Cold Regions Science and Technology 96 (2013) 86–95
Contents lists available at ScienceDirect
Cold Regions Science and Technology
journal homepage: www.elsevier.com/locate/coldregions
Bioprospecting a glacial river in Iceland for bacterial
biopolymer degraders
Jón Pétur Jóelsson, Heiða Friðjónsdóttir, Oddur Vilhelmsson ⁎
Department of Natural Resource Sciences The University of Akureyri Borgir vid Nordurslod IS-600 Akureyri Iceland
a r t i c l e
i n f o
Article history:
Received 20 November 2012
Received in revised form 15 February 2013
Accepted 5 March 2013
Keywords:
Glacial water microbiota
Culturable diversity
Pseudomonas spp.
Flavobacterium spp.
Cold-active enzymes
Laccase
a b s t r a c t
The glacial river Jökulsá á Fjöllum, which originates in the Vatnajökull ice cap and flows through a large basaltic tephra desert on its way to discharge into the Arctic Ocean, presents a number of unique microbial habitats heretofore
unexplored. We sampled river water, sediment and selected other biotopes at 12 sampling points along the river
from source to mouth and generated a collection of 382 purified and confirmed reculturable psychrotrophic bacterial strains. Partial 16S rDNA sequencing yielded 19 genera and 4 non-genus specific assignments in 4 bacterial
phyla, with pseudomonads and flavobacteria being particularly well represented. A large portion of the isolates produced extracellular enzymes at 15 °C, including amylase, betaglucanase, cellulase, protease and laccase.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
Jökulsá á Fjöllum (JáF) is among the largest glacial rivers in Iceland. It originates in the Dyngjujökull, Brúarjökull and Kverkfjöll
areas of the Vatnajökull ice cap and flows north for 206 km to discharge to the sea in Öxarfjörður bay (Fig. 1). It is located in a volcanically active area and, thus it flows for most of its length through
recent basalt formations. JáF is highly loaded with suspended
solids, discharging about 8 × 10 6 metric tons of solids per annum
(Tómasson et al., 1996). Suspended solids are an important habitat
for bacteria in flowing water, presenting a solid surface for attachment (Logan and Hunt, 1987). In the highland region, the JáF riverbed
is shallow and covered by a thick layer of sandy sediment, but as the
river exits the highlands it flows for 30 km through a deep canyon
until it fans out on the Öxarfjörður alluvial cone. While JáF is characterized as a glacial river and is to a large extent glacier-fed, it also contains significant amounts of spring water. The river water is modestly
alkaline, with typical pH values in the 7.4 to 8.1 range (Óskarsdóttir,
2007). Dissolved electrolyte content is comparatively high, with typical conductivity values of river water about 100 to 150 μS cm −1
(Óskarsdóttir, 2007). The average flow rate is about 200 m 3/s
(Sigurðsson, 1990). Conductivity, flow rate and water table height
are continuously monitored by the Icelandic Meteorolical Office at
three points in JáF and the large eastern tributary Kreppa (http://
en.vedur.is/#tab=vatnafar).
⁎ Corresponding author. Tel.: +354 697 4252; fax: +354 460 8999.
E-mail address: [email protected] (O. Vilhelmsson).
0165-232X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.coldregions.2013.03.001
Among likely sources of bacteria in the river are glacial ice, highland
desert soil and soil from vegetated oases such as Bæjarlönd and
Herðubreiðarlindir. Human habitation is very scarce in the JáF watershed
and almost exclusively restricted to the alluvial cone. Significant human
impact on the microbiota is therefore highly unlikely, particularly in the
highland region. The microbiota of a flowing river reflects the microbial
communities in the river's watershed as microbes from surrounding biotopes are washed into the river by riverbed erosion, groundwater, snowmelt, glacial melt, rainwater and winds (Atlas and Bartha, 1993). A large
part of the river microbiota is thus of allochthonous origin. Nevertheless,
a significant part of the river microbiota, regardless of its origin, is not
simply carried along in a passive state but rather contribute to the chemical processes occurring within the river water. Bacterial metabolic activity can therefore be considerable and is generally the driving force of
organic carbon solubilization and biopolymer degradation (del Giorgio
and Davis, 2003; Guo et al., 2007). Among the most commonly observed
phyla and classes of bacteria in river water are the Alphaproteobacteria,
Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes,
Cyanobacteria, Verrucomicrobia and Planctomycetes (Crump et al., 2009;
Logue et al., 2008; Methe et al., 1998; Vallieres et al., 2008; Zwart et al.,
2002). Crump et al. found that the composition of the microbiotas of
the six largest river systems in the Arctic followed predictable seasonal
patterns (Crump et al., 2009).
