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 I I 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: kurtzman@mail.ncaur.usda.gov 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: S I 5 6 7 - I 3 5 6 ( 0 I ) 0 0 0 2 I - 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

Yeast species compared
liable separation of the new species from the closely re-
lated Zygosaccharomyces lentus [7], so a rapid diagnostic Species Strain designation a .b 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-2227 T 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
Y-7259 Salad dressing, USA
and all are maintained in the Agricultural Research Serv-
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
Y-7253 Salad dressing, USA
er culture of Russian origin that was received from
Y-7684 1083 Kombucha tea, Java
Eduard Stadelmann of Botanisches Institut, Universitat Y-12626 T 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,
Russia
from locally produced Kombucha tea. 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
Y-27276 T 8574 Orange juice, K
tubes were incubated on a reciprocal shaker for 4 weeks,
Z. mellis Y-58 nknown
as is customarily done at CAUR. Tolerance to acetic Y-12628 T 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,
Russia
plates containing ca. 20 ml of the medium were inoculated
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; RR L = 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 D A 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-
D A 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 D A 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
under the accession numbers given in Fig. 1.
2.3. Species separation by restriction fragment length
polymorphisms (RFLPs)
Amplicons were prepared from the following five re-
gions of the rD A repeat: 18S+ITS 1 (primers S-
I+ITS-2 (5'-GCT GCG TTC TTC ATC GAT GC)),
18S (primers S-I + S-8A), ITS (primers S-7A + ITS-4
(5'-TCC TCC GCT TAT TGA TAT GC)), ITS2+5'-half
of 26S (primers ITS-3 (5'-GCA TCG ATG AAG AAC
GCA GC)+ L-I6IIAR (5'-CAC CTT GGA GAC CTG
CTG CGG)) and the 3'-half of 26S (primers L-I6IIAF
(5' -CCG CAG CAG GTC TCC AAG GTG)+ L-ETS2-
IAR (5'-GGC TTA ATC TCA GCA GAT CG)).
The amplicons were purified with Geneclean II (Bio 101,
La Jolla, CA, USA) and digested for 24 h at 37°C with the
restriction enzymes DdeI and MboI, either singly or in
combination, using the manufacturer's (Stratagene, La
Jolla, CA, USA) protocol, except that the enzyme concen-
trations were doubled. Aliquots of the enzyme digests were
electrophoresed on 4% agarose gels (3: 1 uSieve-SeaKem,
FMC Corp., Rockland, ME, USA) in 1 X TAE buffer (40
mM Trizma base, 20 mM sodium acetate, 1 mM disodium
EOTA, pH 8.0). The molecular mass marker was ~XI74/
HaeIII (Stratagene) with fragments ranging from 72 to
1353 bp. Gels were stained with ethidium bromide and
photographed under UV illumination.
3. Results and discussion
3.1. Detection, phylogenetic placement and description of
the new species
Analysis of the variable ca. 600-nucleotide domain 01/
D2 of 26S rD A showed that RRL YB-481I, and con-
specific strains, differ from all currently recognized asco-
mycetous yeasts [6]. The closest known species is Z. lentus,
from which RRL YB-48 11 differs at nine of 585 nucle-
otide positions (1.5%). Kurtzman and Robnett [6] demon-
strated for ascomycetous yeasts that strains differing by
more than 1% substitutions in domain DI/D2 represent
separate species. The genus Zygosaccharomyces has been
found to be polyphyletic from phylogenetic analysis of
seq uences from domain D I/D2 [6], as well as from 18S
rO A [10,11]. Species that form a clade with Z. rouxii,
the type species, are Z. bailii, Z. bisporus, Z. lentus, Zy-
gosaccharomyces mellis, and the new species detected in
the present study. Species currently assigned to Zygosac-
charomyces that are not part of the Z. rouxii clade on the
basis of rD A analysis are Z. cidri, Z. fermentati, z.
florentinus, z. microellipsoides and Z. mrakii. Conse-
quently, in view of the genetic isolation predicted for
RRL YB-4811 from rD A analysis and its phylogenetic
placement in the Z. rouxii clade (Fig. 1), the following new
species is proposed.

