yeast diversity in the acidic rio agrio–lake caviahue volcanic environment (patagonia, argentina)

R E S E A R C H A R T I C L E

Yeastdiversity in theacidicRioAgrio^LakeCaviahuevolcanicenvironment (Patagonia, Argentina)Gabriel Russo1, Diego Libkind1, Jose P. Sampaio2 & Maria R. van Broock1

1Laboratorio de Microbiologıa Aplicada y Biotecnologıa, Centro Regional Universitario Bariloche, Universidad Nacional del Comahue (UNComa),

Bariloche, Argentina; 2Centro de Recursos Microbiologicos (CREM), Seccao Autonoma de Biotecnologıa (SABT), Faculdade de Ciencias e Tecnologia,

Universidade Nova de Lisboa, Caparica, Portugal

Correspondence: Gabriel Russo, Laboratorio

de Microbiologıa y Biotecnologıa Aplicada,

Centro Regional Universitario Bariloche,

Universidad Nacional del Comahue

(UNComa), Quintral 1250, Bariloche,

R8400FRF, Argentina. Tel.: 10054 2944 522

111; fax: 10054 02944 42 2111; e-mail:

[email protected]

Received 26 September 2007; revised 27 March

2008; accepted 11 April 2008.

First published online 4 June 2008.

DOI:10.1111/j.1574-6941.2008.00514.x

Editor: Max Haggblom

Keywords

acidic aquatic environments; acidophilic yeasts;

biodiversity; extreme environments.

Abstract

The Rio Agrio and Lake Caviahue system (RAC), in Northwestern Patagonia, is a

natural acidic environment. The aims of this study were to characterize the yeast

community and to provide the first ecological assessment of yeast diversity of this

extreme aquatic environment. Yeast occurrence and diversity were studied at seven

sites where the water pH varied between 1.8 and 6.7. Yeast CFU counts in the river

ranged from 30 to 1200 CFU L�1, but in the Lake the values were lower

(30–60 CFU L�1). A total of 25 different yeast species were found, 11 of which

belonged to undescribed taxa. Among these was an unusual strongly acidophilic

Cryptococcus species. The RAC yeast community resembles that of acidic aquatic

environments resulting from anthropic activities such as the Sao Domingos mines

in Portugal and the Rio Tinto in Spain, respectively. The isolated yeast species were

organized into different grades of adaptation to the RAC aquatic system. Based on

the proposed grades, Rhodotorula mucilaginosa, Rhodosporidium toruloides and

two novel Cryptococcus species were the most adapted species. These Cryptococcus

species are apparently specialists of acidic aquatic environments, and might bear

physiological features that possibly account for their ability to thrive in such

extreme environments.

Introduction

Extremophiles are organisms that live under harsh environ-

mental conditions, such as those with extreme pH, tempera-

ture, pressure or high concentrations of toxic substances

(Raspor & Zupan, 2006). The interest in acidophilic micro-

organisms that thrive in acidic aquatic environments below

pH 3 is increasing because of their potential application to

biotechnological processes (Nakatsu & Hutchinson, 1988;

Johnson, 1998; Gross & Robbins, 2000; Zettler et al., 2002;

Gonzalez-Toril et al., 2003). In the past few years, prokar-

yotic acidophilic microorganisms, as well as some algae, and

filamentous fungi, have been extensively studied (Johnson

et al., 1992; Johnson, 1998; Pedrozo et al., 2001; Yahya &

Johnson, 2002; Zettler et al., 2002; Okibe et al., 2003; Lopez-

Archilla et al., 2004; Baumler et al., 2005). However, the

diversity of yeasts in acidic environments has been the

subject of considerably less study (Lopez-Archilla et al.,

2004; Gadanho et al., 2006). Furthermore, these studies have

been restricted to the Iberian Pyrite Belt acidic water bodies,

which are acidic aquatic environments resulting from

anthropogenic activities i.e. mining or industrial waste

effluents that contain significantly large quantities of HCl,

which give rise to high concentrations of dissolved heavy

metals in the water (Nakatsu & Hutchinson, 1988; Johnson,

1998; Cossa et al., 2001).

