Vol. 88, No. 6, 617-621. 1999
Production of Canthaxanthin by Extremely Halophilic Bacteria DALAL ASKER AND YOSHIYUKI OHTA* Faculty of Applied Biological Science, Hiroshima University, l-4-4 Kagamiyama, Higashi-Hiroshima 7394.528, Japan Received 25 June 1999IAccepted 14 September 1999
Soil samples from a salt farm were used as a source for the isolation of carotenoid-producing bacteria. The conditions for optimum growth and carotenoid production were established for the isolated bacteria. Carotenoids were analysed by spectrophotometry and High Performance Liquid Chromatography (I-IPLC). Thirty-one red extremely halophilic bacteria were isolated from saline soil samples collected from a salt farm in Alexandria, Egypt. Among the isolated strains, strain TM exhibited the highest carotenoid-producing ability. Maximum growth of strain TM occurred in the presence of high concentrations of sodium chloride and magnesium sulfate. Growth did not occur when NaCl concentration was lower than 10% and the cells lysed at this concentration. Optimum growth of and carotenoid production by strain TM were realized at 37’C in the presence of 1% yeast extract, 0.75% casamino acids, 25% NaCl, 4% MgS04, 0.2% KC1 and at pH 7.2 with shaking for 6 d. Strain TM produced 2.06 mg total carotenoids g-l dry cells, including 0.06 mg of ,&carotene and 0.70 mg of canthaxanthin. This is the first report of an extremely halophilic bacterium that produces canthaxanthin. bacteria, Halobacterium sp., carotenoid, spectrophotometer]
[Key words: extremely halophilic terioruberins,
Isolation of halobacteria Soil samples were obtained from the seawater evaporation pond (El-Malahat) near Alexandria City in Egypt in April 1996. This farm is used in the production of common salt. Approximately 10 g (fresh weight) each of soil samples was suspended * Corresponding
in 90ml of complex medium (CM) containing (per liter): log yeast extract, 7.5 g casamino acids, 250g NaCl, 40g MgS04.7Hz0, 2 g KCl, and 3 g trisodium citrate. The pH was adjusted to 7.2 (18). The flasks were incubated on a shaker at 37°C until red turbidity appeared. The turbid cultures were streaked on CM broth containing 1.2% (w/v) agar and incubated at 37°C. After 7 d of incubation, red and yellow colonies were selected and purified. Screening of highest carotenoid-producing strain Each of the isolated strains was grown in 100ml CM broth in a 500ml Erlenmeyer flask and incubated on a rotary shaker at 240 rpm (200; Takasaki Co., Saitama) for 7 d at 37°C. One milliliter of the culture was diluted to 10 ml using 25% NaCl solution, to measure growth in terms of optical density at 660 nm with a spectrophotometer (U 2001; Hitachi, Tokyo). The results were expressed as (OD values x 10). Carotenoid extraction and analysis Ten ml aliquots of cultures were centrifuged at 6000 xg for 10 min. The harvested cells were resuspended in distilled water. Spontaneously, cellular lysis occurred, and then the pigments were extracted by acetone and transferred to hexane (9). The carotenoid extracts were analysed by scanning the absorbance in the wavelength region of 400-600 nm using the spectrophotometer. The total carotenoid content in the hexane extract was estimated by measuring the absorbance at R,, (490nm) (19). The results are given as OD per 100ml culture. The highest carotenoid-producing strain was selected on the basis of high growth, carotenoid and degree of cell pigmentation (20). The degree of cell pigmentation was calculated as the ratio of OD of the carotenoid to OD of the growth (OD,dODm). The selected strain was used in subsequent experiments. HPLC analysis of carotenoid Red pigments obtained were separated and quantified using HPLC (L6200; Hitachi) equipped with a UV-VIS detector (L-4200; Hitachi), according to the method of Sedmak et al. (21) and Calo et al. (9) with slight modification. Carotenoid extracts were subsequently filtered through a 0.5 pm hydrophobic Teflon membrane (Advantec, Tokyo). Chromato-
