- Email: [email protected]
Use of several waste substrates for carotenoid-rich yeast biomass production
Journal of Environmental Management 95 (2012) S338eS342
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Use of several waste substrates for carotenoid-rich yeast biomass production I. Marova a, b, *, M. Carnecka b, A. Halienova b, M. Certik c, T. Dvorakova b, A. Haronikova a, b a
Brno University of Technology, Faculty of Chemistry, Centre for Materials Research, Purkynova 118, 612 00 Brno, Czech Republic Brno University of Technology, Faculty of Chemistry, Department of Food Chemistry and Biotechnology, Purkynova 118, 612 00 Brno, Czech Republic c Slovak University of Technology, Faculty of Chemical and Food Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2009 Received in revised form 1 April 2011 Accepted 8 June 2011 Available online 8 July 2011
Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/ L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Waste substrates Yeast biomass Rhodotorula sp. Sporobolomyces sp.
1. Introduction Carotenoids are naturally occurring lipid-soluble pigments, the majority being C40 terpenoids. They act as membrane-protective antioxidants that efficiently scavenge 1O2 and peroxyl radicals; the antioxidant efficiency is apparently related to their structure. Carotenoid pigments occur universally in photosynthetic systems of higher plants, algae and phototrophic bacteria. In nonphotosynthetic organisms, carotenoids are important in protecting against photo-oxidative damage. Thus, many non-phototrophic bacteria and fungi rely on carotenoids for protection when growing in light and air (Britton et al., 1998).
* Corresponding author. Brno University of Technology, Faculty of Chemistry, Centre for Materials Research, Purkynova 118, 612 00 Brno, Czech Republic. Tel.: þ420 541 149 419; fax: þ420 541 211 697. E-mail address: [email protected] (I. Marova). 0301-4797/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2011.06.018
Commercially, carotenoids are used as food colorants and nutritional supplements, with an estimated global market of some $935 million by 2005 (Fraser and Bramley, 2004). There is an increased interest in carotenoids as natural antioxidants and free radical scavengers for their ability to reduce and alleviate chronic diseases, various pathological stages and aging. However, the application of chemical synthetic methods to prepare carotenoid compounds as food additives has been strictly regulated in recent years. Therefore attention is paid on the finding of suitable natural sources including biotechnological processes. The number of red yeasts species Rhodotorula, Rhodosporidium, Sporidiobolus, Cystofilobasidium and Phaffia are known as producers of carotene pigments. Among yeasts, Rhodotorula species is one of main carotenoid-forming microorganisms with predominant synthesis of b-carotene, torulene and torularhodin (Davoli et al., 2004). Nevertheless, although there are many strategies for stimulation of carotene biosynthetic machinery in yeasts, attention is still focused on unexplored yeast’s habitats for selection of hyper-
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
producing strains what is the important step toward the design and optimization of biotechnological process for pigment formation (Libkind and van Broock, 2006; Maldonade et al., 2008). In order to improve the yield of carotenoid pigments and subsequently decrease the cost of this biotechnological process, diverse studies have been performed by optimizing the culture conditions including nutritional and physical factors. Factors such as nature and concentration of carbon and nitrogen sources, minerals, vitamins, pH, aeration, temperature, light and stress have a major influence on cell growth and yield of carotenoids. Numerous substrates have been considered as potential carbon sources for biotechnological production of carotenoids (Bhosale and Gadre, 2001; Lukacs et al., 2005, 2006). Work of Tinoi et al. (2005) demonstrates the effectiveness of using a widely available agro-industrial waste product as substrate and the importance of the sequential simplex optimization method in obtaining high carotenoid yields. Because overall yield of carotenoids is directly related to the total biomass yield, to keep both high growth rates and high flow carbon efficiency to carotenoids by optimal cultivation conditions is essential in order to achieve the maximal pigment productivity. During growth, different types of environmental and physiological stress conditions constantly challenge all organisms. In microorganisms, the environment of which is highly variable, stress responses are of particular importance. Under stress, different classes of substances are overproduced. Carotenoids are also involved in stress responses of microorganisms (Marova et al., 2004, 2010). Study of the molecular basis of these effects could make it possible to realize regulated overproduction of selected metabolites above all in such industrial applications, when it is not acceptable to use genetically modified strains. Moreover, biotechnological use of specific yeasts strains takes advantage of the utilization of the whole biomass, efficiently enriched for particular metabolites. The aim of the presented work is to study the influence of several types of waste substrates and stress factors on the production of carotenoids by yeast strains Rhodotorula glutinis, Rhodotorula mucilaginosa and Sporobolomyces roseus. 2. Experimental 2.1. Strains In the study following red yeast strains were tested: R. glutinis CCY 20-2-26, S. roseus CCY 19-4-8 and R. mucilaginosa CCY 20-7-31. These strains were obtained from the Culture Collection of Yeasts (CCY), Bratislava, Slovakia. The strain was conserved at malt-agar in darkness at 4 C. 2.2. Cultivation of microorganisms Red yeasts were cultivated in a simple glucose medium aerobically at 28 C. Physiological stress was induced by nutrition components (C and N source) and by addition of 5 mM peroxide and 2% and/or 5% NaCl. Three series of cultivations were realized with each strain. Twostep inoculation was done. All strains were firstly inoculated into a medium containing yeast extract (7 g), (NH4)2SO4 (5 g), glucose (40 g), KH2PO4 (5 g), MgSO4 (0.34 g) per liter (INO I) and cultivated at 28 C for 24 h at permanent shaking and lighting. Second inoculum (INO II) was prepared similarly, in 1st series was used the same medium as INO I, in 2nd series lyophilized whey was added (7 g/L) and in 3rd series potato extract (7 g/L) was added into INO II. Cultivation in INO II undergo at 28 C for 24 h at permanent shaking and lighting. Production media contained (NH4)2SO4 (5 g), glucose