Acidic aquatic environments also occur naturally. Often

they are caused by geothermal activity that releases H2SO4.

Such is the case of the Rio Agrio and Lake Caviahue (RAC)

aquatic system, in Northwestern Patagonia, which has a

wide pH range, from highly acidic (pH = 1.5) to near neutral

(pH = 6.7). Because of the acidic conditions caused by the

release of H2SO4, the RAC system contains large amounts

of dissolved heavy metals in its waters [e.g. 69.5 g L�1 of Fe

and 0.2 g L�1 of As (Pedrozo et al., 2001)]. The proton

and metal concentrations gradually decrease downstream

(Pedrozo et al., 2001). The diversity of yeasts in these naturally

occurring acidic aquatic environments has not been studied.

The present work provides the first report of yeast

diversity for a natural acidic aquatic environment, namely

FEMS Microbiol Ecol 65 (2008) 415–424 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

the RAC (Patagonia, Argentina). It describes changes in the

yeast community along the naturally occurring proton and

heavy metal concentration gradient along the RAC system,

and reports the isolation of yeasts (possibly autochthonous)

that are well adapted to the extreme environmental condi-

tions present at upper Rio Agrio (URA).

Materials and methods

Study area and sampling procedures

RAC is located at 1600 m a.s.l., in the northwestern of

Neuquen Province (371520S 711020W) in Patagonia, Argen-

tina (Fig. 1). The source of the Rio Agrio is on the eastern

slope of Copahue Volcano (2900 m a.s.l), where the water

temperature is 75 1C and pH values range from 0.5 to 1

(Pedrozo et al., 2001). The RAC aquatic system can be

divided into three sections (see Fig. 1): the URA, which runs

from the hot spring to the river inflow into Lake Caviahue

(LC), the LC and the lower Rio Agrio (LRA), which

represents the outflow from the LC. Samples were drawn at

three sites along the upper Rio Agrio (URA1, URA2 and

URA3), one site on the surface of Lake Caviahue (LC4) and

at two sites on the lower Rio Agrio (LRA5 and LRA6). For

comparison, Rio Dulce (RD7) was also sampled at one site,

just before the LC inflow. With the exception of this last site

(from which a 1-L sample was collected), three independent

1-L samples were taken in sterile bottles from each site and

processed in the laboratory. Data for temperature, pH

and water conductivity were obtained in situ using an Orion

138 device. Site locations were determined using a GPS

(Magellan 315).

Yeast isolation and quantitative analysis

A water volume of 400–750 mL was filtered through a

nitrocellulose Millipores membrane (0.45mm pore size

and 47 mm diameter; Millipore, Bedford, MA) by means of

a sterilized Nalgenes device.

For yeast isolation, three culture media were used as

already described in Gadanho et al. (2006). Firstly, the

conventional MYP agar (g L�1: malt extract 7.0; yeast extract

0.5; peptone–soytone 2.5; agar 20.0) with a final pH of 5.0

and supplemented with 0.05% w/v of chloramphenicol to

inhibit bacterial growth. The second medium, MYP3, had

the same composition as MYP but the pH was adjusted to

3.0 with HCl (2 N). The third medium also had the same

composition but was prepared with water collected from

each of the study sites (sterilized by filtration through

0.2-mm pore size membranes) and was designated MYPW

(pH 1.8–6.7). For the last two culture media, sterile agar

(3% w/v) was poured directly on the Petri dish and mixed

with the acid culture media to avoid hydrolysis.

Fig. 1. Location of the RAC study area.

FEMS Microbiol Ecol 65 (2008) 415–424c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

416 G. Russo et al.

For each sampling site, each of the three water samples

was filtrated separately and each filter was placed on the

surface of MYP, MYP3 or MYPW agar-containing plates.

Plates were incubated at 16 1C until colony emergence.

Colony numbers on each filter surface were determined by

examining them with a stereoscopic microscope (Olympus

SZX9), and CFU L�1 were registered. For comparison pur-

poses, the yeast counts obtained in the three culture media

for samples collected in the river (URA and LRA sites) were

averaged and SDs were calculated. The same calculations

were performed for yeast counts of LC4.