Carotenoids are yellow to orange-red pigments that are present in a wide variety of bacteria, algae, fungi and plants (1). The functions of carotenoids include the following: food colorants, absorbers of light energy, oxygen transporters, provitamin A, scavengers of active oxygen, antitumor and enhancers of in vitro antibody production (2-6). The use of colorants in food has been acknowledged to play a part in consumer acceptability of processed foods. Canthaxanthin as a ketocarotenoid pigment is a useful food colorant in the red wavelength range; furthermore, it is used as an agent for pigmenting cultured salmonids and crustaceans (7). The red extremely halophilic bacteria (non-photosynthetic bacteria) produce phytoene, p-carotene, lycopene and derivatives of acyclic Cso bacterioruberin (8). Recently, the production of ketocarotenoids such as 3-hydroxy echinenone or transastaxanthin from Halobacterium salinarium was reported by Calo et al. (9). These bacteria grow in highly saline environments, giving a pink to red color to these habitats. In general, they require at least 1.5 M (9%, w/v) NaCl for growth, and 2-4M (12-23x, w/v) NaCl for optimal growth (10-14). With the rising global concern to avoid the undesirable effects of synthetic food colorants such as allergy, hypersensitivity, intolerance and childhood hyperactivity (15-17), an attempt was made to find a new source of canthaxanthin by isolating red halobacteria strains from a salt farm in Egypt. To maximize the yield of carotenoid production by the isolated strains, the optimum conditions for the production of carotenoid were also investigated. MATERIALS
graphic separation was performed on a normal-phase column (Silica, 250 x 4.6 mm, Inertsil SIL, GL Sciences Inc., Tokyo). The mobile phase was hexane: ethyl acetate (50 : 50, v/v) at a flow rate of 1 ml/min. This was later modified to 30 : 70 (v/v) of hexane: ethyl acetate. The eluant was monitored at 490nm. The results are given as the percentage of total carotenoids. Optimization of culture conditions Determination of optimum conditions for growth and carotenoid production was carried out by inoculating the selected strain in 100ml CM broth in a 5OOml Erlenmeyer flask and incubating on a rotary shaker at 240 rpm for 7 d at 37°C. Growth and carotenoid production were determined as described above. The tested parameters were: NaCl concentration between 0 and 35% in 5% increments; MgS04. 7HzO concentration at 0, 1, 2, 3, 4, 5 and 6%; using MgCIZ or sodium sulfate instead of magnesium sulfate; KC1 concentration at 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 1%; trace elements (FeC12, 0.23 mg; CaC12. 7H20, 0.7 mg; MnS02.Hz0, 0.03 mg; ZnS04, 0.044 mg; CuS04. 5Hz0, 5 jig per 100 ml) (22); incubation time, 1 to 8 d; temperature, 15 to 55°C in 5” increments; different levels of pH, 4.5 to 9; shaking; medium volumes, 100, 200, 300 and 400ml of medium per 500ml Erlenmeyer flask; and light illumination. The optimum parameter from each experiment was utilized in the next experiment and so on until all the parameters were optimized. RESULTS Isolation and screening of carotenoid-producing halobacteria In summer, the temperature of the salt farm in Alexandria City ranges from 37 to 40°C. As a result, the total dissolved salt concentration increases to saturate at pH 7.2. Under these conditions, the surface
of the soil around the salt farm is characterized by an orange-red color, corresponding to the growth of pigmented microorganisms. Soil samples were collected from this environment as a source for the isolation of carotenoid-producing halobacteria. Thirty-one red and six yellow pure halobacteria were isolated. All the red halobacteria strains isolated gave identical absorption spectra of carotenoid (Fig. lc). The spectra are characterized by maximum peaks at 493 and 527 nm with a broad shoulder at 467 nm, indicating that the main carotenoid is w-bacterioruberin (20, 23). However, the yellow-pigment-producing isolates did not produce any carotenoid. Table 1 summarizes the growth and carotenoid production by red halophilic bacterial isolates. It was observed that strain TM had the highest value of the degree of pigmentation, indicating that it exhibited the highest carotenoid-producing ability. Figure 2 shows the HPLC chromatogram of carotenoids produced