Zygosaccharomyces Kombuchaensis, a New Ascosporogenous Yeast from 'Kombucha Tea'

8542

FEMS Yeast Research

ELSEVIER FEMS Yeast Research I (2001) 133-138 www.fems-microbiology.org

Zygosaccharomyces kombuchaensis, a new ascosporogenous yeast from 'Kombucha tea'

Cletus P. Kurtzman *, Christie J. Robnett, Eleanor Basehoar-Powers

Microbial Properties Research Unit, National Center for Agricultural Utili::ation Research, Agricultural Research Service, U. S. Department of Agriculture, 1815 N. University Street, Peoria, lL 61604, USA

Received I February 2001; received in revised form 4 April 2001; accepted 10 April 2001

First published online 27 April 2001

Abstract

A new ascosporogenous yeast, Zygosaccharomyces kombuchaensis sp. n. (type strain RRL YB-4811, CBS 8849), is described; it was isolated from Kombucha tea, a popular fermented tea-based beverage. The four known strains of the new species have identical nucleotide sequences in domain 01/D2 of 265 rO A. Phylogenetic analysis of D 1/02 and 185 rO A sequences places Z. kombuchaensis near Zygosaccharomyces lenlus. The two species are indistinguishable on standard physiological tests used for yeast identification, but can be recognized from differences in restriction fragment length polymorphism patterns obtained by digestion of 18S-ITS I amplicons with the restriction enzymes DdeI and MboI. © 200 I Federation of European Microbiological Societies. Published by Elsevier Science B. V. All rights reserved.

Keywords: ew yeast; Molecular systematics; Ribosomal D A; Kombucha tea; Food microbiology; Zygosaccharomyces kombuchaensis