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-
gation, 7 days, YM agar, 25°C. C: Ascosporulation following heteroga-
tyl-o-glucosaminum, methanolum, erythritolum, ribito-
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
pairs, and in small clusters (Fig. 2A). Growth is white to
tannish-white, dull to semi-glistening and butyrous.
3.1.2.2. Growth on the surface of assimilation media.
Pellicles are not formed.
3.1.2.3. Dalmau plate culture on yeast morphology
agar. After 7 days at 25°C, neither pseudohyphae nor
true hyphae are formed under the coverglass. Aerobic
growth is semi-glistening, white to tannish-white, butyr-
ous, and raised, but with a central depression. Colony
margins may be entire or with small, irregular lobes.
3.1.2.4. Ascosporulation. Ascospores were produced
on YM and 5% ME agar media after 3-7 days at 25°C.
Asci are conjugated and many show the 'dumbbell' con-
figuration typical of Zygosaccharomyces species in which
the equally sized rounded conjugants are joined by a con-
jugation tube. In this configuration, each conjugant forms
one to two spherical ascospores (Fig. 2B). Other asci lack
this configuration and form two to four ascospores in just
one of the conjugating cells, which is larger than the other
conjugant (Fig. 2C). Species showing the type of conjuga-
tion described are ordinarily homothallic. Ascospores were
observed in all four known strains of Z. kombuchaensis,
but RRL YB-4810 failed to form ascospores in the
present study, although ascosporulation was observed on
two earlier occasions.
galactose and assimilate raffinose, and can be separated
from the sensu stricto group by these reactions. Earlier
work [12,13] showed that Z. rouxii and Z. mellis did not
grow on media containing 1% acetic acid and could be
separated from Z. bailii and Z. bisporus by this test.
Z. kombuchaensis grows in the presence of 1% acetic
acid, but some strains of Z. lentus do not ([7], this study).
However, Z. lentus does grow in the presence of 0.9%
acetic acid, and this allows separation of Z. bailii, Z. bis-
porus, Z. kombuchaensis and Z. lentus from Z. mellis and
Z. rouxii. The latter two species can be separated from
each other on sodium chloride media [12]. Of the four
acetic acid tolerant species, Z. bailii assimilates trehalose,
whereas the other three taxa do not. Z. bisporus does not
ferment sucrose and thereby can be separated from
Z. kombuchaensis and Z. lentus. However, there appear
to be no fermentation or assimilation reactions on stan-
dard tests that will separate Z. kombuchaensis from
Z. lentus. Steels et al. [7] reported that Z. lentus did not
assimilate galactose, L-sorbose and sucrose, but these three
sugars permitted growth of Z. lentus in our tests, and
strains of Z. kombuchaensis gave variable growth reactions
on the sugars.
To provide a rapid diagnostic method for separation of
Z. kombuchaensis and Z. lentus, we examined RFLPs for
five regions of the rD A repeat using the enzymes DdeI
and MboI, either singly or in combination. RFLP patterns
from ITS, I8S, ITS2+5' -half of 26S and 3'-half of 26S

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')
C\J L{) CD
physiological tests included all strains listed in Table 2, ~ ~
CD CD
as well as those reported earlier [12].
CO
-.;:;t
CO
-.;:;t
~ ~
" " C\J C\J

3.1.2.6. Source of cultures. Origins of the four strains

I

en en
>- >-
I

">- >-" >-" >-"

C\J C\J C\J C\J

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
3.1.2.7. Type. RRL YB-4811 (=CBS 8849) is pre-
872 -
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
or to a lack of uniformity of patterns among strains of a
species. The amplicon comprised of I8S and ITS 1 pro-
vided resolution of Z. kombuchaensis and Z. lentus, with
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-
nor bands present in single enzyme digests disappeared
leaving a uniform pattern of major bands (Fig. 3). A dou-
ble enzyme digest of the I8S-ITS 1 amplicon also resolved
the type strains of Z. bailii and Z. bisporus from each
other and from Z. kombuchaensis and Z. lentus, but addi-
tional strains of Z. bailii and Z. bisporus were not tested
because these two species can be separated using growth
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.
quently, it appears that all of these species share an ability
to grow in high sugar media at low pH. In view of this,
Z. kombuchaensis could potentially occur as a spoilage
organism in acidic high sugar foods and beverages.
Acknowledgements
Larry W. Tjarks is gratefully acknowledged for skillful
operation of the nucleic acid sequencer. The mention of
firm names or trade products does not imply that they are
endorsed or recommended by the U.S. Department of
Agriculture over other firms or similar products not men-
tioned.
References

[I] Alexopoulos, C.l., Mims, CW. and Blackwell, M. (1996) Introduc-

3.2.1. Diagnostic key to species of Zygosaccharomyces
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 Sucrose is fermented Z. kombuchaensisa eny of ascomycetous yeasts from analysis of nuclear large subunit
Z. lenlUs" (26S) ribosomal D A 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
gene sequences: description of Saccharomyces kunashirensis sp. nov.
kombuchaensis. RRL YB-4882, which was isolated from
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
Taxonomic Study, 3rd edn. (Kreger-van Rij, .l.W., Ed.), pp. 449-
RRL Y-27163 (z. kombuchaensis), but proved to be Z.
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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