by strain TM in comparison with standard carotenoids. It was found that the separation of total carotenoids from strain TM with ethyl acetate: hexane (50 : 50, v/v) as a mobile phase required a longer time than that from Halobacterium salinarium (9). The separation time reached 40min at least until all pigments were eluted (Fig. 2~). Therefore, an increase in solvent polarity seemed to be essential for eluting the polar red pigments from the column (Fig. 2b). Retention times (R,) of peaks 1 and 3 were identified to be those of j-carotene and canthaxanthin, since their R, values were identical to those of authentic standards (Fig. 2a). This bacterium also produced 3-hydroxy echinenone (peak 2) and bacterioruberins (peaks 4, 5, 6 and 7), as shown in Fig. 2b (9). Consequently, strain TABLE Isolate
FIG. 1. Absorption spectra of (a) p-carotene thaxanthin standard and (c) carotenoid produced
standard, by strain
of and carotenoid production bacterial isolates Degree
by red halophihc of pigmentation KQxmd
R H T DT DM X MT Z TM N U 0 S B D I C G A w F E P Y
Growth (O&d 3.35 3.35 3.53 3.43 3.40 3.05 2.90 2.73 2.95 2.90 2.55 2.40 2.30 2.55 2.40 2.50 2.55 3.00 2.95 2.85 2.95 3.10 2.55 2.85
7.25 5.08 6.94 6.49 4.86 6.73 6.90 5.26 10.31 6.16 5.30 4.45 4.62 4.09 5.51 4.66 3.41 6.98 4.08 3.15 3.96 5.16 4.41 3.27
2.16 1.52 1.97 1.89 1.43 2.21 2.38 i .93 3.56 2.12 2.08 1.89 2.01 1.60 2.30 1.86 1.34 2.19 1.38 1.11 1.34 2.10 1.75 1.15
t V M TD K MD
2.90 2.80 2.85 2.90 3.15 2.95 2.75
4.18 3.85 4.70 4.93 3.37 7.48 4.95
1.38 1.44 1.65 1.70 1.07 2.54 1.80
^o g 2.0 e S 5 e u
$ e J
1.5 4.0 1.0
0.0 L 0
NaCl cone. (%)
30 35 Time (mm)
FIG. 2. HPLC profiles of (a) p-carotene and canthaxanthin standards, (b) and (c) carotenoids produced by strain TM were separated by hexane: ethyl acetate 50 : 50 and 30 : 70 (v/v), respectively. Peaks: 1, p-carotene; 2, 3-hydroxy echinenone; 3, canthaxanthin; 4, 5, 6 and 7, bacterioruberins.
TM was selected for further studies. As shown in Optimization of culture conditions Fig. 3, the growth curve of strain TM was characterized by a long lag phase (about 2d). Pigment production by the strain was observed after 4d of incubation, as the culture became slightly orange in color. Growth and carotenoid production increased to a maximum after 6 d of incubation, followed by a decrease in both growth and carotenoid production. Figure 4 shows that the growth of and carotenoid production by strain TM did not occur at less than 10% NaCI. The maximum growth and production of carotenoid were observed at 25% NaCl. Growth of and carotenoid production by strain TM did not occur at less than 1% MgS04, as shown in Fig. 5. Increasing the concentration of MgS04 enhanced the growth and production of carotenoid by the strain. It was found that this strain required 4% (w/v) of MgS04 for maximum growth and carotenoid production. Increasing MgS04 concentration above 4% resulted in
FIG. 4. Effect of NaCl concentration on growth of and carotenoid production by strain TM. Symbols: bar, growth (OD&; line, carotenoid (OD,,).
both low growth and carotenoid production. Replacing magnesium sulfate with magnesium chloride caused very slow growth even at a high concentration of MgC12 (3X), and also, led to low growth with a very low content of carotenoid. The experiment was repeated with Na2S04 to determine whether the cells require sulfate ion or magnesium cation; however no growth was observed at all. This indicated that strain TM required high concentrations of magnesium and sulfate ions. The maximum growth of and carotenoid production by the strain were found at 0.2% KCl. However, without the addition of KCl, strain TM could grow but with little pigment production (24). By increasing the concentration of KCl, both growth and pigment production increased. However, increasing the concentration of KC1 above 0.2% caused a slight decrease in the growth and carotenoid production. The addition of trace elements increased both growth of and carotenoid production by strain TM.
.% x 2 e 9
P 2 f u 0.0
Incubation time (d) FIG. 3. Growth and carotenoid production by strain TM. Symbols: 0, growth (OD.&; 0, carotenoid (OD,,).