1. Introduction and two yeasts RRL YBA81 0, RRL YB-4882) from a 'tea fungus' sample received from Switzerland. Hesseltine Kombucha tea, also known as Manchurian tea, Russian [2] used these microorganisms to produce Kombucha tea tea and Kargasok tea, is a fermented tea-based beverage in the following manner. 34 g of dry tea leaves were that, with regular consumption, reportedly leads to long steeped in II of water, the leaves were removed by filtra life because of the reversal of aging processes. According tion, and 100 g of sucrose was added to the steep water. to some reports ([1,2], website http://kel.otago.ac.nz/maa Following autoclaving, the sugared tea was inoculated ka/ktea.html), this now popular home-fermented beverage with pure cultures of the preceding three strains, either has been consumed in Asia for over two millennia. The singly or in combination, and incubated at room temper U.S. Food and Drug Administration has evaluated the ature for several days. As Hesseltine [2] related, persons practices of several commercial producers of the starter, familiar with Kombucha tea reported a typical product if which is often termed 'Kombucha mushroom' or 'tea fun all three microorganisms were used in the fermentation. gus', and found no pathogenic organisms or other hygiene Kurtzman and Robnett ([6], unpublished data) showed violations [3]. However, there are reports of various phys from analyses of large subunit (26S) rD A domain Dl/ ical ailments, as well as death, attributed to consumption D2 sequences that RRL YB-4810 represented a new of Kombucha tea [4,5]. These adverse effects have been species of Zygosaccharomyces and that RRL YBA882 tentatively attributed to toxic metabolites from contami is a strain of Pichia fluxuum (anamorph Candida vini). nating molds [3]. RRL YB-4811, isolated from the same starter sample One of the clearer accounts of the microorganisms as RRL YBA810, proved to be a second strain of the found in Kombucha tea starter is from Hesseltine [2], new species, and more recently, two additional strains of who reported isolating an Acetobacter sp. ( RRL B-2357) this taxon were received that had been isolated in the USA from Kombucha tea. Consequently, a new genetically dis- . tinct species of Zygosaccharomyces is described here that is * Corresponding author. Tel.: +1 (309) 681-6561; 4:. ~,::.})~,;Jo. ;:; r~c:.o.~~iz~?Jr9II,1 !he extent of nucleotide divergence in Fax: + I (309) 681-6672; E-mail: [email protected] 4. - ..' ft ...... Jw~'t26S domain D1/D2 and 18S rD A sequences. Stan- ~.. ',' "'''fW~- (.if 11'::.'(' . 1567-1356/01/$20.00 © 2001 Federation of European _~~Mies!~~1fY~~ie~, ~~iep.ce B.Y. All rights reserved. PII: SI567-I356(0I)0002I-6 "1- ,yo \> - .. 134 CP. Kurl::man el al./FEMS Yeasl Research 1 (2001) 133-138 dard fermentation and assimilation tests do not allow re Table I liable separation of the new species from the closely re Yeast species compared a b lated Zygosaccharomyces lentus [7], so a rapid diagnostic Species Strain designation . Source of strain method has been developed that allows their recognition NRRL CBS from restriction fragment analysis of a region of the Z. bailii Y-2227T 680 Unknown substrate, rD A repeat. Asia Y-2228 1097 Apple juice, The etherlands Y-7239 Salad dressing, USA 2. Materials and methods Y-7254 Salad dressing, USA Y-7255 Salad dressing, USA 2. J. Yeast strains and growth tests Y-7256 Salad dressing, USA Y-7257 Salad dressing, USA Strains of the new species studied are listed in Table 1, Y-7258 Salad dressing, 'SA and all are maintained in the Agricultural Research Serv Y-7259 Salad dressing, USA Y-7260 Salad dressing, USA ice Culture Collection ( RRL), ational Center for Agri Y-726 1 Salad dressing, USA cultural Utilization Research, Peoria, IL, USA. RRL Y-27164 Kombucha tea, USA YB-4810 and RRL YB-48ll were isolated in 1959 by Z. bisporus Y-I228 nknown substrate, C.W. Hesseltine, CAUR, from a dried 'tea fungus' start USA er culture of Russian origin that was received from Y-7253 Salad dressing, USA Y-7684 1083 Kombucha tea, Java Eduard Stadelmann of Botanisches Institut, Universitat Y-12626T 702 Unknown substrate, Freiburg, Switzerland. RRL Y-27l62 and RRL Asia Y-27l63 were received in 1997 from Joan Bennett, Tulane Y-12627 1082 Kombucha tea, Java University, ew Orleans, LA, USA, and had been isolated Z. kombuchaensis YB-4810 Kombucha tea, from locally produced Kombucha tea. Russia YB-481 IT 8849 Kombucha tea, The composition of culture media used in this study as Russia well as procedures for fermentation, assimilation and other Y-27162 Kombucha tea, SA growth tests standard to yeast taxonomy are given by Y-27163 8850 Kombucha tea, SA Yarrow [8], with the modification that the assimilation Z. len/us Y-27275 8517 Tomato ketchup, UK T tubes were incubated on a reciprocal shaker for 4 weeks, Y-27276 8574 Orange juice, K Z. mellis Y-58 nknown as is customarily done at CAUR. Tolerance to acetic Y-12628T 736 Honey, USA acid was determined on the agar medium