Commercial interest in the use of natural pigments isolated from microorganisms has increased in recent years; hence, molecules belonging to the polyisoprenoid group (i.e. β-carotene, astaxanthin, and canthaxanthin) have been the focus of much attention. The bacterium Gordonia jacobaea readily synthesizes and accumulates large amounts of canthaxanthin (β-β´-carotene-4,4´-dione), which is widely used in the food and cosmetics industries. In the present work, the effects of different low-cost raw materials on fermentation and canthaxanthin accumulation by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin production and peak levels of accumulation varied according to the different media used. [Int Microbiol 2005; 8(1):55-58

Fernando Rosa dos Santos

Lucía Blasco

Margarita Poza

Patricia Veiga-Crespo

Tomás G. Villa

Veiga-Crespo, Patricia

Blasco, Lucía

Rosa dos Santos, Fernando

Poza, Margarita

Villa, Tomás G.

Hemeroteca Cientifica Catalana

RESEARCH NOTESummary. Commercial interest in the use of natural pigments isolated frommicroorganisms has increased in recent years; hence, molecules belonging to thepolyisoprenoid group (i.e. β-carotene, astaxanthin, and canthaxanthin) have beenthe focus of much attention. The bacterium Gordonia jacobaea readily synthesizesand accumulates large amounts of canthaxanthin (β-β´-carotene-4,4´-dione), whichis widely used in the food and cosmetics industries. In the present work, the effectsof different low-cost raw materials on fermentation and canthaxanthin accumula-tion by a hyperpigmented strain of G. jacobaea were studied. Canthaxanthin pro-duction and peak levels of accumulation varied according to the different mediaused. [Int Microbiol 2005; 8(1):55-58]Key words: Gordonia jacobaea · canthaxanthin · carotenoids · soy-meal · fer-mentation Influence of culture conditions of Gordoniajacobaea MV-26 oncanthaxanthin productionIntroductionCanthaxanthin (β-β´-carotene-4,4´-dione) is a ubiquitousketo-carotene that is of considerable industrial interestbecause of its widespread use in both the food and cosmeticindustries [12]. While synthetic forms of canthaxanthin andmany other pigments are currently available, their use in thefood industry has been questioned by consumer agencies, sothat the development of new sources of natural pigments hasbecome essential to meet the increasing demand. Due to their ease of manipulation, microorganisms pro-vide an excellent system for the large-scale production ofcarotenoids, as has been shown with the yeast Phaffiarhodozyma [13] and the bacterium Brevibacterium KY-4313[14], which accumulate the natural carotenoids astaxanthinand canthaxanthin, respectively. However, the inability ofthese two sources to meet world-wide demand has spurredresearch aimed at finding new sources (Table 1) and at opti-mizing fermentation technologies. Gordonia jacobaea MV-1 (gram-positive, catalase nega-tive, G + C 61%), which was isolated in this laboratory duringa routine screening of pigmented microorganisms, is able toaccumulate several carotenoids, including the keto-carotenoidtrans-canthaxanthin [8]. However, its low carotenoid content(200 µg/g dry weight) does not support its industrial applica-tion. After several rounds of mutations, a hyperpigmentedmutant (MV-26) with enhanced canthaxanthin and β-caroteneaccumulation was obtained. This mutant accumulates six-foldmore canthaxanthin than the wild-type strain [9].The influence of growth conditions and medium compo-sition on the carotenoid synthesis pathway has been reportedpreviously. This pathway begins with isoprenyl pyrophos-phate (IPP), which may be formed either from mevalonicacid (MVA) or through the glyceraldehyde phosphate/pyru-vate pathway [4,11]. Thus, media rich in these or related pre-Patricia Veiga-Crespo1Lucía Blasco2Fernando Rosa dos Santos3Margarita Poza1Tomás G. Villa1,2*1Department of Microbiology,Faculty of Pharmacy, University of Santiago de Compostela,Spain2School of Biotechnology,University