Although the bacterial biota of glacial ice has received considerable attention in the last several years (Loveland-Curtze et al., 2009;
Pradhan et al., 2010; Reigstad et al., 2008; Simon et al., 2009; Zhang
et al., 2008), the supraglacial and englacial microbiotas of the
Vatnajökull ice cap have not been studied to date. Subglacial-lake
and tephra microbial communities have, however, been investigated
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
25°W
87
20°W
15°W
1
66°N
66°N
2
3
4
5
6
7
9
8
65°N
65°N
10
11
64°N
64°N
0
25
50
100 km
20°W
15°W
Fig. 1. The river Jökulsá á Fjöllum is located in NE Iceland, originating in the northern part of Vatnajökull ice cap. Major glaciers in Iceland are shaded dark grey, the main Volcanic
Rift Zone is shaded light grey. Sampling locations along the river are indicated by dots and numbered as in Table 1.
in the Grímsvötn and Skaftárkatlar areas in the western and southern
parts of the ice cap (Gaidos et al., 2004, 2009; Marteinsson et al.,
2013). These studies revealed on the one hand diverse, Proteobacteriadominated communities of psychrotolerant bacteria in the Grímsvötn
area (Gaidos et al., 2004), and on the other a strongly oligarchic
chemotrophic community dominated by Acetobacterium, Thermus and
Paludibacter in the Skaftárkatlar area (Gaidos et al., 2009). Comparing
these studies illustrates the somewhat conflicting effects of glacial and
geothermal effects on these habitats, making Vatnajökull, which straddles Iceland's main volcanic rift zone (Fig. 1), and its rivers particularly
intriguing from a microbial ecology perspective.
The JáF microbiota has not been investigated before to our knowledge, but recently the microbiota of several habitats along Glerá, a
smaller semiglacial river in northern Iceland, was investigated
(Markúsdóttir et al., 2012). The culturable microbiota of the pristine,
upper part of that river was found to be characterized by proteindegrading pseudomonads similar to members of the Pseudomonas
fluorescens species group, with members of the Bacilli, Actinobacteria,
Alphaproteobacteria and Sphingobacteria also present.
The main objective of the present study was to bioprospect this cold
and barren environment for bacteria of biotechnological interest, specifically those displaying extracellular enzymatic activity. The main objectives of the present study were to (1) assess the diversity of the
culturable bacterial microbiota of JáF and its immediate surroundings,
(2) establish a culture collection of psychrotrophic JáF isolates, and
(3) screen the culture collection for production of cold-active enzymes,
including laccase, amylase, beta-glucanase, cellulase and protease.
2. Methods
2.1. Sampling and physicochemical measurements
Samples of river water and riverbed sediment were collected from
eleven sampling sites along Jökulsá á Fjöllum river during June and
August 2011 (Fig. 1, Table 1). River water samples were collected in triplicate in sterile 500-ml plastic bottles and were obtained from circa 20 cm
under the surface of rapidly flowing river water. During sampling, water
temperature was measured with a hand-held thermometer. Riverbed
sediment and soil samples were collected with sterile spatulas into sterile
50-ml plastic centrifuge tubes. Exposed riverbed samples (JF16, JF37 and
JF44) were taken from the riverbank at a distance of less than 2 m from
flowing water and a depth of approximately 5 cm below the surface.
Samples were transported on ice to the laboratory at Akureyri where all
further processing was carried out. Electrical conductivity in thawed
river water and glacial meltwater samples was measured at 21 °C with
a CON 510 series conductivity meter (Oakton Instruments, Vernon Hills,
IL, USA) calibrated against 84 and 1140 μS cm−1 KCl standards; pH was
measured at 21 °C with an Orion Dual Star benchtop pH meter (Thermo
Fisher Scientific, Waltham, MA, USA) fitted with a Ross pH electrode
and calibrated against standard buffers in the pH 4.0 to 10.0 range.
2.2. Media, culture conditions and strain isolation
Water samples were plated in duplicate directly onto R2A (Becton
Dickinson, Franklin Lakes, NJ, USA) and PCA (Becton Dickinson), 0.1
88
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
Table 1
Locations, samples, hydrochemical measurements and plate counts.
Location
1
2
3
4
5
6
7
8
9
10
11
Sample no.
Distance from
source (km)
Altitude
(m)
T (°C)
Conductivity
(μS cm−1)
pH
Description
Plate count at 15 °C
(CFU g−1)
Isolates
PCA
R2A
1.9 × 102
4.7 × 102
JF0101 to JF0131
5.8 × 105
3.4 × 101
nd
1.5 × 102
JF0201 to JF0222
JF0301 to JF0335
7.2 × 102
2.0 × 104
JF0401 to JF0425
66.17 N 16.46
W. River delta near
Núpsmýri at an
aquaculture runoff.