S339
(40 g), KH2PO4 (5 g), MgSO4 (0.34 g) per liter. Several waste substrates were added and cultivation was done for 80 h at 28 C under permanent lighting and shaking. Production media were prepared according to following scheme: a) 1st series: INO IdINO IIdproduction: 1 e control, 2 e 5 mM peroxide, 3 e 2% NaCl, 4 e 5% NaCl, 5 e lyophillized whey nonprocessed (7 g/L), 6 e lyophillized whey processed by deproteination agent (7 g/L), 7 e potato extract (Hi Media; 7 g/L) b) 2nd series: INO IdINO II (þwhey, 7 g/L)dproduction: 1 e control, 2e5 mM peroxide, 3e2% NaCl, 4e5% NaCl, 5 e lyophilized whey non-processed (7 g/L), 6 e lyophillized whey processed by deproteination agent (7 g/L) c) 3rd series: INO IdINO II (þpotato extract 7 g/L)dproduction: 1 e control, 2 e 5 mM peroxide, 3 e 2% NaCl, 4 e 5% NaCl, 5 e potato extract (7 g/L). Whey waste substrate was obtained from dairy industry (Pribina Ltd., Pribyslav, Czech Republic) and its composition was as followed: water (94%), dry weight 60 g/L; ash 31 g/L; lactose 40 g/L; glucose 0.4 g/L; phosphorus 63 mg/L; soluble proteins 2 g/L; total nitrogen 0.12%. Whey substrate was either lyophilized without processing or processed by deproteination. Whey was acidified by 0.1 mol/L H2SO4 to pH 4.6, proteins were precipitated by boiling for 20 min and removed by centrifugation (5000 rpm; 10 min). Before cultivation was pH adjusted to neutral by 1 mol/L NaOH. Potato extract was obtained as microbial food supplement (HiMedia). Other chemicals were of analytical grade purity and obtained from local distributors. 2.3. Cultivation in a laboratory fermentor Pilot experiments with R. glutinis CCY 20-2-26, R. mucilaginosa CCY 20-7-31 and S. roseus CCY 19-4-8 were carried out in a 2-l laboratory fermentor Biostat B (B. Braun Biotech International, SRN). Two-step inoculation was performed in Erlenmeyer flasks in optimal inoculation medium (see above). The first inoculum (50 ml) was cultivated for 24 h at 28 C under permanent lighting and shaking. INO I was transferred into 240 ml of fresh inoculation medium (INO II) with or without waste substrate. INO II was grown at the same conditions as INO I. After 24 h, INO II was transferred into a laboratory fermentor containing sterile production medium. Cultivation in a fermentor was carried out at 28 C under permanent lighting, shaking (150e200 min1) and aeration (6 L of air/ min). Ten types of test stress experiments in 2 L fermentor were performed according to following scheme: 1) INO II (flask) e control; production medium (fermentor) e control glucose medium, 2) INO II (flask) e control; production medium (fermentor) e whey processed by protein precipitation 7 g/L, 3) INO II (flask) e control medium; production medium (fermentor) e potato extract 7 g/L, 4) INO II (flask) e whey processed; production medium (fermentor) e 5% NaCl, 5) INO II (flask) e whey processed; production medium (fermentor) e processed whey, 7 g/L, 6) INO II (flask) e potato extract; production medium (fermentor) e 5% NaCl, 7) INO II (flask) e potato extract; production medium (fermentor) e potato extract. 2.4. Extraction and analysis of carotenoids and other metabolites Cells were collected by centrifugation (3000 rpm; 30 min). For the subsequent isolation of carotenoids, the whole biomass obtained from 250 ml of medium was used. Yeast cells were disintegrated using a mechanical disruption by shaking with glass beads (70e100 U.S sieve). A mixture of pigments, sterols and other organic compounds was extracted from the cell homogenate using
S340
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
Table 1 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Rhodotorula glutinis CCY 20-2-26. Medium composition INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein. Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
Biomass (g/L) Beta-carotene (mg/g d.w.) 9.28 8.12 7.78 5.91 9.16 7.60 6.10 8.25 9.32 8.91 9.06 7.83 8.07 5.93 5.70
2.35 1.92 1.65 2.58 2.02 2.36 1.35 1.96 2.58 1.96 1.58 2.14 1.98 1.85 1.59
425.50 210.00 455.60 1068.20 1268.50 1055.30 365.20 241.30 289.60 1042.50 1012.50 425.60 175.20 911.50 1085.50
134.90 45.60 104.67 196.74 269.15 225.97 45.69 61.76 45.15 176.97 194.33 85.25 39.55 125.20 194.33
50 ml of acetone. After saponification of the extract by ethanolic KOH, carotenoids were extracted twice with 50 ml of diethyl ether. The diethyl ether extracts were collected and dried under vacuum. After evaporation, the residue was dissolved by 1e2 ml of methanol (gradient grade) and used for HPLC chromatographic analysis. Carotenoid pigments extracted from yeast cells were individually identified and quantified by RP-HPLC using a chromatographic system described previously (Marova et al., 2004, 2010). Samples (10 mL valve) were filtered through PTFE filters and injected onto Zorbax EclipsePlus C18 column (150 4.6 mm, 5 mm; Agilent Technologies) that had been equilibrated with a mobile phase (methanol/water; 95:5). Isocratic elution was carried out at 45 C by a flow rate of 1.0 ml/min. Detection of carotenoids was achieved at 450 nm. Data processing of analyses was assessed using Clarity software (DataApex, CZ). Individual carotenoids were verified by on-line LC/MS/ESI analysis (Mass spectrometer LCQ Advantage Max, Thermo Finnigan). 2.5. Statistical analysis In flask experiments three parallel cultivations were carried out with each strain and each substrate combination. In fermentor experiments three cultivations were performed in control experiments as well as in all cultivations with both Rhodotorula strains. Average values and standard deviations were evaluated. Biomass yields and carotenoid concentrations in individual cultivations were compared using Student t-test (p < 0.05). In S. roseus two parallel experiments with each substrate were carried out.