All yeast colonies appearing on the plate’s surface were

transferred to fresh media for purification. If highly repre-

sented morphotypes were observed, only 50% of the total

colonies were purified. Pure cultures were preserved on

potato dextrose agar slants at 4 1C in the Centro Regional

Universitario Bariloche (CRUB) culture collection.

Conventional characterization

Morphological characteristics (colony color, margin and

texture) and physiological tests (assimilation of carbon

sources, starch production, urea hydrolysis, glucose fermen-

tation and assimilation of NO3 as the sole nitrogen source)

were performed according to Yarrow (1998). Sexuality

studies were performed on GSA media (g L�1: glucose 2.0;

peptone–soytone 2.0; agar 15.0).

DNA extraction and molecular characterization

DNA was extracted from yeast biomass according to Libkind

et al. (2003). The mini/microsatellite primed-PCR finger-

printing technique (MSP-PCR) was used according to

Libkind et al. (2003). The minisatellite primer M13 (50-

GAG GGT GGC GGT TCT) and the synthetic microsatellite

oligonucleotide (GTG)5 were used to obtain species-specific

fingerprints (Sampaio et al., 2001).

For sequence analysis, DNA was amplified using rRNA

primers ITS5 and LR6. Cycle sequencing of the region D1/

D2 at the 50 end of the 26S rRNA gene domain was

performed following Libkind et al. (2003). The sequences

obtained were compared with those included in the Gen-

Bank database with the Basic Local Alignment Search Tool

(BLAST at http://www.ncbi.nlm.nih.gov) (Altschul et al.,

1997). Sequence alignments were made with MEGALIGN

(DNAStar) and visually adjusted. Phylogenetic trees were

computed with PAUP�4.0b10 (Swofford, 2002) using the

neighbor-joining method (Saitou & Nei, 1987). Bootstrap

analyses were based on 1000 random resamplings.

Effect of pH on growth of selected yeast species

Two yeast species showing the broadest distribution range

(Rhodotorula mucilaginosa CRUB1441 and Cryptococcus sp. 1

CRUB1310), and two species showing the narrowest distribu-

tion range (Cryptococcus sp. 2 and Cryptococcus festucosus),

were selected. Two strains of Cryptococcus sp. 2 (CRUB1564

and 1565) and two of Cr. festucosus (CRUB1358 and 1400)

were studied. Growth experiments were performed in dupli-

cate on MYP media with the pH ranging from 1.5 to 6.5.

Culture media were acidified by adding H2SO4 (5 N). Tubes

(25 mL) containing 5 mL of culture media were inoculated

with 100mL of a dense cell suspension (2 days old), and

incubated in an Innova 4000 Shaker (New Brunswick) at

21 1C and 100 r.p.m. for 7 days. The final dry cell biomass was

obtained by centrifugation at 1500 g for 5 min and drying at

105 1C until a constant weight was established. The final pH

was measured in the supernatant.

Yeast tolerance in acidic Rio Agrio water

Plastic 1.5-mL tubes (Eppendorfs) containing 950mL sterile

water (filtered through 0.2-mm pore size membranes) from

URA1 or URA3 sampling sites were inoculated with 50mL of

a dense suspension of 2-day-old cells and incubated at

18–20 1C with periodical shaking. In every case, at least 20%

of the isolates of each identified species were studied. The

type strain of Saccharomyces cerevisiae PYCC 4455T was

included for comparison purposes. For controls, cultures

were suspended in sterile-distilled water under the same

conditions. After 2, 6, 15, 30 and 60 days of incubation,

10mL was taken from each tube and spread onto MYP agar

plates. Plates were incubated for 1 week and colony growth

was registered. If colony growth was positive and comparable

to that observed for controls, it was considered that yeasts

were still viable, whereas if negative growth was obtained in

MYP plates, it was considered that yeasts had lost viability.

Results and discussion

Physical and chemical parameters of RAC water

The most relevant physical and chemical parameters from

the RAC system are summarized in Table 1. The aquatic

system had a marked pH gradient along the surveyed

distance (c. 21 km), from hyperacidic (1.8) at the first site

to almost neutral (6.7) at the last site. A simultaneous

decreasing gradient of water conductivity was also observed.

