Indian Journal of Biotechnology

Vol 14, January 2015, pp 81-86

Simultaneous production of ligninolytic enzymes by a temperature and

pH tolerant strain of Aspergillus niger under different
cultural conditions
Kusum Dhakar1, Rinu Kooliyottil1, Archana Joshi2 and Anita Pandey1*
1
Biotechnological Applications, G B Pant Institute of Himalayan Environment and Development, Kosi-Katarmal
Almora 263 643, India
2
Mody Institute of Technology and Science, Lakshmangarh, Sikar 332 311, India

Received 14 March 2013; revised 27 September 2013; accepted 3 November 2013

Ligninolytic activity, represented by laccase, lignin peroxidase (LiP) and manganese peroxidase (MnP), of a temperature
and pH tolerant strain of Aspergillus niger isolated from a temperate location in the Indian Himalayan Region (IHR), has
been studied under different cultural (physico-chemical & nutritional) conditions. In plate assays, the fungus exhibited
ligninolytic activity at wide range of temperature (5-45°C) and pH (3.5-9.5). In quantitative estimations, carried out at 15, 25
and 35°C, production of laccase was favoured by low temperature (15°), while production of LiP and MnP were favoured by
higher temperatures (25 & 35°C). At optimum growth temperature (25°C), laccase production was the maximum at 7.5 pH.
LiP and MnP production was favoured between 7.5 to 9.5, and 5.5 to 9.5 pH, respectively. Amongst nutritional sources,
nitrogen sources were recorded as better enhancers for enzyme production, followed by vitamins and carbon sources. Folic
acid (0.01%) was also found to be a good enhancer for production of all the three enzymes.

Keywords Aspergillius niger, ligninolytic, Indian Himalayan Region (IHR), temperature/pH tolerant

Introduction the complex and the recalcitrant polymer lignin.

Decomposition is broadly defined as the physical,
chemical and biological process that transforms
complex organic materials into increasingly simpler
forms. In forest ecosystems, decomposition of leaf
litter is an important factor controlling nutrient
cycling and formation of soil. In high altitudes of
mountain ecosystems, the degradation process is slow
and takes longer period due to the prevalence of low
temperature. Thus, occurrence of temperature tolerant
microorganisms, psychrotolerants in particular, in
such environments plays important role toward
sustainability of soil health1-3. Fungi, mainly
basidiomycetes, are well known for their contribution
in litter decomposition and nutrient release. However,
dominance and contribution of ascomycetous fungi
has been recognized for various biological processes
under low temperature environments in recent
literature4-6.
Ligninolytic enzymes, a group of highly versatile
enzymes, are known for their role in degradation of
——————
*Author for correspondence
Tel: +91-5962-241041; Fax: +91-5962-241150
anita@gbpihed.nic.in
Ligninolytic activity is associated with three major
enzymes, viz., lignin peroxidase (LiP), manganese
peroxidase (MnP) and laccase. Peroxidases possess
heme structure and require H2O2 for catalysis, while
LiP has the ability to oxidize non-phenolic structures
related to lignin. Moreover, MnP facilitates the
decomposition by generating phenoxy radicals
from phenolic rings. Laccases are multicopper
oxido-reductase glycoproteins with the ability to
oxidize phenolics as well as the non-phenolic
compounds in presence of the mediators, such
as, ABTS (2,2'-azino-bis 3-ethylbenzothiazoline-6-
sulphonic acid) and hydroxybenzotriazole (HBT)7.
Besides ecological importance, these enzymes are
also known for their biotechnological applications,
such as, in food, pharmaceuticals, textiles and
synthetic chemistry8. Such applications have led to the
increasing demand of these enzymes at industrial
scale. The production of these enzymes can be greatly
affected by modifying physico-chemical and
nutritional conditions9.
The aim of the present study was to determine the
ligninolytic activity (due to laccase, LiP & MnP) of a
temperature tolerant fungus, initially isolated from the
82 INDIAN J BIOTECHNOL, JANUARY 2015
soil sample collected from a temperate location in
Indian Himalayan Region (IHR), under different
cultural conditions.