Table 2 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Sporobolomyces roseus CCY 19-4-8. Medium composition INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein Whey deprotein Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
3.1. Growth and production of metabolites by red yeasts in optimal conditions The growth curve of R. glutinis CCY 20-2-26 as well as other studied red yeast strains (data not shown) exhibited similarly typical two-phase character with prolonged stationary phase probably due to the ability of the yeast cells to utilize lipid storages formed during growth as additional energy source (Marova et al., 2010). The production of carotenoids during growth fluctuated and some local maxima and minima were observed. The maximum of beta-carotene production was obtained in all strains in stationary phase after about 80 h of cultivation.
5.82 4.84 4.26 5.20 5.25 5.04 5.37 4.92 3.86 4.71 5.35 3.34 4.77 3.14 4.61
0.93 1.36 1.05 0.80 1.15 0.74 0.89 0.66 0.65 0.59 1.11 0.98 1.31 0.45 0.88
Beta-carotene (mg/g d.w.) 65.60 440.80 1500.60 2520.30 2780.50 1745.90 625.50 930.00 1350.80 2580.00 1710.20 532.60 1300.70 981.60 594.42
23.27 120.00 123.40 225.36 331.12 289.00 147.20 220.10 369.82 258.80 450.10 162.14 364.40 230.80 123.12
3.2. Cultivation and production of biomass and pigments in modified conditions In this work the growth of some red yeast strains on selected waste substrates and subsequent effect of these substrates on betacarotene production was studied. It was observed that addition of non-processed or processed whey or potato extract to media can increase beta-carotene production, while biomass production changed relatively slightly. In R. glutinis addition of whey substrate into production medium led to 3.5-times increased production of beta-carotene without substantial changes in biomass. Non-processed whey or potato extract added to production media led to about 3-times increase of beta-carotene production accompanied by biomass loss. The highest yield was reached after addition of lyophillized nonprocessed whey to INO II as well as to production media. Liquid whey exhibited in all strains negative effect. Also potato extract added into INO II led to increased beta-carotene production while biomass yield was lower (Table 1). S. roseus (Table 2) exhibited significant changes in biomass:carotene ratio dependent on whey substrate addition. Substantial biomass decrease in presence of lyophilized whey in INO II (under 5 g/L) was accompanied by very high beta-carotene yields. Also potato extract addition into production medium led
Table 3 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Rhodotorula mucilaginosa CCY 20-7-31. Medium composition
3. Results and discussion
Biomass (g/L)
INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein Whey deprotein Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
Biomass (g/L) 8.12 8.18 5.64 6.48 6.93 7.90 7.24 7.46 4.51 4.72 6.78 8.02 8.45 8.11 7.70
Beta-carotene (mg/g d.w.)
1.85 62.50 1.14 51.70 1.58 432.60 2.01 200.20 1.55 228.60 1.82 384.80 0.98 45.20 1.44 51.20 1.12 422.30 1.54 384.40 1.88 140.50 2.14 42.60 1.38 115.70 2.30 1535.60 1.58 415.20
28.20 14.20 56.65 20.11 42.10 63.82 11.20 9.36 14.88 48.00 29.14 10.10 8.15 156.52 25.36
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
S341
Figure 1. Production of biomass (A) and beta-carotene (B) by studied red yeasts in whey and control media A. Notes: RG c e Rhodotorula glutinis, control cultivation; RG w e R. glutinis, INO II with deproteined whey; SR c e Sporobolomyces roseus, control cultivation; SR w e S. roseus, INO II with deproteined whey; RM c e Rhodotorula mucilaginosa, control cultivation; RM w e R. mucilaginosa, INO II with deproteined whey.
to about 11-fold increase of b-carotene production, while production of biomass was lower than in control. Preincubation of S. roseus cells with potato extract and following cultivation in production medium with 5% hydrogen peroxide led to about 20-times higher b-carotene production as in control, in this cultivation conditions biomass decreased only slightly. In general, total production of biomass by S. roseus was about 2-times lower as in R. glutinis. So, this is the reason why S. roseus CCY 19-4-8 strain is less suitable to enriched biomass production.
In optimal conditions R. mucilaginosa CCY 20-7-31 seems to be relatively poor producer of carotenoids when compared with the other two strains (Table 3). Production of biomass in this strain was more similar to R. glutinis (about 8 g/L). However, cultivation in presence of some complex waste substrate either in INO II or in production medium led to substantial increase of pigment production. Addition of potato extract into INO II combined with salt stress in production medium enabled to reach the highest biomass as well as b-carotene production observed in this strain yet
S342
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
Table 4 Production of beta-carotene enriched biomass in 2 L laboratory fermentor. Substrate/stress factor
Control 0/0 0/whey deprota 0/potato Wheya/salt Wheya/whey Potato/salt Potato/potato a
Biomass
S. roseus
R.g. (g/L)
S.r. (g/L)
37.14 44.56 28.12 40.86 34.60 26.10 18.56
17.00 9.59 10.80 8.16 10.15 7.14 6.28
R. mucilaginosa
R.m. (g/L)
b-carotene (mg/g d.w.)
b-carotene (mg/g d.w.)
b-carotene (mg/g d.w.)
b-carotene
b-carotene (mg/g d.w.)
b-carotene
(mg/L)
26.55 27.06 38.50 18.35 29.82 30.12 28.48
482.76 1025.12 905.10 685.15 1480.22 852.10 1211.10
17.93 45.68 25.45 28.00 51.22 22.23 22.48
191.00 2436.00 1825.06 1744.00 2896.16 1058.00 967.11
3.25 23.36 17.50 14.23 29.40 7.55 6.13
162.26 325.16 680.22 589.06 380.00 1856.40 956.22
4.31 8.80 26.18 10.81 11.33 55.91 27.23
(mg/L)
In all fermentor experiments whey processed by deproteinization was used.