The pH gradient along the RAC aquatic system makes it a

unique environment for studying the effect of pH (and

consequently, metal concentration) on the composition of

the native yeast community.

Yeast quantification

The yeast densities observed in the water samples were

variable, as expected in aquatic environments (Simard &

Blackwood, 1971; Slavikova & Vadkertiova, 1997; Libkind


417Yeasts from volcanic acidic environments

et al., 2003), especially in very dynamic systems like lotic

water bodies. However, some general trends for yeasts

CFU L�1 in the RAC system (Table 1) were detected. Yeast

densities increased along the lotic sections of the aquatic

system (URA and LRA), and a drastic reduction of the total

yeast counts was observed from URA3 to the lake (LC4). The

pH increase and the consequent decrease in the concentra-

tion of heavy metals in the water, along with eventual yeast

inputs from the surrounding area, could explain the increase

in CFU L�1 along the URA. On the other hand, the decrease

in yeast CFU L�1 in the lake could be explained by a decrease

in the available nutrient concentration. Baffico (2006)

reported a significant decrease in macronutrients such as

phosphorus (from 0.75 to 0.12 mg L�1) and nitrogen (from

0.12 to 0.02 mg L�1) between URA and LC. Micronutrients

like sulfur, zinc, nickel and manganese were found to behave

similarly (Baffico, 2006). Reduced availability of nutrients

could affect yeast propagation and survival in the lake.

However, given that the lake has a second important

tributary, the Rio Dulce (RD) at the northwest shore (see

Fig. 1), which has an almost neutral pH water and a low

content of yeasts (Table 1), a dilution effect should not be

excluded as an alternative hypothesis.

The average yeast number in river samples (URA1–3 and

LRA5–6) was 500� 350 CFU L�1, similar to what may be

expected for nonacidified rivers [100–500 cells L�1, (Hagler

& Ahearn, 1987)]. Likewise, the LC water samples showed

an average of 50� 20 CFU L�1, typical of nonacidified lakes

[o 100 cells L�1 (Hagler & Ahearn, 1987)]. These results

indicate that extreme conditions in the RAC environment

do not seem to reduce yeast numbers compared with similar

nonacidified aquatic environments. Although numerous

reports have dealt with the quantification of yeast in aquatic

systems, the present work is the first one to provide yeast

quantitative data for an acidic aquatic environment of

volcanic origin.

Yeast identification

A total of 202 yeast strains were isolated from RAC water

samples. These were sorted into 47 preliminary groups based on

morphological and physiological characteristics, and subse-

quently subjected to MSP-PCR fingerprinting. The initial

characterization showed that Basidiomycetes accounted for

99% of total isolates, greatly outnumbering Ascomycetes. The

MSP-PCR technique allowed the arrangement of isolates into

36 groups based on DNA-banding patterns. Most of the

preliminary groups (66%) obtained from the conventional

characterization were coincident with those from MSP-PCR

assays. Representative strains belonging to the various MSP-

Table 1. Physicochemical parameters and culturable yeasts CFU L�1 of the RAC

Site Location Altitude (m a.s.l.) PH Conductivity� (mS cm�1) Metal concentration (mg L�1)w

Total yeast cell number in CFU L�1

MYP MYP3 MYPW

URA1 3715200800S

71106 03700W

1891 1.8 17.86 Fe: 191–2650

Mg: 176–1193

68 28 80

URA2 37153 00500S

71104 04900W

1779 2.2 8.36 Mn: 8.6–79.8

Zn: 0.3–7.7

712 440 497

URA3 37152 04400S

71103 03500W

1609 2.2 7.74 As: 0.3–4.8

Al: 79–921

190 825 1192

LC4 37152 04000S

71102 02300W

1606 2.7 1.18 Fe: 23.4

Mg: 18.7

Mn: 0.99

Zn: 0.03

As: 0.01

Al: 20.2

65 25 60

LRA5 37149 05000S

70158 01300W

ND 2.8 0.76 Fe: ud-17

Mg: 10.8–14.7

Mn: 0.1–0.7

ND 452 ND

LRA6 37145 03200S

70139 00800W

ND 6.7 0.24 Zn: ud-1

As: ud-0.01

Al: 2.2–15.2

ND 825 ND

RD7 37151 04600S

71102 04900W

1610 6.2 0.29 Mg: 12.8

Mn: 0.2

Fe, Zn, As, Al: ud

ND 106 ND

�Conductivity was measured in mille-Siemens.wData adapted from Gammons et al. (2005). For lotic sections of the RAC system (URA and LRA) ranges of metal concentrations are provided: maximum

