World J Microbiol Biotechnol (2012) 28:2249–2256 DOI 10.1007/s11274-012-1032-3
ORIGINAL PAPER
Extracellular enzymes produced by microorganisms isolated from maritime Antarctica Lyliam Loperena • Vero´nica Soria • Hermosinda Varela • Sandra Lupo • Alejandro Bergalli • Mairan Guigou • Andre´s Pellegrino • Angela Bernardo Ana Calvin˜o • Federico Rivas • Silvia Batista
•
Received: 18 September 2011 / Accepted: 25 February 2012 / Published online: 14 March 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Anta´rtico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities
present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.
Electronic supplementary material The online version of this article (doi:10.1007/s11274-012-1032-3) contains supplementary material, which is available to authorized users.
Introduction
L. Loperena (&) V. Soria H. Varela A. Bergalli M. Guigou A. Pellegrino A. Bernardo A. Calvin˜o F. Rivas Departamento de Bioingenierı´a, Facultad de Ingenierı´a, Instituto de Ingenierı´a Quı´mica, Julio Herrera y Reissig 565, 11300 Montevideo, Uruguay e-mail:
[email protected] S. Lupo Laboratorio de Micologı´a, Facultad de Ciencias, Instituto de Biologı´a, UdelaR, Igua´ 4225, 11400 Montevideo, Uruguay S. Batista Unidad Microbiologı´a Molecular, Instituto de Investigaciones Biolo´gicas Clemente Estable (MEC), Avenida Italia 3318, 1600 Montevideo, Uruguay
Keywords Maritime Antarctica Psychrophile microorganism Psychrotroph microorganism Enzymatic activity
Developments in genetics and microbial physiology have had a profound impact on enzyme production technologies and the search for new organisms with unusual activities remains an important area in process engineering and biotechnology. Enzymes from extremophilic microorganisms offer versatile tools for sustainable development in a variety of industrial applications as they show important environmental benefits due to their biodegradability, specific stability under extreme conditions, improved use of raw materials and decreased amounts of waste products (Brenchley 1996; Antranikian et al. 2005; Margesin et al. 2005; Tosi et al. 2010). The classification in facultative and obligate microorganisms has been established according to their growth
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temperature requirements. Those organisms able to grow only in a narrow range of low temperature (stenothermal) are usually designated obligate psychrophiles. Another group, facultative psychrophiles or psychrotrophs, are able to grow at wider range of temperatures (eurythermal). Psychrotrophs are thought to be more abundant than obligate psychrophiles in cold environments like maritime Antarctica, possibly because they have developed the ability to tolerate large variations in temperature. Several psychrophilic or psychrotrophic bacteria from Antarctica were previously identified as belonging to the genera Pseudoalteromonas, Moraxella, Psychrobacter, Polaromonas, Psychroflexus, Polaribacter, Moritella, Vibrio, Arthrobacter, Bacillus and Micrococcus. Methanogenium, Methanococcoides and Halorubrum were also identified in Antarctica as psychrophilic archaea. Maritime Antarctica has a rich mycoflora including fungi of the genera Candida, Cryptococcus, Geomyces, Penicillium, Mortierella, Cladosporium, Thelebolus, Phoma among others (Feller and Gerday 2003; Ruisi et al. 2007). The mechanisms used by these microorganisms to grow at low temperatures are associated with diverse cellular processes. Microorganisms isolated from low temperature environments typically have increased proportions of unsaturated fatty acids, particularly PUFAs, methyl branched fatty acids, polar carotenoids and the ratio of anteiso- to iso-branched fatty acids, as well as a decrease in the average chain length of fatty acids and in the ratio of sterols to phospholipids. These alterations in the relative composition in these microbes are thought to be necessary for maintaince of membrane fluidity at lower temperatures (Margesin and Miteva 2011; Fogliano et al. 2010). The expression of coldadapted enzymes has been also observed. These enzymes can exhibit ten times higher activity at low temperatures compared to their mesophilic counterparts (Aghajari et al. 1996; Feller and Gerday 2003; Margesin et al. 2005). This study describes screening of Antarctic microorganisms for the ability to produce extracellular enzymes for specific applications in industry and is an attempt to contribute with the research on extremophilic organisms as a source of cold active enzymes for industrial use. Bacteria, yeasts and filamentous fungi isolated were tested for proteases, amylases, pectinases, xylanases, cellulases, lipases and ligninases.