recommended Z. rouxii Y-229~T 732 Grape must, Italy by Yarrow [8], in which 1% acetic acid refers to 1 ml of Y-998 742 Honey, Canada glacial acetic acid (l00 mlr 1 of the agar medium. Petri P. jluxuum YB-4882 8851 Kombucha tea, plates containing ca. 20 ml of the medium were inoculated Russia Saccharomyces Y-12632' T 1171 Brewery, by loop as two parallel streaks with growth from a 2-day cerevisiae The etherlands YM slant culture. The plates were sealed with a single Saccharomyces Y-12651 T 3082 Drosophila pinicola, thickness of Parafilm (American ational Can Co., Green kluyveri USA wich, CT, USA) and incubated at 25°C for up to 2 weeks. a RRL Y-27275 = CYC 2406, RRL Y-27276 = CYC 02627. bT =type strain; NT =neotype strain; RRL =Agricultural Research 2.2. rDNA sequencing and phylogenetic analysis Service Culture Collection, ational Center for Agricultural Utilization Research, Peoria, IL, USA; CBS =CentraalBureau voor Schimmelcul tures, Utrecht, The etherlands; CYC = ational Collection of Yeast Methods for nuclear DA isolation, amplification of Cultures, Torwich, K. 26S rD A domain Dl/D2 by polymerase chain reaction (PCR), and sequencing with the ABI TaqDyeDeoxy Ter minator Cycle sequencing kit/ABI Model 377 automated CGC ACG CGC GCT ACA CTG AC). Reverse sequenc DA sequencer (Applied Biosystems, Inc., Foster City, ing primers were S-2 (5'-GGC TGC TGG CAC CAG CA, USA) were previously described [6]. All sequences ACT TGC), S-4 (5'-CTT CCG TCA ATT CCT TTA reported are based on sequencing both DA strands. AG), S-6B (5'-GCA GAC AAA TCA CTC CAC CAA Methods for l8S rD A sequencing were as above using C) and S-8A. the following primers. Primers S-l (5'-GTA GTC ATA Sequence data were visually aligned with QEdit 2.15 TGC TTG TCT C) and S-8A (5'-CCT TCC GCA GGT (SemWare, Marietta, GA, USA), and phylogenetic rela TCA CCT ACG GAA ACC) were used to synthesize the tionships were calculated using the maximum parsimony nearly complete l8S amplicon. Forward sequencing prim program of PAUP* 4.063a [9] with heuristic searches em ers were S-l, S-3 (5'-GCA AGT CTG GTG CCA ploying both simple and random sequence additions. Con GCA GCC), NS-5B (5'-GGG TTC TGG GGG GAG fidence limits for phylogenetic trees were estimated from TAT GGT CGC AAG GC) and NS-7A (5'-CTG GGC bootstrap analyses (1000 replications). All nucleotide se- c.P. Kurtzman et al./FEMS Yeast Research 1 (2001) 133-138 135 quences analyzed have been deposited with GenBank 3. Results and discussion under the accession numbers given in Fig. 1. 3.1. Detection, phylogenetic placement and description of 2.3. Species separation by restriction fragment length the new species polymorphisms (RFLPs) Analysis of the variable ca. 600-nucleotide domain 01/ Amplicons were prepared from the following five re D2 of 26S rD A showed that RRL YB-481I, and con gions of the rD A repeat: 18S+ITS1 (primers S specific strains, differ from all currently recognized asco I+ITS-2 (5'-GCT GCG TTC TTC ATC GAT GC)), mycetous yeasts [6]. The closest known species is Z. lentus, 18S (primers S-I + S-8A), ITS (primers S-7A+ITS-4 from which RRL YB-48 11 differs at nine of 585 nucle (5'-TCC TCC GCT TAT TGA TAT GC)), ITS2+5'-half otide positions (1.5%). Kurtzman and Robnett [6] demon of 26S (primers ITS-3 (5'-GCA TCG ATG AAG AAC strated for ascomycetous yeasts that strains differing by GCA GC)+ L-I6IIAR (5'-CAC CTT GGA GAC CTG more than 1% substitutions in domain DI/D2 represent CTG CGG)) and the 3'-half of 26S (primers L-I6IIAF separate species. The genus Zygosaccharomyces has been (5'-CCG CAG CAG GTC TCC AAG GTG)+ L-ETS2 found to be polyphyletic from phylogenetic analysis of IAR (5'-GGC TTA ATC TCA GCA GAT CG)). sequences from domain D I/D2 [6], as well as from 18S The amplicons were purified with Geneclean II (Bio 101, rO A [10,11]. Species that form a clade with Z. rouxii, La Jolla, CA, USA) and digested for 24 h at 37°C with the the type species, are Z. bailii, Z. bisporus, Z. lentus, Zy restriction enzymes DdeI and MboI, either singly or in gosaccharomyces mellis, and the new species detected in combination, using the manufacturer's (Stratagene, La the present study. Species currently assigned to Zygosac Jolla, CA, USA) protocol, except that the enzyme concen charomyces that are not part of the Z. rouxii clade on the trations were doubled. Aliquots of the enzyme digests were basis of rD A analysis are Z. cidri, Z. fermentati, z. electrophoresed on 4% agarose gels (3: 1 uSieve-SeaKem, florentinus, z. microellipsoides and Z. mrakii. Conse- FMC Corp., Rockland, ME, USA) in 1 X TAE buffer (40 quently, in view of the genetic isolation predicted for mM Trizma base, 20 mM sodium acetate, 1 mM disodium RRL YB-4811 from rD A analysis and its phylogenetic EOTA, pH 8.0). The molecular mass marker was ~XI74/ placement in the Z. rouxii clade (Fig. 1), the following new HaeIII (Stratagene) with fragments ranging from 72 to species is proposed. 1353 bp. Gels were stained with ethidium bromide and photographed under UV illumination.