values correspond to upper sites of the river a minimum ones to lower sites.

ND, not determined; ud, under detection levels.


418 G. Russo et al.

PCR classes were selected for sequence analysis of the D1/D2

domains of the 26S rRNA gene. The sequence-based identifica-

tion indicated the presence of 25 species (Table 2). Assignment

to species level was not always possible when using the D1/D2

region sequence. According to Kurtzman & Robnett (1998) and

Fell et al. (2000), a three to four nucleotide difference in the D1/

D2 domain of the 26S rRNA gene could be the result of

intraspecific variations. In these cases, when less than five base

substitutions were observed in the D1/D2 region sequence, the

name of the most closely related species found in the BLAST

search was applied. Eleven species showed five or more nucleo-

tide differences when compared with the most closely related

known species and hence were considered novel yeast taxa.

Given that more than one novel species was found for genera

Cryptococcus and Rhodotorula, consecutive numbers were as-

signed to these species. The phylogenetic placement of the new

species isolated from RAC, as well as the placement of the other

basidiomycetous yeasts found, is depicted in Fig. 2. The

basidiomycetous yeasts were distributed within the Agarico-

mycotina (13 species) and Pucciniomycotina (11 species)

subphyla. The former yeasts were distributed in the orders

Cystofilobasidiales, Filobasidiales and Tremellales while the latter

were distributed in the orders Microbotryales and Sporidiobo-

lales. The ascomycetous yeasts were represented solely by

Candida austromarina EF585155 and therefore no phylogenetic

tree was generated for this phylum.

Yeast diversity

Cryptococcus and Rhodotorula genera represented 89% of the

isolates. Similar results were reported in other acidic and

nonacidic aquatic environments (Johnson, 1998; Libkind

et al., 2003; Lisichkina et al., 2003; Lopez-Archilla et al.,

2004; Gadanho et al., 2006).

Cryptococcus sp. 1 and Rh. mucilaginosa represented the

most abundant yeast species (56% of the isolates) and those

with the broadest distribution. The undescribed species

Cryptococcus sp. 1 was the only one present in all sampling

sites of the RAC system, although its isolation frequency

markedly decreased after URA3. This suggests that

Table 2. Yeast species found in RAC, distribution, molecular identification and comparison with other acidic and nonacidic environments

Identification

Site

S. Domingos

mine� R. Tinto�

Lakes, rivers and

ponds from

PatagoniawNo. of nucleotide

differenceszURA1 URA2 URA3 LC4 LRA5 LRA6 RD7

C. austromarina 1 � � 1 0

C. albidisimilis 1 1 � � 0

C. antarcticus 1 3 1 3 � � 1 3

C. bhutanensis 1 � � � 2

Cr. festucosus 1 1 � � � 0

C. laurentii 1 � � 1 1

C. macerans 1 � � 1 0

C. saitoi 1 1 � � 1 0

C. victoriae 1 8 � � � 2

Cryptococcus sp. 1 5 17 43 2 2 2 � � � 40

Cryptococcus sp. 2 7 1 � � 8

Cryptococcus sp. 3 1 � � � 21

Cryptococcus sp. 4 1 1 � � � 8–9

Cryptococcus sp. 5 1 � � � 6

Cys. capitatum 2 � � 1 0

Microbotryales yeast 1 � � � 5

R. colostri 1 � � 1 0

Rh. mucilaginosa 10 27 1 2 3 1 1 1 0

R. babjevae 1 � � 1 0

R. toruloides 1 13 1 1 1 � 2

Rhodotorula sp. 1 1 1 22 3 1 1 � 14

Rhodotorula sp. 2 1 � � � 12



Sporobolomyces sp. 1 � � � 5

Numbers represent the amount of strains isolated from each sampling site.�Gadanho et al. (2006).wLibkind et al. (2003) and unpublished data.zNumber of nucleotide differences in comparison to the closest species (type strain).