(1536 mg/g d.w.). It seems that this strain needs for optimal pigment/biomass production some additional nutrition factors which are no present in simple (but cheap) inorganic medium, but can be obtained from different waste substrates (also cheap). Further test experiments with this strain are needed before cultivation on fermentor to find optimal cultivation conditions and production potential. Comparison of biomass and b-carotene yields produced by all three strains in whey media is demonstrated in Fig. 1. 3.3. Results of cultivation in laboratory fermentor In next series of preliminary experiments, an orientation batch cultivation of R. glutinis CCY 20-2-26, S. roseus CCY 19-4-8 and R. mucilaginosa CCY 20-7-31 cells was carried out in a 2-L laboratory fermentor. In these conditions several combinations of stress factors and waste substrates were tested. In experiments with R. glutinis the production of yeast biomass in a laboratory fermentor was in most types of cultivation more than 30 g per liter (about 3 higher yield than in Erlenmeyer flasks; Table 4). The balance of cultivation in a fermentor in optimum conditions is as follows: we obtained about 37.1 g/L of biomass containing 17.19 mg per liter of b-carotene (see Table 4). The production of b-carotene was induced in most types of media combinations. High total yield of b-carotene was obtained in whey production medium (44.56 g/L of biomass; 45.68 mg of b-carotene per liter of culture). The highest total yield of b-carotene was obtained using combined whey/whey medium (51.22 mg/L); this cultivation was accompanied also with relatively high biomass production (34.60 mg/L). In experiments with S. roseus CCY 19-4-8 substantially higher production of biomass was obtained in fermentor when compared with cultivation in flasks. Mainly in whey medium about 3-times biomass increase (about 12 g/L) was reached and production of bcarotene was mostly higher than in R. glutinis. Because of low biomass production, total yields were in S. roseus mostly lower than in R. glutinis cells. The best b-carotene production (29.4 g/L) was obtained on whey medium (see Table 4). Yeast strain R. mucilaginosa CCY 20-7-31 exhibited in most cases similar biomass production characteristics as R. glutninis, while pigment production was substantially lower (see Table 4). As the only substrate suitable for b-carotene production was found potato extract in INO II combined with 5% salt in production medium. Under these conditions 55.91 mg/L of b-carotene was produced in 30.12 g of cells per liter of medium. The aim of all preliminary experiments carried out in laboratory fermentor was to obtain basic information about potential biotechnological use of the tested strains to the industrial production of b-carotene enriched biomass. The results of both Rhodotorula strains are very promising. The yield of R. glutinis CCY 20-2-26 biomass (37e44.5 g/L) produced in minimal cultivation
medium was similar to the maximal biomass yield obtained in fedbatch cultivation of Phaffia rhodozyma (36 g/L), which is widely used as an industrial producer of astaxanthin (Lukacs et al., 2006). The maximal production of total carotenoids by used P. rhodozyma mutant strain was 40 mg/L, which is also similar to the yields obtained in R. glutinis CCY 20-2-26 cells grown in whey medium. 4. Conclusions Changes in medium composition can lead to substantial changes in biomass as well as carotenoid production. Waste substrates can be used as medium component, which can in particular strains and conditions induce carotenoid as well as biomass production. Thus, cheap waste substrates could be used industrially for carotenoidrich biomass production. From tested red yeast strains predominantly R. glutinis CCY 20-226 can be used for industrial production of carotenoid-rich biomass using processed waste substrates and/or mild physiological stress. Acknowledgments This work has been supported by project "Centre for Materials Research at FCH BUT" No. CZ.1.05/2.1.00/01.0012 from ERDF and project MSM0021630501 of the Czech Ministry of Education. References Britton, G., Liaaen-Jensen, S., Pfander, H., 1998. Carotenoids. In: Biosynthesis and Metabolism, vol. 3. Birkhäuser Verlag Basel, pp. 13e140. Bhosale, P., Gadre, R.V., 2001. Beta-carotene production in sugar cane molasses by a Rhodotorula glutinis mutant. Indian Journal of Microbiology and Biotechnology 26, 327e332. Davoli, P., Mierau, V., Weber, R.W.S., 2004. Carotenoids and Fatty Acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis. Applied Biochemistry and Microbiology 40, 392e397. Fraser, P.D., Bramley, P.M., 2004. The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research 43, 228e265. Libkind, D., van Broock, M., 2006. Biomass and carotenoid pigment production by patagonian native yeasts. World Journal of Microbiology & Biotechnology 22, 687e692. Lukacs, G., Kovacs, N., Papp, T., Vagvolgyi, C., 2005. The effect of vegetable oils on carotenoid production of Phaffia rhodozyma. Acta Microbiologica Immunologica Hungarica 52, 267. Lukacs, G., Linka, B., Nyilasi, I., 2006. Phaffia rhodozyma and Xanthophyllomuces dendrorhous: astaxanthin-producing yeasts of biotechnological importance. Acta Alimentaria 5, 99e107. Maldonade, I.R., Rodriguez-Amaya, D.B., Scamparini, A.R.P., 2008. Carotenoids of yeasts isolated from the Brazilian ecosystem. Food Chemistry 107, 145e150. Marova, I., Breierova, E., Koci, R., Friedl, Z., Slovak, B., Pokorna, J., 2004. Influence of exogenous stress factors on production of carotenoids by some strains of carotenogenic yeasts. Annals of Microbiology 54, 73e85. Marova, I., Carnecka, M., Halienova, A., Koci, R., Breierova, E., 2010. Production of carotenoid/ergosterol supplemeted biomass by red yeast Rhodotorula glutinis grown under external stress. Food Technology and Biotechnology 48, 56e61. Tinoi, J., Rakariyatham, N., Deming, R.L., 2005. Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochemistry 40, 2551e2557.