JF01
200
20
9.0
6230.0
7.25
JF02
JF03
200
200
20
20
7.5
243.0
7.61
JF04
200
20
66.03 N 16.45 W.
Approximately 50 m
downstream of the
Ásbyrgi bridge.
JF06
JF07
186
186
40
40
6.0
nd
nd
River water.
Sandy riverbed
sediment (submerged).
2.1 × 101
8.0 × 102
2.4 × 102
2.1 × 103
JF0601 to JF0639
JF0701 to JF0720
65.94 N 16.53 W.
At Hljóðaklettar.
JF08
164
200
6.0
119.5
7.48
River water near
the ‘Troll’ cliff.
Sandy riverbed
sediment (submerged).
1.0 × 101
9.6 × 101
JF0801 to JF0828
3
4
JF0901 to JF0930
River water.
Sandy sediment (exposed).
Sandy riverbed
sediment (submerged).
1.3 × 101
9.6 × 105
nd
1.7 × 102
1.2 × 106
7.3 × 103
JF1501 to JF1537
JF1601 to JF1640
JF1701 to JF1717
Stone with algae.
Sand (submerged).
River water.
3.2 × 106
4.6 × 104
4.0 × 101
2.4 × 107
nd
1.7 × 102
JF2001 to JF2017
JF2101 to JF2126
JF2201 to JF2220
River water—mixed
spring and glacial water
Sand
Stone (submerged in
spring water)
1.9 × 101
9.2 × 102
JF4301 to JF4324
nd
8.3 × 106
1.0 × 104
1.8 × 107
JF4401 to JF4405
JF4201 to JF4228
Herðubreiðarlindir soil
JáF water at
Gljúfrasmiður waterfall
Sand at Gljúfrasmiður
waterfall
3.6 × 105
5.9 × 101
1.2 × 106
3.5 × 102
JF4001 to JF4011
JF3601 to JF3619
nd
2.5 × 103
JF3701 to JF3706
River water downstream
of aquaculture runoff.
Green algal mat.
River water upstream
of aquaculture runoff.
Silty riverbed sediment
(submerged).
164
200
65.80 N 16.38 W.
At Dettifoss waterfall.
JF15
JF16
JF17
141
141
141
340
340
340
118
118
118
370
370
370
7.0
105.4
7.73
86
420
7.6
77.3
7.82
JF44
JF42
86
80
420
440
65.15 N 16.21 W. Near
Herðubreiðarlindir.
JF40
JF36
63
61
460
480
JF37
61
480
34
600
3.3
101.5
7.81
River water
1.7 × 101
2.3 × 102
JF3501 to JF3505
27
27
27
600
600
600
3.2
43.9
7.39
River water
Clay, about 50 m from the river
Sand, about 50 m from the river
4.0 × 101
4.6 × 103
2.4 × 104
5.3 × 102
nd
1.8 × 104
JF3201 to JF3209
JF3301 to JF3308
JF3401 to JF3419
0
780
0.5
125.8
7.69
River water
2.2 × 101
3.8 × 102
JF3001 to JF3014
Glacial meltwater at Kverkjökull.
Ice (snow).
Glacial moraine sand
at Langafönn.
Glacial meltwater at Langafönn
3.4 × 102
1.3 × 101
nd
1.6 × 102
9.0 × 101
nd
JF2501 to JF2513
JF2601 to JF2614
JF2801 to JF2806
1.2 × 101
2.6 × 103
JF2901 to JF2906
65.65 N 16.20 W.
Approximately 200 m
upstream from
highway 1 bridge.
JF20
JF21
JF22
65.39 N 16.14 W.
At Miðfell.
JF43
7.5
3.9
108.3
102.4
7.64
7.77
65.01 N 16.24 W.
At Upptyppingar.
JF35
65.02 N 16.16 W.
Kreppa river near
Lónshnjúkur.
JF32
JF33
JF34
64.75 N 16.63 W
At Dyngjujökull glacier.
JF30
64.71 N 16.63 W.
At Kverkjökull glacier.
JF25
JF26
JF28
−5
−5
−5
1100
1100
1300
8.0
nd
nd
JF29
−5
1300
nd
13.5
7.50
5.7 × 10
2.2 × 10
JF09
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
89
and 1.0 ml per plate. Soil and sediment samples were weighed (5–
10 g) into sterile stomacher bags with 45–90 ml of 0.25 M KH2PO4
and stomached in a LabBlender 400 (Seward, Worthing, UK) or shaken vigorously by hand for 1 min. The wash was then serially diluted
in 0.25 M KH2PO4 to 10 −6. Stone samples were scraped with a sterile
scalpel blade into a sterile mortar and the scrapings mashed with a
sterile pestle into 1.0 ml of 0.25 M KH2PO4 and serially diluted to
10 −6. Finally, 100 μl of all dilutions were plated in duplicate onto
R2A and PCA plates. All plates were incubated in the dark at 4 °C
and 15 °C. Growth was monitored periodically for up to eight
weeks. The plates were examined for colony morphotypes based on
color, sheen, convexity and other visible features. Representatives of
each morphotype were aseptically picked, streaked onto fresh
media and incubated under identical conditions as the original isolation plates. In order to obtain pure cultures, the isolates were
re-streaked and incubated at least once more. Stocks of purified isolates were prepared by suspending a loopful of growth in 0.5 ml
28% (v/v) glycerol and stored at − 70 °C.