1, present; � , absent.



Cryptococcus sp. 1 is a typical component of RAC system,

mostly associated with the acidic parts of the river, and

possibly an autochthonous species.

Rhodotorula mucilaginosa is the best-known example of a

ubiquitous yeast. It has been found in all kinds of natural

and artificial substrates, and has also been reported in acidic

Fig. 2. Phylogenetic placement of basidiomycetous yeast isolates obtained by neighbor joining (distance K2P method) of D1/D2 domains of the 26S

rRNA gene. The tree was rooted with Taphrina deformans. Names in boldface correspond to the sequences determined in this study. The scale indicates

the number of substitutions accumulated every 100 nucleotides. Bootstrap values higher than 50% are shown (1000 replicates).


420 G. Russo et al.

aquatic environments, namely Rio Tinto and Sao Domingos

mines (Lopez-Archilla et al., 2004; Gadanho et al., 2006).

The occurrence of Rh. mucilaginosa in RAC further supports

its ubiquitous nature.

Gadanho et al. (2006) and Lopez-Archilla et al. (2004)

suggested that the use of water from the environment when

culture media are being formulated and prepared enhances

the growth of autochthonous or well-adapted species of that

environment. The use of this strategy in the present work

allowed the isolation of two yeast species that would have

been less frequently isolated (Rhodosporidium toruloides) or

absent (Cryptococcus sp. 2) using artificially acidified media.

Interestingly, Cryptococcus sp. 2 and R. toruloides were also

isolated from Sao Domingos mines (pH 2.6–2.9) mostly

using acidified media (MYP3 or MYPW) (Gadanho et al.,

2006). These results suggest that certain substances (possibly

heavy metals) present in URA acidic waters as well as in Sao

Domingos mines favor growth in Cryptococcus sp. 2 and

R. toruloides, while inhibiting growth in most of the other

species. For the case of Cryptococcus sp. 2, this hypothesis is

further supported by the fact that growth in MYPW was

higher than in MYP3 (data not shown) and by recent

findings that show that this species has high tolerance to

heavy metals (unpublished data).

Sixteen out of 25 identified species were isolated from

acidic samples (URA1–3, LC4 and LRA5). From these, only

Cryptococcus sp. 1, Rhodotorula sp. 1 and Rh. mucilaginosa

were also present in nonacidic samples (LRA6) but in low

numbers (fewer than three isolates). The nine remaining yeast

species were isolated solely from nonacidic sampling loca-

tions (LRA6 and RD7). Furthermore, nine of the 11 yeast

species that were considered to be novel taxa were found

exclusively from acidic sampling sites and they were isolated

on acidified media, either MYP3 or MYPW. This result,

together with the difference in yeast species composition

observed between acidic and nonacidic samples, suggests that

pH and heavy metals are important environmental factors

affecting yeast’s community structure in aquatic habitats, and

the existence of a specific yeast flora adapted to the extreme

environmental conditions of the RAC aquatic system.

A comparison of the yeast species of RAC volcanic acidic

environment (Patagonia, Argentina) and those of other

acidic and nonacidic aquatic environments is shown in

Table 2. Interestingly, at least five yeast species, isolated

mostly in the acidic parts of the RAC system, were also

found in Sao Domingos mines and/or Rio Tinto (Lopez-

Archilla et al., 2004; Gadanho et al., 2006). Ten out of 25

species were common to nonacidic lakes of Patagonia, and

eight of them were isolated from the URA6 and the RD7,

which have near neutral (6.7) or slightly acidic (6.2) pH.

These results indicate that many of the species obtained

from the acidic part of the RAC aquatic system represent

yeasts adapted to acidic environments in general.

