Journal of Experimental Marine Biology and Ecology 524 (2020) 151290

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Journal of Experimental Marine Biology and Ecology

journal homepage: www.elsevier.com/locate/jembe

Siderophore production by bacteria isolated from mangrove sediments: A T

microcosm study
⁎
Alok K. Sinha, Bhaskar V. Parli
National Centre for Polar and Ocean Research (NCPOR), Goa, 403804, India

A R T I C LE I N FO A B S T R A C T

Keywords: Mangroves are one of the most productive ecosystems worldwide covering up to 75% of the coastline in the
Mangroves tropics and subtropics. They support a highly diverse community (marine and terrestrial) and serves as reservoirs
Iron of nutrients for coastal and shelf waters. Bacterial diversity in mangroves includes heterotrophs, autotrophs
Siderophore (nitrogen fixation) and pathogens (phytopathogens, marine, and human). All these bacterial groups require
Microcosm
sequestration of bioavailable iron, which is largely done by the production of siderophores. In this study, mi-
Sediment
crocosm experiments were conducted to test the effect of incubation conditions (temperature, iron concentra-
tion, pH, and carbon source) on growth and siderophore production in four mangrove sediment bacterial iso-
lates- Escherichia vulneris, Enterobacter cancerogenus, Pantoea agglomerans, and Enterobacter bugandensis. Our study
showed that all isolates produce more siderophores (30 to 60%) at low iron concentrations (10 nM to 1 μM)
during lag-phase and early log-phase of growth. Low temperature suppressed bacterial growth without sig-
nificantly altering the siderophore production, whereas low pH suppressed both growth and siderophore pro-
duction in these isolates. Although all isolates could produce siderophores when using different carbon sources,
glucose served as an ideal carbon source. The observed changes in growth and siderophore production may be
attributed to species-specific physiological traits, changes in bioavailability of iron and/or combination of both.
Our results suggest that in a changing global environment, warming of the surrounding waters may not reduce
the siderophore production and hence, they will be essential in sustaining bacterial activity in sediments.

1. Introduction exposed to diverse carbon sources, pH changes (result of sediment-

Mangrove ecosystems are the most productive wetlands located
along tropical and subtropical coastlines. Approximately 137,760 km2
area of coastline is covered by mangroves globally across 118 countries
(Giri et al., 2011). Mangroves serve as breeding and feeding grounds to
various marine and terrestrial species and host a variety of organisms,
including birds, mammals, fish, and invertebrates (Feller et al., 2010;
Holguin et al., 2001). Mangroves also act as a barrier against tidal
currents and natural catastrophic events such as tsunamis (Dahdouh-
Guebas et al., 2005) and serve also as a carbon sink (Alongi, 2014).
Therefore, they are essential in the global carbon cycle (Lacava et al.,
2008). Apart from serving as a carbon sink, mangrove ecosystems are
important in sustaining coastal human populations and their economy
(Alongi, 2002; Durand et al., 2018; Mallick et al., 2018).
Mangroves are responsible for the net export of organic carbon and
nutrients to estuarine and coastal waters (Alongi, 2014). The sediments
are usually silty and poorly oxygenated (Kathiresan and Bingham,
2001) and are influenced by tidal flushing. As a result, microbes are
water exchange), and exchange of metals (especially iron) (Boto and
Wellington, 1988; Robertson and Phillips, 1995). High nutrient and
organic carbon concentrations coupled with high microbial turnover
rates and low porosity prevent gaseous exchanges. This results in in-
creased biological oxygen demand (BOD) in sub-surface sediment
layers, resulting in reduced bioavailability of iron (Fe3+). Various or-
ganisms have evolved to overcome the limitation in the bioavailability
of iron by producing siderophores and siderophore-like organic com-
pounds (Amin, 2010).
Siderophores are low molecular weight organic compounds pro-
duced by a variety of microorganisms, fungi, and plants, while growing
under iron limiting conditions (Gaonkar, 2015; Gaonkar and Borkar,
2016; Schalk and Braud, 2011). These compounds primarily chelate the
ferric iron (Fe3+) from different sources and thereby, make it available
for the cells. Apart from facilitating supply of iron for diverse cellular
functions including photosynthesis, respiration, bioremediation,
growth-promoting factors, and complexation with other essential ele-
ments such as Mo, Mn, Co, and Ni (Morel and Price, 2003; Saha et al.,

⁎
Corresponding author.
E-mail address: bhaskar@ncaor.gov.in (B.V. Parli).