Materials and Methods
Fungal Culture
Fungal culture was taken from the microbial
culture collection that has been developed in the
Microbiology Laboratory of G B Pant Institute of
Himalayan Environment and Development, Almora
(Uttarakhand), India. The fungus was originally
isolated from the soil sample collected from a
temperate location in IHR. The soil pH at sampling
site ranged from 4.5 to 6.5. The location experiences
heavy rainfall and snowfall as well, maintaining low
temperature up to subzero levels. The fungal culture
was maintained on potato dextrose agar (PDA) slants
at 4°C, following sub-culture at prescribed intervals.
The fungal isolate (Aspergillus niger) under study has
been accessioned by Indian Type Culture Collection,
Indian Agricultural Research Institute, New Delhi,
India (Acc. No. ITCC2546). The description of the
site and properties of the fungal isolate have been
reported earlier6.
Qualitative and Quantitative Estimation of Ligninolytic
Activity
Modified Kirk and Farrell medium10 was used for
the production of lignin degrading enzymes. The
medium contained (g/L): malt extract 2.0, glucose
2.0, NH4NO3 2.0, Na2HPO4 0.26, KH2PO4 0.26,
MgSO4.7H2O 0.5, CuSO4.5H2O 0.01, CaCl2.2H2O
0.006, FeSO4.7H2O 0.005, ZnSO4.7H2O 0.0005,
Na2MoO4 0.00002, MnSO4.H2O 0.00009 and H3BO3
0.00007. Plate assay was done by supplementing
0.35 g/L ABTS. The inoculated plates were observed
for development of green colour zone around the
colony, following 7 d of incubation.
For quantitative estimation, 50 mL medium
(pH=5.0±0.5) was prepared in 250 mL Erlenmeyer
flasks and 5 mm disc of 7-d-old fungal culture grown
on PDA was used for inoculation. Laccase activity
was determined by using ABTS (ε420=36000 M-1cm-1)11
at 420 nm in citrate-phosphate buffer (pH=3.0).
LiP activity was assayed by veratryl alcohol
(ε310=9300 M-1 cm-1)12. MnP activity was determined
by reading the formation of stable tartrate
complex through the oxidation of Mn+2 to Mn+3
(ε238= 6500 M-1 cm-1)12 in tartrate buffer (pH=4.5).
Enzyme activity was defined as 1 µM of substrate
oxidized per minute under the given conditions.
Effect of Physico-chemical Conditions on Production of
Ligninolytic Enzymes
The production of ligninolytic enzymes was
investigated at 3 temperatures (15, 25 & 35°C). At
15°C, the readings were recorded at weekly intervals
up to 5 wk. At 25 and 35°C, the readings were
recorded at every 3rd d up to 15 d. For determination
of effect of pH on production of ligninolytic enzymes,
the experiments were conducted at 25°C in 4 sets,
maintaining pH of the media at 3.5, 5.5, 7.5 and 9.5.
Enzyme activity was estimated at 12th d of incubation,
in each case.
Effect of Nutritional Supplements on Production of
Ligninolytic Enzymes
Effect of carbon sources, replacing glucose by
arabinose, fructose, galactose, lactose, maltose,
trehalose, pectin, starch, cellulose and chitin; nitrogen
sources, replacing ammonium nitrate by ammonium
sulfate, ammonium acetate, ammonium ferrous
sulfate, potassium nitrate, urea, casein, yeast extract,
peptone and allylthiourea (0.2% each, separately); and
five vitamin supplements [pyridoxine, B1, biotin,
nicotinic acid & folic acid (0.01% each, separately)]
were studied on the production of lignin degrading
enzymes. Vitamins were added in the medium at 5th d
of incubation. The readings were taken following
12 d incubation at 25°C.
All the experiments were conducted in triplicate
and the mean values with standard deviation were
shown as the error bars.
Results and Discussion
Plate assays revealed the production of ligninolytic
enzymes at wide range of temperature (4-35°C) by
A. niger. In quantitative estimations conducted at
3 temperatures, the production of laccase was favoured
by low temperature. The maximum (9.2±1.8 U/L)
production of laccase was estimated at 15°C on 21st d
of incubation, following decline to 4.2±1.1 U/L
(35th d). At 25 and 35°C, the production of laccase
was the maximum (8.0±1.0 & 2.4±0.7 U/L,
respectively) at 12th and 6th d, respectively. Contrary
to these results, production of LiP and MnP was
favoured by higher temperatures (25 & 35°C).