Effect of pH on yeast growth

Both Rh. mucilaginosa and Cryptococcus sp. 1 were able to

grow at pH 3.5, having a wider pH range than Cr. festucosus

(Fig. 3a), although the optimum pH for growth for all three

species was between 4.5 and 5.5 (Fig. 3a and b). These results

are consistent with the observed distribution of these species.

Interestingly, Cryptococcus sp. 2 could grow at a pH ranging

between 2.5 and 4.5 with an optimum pH of 3.5 (Fig. 3b).

Gross & Robbins (2000) suggested that yeasts should not be

included in the acidophilic group of organisms because the

optimal pH for growth ranges between 4.5 and 5 (Kurtzman

& Fell, 1998). However, the optimal pH and growth range in

Cryptococcus sp. 2, both in the present study, and in the study

of Gadanho et al. (2006), indicate that this novel species is the

first case of an acidophilic yeast species.

Cryptococcus sp. 1 and Rh. mucilaginosa were able to

increase the final pH in culture media from an initial pH of

3.5. For example, Rh. mucilaginosa increase the pH from 3.5

to 8.2 while Cryptococcus sp. 1 increase it from 3.5 to 6.2

(Fig. 3a). Cryptococcus festucosus did not alter the pH.

Cryptococcus sp. 2 raised the pH of the culture medium only

when the initial pH was between 3.5 and 5.5. Nguyen et al.

(2001) observed a similar phenomenon in Rhodotorula

glutinis R1 isolated from a hot spring. The mechanisms

involved in this process are still not understood.

Yeast tolerance to acidic Rio Agrio water

Yeast isolates, suspended in river water (without added

nutrients) from two acidic sites of URA (URA1, pH 1.8 and

4

3

2

1

0

Dry

wei

ght (

g L

–1)

Dry

wei

ght (

g L

–1)

4

3

2

1

0

1.5

1.5

2.5 3.5pH

pH

4.5 5.5 6.5

1.5

1.5

2.5

2.5

3.5

3.5

4.5

4.5

7.0

4.7

8.0

6.1

6.5

6.0

8.0

6.8

8.2

6.9

8.1

6.2

3.5

2.52.5

5.5 6.5

(a)

(b)

7.1

Fig. 3. Growth of selected yeast species at different pH. (a) Species with a

wide distribution range. Gray bars, Rhodotorula mucilaginosa (CRUB 1441);

black bars, Cryptococcus sp. 1 (CRUB 1305). (b) Species with a narrow

distribution range. Gray bars, Cryptococcus sp. 2 (CRUB 1564 and CRUB

1565); Black bars, Cryptococcus festucosus (CRUB 1358 and CRUB 1400).

SDs for all strains are included. The final pH is shown above each bar.



URA3, pH 2.2), showed different survival rates, as shown in

Fig. 4. With the exception of R. toruloides and Cryptococcus

victoriae, all strains of the same species showed similar

survival times. The variability registered for R. toruloides

and Cr. victoriae could be related to the genetic intraspecific

heterogeneity observed for these species (see Fig. 2).

Fewer species survived in URA1 water than in URA3

water, especially during the first 15 days of the experiment,

indicating that URA1 is a more stressful habitat. This

confirms that less extreme environmental conditions along

the river are related to higher pH and lower concentration of

heavy metals. Several yeast species were able to survive for

up to 2 months in the river water, whereas others failed to

survive beyond 2 days at most. Species such as Cryptococcus

sp. 2 and Cryptococcus sp. 1 were highly tolerant, as were

Rh. mucilaginosa and Rhodotorula sp. 1. Previous research

showed that Rh. mucilaginosa could survive for more than

30 days in the acid leachate water at pH 2 that occurs within

a uranium-mineral heap (de Siloniz et al., 2002).

The species Cystofilobasidium capitatum, Cryptococcus

macerans and Cr. festucosus displayed the lowest survival in the

river water samples, and interestingly were originally isolated in

the near-neutral part of the river (LRA6, pH 6.7). These species

are clearly not adapted to survive in the acidic conditions of the

river and are most certainly allochthonous. Cryptococcus laur-

entii and Rhodotorula colostri showed an unexpectedly high

tolerance to the river water samples, based on their distribution

in the river. Candida austromarina was highly susceptible,

resisting up to 2 days in URA1 and 6 days in URA3 water. This

result, together with the fact that only one isolate was obtained

from URA1, indicates that this species is allochthonous to the

aquatic system. The rest of the species showed intermediate

survival times, and in general, species isolated from the upper

part of the river were more tolerant than those from the lower

part, which is in agreement with the different environmental

conditions present at both sites.