Materials and methods Sampling sites and microorganism isolation Samples from which microorganisms isolated were collected during Antarctic expeditions organized by the
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Instituto Anta´rtico Uruguayo in December 2006 and February 2007. One hundred seventy-four samples were collected at different sites on the Fildes peninsula, King George Island (KGI) (62°020 S, 58°210 W), Deception Island (62°580 S, 60°390 W, Port Lockroy (64°490 S, 63°300 W, Cuverville Island (64°410 S, 62°380 W) and Cape Kaiser (64°140 S, 62°000 W). Samples of air with suspended material collected on plates with solid media sea and freshwater ice, soil, sediment, bird and marine animal faecal sediments, dead animals, algae, plants, rocks and microbial mats were aseptically collected. Aliquots of 0.2 ml of seawater, freshwater and melted ice were streaked directly on plates with Marine Agar medium or Tryptone Soy Agar (TSA). Each solid sample (ca. 10 g) was suspended in sterile water or Tryptone Soy Broth and incubated at 4°C. Aliquots of the suspension were spread on the surface of TSA and Malt Yeast extract agar plates. The plates were incubated at 4°C and the bacterial and fungal colonies with distinct morphologies were repeatedly isolated until pure.
Enzymatic activity The production of extracellular enzymes was determined using a diffusion method involving colonies grown on solid media with a specific substrate. Each isolate was inoculated and tested at 4 and 20°C, assays were performed by duplicate. Zones of clearing around the colonies were used as an indication of enzymatic activities and measured in mm as the difference between the diameter of the halo and the colony. In some cultures, the halos could be measured daily. If the addition of a chemical compound was required, the plates were incubated for at least a week before the incorporation of the developing agent. The production of extracellular proteases was determined using two culture media, one with casein (Wang et al. 2007) and the other with gelatin (Seeley et al. 1991). Measures of extracellular amylases, xylanases and cellulases were done incorporating, respectively, starch, xylan or carboxymethylcellulose into the growth medium (Seeley et al. 1991; Paterson and Bridge 1994; Gonzales et al. 2004; Villalba et al. 2004; Rivas et al. 2007). Pectinolytic activity was analyzed including pectin in the growth media prepared at two different pHs because exopectinases are usually most active at pH 5.0 and endopectinases at pH 7.0 (Paterson and Bridge 1994; Rodrı´guez et al. 2007). Tributyrin was included in the medium for the detection of lipolytic activity in bacteria and Tween 80 in the case of yeasts and fungi (Paterson and Bridge 1994). Lignilolitic activity was determined in a medium containing Remazol Brilliant Blue R (Mtui and Nakamura 2004).
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Isolate identification
GenBank database noted above. Filamentous fungal isolates were identified using micro-morphological criteria.