5 min at 94 °C, followed by 30 cycles of 94 °C for 30 s, 50 °C for
30 s and 72 °C for 90 s; final extension was performed at 72 °C for
7 min. PCR products were purified with the Nucleospin®Extract II
(Macherey-Nagel GmbH and Co.KG, Düren, Germany) kit according to
the manufacturer's protocol. Partial sequencing of the purified PCR
products was performed with a BigDye terminator kit and run on
Applied Biosystems 3130XL DNA analyzer (Applied Biosystems, Foster
City, USA) at Macrogen Europe, Amsterdam, The Netherlands.
For amplification of laccase gene fragments, the following primer pairs
were used: LaccCuF (5′-ACMWCBGTYCAYTGGCAYGG-3′) and LaccCuR
(5′-CRCTGTGGTACCAGAANGTNCC-3′) (Kellner et al., 2008), LaccSlF
(5′-TGGTAYCAYGAYCAYGCSATG-3′) and LaccSlR (5′-ADATRTGCATSG
GRTG-3′) (Suzuki et al., 2003), and CotAF (5′-CAGATGCATATATCATGCA
ATTCAGAGTC-3′) and CotAR (5′-TCATGTAGATCTTGTGTGAGCATAAAAA
GCAGCTCC-3′) (Martins et al., 2002). Other reaction conditions were as
described above.
2.3. Enzyme activity
The 16S rDNA sequences obtained were subjected to multiple
alignments at the Ribosomal Database Project (RDP) (Cole et al.,
2009) using the INFERNAL sequence- and structure-based multiple
alignment algorithm (Nawrocki et al., 2009). Reference sequences
were selected from the RDP database based on their quality and taxonomy, using as a guide the results of a preliminary Megablast against
the refseq_rna database within GenBank. The firmicute B. subtilis DSM
10 provided the outgroup sequence (AJ276351). A maximum likelihood phylogenetic tree was then constructed using MEGA 5.1
(Tamura et al., 2011).
Degradation assays were performed on 1.5% agar plates as follows.
In all cases, plates were incubated for two days or until growth was visible. Casein degradation was assayed by the appearance of clear halos
on a 50% (v/v) skim milk in Nutrient Agar (NA; Becton Dickinson) plates
(Tindall et al., 2007). Bacillus subtilis DSM 10 and sphingomonad
UA-AR0413 were employed as positive and negative controls, respectively. Starch degradation was assayed on 0.2% (w/v) soluble starch
half-strength Tryptic Soy Agar (TSA; Becton Dickinson) plates. Colonies
were rinsed off with sterile water and the plate flooded with Gram's
iodine. A clear halo in the black iodine-stained medium was indicative
of starch hydrolysis (Tindall et al., 2007). Positive and negative controls
were, respectively, B. subtilis DSM 10 and Escherichia coli DSM 1103.
Degradation of barley beta-glucan, birch xylan and chitosan was
assayed by the appearance of blue halos on half-strength TSA plates
supplemented with the appropriate 0.2% (w/v) AZCL-cross-linked
polymers (Megazyme, Wicklow, Ireland). Negative control was provided by E. coli DSM 1103. Positive controls were Saccharophagus degradans
DSM 17024, Bacillus sp. UA-KA0902 and Cellulophaga chitinilytica DSM
17922 for, respectively, betaglucan, xylan and chitosan. Carboxymethylcellulose degradation was assayed on half-strength TSA plates
supplemented with the appropriate 0.2% (w/v) carboxymethylcellulose. Colonies were rinsed off with sterile water and the plate flooded
with 0.1% (w/v) Congo Red followed by a 3% (w/v) NaCl rinse. A clear
halo in the red stained medium was indicative of carboxymethylcellulose hydrolysis (Teather and Wood, 1982). Positive and negative
controls were, respectively, Bacillus sp. UA-AR0245 and E. coli DSM
1103. Dye decolorization was assayed by appearance of halos in halfstrength TSA supplemented with 0.2 g/L Azure B or 0.4 g/L Remazol
Brilliant Blue (RBB) (Pangallo et al., 2007). Positive and negative
controls were, respectively, Rhodococcus sp. UA-KA0206 and E. coli
DSM 1103.
