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A microbial consortium involving the astaxanthin producer Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany
mycologist 20 (2006) 57–61
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycol
A microbial consortium involving the astaxanthin producer Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany Roland W. S. WEBERa,*, Paolo DAVOLIb, Heidrun ANKEc a
Department of Biotechnology, University of Kaiserslautern, Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy c Institute of Biotechnology and Drug Research (IBWF) e.V., Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany b
abstract Keywords:
The astaxanthin-producing Xanthophyllomyces dendrorhous was the dominant pigmented
Astaxanthin
yeast in yellow- and orange-coloured microbial mats formed on exudates from freshly
Betula
cut birch stumps in Kaiserslautern (Germany) in April 2005. Other ubiquitous members
Consortium
of the consortium included Mucor hiemalis, two Fusarium spp., the yeast Hanseniaspora
Phaffia rhodozyma
uvarum and two rod-shaped bacteria.
Slime-flux
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Xanthophyllomyces dendrorhous
1.
Introduction
The logging of trees in residential areas is not usually accepted with enthusiasm but can have its compensations if it is carried out in early spring and the resulting slime-fluxes on the cut stumps become colonized by interesting microbial communities. Such was the case in Kaiserslautern (Germany) in late March 2005 when a plot of mixed forest (Pinus sylvestris and Fagus sylvatica) was cleared for development. The forest, about 100 200 m in size, was surrounded by roads and pavements and located at an altitude of 230 m above sea-level. Several birch trees (Betula pendula), 15-25 y old, were also cut at the boundaries of the plot. Birch stumps are prolific producers of slime-flux, and several of them were observed on 3 April to be covered with a thick (2-10 mm) slimy or rubbery mat of striking orange and yellow colours (Figs 1 and 2). Liquid continued to be produced throughout the month of April and was so plentiful that it ran down from the cut stump surface along the bark to the ground; spectacular stalactite-like outgrowths were formed on occasions (Fig. 3). The observation of a lawn of zygomycete sporangia growing on the microbial
mat, apparently without damaging its pigmentation (Fig. 4), prompted us to analyse the overall composition of the consortium by isolating its members. With the onset of warm dry weather in early May, exudate production declined and the mats dried up, with no obvious trace left by mid-May.
2.
Material and methods
Samples were collected from four birch stumps. Stumps 1 and 2 were about 5 m apart from each other, and 3 and 4 10 m; the distance between these two pairs was about 100 m. Fungi and bacteria were isolated by using selective agar media, viz. YMG agar (4 g yeast extract, 10 g malt extract, 4 g glucose l1) augmented with 200 mg l1 each of penicillin G and streptomycin sulphate, and LB agar (10 g tryptone, 5 g NaCl, 5 g yeast extract l1), respectively. Isolations were made both from material streaked out directly onto the agar plates, and by plating out 0.1 ml aliquots of a dilution series obtained by shaking 1 g (fresh weight) of microbial mat in 10 ml 0.9% sterile aqueous NaCl, and stepwise dilution of the resulting suspension in
* Corresponding author. E-mail addresses: [email protected] (R.W.S. Weber), [email protected] (P. Davoli), [email protected] (H. Anke). 0269-915X/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycol.2006.03.010
58
R. W. S. Weber et al.
Figs 1–4 – The microbial mat on freshly cut birch stumps in early April 2005. Fig. 1 Large stump (about 30 cm diam.) producing prolific exudates which have run down and seeped into the soil, supporting a thick microbial mat of yellow, orange and white colours. Fig. 2 Removal of the microbial mat from soil near the base of the same stump. The mat is about 3 mm thick at this point. Fig. 3 Stalactite-like outgrowths of a microbial mat on a younger stump. Drops of birch juice can be seen at the stalactite tips. Fig. 4 Sporangial lawn of Mucor hiemalis colonizing the surface of a microbial mat.