Contents lists available at ScienceDirect
Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Use of several waste substrates for carotenoid-rich yeast biomass production I. Marova a, b, *, M. Carnecka b, A. Halienova b, M. Certik c, T. Dvorakova b, A. Haronikova a, b a
Brno University of Technology, Faculty of Chemistry, Centre for Materials Research, Purkynova 118, 612 00 Brno, Czech Republic Brno University of Technology, Faculty of Chemistry, Department of Food Chemistry and Biotechnology, Purkynova 118, 612 00 Brno, Czech Republic c Slovak University of Technology, Faculty of Chemical and Food Technology, Radlinského 9, 812 37 Bratislava, Slovak Republic b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 September 2009 Received in revised form 1 April 2011 Accepted 8 June 2011 Available online 8 July 2011
Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/ L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Carotenoids Waste substrates Yeast biomass Rhodotorula sp. Sporobolomyces sp.
1. Introduction Carotenoids are naturally occurring lipid-soluble pigments, the majority being C40 terpenoids. They act as membrane-protective antioxidants that efficiently scavenge 1O2 and peroxyl radicals; the antioxidant efficiency is apparently related to their structure. Carotenoid pigments occur universally in photosynthetic systems of higher plants, algae and phototrophic bacteria. In nonphotosynthetic organisms, carotenoids are important in protecting against photo-oxidative damage. Thus, many non-phototrophic bacteria and fungi rely on carotenoids for protection when growing in light and air (Britton et al., 1998).
* Corresponding author. Brno University of Technology, Faculty of Chemistry, Centre for Materials Research, Purkynova 118, 612 00 Brno, Czech Republic. Tel.: þ420 541 149 419; fax: þ420 541 211 697. E-mail address: [email protected] (I. Marova). 0301-4797/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jenvman.2011.06.018
Commercially, carotenoids are used as food colorants and nutritional supplements, with an estimated global market of some $935 million by 2005 (Fraser and Bramley, 2004). There is an increased interest in carotenoids as natural antioxidants and free radical scavengers for their ability to reduce and alleviate chronic diseases, various pathological stages and aging. However, the application of chemical synthetic methods to prepare carotenoid compounds as food additives has been strictly regulated in recent years. Therefore attention is paid on the finding of suitable natural sources including biotechnological processes. The number of red yeasts species Rhodotorula, Rhodosporidium, Sporidiobolus, Cystofilobasidium and Phaffia are known as producers of carotene pigments. Among yeasts, Rhodotorula species is one of main carotenoid-forming microorganisms with predominant synthesis of b-carotene, torulene and torularhodin (Davoli et al., 2004). Nevertheless, although there are many strategies for stimulation of carotene biosynthetic machinery in yeasts, attention is still focused on unexplored yeast’s habitats for selection of hyper-
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
producing strains what is the important step toward the design and optimization of biotechnological process for pigment formation (Libkind and van Broock, 2006; Maldonade et al., 2008). In order to improve the yield of carotenoid pigments and subsequently decrease the cost of this biotechnological process, diverse studies have been performed by optimizing the culture conditions including nutritional and physical factors. Factors such as nature and concentration of carbon and nitrogen sources, minerals, vitamins, pH, aeration, temperature, light and stress have a major influence on cell growth and yield of carotenoids. Numerous substrates have been considered as potential carbon sources for biotechnological production of carotenoids (Bhosale and Gadre, 2001; Lukacs et al., 2005, 2006). Work of Tinoi et al. (2005) demonstrates the effectiveness of using a widely available agro-industrial waste product as substrate and the importance of the sequential simplex optimization method in obtaining high carotenoid yields. Because overall yield of carotenoids is directly related to the total biomass yield, to keep both high growth rates and high flow carbon efficiency to carotenoids by optimal cultivation conditions is essential in order to achieve the maximal pigment productivity. During growth, different types of environmental and physiological stress conditions constantly challenge all organisms. In microorganisms, the environment of which is highly variable, stress responses are of particular importance. Under stress, different classes of substances are overproduced. Carotenoids are also involved in stress responses of microorganisms (Marova et al., 2004, 2010). Study of the molecular basis of these effects could make it possible to realize regulated overproduction of selected metabolites above all in such industrial applications, when it is not acceptable to use genetically modified strains. Moreover, biotechnological use of specific yeasts strains takes advantage of the utilization of the whole biomass, efficiently enriched for particular metabolites. The aim of the presented work is to study the influence of several types of waste substrates and stress factors on the production of carotenoids by yeast strains Rhodotorula glutinis, Rhodotorula mucilaginosa and Sporobolomyces roseus. 2. Experimental 2.1. Strains In the study following red yeast strains were tested: R. glutinis CCY 20-2-26, S. roseus CCY 19-4-8 and R. mucilaginosa CCY 20-7-31. These strains were obtained from the Culture Collection of Yeasts (CCY), Bratislava, Slovakia. The strain was conserved at malt-agar in darkness at 4 C. 2.2. Cultivation of microorganisms Red yeasts were cultivated in a simple glucose medium aerobically at 28 C. Physiological stress was induced by nutrition components (C and N source) and by addition of 5 mM peroxide and 2% and/or 5% NaCl. Three series of cultivations were realized with each strain. Twostep inoculation was done. All strains were firstly inoculated into a medium containing yeast extract (7 g), (NH4)2SO4 (5 g), glucose (40 g), KH2PO4 (5 g), MgSO4 (0.34 g) per liter (INO I) and cultivated at 28 C for 24 h at permanent shaking and lighting. Second inoculum (INO II) was prepared similarly, in 1st series was used the same medium as INO I, in 2nd series lyophilized whey was added (7 g/L) and in 3rd series potato extract (7 g/L) was added into INO II. Cultivation in INO II undergo at 28 C for 24 h at permanent shaking and lighting. Production media contained (NH4)2SO4 (5 g), glucose
