What is the effect of temperature on the astaxanthin of Rhodococcus rainieri?

 Astaxanthin has high economic value and many functions. Astaxanthin has a unique coloring function, and can be used in aquatic animals such as salmon and salmon, as well as poultry and livestock breeding, to improve product quality; astaxanthin can scavenge free radicals, quench unilinear oxygen, and has a stronger antioxidant capacity than β-carotene, which can slow down the aging of the skin, protect the body from chemically-induced carcinogenesis, and enhance the body's immunity, so astaxanthin has a very high potential for use in the production of pharmaceuticals and cosmetics. 

In addition, astaxanthin has been gradually developed as a function
al food and has been approved for use in human consumer products in some European countries[1] . Currently, there are four main sources of astaxanthin[2]: one is chemical synthesis, and the most important company in chemical synthesis is the Swiss company Roche, which has synthesized a commercial product called Carophyll pink[3]. The absorption efficiency of chemically synthesized astaxanthin is lower than that of natural astaxanthin, and its antioxidant activity is weaker than that of natural astaxanthin. However, due to the limited sources of natural astaxanthin, only synthetic astaxanthin can be used in aquaculture; secondly, astaxanthin can be extracted from crustaceans, which is limited by the fact that the shells of these crustaceans contain high levels of ash, chitin, low levels of protein, and other nutrients; thirdly, the use of yeast is used; thirdly, the use of yeast is used, which is limited by the fact that it is not a good source of astaxanthin. Thirdly, the production of astaxanthin by using yeast, the content of astaxanthin in these fungi is very low (o.o5%~o.3%), and the fermentation cost is also very high; fourthly, the production by using algae. The natural astaxanthin content in the cells of red algae is relatively high, generally up to 1.5%~3.o% of the dry weight, and the structure is identical with that of the natural salmon. Therefore, the production of astaxanthin from rainbow red algae has great prospects for development.

 

Temperature has a great influence on the growth of H. lacuSTriS and the accumulation of astaxanthin, a lot of research work has been done, but the conclusions of different researchers are quite different. Harker et al. [4] and Borowizka et al. [5] considered that the optimal growth temperature of H. lacuSTriS was about 14~15℃; Lu et al. [6] considered that the optimal growth temperature of H. lacuSTriS was 24~28℃, and its growth was inhibited when the environmental temperature was higher than 28℃; Ding et al. [7] believed that the growth temperature range of H. lacuSTriS was 21~27℃, and the optimal growth was inhibited. Lu et al.[6] concluded that the optimum growth temperature of H. lacuSTriS was 24~28℃, and when the ambient temperature was higher than 28℃, the growth of H. lacuSTriS was inhibited; Ding et al.[7] concluded that the growth temperature of H. lacuSTriS ranged from 21~27℃, and the optimum temperature was 25℃; Zhang Jingpu et al.[8] concluded that the temperature of H. lacuSTriS was 33℃, and the experimental results of Yin Mingyan et al.[9] proved that the growth of H. lacuSTriS was better at around 2℃, and that the cells were inactive at more than 28℃. Tjahjono et al.[1o] concluded that the production of astaxanthin in red algae incubated at 3o C was 2.5 times higher than that at 2o C. The results of the experiments by Yin Mingyan et al.

 

The author selected a suitable strain of Rhodococcus rainbowii selected in the laboratory of algae and water purification for large-scale culture [11], and utilized the simulation system of circular culture tanks to cultivate it, to study the effects of different temperatures on the biomass and astaxanthin production of Rhodococcus rainbowii, and to find out the effect of temperature on the accumulation of astaxanthin of this strain, so as to provide a basis for the control of the temperature in the process of large-scale production.

 

1 Materials and Methods

1.1 Algae species

The test alga was H. pluvialiS 26#, which was supplied by the Algae Species Bank of Wuhan Botanical Garden, Chinese Academy of Sciences. The incubation temperature was (22±1)°C, the light intensity was 5oμmol.m- 2.s - 1, and the light-dark cycle was 12h:12h.