2.4. DNA extraction, PCR amplification and partial 16S rDNA sequencing
Isolates were selected for identification based on colony morphology and/or enzyme activity. For the extraction of DNA from pure cultures
the UltraClean™ DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA,
USA) was used according to the manufacturer's protocol. Extracted
DNA was PCR amplified in an MJR PTC-200 thermocycler (MJ Research
Inc., MA, USA) using the universal primer pair 27F (5´-AGAGT
TTGATCCTGGCTCAG-3´) and 1492R (5´-TACCTTGTTACGACTT-3´)
(Lane, 1991) at a final concentration of 1.0 μM in a total volume of
25 μl of PCR mixture containing approximately 10 ng purified DNA
and 0.3 μl of Taq polymerase (New England Biolabs, Ipswich MA). The
PCR reaction was performed as follows: initial denaturation was for
2.5. Phylogenetic analysis
3. Results and discussion
3.1. Diversity of the cultured microbiota
A total of 382 isolates from the samples listed in Table 1 were
subcultured and deposited into the University of Akureyri culture collection where they were stored in 28% (v/v) glycerol at − 70 °C.
Reculturability from frozen stocks was confirmed for all isolates.
DNA was extracted, 16S rDNA amplified and sequenced from 104 isolates in the culture collection. After removal of ambiguously called sequence (average sequence length 1036 nucleotides), the sequences
were analyzed by MegaBLAST against the refseq_rna data, and classified by the RDP Classifier at a confidence of 70% or better (Table 2).
The 104 sequenced isolates yielded 19 genera and 4 non-genus specific assignments in 4 bacterial phyla (Actinobacteria, Firmicutes,
Bacteroidetes and Proteobacteria). Most isolates (65) were found to
belong to the Proteobacteria, with pseudomonads (46 isolates) being
particularly conspicuous.
3.1.1. Gammaproteobacteria
Markúsdóttir et al. (2012), who described pseudomonads in the Icelandic semiglacial river Glerá, surmised that these bacteria most likely
originated from soil and vegetation surrounding in that river's pristine
origin, and, with their metabolic versatility and biodegradative properties, were important contributors to the self-purification capacity of
Glerá river. In Fig. 2 we compare the 16S rDNA phylogeny of the pseudomonads isolated in the present study with those studied by
Markúsdóttir et al. As in the previous study, most of the isolates in the
present study show a high degree of similarity to members of the
P. fluorescens group, such as P. antarctica, P. thivervalensis, P. marginalis
and P. brenneri. Several members of the P. fluorescens species group,
including P. migulae and P. veronii were originally isolated from
spring water (Elomari et al., 1996, 1997; Verhille et al., 1999) and
some are psychrotrophic, such as the three Antarctic species P. antarctica,
P. meridiana and P. proteolytica (Reddy et al., 2004). The non-speciesgroup-assigned P. trivialis also frequently appears among top BLAST
90
Table 2
Isolates identified by partial 16S rDNA sequencing and biodegradatory tests on selected representatives.
Strains
GenBank accession numbers
Degradation tests of selected strainsa
RDP classification at 70% confidence threshold
Phylum
Class
Order
Family
Starch
β-glucan
Cellulose
Xylan
Casein
Azure B
RBB
Arthrobacter
+
+
−
−
−
−
−
Frigoribacterium
“Unclassified
Microbacteriaceae”b
+
+
−
(+)
−
−
−
−
−
−
−
−
−
−
Staphylococcus
+
−
−
−
+
−
−
Flavobacterium
++
+
+
−
+
−
−
Chryseobacterium
−
nd
−
−
−
−
−
Pedobacter
+
+
−
−
−
+
−
Rhizobium
−
−
−
−
−
−
−
Deefgea
Iodobacter
−
(+)
−
−
−
−
−
−
−
++
−
nd
−
−
Actinobacteria
Actinomycetales
Micrococcaceae
JF2601, JF2608, JF3201,
JF3701, JF3703
KC108912–KC108915,
KC583337
JF1532
JF4222
KC108916
KC108917
Microbacteriaceae
Firmicutes
Bacilli
Bacillales
Staphylococcaceae
JF4218
KC108918
Bacteroidetes
Flavobacteria
Flavobacteriales
Flavobacteriaceae
JF0634, JF0801, JF0827,
JF0927, JF1503, JF1504,
JF1506, JF1510, JF1511,
JF1513, JF1515, JF1530,
JF1537, JF1603, JF1604,
JF3206, JF3505, JF3608,
JF4207, JF4313, JF4316