S339
(40 g), KH2PO4 (5 g), MgSO4 (0.34 g) per liter. Several waste substrates were added and cultivation was done for 80 h at 28 C under permanent lighting and shaking. Production media were prepared according to following scheme: a) 1st series: INO IdINO IIdproduction: 1 e control, 2 e 5 mM peroxide, 3 e 2% NaCl, 4 e 5% NaCl, 5 e lyophillized whey nonprocessed (7 g/L), 6 e lyophillized whey processed by deproteination agent (7 g/L), 7 e potato extract (Hi Media; 7 g/L) b) 2nd series: INO IdINO II (þwhey, 7 g/L)dproduction: 1 e control, 2e5 mM peroxide, 3e2% NaCl, 4e5% NaCl, 5 e lyophilized whey non-processed (7 g/L), 6 e lyophillized whey processed by deproteination agent (7 g/L) c) 3rd series: INO IdINO II (þpotato extract 7 g/L)dproduction: 1 e control, 2 e 5 mM peroxide, 3 e 2% NaCl, 4 e 5% NaCl, 5 e potato extract (7 g/L). Whey waste substrate was obtained from dairy industry (Pribina Ltd., Pribyslav, Czech Republic) and its composition was as followed: water (94%), dry weight 60 g/L; ash 31 g/L; lactose 40 g/L; glucose 0.4 g/L; phosphorus 63 mg/L; soluble proteins 2 g/L; total nitrogen 0.12%. Whey substrate was either lyophilized without processing or processed by deproteination. Whey was acidified by 0.1 mol/L H2SO4 to pH 4.6, proteins were precipitated by boiling for 20 min and removed by centrifugation (5000 rpm; 10 min). Before cultivation was pH adjusted to neutral by 1 mol/L NaOH. Potato extract was obtained as microbial food supplement (HiMedia). Other chemicals were of analytical grade purity and obtained from local distributors. 2.3. Cultivation in a laboratory fermentor Pilot experiments with R. glutinis CCY 20-2-26, R. mucilaginosa CCY 20-7-31 and S. roseus CCY 19-4-8 were carried out in a 2-l laboratory fermentor Biostat B (B. Braun Biotech International, SRN). Two-step inoculation was performed in Erlenmeyer flasks in optimal inoculation medium (see above). The first inoculum (50 ml) was cultivated for 24 h at 28 C under permanent lighting and shaking. INO I was transferred into 240 ml of fresh inoculation medium (INO II) with or without waste substrate. INO II was grown at the same conditions as INO I. After 24 h, INO II was transferred into a laboratory fermentor containing sterile production medium. Cultivation in a fermentor was carried out at 28 C under permanent lighting, shaking (150e200 min1) and aeration (6 L of air/ min). Ten types of test stress experiments in 2 L fermentor were performed according to following scheme: 1) INO II (flask) e control; production medium (fermentor) e control glucose medium, 2) INO II (flask) e control; production medium (fermentor) e whey processed by protein precipitation 7 g/L, 3) INO II (flask) e control medium; production medium (fermentor) e potato extract 7 g/L, 4) INO II (flask) e whey processed; production medium (fermentor) e 5% NaCl, 5) INO II (flask) e whey processed; production medium (fermentor) e processed whey, 7 g/L, 6) INO II (flask) e potato extract; production medium (fermentor) e 5% NaCl, 7) INO II (flask) e potato extract; production medium (fermentor) e potato extract. 2.4. Extraction and analysis of carotenoids and other metabolites Cells were collected by centrifugation (3000 rpm; 30 min). For the subsequent isolation of carotenoids, the whole biomass obtained from 250 ml of medium was used. Yeast cells were disintegrated using a mechanical disruption by shaking with glass beads (70e100 U.S sieve). A mixture of pigments, sterols and other organic compounds was extracted from the cell homogenate using
S340
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
Table 1 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Rhodotorula glutinis CCY 20-2-26. Medium composition INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein. Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
Biomass (g/L) Beta-carotene (mg/g d.w.) 9.28 8.12 7.78 5.91 9.16 7.60 6.10 8.25 9.32 8.91 9.06 7.83 8.07 5.93 5.70
2.35 1.92 1.65 2.58 2.02 2.36 1.35 1.96 2.58 1.96 1.58 2.14 1.98 1.85 1.59
425.50 210.00 455.60 1068.20 1268.50 1055.30 365.20 241.30 289.60 1042.50 1012.50 425.60 175.20 911.50 1085.50
134.90 45.60 104.67 196.74 269.15 225.97 45.69 61.76 45.15 176.97 194.33 85.25 39.55 125.20 194.33
50 ml of acetone. After saponification of the extract by ethanolic KOH, carotenoids were extracted twice with 50 ml of diethyl ether. The diethyl ether extracts were collected and dried under vacuum. After evaporation, the residue was dissolved by 1e2 ml of methanol (gradient grade) and used for HPLC chromatographic analysis. Carotenoid pigments extracted from yeast cells were individually identified and quantified by RP-HPLC using a chromatographic system described previously (Marova et al., 2004, 2010). Samples (10 mL valve) were filtered through PTFE filters and injected onto Zorbax EclipsePlus C18 column (150 4.6 mm, 5 mm; Agilent Technologies) that had been equilibrated with a mobile phase (methanol/water; 95:5). Isocratic elution was carried out at 45 C by a flow rate of 1.0 ml/min. Detection of carotenoids was achieved at 450 nm. Data processing of analyses was assessed using Clarity software (DataApex, CZ). Individual carotenoids were verified by on-line LC/MS/ESI analysis (Mass spectrometer LCQ Advantage Max, Thermo Finnigan). 2.5. Statistical analysis In flask experiments three parallel cultivations were carried out with each strain and each substrate combination. In fermentor experiments three cultivations were performed in control experiments as well as in all cultivations with both Rhodotorula strains. Average values and standard deviations were evaluated. Biomass yields and carotenoid concentrations in individual cultivations were compared using Student t-test (p < 0.05). In S. roseus two parallel experiments with each substrate were carried out.