The maximum LiP was produced (165.3±15.7 U/L)
at 25°C at 9th d of incubation, then declined to
91.1±10.6 U/L (15th d). A similar trend of LiP activity
was recorded at 35°C. At 15°C, the maximum LiP
activity (137.0±7.8 U/L) was recorded at 21st d,
declined to 96.6±9.5 U/L at 35th d of incubation. The
production of MnP was also favoured by higher
 DHAKAR et al: LIGNINOLYTIC ENZYMES BY TEMPERATURE AND pH TOLERANT A. NIGER 83

temperatures, being maximum (2326.9±226.7 U/L) at the minimum MnP production (1439.1±164.5 U/L)
35°C at 12th d of incubation and then declined to was recorded at pH 3.5 (Table 2). The pH of the
2022.4±217.2 U/L at 15th d. The production of MnP medium is known to alter the structure of catalytic site
was higher at 25° in comparison to 15°C. Results on of enzymes affecting the activity. In Rhizoctonia
the production of all the 3 enzymes are presented in praticola, pH 7.5 has been reported optimum for the
Table 1. production of laccase16. In general, acidic pH is
The fungus under study (A. niger) has been known to be more suitable for the production of the
reported to tolerate a wide range of temperature enzymes17. In the present study, the production of
with its optimum growth in mesophilic range6. ligninolytic enzymes has been recorded at a much
Importance of temperature in production of wider range of pH.
bioactive molecules by various fungi, initially The effect of carbon sources on production of
isolated from low temperature environments, ligninolytic enzymes is presented in Fig. 1. Among
has been reported in recent years13-14. In the present 10 carbon sources used at 0.2 % concentration,
study, the production of different ligninolytic arabinose, trehalose and cellulose were found to be
enzymes varied with respect to temperature. While the best enhancers of laccase production (10.6±1.5,
the production of laccase was favoured by 8.9±1.6 & 10.0±2.1 U/L, respectively). Other carbon
psychrophilc range, production of LiP and MnP sources, namely, fructose, galactose, lactose, maltose,
was better under mesophilic temperature range. pectin, starch and chitin resulted in inhibitory effect
Temperature for optimum production of laccase on laccase production. Fructose, maltose, trehalose,
has been reported in between 25 to 30°C15. pectin and chitin were found to be the effective
The production of the ligninolytic enzymes enhancers (100.5±7.1, 97.3±9.2, 91.2±10.6, 92.1±12.9
also varied with respect to pH, most pronounced in & 91.7±11.7 U/L, respectively) of LiP; while
case of laccase. The minimum production of laccase arabinose, galactose, lactose, starch and cellulose
(0.8±0.3 U/L) was recorded at pH 3.5, which exhibited inhibitory effects on LiP production. All the
increased with the increase of pH up to pH 7.5
(8.9±0.6 U/L) and then it declined to pH 9.5 Table 2—Effect of pH on production of ligninolytic enzymes
(4.1±0.5 U/L). The production of LiP was recorded pH Laccase LiP MnP
almost stable at pH 7.5 and 9.5, showing the activities (U/L) (U/L) (U/L)
at 211.6±14.5 and 214.9±15.2 U/L, respectively. The 3.5 0.8±0.3 110.0±28.2 1439.1±164.5
minimum production (110.0±28.2 U/L) of LiP was 5.5 7.6±1.4 149.3±13.2 1737.4±74.5
recorded at pH 3.5. The maximum production 7.5 8.9±0.7 211.6±14.5 1642.8±177.0
(1737.4±74.5U/L) of MnP was recorded at pH 5.5, 9.5 4.1±0.5 214.9±15.2 1618.3±96.3
showing stability at pH 9.5 (1618.3±96.3 U/L), while Values are mean±SD (n= 3)
Table 1—Effect of temperature on production of ligninolytic enzymes
Incubation (d) Laccase LiP MnP
(U/L) (U/L) (U/L)
A 15°C 15°C 15°C
7 0.9±0.3 86.5±17.7 1667.1±133.1
14 3.3±0.5 126.7±8.5 1782.4±105.2
21 9.2±1.8 137.0±7.8 2131.7±288.5
28 5.9±0.9 104.5±14.5 2028.6±262.4
35 4.2±1.1 96.6±9.5 1974.8±197.3
B 25°C 35°C 25°C 35°C 25°C 35°C
3 1.4±0.4 1.3±0.3 52.8±6.9 79.7±4.1 1783.9±87.5 1779.4±104.3
6 1.6±0.6 2.4±0.7 85.8±7.6 94.3±6.1 1751.9±124.0 2028.8±192.1
9 3.9±0.7 2.1±0.2 165.3±15.7 168.4±5.7 2193.4±261.4 2205.1±165.8
12 8.0±1.0 1.7±0.5 92.1±9.3 89.7±10.0 1909.6±145.5 2326.9±226.7
15 4.3±1.0 1.9±0.3 91.1±10.6 80.6±5.7 1933.3±203.1 2022.4±217.2
Values are mean ± SD (n= 3)
84 INDIAN J BIOTECHNOL, JANUARY 2015
Fig. 1 (A-C)—Effect of carbon sources on production of
ligninolytic enzymes: A, Laccase; B, LiP; & C, MnP. [G, Glucose;
F, Fructose; Gal, Galactose; Lac, Lactose; M, Maltose; Tre,
Trehalose; P, Pectin; St, Starch; Cel, Cellulose; & Chi, Chitin]
carbon sources enhanced the production of MnP;
maximum being in case of chitin (2307.9±284.1 U/L).
