Extremophiles (2003) 7:451–458

DOI 10.1007/s00792-003-0347-2

O R I GI N A L P A P E R

Rosa Margesin Æ Silvia Gander Æ Gabriele Zacke

Anne Monique Gounot Æ Franz Schinner

Hydrocarbon degradation and enzyme activities

of cold-adapted bacteria and yeasts

Received: 1 February 2003 / Accepted: 27 June 2003 / Published online: 26 August 2003

Springer-Verlag 2003

Abstract The potential of 89 culturable cold-adapted degrading strains produced catechol 1,2 dioxygenase
isolates from uncontaminated habitats, including 61 activity.
bacterial and 28 yeast strains, to utilize representative
fractions of petroleum hydrocarbons (n-alkanes, Keywords Bacteria Æ Biodegradation Æ Cold-adapted Æ
monoaromatic and polycyclic aromatic hydrocarbons) Enzymes Æ Hydrocarbons Æ Phenol Æ Psychrophilic Æ
for growth and to produce various enzymes at 10C Yeasts
was investigated. The efficiency of bacterial and yeast
strains was compared. The growth temperature range
of the yeast strains was significantly smaller than that
of the bacterial strains. Sixty percent of the yeasts but Introduction
only 8% of the bacteria could be classified as true
psychrophiles, showing no growth above 20C. A high The potential of hydrocarbon-degrading microorgan-
percentage (89%) of the yeast strains showed lipase isms has led to the development of bioremediation
activity. More than one-third of the 61 bacterial techniques for contaminated soil and water (Dua et al.
strains produced amylase, b-lactamase, b-galactosidase 2002). Cold-adapted indigenous microorganisms play a
or lipase; more than two-thirds were protease pro- significant role in the in-situ biodegradation of hydro-
ducers. Only 6% of the bacterial strains but 79% of carbons in cold environments, where temperatures often
the yeast strains utilized n-hexadecane for growth; coincide with their growth temperature range. The aer-
13% of the bacterial strains and 21–32% of the yeast obic biodegradation of many components of petroleum
strains utilized phenol, phenanthrene or anthracene for hydrocarbons, including n-alkanes, aromatic and poly-
growth. Only four yeast strains but none of the bac- clic aromatic hydrocarbons (PAHs), at low temperatures
terial strains could grow with all hydrocarbons tested. has been reported in arctic, alpine and antarctic envi-
The biodegradation of phenol was investigated in fed- ronments (for a review, see Margesin and Schinner
batch cultures at 10C. Three yeast strains degraded 2001).
phenol concentrations as high as 10 mM (one strain) A wide variety of bacteria, fungi and algae have the
or 12.5 mM (two strains). Of eight bacterial strains, ability to metabolize aliphatic and aromatic hydrocar-
two strains degraded up to 10 mM phenol. The opti- bons (Alexander 1999). A large number of degradative
mum temperature for phenol degradation was 20C cold-tolerant bacteria have been identified, including
for all eight bacterial strains and for two yeast strains. representatives of heterotrophic aerobic Gram-negative
Biodegradation by five yeast strains was optimal and Gram-positive genera (Whyte et al. 1998; Yakimov
at 10C and faster at 1C than at 20C. All phenol- et al. 1999; Aislabie et al. 2000; Bej et al. 2000;
Baraniecki et al. 2002). Filamentous fungi are known for
their potential to degrade PAHs (Cerniglia 1992;
Communicated by K. Horikoshi Gramss et al. 1999). There is, however, little information
about the hydrocarbon-degradative potential of yeasts.
R. Margesin (&) Æ S. Gander Æ G. Zacke Æ F. Schinner
Institute of Microbiology, University of Innsbruck, Culturable cold-adapted yeasts have been isolated from
Technikerstrasse 25, 6020 Innsbruck, Austria oil-contaminated antarctic (Aislabie et al. 2001) and
E-mail: rosa.margesin@uibk.ac.at alpine soils (Margesin and Schinner 1997a), and from
Fax: +43-512-5072929 glacier cryoconite (Margesin et al. 2002).
A. M. Gounot Cold-adapted microorganisms are a potentially
University Claude Bernard Lyon 1, Villeurbanne, France exploitable source for cold-active enzymes (Gerday et al.
452

