ORIGINAL ARTICLE

Lipolytic activity of Antarctic cold-adapted marine bacteria(Terra Nova Bay, Ross Sea)A. Lo Giudice1, L. Michaud1, D. de Pascale2, M. De Domenico1, G. di Prisco2, R. Fani3 and V. Bruni1

1 Dipartimento di Biologia Animale ed Ecologia Marina, Universita di Messina, Messina, Italy

2 Istituto di Biochimica delle Proteine, CNR, Napoli, Italy

3 Dipartimento di Biologia Animale e Genetica, Universita di Florence, Firenze, Italy

Introduction

A great portion of the marine biosphere is characterized

by permanent low temperatures. Despite their inhospit-

able character, cold environments are successfully colon-

ized by numerous organisms which have to cope with

extreme conditions. The cold-adaptation of both psychro-

philic and psychrotrophic micro-organisms (Morita 1975)

is partly due to their ability to produce cold-active

enzymes exhibiting higher catalytic activity at low tem-

perature than their mesophilic and thermophilic counter-

parts (Gerday et al. 2000). The considerable potential of

cold enzymes for biotechnological exploitations at low

temperature has been recognized. They could be utilized

as additives in the detergent and food industries, as bio-

catalysts or in bioremediation processes, minimizing

energy consumption, reducing the risk of microbial con-

tamination and avoiding the temperature instability of

reactants or products (Margesin and Schinner 1994). Lip-

ases and esterases are among biotechnologically relevant

enzymes with wide-ranging versatility in industrial appli-

cations, especially in the cosmetic and food fields (Pandey

et al. 1999). They could be distinguished on the basis of

their substrate specificity. Lipases are, by definition, carb-

oxylesterases displaying maximal activity towards water-

insoluble long-chain acylglycerols (‡C10), while esterases

are able to hydrolyze ester substrates with short-chain

fatty acids (£C10) at least partly soluble in water.

Keywords

Antarctica, bacterial characterization, cold-

adapted bacteria, environmental factors,

lipolytic activity.

Correspondence

Vivia Bruni, Dipartimento di Biologia Animale

ed Ecologia Marina, University of Messina,

Salita Sperone 31, 98166 Messina, Italy.

E-mail: vivia.bruni@unime.it

2005/1072: received 19 September 2005,

revised 23 January 2006 and accepted 3

March 2006

doi:10.1111/j.1365-2672.2006.03006.x

Abstract

Aims: The aim of this study was to investigate the lipolytic activity of cold-

adapted Antarctic marine bacteria and, furthermore, the combined effect of

some environmental factors on this enzymatic process.

Methods and Results: Strains were assayed for lipolytic activity on a basal med-

ium amended with seven individual fatty acid esters. A significant activity was

observed for 148 isolates (95Æ5% of the total screened). The interactive effect of

pH, temperature and NaCl concentration on the substrates was tested for six

representative isolates, identified as Pseudoalteromonas, Psychrobacter and Vib-

rio. Differences between strains according to NaCl and pH tolerances were

observed. Only one strain degraded the substrate more efficiently at 4�C than

at 15�C.

Conclusions: Our findings demonstrate that the lipolytic activity of Antarctic

marine bacteria is rather variable, depending on culture conditions, and occurs

in a wide range of salt concentration and pH.

Significance and Impact of the Study: Isolation and characterization of bacteria

that are able to efficiently remove lipids at low temperatures will provide

insight into the possibility to use cold-adapted bacteria as a source of exploita-

ble enzymes. Moreover, research on the interactive effects of salt concentration,

pH and temperature will be useful to understand the true enzyme potentialities

for industrial applications.

Journal of Applied Microbiology ISSN 1364-5072

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048 1039

However, lipases are perfectly able to hydrolyze esterase

substrates (Jaeger et al. 1999).

Lipolytic enzymes have been also proposed as inter-

esting alternative to the commercially available micro-

bial agents of mesophilic origin for the removal of fats

from aqueous systems (Suzuki et al. 2001). Fats and

oils present in wastewaters from food-processing plants

and restaurants, for example, are difficult to degrade by

ordinary micro-organisms. As reported by Yumoto et al.

(2003), grease-trap temperatures for the treatment of

lipid-containing wastewaters are relatively low (approx.