https://doi.org/10.1016/j.jembe.2019.151290
Received 3 July 2019; Received in revised form 3 December 2019; Accepted 4 December 2019
0022-0981/ © 2019 Elsevier B.V. All rights reserved.
A.K. Sinha and B.V. Parli
2016), siderophores are also linked to pathogenicity in various bacteria
(Dale et al., 2004; Haldar and Nazareth, 2018).
Siderophore production is species-specific and varies with the mi-
crobial physiological and growth conditions (Braud et al., 2006; Sinha
et al., 2019). There are numerous reports on microbial communities
from mangroves sediments (Gaonkar and Borkar, 2016; Holguin et al.,
2001; Sayed et al., 2019; Vala et al., 2000) that primarily discuss the
microbial diversity and abundance (Haldar and Nazareth, 2018; Mallick
et al., 2018; Mukherji et al., 2019; Zhu et al., 2018). In this study, we
used a series of microcosm experiments to test the influence of in-
cubation conditions on growth and siderophore production by bacteria
isolated from mangrove sediments. Experiments were done to assess the
influence of different Fe3+ concentrations, temperature, pH, and
carbon forms on growth and siderophore production under laboratory
conditions.
2. Materials and methods
2.1. Sampling location and bacterial isolation
Duplicate sediment core (25 cm) samples were collected aseptically
from two different mangrove locations of Goa, namely Ribandar (Rib)
(15.505 N, 73.863 E) and Cortalim (Cot) (15.400 N, 73.908 E) (Fig. 1).
Ambient temperature, salinity, and pH of surface sediments were
measured at the sampling site. Samples were brought back to the la-
boratory at 4 °C and further processed for isolation of siderophore
producing bacteria. Each sediment core was sectioned at 2 cm interval
and from each section, 10 g of sediment was serially diluted and spread
plated onto Zobell Marine Agar plates at ambient temperature
(24 °C ± 2 °C) for 24–48 h. Morphologically different isolates based on
colony characteristics were selected and then further screened for
siderophore production and other biochemical analysis.
2.2. Identification of siderophore positive isolates
Chrome Azurol S (CAS) agar plate method (Schwyn and Neilands,
1987) was used to pick siderophore positive isolates. Siderophore
Fig. 1. Map of the study area showing Ribandar and Cortalim mangroves, Goa,
India.
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Journal of Experimental Marine Biology and Ecology 524 (2020) 151290
positive bacterial isolates were separated from total isolated colonies
based on an orange halo formed around colonies on CAS blue agar
background. CAS liquid assay method was used to quantify siderophore
in terms of percentage of iron utilization and colour change from blue
to orange was measured at 630 nm (UV–Vis spectrophotometer, Ep-
pendorf, Germany) (Granger and Price, 1999; Heui et al., 2001; Sinha
et al., 2019). In this assay, the amount of CAS replaced by siderophores
is measured spectrophotometrically and expressed as percentage of
siderophore. The reaction can be summarised as follows: FeCAS (Blue
colour) + L (Siderophore) → Fe.L + CAS (Orange or yellow). Various
assays as described in Sinha et al. (2019) were done with siderophore-
positive bacteria to ascertain the type of siderophore produced. In brief,
Cśaky assay (Csaky, 1948) was used to test for hydroxamate type
siderophores, wherein 1 ml of sulfanilic acid (1% w/v) prepared in
acetic acid 30% (v/v) with 0.5 ml iodine (1.3% w/v) was mixed to
equal volumes of sample and 6 N sulphuric acid and then hydrolyzed at
121 °C for 30 min. This mixture was then treated with 1 ml α-naph-
thylamine (0.3% w/v) for 5 min at room temperature and excess iodine
was removed by aqueous trisodium arsenite (Na3AsO2) (2% w/v). The
absorbance was measured at 256 nm after 30 min of incubation at
ambient temperature. Tetrazolium assay (Snow, 1954) was used as a
confirmatory test of hydroxamate siderophores (Sinha et al., 2019).
Neilands' method was used to identify the catecholate type of side-
rophore, wherein the sample formed wine colour complex with FeCl3
and was measured at 495 nm (Neilands, 1981). Arnow's assay was used
as a confirmatory test of catecholate production, wherein nitrous acid
reacts with catecholate siderophores and gives yellow colour, which
turns orange-red with an excess of NaOH. The mixture solution was
measured spectrophotometrically at 510 nm (Arnow, 1937; Lacava
et al., 2008). Vogel's assay was used for the qualitative analysis of
carboxylate siderophores. In this assay, three drops of 2 N NaOH were
mixed with one drop of phenolphthalein and water to make a pink
coloured working solution to which 1 ml of sample was added. The
disappearance of the pink colour indicated the carboxylate nature of
siderophore (Payne, 1994). Copper sulphate assay was also used to
identify the carboxylate type of siderophores. In this assay, 1 ml of
siderophore sample was mixed with 1 ml 250 μM CuSO4 and 2 ml
acetate buffer (pH 4). The presence of carboxylate siderophores was
confirmed by scanning absorbance between 190 and 280 nm (Shenker
et al., 1992).
2.3. Culture conditions and media for siderophore production
Prior to the preparation of growth media for the microcosm ex-
periments, all glassware was soaked overnight in 2 N HCl followed by
rinsing with deionised water. Two different types of media were chosen
for this study: (a) Iron deficient seawater based media (IDSM) (Guan
et al., 2000) to assess the effect of iron concentrations and (b) Minimal
media 9 (MM-9) for all other growth experiments that require fixed iron
concentrations and faster growth rate (Alexander and Zuberer, 1991).
The composition of MM-9 (g/l) included KH2PO4 0.3 g, NaCl 0.5 g,
NH4Cl 1 g, MgSO4.7H2O 493 mg, CaCl2 11 mg, MnSO4.H2O 1.17 mg,
H3BO3 1.4 mg, CuSO4.5H2O 0.04 mg, ZnSO4.7H2O 1.2 mg,
Na2MoO4.2H2O 1 mg, Cas-amino acids 3 g, PIPES 30.24 g, FeCl3.6H2O
10 M, EDTA 3.27 mg, and glycerol 5 g and 0.1% glucose. The compo-
sition of IDSM (g/l) included NH4NO3 0.25; NaCl 7.5; MgSO4.7H2O
0.125; KCl 0.075; K2HPO 4 0.375; CaCl2 0.05 and C8H18N2O4S (HEPES)
2.38 (Cabaj and Kosakowska, 2009; Murugappan et al., 2012). The
medium was adjusted for pH and de-ferreted by passing through
Chelex-100 (Sigma Aldrich, USA) (Price et al., 1989). After sterilisation,
0.1% glucose and 0.1 mM FeCl3.6H2O (dissolved in 10 mM HCl) filtered
through a 0.2 μm membrane were added. The overall composition of
both media is similar except that IDSM is deferrated and MM9 is more
enriched medium.
A.K. Sinha and B.V. Parli Journal of Experimental Marine Biology and Ecology 524 (2020) 151290