of Santiago deCompostela, Spain3Department of PharmaceuticalTechnology, Faculty of Pharmacy,University of Santiago deCompostela, SpainINTERNATIONAL MICROBIOLOGY (2005) 8:55-58 www.im.microbios.orgReceived 3 November 2004Accepted 11 January 2005*Corresponding author:T.G. VillaDepartment of MicrobiologyFaculty of Pharmacy, Campus Sur15782 Santiago de Compostela, SpainTel.: +34-981563100 ext.14949. Fax: +34-981592490E-mail: mpvilla@usc.es56 INT. MICROBIOL. Vol. 8, 2005cursors can be used to increase the carotenoid yield in industrialfermentations. In the present study, different media and low-costraw materials were assayed in order to determine the optimalconditions of canthaxanthin production by G. jacobaea MV-26.Materials and methodsStrains and culture conditions. The strain employed was the hyper-pigmented Gordonia jacobaea MV-26 [9]. The bacterium was grown in dif-ferent commercial media: yeast extract peptone dextrose (YPD) (20 g pep-tone/l, 10 g yeast extract/l, 20 g glucose/l), tryptone soy-meal broth (TSB) (3g soy-meal peptone/l, 2.5 g glucose/l, 17 g casein peptone/l, 5 g dipotassiumhydrogen phosphate/l, 5 g NaCl/l) and brain heart infusion broth (BHIB)(12.5 g calf-brain infusion solids/l, 5 g beef-heart infusion solids/l, 10 g pro-tease peptone/l, 2 g glucose/l, 5 g NaCl/l, 2.5 g di-sodium phosphate/l). Lowcost media consisting of different proportions of soy meal (0.5%–2%), beetmolasses (0.5%–5%), and a mixture of soy meal (0%–5%) and glucose(0%–10%) were also assayed.G. jacobaea MV-26 was grown in 1-l flasks containing 250 ml of theappropriate medium at 30ºC or 37ºC in a rotary shaker (150 rpm) for 8 days.In the case of YPD medium, screening was implemented for 10 days. Everyother day, aliquots were withdrawn, and the levels of β-carotene and can-thaxanthin (Fig. 1) were evaluated. After this first screening, G. jacobaeawas grown in 2 l of each medium: BHIB, TSB, YPD, 0.5% soy meal, and1.5% glucose/2% soy meal at 30ºC in a rotary shaker (150 rpm) since thesemedia resulted in the highest levels of canthaxanthin production. When nec-essary, G. jacobaea was plated on medium supplemented with agar (2%).Growth curve of Gordonia jacobaea MV-26. Growth curveswere established either by determining the optical density at 600 nm in aBeckman DU-40 spectrophotometer or by plate counting (colony-formingunits, CFU) when complex media were used (i.e. soy-meal-based).Analysis of pigment production. Samples (3 ml) were withdrawnevery 24 h and 1 ml of 0.1 M potassium phosphate buffer (pH 7) and 3 mlhexane (Merck) were added. After vigorous vortexing, the samples werecentrifuged for 10 min at 5000 rpm to allow phase formation. The organicphase was filtered through a 0.22-µm filter (Gelman Sciences) and thepigment content was evaluated. When pigment production was analyzedin 2-liter cultures, an additional ethanol extraction was performed. In thiscase, following maximum canthaxanthin production, the biomass was harvest-ed at 4ºC by means of continuous-flow centrifugation at 15000×g. The pig-ments were subsequently extracted by resuspending the cells in pure ethanol.Carotenoid pigments were analyzed by HPLC using a silica-gel column(Teknokroma, 5-µm pore size, 25-cm length and 45-mm diameter). Themobile phase was hexane/ethyl acetate (1:1 v/v) (Romil). The flow was 1ml/min and the pressure was 0.4 kpsi; the injection volume was 30 µl andthe temperature was 25ºC. HPLC analysis of carotenoid pigments extractedwith ethanol was carried out by adding one volume of hexane plus 1 ml 0.1M potassium phosphate buffer (pH 7). After centrifugation for 10 min at5000 rpm, the hexane phase was recovered and filtered as described previ-ously. The peaks were evaluated based on their absorption at 480 nm.Retention times and concentrations of the samples were compared with purestandards of β-carotene and canthaxanthin.Statistical analysis. The results of the influence of the growth mediaon canthaxanthin production were subjected to