0.9% NaCl. An additional isolation method for yeasts consisted of enrichment by incubating small samples of consortium in shaken flasks (100 ml liquid YMG medium with antibiotics) on an orbital shaker (120 rpm) at 24 C for 48 h, followed by filtration through sterile glass wool, establishment of a dilution series in sterile YMG medium, and plating-out on YMG agar as above. Fungi were identified by microscopic examination and comparison of their ITS ribosomal DNA sequences with those available in GenBank (see Schwarz et al. 2004). Carotenoid pigments were extracted according to Weber and Davoli (2003), and identified by HPLC and HPLC-MS analysis, using methods and equipment described by Davoli et al. (2004).
3.
Members of the microbial consortium
Nine fungi and two bacteria were isolated from consortia on the four birch stumps examined. These have been incorporated into the Culture Collection, Dept. of Biotechnology, University of Kaiserslautern as strains Car223-233. Details of their identification and occurrence in microbial mats on the four birch stumps examined are given in Table 1. Dominant consortium members isolated from all four stumps were the sporangium-forming zygomycete identified as Mucor hiemalis, the yeasts Xanthophyllomyces dendrorhous and Hanseniaspora uvarum, Fusarium merismoides, F. sporotrichioides and two rod-shaped
Microbial consortium involving Xanthophyllomyces dendrorhous on birch stumps
59
Table 1 – Identity and frequency of consortium members isolated from pigmented microbial mats on freshly cut birch stumps Strain
Identity
Car223 Car224
Mucor hiemalis Xanthophyllomyces dendrorhous
Car225
Hanseniaspora uvarum
Car226 Car227
Galactomyces geotrichum Pichia sp.
Car228
Aureobasidium pullulans
Car229 Car230
Fusarium merismoides Cystofilobasidium infirmo-miniatum
Car231 Car232 Car233
Fusarium sporotrichioides Rod-shaped bacterium Rod-shaped bacterium
Details of identification1
microscopy (Zycha et al. 1969; Domsch et al. 1980) ITS seq. (identical with AF139628 and AF139632; Fell & Blatt 1999) ITS seq. (identical with AY796120 and others; Cadez et al. 2003) microscopy (de Hoog et al. 1998) microscopy and ITS seq. (98.7% similarity with AF218969) ITS seq. (identical with AF455533 and others; Yurlova et al. 1999) microscopy (Booth 1971) ITS seq. (identical with AF444400 and AY264716; Scorzetti et al. 2002) microscopy (Booth 1971)
Sampled tree2 1
2
3
4
D D
D D
D D
D D
D
D
D
D
D -
-
D
D -
-
D
-
-
D -
D D
tr D
tr -
D D D
D D D
D D D
tr D D
1 Numbers given refer to GenBank accession numbers of reference sequences. 2 An indication of abundance is given as þ (prominent), tr (trace) and (absent).
bacteria. Several of these members were recognized in handsections and squash preparations of fresh samples, including M. hiemalis, rod-shaped bacteria, F. merismoides and actively budding yeast cells similar to X. dendrorhous (Figs 5–7). When fragments of microbial mats were shaken vigorously in water,
yeast and bacterial cells were washed out, leaving behind a tight mesh of fungal hyphae. It is likely that these provide structural support to the consortium in its natural habitat. Two pigmented yeasts were found, of which only X. dendrorhous was ubiquitous. An analysis of its carotenoid content in
Figs 5–8 – Microscopic details of members of the microbial consortium making up the mats. All figures to same scale. Fig. 5 Swollen branched hyphae of Mucor hiemalis in a matrix of yeast cells. Fig. 6 Fusarium sp. Car229 associated with Xanthophyllomyces dendrorhous and bacterial cells. Fig. 7 Budding cells of X. dendrorhous from a consortium. Fig. 8 Pure culture of X. dendrorhous. Budding yeast cells and pseudohyphal growth are visible.
60
R. W. S. Weber et al.
pure cultures grown under well-aerated standard conditions (see Weber & Davoli 2003) revealed astaxanthin as the dominant carotenoid, identified by its smooth bell-shaped absorption spectrum in visible light with lmax at 480 nm in acetone, its HPLC retention time in comparison with a pure astaxanthin standard, and its mass spectra showing peaks at m/z 596 (molecular ion, M; APCI-negative mode) and 597 ([M þ 1]þ; APCI-positive mode).