Yeast community structure

A relative grade of adaptation was assigned to each species

based on its abundance and distribution in the studied acidic

aquatic system, presence or absence in other acidic and

nonacidic environments, culture media of isolation, optimum

pH and survival in URA water. Species such as Cryptococcus

sp. 2 and Cryptococcus sp. 1 are the most adapted, followed by

Rh. mucilaginosa and R. toruloides, being possibly autochtho-

nous members of the acidic sections of RAC system. Both

novel Cryptococcus species are more likely to be especially

adapted to acidic aquatic environments, because they were not

registered in other nonacidic environments. Gadanho et al.

(2006) had already suggested that Cryptococcus sp. 2 was an

acidophilic yeast of acidic environments, and our study

provides further support for this hypothesis.

Additionally, in this report we have detected a second

yeast (Cryptococcus sp. 1), which seems to be specific of the

RAC volcanic ecosystem. The distinctive physiological char-

acteristics of these yeasts, which allow them to thrive in

acidic extreme environments, are currently being investi-

gated in our laboratory.

The particular distribution of yeasts along the pH gradient

of the aquatic system showed agreement with the physiologi-

cal data of each species. We observed that as the proton and

heavy metal concentration of the water gradually decreases,

environmental conditions become less extreme and highly

extremophilic yeast species are replaced by less extremophilic

ones. As water pH reaches near-neutral values, the yeast flora

changes considerably, becoming similar to that of nonacidic

rivers and lakes of the Patagonian region (see Table 2).

Acidic water bodies of volcanic origin, such as the one

studied here, with a chemical composition similar to that of

mining waters, are promising habitats to search for acid-

adapted microorganisms. In fact, in our study we obtained

several yeast species with such characteristics. The biopros-

pecting of these organisms represents an important strategy

in the task of obtaining agents for bioremediation processes

and other fundamental and applied purposes.

Acknowledgements

This work was funded by the Universidad Nacional del

Comahue (Project B121), CONICET (Project PIP6536)

25

20

15

10

Num

ber

of s

urvi

ving

spe

cies

5

00 10 20 30 40 50 60

URA1 URA3 Ctrl

Time (days)

Fig. 4. Yeasts survival in water from the Rio Agrio. Numbers in parenth-

eses indicate those yeasts that did not survive, in the following order: (1)

Saccharomyces cerevisiae; (2) Rhodotorula sp. 4; (3) Rhodotorula sp. 3;

(4) Cryptococcus sp. 5; (5) Cystofilobasidium capitatum; (6) Cryptococcus

festucosus; (7) Candida austromarina; (8) Microbotryales yeast; (9)

Cryptococcus bhutanensis; (10) Cryptococcus macerans; (11) Sporobo-

lomyces sp.; (12) Cryptococcus saitoi; (13) Cryptococcus victoriae; (14)

Cryptococcus sp. 2; (15) Cryptococcus albidisimilis; (16) Rhodosporidium

toruloides; (17) Rhodosporidium babjevae; (18) Cryptococcus antarcti-

cus; and (19) Cryptococcus sp. 4.


422 G. Russo et al.

grants to M.R.v.B. and ANPCYT (Project PICT 22200).

Bilateral cooperation between Argentina and Portugal was

supported by a SECYT-ICCTI cooperation agreement (PO/

PA02-BI/002). G.R. was supported by CONICET PhD

fellowships. We would like to thank the authorities of

Parque Provincial Caviahue-Copahue (Argentina) for their

courtesy and cooperation, and Professor Michael Vaile, Dr

Celia Tognetti and Dr Allan Phillips for their critical reviews

of the manuscript.

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yeast diversity in the acidic rio agrio–lake caviahue volcanic environment (patagonia, argentina)

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