5–25�C). Thus, the isolation and characterization of

lipolytic-enzyme producers that are able to efficiently

remove lipids at low temperatures will provide insight

into the possibility to use different cold-adapted bac-

teria as a source of extremophilic enzymes. Antarctica

is a promising environment as a source of psychro-

trophic and psychrophilic lipolytic bacteria. Members of

several bacterial genera have been reported as producers

of diverse typologies of cold-active lipolytic enzymes

and characterized to date (Suzuki et al. 2001; Martın

et al. 2003; Yumoto et al. 2003; Zeng et al. 2004); some

of these enzymes have been isolated from Antarctic

micro-organisms (Feller et al. 1994; Kulakova et al.

2004).

The activity of the enzymes suitable for industrial

applications cannot often be optimized by adjusting

physical and chemical conditions under which industrial

processes are performed (Sanchez-Porro et al. 2003).

Thus, the availability of enzymes showing optimal activit-

ies at different values of salt concentration, pH and tem-

perature becomes of great importance, and research on

the interactive effects of these environmental factors will

be useful to understand the true enzyme potentialities

(Gobbetti et al. 1999).

The present paper reports on the lipolytic activity of

Antarctic marine bacteria. The most promising isolates

were utilized for studying in vivo the combined effect of

three abiotic factors (pH, temperature and NaCl concen-

tration) on this enzymatic process, in order to establish

the range of pH and NaCl for the expression of lipolytic

activity, as well as the optimal conditions for the produc-

tion of lipolytic enzymes to be chemically and structurally

analyzed in the near future.

Materials and methods

Bacterial strains

Bacterial strains were isolated from Antarctic samples of

different origin collected in Terra Nova Bay (Ross Sea,

Antarctica) during two oceanographic campaigns. A total

of 112 strains were isolated from seawater samples collec-

ted along the water column at the fixed station Tiburtina

(Stn. TIB; coordinates: 74�42¢3¢¢S–164�10¢0Æ5¢¢E) during

the 1994–95 season (Bruni et al. 1999); 43 additional

strains were isolates from samples of sea-surface micro-

layer (20 lm), sediment (by using sedimentation traps),

mollusk faecal pellets and intestinal content of two

Antarctic fish (Trematomus bernacchii and Cygnodraco

mawsoni) during the 1997–98 season.

All the isolates were from the bacterial collection of the

National Antarctic Museum kept at the University of

Messina. They are maintained on Marine Agar (MA,

Difco) slopes at 4�C and they are routinely streaked on

agar plates from tubes every 6 months to control purity

and viability. Antarctic strains are also preserved by freez-

ing cell suspensions at )80�C in Marine Broth (MB; Dif-

co) to which 20% (v/v) glycerol is added.

All media used in this study were sterilized by autocla-

ving at 121�C for 15 min, plates were incubated at 15�C

for 14 days, and the analyses were performed at least

twice, unless otherwise stated.

Lipolytic activity assay

Lipolytic activity was detected by screening for zone of

hydrolysis around colonies growing on a solid basal med-

ium containing (w/v): 1% Bacto-peptone, 0Æ01% CaCl22H2O and 2% Bacto-agar. The medium was prepared in

a mixture of 75% (v/v) natural seawater and 25% (v/v)

distilled water (pH 7Æ4) (Sierra 1957). The medium was

amended with seven individual fatty acid esters: Tween 20

(1%, v/v), Tween 40 (1%, v/v), Tween 60 (1%, v/v),

Tween 80 (1%, v/v), Tween 85 (0Æ2%, v/v), tributyrin

(0Æ2%, v/v), and triolein (0Æ2%, v/v) (Bruni et al. 1982).

The inoculation of the medium was performed by stab-

bing and plates were incubated at 4, 15 and 30�C for

21 days. Rhodamine B (0Æ001%, w/v) was added to the

medium containing triolein and production of lipase was

monitored under UV light (350 nm). Appearance of clou-

diness or clearness around the colonies was the principal

indicator for the production of lipolytic enzymes. Total

halo diameter, minus the diameter of the colony, was

considered to be proportional to the lipolytic activity. All

assays were performed in duplicate.

Influence of abiotic factors on lipolysis

The combined influence of pH, temperature and NaCl

concentration on lipolysis was analyzed on agar plates for

six isolates less active at high temperature. Each strain

was used to investigate the effect of abiotic factors on the

hydrolysis of the substrate that it better utilized during

the preliminary screening. The assays were performed by

inoculating a modified basal medium containing distilled

Lipolysis of Antarctic bacteria A. Lo Giudice et al.