Nineteen bacterial isolates were identified by sequence analysis of a portion of the 16S rDNA gene. Genomic DNA was extracted from cultures grown on TSA plates for seven days at 4°C by a phenol–chloroform method (Alippi and Aguilar 1998). PCR amplification was done in 20 ll reaction mixtures containing 10 ll of ‘‘Fast PCR Master Mix’’ (2X) (Fermentas), 2 ll of each primer solution (10 lM of each universal primer 27F and 1492R), 1 lg of DNA and water up to final volume. PCR reaction was done in a Palm-Cycler TM (Corbett Research UK Ltd). The amplification conditions were: incubation at 95°C for 2 min, followed by 35 cycles of 95°C, 15 s, 50°C, 30 s, 70°C, 1 min 30 s. The reaction included a final extension at 70°C, 5 min. Amplified products of expected size were purified and sequenced at Macrogen Inc. (Korea). DNA sequences obtained from each isolate using primers 27F, 518F and 1492R were aligned by CLUSTALW (Higgins et al. 1994) using MEGA version 5.1 (Tamura et al. 2007). Assembled DNA sequence data was analyzed by BLASTn (Altschul et al. 1990) and compared with non-redundant nucleotide sequence database (nr/nt) at the National Center for Biotechnology Information (NCBI) (http://blast.ncbi. nlm.nih.gov/Blast.cgi). The identification of yeast isolates was done by DNA sequence analysis of the ITS region. DNA extraction was performed from cells growing in Yeast Extract Peptone Dextrose Agar (Lupo et al. 2006). The ITS region was amplified by PCR using the primers ITS4 and ITS5 (White et al. 1990) and reaction conditions were described by Lupo et al. (2006). The amplification product was purified and sequenced by Macrogen, Inc. (Korea). The analysis was done using BLASTn (http://www.ncbi.nlm.nih.gov) and compared with the sequences deposited in the
Phylogenetic analysis Multiple alignments were generated using CLUSTALW (Higgins et al. 1994). Phylogenetic distance trees were inferred by Maximum-Parsimony (MP, heuristic search factor of 2) and Neighbour-Joining (NJ, p-distance matrix) analyses, using MEGA 5.1 (Tamura et al. 2007). Confidence in topologies was assessed using bootstrapping (1,000 replicates).
Results and discussion A total of 518 microorganisms were isolated: 421 bacteria, 46 yeasts and 51 filamentous fungi. The collection of isolates used in this work was deposited in the Culture Collection of Microorganisms of the Bioengineering Department and Mycology Laboratory at the Faculty of Engineering (UdelaR, Uruguay). For screening of enzymatic activities we worked with 120 bacteria, 31 yeasts and 10 filamentous fungal isolates (Table 1). The size of halos and colonies were used to compare enzymatic activities and colony growth. Several investigators have described the sensitivity of this method for semi-quantitative determinations and it has been used successfully for identification of novel activities in environmental isolates of microorganisms (Wickstro¨m 1983; Leo´n et al. 2007; Li et al. 2009). The enzymatic activities most frequently found among bacterial isolates were: amylase, caseinase, lipase and gelatinase. Some bacterial clones had ligninase, xylanase and cellulase activities. Yeasts and filamentous fungal isolates more frequently had
Table 1 Percentage of clones with enzymatic activity detected at 4 and 20°C Temperature
Bacteria
Yeasts
Fungi
4°C
20°C
Both temp.
4°C
20°C
Both temp.
4°C
20°C
Both temp.
Proteolytic activity with casein
