Geomicrobiology Journal Siderophore production by microorganisms isolated from a podzol soil ...

46 Pages·2014·1.65 MB·English

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Siderophore production by microorganisms isolated
from a podzol soil profile
Engy Ahmeda & Sara J. M. Holmstromb
a Postal address: Department of Geological Sciences, Stockholm University, SE-10691
Stockholm, Sweden
b Postal address: Department of Geological Sciences, Stockholm University, SE-10691
Stockholm, Sweden, Phone: [phone], Fax: [phone], E-mail:
Accepted author version posted online: 10 Sep 2014.
To cite this article: Engy Ahmed & Sara J. M. Holmstrom (2014): Siderophore production by microorganisms isolated from a
podzol soil profile, Geomicrobiology Journal
To link to this article: http://dx.doi.org/10.1080/01490451.2014.925011
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Title: Siderophore production by microorganisms isolated from a podzol soil profile
Author names and affiliations:
Engy Ahmed (Corresponding author)
Postal address: Department of Geological Sciences, Stockholm University, SE-10691
Stockholm, Sweden
4 E-mail: [email protected]
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Abstract
Siderophore producing bacteria/actinobacteria and fungi were isolated from O- (organic), E-
(eluvial), B- (upper illuvial), and C- (parent material) horizons of podzol soil. Siderophores were
isolated and hydroxamate type siderophores were detected and quantitated by high-performance
liquid chromatography coupled to electrospray ionization mass spectrometry. The molecular
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2 identification of siderophore producing isolates showed that there was a high diversity of fungal
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1. Introduction
Iron (Fe) is an essential element for the growth of almost all living microorganisms since it acts
as a catalyst in enzymatic processes, oxygen metabolism, electron transfer, and DNA and RNA
synthesis (Hayat et al., 2010; Aguado-Santacruz et al., 2012). Due to the low bioavailability of
Fe in the environment, microorganisms have developed specific uptake strategies like production
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2 of siderophores (Ahmed and Holmström, 2014a). Siderophores are metal chelating agents with
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e low molecular masses (200 to 2000 Daltons), which provide the microorganisms with an
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y The production of the siderophores could help the microorganisms in their competition for
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a Soil microorganisms produce a wide range of siderophores. Bacteria mainly produce three
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Catecholate or phenolate siderophores are cyclic tri-ester of 2,3-dihydroxybenzoylserine, which
is characterized by extremely high stability constants (Pollack et al., 1970; Patel et al., 2009).
The main catecholate type siderophores, enterobactin, is produced by Escherichia coli and
Enterobacter, which are commonly associated with plants (Crowley, 2006). There are also
various catecholate siderophores produced by soil bacteria like agrobactin, which is produced by
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Agrobacterium tumefaciens; dihydroxybenzoic acid by Erwinia sp. and Bacillus subtilis;
mycobactins by Mycobacterium, Nocardia, and Rhodococcus, and pyochelins by Pseudomonas
spp. (Ratledge, 1987). The second major group of bacterial siderophores is hydroxamates, which
have both linear and cyclic compounds containing 1-amino-5-hydroxyaminopentane (Dhungana
et al., 2001). Hydroxamates are resistant to hydrolysis and enzymatic degradation in the natural
environment due to their hexadentate structure (Winkelmann, 2007). Bacterial hydroxamates
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2 such as schizokinen, aerobactin, and ferrioxamines are produced for example by Streptomyces
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e spp., Enterobacteriaceae, and Arthrobacter spp. (Lee et al., 2012). The third group of bacterial
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y a mix of hydroxamate and carboxylate groups like pyoverdines, which are commonly produced
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Soil fungi mainly produce four different groups of siderophores included in the hydroxamate
family, i.e. ferrichromes, coprogens, fusarinines, and rhodotorulic acids (Winkelmann, 2007).
Ferrichromes, the most common type of siderophores produced by soil fungi, are based on a
cyclic hexapeptide structure (Leong and Nielands, 1982; Deml et al., 1984). Ferrichromes are
further divided into five groups depending on the side chain of the hydroxamate functional
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group: acetyl (ferrichrome, ferrichrome C, ferricrocin, and ferrichrysin), malonyl
(malonichrome), trans-b-methylglutaconyl (ferrichrome A), trans-anhydromevalonyl (ferrirubin),
and cis-anhydromevalonyl (ferrirhodin) (Renshaw et al., 2002; Winkelmann, 2007). Common
soil fungi that produce ferrichrome type siderophores are U. sphaerogena for ferrichrome
(Emery, 1971), Aspergillus fumigatus for ferricrocin (Wallner et al., 2009), Neurospora crassa
for tetraglycylferrichrome (Winkelmann, 2007) and Cenococcum geophilum and Hebeloma
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2 crustuliniforme for ferrichrysin (Martino and Perotto, 2010). Coprogens consist of a
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e diketopiperazine ring formed by two N5-acyl-N5-hydroxy-L-Orn units (Budzikiewicz, 2010).
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y and Challis, 2009). Rhodotorulic acid contains two acetyl groups and has previously been
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Given the great impact of siderophore producing soil microorganisms in mineral weathering,
soil formation and plant growth promotion, the present study aims to: a) study the diversity of
siderophore producing microorganisms within the horizons of podzol soil, and b) determine the
types and concentrations of hydroxamates produced by the isolated microorganisms.