2000; Margesin 2002). Screenings for cold-active enzyme Screening for the microbial utilization of hydrocarbons
producers have been mainly focused on bacteria, while for growth
yeasts have been rarely considered (Birgisson et al. All 89 isolates were screened for their ability to utilize aerobically
2003). aliphatic and aromatic hydrocarbons in liquid media for growth.
It was the objective of this study to investigate the Inoculated medium without hydrocarbons, as well as sterile
potential of 89 culturable cold-adapted isolates to utilize hydrocarbon-containing medium, served as negative controls.
Growth was determined spectrophotometrically at 600 nm. Cul-
aerobically representative fractions of petroleum tures with an OD(600 nm)>0.2 were scored as positive for growth
hydrocarbons (n-alkanes, aromatic hydrocarbons, (Bej et al. 2000). Two to three replicates were used for all experi-
PAHs) for growth, and to produce various enzymes at ments and the mean values obtained are reported. The standard
low temperatures. The efficiency of bacterial and yeast deviations obtained were £10%.
strains and their growth temperature ranges were com-
pared. The biodegradation of phenol was characterized
n-alkanes (n-dodecane, n-hexadecane)
by determining the effect of temperature and the phenol
concentration on biodegradation and by determining the The screening was performed in 100-ml Erlenmeyer flasks
presence of catechol dioxygenases. containing 10 ml mineral medium. The medium was contami-
nated with n-hexadecane or n-dodecane (99% purity, 1 g l)1;
corresponding to 4.4 mM hexadecane and 5.9 mM dodecane) and
inoculated with 250 ll of a dense suspension of microbial cells (pre-
Materials and methods grown on R2A agar plates at 10C) in 0.9% NaCl. After an
incubation period of 14–21 days at 10C, growth was determined.
Strains The residual hydrocarbon concentration was quantified by infra-
red-spectroscopy after extraction with trichoro-trifluoro-ethane
(McLean et al. 1995).
Eighty-nine isolates, 61 bacterial and 28 yeast strains, were tested.
These strains were isolated on complex medium as described from
three alpine glacier cryoconite samples (Margesin et al. 2002) and
various alpine, Sibirian and antarctic cold environments, such as Aromatic hydrocarbons (phenol)
ice caves, continental ice and mud in the thawing zone at the foot of
glaciers. To distinguish between bacterial and yeast cells, the effect The screening was done in 100-ml Erlenmeyer flasks containing
of cycloheximide (inhibitor of fungal growth) on growth was 10 ml mineral medium. The medium was contaminated with
determined. Strains were grown on R2A agar plates containing 2.5 mM (235 mg l)1) phenol (99.5% purity) and inoculated with
400 lg cycloheximide ml)1 at 10C for 14 days. Phenol-degrading 250 ll of a dense suspension of microbial cells (pre-grown on R2A
strains were identified by Margesin et al. (2002) and by DSMZ agar plates at 10C) in 0.9% NaCl. After an incubation period of
(German Culture Collection of Microorganisms, Braunschweig, 7 days at 10C, growth was determined. The residual phenol con-
Germany). centration was determined according to Bastos et al. (2000a) by
measuring the optical density at 270 nm in the culture supernatants
using a calibration curve (0–1.4 mM phenol) prepared in mineral
medium.
Media

The strains were routinely cultivated on R2A agar plates. The

ability to utilize various hydrocarbons for growth was tested using PAHs (phenanthrene, anthracene)
a pH-neutral phosphate-buffered mineral medium (Margesin and
Schinner 1997b). This screening was performed in microtiter plates (flat bottom, 96
wells), using 200 ll mineral medium per well. Each well received
10 ll of a stock solution of anthracene or phenanthrene (>96%
purity) dissolved in acetone to give a final concentration of 10 mg
Growth temperature range PAH l)1 (5.6 lM). The acetone was allowed to evaporate at room
temperature. Afterwards, the PAH-containing medium was inoc-
Suspensions of microbial cells (pre-grown on R2A agar plates at ulated with 10 ll of a dense suspension of microbial cells (pre-
10C) in 0.9% NaCl were used to inoculate R2A agar plates which grown on R2A agar plates at 10C) in 0.9% NaCl. Microtiter
were incubated at 2, 10, 15, 20, 25, 30 and 35C, using two repli- plates were wrapped in plastic bags. After an incubation period of
cates per strain and temperature. Growth was monitored up to an 14 days at 10C, growth was determined using a microplate
incubation time of 7–21 days. reader.