1040 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048

ª 2006 The Authors

water instead of seawater. The pH range for hydrolysis

was assayed by adjusting the pH of the medium to 5, 6,

7Æ4, 8 and 9. The influence of NaCl was tested in the

range 0–11% (w/v). The experiments were performed as

previously indicated for the preliminary screening. Plates

were incubated at 4 and 15�C for 21 days. A total of 70

combinations were set up for each isolate. Two replicates

of each combination were used.

Cell extract preparation and enzyme assays

Bacterial strains (41, 63, 69, 124, 25/3 and 50/3) were

grown on MB at 15�C in horizontally shaken cultures, in

a dry-air incubator for 14 days. Cells were grown up to

early stationary phase, harvested by centrifugation at

4000 g for 10 min and stored at )70�C. Cell extracts were

prepared as described by Manco et al. (1994). Lipase and

esterase activity in cell extracts were measured spectro-

photometrically using p-nitrophenyl esters as substrates.

All procedures were performed at 10�C. The activity was

assayed by measuring the absorbance of released p-nitro-

phenol at 405 nm in 1-cm pathlength cells using a

single-beam HP spectrophotometer. The standard assay

contained 40 mmol l)1 Na-phosphate buffer pH

7Æ1, 0Æ36% Triton X-100, 2%, propan-2-ol, 0Æ2 mmol l)1

p-nitrophenyl-ester. Stock solutions of p-nitrophenyl-

esters were prepared by dissolving substrates in propan-2-

ol, ethanol or acetonitrile. Background substrate hydroly-

sis was deducted by using a reference sample of identical

composition to the incubation mixture, without homo-

genate. The pH was adjusted for the appropriate working

temperature. Lipase and esterase activity in cell-free

culture supernatants was analyzed towards only one

substrate, p-nitrophenyl-caproate, in the same condition

described above.

PCR amplification and analysis of 16S rDNA

PCR-amplification, sequencing and phylogenetic analysis

of 16S rDNA from bacterial isolates were carried out as

previously described by Michaud et al. (2004).

Morphological, biochemical and physiological

characterization

Isolates were characterized by means of a combination of

phenotypic tests as previously described in Bruni et al.

(1999). Colony morphology and pigmentation were

recorded from growth on MA at 4�C. The growth of iso-

lated bacteria at different temperatures was tested in MB

incubated at 4, 20, 30 and 37�C for up to 4 weeks. The

pH range for growth was determined in MB with pH val-

ues of separate batches of medium adjusted to 4, 5, 6, 7,

8 and 9 by the addition of HCl and NaOH (0Æ01 g l)1,

0Æ1 g l)1 and 1 g l)1 solutions). Salt tolerance tests were

performed on nutrient agar (NA) with NaCl concentra-

tions ranging from 0% to 17% (w/v). Isolates were tested

for the ability to grow on various solid media such as NA

(Oxoid), NA + 2Æ5% (w/v) NaCl, trypticase soy agar

(TSA; Oxoid), TSA + 2Æ5% (w/v) NaCl and TCBS agar

(Difco). Agarolytic activity was tested on the medium of

Vera et al. (1998).

Statistical analysis

The analysis of variance (one-way anova) was per-

formed on the entire data set from the initial screening

to test differences between substrates in relation to the

incubation temperature. Comparison between groups

for a significant difference of mean or rank values was

performed after normality and variance tests. Statistical

calculations were performed with sigmastat software

for Windows, version 2Æ0 (copyright 1992–1995 Jandel

Corporation). Differences were considered significant

when P < 0Æ05.

Results

Lipolytic activity assay

Each of the 155 isolates was tested for its lipolytic activity

at 4, 15 and 30�C, as described in Materials and methods.

Most of the isolates screened for lipolysis showed esterase

and lipase activities as indicated by the comparison of

degradation halos on agar plates. The degradation of trio-

lein was observed for seven strains producing on agar

plates halos ranging from 2- to 4-mm diameter and often

difficult to detect. Thus, triolein was not utilized for fur-

ther analysis.

A significant lipolytic activity was observed for 148 iso-

lates (95Æ5% of the total screened) at least at one of the

three temperatures; 93 of them (62Æ8% of the positive

strains) were able to utilize all the substrates (without

considering triolein), while 20 hydrolyzed five substrates

and 14 were active on four substrates; both three and two

substrates were utilized by 10 isolates; finally, only one

strain (isolate 22/3) was selective for tributyrin. As shown

in Table 1, all substrates were degraded by a quite similar

number of Antarctic isolates.

