RESEARCH ARTICLE
Spathaspora arborariae sp. nov., a D -xylose-fermenting yeast
species isolated from rotting wood in Brazil
Raquel M. Cadete1, Renata O. Santos1, Monaliza A. Melo1, Adriane Mouro2, Davi L. Gonçalves2, Boris U.
Stambuk2, Fátima C.O. Gomes3, Marc-André Lachance4 & Carlos A. Rosa1
1
Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 2Departamento de
Bioquı́mica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil; 3Departamento de Quı́mica, Centro Federal
de Educação Tecnológica de Minas Gerais, Belo Horizonte, MG, Brazil; and 4Department of Biology, University of Western Ontario, London, ON, Canada
Correspondence: Carlos A. Rosa,
Departamento de Microbiologia, Instituto de
Ciências Biológicas, Universidade Federal de
Minas Gerais, C.P. 486, Belo Horizonte, MG
31270-901, Brazil. Tel.: 155 31 3409 2751;
fax: 155 31 3409 2630; e-mail:
[email protected]
Received 3 July 2009; revised 27 August 2009;
accepted 29 August 2009.
Final version published online 10 October 2009.
Abstract
Four strains of a new yeast species were isolated from rotting wood from two sites
in an Atlantic Rain Forest and a Cerrado ecosystem in Brazil. The analysis of the
sequences of the D1/D2 domains of the large-subunit rRNA gene showed that this
species belongs to the Spathaspora clade. The new species ferments D-xylose
efficiently and is related to Candida jeffriesii and Spathaspora passalidarum, both
of which also ferment D-xylose. Similar to S. passalidarum, the new species
produces unconjugated asci with a single greatly elongated ascospore with curved
ends. The type strain of Spathaspora arborariae sp. nov. is UFMG-HM19.1AT
(= CBS11463T = NRRL Y-48658T).
DOI:10.1111/j.1567-1364.2009.00582.x
Editor: Cletus Kurtzman
Keywords
new yeast species; Spathaspora arborariae;
D-xylose-fermenting yeast; rotting wood;
tropical ecosystems.
YEAST RESEARCH
Introduction
The Spathaspora clade contains several D-xylose-fermenting
yeast species isolated from rotting wood or wood-boring
insects. Spathaspora passalidarum, the single teleomorph
species of the clade, was isolated from the passalid beetle
Odontotaenius disjunctus in Louisiana (Nguyen et al., 2006).
The D-xylose-fermenting asexual species Candida jeffriesii and
Candida lyxosophila are phylogenetically related to S. passalidarum, and were isolated from the surface of a wood-boring
beetle and woodland soil, respectively (van der Walt et al.,
1987; Nguyen et al., 2006). Candida insectamans and Candida
materiae, two species that do not ferment this pentose, also
belong to the Spathaspora clade, although with little or no
support, and were isolated from the frass of beetle larvae in
trees in South Africa and from rotting wood samples in Brazil,
respectively (van der Walt et al., 1972; Barbosa et al., 2009).
We have isolated four strains of a new yeast species
belonging to the Spathaspora clade from rotting wood
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samples collected in the Atlantic Rain Forest and the
Cerrado ecosystem in Brazil. The strains formed asci without conjugation and asci containing a single elongated
ascospore resembling that of S. passalidarum. Analysis of
the sequences of the D1/D2 domains of the large-subunit
rRNA gene showed that these strains represented a single
species that is closely related to C. jeffriesii, S. passalidarum
and C. materiae. The new species is described as Spathaspora
arborariae sp. nov.
Materials and methods
Yeast isolation and identification
The yeasts were isolated from rotting wood samples collected in the National Park of Serra do Cipó and in the Rio
Doce State Park. The National Park of Serra do Cipó is
located in Minas Gerais state, Brazil, at the southern part of
Serra do Espinhaço, and extends over a total area of
FEMS Yeast Res 9 (2009) 1338–1342
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Spathaspora arborariae sp. nov.