4.
Xanthophyllomyces dendrorhous
The finding of an astaxanthin-producing yeast on birch slime-flux is noteworthy because only two yeasts of this kind have been found, the strictly anamorphic Phaffia rhodozyma on Fagus crenata, and a budding yeast with a basidial teleomorph, Xanthophyllomyces dendrorhous, on slime-fluxes mainly of Betula spp. (Fell & Blatt 1999; Golubev 2000). Initially thought to be conspecific, X. dendrorhous is now believed to represent a different, albeit closely related species (Fell & Blatt 1999; Kucsera et al. 2000). Basidium formation can be induced in X. dendrorhous, but not P. rhodozyma, by streaking well-grown yeast cells onto a sporulation agar containing 0.5% ribitol (Kucsera et al. 1998). Although we have observed basidium formation in one of our Xanthophyllomyces strains isolated from a different habitat (R.W.S. Weber, unpublished), basidia were not observed in strain Car224. Instead, this isolate showed pseudohyphal growth as swollen, branched segments, in addition to unipolar enteroblastic budding (Fig. 8). Nonetheless, the identity of its ITS sequence with those of several GenBank accessions confirmed it as belonging to X. dendrorhous, especially when bearing in mind the unusually high sequence variability observed within that species (Fell & Blatt 1999). The known geographic distribution of Xanthophyllomyces and Phaffia has been summarized by Golubev (2000) and Barnett et al. (2000) and is limited to Alaska, Finland, Japan and Russia, making the present report the first one from Western Europe. It also seems to be the first account of Xanthophyllomyces growing in a microbial consortium, and of its associated fungi.
5.
Astaxanthin and environmental stress
Numerous pure-culture studies have provided evidence that astaxanthin may play a role in protecting its producer (Phaffia or Xanthophyllomyces) against oxidative stress. In liquid culture, astaxanthin production is stimulated by enhanced aeration or by exposure to generators of reactive oxygen species such as singlet oxygen (1O2) or the hydroxyl (HOc) or superoxide (O2c) radicals (Johnson & Lewis 1979; Schroeder & Johnson 1993, 1995a; Johnson 2003). A particularly interesting report is that by Schroeder & Johnson (1995b) who provided evidence that an as yet unidentified substance contained in birch juice may act as a photosensitizer, i.e. it is stimulated by UV light to generate reactive oxygen species. This might explain the occurrence of Xanthophyllomyces in birch exudates in nature. It is possible that this species acts as a primary colonizer, detoxifying birch exudate as a pre-requisite for colonization by other members of the consortium observed. We are currently
examining this possibility. Of all dominant consortium members, the only fungus known to be principally associated with tree slime-fluxes is X. dendrorhous, all other fungi being generally found in soil situations (Domsch et al. 1980) or, in the case of H. uvarum, also in association with a wide range of plants (Barnett et al. 2000). There may be additional metabolic interactions between Xanthophyllomyces and other consortium members. For instance, Echavarri-Erasun and Johnson (2004) observed that astaxanthin biosynthesis was stimulated by a chance contaminant of a Phaffia culture, Epicoccum nigrum. Similarly, extracts of different fungi, including Mucor mucedo and the red yeast Rhodotorula glutinis, were found to increase total carotenoid and astaxanthin production in X. dendrorhous (Wang et al. 2006). It would be interesting to examine the effects of the other consortium members on growth and carotenoid biosynthesis in Xanthophyllomyces. Much is known about physiological aspects of astaxanthin production in Phaffia and Xanthophyllomyces in pure-culture fermentor studies, due mainly to the industrial importance of astaxanthin as a pigment for fish farming and in nutraceuticals (Johnson & An 1991). However, it is apparent that we are only beginning to scratch at the surface of its ecophysiological functions in nature.