Table 2 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Sporobolomyces roseus CCY 19-4-8. Medium composition INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein Whey deprotein Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
3.1. Growth and production of metabolites by red yeasts in optimal conditions The growth curve of R. glutinis CCY 20-2-26 as well as other studied red yeast strains (data not shown) exhibited similarly typical two-phase character with prolonged stationary phase probably due to the ability of the yeast cells to utilize lipid storages formed during growth as additional energy source (Marova et al., 2010). The production of carotenoids during growth fluctuated and some local maxima and minima were observed. The maximum of beta-carotene production was obtained in all strains in stationary phase after about 80 h of cultivation.
5.82 4.84 4.26 5.20 5.25 5.04 5.37 4.92 3.86 4.71 5.35 3.34 4.77 3.14 4.61
0.93 1.36 1.05 0.80 1.15 0.74 0.89 0.66 0.65 0.59 1.11 0.98 1.31 0.45 0.88
Beta-carotene (mg/g d.w.) 65.60 440.80 1500.60 2520.30 2780.50 1745.90 625.50 930.00 1350.80 2580.00 1710.20 532.60 1300.70 981.60 594.42
23.27 120.00 123.40 225.36 331.12 289.00 147.20 220.10 369.82 258.80 450.10 162.14 364.40 230.80 123.12
3.2. Cultivation and production of biomass and pigments in modified conditions In this work the growth of some red yeast strains on selected waste substrates and subsequent effect of these substrates on betacarotene production was studied. It was observed that addition of non-processed or processed whey or potato extract to media can increase beta-carotene production, while biomass production changed relatively slightly. In R. glutinis addition of whey substrate into production medium led to 3.5-times increased production of beta-carotene without substantial changes in biomass. Non-processed whey or potato extract added to production media led to about 3-times increase of beta-carotene production accompanied by biomass loss. The highest yield was reached after addition of lyophillized nonprocessed whey to INO II as well as to production media. Liquid whey exhibited in all strains negative effect. Also potato extract added into INO II led to increased beta-carotene production while biomass yield was lower (Table 1). S. roseus (Table 2) exhibited significant changes in biomass:carotene ratio dependent on whey substrate addition. Substantial biomass decrease in presence of lyophilized whey in INO II (under 5 g/L) was accompanied by very high beta-carotene yields. Also potato extract addition into production medium led
Table 3 Production of biomass and pigments by red yeasts in Erlenmeyer flasks by Rhodotorula mucilaginosa CCY 20-7-31. Medium composition
3. Results and discussion
Biomass (g/L)
INO I
INO II
Production
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Control Control Control Control Control Control Whey deprotein. Whey deprotein. Whey deprotein. Whey deprotein Whey deprotein Potato extract. Potato extract Potato extract Potato extract
Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Potato extract Control Peroxide 5 mmol/L Salt 5% Whey lyophilized Whey deprotein. Control Peroxide 5 mmol/L Salt 5% Potato extract
Biomass (g/L) 8.12 8.18 5.64 6.48 6.93 7.90 7.24 7.46 4.51 4.72 6.78 8.02 8.45 8.11 7.70
Beta-carotene (mg/g d.w.)
1.85 62.50 1.14 51.70 1.58 432.60 2.01 200.20 1.55 228.60 1.82 384.80 0.98 45.20 1.44 51.20 1.12 422.30 1.54 384.40 1.88 140.50 2.14 42.60 1.38 115.70 2.30 1535.60 1.58 415.20
28.20 14.20 56.65 20.11 42.10 63.82 11.20 9.36 14.88 48.00 29.14 10.10 8.15 156.52 25.36
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
S341
Figure 1. Production of biomass (A) and beta-carotene (B) by studied red yeasts in whey and control media A. Notes: RG c e Rhodotorula glutinis, control cultivation; RG w e R. glutinis, INO II with deproteined whey; SR c e Sporobolomyces roseus, control cultivation; SR w e S. roseus, INO II with deproteined whey; RM c e Rhodotorula mucilaginosa, control cultivation; RM w e R. mucilaginosa, INO II with deproteined whey.
to about 11-fold increase of b-carotene production, while production of biomass was lower than in control. Preincubation of S. roseus cells with potato extract and following cultivation in production medium with 5% hydrogen peroxide led to about 20-times higher b-carotene production as in control, in this cultivation conditions biomass decreased only slightly. In general, total production of biomass by S. roseus was about 2-times lower as in R. glutinis. So, this is the reason why S. roseus CCY 19-4-8 strain is less suitable to enriched biomass production.
In optimal conditions R. mucilaginosa CCY 20-7-31 seems to be relatively poor producer of carotenoids when compared with the other two strains (Table 3). Production of biomass in this strain was more similar to R. glutinis (about 8 g/L). However, cultivation in presence of some complex waste substrate either in INO II or in production medium led to substantial increase of pigment production. Addition of potato extract into INO II combined with salt stress in production medium enabled to reach the highest biomass as well as b-carotene production observed in this strain yet
S342
I. Marova et al. / Journal of Environmental Management 95 (2012) S338eS342
Table 4 Production of beta-carotene enriched biomass in 2 L laboratory fermentor. Substrate/stress factor
Control 0/0 0/whey deprota 0/potato Wheya/salt Wheya/whey Potato/salt Potato/potato a
Biomass
S. roseus
R.g. (g/L)
S.r. (g/L)
37.14 44.56 28.12 40.86 34.60 26.10 18.56
17.00 9.59 10.80 8.16 10.15 7.14 6.28
R. mucilaginosa
R.m. (g/L)
b-carotene (mg/g d.w.)
b-carotene (mg/g d.w.)
b-carotene (mg/g d.w.)
b-carotene
b-carotene (mg/g d.w.)
b-carotene
(mg/L)
26.55 27.06 38.50 18.35 29.82 30.12 28.48
482.76 1025.12 905.10 685.15 1480.22 852.10 1211.10
17.93 45.68 25.45 28.00 51.22 22.23 22.48
191.00 2436.00 1825.06 1744.00 2896.16 1058.00 967.11
3.25 23.36 17.50 14.23 29.40 7.55 6.13
162.26 325.16 680.22 589.06 380.00 1856.40 956.22
4.31 8.80 26.18 10.81 11.33 55.91 27.23
(mg/L)
In all fermentor experiments whey processed by deproteinization was used.