265 01/02 185 S. cerevisiae U44806 .------S. cerevisiae Z75578

Z. rouxii U72163 Z. rouxii X90758 100

Z. l11ellis U72164 Z. l11ellis AF339891

98 100 Z. bailii U72161 Z. bailii X91083 92 65

Z. bisporus U72162 Z. bisporus X91084 89 89

Z. kOl11buchaensis AF339904 Z. kombuchaensis AF339890 99 86 5 10 Z. len/us AF339888 Z. lentus AF339889

'--- S. kluyveri U68552 S. kluyveri Z75580 Fig. I. Phylogenetic trees calculated from maximum parsimony analysis depicting the relationship of Z. kombuchaensis among species of Zygosaccharo myces and reference species S. cerevisiae and S. kluyveri (the outgroup species in the analyses) as analyzed from LSU 26S domain 01/02 rO A and from 18S rONA. Branch lengths are proportional to nucleotide differences, as indicated by the bars, and the numbers given at nodes are the percentage of frequencies with which a given branch appeared in 1000 bootstrap replications. 26S 01/02: single most parsimonious tree, tree length =136, consis tency index (CI) =0.868, retention index (RI) =0.723, rescaled consistency index (RC) =0.627, homoplasy index (HI) =0.132, number of parsimony-infor mative characters =50. 18S: single most parsimonious tree, tree length =58, CI =0.931, RI =0.892, RC =0.830, HI =0.069, number of parsimony-infor mative characters =26. All sequences are from the type strains of the species shown. Sequences prefixed by the letter U are from [6], sequences with X and Z prefixes are from [11], and those prefixed by AF are from the present study. 136 c. P. Kurtzman et al. / FEMS Yeast Research 1 (2001) 133 138

3.1.1. Latin diagnosis of Zygosaccharomyces kombuchaensis Kurtzman, Robnett & Basehoar A c Powers, sp. n. Asci liberi, conjugati, 2-4 ascosporae globosae, non lib eri. Species homothallica. In agaro malti post dies 3 ad 25°C, cellulae vegetativae globosae (3.2-7.5 )J.m) aut ellip soideae (3.0-6.7 X 3.8-8.7 )J.m), singulae, binae et fascicu latae. In agaro morphologico post dies 7 ad 25°C, incre mentum fuscum pallidum, nitens, butyrosum; centrum colonia sublatum; margo glabro vel undulato. Pseudohy phae et hyphae verae non fiunt. Glucosum et sucrosum fermentantur. Galactosum, maltosum, lactosum, raffino sum et trehalosum non fermentantur. Assimilantur gluco sum, galactosum, L-sorbosum (variabile), sucrosum (vari abile), maltosum (variabile), ethanolum, glycerolum, o-mannitolum (variabile), o-glucitolum (variabile) et ca daverinum. on assimilantur cellobiosum, trehalosum, lactosum, melibiosum, raffinosum, melezitosum, inulinum, amylum solubile, o-xylosum, L-arabinosum, o-arabino Fig. 2. Z. kombuchaensis RRL YB-4811. A: Budding cells, 3 days, 5% sum, o-ribosum, L-rhamnosum, o-glucosaminum, N-ace malt extract agar, 25°C. B: Ascosporulation following isogamous conju tyl-o-glucosaminum, methanolum, erythritolum, ribito gation, 7 days, YM agar, 25°C. C: Ascosporulation following heteroga mous conjugation, 7 days, YM agar, 25°C. Bar =5 !lm for A-C. lum, galactitolum, a-methyl-o-glucosidum, salicinum, o-gluconas, 2-keto-o-gluconas, 5-keto-o-gluconas, saccha ratas, oL-acidum lacticum, acidum succinicum, acidum cit 'Kombucha tea' ex Rossia, depositata in Collectione Cul ricum, inositolum, hexadecanum et potasii nitras. Amylum turarum ARS ( RRL), Peoria, IL, USA. non formatur. Vitaminae externae ad crescentiam necessa ria sunt. Non crescit in medio 0.01 % cycloheximido addi 3.1.2. Description of z. kombuchaensis Kurtzman, Robnett to; crescit in medio 1% acido acetico addito. Gelatinum & Basehoar-Powers, sp. n. liquescit (infirme, variabile); pellicula non fiunt. Augmen tum non fiunt in temperatura 37°C. Typus: RRL YB 3.1.2.1. Growth on 5% malt extract agar. After 3 days 4811 (= CBS 8849), designat stirpem typicum. Isolata at 25°C, the cells are spherical (3.2-7.5 )J.m diameter) to

