Supplementary fermentation studies on astaxanthin production by Fife yeasts

 Abstract: The supplemental fermentation of astaxanthin production was carried out with Fife yeast (pha『ia rhodozyma) Wss-FF6 as the producer bacterium at an aeration rate of 250 L/h, pH = 6 . 0 ± 0 . 5, the medium was first flow-added with a high sugar concentration, followed by the addition of 0 .0% ethanol, and the fermentation was carried out in batches. After 130 h of fermentation, the biomass and carotenoid yields were 27.4 mg/mL and 26.4 mg/mL, respectively. After 130 h of fermentation, the biomass and carotenoid production were 27.4 mg/mL and 26.12 μg/mL, respectively. After 130 h of fermentation, the biomass and carotenoid yields were 27.4 mg/mL and 26.12 μg/mL, respectively. The growth rate, product yield and mass fraction of yeast pigment were 0.46, 0.44 and 0.12 μg/mL respectively. 0.46, 0.44 and 0.95 . 95 .

 

Fife yeast (pha'ia rhodozyma) is considered to be the most likely to realize the industrial fermentation method of astaxanthin production of excellent strains, pha'ia rhodozyma production of astaxanthin in recent years, the study of astaxanthin production by pha'ia rhodozyma yeast has received domestic and foreign researchers to pay attention to. Fife yeast shows CRABTREE EFFECT in the fermentation process [1, 2].

 

Therefore, for Fife yeast, the biomass per unit of fermentation broth can be realized by simple batch fermentation, which is an excellent strain for the production of astaxanthin by industrial fermentation method, and the study of astaxanthin production by Fife yeast has been paid attention by domestic and foreign researchers in recent years. Fife yeast shows CRABTREE EFFECT in the fermentation process [1, 2], therefore, for Fife yeast, the biomass and pigmentation per unit of fermentation broth are lower with simple batch fermentation, and it is difficult to increase the coefficient of substrate growth rate and the coefficient of product yield.

The authors used the mutant strain Wss-FF6 obtained from laboratory screening as the starting bacterium [3], and studied its replenishment and batch fermentation process, hoping to improve the fermentation efficiency, so as to provide a certain guidance for industrialized production.

 

1 Materials and Methods

1 . 1 Main instruments

2 L volume benchtop fermenter, manufactured by INFER, Switzerland .

 

1 . 2 Strains

Fife yeast WSS-FF6 [3], in the custody of the author's laboratory . 1 . 3 Culture medium

 

1 . 3 . 1 Inclined medium YM

Medium (g/L): glucose 10, wort 3, peptone 5, yeast paste 3, agar 20; pH 6 . 0 .

 

1 .3 . 2 Seed culture medium

Glucose 10 g/L, wort 3 g/L, peptone 5 g/L, yeast paste 3 g/L; pH 6 . 0 .

 

1 . 3 . 3 Shake flask medium

glucose 20 g/L, (NH4 )2 SO4 5 g/L, KH2 PO4 1 g/L, MgSO4 - 7H2 O 0 . 5 g/L, cacl2 -2H2 O 0 . 1 g/L, yeast paste 1 g/L; PH 6 . 0 .

 

1 .3 . 4 Flow-added media  

Expansion of the mass concentration of each component of the above shake flask medium by 7.5 times . 5 times .

 

1 . 4 Cultivation methods

1 . 4 . 1 Seed culture methods

The fermentation was carried out in a 250 mL volumetric triangular flask containing 25 mL of seed medium at 22 ℃, 220 r/min for 48 h. 2 mL of this fermentation broth (10% of the inoculum) was added to a 250 mL volumetric triangular flask with 23 mL of shaking medium at 22 ℃, 220 r/min for 48 h. The fermentation was carried out at 22 ℃ for 48 h. The fermentation was carried out in a 250 mL volumetric triangular flask with 23 mL of shaking medium at 22 ℃, 220 r/min.

 

1 .4 . 2 Shake flask culture methods  

The inoculum was 10%, 250 mL volume, 25 mL of solution in a triangular bottle, 22 ℃, 220 r/min, and incubated for 72 hours.

1 . 4 . 3 Fermenter culture methods  

The inoculum volume is 10% and the loading volume is 1 . 2 L, 20 ~ 22°C . 1 . 5 Measurement method

 

1 . 5 . 1 Biomass determination  

For the dry weight method, the culture medium was centrifuged, washed once with distilled water, centrifuged again, and dried in an oven at 105 ℃ until constant weight.