JF4202
KC108919–KC108936,
KC583338–KC583340
KC108937
Sphingobacteria
Sphingobacteriales
Sphingobacteriaceae
JF0115, JF0701, JF1508
KC108938–KC108940
Proteobacteria
Alphaproteobacteria
Rhizobiales
Rhizobiaceae
JF3307
KC108941
Betaproteobacteria
Neisseriales
Neisseriaceae
JF1505
JF0906, JF2201, JF2216
KC108942
KC108943–KC108945
Burkholderiales
Burkholderiaceae
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
Genus
Actinobacteria
JF1514, JF3001
KC108946, KC583341
Janthinobacterium
(+)
−
−
−
−
−
−
Polaromonas
“Unclassified
Comamonadaceae”
−
−
−
−
−
−
−
nd
−
−
−
nd
−
nd
Paucibacter
nd
−
−
−
−
nd
−
Aeromonas
+
−
+
−
++
−
−
Rugamonas
Pseudomonas
−
−
−
−
−
++
−
−
+
++
−
−
nd
−
Serpens
−
−
−
−
+
+
−
Stenotrophomonas
−
−
−
−
+
−
−
Comamonadaceae
JF3006, JF3504
JF3607
KC583342, KC583343
KC583344
JF0116
KC108947
Incertae sedis
Gammaproteobacteria
Aeromonadales
Aeromonadaceae
JF4209, JF4213
KC108948, KC108949
Pseudomonadales
Pseudomonadaceae
KC108950
KC108951–KC108983,
KC583345–KC583352
KC108984
Xanthomonadales
Xanthomonadaceae
JF3405, JF3410, JF3417,
JF3418
KC108985–KC108988
Enterobacteriales
Enterobacteriaceae
JF0101, JF0212
JF0617, JF0918, JF0919,
JF1507, JF1709, JF4206,
JF4208
JF2207
KC108989, KC108990
KC108991–KC108997
Serratia
Yersinia
−
−
−
−
−
−
−
−
+
−
+
−
−
+
KC108998
−
−
−
−
−
+
−
JF1512
KC108999
“Unclassified
Enterobacteriaceae”c
“Unclassified
Proteobacteria”d
−
−
−
−
−
−
−
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
JF2904
JF0106, JF0302, JF0402,
JF0601, JF0705, JF0707,
JF0712, JF0804, JF1501,
JF1502, JF1518, JF2002,
JF2005, JF2501, JF2512,
JF2513, JF2806, JF2901,
JF2902, JF2906, JF3003,
JF3202, JF3203, JF3207,
JF3208, JF3302, JF3306,
JF3601, JF3602, JF3606,
JF4004, JF4006, JF4011,
JF4201, JF4205, JF4215,
JF4216, JF4220, JF4227,
JF4308, JF4310
JF3308
nd = not determined.
a
Selected representative strains are indicated in bold.
b
A MegaBLAST on the refseq_rna database at NCBI yielded greatest similarity (92% identity over 767 nucleotides) against Cryobacterium psychrotolerans strain 0549 (acc. no. NR_043892).
c
A MegaBLAST on the refseq_rna database at NCBI yielded greatest similarity (98% identity over 1172 nucleotides) against Serratia proteamaculans strain DSM 4543 (acc. no. NR_025341).
d
A MegaBLAST on the refseq_rna database at NCBI yielded greatest similarity (98% identity over 891 nucleotides) against Duganella zoogloeoides strain IAM 12670 (acc. no. NR_025833).
91
92
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
hits on both JáF and Glerá pseudomonad 16S rDNA sequences. It was
originally isolated from grass phyllosphere (Behrendt et al., 2003).
Among other gammaproteobacterial strains isolated in the present
study were several strains assigned to the genera Yersinia and Serratia,
both of which have long been known to include psychrotrophic
and psychrophilic strains, such as the common foodborne pathogen
Y. enterocolitica (Stern and Pierson, 1979) and Serratia marcescens, an
important source of cold-active proteases and lipases (Abdou, 2003;
Morita et al., 1997). Similar bacteria have been isolated from glacial environments before. For example Yersinia intermedia has been isolated
from glacial moraine soil (Huang et al., 2006) and members of Yersinia,
Serratia and other Enterobacteriaceae have been isolated from High
Arctic glacier streams (Dancer et al., 1997).
3.1.2. Betaproteobacteria
Although few in number, the betaproteobacterial isolates are fairly
diverse, five genera and one non genus-assigned isolate in two orders
being represented (Table 2). These include Iodobacter, a genus of
purple-pigmented facultative anaerobes often found in freshwater
(Logan, 1989). A recently described species, I. limnosediminis, was isolated from an Arctic lake sediment (Su et al., 2012). Another purple
betaproteobacterium isolated in the present study was found to be
similar to members of the genus Janthinobacterium, which have also
been found by others in glacial environments (Lee et al., 2011; Lu et
al., 2009).