Table 2 Responses of Z. kombuchaensis on fermentation, assimilation and other growth test a.b

Fermentation: Glucose + Maltose Trehalose Galactose Lactose Sucrose + Raffinose Assimilation: Glucose + L-Ara binose a-Methyl-o-glucoside Galactose + o-Arabinose Salicin L-Sorbose v o-Ribose o-Gluconate Sucrose v L-Rhamnose 2-Keto-o-gluconate Maltose v o-Glucosamine 5-Keto-o-gluconate Cellobiose N-Acetyl-o-glucosamine Saccharate Trehalose Methanol oL-Lactate Lactose Ethanol + Succinate Melibiose Glycerol Citrate Raffinose Erythritol Inositol elezitose Ribitol Hexadecane Inulin Galactitol . itrate Soluble starch o-Mannitol v Vitamin-free o-Xylose o-Glucitol v Additional growth tests: 10% NaCl/5% glucose v Cycloheximide, 100 !lg ml-\ Gelatin liquefaction w/- Starch formation Cadaverine + Growth at 37°C aCombined test results from RRL YB-4810, YB-4811, Y-27162, and Y-27163. b_ negative, + positive, w weak, v variable, i.e. + or -. c.P. Kurtzman et al./FEMS Yeast Research 1 (2001) 133 138 137 ellipsoidal (3.0-6.7 X 3.8-8.7 Ilm), and occur singly, in galactose and assimilate raffinose, and can be separated pairs, and in small clusters (Fig. 2A). Growth is white to from the sensu stricto group by these reactions. Earlier tannish-white, dull to semi-glistening and butyrous. work [12,13] showed that Z. rouxii and Z. mellis did not grow on media containing 1% acetic acid and could be 3.1.2.2. Growth on the surface of assimilation media. separated from Z. bailii and Z. bisporus by this test. Pellicles are not formed. Z. kombuchaensis grows in the presence of 1% acetic acid, but some strains of Z. lentus do not ([7], this study). 3.1.2.3. Dalmau plate culture on yeast morphology However, Z. lentus does grow in the presence of 0.9% agar. After 7 days at 25°C, neither pseudohyphae nor acetic acid, and this allows separation of Z. bailii, Z. bis true hyphae are formed under the coverglass. Aerobic porus, Z. kombuchaensis and Z. lentus from Z. mellis and growth is semi-glistening, white to tannish-white, butyr Z. rouxii. The latter two species can be separated from ous, and raised, but with a central depression. Colony each other on sodium chloride media [12]. Of the four margins may be entire or with small, irregular lobes. acetic acid tolerant species, Z. bailii assimilates trehalose, whereas the other three taxa do not. Z. bisporus does not 3.1.2.4. Ascosporulation. Ascospores were produced ferment sucrose and thereby can be separated from on YM and 5% ME agar media after 3-7 days at 25°C. Z. kombuchaensis and Z. lentus. However, there appear Asci are conjugated and many show the 'dumbbell' con to be no fermentation or assimilation reactions on stan figuration typical of Zygosaccharomyces species in which dard tests that will separate Z. kombuchaensis from the equally sized rounded conjugants are joined by a con Z. lentus. Steels et al. [7] reported that Z. lentus did not jugation tube. In this configuration, each conjugant forms assimilate galactose, L-sorbose and sucrose, but these three one to two spherical ascospores (Fig. 2B). Other asci lack sugars permitted growth of Z. lentus in our tests, and this configuration and form two to four ascospores in just strains of Z. kombuchaensis gave variable growth reactions one of the conjugating cells, which is larger than the other on the sugars. conjugant (Fig. 2C). Species showing the type of conjuga To provide a rapid diagnostic method for separation of tion described are ordinarily homothallic. Ascospores were Z. kombuchaensis and Z. lentus, we examined RFLPs for observed in all four known strains of Z. kombuchaensis, five regions of the rD A repeat using the enzymes DdeI but RRL YB-4810 failed to form ascospores in the and MboI, either singly or in combination. RFLP patterns present study, although ascosporulation was observed on from ITS, I8S, ITS2+5'-half of 26S and 3'-half of 26S two earlier occasions.