 

1 .5 . 2 Determination of total carotenoids  

Dimethyl sulfoxide (DMSO) method [4]. 2 mL of fermentation broth was broken with 1 mL of DMSO. 2 mL of fermentation broth was broken with 1 mL of DMSO, 5 mL of acetone was added for direct extraction, and the absorbance at λmax = 480 nm was measured with V(acetone): V(DMSO) = 5 : 1 as blank.

 

2 Results and Discussion

2 . 1 Controlling the effect of PH on fermentation

Environmental pH affects the charge of cell membranes and the degree of ionization of nutrients in the medium, thus influencing the growth and production of microorganisms. Unlike shake flask cultures, fermenter cultures are more effective in controlling the pH of the fermentation process so that the yeast grows in an optimal pH environment. Fig. 1 shows the pH value of the fermentation tank culture with 0.2 mol/L sodium dihydrogen phosphate (NaOH). Fig. 1 shows the pH value of yeast grown in 0.2 mol/L sodium dihydrogen phosphate and 0.1 mol/L citric acid. Figure 1 shows the results of shaker culture with 0.2 mol/L sodium dihydrogen phosphate and 0.1 mol/L citric acid buffer system [5], which adjusted the pH value of shaker culture medium to 3, 4, 5, 6, 7 and 8, respectively.

 

It can be seen that maintaining a relatively stable pH value during the fermentation process is beneficial to the increase of biomass and pigmentation, and the effect on biomass is more obvious. At the optimum PH value of 6, the biomass of the "buffered PH system" was nearly doubled compared to the "starting PH system", while there was no significant difference in the amount of pigmentation. This effect of pH on the biomass of Saccharomyces cerevisiae is consistent with the study of HO et al.  2 . 2.2 Effect of Aeration on Fermentation The fermentation experiment was carried out using shake flask medium as the basal medium, and the fermentation progress curves at different aeration levels are shown in Fig. 2. The results of the effect of aeration on fermentation obtained from Fig. 2 are summarized in Table 1.

 

The process curves showed that the fermentation of the yeast was similar at all three aeration rates, but at 75 L/h, the yeast entered the logarithmic growth phase earlier, and the amount of yeast pigmentation started to decrease after 90 h, which may be related to the autolysis of the yeast [7]. Comparing the data in Table 1, it is easy to see that it is more effective to use the aeration rate of 250 L/h or more.

 

2 . 3 Effect of starting glucose mass concentration on fermentation

YAMANE et al. (1997) showed that fermentation of Fife yeast ATCC24202 at a high concentration of dissolved oxygen (DO = 5.0 mg/L) inhibited the effects of the krebuterol effect. (DO = 5.0 mg/L) could inhibit the Krebtree effect, so that the yeast did not produce metabolic by-products such as ethanol, glycerol, acetate, pyruvate, etc., when cultured in different mediums with different starting glucose mass concentrations ranging from 10 to 120 g/L. However, the maximal specific growth rate of the yeast decreased with the increase of the starting sugar mass concentration, and it was also found that there was a relationship between the mediums with different mass concentrations of sugar, that is, the medium with low mass concentration of sugar was favorable to the fermentation of Fife yeast ATCC24202. A relationship was found between different sugar concentrations, i.e., the medium with low mass concentration of sugar favored the growth of yeast, while the medium with high mass concentration of sugar favored the increase of pigment production. Therefore, the authors used a low sugar concentration (i.e., a low carbon-to-nitrogen ratio of 2.8 mol glucose/1 mol ammonium sulfate) in conjunction with the nitrogen changes in the medium. 8 mol glucose/1 mol ammonium sulfate) followed by high sugar (11.2 mol glucose/1 mol ammonium sulfate). The authors used a two-step batch fermentation with low sugar (i.e., low carbon to nitrogen ratio of 2.8 mol glucose/1 mol ammonium sulfate) followed by high sugar (11.2 mol glucose/1 mol ammonium sulfate), and obtained good results [1]. The authors carried out fermentation experiments with different starting glucose concentrations under 250 L/h aeration, and the process curves are shown in Fig. 3, and the effects of different starting glucose concentrations on fermentation are shown in Table 2.

The process curves showed that under the condition of 250 L/h aeration, the fermentation state was similar for different starting sugar concentrations, but the fermentation time was prolonged with the increase of sugar concentration, which was consistent with the experimental results of Yamane et al. (1997) [1].