references
Barnett JA, Payne RW, Yarrow D, 2000. Yeasts. Characteristics and Identification, third edn. Cambridge University Press., Cambridge. Booth C, 1971. The Genus Fusarium. Commonwealth Agricultural Bureaux, Kew. Cadez N, Poot GA, Raspor P, Smith MT, 2003. Hanseniaspora meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate yeast species. International Journal of Systematic and Evolutionary Microbiology 53: 1671–1680. Davoli P, Mierau V, Weber RWS, 2004. Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis. Applied Biochemistry and Microbiology 40: 392–397. de Hoog GS, Smith MT, Gue´ho E, 1998. Galactomyces Redhead & Malloch. In: Kurtzman CP, Fell JW (eds), The Yeasts: A Taxonomic Study, fourth edn. Elsevier, Amsterdam, pp. 209–213. Domsch KH, Gams W, Anderson T-H, 1980. Compendium of Soil Fungi. Academic Press, London. Echavarri-Erasun C, Johnson EA, 2004. Stimulation of astaxanthin formation in the yeast Xanthophyllomyces dendrorhous by the fungus Epicoccum nigrum. FEMS Yeast Research 4: 511–519. Fell JM, Blatt GM, 1999. Separation of strains of the yeasts Xanthophyllomyces dendrorhous and Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis. Journal of Industrial Microbiology and Biotechnology 23: 677–681. Golubev WI, 2000. Collection resources of Xanthophyllomyces dendrorhous (Phaffia rhodozyma, Rhodomyces dendrorhous). Yeast – A Newsletter for Persons Interested in Yeast 49: 47–48. Johnson EA, 2003. Phaffia rhodozyma: colorful odyssey. International Microbiology 6: 169–174. Johnson EA, An GH, 1991. Astaxanthin from microbial sources. Critical Reviews in Biotechnology 11: 297–326. Johnson EA, Lewis MJ, 1979. Astaxanthin formation by the yeast Phaffia rhodozyma. Journal of General Microbiology 115: 173–183. Kucsera J, Pfeiffer I, Ferenczy L, 1998. Homothallic life cycle in the diploid red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Antonie van Leeuwenhoek 73: 163–168.
Microbial consortium involving Xanthophyllomyces dendrorhous on birch stumps
Kucsera J, Pfeiffer I, Takeo K, 2000. Biology of the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Mycoscience 41: 195–199. Schroeder WA, Johnson EA, 1993. Antioxidant role of carotenoids in Phaffia rhodozyma. Journal of General Microbiology 139: 907–912. Schroeder WA, Johnson EA, 1995a. Singlet oxygen and peroxyl radicals regulate carotenoid biosynthesis in Phaffia rhodozyma. Journal of Biological Chemistry 270: 18374–18379. Schroeder WA, Johnson EA, 1995b. Carotenoids protect Phaffia rhodozyma against singlet oxygen damage. Journal of Industrial Microbiology 14: 502–507. Schwarz M, Ko¨pcke B, Weber RWS, Sterner O, Anke H, 2004. 3-Hydroxypropionic acid as a nematicidal principle in endophytic fungi. Phytochemistry 65: 2239–2245. Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A, 2002. Systematics of basidiomycetous yeasts: a comparison of large
61
subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Research 2: 495–517. Wang W, Yu L, Zhou P, 2006. Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. Bioresource Technology 97: 26–31. Weber RWS, Davoli P, 2003. Teaching techniques for mycology: 20. Astaxanthin, a carotenoid of biotechnological importance from yeast and salmonid fish. Mycologist 17: 30–34. Yurlova NA, de Hoog GS, Gerrits van den Ende AHG, 1999. Taxonomy of Aureobasidium and allied genera. Studies in Mycology 43: 63–69. Zycha H, Siepmann R, Linnemann G, 1969. Mucorales. Eine Beschreibung aller Gattungen und Arten dieser Pilzgruppe. J. Cramer, Lehre, Germany.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/mycol
A microbial consortium involving the astaxanthin producer Xanthophyllomyces dendrorhous on freshly cut birch stumps in Germany Roland W. S. WEBERa,*, Paolo DAVOLIb, Heidrun ANKEc a
Department of Biotechnology, University of Kaiserslautern, Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41100 Modena, Italy c Institute of Biotechnology and Drug Research (IBWF) e.V., Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany b
abstract Keywords:
The astaxanthin-producing Xanthophyllomyces dendrorhous was the dominant pigmented
Astaxanthin
yeast in yellow- and orange-coloured microbial mats formed on exudates from freshly
Betula
cut birch stumps in Kaiserslautern (Germany) in April 2005. Other ubiquitous members
Consortium
of the consortium included Mucor hiemalis, two Fusarium spp., the yeast Hanseniaspora
Phaffia rhodozyma
uvarum and two rod-shaped bacteria.