Addition of nitrogen sources was more effective in
enhancing the production of ligninolytic enzymes
(Fig. 2). Out of 10 sources, ammonium ferrous
sulfate, urea, yeast extract and peptone were found to
enhance the laccase production (10.3±1.8, 10.5±1.2,
9.5±1.5 & 9.9±1.6 U/L, respectively). However,
ammonium sulfate, ammonium acetate, potassium
nitrate, casein and allylthiourea exhibited inhibitory
effects on laccase production. Seven of the nitrogen
sources (ammonium acetate, ammonium ferrous
sulfate, urea, casein, yeast extract, peptone and
allylthiourea) resulted in enhancement of LiP
production, which was recorded as 109.2±13.8,
360.2±74.1, 174.3±12.1, 116.4±12.5, 94.3±14.5,
191.5±27.7, 170.1±12.4 and 145.2±11.8 U/L,
Fig. 2 (A-C)—Effect of nitrogen sources on produciton
of ligninolytic enzymes: A, Laccase; B, LiP; & C, MnP.
[AN, Ammonium nitrate; AS, Ammonium sulfate; AA,
Ammonium acetate; AFS, Ammonium ferrous sulfate; KN,
Potassium nitrate; U, Urea; Cs, Casein; Ye, Yeast extract; Pep,
Peptone; & ATU, Allythiourea]
respectively. On the contrary, ammonium sulfate
and potassium nitrate were found to be inhibitory for
LiP production. In case of MnP, allylthiourea was
found to be the best enhancer that resulted in approx
3-fold enhancement (6499.9±701.8 U/L). Ammonium
ferrous sulfate, casein, yeast extract, peptone and
allylthiourea were the other enhancers (5307.6±780.2,
2576.9±378.8, 2532.1±640.2, 2436.14±390.3 &
6499.6±701.8 U/L, respectively) of MnP. However,
MnP production was inhibited by 4 nitrogen sources,
namely, ammonium sulfate, ammonium acetate,
potassium nitrate and urea. Addition of different
carbon and nitrogen sources for regulating the
production of ligninolytic enzymes has been well
reported18-20. In the present study, among various
nitrogen sources, organic nitrogen sources, in
 DHAKAR et al: LIGNINOLYTIC ENZYMES BY TEMPERATURE AND pH TOLERANT A. NIGER
Fig. 3 (A-C)—Effect of vitamins on production of ligninolytic
enzymes: A, Laccase; B, LiP; & C, MnP. [C, Control; P,
Pyridoxine; B1, Thiamine; B, Biotin; N, Nicotinic acid; & F,
Folic acid]
particular, was found to be more effective in
comparison to the addition of carbon sources.
Vitamin supplements also affected production of
ligninolytic enzymes. Pyridoxine, biotin and folic acid
resulted in enhanced production (10.3±1.6, 10.1±1.5
& 11.4±1.2 U/L, respectively) of laccase. On the
contrary, addition of B1 and nicotinic acid was found
to be inhibitory for the production of laccase. While
all the vitamins showed strong positive effect on
production of LiP and MnP, maximum enhanced
production (662.3± 108.7 & 3525.6±483.9 U/L,
respectively) was recorded in case of folic acid
(Fig. 3). Effect of biotin and pyridoxine in
85
enhancement of laccase production by Cyathus
bulleri21, and effect of physico-chemical and
nutritional parameters as well as other supplements on
production of ligninolytic enzymes from Trametes sp.
have also been reported earlier19.