33 800 ha. The local climate is altitudinal tropical with fresh
and rainy summers and a well-established dry season, with
annual mean temperatures oscillating between 17 and
18.5 1C. The vegetation is varied, although dominated by
grasslands (Cerrado) and rupestrian fields. The Rio Doce
State Park has an area of approximately 36 000 ha and is the
largest preserved semi-deciduous Atlantic Rain Forest found
in Minas Gerais state. This state park is characterized by a
transition of mesothermic humid tropical climate and
mesothermic rainy tropical climate, with annual average
temperatures ranging from 21 to 24 1C.
Five decayed wood samples were collected from the
National Park of Serra do Cipó in April 2008 and 20 samples
from the Rio Doce State Park in August 2008. The samples
were stored in sterile plastic bags and transported under
refrigeration to the laboratory over a period of no more than
24 h. One gram of each sample was placed separately in
flasks with 20 mL sterile D-xylose medium (yeast nitrogen
base 0.67%, D-xylose 0.5%, chloramphenicol 0.02%) and
20 mL sterile xylan medium (yeast nitrogen base 0.67%,
xylan 1%, chloramphenicol 0.02%, pH 5.0 0.2), respectively. The flasks were incubated at 25 1C on an incubator
shaker (New Brunswick) at 150 r.p.m. for 3–10 days. When
growth was detected, 0.5 mL of a culture was transferred to a
tube containing 5 mL sterile D-xylose or xylan media, and
the tubes were incubated on an incubator shaker as
described above. One loopful of each tube was streaked on
D-xylose or xylan agar media. The plates were incubated at
25 1C until yeast colonies developed. The yeasts isolated
were chosen based on colony morphology by repeated
streaking on yeast extract–malt extract agar (YMA, glucose
1%, yeast extract 0.3%, yeast malt 0.3%, peptone 0.5%, agar
2% and chloramphenicol 0.02%) and preserved at 80 1C
or in liquid nitrogen for later identification. The yeasts were
characterized using standard methods (Yarrow, 1998). The
ability to ferment D-xylose was tested in Durham tubes
containing a 2% (p/w) solution of the sugar. The tubes were
incubated at 25 1C on an incubator shaker (New Brunswick)
at 100 r.p.m. for 25 days. Candida shehatae var. shehatae CBS
5813, C. shehatae var. insectosa CBS 4286, C. shehatae var.
lignosa CBS 4705 and Pichia stipitis CBS 5773 were used as
positive controls for D-xylose fermentation (Barbosa et al.,
2009). Preliminary identifications followed the taxonomic
keys of Kurtzman & Fell (1998).
DNA sequencing and sequence analysis
The D1/D2 variable domains of the large-subunit rRNA gene
were amplified by PCR directly from whole cells as described
previously (Lachance et al., 1999). The amplified DNA was
concentrated, cleaned (Wizard Plus SV Minipreps DNA
Purification System, Promega) and sequenced in a MegaBACETM 1000 automated sequencing system (Amersham
FEMS Yeast Res 9 (2009) 1338–1342
Biosciences). The sequence was edited with the program
DNAMAN, version 6 (Lynnon BioSoft, Vaudreuil, QC, Canada).
Existing sequences for other yeasts were retrieved from
GenBank. The CLUSTAL W software (Thompson et al., 1994)
was used to align the sequences and to construct a neighborjoining tree with 1000 bootstrap iterations.
Growth conditions and fermentation assays
Cells were grown on YP medium (1% yeast extract and
2% peptone), adjusted to pH 5.0 with HCl and supplemented with 2% glucose or D-xylose. Cells were grown with
shaking at 28 1C (160 r.p.m.) in cotton-plugged Erlenmeyer
flasks filled to 1/5 of the volume with medium. The
inoculum for growth assays was prepared by transferring a
single colony aseptically from a plate into 5 mL of the
medium containing glucose or D-xylose, and allowing
growth to proceed to the stationary phase for 2–3 days
before inoculating cells (by a 100 or a 1000 dilution
factor) to new media of a similar composition. Culture
samples were harvested regularly, centrifuged (5000 g,
1 min) and their supernatants were used for the determination of sugars and ethanol. For batch fermentations, the
yeasts were pregrown on YP-2% sugar to the late exponential phase (1 g dry yeast L1), centrifuged (3500 g, 3 min),
washed twice with cold water and inoculated at a high cell
density (10 g dry yeast L1) into a YP medium containing
the amounts of glucose and/or D-xylose indicated. Batch
fermentations were incubated as described above for growth
assays, and samples were collected regularly, centrifuged and
their supernatants were analyzed as described below.