The diameters of halos on agar plates after substrate

hydrolysis were generally proportional to the diameter

of the colonies. The mean diameter of each halo was

calculated for each substrate at the three temperatures

utilized for the preliminary assay. Data obtained were

analyzed by using anova and revealed that lipolytic

activity generally occurred more efficiently at 30�C

A. Lo Giudice et al. Lipolysis of Antarctic bacteria

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Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048 1041

(anova, P < 0Æ05 for all substrates); no significant dif-

ferences were observed between the mean dimensions

of halos at 4 and 15�C (anova, P > 0Æ05). The halos

were generally larger using Tween 20 and Tween 40

than on the other substrates.

Influence of abiotic factors on lipolysis

Six representative isolates (namely 41, 63, 69, 124, 25/3

and 50/3) more active at low temperature (4 and 15�C),

as indicated by the degradation halos, were selected and

further characterized. Strains 41, 63, 69 and 124 were iso-

lated from the Stn. TIB at 100-, 150-, 25- and 0-m depth,

respectively. Strains 25/3 and 50/3 were isolated from sea-

surface microlayer and intestinal content of T. bernacchii,

respectively. Except for the strain 50/3, which utilized

only Tween 20 and Tween 80, the other five strains (41,

63, 69, 124 and 25/3) were able to hydrolyze each of the

six substrates utilized for the lipolytic assays. Measures of

the degradation halos produced by each isolate on differ-

ent substrates are reported in Table 2.

The combined influence of temperature, pH and NaCl

on lipolysis was studied for these six isolates on the sub-

strate they hydrolyzed more efficiently at low tempera-

ture, strain namely 50/3 on Tween 20, strain 124 on

Tween 40, strain 41 on Tween 60, strain 69 on Tween 80,

strain 63 on Tween 85 and strain 25/3 on tributyrin.

Lipolysis generally occurred in a wide range of pH and

NaCl concentration and was observed at pH 5 only for

strains 41 and 25/3. Four isolates (50/3, 124, 41 and 69)

degraded the substrate more efficiently at 15�C than at

4�C. Strain 25/3 produced degradation halos of compar-

able diameters at the two temperatures tested, whereas

strain 63 was more active at 4�C. The optimal conditions

Table 1 Number of isolates hydrolyzing one to six substrates at least

at one temperature

No. of isolates TW 20 TW 40 TW 60 TW 80 TW 85 TB

93 + + + + + +

8 + + + + + –

6 + + + + – +

5 + + + – + +

1 + – + + + +

1 + + + + – –

1 + + + – + –

8 + + – + – +

1 + + – – + +

1 + – – + + +

1 – – + + + +

1 – + + – + +

1 + + + – – –

3 + + – – – +

1 – + + – – +

1 – – + + – +

1 – – + – + +

1 – – – + + +

1 + – – + – +

1 + + – + – –

5 + – – + – –

3 + – – – – +

2 – – – – + +

1 – – – – – +

Tot. 148 139 130 121 128 116 131

+, substrate hydrolysis; –, no substrate hydrolysis

Table 2 Diameter of the degradation halos (mm ± SD) produced by six Antarctic isolates on medium containing tween 20, 40, 60, 80 and 85 or

