched to an aerobic batch culture (Fig. 6). As shown in Fig. 6, the aerobic batch culture was switched to the anaerobic fed-batch culture before an increase in the DO concentration occurred, and the anaerobic fed-batch and aerobic batch cultures were repeated twice alternately. Throughout the experiment, the DO concentration remained constant at approximately zero, and the propionate concentration was lower than the concentration at which DHNA production is inhibited. However, DHNA production stopped immediately after the start of the second fed-batch culture, and the maximum DHNA concentration in this cultivation was 0.52 mM; this concentration was almost the same as that observed in the cultivation in which an anaerobic fed-batch culture was switched to an aerobic batch culture, as described above.

• Open in new tab
• Download powerpoint

FIG. 6.

Profiles of cultivation in which an anaerobic fed-batch culture was repeatedly alternated with an aerobic culture. During the fed-batch culture, the lactose feeding rate was maintained at 0.90 ml h⁻¹. During the first and second feeding periods, it took 178 and 90 h to feed 0.24 and 0.12 mol of lactose, respectively. The concentration of the lactose solution fed was 1.5 M. The vertical arrow indicates the start time of lactose feeding. The solid and dotted horizontal arrows indicate the periods of lactose feeding and aerobic culture, respectively. DCW, dry cell weight.

At the time that DHNA production stopped in this cultivation, the acetate concentration was 0.45 M. Acetate also inhibits DHNA production, although the inhibitory effect of acetate is much weaker than that of propionate (4). To investigate the effect of acetate accumulation on DHNA production, 0.84 mol of acetate was added 48 h after the start of an anaerobic batch culture with a 2.0-liter working volume (data not shown). In this culture, DHNA production stopped immediately after the addition of acetate, whereas DHNA was produced continuously for 120 h in the anaerobic batch culture to which acetate was not added (Fig. 3).