Literature on ascomycetous fungi in lignin
degradation, specifically from low temperature
environments, is limited. Various species from
the ascomycetous genera, mainly Aspergillus,
Paecilomyces and Penicillium, have been of interest
because of their presence and activities in various
geographical locations, including low temperature
environments under mountain ecosystem4-6,22. The
psychrotolerant strain of A. niger, used in the present
study, was originally isolated from the mixed forest of
Cedrus-Taxus in IHR, where litter degradation is slow
due to the prevalence of low temperature. The present
study highlights the importance of temperature and
pH in production of the ligninolytic enzymes. The
fungus showed its ability to produce ligninolytic
enzymes at a wide and different range of temperature
and pH. Because of the stability at low temperature,
laccase is likely to be more applicable for degradation
in the low temperature environments. Research on
microorganisms dominating the low temperature
environments, such as, Arctic, Antarctic, Andes and
IHR, is getting increasing attention of the scientific
community. The focus in these studies is mainly on
the phylogeny, adaptation mechanisms, biogeography,
bioremediation, biotechnological applications and
ecological resilience23-28.
Acknowledgment
Authors are thankful to the Director, G B Pant
Institute of Himalayan Environment and
Development, Almora for encouragement and
providing the facilities. The Department of Science
and Technology, and the Union Ministry of
Environment, Forests and Climate Change,
Government of India, New Delhi, are acknowledged
for financial support. Senior author (DK) is also
thankful to Indian Council of Medical Research, New
Delhi for the award of fellowship.
References
1 Sharma G, Pandey R R & Singh M S, Microfungi associated
with surface soil and decaying leaf litter of Quercus serrata
in a subtropical natural oak forest and managed plantation in
Northeastern India, Afr J Microbiol Res, 5 (2011) 777-787.
2 Pandey A & Palni L M S, The rhizosphere effect in trees of
the Indian Central Himalaya with special reference to
altitude, Appl Ecol Environ Res, 5 (2007) 93-102.
86 INDIAN J BIOTECHNOL, JANUARY 2015
3 Kaira G S, Dhakar K & Pandey A, A psychrotolerant strain
of Serratia marcescens (MTCC 4822) produces laccase at
wide temperature and pH range, AMB Express 5 (2015) 1.
[DOI: 10.1186/s13568-014-0092-1]
4 Dhakar K, Sharma A & Pandey A, Cold, pH and salt tolerant
Penicillium spp. inhabit the high altitude soils in Himalaya,
India, World J Microbiol Biotechnol, 30 (2014) 1315-1324.
5 Kellner H, Zak D R & Vandenbol M, Fungi unearthed:
Transcripts encoding lignocellulolytic and chitinolytic enzymes
in forest soil, PLoS One, 5 (2010) e10971. [DOI: 10.1371/
annotation/84b7b537-84f6-49e6-ac7c-9a2f0ad3f862]
6 Rinu K & Pandey A, Temperature-dependent phosphate
solubilization by cold- and pH-tolerant species of Aspergillus
isolated from Himalayan soil, Mycoscience, 51 (2010)
263-271.
7 Hatakka A, Biodegradation of lignin, in Biopolymers: Lignin,
humic substances and coal, vol 1, edited by M Hofrichter &
A Steinbuchel (Wiley-VCH, Weinheim) 2001, 129-180.
8 Maciel M J M, Silva A C e & Ribeiro H C T, Industrial and
biotechnological applications of ligninolytic enzymes of the
basidiomycota: A review, Electron J Biotechnol, 13 (2010)
14-15. [DOI: 10.2225/vol13-issue6-fulltext-2]
9 Kanwal H K & Reddy M S, Effect of carbon, nitrogen
sources and inducers on ligninolytic enzyme production by
Morchella crassipes, World J Microbiol Biotechnol, 27
(2011) 687-691.
10 Kirk T K & Farrell R L, Enzymatic “combustion”:
The microbial degradation of lignin, Annu Rev Microbiol,
41 (1987) 465-505.
11 Han M J, Choi H T & Song H G, Purification and
characterization of laccase from the white rot fungus
Trametes versicolor, J Microbiol, 43 (2005) 555-560.