Slime-flux
ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Xanthophyllomyces dendrorhous
1.
Introduction
The logging of trees in residential areas is not usually accepted with enthusiasm but can have its compensations if it is carried out in early spring and the resulting slime-fluxes on the cut stumps become colonized by interesting microbial communities. Such was the case in Kaiserslautern (Germany) in late March 2005 when a plot of mixed forest (Pinus sylvestris and Fagus sylvatica) was cleared for development. The forest, about 100 200 m in size, was surrounded by roads and pavements and located at an altitude of 230 m above sea-level. Several birch trees (Betula pendula), 15-25 y old, were also cut at the boundaries of the plot. Birch stumps are prolific producers of slime-flux, and several of them were observed on 3 April to be covered with a thick (2-10 mm) slimy or rubbery mat of striking orange and yellow colours (Figs 1 and 2). Liquid continued to be produced throughout the month of April and was so plentiful that it ran down from the cut stump surface along the bark to the ground; spectacular stalactite-like outgrowths were formed on occasions (Fig. 3). The observation of a lawn of zygomycete sporangia growing on the microbial
mat, apparently without damaging its pigmentation (Fig. 4), prompted us to analyse the overall composition of the consortium by isolating its members. With the onset of warm dry weather in early May, exudate production declined and the mats dried up, with no obvious trace left by mid-May.
2.
Material and methods
Samples were collected from four birch stumps. Stumps 1 and 2 were about 5 m apart from each other, and 3 and 4 10 m; the distance between these two pairs was about 100 m. Fungi and bacteria were isolated by using selective agar media, viz. YMG agar (4 g yeast extract, 10 g malt extract, 4 g glucose l1) augmented with 200 mg l1 each of penicillin G and streptomycin sulphate, and LB agar (10 g tryptone, 5 g NaCl, 5 g yeast extract l1), respectively. Isolations were made both from material streaked out directly onto the agar plates, and by plating out 0.1 ml aliquots of a dilution series obtained by shaking 1 g (fresh weight) of microbial mat in 10 ml 0.9% sterile aqueous NaCl, and stepwise dilution of the resulting suspension in
* Corresponding author. E-mail addresses: [email protected] (R.W.S. Weber), [email protected] (P. Davoli), [email protected] (H. Anke). 0269-915X/$ – see front matter ª 2006 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycol.2006.03.010
58
R. W. S. Weber et al.
Figs 1–4 – The microbial mat on freshly cut birch stumps in early April 2005. Fig. 1 Large stump (about 30 cm diam.) producing prolific exudates which have run down and seeped into the soil, supporting a thick microbial mat of yellow, orange and white colours. Fig. 2 Removal of the microbial mat from soil near the base of the same stump. The mat is about 3 mm thick at this point. Fig. 3 Stalactite-like outgrowths of a microbial mat on a younger stump. Drops of birch juice can be seen at the stalactite tips. Fig. 4 Sporangial lawn of Mucor hiemalis colonizing the surface of a microbial mat.