Analytical methods
Glucose was measured by the glucose oxidase and peroxidase method using a commercial kit (BioDiagnostica-Laborclin, Brazil), and D-xylose was determined as described
by Miller (1959). Ethanol was determined with alcohol
oxidase (Sigma) and peroxidase (Toyobo do Brasil, Brazil)
as described previously (Alves et al., 2007). Growth was
followed by turbidity measurements at 570 nm after appropriate dilution, and yeast cell dry weight was determined as
described elsewhere (Badotti et al., 2008). Briefly, from 1 to
3 mL of fermentation broth was filtered through preweighed
filters (0.45-mm mixed nitrocellulose and cellulose acetate
filters), washed with 5 mL of distilled water and, after
placing in a small (5 cm diameter) covered Petri dish, the
filter membranes were dried for 1 min in a microwave oven
at maximum power (900 W) and overnight at 80 1C. The
sugar consumption rates were calculated using samples
harvested at intervals during which maximal rates were
attained. Mean values of dry weight in the specified time
intervals were used in the rate calculations. The specific
growth rate (m, h1) was determined as the slope of a straight
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1340
line between ln OD570 nm and time (h) during the initial
(12 h) exponential phase of growth. Ethanol yield coefficients (Ye/s) were obtained at the end of ethanol production.
Results and discussion
Species delineation, classification and ecology
The four strains of the new species were isolated from media
with xylan as the sole carbon source. One yeast strain
(UFMG-HM19.1A) was isolated from the National Park of
Serra do Cipó and the three other strains (UFMG-HM32.1,
HM33.2a and HM34.2) were obtained from samples collected in different locations in the Rio Doce State Park. The
sequences of the D1/D2 domains of the large-subunit rRNA
gene were identical in all four strains and showed that the
new species belongs to the Spathaspora clade (Fig. 1). The
new species (GenBank accession no. GQ149081) differs by
11 substitutions and four gaps from C. jeffriesii and 13
substitutions and four gaps from C. materiae in the D1/D2
region of the large-subunit rRNA gene. In relation to
S. passalidarum, the three species differ by 17 substitutions
and four gaps. We conclude that the strains represent a new
species of the Spathaspora clade. Similar to S. passalidarum,
this new species produced asci with a single elongated
ascospore with curved ends (Fig. 2). The name S. arborariae
sp. nov. is proposed for the new species.
Spathaspora arborariae can be distinguished from
S. passalidarum based on the assimilation of L-sorbose and
growth on 50% glucose, which does not occur in the latter.
Spathaspora arborariae can grow at 37 1C, but C. jeffriesii
does not. Spathaspora arborariae is easily separated from
C. materiae based on the ability to ferment D-xylose and the
production of ascospores by the former species.
The isolation of S. arborariae from rotting wood of two
different localities suggests that the yeast is associated with
this kind of substrate. Spathaspora arborariae assimilates
and ferments D-xylose efficiently and is therefore able to
utilize this and related compounds from rotting wood. Gas
formation from D-xylose fermentation in Durham tube tests
was observed after 4 days with the type strain (UFMGHM19.1A), and after 10 days with the other three strains.
Growth and fermentation of D -xylose by
S. arborariae
Figure 3 shows the kinetics of growth on glucose and
D-xylose by strain UFMG-HM19.1A. The strain exhibited a
typical growth curve where the sugar is efficiently fermented, and after the sugar is exhausted from the media, the
ethanol produced starts to be consumed and used as a
carbon source. The strain grew well on both carbon sources
(m = 0.35 h1), producing practically the same amount
of biomass and ethanol (Fig. 3) from glucose or D-xylose
2009 Federation of European Microbiological Societies
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R.M. Cadete et al.