Screening for enzyme activities

Phenol biodegradation
Amylase, protease, pectolytic enzymes, cellulase, b-galactosidase,
lipase and b-lactamase activities were tested on R2A agar plates Fed-batch cultivation
supplemented with starch, skim milk, polygalacturonic acid (each
compound 0.4% w/v), carboxymethylcellulose and trypan blue Phenol-degrading strains were cultivated at 10C and 180 rpm in
(0.4% and 0.01% w/v, respectively), lactose and X-Gal (0.2% 100-ml Erlenmeyer flasks containing 10 ml mineral medium that
and 0.004% w/v, respectively), tributyrin (0.5% v/v) or ampi- was contaminated with 1 mM phenol. After 7 days of cultivation
cillin (50 lg ml)1). After 3–6 days at 10C, a positive reaction and after the disappearance of the contamination, the same culture
was noticed when transparent zones around the colonies were was re-contaminated with increasing phenol concentrations, rang-
directly visible or detected after precipitation or coloration of the ing from 2.5 to 15 mM phenol. With each phenol amendment, the
undegraded substrate. In the case of b-lactamase activity, a volume was adjusted to 10 ml by adding fresh mineral medium
positive reaction was indicated by growth in the presence of (2–3 ml). Growth and the residual phenol concentration were
ampicillin. monitored at regular time intervals.
 453

Effect of temperature on phenol biodegradation percentage (89%) of the yeast strains showed lipase
activity, but none of them produced b-galactosidase or
Phenol-degrading strains were cultivated in phenol-containing
mineral medium at 1, 10 and 20C (15C for strain AG17, which pectolytic enzymes. More than one-third of the 61 bac-
cannot grow at 20C) and 180 rpm. Growth and the residual terial strains produced amylase, b-lactamase, b-galac-
phenol concentration were monitored up to an incubation time of tosidase or lipase; more than two-thirds were protease
16 days. producers (Table 1).

Presence of catechol dioxygenases

The presence of catechol dioxygenases, which catalyze the ring

Screening for the microbial utilization
cleavage of catechol, was tested using qualitative assays in mi- of hydrocarbons for growth
crotiter plates. Catechol 1,2 dioxygenase activity (C1,2D) was tes-
ted as described by Neidle and Ornston (1986) and Birger et al. n-alkanes (n-dodecane, n-hexadecane)
(1997). A solution (150 ll) containing 0.004% phenol red, 1 mM
EDTA and 10 mM catechol (pH 7.5 adjusted with ammonium Due to the different melting points of the two tested
hydroxide) was added to 50 ll of the liquid culture of phenol-
degrading strains [OD(600 nm) adjusted to 1]. The presence of n-alkanes, n-dodecane with a melting point of )12C
C1,2D resulted in a color change from red to yellow-orange within remained liquid at 10C, whereas n-hexadecane with a
10 min in the dark at room temperature. Catechol 2,3 dioxygenase melting point of 18.2C became solid at 10C. This may
activity (C2,3D) was determined as described by Morgan et al. have influenced the bioavailability of the compounds to
(1989) and Birger et al. (1997). A solution (150 ll) containing
90 mM catechol in 50 mM Tris-acetate buffer (pH 7.5) was added to the microorganisms. There was also a significantly
50 ll of the liquid culture of phenol-degrading strains higher abiotic loss of n-dodecane (14, 28, 48 and 67%
[OD(600 nm) adjusted to 1]. The presence of C2,3D resulted in the after 1, 3, 6 and 10 days, respectively, at 10C) than of
formation of a green-brownish color within 2 h in the dark at room n-hexadecane (8 and 10% after 8 and 14 days, respec-
temperature.
tively, at 10C) in sterile controls.
In spite of the solidification of hexadecane at 10C, 26
of the 89 tested strains could utilize this n-alkane for
Results growth. Among these strains were 4 bacterial and 22
yeast strains (6.6% of the tested bacterial strains and
Growth temperature range of bacterial and yeast strains 78.6% of the 28 yeast strains). However, only 7 of the 89
strains showed an OD(600 nm)>0.2 when n-dodecane
All strains could grow at temperatures ranging from 2 to was present (Table 2). The n-dodecane utilizers were
15C; only few (3% of the bacteria and yeasts, respec- yeast strains and were also able to utilize n-hexadecane
tively) showed no growth at 20C. Interestingly, the for growth.
growth temperature range of the yeast strains was sig- The degradation of n-dodecane and n-hexadecane by
nificantly smaller than that of the bacterial strains. a selected yeast strain that showed significantly better
Compared to bacteria, only a small proportion of the growth than any other strain [OD(600 nm)>1], was
yeasts could grow at 25 and 30C (Fig. 1). Sixty percent investigated. This strain degraded 39.9% and 35.4% of
of the yeasts but only 8% of the bacteria could be hexadecane and dodecane (1 g l)1), respectively, after
classified as true psychrophiles, showing no growth 8 days at 10C. After 5 days at 15C, 50% and 73% of
above 20C. hexadecane and dodecane were degraded. The strain has
been identified as Yarrowia lipolytica RM7/11 and has
been found to be an efficient diesel-oil degrader at low
Screening for enzyme activities temperatures (Margesin and Schinner 1997a), showing
maximum oil-degradation activity at 0–20C and no
A considerable number of isolates produced enzymes. growth above 30C.
All 28 yeast strains had b-lactamase activity, and a high