Fig. 2. Molecular phylogeny of pseudomonad sequences generated in MEGA5 (Tamura
et al., 2011). The tree was inferred by using the maximum likelihood method based on
the Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (−3580.4357) is shown. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood (MCL) approach, and
then selecting the topology with superior log likelihood value. The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. The analysis involved 66 nucleotide sequences. All positions containing gaps and missing data
were eliminated. There were a total of 851 positions in the final dataset.
3.1.3. Bacteroidetes
Flavobacteria are found in various habitats, including soil, freshwater, sea water, seashore environments, food and dairy products, various
diseased animals and more. They have been found to be very important
in freshwater environments (Brümmer et al., 2000; Kirchman, 2002)
and in marine environments, particularly in the polar regions
(Bowman et al., 1997; Ravenschlag et al., 2001). Among reported habitats are particle-attached bacteria in a river estuary (Crump et al., 1999),
in a glacier in China (Zhu et al., 2003) and in Antarctic lake microbial
mats (Van Trappen et al., 2002). The isolates in the present study appear
to reflect a fairly diverse Flavobacterium population, as the 21
flavobacterial isolates are distributed widely among the reference sequences in the 16S rDNA phylogenetic analysis (Fig. 3). It should be
borne in mind, however, that species assignment based on 16S rDNA sequence data within the family Flavobacteriaceae is notoriously
unreliable (Bernardet and Nakagawa, 2006). Nevertheless, a brief discussion of the isolates' closest neighbors on the tree presented in
Fig. 3 is warranted. Most of the flavobacterial isolates turned out to be
most similar to flavobacteria from cold environments. These include
the Antarctic-lake isolate F. hibernum, a psychrotrophic, gliding, probable bacteriovore (McCammon et al., 1998), F. glaciei and F. xinjiangense,
both of which were originally isolated from a Chinese glacier (Zhang et
al., 2006; Zhu et al., 2003), and F. psychrolimnae which was originally
isolated from a microbial mat in an Antarctic lake (Van Trappen et al.,
2005). River water species are present among the closest relatives,
such as F. aquidurense and F. hercynium which were originally isolated
from a hardwater creek in Germany (Cousin et al., 2007). A single isolate, JF4202, was assigned to the flavobacterial genus Chryseobacterium,
often found in freshwater habitats (Bernardet et al., 2006). Three
flavobacterial isolates, UA-JF1537, 1603 and 0827 display comparatively
deep branching (0.038, 0.025 and 0.025 substitutions per site, respectively; Fig. 3) and when subjected to BLAST yielded sequence identities
of 95, 97 and 98% over sequences of 970, 1002 and 1060 nucleotides, respectively, against their best hit sequences in the refseq_rna database.
They may therefore represent novel species.
Members of the Sphingobacteriales are often found in great numbers in aquatic habitats that are rich in organic materials, where
they probably subsist on various biopolymers, which they are specialized to degrade (Reichenbach, 2006). Three sphingobacterial isolates
were identified, all assignable to the genus Pedobacter (Table 2).
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
93
Table 3
Summary results of biodegradative tests.
Substrate
Positives/Total screened
Starch
CM-cellulose
Barley betaglucane
Beechwood xylan
Caseine
Azure B
RBB
133/238
14/234
31/244
4/236
114/213
19/192
2/198
for production of several extracellular enzymes at 15 °C. A summary
of the screen can be found in Table 3, and some representative profiles
are presented in Table 2. Amylase and protease production, indicated by
starch and casein degradation, respectively, was common among the
JáF isolates, as indicated by the appearance of clear halos around the colonies within three days of culturing at 15 °C for more than half of the
strains assayed. Production of cellulase, betaglucanase and xylanase
was less common. Strong activity with 1 cm or wider clearance halos
appearing within a day after visible growth had developed was noted
for some strains, indicating enzyme production of potential biotechnological interest. These include amylase from Flavobacterium sp.
UA-JF1504, cellulase from Pseudomonas sp. UA-JF4216, and protease
from Iodobacter sp. UA-JF0906, Aeromonas sp. UA-JF4209 and Pseudomonas sp. UA-JF4216 (Table 2).