FIG. 5. Effect of MgS04 concentration on growth of and carotenoid production by strain TM. Symbols: 0, growth (OD,&; 0, carotenord (OD&.
ASKER AND OHTA
FIG. 6. Effect of temperature on growth of and carotenoid production by strain TM. Symbols: 0, growth (ODsso); 0, carotenoid (OD,,).
Figure 6 demonstrates that the growth of and carotenoid production by strain TM did not occur below 20°C and above 50°C. The maximum growth and production of carotenoid occurred at 37°C. At temperatures above 37”C, low growth and pigment content was observed. The growth of and carotenoid production by strain TM did not occur below pH 5 and above 7.5, as shown in Fig. 7. Maximum growth and production occurred at pH 7.2. The strain when cultivated under shaking conditions exhibited high growth and carotenoid production. Without shaking, little growth with no carotenoid production was observed. Maximum growth and carotenoid production by strain TM occurred in 100 ml medium per 500 ml Erlenmeyer flask. No effect of light illumination was found on growth of and carotenoid production by the strain. Strain TM, grown in CM broth under optimum conditions, produced 2.06mg total carotenoids gg’ dry cells. The relative percentages of p-carotene, canthaxanthin and bacterioruberins to the total carotenoids were 2.94%, 33.88%, and 63.17%, respectively. DISCUSSION It is important to isolate not only a new biological source which produces a natural colorant which can be
35 1.5 6
-3 a" o_ = E E B
used in both food and feed industries, but also a source which has features to facilitate its industrial application. Extremely halophilic bacteria producing red pigments were isolated. Among the thirty-one strains isolated, strain TM was selected as the highest carotenoid-producing strain. It was interesting to find that some ketocarotenoids produced by the strain were similar to those produced by the marine bacterium, Agrobacterium auranticum (25, 26), as well as the yeast, P. rhodozyma (27, 28). Strain TM produced 2.06mg total carotenoids g I. dry cells. In comparison, Phafia rhodozyma produces 0.41 mg.g ’ dry yeast and Agrobacterium auranticum produces 0.46 mg total carotenoids.1 I culture. The microbial sources of canthaxanthin are limited to only a few microorganisms (29). The presence of canthaxanthin (33.88% of the total carotenoids) and 3hydroxy echinenone in strain TM, especially astaxanthin (data not shown) indicates its potential industrial applicability. The unique features of strain TM are as follows: (i) the extremely high NaCl concentration used in the growth medium, which is useful to prevent contamination by other organisms. Because of this, no sterilization procedures are required (data not shown); (ii) NaCl concentration below 10% induces cell lysis (unpublished data), so no cell disrupting devices are required as cells lyse spontaneously in freshwater; and (iii) furthermore, the procedures for extraction and purification of carotenoids from strain TM seem to be simpler than those from other sources, as bacterial carotenoids are usually easier to isolate than carotenoids from other sources (25). The strain requires extremely high concentrations of NaCl and MgS04 for optimum growth. The concentration of the latter was about twice that reported for Halobacterium sp. which required 2% MgS04. Magnesium has been reported previously to be an essential element for red halophiles; it is possible that Mg” is required for cell division (12). The addition of trace elements solution increased both growth of and carotenoid production by the strain, because it fulfills the requirements of iron, manganese, and zinc of the strain (18). Shaking the culture significantly increased the growth of and carotenoid production by the strain, because it is a strict aerobe. Increasing the volume of the medium above 100ml in a 500ml Erlenmeyer flask decreased the amount of dissolved oxygen; therefore, growth of and carotenoid production by the cells decreased under these conditions. Yokoyama and Miki (26) reported that varying the medium volume controlled the growth of and carotenoid production by Agrobacterium auranticum. In conclusion, strain TM is a promising source of canthaxanthin for fish and poultry industries, and could be used as a colorant in the food industry. Improved production can be expected by obtaining a mutant with higher productivity or by isolating a cloned gene related to a specific carotenoid biosynthesis from the strain. ACKNOWLEDGMENTS
We would like to thank Professor Ahmed R. El-Mahdy of Alexandria University for useful suggestions and Mr. M. Adjei for critical reading of the manuscript.
FIG. 7. Effect of pH on growth of and carotenoid production by strain TM. Symbols: 0, growth (OD,&; 0, carotenoid (ODdw).
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