Aromatic hydrocarbons (phenol)

Fifteen of the tested 89 strains showed an OD

(600 nm)>0.2 when grown with 2.5 mM phenol. Eight
of these strains were bacteria (13.1% of 61 bacterial
strains) and 7 were yeasts (25% of 28 yeast strains)
(Table 2). All strains degraded 2.5 mM phenol within
3–7 days at 10C. Bacterial strains were identified as
Arthrobacter spp. (two strains) or Pseudomonas spp. (six
strains) (Margesin et al. 2002). Yeast strains were clas-
sified as Rhodotorula spp. (three strains), Candida sp.
Fig. 1 Growth temperature range of cold-adapted bacteria (one strain) or Cryptococcus spp. (two strains) (Table 3).
(61=100%) and yeasts (28=100%) Both Cryptococcus spp. strains were tentatively
454

Table 1 Potential of 61
bacterial (=100%) and 28 yeast Enzyme activity Bacteria Yeasts Total
(=100%) strains to produce (100%=61) (100%=28) (100%=89)
enzymes at 10C
No. % No. % No. %

Amylase 23 37.7 7 25.0 30 33.7

Protease 43 70.5 6 21.4 49 55.1
Cellulase 7 11.5 7 25.0 14 15.7
b-galactosidase 22 36.1 0 0 22 24.7
b-lactamase 31 50.8 28 100 59 66.3
Lipase 23 37.7 25 89.3 48 53.9
Pectolytic enzymes 4 6.6 6 21.4 10 11.2

Table 2 Potential of 61 bacterial (=100%) and 28 yeast (=100%) bacterial strains could grow with all hydrocarbons; all of
strains to utilize hydrocarbons for growth at 10C them were isolated from alpine glacier cryoconite and
[OD(600 nm)>0.2] did not grow at temperatures above 20C.
Hydrocarbons Bacteria Yeasts Total
(100%=61) (100%=28) (100%=89)
Biodegradation of phenol
No. % No. % No. %