Laccases, lignin peroxidases and other lignolytic enzymes hold
great potential in several areas of biotechnology (Bugg et al., 2011;
Osma et al., 2010; Riva, 2006; Rodríguez Couto and Toca Herrera,
2006), including the food sector and bioremediation where
cold-active enzymes may be required. Plate-based dye decolorization
assays are a convenient, albeit not highly specific method to screen
for production of lignolytic enzymes. Azure B decolorization is usually
taken to be indicative of the presence of lignin peroxidase (Archibald,
1992; Pangallo et al., 2007), although many laccases are also capable
of decolorizing Azure B in the presence of redox mediators (Camarero
et al., 2005). Remazol Brilliant Blue (RBB) degradation is considered
indicative of the presence of laccase, also requiring the presence of
redox mediators (Soares et al., 2001). Nevertheless, dyes such as
Azure B and RBB have been used in growth media without mediator
Fig. 3. Molecular phylogeny of Bacteroidetes sequences generated in MEGA5 (Tamura
et al., 2011). The tree was inferred by using the Maximum Likelihood method based
on the Tamura-Nei model (Tamura and Nei, 1993). The tree with the highest log likelihood (−6198.7580) is shown. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise
distances estimated using the Maximum Composite Likelihood (MCL) approach, and
then selecting the topology with superior log likelihood value. The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. The analysis involved 48 nucleotide sequences. All positions containing gaps and missing data
were eliminated. There were a total of 635 positions in the final dataset.
3.1.4. Actinobacteria
The actinobacterial isolates in the present study were most similar
to Arthrobacter, Frigoribacterium and Cryobacterium spp. which appear
to be fairly commonly encountered in pristine, cold environments
(Gupta et al., 2004; Lee et al., 2011; Peeters et al., 2011), being nutritionally versatile and highly resistant to drying and starvation (Jones
and Keddie, 2006). The actinobacterial isolates in the present study
were isolated from ice, sand and riverwater samples in and along
the pristine, highland part of the river.
3.2. Bioprospecting for extracellular enzymatic activities
Cold-active enzymes, such as those produced by psychrophilic and
psychrotrophic bacteria, are of interest due to their applicability in biotechnology, agriculture, medicine and bioremediation (Cavicchioli et al.,
2011; Feller and Gerday, 2003). We therefore screened the JáF isolates
Table 4
Dye decolorization and laccase-specific PCR products for selected strains.a.
Isolate
JF0101
JF0212
JF0402
JF0408
JF0601
JF0607
JF0612
JF0617
JF0701
JF0705
JF0707
JF0804
JF0807
JF0827
JF0914
JF0918
JF0919
JF0920
JF0927
JF1622
JF1709
JF2207
Dye decolorization
PCR
RBB
Azure B
cotA
laccSI
laccCu
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
−
+
+
+
−
+
+
+
−
+
+
+
+
+
−
+
+
+
+
+
−
−
+
+
−
−
+
+
+
−
+
+
−
−
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
+
−
−
+
−
−
−
+
+
−
+
−
+
−
−
−
−
+
a
Results are shown for strains that displayed dye decolorization and/or yielded PCR
products with one or more of the indicated primer pairs.
94
J.P. Jóelsson et al. / Cold Regions Science and Technology 96 (2013) 86–95
supplementation for screening for bacterial lignolytic enzyme production, including both lignin peroxidase and laccase (Bandounas et
al., 2011; Pangallo et al., 2007).
Several isolates that tested positive for decolorization of Azure B
or RBB were tested for the presence of laccase-like genes by PCR
using three laccase-specific primer pairs, CotA, LaccSI and LaccCu,
and yielded PCR products from one or more of the primer pairs
(Table 4). While most dye-decolorizing strains yielded the expected
bands from one or more primer pairs, eight isolates (UA-JF0212,
0402, 0612, 0705, 0707, 0927, 1622 and 1709) positive for dye decolorization did not yield PCR products from any of the primer pairs, indicating either the production of a laccase not suffiently conserved to
yield products from the selected primer pairs, non-laccase-mediated
dye degradation or non-degradative dye sequestration. Several
strains negative for dye decolorization were also tested for laccase
by PCR. While most did not yield PCR products, three strains
(UA-JF0408, 0617 and 0827) yielded products from one or more
primer pairs, suggesting that while decolorization of RBB and Azure
B presents a useful tool for laccase bioprospecting, it should not be
considered an exhaustive screen.
4. Conclusions
The isolates cultured from glacial river water and other biotopes
along the JáF river form a diverse collection, identified strains being
assignable to four phyla. Although dominated by psychrotrophic
pseudomonads and flavobacteria, several other taxa were observed
and four isolates could not be confidently assigned to genera, indicating potentially novel taxa. Several strains were found to produce extracellular cold-active enzymes, including laccase, amylase, cellulase,
beta-glucanase and protease and may thus be of potential biotechnological interest.
Acknowledgments
This work was supported by grants from the University of
Akureyri Research Fund and the Landsvirkjun Energy Research
Fund. The authors also wish to thank Helga Árnadóttir and the
rangers of Vatnajökull National Park (www.vjp.is) for their valuable
input and discussions pertaining to the selection of and access to sampling sites. We also thank Guðrún Sigríður Jónsdóttir of Iceland
GeoSurvey (www.isor.is) for drawing the map in Fig. 1.
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