tributyrin during the preliminary screening

Strain Tween 20 Tween 40 Tween 60

4�C 15�C 30�C 4�C 15�C 30�C 4�C 15�C 30�C

41 26 ± 1Æ41 27 ± 5Æ66 52 ± 7Æ07 26 ± 5Æ66 17 ± 1Æ41 42 ± 5Æ65 28 ± 4Æ24 27 ± 2Æ83 20 ± 1Æ41

63 27 ± 2Æ83 28 ± 1Æ41 38 ± 2Æ83 28 ± 2Æ83 16 ± 2Æ83 38 ± 2Æ83 13 ± 1Æ41 26 ± 1Æ41 26 ± 4Æ24

69 25 ± 4Æ24 26 ± 2Æ83 33 ± 2Æ83 21 ± 4Æ24 15 ± 4Æ24 31 ± 4Æ24 19 ± 0Æ00 13 ± 1Æ41 30 ± 2Æ83

124 10 ± 2Æ83 22 ± 5Æ66 30 ± 2Æ83 27 ± 1Æ41 23 ± 4Æ24 10 ± 2Æ83 22 ± 2Æ83 14 ± 4Æ24 30 ± 2Æ83

25/3 9 ± 2Æ83 8 ± 1Æ41 3 ± 2Æ83 8 ± 4Æ24 8 ± 1Æ41 10 ± 1Æ41 9 ± 1Æ41 9 ± 1Æ41 9 ± 0Æ00

50/3 29 ± 1Æ41 22 ± 2Æ83 ng – – ng – – ng

Tween 80 Tween 85 Tributyrin

41 26 ± 4Æ24 26 ± 4Æ24 27 ± 4Æ24 25 ± 1Æ41 26 ± 0Æ00 39 ± 2Æ83 w 9 ± 0Æ00 12 ± 1Æ41

63 20 ± 1Æ41 25 ± 2Æ83 9 ± 1Æ41 28 ± 1Æ41 19 ± 1Æ41 3 ± 1Æ41 w 14 ± 1Æ41 5 ± 1Æ41

69 32 ± 0Æ00 28 ± 1Æ41 19 ± 1Æ41 14 ± 2Æ83 12 ± 1Æ41 11 ± 0Æ00 9 ± 1Æ41 5 ± 0Æ00 11 ± 2Æ83

124 w w 19 ± 1Æ41 w 8 ± 0Æ00 30 ± 2Æ83 3 ± 0Æ00 2 ± 1Æ41 4 ± 1Æ41

25/3 w w – w 9 ± 1Æ41 w 12 ± 2Æ83 9 ± 1Æ41 4 ± 1Æ41

50/3 26 ± 4Æ24 13 ± 2Æ83 ng – – ng – – ng

w, weak activity;

ng, no growth;

–, no substrate degradation.

Lipolysis of Antarctic bacteria A. Lo Giudice et al.

1042 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048

ª 2006 The Authors

for lipolytic activity of each strain are reported in

Table 3.

Enzymatic activity

The relative lipase and esterase activities were investigated

using several p-nitrophenyl esters as substrates at 10�C at

pH 7Æ0 (Table 4). The crude extracts were assayed

towards pNP esters with aliphatic acyl-chain length of

C2-C12. Short-chain p-nitrophenyl esters were good

substrates for strain 63 (maximum activity towards C2

and C5); medium acyl-chain length C6 was a very good

substrate for strain 25/3. These results may indicate the

presence of esterase activity in the crude extract of these

strains. Strains 41 and 69 showed highest activity towards

p-nitrophenyl-caprate (C10), whereas very high activity

towards a broad range of substrates with acyl-chain length

from C6 to C12 was assaying in strain 124, and the

relative activity percentage increased in this order:

C6 > C8 > C10 > C12. The broader substrate specificity of

strain 124 is probably due to the simultaneous presence

of lipase and esterase activity in the crude extract. Very

low activity was detected when strain 50/3 was assayed

towards all substrates; the highest relative activity being

found using p-nitrophenyl acetate (C2), suggesting that,

in the crude extract, hydrolytic activity may come from a

single esterase. The lipolytic activity of the culture supern-

atants was analyzed towards the p-NP-caproate (C6). All

the culture supernatants showed high activity towards this

substrate (data not shown). As a preliminary step of this

research, the attention was focused on intracellular

enzymes.

Phenotypic and molecular characterization

The six isolates were further characterized by using a

combination of classical phenotypical tests and sequen-

cing of 16S rDNA. The 16S rDNA was successfully

amplified via PCR as described in Materials and meth-

ods. The nucleotide sequences of each amplified frag-

ment was determined; sequences were submitted to

GenBank and were assigned the accession numbers

AY657017, AY660956, AY660954, AY656801, AY660955

and AY660952. Each sequence was aligned using the

clustal w program with the most similar ones

retrieved from databases; the alignments were then used

to construct the phylogenetic trees reported in Fig. 1.

The phylogenetic analysis revealed that the six isolates

belong to three bacterial genera: Pseudoalteromonas

(isolates 41, 124 and 69), Psychrobacter (isolates 63 and

25/3) and Vibrio (isolate 50/3). The six strains were

then analyzed for morphological, biochemical and

physiological properties summarized in Table 5. On

MA, they produced creamy to white circular opaque

colonies, with an entire margin. Cells were rod-shaped,

nonspore-forming and Gram-negative. Pseudoalteromonas

and Vibrio isolates were motile by means of a single

polar flagellum. Isolates were halotolerant (up to 7%

NaCl) and, except for strain 25/3, required NaCl for

growth. The temperature range for growth led to

Table 3 Ranges of pH and NaCl, and optimal

environmental conditions for the hydrolysis of

the substrate on agar plates. The dimensions

of the degradation halos produced under

optimal conditions are reported in the last

column

Isolate Substrate

Range

Optimal conditions for

hydrolysis Halo diameter (mm)