statistical analysis with theSPSS 12.0 program.Results and DiscussionG. jacobaea MV-26 grew in all of the media, except those contain-ing only beet molasses. While in all media supporting growth theproduction of carotenoids varied, maximum canthaxanthin accu-mulation consistently occurred in the stationary phase of growth(Fig. 2). Moreover, pigment production in G. jacobaea followed apattern in which peak canthaxanthin production was inversely cor-related with peak β-carotene production. Thus, as observed in otherorganisms [5], the keto-carotene canthaxanthin is the end-productof the carotenoid pathway, and β-carotene is an intermediate. VEIGA-CRESPO ET AL.Int. Microbiol.Table 1. Microbial sources of carotenoidsMicroorganism Main carotenoid Yield ReferenceHaematococcus pluvialis Astaxanthin 1.30 mg/l [16]Phaffia rhodozyma Astaxanthin 30 µg/g [13]Halobacterium salinarium Astaxanthin 265 µg/g [7]Dictyococcus cinnabarinus Canthaxanthin 1 mg/g [14]Brevibacterium KY-4313 Canthaxanthin 2 mg/l [14]Haloferax alexandrinus Canthaxanthin 2156.67 µg/g [2]Muriellopsis sp. Lutein 22.7 mg/g [10]Blakeslea trispora Lycopene 40 mg/l [6]Flavobacterium sp. Zeaxanthin 0.09 µg/l [1]Dunaliella salina ß-Carotene 2.12 mg/l [15]Dunaliella bardawil ß-Carotene 20.1 pg/cell [3]Fig. 1. β-Carotene (A) andcanthaxanthin (B) molecules.57INT. MICROBIOL. Vol. 8, 2005Standard calibration curves prepared with known amountsof pure β-carotene and trans-canthaxanthin allowed quantifica-tion of these pigments in the different media (Table 2). Thehighest levels of canthaxanthin were produced in soy-meal-based media, especially when supplemented with glucose ascarbon source. In a previous study [8], an effect of the carbonsource on carotenoid synthesis and accumulation was observed.Among all the carbon sources tested, glucose induced the high-est level of canthaxanthin synthesis, probably by favoring theoverproduction of mevalonic acid, which is a key metabolite inthe synthesis of polyprenoid-derived carotenoids. Accordingly,beet molasses should have been a good medium for canthaxan-thin production. Thus, the inability of G. jacobaea to grow onmolasses-based media was most likely due to the presence of aninhibitor in the industrial raw material. The production profile varied both in quantity and timingdepending on the medium used. Thus, in BHIB media themaximum pigment concentration (2600 µg/ml) was meas-ured on day 3 of fermentation (stationary phase), whereas inTSB medium the maximum (1500 µg/ml) was attained onday 6 (stationary phase). In the case of YPD medium, a max-imum yield was obtained on day 9 (stationary phase), but itwas always lower than the yield from BHIB medium. Whensoy-meal-containing media were compared, 5% soy meal/1%glucose was found to elicit a peak of around 13000 µg/ml,while 2% soy meal/1.5% glucose afforded a peak of nearly3500 µg/ml. However, despite the higher level of canthaxan-thin production, the use of 5% soy meal/1% glucose mediumwas ruled out because of the difficulties involved in handlingthis mixture. Instead, it was concluded that the addition of0.5% soy meal/0% glucose could be implemented in order toincrease canthaxanthin production. The ability of soy meal toincrease pigment production was most likely due to the pres-ence of precursors of the carotenoid pathway, such as meval-onic acid or related substances, which, together with the extraamounts of glucose present in the media, could haveincreased canthaxanthin production. These putative precur-sors might also have been responsible for the increases inβ-carotene and canthaxanthin that occurred during the firstfew days of the stationary phase of growth, an effect that wasparticularly observable using BHIB medium, which is rich interpenoid precursors, this leading to the emergence of an ear-lier peak of pigment production.Varying the temperature from 30ºC to 37ºC had little effecton canthaxanthin production (data not shown). However, a larg-er range of temperatures should