n-dodecane 0 0 7 25.0 7 7.9

Fed-batch cultivation
n-hexadecane 4 6.6 22 78.6 26 29.2
Phenol 8 13.1 7 25.0 15 16.9 The 15 strains that utilized phenol for growth were se-
Phenanthrene 8 13.1 6 21.4 14 15.7 lected to study the effect of the phenol concentration on
Anthracene 8 13.1 9 32.1 17 19.1 biodegradation using fed-batch cultivation, whereby the
same culture was re-contaminated with increasing phe-
nol concentrations as soon as the phenol from the pre-
vious amendment had disappeared. The same pattern
identified as Cryptococcus aerius Nannizzi within the was observed with all strains: growth increased as long
Cryptococcus albidus group. One yeast strain could not as phenol was degraded but decreased rapidly when the
be classified as belonging to any of the described yeast phenol concentration became toxic. With increasing
species; however, the strain could be identified as a phenol concentration, the lag-phases increased due to
heterobasidiomycete. substrate inhibition by the toxic substrate phenol. Figure
2 shows the changes in OD and the remaining phenol
PAHs (phenanthrene, anthracene) concentration during the cultivation of two yeast strains
that tolerated high phenol concentrations. The drop in
The two tested three-ring aromatics were utilized to a OD at the time of phenol amendments was caused by the
similar extent. After 14 days at 10C, 14 and 17 of the addition of fresh medium (volume adjustment). Since
tested 89 strains utilized phenanthrene or anthracene, there was no abiotic loss of phenol in sterile controls,
respectively, for growth [OD(600 nm)>0.2]. Among phenol disappearance had to be attributed to biodegra-
these strains were 13% of the 61 bacterial strains, and 21 dation.
or 32% (phenanthrene or anthracene) of the 28 yeast Three yeast strains (two Cryptococcus spp. and one
strains. All these strains originated from alpine glacier heterobasidiomycete; Table 3) degraded phenol con-
cryoconite. The best growth was observed with two centrations as high as 10 mM (one strain; Fig. 2, upper
bacterial strains showing an OD(600 nm)>0.8, which part) or 12.5 mM (two strains; Fig. 2, lower part). Up to
was higher than the OD measured in controls containing 5 mM phenol was degraded within 3 days, while 7 days
glucose as carbon source (data not shown). Both strains were needed for the degradation of 7.5 and 10 mM
were isolated from different samples of alpine-glacier phenol. The degradation of 12.5 mM phenol was com-
cryoconite and were tentatively identified as belonging plete after 18 days (Fig. 2). The rapid decrease in
to the genus Chryseomonas sp. (Margesin et al. 2002). microbial growth after the last phenol addition might be
Considering the data given here on the microbial caused by the toxic effects of high phenol concentrations
utilization of aliphatic and aromatic hydrocarbons for or by the accumulation of toxic intermediates.
growth, the following pattern was obtained. The ability Four yeast strains from alpine glacier cryoconite
to utilize at least one of the tested hydrocarbons (Table 3) degraded up to 5 mM phenol over a total
(hexadecane, phenol, phenanthrene or anthracene) for cultivation period of 24 days (7 days each with 1 and
growth [OD(600 nm)>0.2], was restricted to 41 of the 2.5 mM, and 10 days with 5 mM). However, no complete
89 strains investigated. Among these strains were 16 degradation was observed; 1.3–1.5 mM phenol remained
bacterial strains (26.2% of 61 strains) and 25 yeast after 10 days of incubation with 5 mM phenol. A
strains (89.3% of 28 strains). Only 4 yeast strains (three brownish color was noticed in the cultures of all four
Rhodotorula spp. and one Candida sp.) but none of the strains, indicating the presence of phenol oxidases.
 455

Table 3 Characterization of phenol-degrading cold-adapted yeasts and bacteria (Tmax maximum temperature at which growth in R2A medium occurred; C1,2D presence of catechol 1,2 Of the eight bacterial strains, six strains (Pseudomo-

[Phenolmax] (mM)
dioxygenase; C2,3D presence of catechol 2,3 dioxygenase;+ positive reaction; (+) weak reaction, ) negative reaction;[Phenolmax] highest phenol concentration degraded at 10C) nas spp.) could not degrade phenol concentrations
higher than 2.5 mM, while two strains (Arthrobacter
spp.) degraded 10 mM phenol within 7 days (Table 3).

12.5
12.5

2.5
2.5
2.5
2.5
2.5
2.5
10

10
10
5
5
5
5
Effect of temperature on phenol biodegradation
C2,3D

The optimum temperature for phenol degradation was

(+)

(+)
(+)
(+)
(+)
+

+
+
20C for all eight bacterial strains and for two yeast
)
)

)
)
)
)