pH NaCl (%) pH NaCl (%) T (�C) mean ± SD

41 Tween 60 5–9 0–11 6 3Æ5 15 50 ± 4Æ24

63 Tween 85 6–9 0–11 7Æ4 1 4 32 ± 2Æ83

69 Tween 80 6–9 0–9 7Æ4 1 15 42 ± 1Æ41

124 Tween 40 6–9 0–11 8 3Æ5 15 37 ± 4Æ24

25/3 Tributyrin 5–9 0–11 9 7 4–15 20 ± 1Æ41

50/3 Tween 20 6–9 3Æ5–7 8 3Æ5 15 36 ± 2Æ83

Table 4 Esterase and lipase activities towards p-NP-esters (U mg)1) in the cell extract

Strain p-NP-acetate (C2) p-NP-valerate (C5) p-NP-caproate (C6) p-NP-caprylate (C8) p-NP-caprate (C10) p-NP-laurate (C12)

41 2Æ38 5Æ22 6Æ07 12Æ90 11Æ52 1Æ60

63 4Æ10 8Æ71 5Æ63 6Æ90 7Æ93 2Æ75

69 1Æ35 2Æ17 2Æ16 6Æ30 7Æ29 0Æ44

124 3Æ07 7Æ49 7Æ23 15Æ00 12Æ80 6Æ40

25/3 1Æ02 6Æ27 5Æ92 0Æ90 0Æ51 0Æ44

50/3 0Æ53 0Æ26 0Æ36 0Æ45 0Æ25 0Æ06

A. Lo Giudice et al. Lipolysis of Antarctic bacteria

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Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048 1043

classify isolates 41, 63, 124, 25/3 and 69 as psychro-

trophs, whereas strain 50/3 was psychrophilic, being

unable to grow above 20�C. Biochemical tests revealed

that all the isolates were negative for H2S and indole

production, arginine dehydrolase, lysine and ornithine

decarboxylases, and for degradation of chitin and agar.

Discussion

Among 155 psychrotrophic isolates, 95Æ5% showed signifi-

cant lipolytic activity. The percentage of lipolytic enzyme

producers detected in Antarctic marine environments

throughout this study is much higher than that reported

100 Vibrio fisheri X74702·1

Vibrio fischeri AY292921·1

Vibrio wodanis AY628647·1

Vibrio salmonicida X70643·1

Vibrio logei AY292929·1

Vibrio lentus AY292927·1

Photobacterium sp. AY781193·1

AB094794·1 Psychrobacter okhotskensis

AJ430827·1 Psychrobacter fozii

AY771717·1 Psychrobacter fozii

AJ310992·1 Psychrobacter sp.

AJ609555·1 Psychrobacter urativorans

Isolate 63

Isolate 25/3

Isolate 124

Isolate 69

Isolate 41

100

69

46

92

0·001

0·005

0·001

98

87

68

85

60

99

91

86

Pseudoalteromonas sp. AY687990·1

Pseudoalteromonas sp. AY227707·1

Pseudoalteromonas gracilis AF038846·1

Pseudoalteromonas agarovorans AJ417594·1

AY864646·1 Psychrobacter sp.

AJ539102·1 Psychrobacter glacialis

AJ440989·1 Antarctic bacterium

Pseudoalteromonas elyakovii AF116188·1

Isolate 50/3

Figure 1 Phylogenetic relationship based on 16S rDNA sequences of representative isolates.

Lipolysis of Antarctic bacteria A. Lo Giudice et al.

1044 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048

ª 2006 The Authors

by Jaeger and Eggert (2002) (20%), Cardenas et al. (2001)

(22%), for soil samples, and by Zeng et al. (2004) (43%)

for deep-sea sediments. Assaying for lipase and esterase

producers on agar plates is a rapid and easy method often

indifferently performed by using one to three substrates.

In this study, lipolysis was determined by using several

lipid substrates, either synthetic or natural. Probably, the

utilization of a set of different media for initial screening

allowed the detection of a higher number of lipolytic bac-

teria. The substrates for the screening of hydrolytic activ-

ity because of esterases (E.C. 3.1.1.1) were Tweens 20, 40,

60 and 80, soluble monoesters of fatty acids (lauric, pal-

mitic, stearic and oleic, respectively). For lipases (E.C.