0.05
Candida lyxosophila NRRL Y-17539 / U76204
Spathaspora passalidarum NRRL Y-27907 / DQ109807
Candida materiae UFMG-07-C15.1B / FJ154790
72
76
54
Spathaspora arborariae UFMG-HM19.1A / GQ149081
Candida jeffriesii NRRL Y-27738 / AY520415
Candida insectamans NRRL Y-7786 / U45753
Fig. 1. Neighbor-joining tree showing the placement of Spathaspora
arborariae among related species in the Spathaspora clade. Bootstrap
values of 50% or above are shown.
Fig. 2. Budding yeast cells and asci of Spathaspora arborariae with a
single elongated ascospore with ends curved on dilute V8 agar after 3
days at 20 1C. Scale bar = 5 mm.
(Ye/s = 0.35–0.37 g ethanol g1 sugar). However, as found typically for other D-xylose-fermenting yeasts (Panchal et al.,
1988; Sanchez et al., 2002; Stambuk et al., 2003), this yeast
has a clear preference for glucose uptake and fermentation.
This characteristic is evident during batch fermentations at
high cell densities (see Fig. 4a), where glucose consumption
rates by the yeasts (0.74 g g1 cell dry weight h1) are
higher than D-xylose consumption rates (0.33 g g1 cell
dry weight h1), and glucose consumption occurs before
D-xylose utilization when both sugars are present at the
beginning of the fermentation (Fig. 4b). Nevertheless, given
the high ethanol yields (Ye/s0.50 g ethanol g1 sugar)
obtained during the batch fermentations described above,
S. arborariae may provide a source of genes, enzymes and/or
sugar transporters to engineer strains for efficient ethanol
production from renewable biomass (Hahn-Hagerdal et al.,
2007; Stephanopoulos, 2007; Stambuk et al., 2008).
Latin diagnosis of Spathaspora arborariae sp.
nov. Cadete, Santos, Melo, Gomes, Mouro,
Goncalves, Stambuk, Lachance & Rosa
In medio liquido post dies tres cellulae singulae aut binae;
cellulae ovoidae aut ellipsoideae (2–3 2–4 mm). Post unum
FEMS Yeast Res 9 (2009) 1338–1342
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Spathaspora arborariae sp. nov.
Fig. 3. Aerobic batch growth of Spathaspora
arborariae on 20 g L1 of glucose (open symbols)
or D-xylose (black symbols). Cell growth (a), the
consumption of sugars (b), and the production
of ethanol (c) by strain UFMG-HM19.1A were
determined during growth in rich YP medium.
Fig. 4. Sugar batch fermentations by 10 g L1
(dry weight) of Spathaspora arborariae yeast
cells in rich YP medium. (a) The consumption of
sugars (circles), and biomass (squares) and
ethanol (triangles) production during batch
fermentations of 20 g L1 of glucose (open
symbols) or D-xylose (black symbols) were
determined with cells of strain UFMG-HM19.1A
pregrown on glucose or D-xylose, respectively.
In (b) D-xylose pregrown cells were used to
ferment a mixture of 20 g L1 of both glucose
plus D-xylose [same symbols as in (a)].
mensem sedimentum formatur. Cultura in agaro malti post
dies 2 (17 1C) candida, butyrosa et teres. In agaro farinae Zea
mays post dies 14 pseudomycelium non formatur. Asci inconjugati et stabiles. Ascosporae alatae constipatae et non liberatio.
Glucosum, galactosum, maltosum et D-xylosum fermentantur.