)
strains. Five yeast strains, however, had an optimum
C1,2D

temperature of 10C, and biodegradation was faster at

(+)
1C than at 20C or 15C. In one case, biodegradation
+
+

+
+
+
+
+

+
+
+

+
+
+
+
performance at 1C and 10C was comparable (Table 3).
Biomass formation (OD) was always greater at 10C
on biomass formation (OD)

than at 20C. Three bacteria and one yeast strain

showed even higher biomass production at 1C
Effect of temperature

(Table 3).
10>1>>20

Presence of catechol dioxygenases

10>1>20
10>20=1

10>1>15
10>20=1

1>10>20
10>20>1

1>10>20
1>10>20
10>1=20
10>1=20
1>10>20
10>20>1
10>1>20
10>1>20
C1,2D was detected with all strains tested, indicating the
oxidation of catechol by the ortho type of ring cleavage.
Some strains also produced C2,3D; these strains are able
on phenol degradation

to oxidize catechol by the meta type of ring cleavage

(Table 3).
temperature (C)

10>1>>15

10>1>>20
1=10>>20

20>10>>1
20>10>1
10>1>20

10>1>20

20>10>1

20>10>1
20>10>1
20>10>1
20>10>1
20>10>1

20>10>1
20>10>1

Discussion
Effect of

A wide variety of bacteria, fungi and algae have the

ability to metabolize aliphatic and/or aromatic hydro-
carbons. Cold-adapted hydrocarbon-degrading micro-
growth (C)

organisms are predominantly bacteria (Chablain et al.

Tmax for

1997; Aislabie et al. 1998, 2000; Whyte et al. 1998;

Yakimov et al. 1999; Bej et al. 2000). We observed a
20
20

15
20
20
20
20

25
25
25
30
25
25
25
25

significantly lower percentage of culturable n-dodecane

utilizers compared to n-hexadecane utilizers, which we
Heterobasidiomycete

attribute to the higher abiotic loss and probably also to

Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Pseudomonas sp.
Cryptococcus sp.
Cryptococcus sp.

Arthrobacter sp.
Arthrobacter sp.

the higher toxicity of n-dodecane. Olivera et al. (1997)

Rhodotorula sp.
Rhodotorula sp.

Rhodotorula sp.
Identification

attributed the low dodecane biodegradation to the rapid

Candida sp.

evaporation of this compound. In our study, we found a

higher percentage of culturable yeast than of bacterial
strains able to utilize n-hexadecane, phenol or two three-
ring aromatics (phenanthrene and anthracene) for
growth at 10C (Table 2). The ability to utilize n-hex-
(A)
(A)
(A)
(A)

(A)
(A)
(A)
(A)
(A)
(A)

adecane was especially widespread among the investi-

Alpine glacier cryoconite
Alpine glacier cryoconite
Alpine glacier cryoconite
Alpine glacier cryoconite

glacier cryoconite
glacier cryoconite
glacier cryoconite
glacier cryoconite
glacier cryoconite
glacier cryoconite

gated yeast strains. Interestingly, only four yeast strains

(mud in the thawing

but none of the bacterial strains could grow with both

Alpine ice cave (A)

Alpine ice cave (A)

ice cave (A)

ice cave (A)
Alpine glacier foot

aliphatic and aromatic hydrocarbons. Thus, cold-

adapted yeasts appear to have a versatility to utilize
Isolated from

hydrocarbons. In comparison, many of the 200 investi-

zone; F)

gated bacterial strains mineralized aliphatic or aromatic

Alpine
Alpine
Alpine
Alpine
Alpine
Alpine
Alpine
Alpine

hydrocarbons but not both (Foght et al. 1990). About

one-third of hydrocarbon-degrading bacteria had both
catabolic pathways for aliphatic and aromatic hydro-
Bacteria

carbons degradation (Sotsky and Atlas 1994).