3.1.1.3) tributyrin, Tween 85 (a synthetic ester of oleic

acid) and triolein were used. Ninety-three isolates

(62Æ8%) displayed lipolytic activity on all the substrates

amending the basal medium, indicating the potential pro-

duction of esterases and lipases with a variable degree of

specificity. The remaining 55 isolates were characterized

Table 5 (I) and (II) Phenotypic features of the six isolates

Feature

Pseudoaltero-

monas

Psychro-

bacter Vibrio

41 69 124 63 25/3 50/3

(I)

Gram reaction ) ) ) ) ) )Motility + + + ) ) +

Cell morphology r r r r r r

Polar flagellum + + + ) ) +

Colony pigmentation ) ) ) ) ) )Endospores ) ) ) ) ) )Growth temperature (�C)

4 + + + + + +

20 + + + + + +

30 + + + + + )37 ) ) ) ) ) )pH range for growth 4–9 5–9 5–9 5–9 4–9 6–9

NaCl conc. for growth (%)

Minimum 1 1 1 1 0 1

Maximum 11 9 11 11 11 9

Growth in the absence of NaCl w w w w + )Utilization as a carbon source

Glucose ) + + ) + )Arabinose ) ) ) ) ) )Mannose ) ) ) ) ) )Mannitol ) + + ) ) )N-acetyl-glucosamine ) ) ) ) ) )Maltose ) + + ) + )Gluconate ) + + ) ) )Caprate ) ) ) ) ) )Adipate ) ) ) ) ) )Malate ) ) + ) + )Citrate ) ) + ) + )Phenyl-acetate ) ) ) ) ) )

Biochemical tests

Urease ) ) + ) + )Lysine decarboxylase ) ) ) ) ) )Ornithine decarboxylase ) ) ) ) ) )b-galactosidase + ) ) ) ) )Arginine dihydrolase ) ) ) ) ) )Tryptophane deaminase ) ) ) + + )Oxidase + + + + + +

Catalase + + + + + +

Indole formation ) ) ) ) ) )Voges–Proskauer reaction + + + ) + )Nitrate reduction ) ) ) ) + )H2S formation ) ) ) ) ) )

Macromolecule hydrolysis

Aesculin + ) ) ) + )Gelatin + + + ) ) +

Chitin ) ) ) ) ) )Agar ) ) ) ) ) ng

Starch ng + ) ) ) )Tween 80 + + + + + +

(II)

Growth on solid media

Trypticase soy agar (TSA) + + + + + )

Table 5 Continued

Feature

Pseudoaltero-

monas

Psychro-

bacter Vibrio

41 69 124 63 25/3 50/3

TSA + 3% NaCl + + + + + +

TCBS ) ) ) ) ) +

Nutrient agar (NA) + + + + + )NA + 3% NaCl + + + + + +

Acid produced from

Glucose + ) ) ) ) +

Mannitol ) ) ) ) ) +

Inositol ) ) ) ) ) )Sorbitol ) ) ) ) ) )Rhamnose ) ) ) ) ) )Sucrose + + + ) ) )Melibiose ) ) ) ) ) )Amygdalin ) ) ) ) ) )Arabinose ) ) ) ) ) )Glycerol ) ) ) ) ) +

Maltose ) ) ) ) ) +

Galactose ) ) ) ) ) +

Mannose ) ) ) ) ) +

Starch ) ) ) ) ) +

Fructose ) ) ) ) ) +

Susceptibility to

Penicilline G ) ) ) ) ) +

Polimixine B + + ) ) + +

O/129 ) ) ) ) ) +

Nalidixic acid + + + + + +

Tobramicine ) ) + + + +

Tetracicline ) ) ) ) ) +

Chloramphenicol + + + + + +

Ng, no growth in the test medium; w, weak reaction;

r, rod-shaped cells.

A. Lo Giudice et al. Lipolysis of Antarctic bacteria

ª 2006 The Authors

Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 101 (2006) 1039–1048 1045

by rather variable lipolytic activity. Thus, the different

responses found with various lipids show that the detec-

tion of lipolytic activity in marine Antarctic bacteria

needs the utilization of several substrates. In contrast to

tributyrin (which was hydrolyzed by 131 strains), triolein,

the typical substrate for true lipases (Jaeger et al. 1999),

was poorly degraded by few isolates, indicating predomin-

ant presence of lipases acting on short-chain triglycerides.