12 Khiyami M A, Pometto A L & Kennedy W J, Ligninolytic
enzyme production by Phanerochaete chrysosporium in
plastic composite support biofilm stirred tank bioreactors,
J Agric Food Chem, 54 (2006) 1693-1698.
13 Ghildiyal A & Pandey A, Isolation of cold tolerant antifungal
strains of Trichoderma sp. from glacial sites of Himalayan
region, Res J Microbiol, 3 (2008) 559-564.
14 Rinu K & Pandey A, Slow and steady phosphate
solubilization by a psychrotolerant strain of Paecilomyces
hepiali (MTCC 9621), World J Microbiol Biotechnol,
27 (2011) 1055-62.
15 Zadrazil F, Gonser A & Lang E, Influence of incubation
temperature on the secretion of extracellular ligninolytic
enzymes of Pleurotus and Dichomitus squalus into soil, in
Proc Conf on Enzymes in the Environment (Granada, Spain),
1999.
16 Janusz G, Roglaski J, Barwinska M & Szczodrak J, Effects
of culture conditions on production of extracellular laccase
by Rhizoctonia praticola, Pol J Microbiol, 55 (2006)
309-319.
17 Elsayed M A, Hassan M M, Elshafei A M, Haroun B M &
Othman A M, Optimization of cultural and nutritional
parameters for the production of laccase by Pleurotus
ostreatus ARC280, Br Biotechnol J, 2 (2012) 115-132.
18 Mikiashvili N, Wasser S P, Nevo E & Elisashvili V, Effects
of carbon and nitrogen sources on Pleurotus ostreatus
ligninolytic enzyme activity, World J Microbiol Biotechnol,
22 (2006) 999-1002.
19 Levin L, Melignani E & Ramos A M, Effect of nitrogen
sources and vitamins on ligninolytic enzyme production by
some white-rot fungi. Dye decolorization by selected culture
filtrates, Bioresour Technol, 101 (2010) 4554-4563.
20 Dhakar K & Pandey A, Laccase production from a
temperature and pH tolerant fungal strain of Trametes hirsuta
(MTCC 11397), Enzyme Res, 2013 (2013) 869062. [DOI:
10.1155/2013/869062]
21 Dhawan S & Kuhad R C, Effect of amino acids and vitamins
on laccase production by the bird’s nest fungus Cyathus
bulleri, Bioresour Technol, 84 (2002) 35-38.
22 Rinu K, Pandey A & Palni L M S, Utilization of
psychrotolerant phosphate solubilizing fungi under low
temperature conditions of the mountain ecosystem, in
Microorganisms in sustainable agriculture and
biotechnology, edited by T Satyanarayana, B N Johri &
A Prakash (Springer Science+Buisiness Media, The
Netherlands) 2011, 77-90.
23 Dhakar K, Jain R, Tamta S & Pandey A, Prolonged laccase
production by a cold and pH tolerant strain of Penicillium
pinophilum (MCC 1049) isolated from a low temperature
environment, Enzyme Res, 2014 (2014) 120708.
24 Yarzábal L A, Cold-tolerant phosphate-solubilizing
microorganisms and agriculture development in mountainous
regions of the World, in Phosphate solubilizing
microorganisms, edited by M S Khan, A Zaidi & J Musarrat
(Springer International Publishing, Switzerland) 2014,
113-136. [DOI 10.1007/978-3-319-08216-5_5]
25 Margesin R & Schinner F, Biodegradation and
bioremediation of hydrocarbons in extreme environments,
Appl Microbiol Biotechnol, 56 (2001) 650-663.
26 Pandey A, Palni L M S & Hebbar K P, Suppression of
damping-off in maize seedlings by Pseudomonas corrugate,
Microbiol Res, 156 (2001) 191-194.
27 Trivedi P, Kumar B, Pandey A & Palni L M S, Growth
promotion of rice by phosphate solubilizing bioinoculants in
a Himalayan location, in Plant and Soil: Developments in
Plant and Soil Sciences, First International Meeting on
Microbial Phosphate Solubilization, vol 102, edited by E
Velazqez & C Rodriguez-Barrueco (Springer, The
Netherlands) 2007, 291-299.
28 Margesin R & Miteva V, Diversity and ecology of
psychrophilic microorganisms, Res Microbiol, 162 (2011)
346-361.