AG 15
AG 21

AG 17

AG 30
AG 31
Yeasts
Strain

A 10
A 11
A 19
A 43

C 16
C 31
C 32
C 34
B 48

Special attention was payed in this study to the deg-

B41

radation of phenol. Phenolic compounds are common

456

to degrade 16 mM phenol (Babu et al. 1995) and

10.6 mM within 5 days (Hinteregger et al. 1992). In our
study, 10 mM phenol was utilized both by bacteria and
yeasts within 7 days at 10C, while 14–18 days were
needed for the degradation of 12.5 mM phenol.
Interestingly, the strains investigated in this study
were isolated from cold environments that were not
hydrocarbon-contaminated, which indicates the ubiq-
uity of hydrocarbon-degrading bacteria and yeasts.
Microorganisms able to efficiently degrade oil hydro-
carbons (Margesin and Schinner 1998) and phenol
(Bastos et al. 2000b) have been isolated from uncon-
taminated environments. However, Aislabie et al. (2001)
detected culturable yeasts only in oil-contaminated
antarctic soils but occasionally in pristine control soils.
These authors attributed the significant enhancement in
numbers of culturable yeasts and filamentous fungi in
oil-contaminated cold soils to the important role of
fungi in the degradation of hydrocarbons or their
metabolites.
Screenings for cold-active enzyme producers have
been mainly focused on bacteria, while yeasts have
been rarely considered (Birgisson et al. 2003). Our data
show that cold-adapted yeast strains are potentially
useful as lipase producers. Lipases from yeasts are
gaining industrial interest with applications in laundry
Fig. 2 Residual phenol concentration (n) and growth (optical
detergent and dairy industries (Burden and Eveleight
density, h) as a function of incubation time at 10C in the fed- 1990). The use of cold-active enzymes in detergents
batch cultures of two psychrophilic yeast strains (upper part: allows colder washing cycles and thus energy-saving by
heterobasidiomycete strain AG 17; lower part: Cryptococcus sp. lowering the temperature without a loss of enzyme
strain AG 21). Phenol amendments are represented by arrows activity.
Many of the yeast strains (60%) investigated in this
constituents of wastewaters from the oil industry. Due to study were true psychrophiles, showing a maximum
their toxicity to microorganisms, phenolic compounds growth temperature of 20C, while the majority of the
can cause the breakdown of wastewater-treatment plants bacteria were cold-tolerant (92%) and could grow at
by inhibition of microbial growth, even at relatively low temperatures above 20C (Fig. 1). Accordingly, the
concentrations such as >2 mM (Li and Humphrey optimum temperature for phenol degradation was gen-
1989). In our study, phenol concentrations as high as erally higher for bacteria (20C) than for yeast strains
12.5 mM could be degraded at 10C by two yeast strains (10C). Five yeast strains degraded phenol even faster at
using fed-batch cultivation. This cultivation method has 1C than at 20C. Psychrophiles are found in perma-
been proven to be efficient for the selection and accli- nently cold habitats such as ice caves, cryoconite on
mation of a phenol-degrading bacterial consortium melting glacier ice (Gounot 1999) and melting sea ice
(Guieysse et al. 2001). There is little information about (Bowman et al. 1997). However, even in permanently
cold-adapted phenol degraders. Kotturi et al. (1991) cold environments, at least 50% of the bacteria (Delille
demonstrated low temperature degradation of 10.6 mM and Perret 1989) are not psychrophilic. Significantly
phenol (1 g l)1) by a cold-tolerant Pseudomonas putida. lower numbers of bacteria that could grow at 2C than
In our study, bacterial strains (Arthrobacter spp.) de- at 20C were found in alpine glacier cryoconite, while
graded up to 10 mM, while yeast strains (Cryptococcus the opposite pattern was observed for yeasts (Margesin
spp.) tolerated and degraded up to 12.5 mM phenol at et al. 2002). Due to this apparently restricted growth
10C. Mesophilic degraders of high phenol concentra- temperature range and the low optimum temperature for
tions include bacterial and yeast strains. Among meso- efficient biodegradation, psychrophilic yeasts may offer
philic yeasts, strains of Candida tropicalis degraded a great potential for bioremediation processes in per-
16 mM phenol within 6 days at 29C (Bastos et al. manently cold climatic regions or environments where
2000a) and even 27 mM phenol at 30C (Krug et al. temperatures of 20C are not exceeded, such as
1985), Trichosporon sp. degraded 18 mM phenol during groundwater and subsoils. They could be useful for the
5 days at 30C (Santos and Linardi 2001). An inhibitory construction of biosensors for the selective, sensitive and
effect on yeasts was observed at concentrations higher rapid monitoring, or in-situ analysis of pollution, since
than 11 mM phenol (Santos and Linardi 2001). Meso- the broad measuring ranges and long-term viabilities of
philic bacteria of the genus Pseudomonas were reported many yeast strains make them good candidates for use
 457

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