 

Analyzing the growth rate and product yield in Table 2, it can be seen that both of them have the same trend of decreasing with the increase of the starting sugar concentration, which means that the fermentation with a low sugar concentration is beneficial to the increase of biomass and pigmentation, which is different from the conclusion of YAMANE mentioned above. Combined with the experimental results from the literature [2], it is easy to find that, due to the difficulty of controlling the amount of aeration, the substrate growth rate and product yield of shake flask culture are 2~3 times lower than that of fermenter culture with the same sugar concentration, which reflects the importance of oxygen in Fife yeast fermentation. However, comparing the mass fraction of carotenoids (ΔP/ΔX per unit of cytochrome) of the two cultures, it was found that the amount of cytochrome per unit of the same sugar concentration in the shake flasks was about 1 times higher than that in the fermentation tanks. This result not only proves that ethanol promotes the pigmentation of the fermented yeasts, but also verifies the conclusion made by YA-Mane et al. that Fife yeasts do not produce ethanol when fermented in a certain range of sugar concentrations under the condition of high dissolved oxygen. This result not only further proves that ethanol can promote pigment accumulation, but also verifies the conclusion of YA- MANE et al.

 

2 . 4 Effect of Ethanol Mass Concentration on Fermentation

While ethanol as the sole carbon source has a strong inhibitory effect on the growth of Fife yeast, the addition of appropriate amounts of ethanol to the culture medium can increase the activity of ethanol dehydrogenase and methylglutarate monoacyl-coenzyme A reductase in the astaxanthin synthesis pathway, and is therefore beneficial in increasing the amount of pigment per unit of cell [8], which has been demonstrated in the experimental results in the literature [2]. Taking advantage of this effect of ethanol, Yamane et al. (1997) [9] added 0.3% ethanol to the cells during the stabilization phase of cell growth. (1997) [9] added 0.3% ethanol during the stabilization phase of cell growth, and Gu et al. (1997) [10] added 0.2% ethanol during the late delayed or logarithmic growth phase. (1997) [10] added 0.3% ethanol at the late delayed or logarithmic growth stage, and Gu et al. (1997) [10] increased the pigment yield by about onefold.

 

In order to investigate the effect of ethanol on WSS-FF6, we used shake flask medium as the base, and replaced all the glucose in the medium with different concentrations of ethanol or added different amounts of ethanol to the original medium to carry out shake flask fermentation, and the results of the experiment are shown in Table 3. The results are shown in Table 3. It can be seen that when ethanol was used solely as a carbon source, and the concentration of ethanol was ≤10 g/L, ethanol only played the role of a general carbon source, and did not promote the accumulation of astaxanthin. On the other hand, the addition of 1.0 g/L ethanol in the presence of glucose had no effect on the accumulation of astaxanthin. However, when 1.0 g/L ethanol was added in the presence of glucose, the inhibitory effect on the growth of WSS-FF6 yeast was eliminated, and the amount of carotenoids was 1.1 times higher than that of the yeast without ethanol. The quality fraction of carotenoids was 1.1 times higher than that in the absence of ethanol.

 

2 . 5 Two-step batch replenishment fermentation

In view of the above results, the fermentation was carried out with 20 g/L glucose in shake flask medium at pH 6 ± 0.5 (2 mol/L NaoH dropwise control) and 250 g/L aeration. Under the conditions of pH 6 ± 0.5 (2 mol/L NaoH drip control) and 250 g/L aeration, a one-step replenishment fermentation with only flow-through medium and a two-step replenishment fermentation with 0.1 g/dL ethanol were conducted. One-step replenishment fermentation with flow-through medium only and two-step replenishment fermentation with 0.1 g/dL ethanol mass concentration were carried out. The first replenishment was started when the residual sugar in the fermentation broth was less than 5.0 g/L [1]. The second addition of ethanol was made at a residual sugar level of less than 2.0 g/L. The second addition of ethanol was made at a residual sugar level of less than 2.0 g/L. The second addition of ethanol was made when the residual sugar content of the fermentation broth was below 2.0 g/L. The results are shown in Figure 4.