be tested before an effect of tem-perature on canthaxanthin synthesis can be completely ruled out.Statistical analysis, based on Student’s t-test (95% confi-dence interval), confirmed the observations on the differen-tial effects of the tested media on pigment production byG. jacobaea MV-26.CANTHAXANTHIN PRODUCTION BY G. JACOBAEAInt. Microbiol.Fig. 2. Concentrations of canthaxanthin (µg/ml) (•) and ß-carotene (µg/ml)(o) produced by Gordonia jacobaea MV-26 in different production media:(A) 0.5% soy meal/0% glucose; (B) 2% soy meal/1.5% glucose; (C) YPD;(D) TSB; (E) BHIB. (A´-E´) represent the evolution of growth ofG. jacobaea (  ) in the different media. The dashed vertical line marks thepeak of production and the bacterial growth phase. Table 2. Yields of canthaxanthin production by Gordonia jacobea MV-26 indifferent production mediaMedium Canthaxanthin (µg/ml)YPD 2140BHIB 2489TSB 176010% glucose/0% soy 18000% glucose/0.5% soy 23405% glucose/1% soy 10002% glucose/1.5% soy 26501.5% glucose/2% soy 34401% glucose/5% soy 1337358 INT. MICROBIOL. Vol. 8, 2005A comparison of the abilities of hexane and ethanol to extractcanthaxanthin showed that the extraction capacity of hexane was50% higher than that of ethanol. However, since hexane is notallowed in foods, ethanol extraction must be optimized for G..jacobaea. Nonetheless, ethanol extraction of carotenoids from thisbacterium is an advantage compared to other carotenoid-produc-ing microorganisms, such as Phaffia rhodozyma, in which thissolvent is ineffective unless the yeast has previously been disrupt-ed. Moreover, the use of ethanol in pigment extraction lowers thepossibility of toxicity and animal intoxication due to contamina-tion with residual organic solvents in downstream processes. Taking into account that higher concentrations of soymeal hampered fermentations because of its insolubility, themedium containing 0.5% of soy meal was chosen as the opti-mum to obtain the highest and the more profitable pigmentproduction. The production profiles in higher volumes weresimilar to those observed for lower volumes. In general, soymeal alone, even without glucose added, promoted pigmen-tation and the amount of carotenoids was also higher. The current widespread concern about the use of geneti-cally modified organisms (GMO) in processes related to thefood industry makes classical approaches, such as the onedescribed here, of commercial relevance as the best andsafest way for increasing the concentrations of important bio-molecules.Acknowledgements. M. Poza wishes to thank the University ofSantiago de Compostela for a post-doctoral fellowship. The authors also wish to thank the Xunta de Galicia for Grant ReferencePGIDO1BI020302PR and PETRI for Grant Reference PTR1995-0572-OP.The authors wish to thank Nick Skinner for his language revisions. References1. Alcántara S, Sánchez S (1999) Influence of carbon and nitrogen sourcesof Flavobacterium growth and zeaxanthin biosynthesis. J Ind MicrobiolBiotechnol 23:697-7002. Asker D, Ohta Y (2002) Production of canthaxanthin by Haloferaxalexandrinus under non-aseptic conditions and a simple, rapid methodfor its extraction. Appl Microbiol Biotechnol 58:743-7503. Ben-Amotz A (1996) Effect of low temperature on the steroisomer com-position of β-carotene in the halotolerant alga Dunaliella bardawil(Chloropyta). J Phycol 32:272-2754. Bhosale P (2004) Environmental and cultural stimulants in the production ofcarotenoids from microorganisms. Appl Microbiol Biotechnol 63:351-3615. Calo P, Velázquez JB, Sieiro C, Blanco P, Longo E, Villa TG (1995)Analysis of astaxanthin and other carotenoids from several Phaffiarhodozyma mutants. J Agric Food Chem 43:1396-13996. Calo P, Villa TG (1995). The yeast Phaffia rhodozyma as an industrialsource of astaxanthin. Microbiología SEM 11:386-3887. Calo P, de Miguel T, Sieiro C, Velázquez JB, Villa TG (1995)Ketocarotenoids in halobacteria: 3´-hydroxy-echinenone and astaxan-thin. J Appl Bacteriol 79:282-2858. de