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2. Materials and Methods
2.1 Sampling site
Soils were sampled in September 2011 near the village Bispgården, central Sweden (63°07′N,
16°70′E). The site is located on a slope (angle 2°) at an altitude of 258 m above sea level and is
forested with 80-yr-old Norway spruce (Picea abies) and Scots pine (Pinus sylvestris). The
4 annual average precipitation is 700 mm. The annual average temperature is +2 °C. The bedrock
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er in the area is granite and gneiss. The soil is a typical haplic podzol (FAO, 1990). The soil
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e horizons in the studied soil profile have the following depths: 12 cm for O (organic horizon), 10
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3 cm for E (elluvial horizon), 9 cm for B (upper illuvial horizon) and finally C (parent material).
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at The soil samples for this study were taken from the bulk soil of each specific soil horizon for the
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b whole profile and kept cold (+4 °C) until further analysis. The characteristics of the soil samples
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ed The aerobic and facultative anaerobic siderophore producing microorganisms were isolated from
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w soil solution. Soil solutions were obtained by shaking 5 g of soil in 15 ml of Milli-Q water with
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glass pearls for 4 h and then centrifuged for 15 min at 5000 rpm. Then the supernatant were
serially diluted to minimize the transfer of Fe from the soil solution to the growth plates. 0.1 ml
of the 1/1000 and 1/10000 dilutions of the soil solution were plated using the spread plate
technique onto two replicates of the appropriate media containing diluted chrome azurol S (CAS)
indicator in order to be selective for the isolation of siderophore producing microorganisms. The
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CAS was diluted in order to avoid a possible growth inhibition caused by its toxicity for some
microbial species (Milagres et al., 1999). Fungi were isolated using modified ½MMN (Melin
Norkans agar): Glucose 5 g/l, Malt extract 3 g/l, (NH ) HPO 0.25 g/l, KH PO 0.5 g/l, NaCl
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0.025 g/l, Agar 15 g/l; the inoculated plates were incubated at 28°C for 4-6 days. Bacteria and
actinobacteria were isolated using soybroth agar (Bacto Tryptone 17 g/l, Bacto soytone 3 g/l,
Glucose 2.5 g/l, NaCl 5g/l, Agar 15g/l) and the inoculated plates were incubated at 28-37°C for
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2 3-7 days. The color change from blue to orange was an indicator of the production of
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e siderophores. When the microorganisms secrete siderophores in the blue colored CAS media, the
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according to the microbial type and were incubated on a rotary shaker (200 rpm) for 2-4 days.
The biomass was harvested by centrifugation at 3000 rpm for 10 min, then washed twice with
sterilized MilliQ water and again centrifuged at 3000 rpm for 10 min to collect the microbial
cells. The microbial genomic DNA was extracted by using Genomic DNA Miniprep Kit
(Axygen Biosciences, USA) according to the manufacturer’s instructions. The quantity of DNA
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was measured using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher Scientific, USA)
at 260 nm. The molecular identification was carried out by amplification of a set of universal
primers. For bacteria and actinomycetes, 518F (5’- CCA GCA GCC GCG GTA ATA CG -3’)
and 800R (5’- TAC CAG GGT ATC TAA TCC -3’) primers were used. For fungi, ITS1f (5’-
CTT GGT CAT TTA GAG GAA GTA A -3’) and ITS4 (5’- TCC TCC GCT TAT TGA TAT
GC -3’) primers were used. The PCR reaction was performed by adding 1µl of template DNA in
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2 50µl of PCR reaction solution (10X MGTM Taq-HF buffer 10µl; 2mM MGTM dNTP mixture
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e 10µl; 10pmol Primer 5µl; MGTM Taq-HF polymerase 1µl; Distilled water). The thermal cycling
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sity by the sequencing service Macrogen inc. (http://www.macrogen.com). The obtained sequences
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evaluated by using the bootstrap test (1000 bootstrap replication) (Felfenstein, 1985). All the
sequences were submitted to the GeneBank and were got accession numbers.

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2.4 Screening for Fe accumulation by the isolated siderophore producing microorganisms
In order to screen the different abilities of the isolated microorganisms for Fe accumulation as a
microbial activity, the isolated microorganisms were inoculated in TSA media plates (Tryptone
15 g/l, Soya peptone 5 g/l, NaCl 5 g/l, agar 15 g/l) amended with a wide range of Fe (FeCl )
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concentrations (40, 60, 80, 100, 140, 160, 180, 200 mg/l) and were incubated for 3-7 days at
4 28°C. The accumulation of Fe was indicated by the formation of dark haloes around the cultures
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be as a result of the diffusion of dissolved Fe towards the microorganisms (Pümpel et al., 1995).
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shaker (200 rpm) for 7 days. Centrifugation at 3000 rpm for 10 min was used to collect the
biomass. A low concentration (1%) of FeCl was added to the microbial filtrates during stirring.
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Then the brown microbial filtrates were filtrated through 0.45 μm filters (Filtropur S, Sarstedt,
Germany). The filtrates were pre-concentrated by freeze-drying (Scanvac cool Safe, 100-9 Pro).
The remaining yellow-brown solid dust was dissolved in 1 ml of MilliQ-water after the freeze-
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