 

1.2 Incubation devices

The simulation system consists of five parts: an annular tank, a stirring system, a light source system, a temperature control system and a CO2 system (see Fig. 1). The main technical parameters are as follows: ① the annular culture tank is made of o.8cm-thick organic glass, with an inner diameter of 35cm, an effective culture area of 62.8dm2 , and the depth of culture liquid is adjustable from 2 to 2ocm; ② the main body of temperature control system is a thermostatic water bath, which is connected to the annular tank with an inlet/outlet pipe and can be used to control the temperature automatically from 5 to 45℃ with an accuracy of o.1℃; ③ the light source consists of 12 H-type energy-saving lamps, with a light intensity of o~1℃ and a light intensity of o~1℃; ③ the light source consists of 12 H-type energy-saving lamps, with a light intensity of o~1℃. The light source consists of 12 H-type energy-saving lamps, the intensity of light is continuously adjustable within o~7ooμmol.m - 2. s - 1, the light and dark cycle is controlled by a timer; ④ The stirring system consists of a power unit and stirring blades, the stirring blades are 25cm long and fixed at the upper end of the vertical shaft, the rotation rate of the vertical shaft is continuously adjustable from o~8or/min, and the flow rate of the culture liquid is about o~12cm/min, and the temperature of the culture liquid is about o~12cm/min. The flow rate of culture liquid was about o~12o cm/s, and the stirring time was automatically controlled; ⑤ CO2 supply system, the CO2 flow rate (o~1ooo L.h-1) was continuously adjustable.

 

1.3 Culture media

were cultured using modified BG11 medium [12].

 

1.4 Cultivation conditions

The temperature was set at 15℃, 18℃, 22℃, 25℃ and 28℃; on the 1st day after inoculation, the light intensity was 1ooμmol.m- 2.s - 1, and from the 2nd day onwards, the light intensity was increased to 2ooμmol.m- 2.s - 1, which was maintained until the end of the culture, and the light-dark cycle was 14h:1oh. From the 2nd day, the light intensity was increased to 2ooμmol.m- 2.s - 1, and was maintained until the end of the incubation period. The rotation speed of the stirring blades was 1 oor/min, and the incubation period was 12d per batch.

 

1.5 Determination of fresh and dry weight of algae

After centrifuging the algal liquid, the supernatant was discarded and the mass of the algal slime was measured, which was the fresh weight of the algae. After measuring the fresh weight, the algal slime was dried in vacuum with LG-3 type multi-function vacuum dryer until constant weight, which was taken as the dry weight of the algae.

 

1.6 Determination of astaxanthin content and calculation of yield

The astaxanthin content was determined by the method of Boussiba et al[13] . Three parallel samples were taken each time and 1oomg of fresh algae was taken for each sample. Equation (1) was used to calculate the concentration of astaxanthin in the extract; Equation (2) was used to calculate the content of astaxanthin; Equation (3) was used to calculate the yield of astaxanthin. C1 is the concentration of astaxanthin in the solution to be tested; A492 is the concentration of astaxanthin in the solution to be tested; A492 is the concentration of astaxanthin in the solution to be tested.

Absorbance value of astaxanthin in liquid; Am = 222O, 1% of astaxanthin.

C2 is the content of astaxanthin in the dried algae; v is the total volume of the diluted extract; M1 is the fresh weight of the algae; M2 is the dry weight of the algae; C3 is the astaxanthin yield.

 

1.7 Data analysis

The experimental data were analyzed by ANOVA using SPSS software.