Miguel T, Sieiro C, Poza M, Villa TG (2000). Isolation and taxonom-ic study of a new canthaxanthin-containing bacterium, Gordoniajacobaea MV-1 sp. nov. Int Microbiol 3:107-1119. de Miguel T, Sieiro C, Poza M, Villa TG (2001) Analysis of canthaxan-thin and related pigments from Gordonia jacobaea mutants. J AgricFood Chem 48:1200-120210. Del Campo JA, Moreno J, Rodríguez H, Angeles Vargas M, Rivas J,Guerrero MG (1999) Carotenoid content of chlorophycean microalgae:factors determining lutein accumulation in Muriellopsis sp. (Chloro-phyta). J Biotechnol 76:51-6911. Disch A, Schwender J, Müller C, Lichtenthaler HK, Rohmer M (1998)Distribution of the mevalonate and glyceraldehyde phosphate/pyruvatepathways for isoprenoid biosynthesis in unicellular algae and thecyanobacterium Synechosystis PCC 6714. Biochem J 333:381-38812. Hannibal L, Lorquin J, D´Ortoli NA, García N, Chaintreuil C, Masson-Boivin C, Dreyfus B, Giraud E (2000) Isolation and characterization ofcanthaxanthin biosynthesis genes from the photosynthetic bacteriumBradyrhizobium sp. Strain ORS278. J Bacteriol 182:3850-385313. Johnson EA, Villa TG, Lewis MJ (1980) Phaffia rhodozyma as an astax-anthin source in salmonid diets. Aquaculture 20:123-13414. Nelis HT, de Leenher AP (1991) Microbial sources of carotenoid pig-ments used in food and feed. J Appl Bacteriol 70:181-19115. Orset SC, Young AJ, (2000) Exposure to low irradiances favors the syn-thesis of 9-cis β, β-carotene in Dunaliella salina (Teod.). Plant Physiol.122:609-61816. Park EK, Seo MW, Lee CG (2001) Effects of medium compositions for thegrowth and the astaxanthin production of Haematococcus pluvialis. SanopMisaengmul Hakhoechi 29:227-233 (In Korean with English abstract)VEIGA-CRESPO ET AL.Influencia de las condiciones de cultivo deGordonia jacobea MV-26 en la producción decantaxantinaResumen. El interés comercial del uso de pigmentos naturales aislados apartir de microorganismos se ha incrementado en los últimos años y lasmoléculas pertenecientes al grupo de los poliisoprenoides (p.e. β-caroteno,astaxantina y cantaxantina) se han convertido en un foco de atención. Labacteria Gordonia jacobaea es capaz de sintetizar y acumular grandes can-tidades de cantaxantina (β-β´-caroteno-4,4´-diona), muy usada en la indus-tria alimentaria y de cosméticos. En este trabajo estudiamos la influencia dediferentes materias primas de bajo coste en la fermentación y la acumulaciónde cantaxantina por una cepa mutante hiperpigmentada de G. jacobaea. Seha observado que la producción de cantaxantina y el momento en el que sealcanza la máxima producción varía según los diferentes medios empleados.[Int Microbiol 2005; 8(1):55-58]Palabras clave: Gordonia jacobaea · cantaxantina · carotenoides · mediode soja · fermentaciónInfluencia das condições de cultivo deGordonia jacobaea MV-26 na produção decantaxantinaResumo. O interesse comercial do uso pela indústria alimentar de pigmen-tos de origem natural isolados a partir de microorganismos experimentou umaumento consideravel nos últimos anos, em especial aqueles com umaestructura carotenóide (β-caroteno, astaxantina e cantaxantina) A bacteriaGordonia jacobaea presenta uma grande capacidade para produzir e acumu-lar grandes quantidades de cantaxantina (β-β´-carotene-4,4´-dione), muitousada pela indústria alimentar e cosmética. O presente trabalho utiliza cepashiperpigmentadas de Gordonia jacobaea modificadas geneticamente paraanalizar a influencia de diferentes matérias primas de baixo custo, na fer-mentação e acumulação de cantaxantina. A produção de cantaxantina e omomento em que esta é máxima variou consoante o meio de cultivo utilizado.[Int Microbiol 2005; 8(1):55-58]Palavras chave: Gordonia jacobaea · cantaxantina · carotenóides · meiode cultivo de soja · fermentação

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production

Influence of culture conditions of Gordonia jacobaea MV-26 on canthaxanthin production

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