Glucosum, galactosum, L-sorbosum (exigue), sucrosum, maltosum, cellobiosum, trehalosum, melezitosum, D-xylosum, Dribosum (lente), ethanolum, glycerolum (variabile), erythritolum, ribitolum, D-mannitolum, glucitolum, salicinum, acidum
succinicum (exigue), D-glucosaminum (exigue), N-acetylglucosaminum (exigue) et xylitolum (variabile) assimilantur, et
non lactosum, melibiosum, raffinosum, inulinum, amylum
solubile, D-arabinosum, L-arabinosum, L-rhamnosum, galactitolum, acidum lacticum, acidum citricum, meso-inositolum,
methanolum, hexadecanum, D-glucosaminum, acetonum, isopropanolum nec ethyl acetas. Ethylaminum, lysinum et cadaverinum assimilantur at non kalium nitricum et natrium
nitrosum. Ad crescentiam vitamina externa necessaria sunt.
Augmentum in 37 1C. Habitat materiam in Brazil. Typus
UFMG-HM19.1A. In collectione zymotica Centraalbureau
voor Schimmelcultures, Trajectum ad Rhenum, sub no.
CBS11463 typus stirps deposita est.
FEMS Yeast Res 9 (2009) 1338–1342
Description of Spathaspora arborariae sp. nov.
Cadete, Santos, Melo, Gomes, Mouro, Goncalves,
Stambuk, Lachance & Rosa
In yeast extract (0.5%), glucose (2%) broth after 3 days at
25 1C, the cells are ovoid to ellipsoidal (2–3 2–4 mm).
Budding is multilateral. A sediment is formed after a month,
but a pellicle is not observed. On YMA after 2 days at 17 1C,
colonies are white, butyrous and glistening. In Dalmau
plates, after 2 weeks on cornmeal agar, pseudomycelia are
not formed. Sporulation occurs on dilute V8 agar at 20 1C
after 3 days (Fig. 2). Unconjugated asci are formed from
single cells with a single highly elongated ascospore with
curved ends. Asci are not dehiscent. Fermentation of glucose, galactose, maltose and xylose is positive. Assimilation
of carbon compounds: glucose, galactose, L-sorbose (slow),
sucrose, maltose, cellobiose, trehalose, melizitose, D-xylose,
D-ribose (slow), ethanol, glycerol (variable), erythritol,
ribitol, D-mannitol, glucitol, salicin, succinic acid (slow),
D-glucosamine (slow), xylitol (variable) and N-acetyl-glucosamine (slow). No growth occurs on lactose, melibiose,
raffinose, inulin, soluble starch, D-arabinose, L-arabinose,
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1342
L-rhamnose,
galactitol, lactic acid, citric acid, m-inositol,
methanol, hexadecane, acetone, isopropanol and ethyl acetate. Assimilation of nitrogen compounds: positive for ethylamine-HCl, lysine and cadaverine, and negative for nitrate
and nitrite. Growth in vitamin-free medium is negative.
Growth in amino acid-free medium is positive. Growth at
37 1C is positive. Growth on YMA with 10% sodium
chloride is negative. Growth in 50% glucose/yeast extract
(0.5%) is positive. Starch-like compounds are not produced.
In 100 mg cycloheximide mL1 the growth is negative. Urease
activity is negative. Diazonium Blue B reaction is negative.
The habitat is rotting wood in the Atlantic Rain Forest
ecosystem, in the state of Minas Gerais, Brazil. The type
strain accession number of S. arborariae is UFMGHM19.1AT. It was isolated from rotting wood in Brazil. It
has been deposited in the collection of the Yeast Division of
the Centraalbureau voor Schimmelcultures, Utrecht, the
Netherlands, as strain CBS11463T ( = NRRL Y-48658T). The
epithet arborariae (ar.bo.ra 0 ri.ae 0 ) L. gen. nov. arborariae, of
or pertaining to trees, and referred to the substrate where
this yeast was found.
Acknowledgements
This work was supported in part by grants from the
Brazilian agencies Conselho Nacional de Desenvolvimento
Cientifico e Tecnologico-CNPq (process no. 552877/2007-7)
and Fundação de Amparo a Pesquisa do Estado de São
Paulo-FAPESP (process no. 04/10067-6), and the Natural
Science and Engineering Research Council of Canada
(M.-A.L.). D.L.G., A.M., B.U.S. and C.A.R acknowledge
research fellowships from FUNCAMP and CNPq.
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