 

2 Results and analysis

The biomass (dry weight), astaxanthin content and production of Rhodococcus rainbowensis underwent a process of increase-maximum-decrease at different temperatures (Figs. 2~4). In terms of biomass, the highest biomass at 25℃ was 2.68g, the second highest biomass at 22℃ was 2.62g, and the lowest biomass at 15℃ was 1.4g (Fig. 2); in terms of astaxanthin content, the highest biomass at 22℃ was 1.52%, the second highest biomass at 25℃ was slightly lower than the second highest biomass at 22℃, which was 1.51%, and the lowest biomass at 15℃ was O.54%, which was 1/3 of that at 22℃ (Fig. 3). /3 (Figure 3); astaxanthin production, 25 ℃ at the highest, 13.53 mg / L, 22 ℃ at the next to 25 ℃, 15 ℃ astaxanthin production is the lowest, 2.49 mg / L (Figure 4).

Analysis of variance (ANOVA) was performed on the experimental data, and the results showed that the astaxanthin production at 25℃ and 22℃ was significantly higher than that at other temperatures (P<O. O1), and the astaxanthin content in the dry weight of Rhodococcus erythropolis was not significantly different between 22℃ and 25℃ (P>O. O5) but was significantly higher than that at other temperatures (P<O. O1). The astaxanthin content in the dry weight of red algae at 22℃ and 25℃ was not significant (P>O. O5), but was significantly higher than that at other temperatures (P<O. O1).

 

3 Discussion

Many authors have reported the effect of temperature on the growth rate of Rhodococcus rainbowensis [46], and the effect of temperature on the accumulation of astaxanthin has also been reported [10]; however, there are few reports on the effect of temperature on biomass, astaxanthin content and yield. We utilized the simulated systematic cultivation of Rhodococcus rainbowensis in an annular culture tank to study the effect of temperature on biomass, astaxanthin content and yield, and deepened our understanding of the effect of temperature on biomass, astaxanthin content and yield. We used a simulation system to study the effect of temperature on biomass, astaxanthin content and yield of Rhodococcus rainbowensis.

 

(1) Effect of temperature on biomass: The maximum biomass (dry weight) of Rhodococcus pyrenoidus was found at 22℃ and 25℃, indicating that these two temperatures are favorable for the trophic growth of Rhodococcus pyrenoidus, which is in agreement with the results reported by Ding et al [7], but not with the results of BoRowizka et al [5] and HaRkeR et al [4]. The biomass (dry weight) of astaxanthin was not high at 15℃ and 28℃, indicating that the nutrient growth of Rhodococcus rainbowensis was inhibited at lower or higher temperatures, resulting in a decrease in its biomass, which is also inconsistent with the results reported by BoRowizka et al [5] and HaRkeR et al [4] and in agreement with the results reported by Lu et al [6]. This is also inconsistent with the reports of BoRowizka and HaRkeR, and Lu et al.

 

(2) The effect of temperature on the content and yield of astaxanthin: the content and yield of astaxanthin in red algae at 22℃ and 25℃ were the largest, and the variability of the yield and content of astaxanthin in the samples was small, which indicated that the optimal temperature range for the accumulation of astaxanthin in rain-grown red algae was between 22~25℃, therefore, in production practice, we can consider controlling the temperature at around 22℃ and 25℃ to maximize the yield of astaxanthin and obtain the best economic benefits. Therefore, in production practice, we can consider controlling the temperature around 22℃ and 25℃ to maximize the astaxanthin production and obtain the best economic benefits.

 

(3) The experimental results showed that both biomass and astaxanthin content went through a process of increase-maximum-decrease with increasing temperature, but the changes were different: compared with the optimal temperature, both increasing and decreasing temperatures resulted in a significant decrease in biomass, which was basically the same at 28℃ and 18℃, and half of the decrease compared with that at 25℃. At 28℃ and 18℃, the biomass was almost the same, but half as much as at 25℃. However, the effect of low temperature on astaxanthin content was greater than that of high temperature, with the astaxanthin content decreasing by 65% at 15℃ as compared to 25℃, and by only 17% at 28℃. The effect of low temperature on astaxanthin production was through the simultaneous reduction of biomass and astaxanthin content, whereas the effect of high temperature on astaxanthin content was not as large, and mainly affected astaxanthin production through the reduction of biomass.