3.1.2.5. Physiological tests. Reactions on the fermenta Z. kombucha- Z.lentus tion, assimilation and other growth tests commonly used ensis in yeast taxonomy are given in Table 2. Comparative 0 ~ C\J C') L{) CD physiological tests included all strains listed in Table 2, ~ ~ CO CO CD CD as well as those reported earlier [12]. -.;:;t -.;:;t ~ ~ C\J "C\J I " I en en "C\J "C\J "C\J "C\J 3.1.2.6. Source of cultures. Origins of the four strains >- >- >- >- >- >- of Z. kombuchaensis studied are given in Table 1. All four strains had identical nucleotide sequences in 26S domain 1353 01/02 rO A. 1078 872 - 3.1.2.7. Type. RRL YB-4811 (=CBS 8849) is pre- served as a lyophilized preparation in the Agricultural Research Service Culture Collection ( RRL), Peoria, IL, 603 - USA, and was isolated from a 'tea fungus' starter used to make Kombucha tea. The starter was reportedly from Russia. 310,- 281> 3.1.2.8. Etymology. The species name kombuchaensis 271 refers to isolation from Kombucha tea. 234- 194 - 3.2. Diagnostic separation of Zygosaccharomyces species

Fig. 3. Diagnostic separation of Z. kombuchaensis and Z. lentus from The five species phylogenetically unrelated to Zygosac RFLPs generated by digestion of 18S-ITSI amplicons using a combina charomyces sensu stricto, i.e. Z. cidri, Z. fermentati, tion of the enzymes DdeI and Mba!. Five smaller bands « 100 bp) ho z. jlorentinus, Z. microellipsoides and Z. mrakii, ferment mologous in size for all strains are not shown. 138 c.P. Kurt::man et al. / FEMS Yeas I Research 1 (2001) 133-138

failed to resolve the two species either due to failure to cut quently, it appears that all of these species share an ability or to a lack of uniformity of patterns among strains of a to grow in high sugar media at low pH. In view of this, species. The amplicon comprised of I8S and ITS1 pro Z. kombuchaensis could potentially occur as a spoilage vided resolution of Z. kombuchaensis and Z. lentus, with organism in acidic high sugar foods and beverages. all strains giving species-specific patterns. Either enzyme alone was adequate for separation of the two species, but when DdeI and MboI were used in combination, mi Acknowledgements nor bands present in single enzyme digests disappeared leaving a uniform pattern of major bands (Fig. 3). A dou Larry W. Tjarks is gratefully acknowledged for skillful ble enzyme digest of the I8S-ITS1 amplicon also resolved operation of the nucleic acid sequencer. The mention of the type strains of Z. bailii and Z. bisporus from each firm names or trade products does not imply that they are other and from Z. kombuchaensis and Z. lentus, but addi endorsed or recommended by the U.S. Department of tional strains of Z. bailii and Z. bisporus were not tested Agriculture over other firms or similar products not men because these two species can be separated using growth tioned. tests. Amplicons that were treated with the Geneclean procedure gave much clearer RFLP patterns, but species could be recognized from digests made using the unpuri fied PCR reaction mixtures. References