33
36
31
3
3
3
0
0
0
Proteolytic activity with gelatin
18
29
17
6
10
6
70
70
50
Lipolytic activity
35
26
17
13
16
10
40
30
30
Amyolytic activity
16
51
8
3
3
3
0
0
0
Pectinolytic activity at pH 5.0
16
12
5
0
6
0
0
0
0
Pectinolytic activity at pH 7.0
0
3
0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Cellulolytic activity
1
1
1
6
10
2
50
40
20
Xylanolytic activity
3
13
2
3
6
3
0
10
0
Lignilolytic activity
18
7
7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Total clones analyzed: 120 bacteria, 31 yeasts and 10 fungi n.d. not determined
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Pseudomonas sp. Gd3F (99%)
Antarctic bacterium GAOF (99%)
Pseudomonas sp. tsz07 (99%)
Pseudomonas fluorescens MS300 (99%)
Pseudomonas sp. tsz07 (99%)
Pseudomonas fluorescens FB25 (99%)
Arthrobacter oxydans S32212 (99%)
Thermoleophilum minutum YS-4 (99%)
Pseudomonas sp. LD126 (99%)
Arthrobacter sp. KOPRI 25519 (99%)
c6-HM165446
c7-HM165445
c8-HM165452
c9-HM165453
c10-HM165454
c11-HM165455
c12-HM165456
c13-HM165457
c14-HM165458
C15-HM165459
Bacillus sp. Nj-19 (99%)
Arthrobacter lactophilus KNOUC403 (99%)
c5-HM165447
C18-HM165462
Psychrobacter luti NF11 (99%)
c4-HM165448
Pseudomonas sp. AW6 (99%)
Pseudomonas sp. KOPRI 25402 (99%)
c3-HM165449
Arthrobacter psychrochitiniphilus JCM 13874 (99%)
Pseudomonas sp. P1 (99%)
c2-HM165450
C16-HM165460
Antarctic bacterium R25 (99%)
c1-HM165451
C17-HM165461
Database microorganism with highest similarity (% identity)
Clone nameAccession number
Sediment and lichens collected next to Artigas Base
Piece of wood collected next to Artigas Base
Microbial mat (KGI)
Bryophyte sample (Deception Island)
Bird remains (KGI)
Bryophytes collected on KGI
Sediment sample collected next to Suffield Point
Bryophytes collected next to Artigas Base
Benthic mat from a creek between Collins glacier and Artigas Base
Grass collected next to Artigas Base
Microbial mat from the beach in front of Drake sea
Ice with algae collected between Drake passage and Artigas Base
Sediment sample collected next to Suffield Point (KGI)
String sample collected next to Artigas Base (KGI)
Krill remains on the beach in front of Drake sea
Bird remains on the beach in front of Drake sea
Bird remains on the beach in front of Drake sea (KGI)
Sediment sample collected next to Collins glacier (KGI)
Origin
Table 2 Enzymatic activity at 4 and 20°C of bacterial isolates
n.a.
n.a.
6±1
n.a.
11 ± 1
15 ± 1
n.a.
10 ± 1
n.a.
14 ± 1
10 ± 1
11 ± 0
8.5 ± 2
n.a.
n.a.
14 ± 2
9±1
11 ± 0
n.a.
n.a.
11 ± 1
2.5 ± 1
24 ± 0
26 ± 1
n.a.
14 ± 0
18 ± 3
20 ± 2
20 ± 1
17,5 ± 2
17 ± 0
n.a.
n.a.
8±1
25 ± 1
23 ± 1
n.a.
n.a.
n.a.
7.5 ± 1
30 ± 0
34 ± 3
n.a.
14 ± 3
26 ± 2
6±1
36 ± 2
17 ± 1
21 ± 0
n.a.
n.a.
18 ± 1
28 ± 1
32 ± 2
4°C
4°C
20°C
Gelatinase
Caseinase
Temperatures
n.a.
n.a.
n.a.
17 ± 2
32 ± 1
36 ± 2
22 ± 3
20 ± 3
33 ± 2
30 ± 2
30 ± 0
22 ± 2
30.5 ± 1
n.a.
n.a.
30 ± 1
30 ± 1
n.a.
20°C
14 ± 2
n.a.
n.a.
22 ± 1
n.a.
n.a.
n.a.
3±
18 ± 1
n.a.
n.a.
n.a.
20 ± 0
n.a.
22 ± 2
n.d.
n.d.
n.d.
4°C
Lipase
n.a.
n.a.
n.a.
23 ± 2
n.a.
n.a.
n.a.
6±0
3±1
n.a.
n.a.
n.a.
15 ± 1
n.a.
n.a.
n.d.
n.d.
n.d.
20°C
n.a.
n.a.
n.a.
n.a.
n.a.
4±1
9±1
5±0
6±1
3±1
4±1
6±1
n.a.
4±1
n.a.
n.a.
n.a.
5±2
4°C
4±0
19 ± 2
7±3
n.a.
4±2
6±1
17 ± 1
4±1
7±1
5.5 ± 0
4±1
8±3
12 ± 3
16 ± 1
n.a.
n.a.
4±2
10 ± 3
20°C
Amylase
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
8±2
n.a.
6±2
n.a.
n.a.
n.a.
n.d.
n.d.
n.d
4°C
n.a.
n.a.