 

(4) Tjahjono et al.[10] reported that the astaxanthin content of Rhodococcus rainbowensis cultured at 30℃ reached 200pg/cell and 20mg/L, which was three times higher than that at 20℃[10] . The high temperature increased both the astaxanthin content per cell and the astaxanthin content per unit volume of algal solution. Our results showed that although high temperature increased the astaxanthin content, i.e., the content of astaxanthin in individual cells increased, the decrease in biomass was greater than the increase in astaxanthin content, and the astaxanthin content per unit volume of algal sap decreased, which was similar to and different from Tjahjono's results, probably due to the different responses of different strains to the temperature change.

H. pluwIalIs 26# is a suitable species for large-scale culture[11] , and the conditions of H. pluwIalIs 26# cultivated in a simulated system of circular culture tanks are close to those of large-scale culture tanks, and the results of the experiments are useful for the large-scale cultivation of H. pluwIalIs 26# in the future.

Optimization of temperature regulation for astaxanthin production by algae is instructive.

 

References.

[ 1] Richmond A. Handbook of Microalgal culture :Biotechnology and applied phycology [M] .  Iowa , USA: Blackwell Science Ltd , 2004. 281 288.

[ 2] Johnson E A, Gil-Hwan An. Astaxanthin from microbial sources[J].  BIotechnology , 1991, 11(4) : 296 326.

[ 3] Fang T J, chiou T Y. Batch cultivation and astaxanthin production by a mutant of the red yeat pha fIa rhodo≈yma NcHU-FS501[ J] .   IndustrIal MIcrobIology , 1996, 16:175 181.

[ 4] Harker M , Alex J, Tsavalos A J. Young use of response surface methodology to optimize carotenogenesis in the microalga , Haematococcus pluwIalIs [J] .   Appl phycol , 1995, 7: 399 406.

[ 5] Borowitzka M A, Huisman JM , osborn HAnn. culture of the astaxanthin-producing green alga Haematococcus pluwIalIs 1. effects of nutrients on growth and cell type [J].  Appl phycol , 1991, 3: 295 304.

[ 6] Lu F, vonshak A, Boussiba S. Effect of temperature and irradiance on growth of Haematococcus pluwIalIs (chlorophyceae)[J].  phycol , 1994, 30:829 833.

[ 7] Ding S Y, Lee Y k. Growth of entrapped Haematococcus lacustrIs in alginate beads in a fluidized bed air-lift bioreactor system[A] .  First Asia-pacific conference on Algal Biotechnology [c] .   Malaysia , kuala Lumpur , 1994.

[ 8] ZHANG Jingpu , LIU Jianguo. Effects of nutrient salts on the photosynthesis of Rhodococcus rainbowensis [J]. Industrial Microbiology , 1997, 27(4): 14 17.

[ 9] YIN Mingyan , LIU Jianguo , ZHANG Jingpu , MENG Zhaocai. A review of the research on red algae and astaxanthin[J]. Marine Lakes and Marshes Bulletin , 1998(2): 53 62.

[10] TjahjonoA E, Hayama Y, kakizonoT, Terada Y, Nishio N, Nagai S. Hyper-accumulation of astaxanthin in a green alga Haematococcus pluwIalIs at elevated temperature[J].  BIotechnol Lett , 1994a , 16: 133 138.

[11] ZHANG Baoyu , LI Yiguang , GENG Yahong , LI Zhongkui , HU Hongjun. Selection of suitable red algae for mass culture[J]. Journal of Aquatic Biology , 2004, 28(3): 289 293.

[12] ZHANG Baoyu , LI Yiguang , GENG Yahong , LI Zhongkui , HU Hongjun . Effects of temperature, light intensity and pH on photosynthesis and growth rate of Rhodococcus pyrenoidus [J]. Ocean and Lake , 2003,34(5): 558 565.

[13] Boussiba S, vonshak A. Astaxanthin accumulation in the green alga Haematococcus pluwIalIs [J] .    Plant cell physIol , 1991, 32(7): 1077-1082.