(1536 mg/g d.w.). It seems that this strain needs for optimal pigment/biomass production some additional nutrition factors which are no present in simple (but cheap) inorganic medium, but can be obtained from different waste substrates (also cheap). Further test experiments with this strain are needed before cultivation on fermentor to find optimal cultivation conditions and production potential. Comparison of biomass and b-carotene yields produced by all three strains in whey media is demonstrated in Fig. 1. 3.3. Results of cultivation in laboratory fermentor In next series of preliminary experiments, an orientation batch cultivation of R. glutinis CCY 20-2-26, S. roseus CCY 19-4-8 and R. mucilaginosa CCY 20-7-31 cells was carried out in a 2-L laboratory fermentor. In these conditions several combinations of stress factors and waste substrates were tested. In experiments with R. glutinis the production of yeast biomass in a laboratory fermentor was in most types of cultivation more than 30 g per liter (about 3 higher yield than in Erlenmeyer flasks; Table 4). The balance of cultivation in a fermentor in optimum conditions is as follows: we obtained about 37.1 g/L of biomass containing 17.19 mg per liter of b-carotene (see Table 4). The production of b-carotene was induced in most types of media combinations. High total yield of b-carotene was obtained in whey production medium (44.56 g/L of biomass; 45.68 mg of b-carotene per liter of culture). The highest total yield of b-carotene was obtained using combined whey/whey medium (51.22 mg/L); this cultivation was accompanied also with relatively high biomass production (34.60 mg/L). In experiments with S. roseus CCY 19-4-8 substantially higher production of biomass was obtained in fermentor when compared with cultivation in flasks. Mainly in whey medium about 3-times biomass increase (about 12 g/L) was reached and production of bcarotene was mostly higher than in R. glutinis. Because of low biomass production, total yields were in S. roseus mostly lower than in R. glutinis cells. The best b-carotene production (29.4 g/L) was obtained on whey medium (see Table 4). Yeast strain R. mucilaginosa CCY 20-7-31 exhibited in most cases similar biomass production characteristics as R. glutninis, while pigment production was substantially lower (see Table 4). As the only substrate suitable for b-carotene production was found potato extract in INO II combined with 5% salt in production medium. Under these conditions 55.91 mg/L of b-carotene was produced in 30.12 g of cells per liter of medium. The aim of all preliminary experiments carried out in laboratory fermentor was to obtain basic information about potential biotechnological use of the tested strains to the industrial production of b-carotene enriched biomass. The results of both Rhodotorula strains are very promising. The yield of R. glutinis CCY 20-2-26 biomass (37e44.5 g/L) produced in minimal cultivation
medium was similar to the maximal biomass yield obtained in fedbatch cultivation of Phaffia rhodozyma (36 g/L), which is widely used as an industrial producer of astaxanthin (Lukacs et al., 2006). The maximal production of total carotenoids by used P. rhodozyma mutant strain was 40 mg/L, which is also similar to the yields obtained in R. glutinis CCY 20-2-26 cells grown in whey medium. 4. Conclusions Changes in medium composition can lead to substantial changes in biomass as well as carotenoid production. Waste substrates can be used as medium component, which can in particular strains and conditions induce carotenoid as well as biomass production. Thus, cheap waste substrates could be used industrially for carotenoidrich biomass production. From tested red yeast strains predominantly R. glutinis CCY 20-226 can be used for industrial production of carotenoid-rich biomass using processed waste substrates and/or mild physiological stress. Acknowledgments This work has been supported by project "Centre for Materials Research at FCH BUT" No. CZ.1.05/2.1.00/01.0012 from ERDF and project MSM0021630501 of the Czech Ministry of Education. References Britton, G., Liaaen-Jensen, S., Pfander, H., 1998. Carotenoids. In: Biosynthesis and Metabolism, vol. 3. Birkhäuser Verlag Basel, pp. 13e140. Bhosale, P., Gadre, R.V., 2001. Beta-carotene production in sugar cane molasses by a Rhodotorula glutinis mutant. Indian Journal of Microbiology and Biotechnology 26, 327e332. Davoli, P., Mierau, V., Weber, R.W.S., 2004. Carotenoids and Fatty Acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis. Applied Biochemistry and Microbiology 40, 392e397. Fraser, P.D., Bramley, P.M., 2004. The biosynthesis and nutritional uses of carotenoids. Progress in Lipid Research 43, 228e265. Libkind, D., van Broock, M., 2006. Biomass and carotenoid pigment production by patagonian native yeasts. World Journal of Microbiology & Biotechnology 22, 687e692. Lukacs, G., Kovacs, N., Papp, T., Vagvolgyi, C., 2005. The effect of vegetable oils on carotenoid production of Phaffia rhodozyma. Acta Microbiologica Immunologica Hungarica 52, 267. Lukacs, G., Linka, B., Nyilasi, I., 2006. Phaffia rhodozyma and Xanthophyllomuces dendrorhous: astaxanthin-producing yeasts of biotechnological importance. Acta Alimentaria 5, 99e107. Maldonade, I.R., Rodriguez-Amaya, D.B., Scamparini, A.R.P., 2008. Carotenoids of yeasts isolated from the Brazilian ecosystem. Food Chemistry 107, 145e150. Marova, I., Breierova, E., Koci, R., Friedl, Z., Slovak, B., Pokorna, J., 2004. Influence of exogenous stress factors on production of carotenoids by some strains of carotenogenic yeasts. Annals of Microbiology 54, 73e85. Marova, I., Carnecka, M., Halienova, A., Koci, R., Breierova, E., 2010. Production of carotenoid/ergosterol supplemeted biomass by red yeast Rhodotorula glutinis grown under external stress. Food Technology and Biotechnology 48, 56e61. Tinoi, J., Rakariyatham, N., Deming, R.L., 2005. Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate. Process Biochemistry 40, 2551e2557.