Our results do not support those recently reported by

Sanchez-Porro et al. (2003) on both culture collection

and environmental isolates. They state that most culture

collection strains are unable to produce hydrolases. This

is not the case of our isolates, as demonstrated by the

high percentage of strains hydrolyzing lipid substrates:

only seven isolates were able to grow on the medium

without utilizing them, while the remaining 148 hydrolyze

at least one substrate, although often weakly.

Cell temperature is strictly dependent on environmen-

tal temperature, which influences metabolic pathways, cell

reactions, nutritional requirements and biomass composi-

tion of micro-organisms (Ruberto et al. 2005). The pre-

liminary assay for the expression of lipolytic activity of

Antarctic marine bacteria was performed at three temper-

atures and the results clearly showed that lipolytic activity

strongly depends on growth temperature and generally

occurs more efficiently at high temperature. No emerging

differences were observed between the halo dimensions at

4 and 15�C. In some cases, the production of smaller deg-

radation halos was observed at 15�C, than at 4 and 30�C.

Thus, for these isolates, the halo dimensions were not

directly linked to the increase in the incubation tempera-

ture, and probably depended on the activation of several

lipolytic enzymes.

The psychrotolerant nature of the isolates gives them

the potential to be used for bioremediation at both low-

and-moderate temperature (4–30�C). However, another

one of main objectives of this study was to establish opti-

mal conditions for the expression of the lipolytic activity

of Antarctic marine bacteria at low temperature.

The 16S rDNA gene fragment of the six lipolytic

Antarctic bacteria chosen for further characterizations was

sequenced. The six strains were affiliated to the c-Proteo-

bacteria group. Psychrobacter species successfully colonize

a number of cold habitats with heterogeneous features,

such as seawaters, sea-ice, sediments, marine organisms

and food (Maruyama et al. 2000; Denner et al. 2001; Ro-

manenko et al. 2002, 2004; Yumoto et al. 2003; Yoon

et al. 2004). From an ecological point of view, their

worldwide occurrence is mainly due to rapid growth at

low temperatures, halotolerance and wide substrate specif-

icity (Yumoto et al. 2003). Psychrobacter strains have been

isolated from Antarctic ornithogenic soils (Bowman et al.

1996), sea-ice (Bowman et al. 1997), seawater (Bozal et al.

2003) and cyanobacterial mats (Shivaji et al. 2005).

Several enzymes (i.e. glutamate dehydrogenase, chitinase,

b-lactamase) from Antarctic Psychrobacter have been

characterized to date (Feller et al. 1995; Di Fraia et al.

2000; Camardella et al. 2002; Kulakova et al. 2004). As

well as Psychrobacter species, Vibrio strains are widely dis-

tributed in aquatic environments, especially in association

with animals. Although they are generally mesophilic,

some cold-adapted Vibrio strains have been isolated (Xu

et al. 1998; Yumoto et al. 1999; Bendt et al. 2001). Mem-

bers of the Pseudoalteromonas genus are recognized as

producers of biologically active compounds (Holmstrom

and Kjelleberg 1999; Isnanseytyo and Kamei 2003) and

enzymes (Feller et al. 1998; Vera et al. 1998; Hoyoux

et al. 2001).

Specific physiological conditions may support the pro-

duction of lipolytic enzymes. Most lipases can act in a

wide range of pH and temperature, although alkaline bac-

terial lipases are the most common (Gupta et al. 2004).

Through the study of the influence of abiotic factors on

lipolysis, differences between strains according to NaCl

and pH tolerances were observed. Our findings demon-

strate that the lipolytic activity of Antarctic marine bac-

teria is rather variable and occurs in a wide range of salt

concentration and pH, in accordance to the bacterial phy-

siological characterization. Thus, the isolates could be

interesting sources of enzymes to be exploited in specific

industrial processes whose environmental conditions can-

not be modified to achieve enzyme optimal activity. Fur-

thermore, nutritional requirements of lipolytic bacteria

could be usefully applied in developing strategies for

bioremediation of fat-contaminated aqueous systems.

Finally, data obtained in this study will be utilized to

optimize the production of lipolytic enzymes to be chem-

ically and structurally analyzed.

Acknowledgements

We are grateful to two anonymous reviewers for their help-

ful comments and valuable suggestions to improve the

manuscript. This research was supported by grants from

PNRA (Programma Nazionale di Ricerche in Antartide),

Italian Ministry of Education and Research (PEA 2002–

2003, Research Project PNRA 2002/1Æ5) and from the PhD

Course in Environmental Sciences (Scienze Ambientali:

Ambiente Marino e Risorse) of the University of Messina.

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