31 ± 4
n.a.
n.a.
n.a.
n.a.
21 ± 2
31 ± 3
n.a.
n.a.
23 ± 2
n.a.
n.a.
n.a.
n.d.
n.d.
n.d.
20°C
Pectinase at pH 5.0
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
24 ± 2
n.a.
n.a.
n.a.
22 ± 2
n.a.
n.a.
n.a.
n.d.
n.d.
n.d.
20°C
Pectinase at pH 7.0
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n.a. n.a. n.a.
The highest activities at 4 or 20°C are indicated in bold
Activities were measured as the difference between the diameter of the enzymatic activity and the colony halo in mm; n.d. not determined, n.a. no activity
11 ± 2 n.a. n.a. n.a. n.a. n.a. n.a. n.a. Carnobacterium sp. Nj-46 (99%) C19-HM165463
Sediment between Artigas Base and Suffield passage
20°C 20°C 4°C 20°C 4°C 4°C 4°C 4°C
20°C
Gelatinase Caseinase
Database microorganism with highest similarity (% identity) Clone nameAccession number
Table 2 continued
Origin
Temperatures
20°C
Lipase
20°C
Amylase
Pectinase at pH 5.0
Pectinase at pH 7.0
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lipase and cellulase activities. Fungal isolates had little or no detectable pectinase, xylanase or amylase activities. The four bacterial isolates with the highest activity for each of the following enzymes: caseinase, gelatinase, amylase, lipase and pectinase, are shown in Table 2. Isolates with highest caseinase, gelatinase and pectinase activities belonged to the genus Pseudomonas, and were isolated from bird remains, algae, bryophyte and microbial mat samples. The isolates with the highest lipase activity were assigned to Psychrobacter, Pseudomonas and Arthrobacter. These bacteria were recovered from krill remains, sediment and bryophyte samples. The highest amylase activities were found in isolates identified as Arthrobacter. These bacteria were recovered from sediments and pieces of wood. Pseudomonas are well known for their high genetic and physiological diversity and ability to produce extracellular enzymes (Zhang and Zeng 2011). In agreement with this, 9 of the 19 isolates analyzed belonged to this genus and had the highest enzyme activities in five of the six tests done in this study. We also selected two yeast and two filamentous fungal clones that showed the highest activities for cellulase, lipase, xylanase, caseinase and gelatinase. Yeast clone 80-2, identified as Trichosporon pullulans, was isolated from a creek. This isolate exhibited high caseinase activity at 4°C (20 ± 1 mm) and gelatinase activity at 4°C and at 20°C (18 ± 1 mm and 21 ± 2 mm, respectively). This clone also had high xylanase activity at 20°C (25 ± 2.5 mm). Yeast clone 65-2, identified as Cryptococcus victoriae, was isolated from water of Lake Uruguay. This clone had lipase, cellulase and xylanase activities, but activity was found only at 20°C (22.5 ± 9, 12 ± 3 and 22.5 ± 1.5 mm respectively). Fungal clones 14-2 and 63-2, identified as Geomyces pannorum, were isolated from decomposing algae collected near Drake Passage and from water of Lake Uruguay. These isolates exhibited high gelatinase activity (20 ± 3.5 and 22.5 ± 2 mm) as well as lipase activity (27.5 ± 2.5 and 24.5 ± 2 mm respectively) at 4°C. The potential use of a number of these isolates in industrial microbial fermentation could be evaluated in more detail, Psychrobacter sp. c4, Arthrobacter sp. c15 and the yeast C. victoriae 65-2) could be considered as a source of lipases, and Arthrobacter ssp. c12 and c17 a source of amylases. The production of gelatinase, caseinase and pectinase could also be analyzed using Pseudomonas spp. c2, c8, c10, c11 and c16. T. pullulans 80-2 and Cryptococcus victoria 65-2 both could be a good source of xylanases. In particular, yeast isolate 65-2 exhibited cellulase and xylanase activities, which are of potential use for the pre-treatment of lignocellulose for ethanol production and of forage crops to improve nutritional quality and digestibility, and also for the treatment of water