3.2.1. Diagnostic key to species of Zygosaccharomyces [I] Alexopoulos, C.l., Mims, CW. and Blackwell, M. (1996) Introduc tory Mycology, 4th edn. John Wiley and Sons, lew York. [2] Hesseltine, CW. (1965) A millennium of fungi, food and fermenta- a Growth on culture medium containing 2 tion. ycologia 57, 149-197. 0.9% acetic acid; maltose is not [3] CDC (1996) CDC editorial note. J. Am. ed. Assoc. 275, 97-98. fermented [4] Perron, A.D., Patterson, J.A. and Yanofsky, .. (1995) Kombucha b Absence of growth on culture medium 4 'mushroom' hepatotoxicity. Ann. Emerg. Med. 26, 660-661. containing 0.9% acetic acid; maltose is [5] Currier, R.W., Goddard, J., Buechler, K., Quinlisk, M.P., Wolfe, fermented S.L., Spencer, M.D., Carroll, T.l., Bennett, T. and Stokes, J. 2 a Trehalose is assimilated (strong or weak Z. bailii (1996) Unexplained severe illness possibly associated with consump growth) tion of Kombucha tea - Iowa 1995. J. Am. Med. Assoc. 275, 96-97. b Trehalose is not assimilated [6] Kurtzman, CP. and Robnett, C.l. (1998) Iden~ification and phylog a a Sucrose is fermented Z. kombuchaensis eny of ascomycetous yeasts from analysis of nuclear large subunit Z. lenlUs" (26S) ribosomal DA partial sequences. Antonie van Leeuwenhoek b Sucrose is not fermented Z. bisporus 73, 331-371. 4 a Growth in yeast nitrogen base liquid Z. rouxii [7] Steels, H., Bond, CJ., Collins, M.D., Roberts, I. ., Stratford, M. medium with 16% NaCI+5'Yo glucose and James, S.A. (1999) Zygosaccharomyces lenlus sp. nov., a new b Absence of growth in yeast nitrogen Z. mellis member of the yeast genus Zygosaccharomyces Barker. Int. J. Syst. base liquid medium with 16% NaCI+5% Bacteriol. 49, 319-327. glucose [8] Yarrow, D. (1998) Methods for the isolation, maintenance and iden "The two species can be separated from one another by RFLP analysis, tification of yeasts. In: The Yeasts, A Taxonomic Study, 4th edn. as illustrated in Fig. 3. Under the conditions described, digestion of the (Kurtzman, CP. and Fell, J.W., Eds.), pp. 77-100. Elsevier Science 18S-lTS I amplicon for Z. kombuchaensis produced five major bands, B.Y., Amsterdam. whereas six are produced for Z. lenlus. [9] Swofford, D.L. (1998) PAUP* 4.0: Phylogenetic Analysis using Par simony. Sinauer Associates, Sunderland, MA. [10] James, S.A., Collins, M.D. and Roberts, I. . (1994) Genetic interre lationship among species of the genus Zygosaccharomyces as revealed 3.3. Other yeasts associated with Kombucha tea by small-subunit rR A gene sequences. Yeast 10,871-881. [II] James, S.A. Cai, J., Roberts, I. . and Collins, M.D. (1997) A phy The yeast flora of Kombucha tea is not restricted to Z. logenetic analysis of the genus Saccharomyces based on 18S rR A kombuchaensis. RRL YB-4882, which was isolated from gene sequences: description of Saccharomyces kunashirensis sp. nov. and Saccharomyces martiniae sp. nov.. Int. J. Syst. Bacteriol. 47, 453 the same starter as RRL YB-48II, was identified as P. 460. fluxuum (zero DI/D2 domain nucleotide differences with [12] Kurtzman, CP. (1990) DNA relatedness among species of the genus the type strain). RRL Y-27164 was isolated from the Zygosaccharomyces. Yeast 6, 213-219. same Kombucha tea sample as RRL Y-27162 and [13] Yarrow, D. (1984) Zygosaccharomyces Barker. In: The Yeasts, A RRL Y-27163 (z. kombuchaensis), but proved to be Z. Taxonomic Study, 3rd edn. (Kreger-van Rij, .l.W., Ed.), pp. 449 465. Elsevier Science B.Y., Amsterdam. bailii from Dl/D2 sequence analysis. In addition, two [14] Kurtzman, CP. (1998) Zygosaccharomyces Barker. In: The Yeasts, A strains of Z. bisporus (Table 1) are listed from Kombucha Taxonomic Study, 4th edn. (Kurtzman, CP. and Fell, J.W., Eds.), tea, as are two strains of Z. microellipsoides [14]. Conse- pp. 424-432. Elsevier Science B.Y., Amsterdam.

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