0.9% NaCl. An additional isolation method for yeasts consisted of enrichment by incubating small samples of consortium in shaken flasks (100 ml liquid YMG medium with antibiotics) on an orbital shaker (120 rpm) at 24 C for 48 h, followed by filtration through sterile glass wool, establishment of a dilution series in sterile YMG medium, and plating-out on YMG agar as above. Fungi were identified by microscopic examination and comparison of their ITS ribosomal DNA sequences with those available in GenBank (see Schwarz et al. 2004). Carotenoid pigments were extracted according to Weber and Davoli (2003), and identified by HPLC and HPLC-MS analysis, using methods and equipment described by Davoli et al. (2004).
3.
Members of the microbial consortium
Nine fungi and two bacteria were isolated from consortia on the four birch stumps examined. These have been incorporated into the Culture Collection, Dept. of Biotechnology, University of Kaiserslautern as strains Car223-233. Details of their identification and occurrence in microbial mats on the four birch stumps examined are given in Table 1. Dominant consortium members isolated from all four stumps were the sporangium-forming zygomycete identified as Mucor hiemalis, the yeasts Xanthophyllomyces dendrorhous and Hanseniaspora uvarum, Fusarium merismoides, F. sporotrichioides and two rod-shaped
Microbial consortium involving Xanthophyllomyces dendrorhous on birch stumps
59
Table 1 – Identity and frequency of consortium members isolated from pigmented microbial mats on freshly cut birch stumps Strain
Identity
Car223 Car224
Mucor hiemalis Xanthophyllomyces dendrorhous
Car225
Hanseniaspora uvarum
Car226 Car227
Galactomyces geotrichum Pichia sp.
Car228
Aureobasidium pullulans
Car229 Car230
Fusarium merismoides Cystofilobasidium infirmo-miniatum
Car231 Car232 Car233
Fusarium sporotrichioides Rod-shaped bacterium Rod-shaped bacterium
Details of identification1
microscopy (Zycha et al. 1969; Domsch et al. 1980) ITS seq. (identical with AF139628 and AF139632; Fell & Blatt 1999) ITS seq. (identical with AY796120 and others; Cadez et al. 2003) microscopy (de Hoog et al. 1998) microscopy and ITS seq. (98.7% similarity with AF218969) ITS seq. (identical with AF455533 and others; Yurlova et al. 1999) microscopy (Booth 1971) ITS seq. (identical with AF444400 and AY264716; Scorzetti et al. 2002) microscopy (Booth 1971)
Sampled tree2 1
2
3
4
D D
D D
D D
D D
D
D
D
D
D -
-
D
D -
-
D
-
-
D -
D D
tr D
tr -
D D D
D D D
D D D
tr D D
1 Numbers given refer to GenBank accession numbers of reference sequences. 2 An indication of abundance is given as þ (prominent), tr (trace) and (absent).
bacteria. Several of these members were recognized in handsections and squash preparations of fresh samples, including M. hiemalis, rod-shaped bacteria, F. merismoides and actively budding yeast cells similar to X. dendrorhous (Figs 5–7). When fragments of microbial mats were shaken vigorously in water,
yeast and bacterial cells were washed out, leaving behind a tight mesh of fungal hyphae. It is likely that these provide structural support to the consortium in its natural habitat. Two pigmented yeasts were found, of which only X. dendrorhous was ubiquitous. An analysis of its carotenoid content in
Figs 5–8 – Microscopic details of members of the microbial consortium making up the mats. All figures to same scale. Fig. 5 Swollen branched hyphae of Mucor hiemalis in a matrix of yeast cells. Fig. 6 Fusarium sp. Car229 associated with Xanthophyllomyces dendrorhous and bacterial cells. Fig. 7 Budding cells of X. dendrorhous from a consortium. Fig. 8 Pure culture of X. dendrorhous. Budding yeast cells and pseudohyphal growth are visible.
60
R. W. S. Weber et al.
pure cultures grown under well-aerated standard conditions (see Weber & Davoli 2003) revealed astaxanthin as the dominant carotenoid, identified by its smooth bell-shaped absorption spectrum in visible light with lmax at 480 nm in acetone, its HPLC retention time in comparison with a pure astaxanthin standard, and its mass spectra showing peaks at m/z 596 (molecular ion, M; APCI-negative mode) and 597 ([M þ 1]þ; APCI-positive mode).