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Fig. 1 Rooted phylogenetic tree showing sequences with highest similarity from the GenBank database of the NCBI and the 19 operational taxonomic units that were used in this analysis. The evolutionary history was inferred using the NeighborJoining method. The percentage of replicate trees ([50%) in which the associated taxa clustered together in the bootstrap test is shown next to the branches. Synechococcus sp. SRODG098 was used as an outgroup taxon
Pseudomonas frederiksbergensis strain DSM 13022 Pseudomonas sp. PR3-5 91
Pseudomonas sp. AW6 Pseudomonas sp. W6 c 16 c1 Pseudomonas sp. INK1
68
Antarctic bacterium R25 Pseudomonas congelans SS157 c 13 c 14
81 Thermoleophilum minutum YS-4 Pseudomonas brenneri FB22 Pseudomonas sp. 8H1 Pseudomonas sp. 3(2008b) Pseudomonas sp. LD126 Antarctic bacterium GA0A 65 Antarctic bacterium GA0F c 10 c7 c9 Pseudomonas sp. J83
99
Pseudomonas fluorescens CCM 2115 ATCC13525 Pseudomonas sp. YXE3-18 c8 65 Pseudomonas fluorescens FB25 Antarctic bacterium R-7616 99
Pseudomonas fluorescens MS300 c 11 Pseudomonas sp. tsz07 c2 c3 Pseudomonas sp. KOPRI 25400 99
Pseudomonas sp. KOPRI 25402 Pseudomonas syringae Lz4W
64 Pseudomonas sp. a101-18-2
99
Pseudomonas sp. Nj-59 Pseudomonas sp. NJ-22 Pseudomonas sp. P1(2010) Pseudomonas sp. ArSA Pseudomonas sp. BAM288
99 69
c6 Pseudomonas sp. Gd3F Psychrobacter frigidicola type strain: DSM 12411
85
Arctic seawater bacterium Bsw20370 99
c4 Psychrobacter okhotskensis MD17 NCIMB 13931
95
Psychrobacter luti NF11 LMG 21276 Psychrobacter sp. BSw21070 Carnobacterium funditum pf3 DSM 5970 99
Carnobacterium. funditum R-36987 1 c 19
Sporosarcina globispora 785 DSM
99
73 Bacillus sp. Nj-19 99 c 18 Sporosarcina globispora AIC11-11 90
Sporosarcina globispora OS-253 Arthrobacter sp. KOPRI 25422 61
Arhtrobacter sp. SS-2009-PA1 PA1 Arthrobacter psychrochitiniphilus JCM 13874 Arthrobacter sp. SH-82B Arthrobacter psychrolactophilus KNOUC403 c5 c 17 Arthrobacter sp. KOPRI 25535
99
Arthrobacter sp. KOPRI 25486 Arthrobacter oxydans BG1-7 c 12 Arthrobacter sp. DSP-S2
99 94
Arthrobacter oxydans S32212 Arthrobacter oxydans Asd M5-7 Arthrobacter oxydans Asd M3-4 c 15
99
Arthrobacter sulfureus DSJ12 Arthrobacter sp. Tibet-ITa1 Synechococcus sp. SRODG098
0,01
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effluents (Pathan et al. 2010; Antranikian et al. 2005; Mika´n Venegas and Castellanos Sua´rez 2004). There is current interest in the use lipases and proteases in laundry detergents and as well as in the dairy industries. Pectinase, amylase and cellulase have been shown to be useful in clearing the appearance of and increasing the sweetness of fruit juices. Amylases are used to hydrolyze starch for the production of ethanol, and also to synthesize high-fructose corn sweeteners and syrups (Nigam and Singh 1995; Pandey et al. 2000; Vihinen and Ma¨ntsa¨la¨ 1989). When activity was analyzed at 4°C, Psychrobacter sp. c4 and Arthrobacter sp. c15 had the highest lipase activity, Arthrobacter sp. c12 the highest amylase activity, Pseudomonas spp. c3 and c9, the highest caseinase activity and Pseudomonas spp. c1 and c8 the highest gelatinase activities. G. pannorum 14-2 and 63-2 could be also considered as a source of gelatinase and lipase. These microorganisms could be evaluated for the design of inoculants for organic matter biodegradation, e.g., for effluent treatments (Gratia et al. 2009). In temperate climates, low temperatures achieved during the cold seasons may negatively affect the metabolic activity of native microorganisms (Brenchley 1996), leading to a poor biotransformation rate of pollutants and to their accumulation in