 

At 120 h of fermentation, the biomass and total carotenoids without ethanol were 28.48 mg/mL, 21.5 mg/mL, 21.5 mg/mL, and 21.5 mg/mL, respectively. 48 mg/mL and 21.34 μg/mL, respectively, after the addition of ethanol. 34 μg/mL without ethanol, and 28.3 mg/mL, 25.3 μg/mL, 25.3 μg/mL, 25.3 μg/mL, and 25.3 μg/mL, respectively, with ethanol. The biomass and total amount of carotenoids were 28.48 mg/mL and 21.34 μg/mL without ethanol, 28.3 mg/mL and 25.5 μg/mL with ethanol. After adding ethanol, the total amount of carotenoids increased to 1.2 times of that without adding ethanol, and the amount of carotenoids increased to 1.2 times of that without adding ethanol. After adding ethanol, the total amount of carotenoids increased to 1.2 times of that before ethanol addition, and the quality fraction of carotenoids increased from 750 μg/g before ethanol addition to 900 μg/g, which was also 1.2 times. It can be seen that the addition of ethanol had a certain effect on the pigment accumulation of WSS-FF6, but the increase was smaller than that reported overseas. A total of 510 mL of flow-through medium was added, which was equivalent to a starting glucose concentration of 59 g/L in the batch fermentation. Based on the biomass and the total amount of carotenoids in the two supplemental fermentations for 130 h, the yeast growth rate ΔX/ΔS, the product yield ΔP/ΔS, and the carotenoid mass fraction ΔP/ΔS were calculated as 0.46, 0.46, 0.001, 0.001, 0.001 and 0.001, respectively. 46, 0 . 0.46, 0.44 and 0.95, respectively. The efficiencies of ΔX/ΔS, product yield ΔP/ΔS, and carotenoid mass fraction ΔP/ΔS were 0.46, 0.44, and 0.95, respectively, which were the same as that of the batch fermentation at a mass concentration of 40 g/L (Table 2), indicating that the efficiency of the fermentation was improved by using the flow-through method.

 

3 CONCLUSIONS

Batch flow-through can increase the growth rate and product yield of WSS-FF6 strain, and the addition of ethanol during flow-through can promote the accumulation of pigment to a certain extent.

 

References:

[1] YAMANE Y, HIGASHIDA K, NAKASHIMADA Y, et al .  Influence of oxygen and glucose on primary metabolism and astaxanthin pro- duction by phaffia rhodozymain batch and fed-batch cultures: kinetic and stoichiometric analysis[J].  Applied and Envir Microbiology, 1997, 63(1):4471 - 4478 .

[2] XU Xue-Ming, JIN Zheng-Yu, LIU Dang-Hui, et al. Shaking bottle process of astaxanthin production by Fife yeast［J］. Journal of Wuxi University of Light Industry, 2000, 19(3): 230 - 235 . ［3] XU Xue-Ming, JIN Zheng-Yu, LU Yu-Hua . Selection and breeding of astaxanthin high-yield mutant strain［J］. Food and Fermentation Industry, 2000, 26(5):22 - 27 .

[4] JAMES S J, DEEpTHI K, Extraction and quantitation of astaxanthin from phaffia rhodozyma [J].  Biotechnology Technoque, 1990, 4

(2): 107 - 112 .

[5] Zhuge Jian, Wang Zhengxiang. Industrial microbiology experimental technology manual [M]. Beijing: China Light Industry Press, 1994 .

[6] HO K p, TAM C Y, ZHOU B .  Growth and carotenoid production of phaffia rhodozyma in fed-batch cultures with different feeding meth- ods [J].  Biotechnol Letters, 1999, 21:175 - 178 .

[7] MEYER p S, Du pREEZ J C .  Effect of culture conditions on astaxanthin production by a mutant of phaffia rhodozylma in batch and chemostat culture［J］.  Appl Microbiol Biotechnol, 1994, 40:780 - 785 .

[8] AN G H, SCHUMAN D B, JOHNSON E A .  Isolation of Pha『ia rhodozyma mutants with increased astaxanthin content［J］.  Appl Envi- ron Microbiol, 1989, 55:116 - 124 .

[9] YAMANE Y, HIGASHIDA K, NAKASHIMADA Y, et al .  Astaxanthin production by phaffia rhodozyma enhanced in fed-batch culture with glucose and ethanol feeding［J］.  Biotechnol Letters, 1997, 19(11):1109 - 1111 .

［10］GU W L, AN G H, JOHNSON E A, Ethanol increases carotenoid production in phaffia rhodozyma［J］.  J Ind Microbiol Biotechnol, 1997, 19(2):114 - 117 .