4.
Xanthophyllomyces dendrorhous
The finding of an astaxanthin-producing yeast on birch slime-flux is noteworthy because only two yeasts of this kind have been found, the strictly anamorphic Phaffia rhodozyma on Fagus crenata, and a budding yeast with a basidial teleomorph, Xanthophyllomyces dendrorhous, on slime-fluxes mainly of Betula spp. (Fell & Blatt 1999; Golubev 2000). Initially thought to be conspecific, X. dendrorhous is now believed to represent a different, albeit closely related species (Fell & Blatt 1999; Kucsera et al. 2000). Basidium formation can be induced in X. dendrorhous, but not P. rhodozyma, by streaking well-grown yeast cells onto a sporulation agar containing 0.5% ribitol (Kucsera et al. 1998). Although we have observed basidium formation in one of our Xanthophyllomyces strains isolated from a different habitat (R.W.S. Weber, unpublished), basidia were not observed in strain Car224. Instead, this isolate showed pseudohyphal growth as swollen, branched segments, in addition to unipolar enteroblastic budding (Fig. 8). Nonetheless, the identity of its ITS sequence with those of several GenBank accessions confirmed it as belonging to X. dendrorhous, especially when bearing in mind the unusually high sequence variability observed within that species (Fell & Blatt 1999). The known geographic distribution of Xanthophyllomyces and Phaffia has been summarized by Golubev (2000) and Barnett et al. (2000) and is limited to Alaska, Finland, Japan and Russia, making the present report the first one from Western Europe. It also seems to be the first account of Xanthophyllomyces growing in a microbial consortium, and of its associated fungi.
5.
Astaxanthin and environmental stress
Numerous pure-culture studies have provided evidence that astaxanthin may play a role in protecting its producer (Phaffia or Xanthophyllomyces) against oxidative stress. In liquid culture, astaxanthin production is stimulated by enhanced aeration or by exposure to generators of reactive oxygen species such as singlet oxygen (1O2) or the hydroxyl (HOc) or superoxide (O2c) radicals (Johnson & Lewis 1979; Schroeder & Johnson 1993, 1995a; Johnson 2003). A particularly interesting report is that by Schroeder & Johnson (1995b) who provided evidence that an as yet unidentified substance contained in birch juice may act as a photosensitizer, i.e. it is stimulated by UV light to generate reactive oxygen species. This might explain the occurrence of Xanthophyllomyces in birch exudates in nature. It is possible that this species acts as a primary colonizer, detoxifying birch exudate as a pre-requisite for colonization by other members of the consortium observed. We are currently
examining this possibility. Of all dominant consortium members, the only fungus known to be principally associated with tree slime-fluxes is X. dendrorhous, all other fungi being generally found in soil situations (Domsch et al. 1980) or, in the case of H. uvarum, also in association with a wide range of plants (Barnett et al. 2000). There may be additional metabolic interactions between Xanthophyllomyces and other consortium members. For instance, Echavarri-Erasun and Johnson (2004) observed that astaxanthin biosynthesis was stimulated by a chance contaminant of a Phaffia culture, Epicoccum nigrum. Similarly, extracts of different fungi, including Mucor mucedo and the red yeast Rhodotorula glutinis, were found to increase total carotenoid and astaxanthin production in X. dendrorhous (Wang et al. 2006). It would be interesting to examine the effects of the other consortium members on growth and carotenoid biosynthesis in Xanthophyllomyces. Much is known about physiological aspects of astaxanthin production in Phaffia and Xanthophyllomyces in pure-culture fermentor studies, due mainly to the industrial importance of astaxanthin as a pigment for fish farming and in nutraceuticals (Johnson & An 1991). However, it is apparent that we are only beginning to scratch at the surface of its ecophysiological functions in nature.
references
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Microbial consortium involving Xanthophyllomyces dendrorhous on birch stumps
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