case of continuous input. In this respect, the use of psychrophilic microorganisms could provide a useful alternative since psychrophiles are adapted to both low and moderate temperatures (Margesin et al. 2002). In addition, such microorganisms have in some cases been shown to produce cold-activated enzymes possessing a much higher specific activity than those of their mesophilic counterparts (Feller and Gerday 2003). We compared the colony diameters of 103 bacterial, 10 yeast and 10 filamentous fungal isolates expressing enzymatic activities, grown on the same solid media at 4 and 20°C. About 80% of the bacteria grew better at 20°C than at 4°C and only 8% had a reduced growth at 4°C. In relation to bacterial enzymatic activities (see Table 1), most of the activities were expressed at 20°C. Our tests suggest that most of bacterial isolates from maritime Antarctica were psychrotrophs, similar results were described in related studies (Feller and Gerday 2003; Lo Giudice et al. 2010; Selbmann et al. 2010), confirming that psychrotrophs are usually more abundant than obligate psychrophiles in cold ecosystems. In this sense, psychrotrophs are usually preferred for enzyme production than psycrophilic microorganisms because industrial fermentations could proceed at ambient temperatures. Comparable results were obtained with yeasts as well: nine grew better at 20°C than at 4°C. However, only 40% of filamentous fungal isolates grew better at 20°C than at 4°C, and three could be considered obligate psychrophiles. Comparative sequence analysis of 16S rRNA gene indicated that some were closely related to previously
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described bacteria, (similarity of 99%). BLASTn analyses of isolates c1, c2, c3, c4, c7, c8, c10, c12, c16, c17, c18 and c19 exhibited high similarity (within the 5 best scores) with other Antarctic bacteria identified previously. The affiliation of each isolate, based on the BLAST analyses, is presented in Table 2: Pseudomonas spp. (c2, c3, c6, c8, c9, c10, c11, c14, c16), Psychrobacter sp. (c4), Arthrobacter spp. (c5, c12, c15, c17), Bacillus sp. (c18), Carnobacterium sp. (c19). Isolate c13 had highest similarity with Thermoleophilum minutum (Genbank locus: HQ223108.1). Some physiological properties described for this species, however, did not match those of the isolate including growth temperature and carbon source requirements. The location of c13 in the phylogenetic tree suggests that it is most similar to Pseudomonas and that the identification of T. minutum should be revised. As such, the NJ distance tree shown in Fig. 1 includes reference sequences with highest similarity from strains of the GenBank database of the NCBI. Both NJ and MP algorithms gave similar topologies (Fig. 1 and Figure 1S as supplemental material). Two of the clones grouped with Arthrobacter, clones c5 and c17, showed a similar pattern of amylase activity, in agreement with the close relation inferred from the distribution of sequences observed in the phylogenetic trees (Fig. 1 and Figure 1S). However, clone c5 exhibited amylase activity at 4 and 20 °C, whereas c17 expressed this activity only at 20°C. Clones related to Pseudomonas showed complex patterns of enzymatic activities. In particular, clones c8, c9 and c11, closely related according to phylogenetic analysis like clones c7 and c10, showed properties that did not correspond the nearest phylogenetic relation (see Table 2). Acknowledgments We are grateful to Dr. Paul R. Gill for English corrections and valuable comments. This study was supported by a grant from the Comisio´n Sectorial de Investigacio´n Cientı´fica— Universidad de la Repu´blica and by Instituto Anta´rtico Uruguayo.
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