2.4. Effect of iron concentration, temperature, pH, and carbon source

Bacterial isolates were grown in different iron concentrations

(10 nM, 100 nM, 1 μM, 10 μM, and 50 μM) with each blank as a ne-
gative control in 250 ml acid cleaned flask containing IDSM. One day
old cultures were inoculated in each flask except for the negative
control and incubated at 24 °C and 200 rpm. The entire experiment was
done in duplicates and sampling for growth and siderophores produc-
tion was done at 24 h interval. To test the effect of different tempera-
ture (5 °C ± 1 °C, 15 °C ± 1 °C and 25 °C ± 1 °C), pH (5.5, 6.5, 7.5
and 8.5) and three different sources of carbon including glucose, xylose
and cellobiose on the growth and siderophores production, the selected
isolates were inoculated in MM9 media (Schwyn and Neilands, 1987) in
duplicate. Except for the temperature experiment, all incubations were
carried out at 24 ± 1 °C. All experiments were done by growing the
isolates at 200 rpm. Sub-samples for growth and siderophore produc-
tion in these experiments were collected only after 120 h. In all the
above experiments, controls were plain media without the cultures
which were also analysed for growth and siderophore production. All
the subsamples for growth and siderophore production were analysed
spectrophotometrically (Basic Spectrophotometer, Eppendorf, Ger-
many) at 540 nm and by CAS liquid assay at 630 nm (Granger and
Price, 1999), respectively.

2.5. DNA isolation and 16S rDNA sequencing

Bacterial DNA was isolated from cultures by Gene-Elute Bacterial

genomic DNA kit (Sigma, USA). The 16S rRNA genes were amplified by
using universal primers 27F (5’AGAGTTTGATCMTGGCTCAG-3′) and
1492R (5’TACGGTTAACCTTGTTACGACTT-3′) (Frank et al., 2008) and
25 μl Emerald Amp GT PCR Master Mix (Takara, India) for polymerase
chain reaction (PCR). DNA amplification was carried out by pre-dena-
turation at 95 °C for 5 min, followed by 25 reactions of denaturation at
95 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for
1 min. At the end of the PCR reaction, 10 min post extensions were
done at 72 °C using CFX96 PCR (Bio-Rad, India). Amplified DNA was
purified by PureLink PCR Purification Kit (Invitrogen, India) and se-
quencing of DNA was outsourced (M/s Agri-genome, India). The simi-
larity of the 16S rRNA gene sequences was checked using BLAST se-
quence similarity search to identify the nearest taxa and verified with
EzBioCloud (Yoon et al., 2017). Sequences were submitted to the na-
tional library of medicine, NCBI. Sequences were aligned with the
nearest taxonomic group using CLUSTAL-W before constructing phy-
logenetic trees using MEGA-7.0.9 (Shivaji et al., 2013).
3. Results
3.1. Bacterial identification and siderophore production
Bacterial colony-forming unit (CFU) abundance at sampling loca-
tion was high (45 × 106 to 48 × 106 CFU/g wet sediment) in the top
3 cm (Ribandar) and 4–5 cm (Cortalim), respectively, and gradually
decreased with depth. In total, 210 morphologically and biochemically
different bacteria from Ribandar and 203 bacteria from Cortalim were
isolated. Siderophore positive bacteria represented 45.8% of bacterial
isolates in Ribandar and 52.7% in Cortalim. From all the isolates, four
bacterial isolates producing > 50% siderophore based on CAS liquid
assay were selected. All four bacteria were Gram-negative producing
hydroxamate siderophore and were selected for growth experiment and
sequencing. The 16S rRNA sequencing results of these bacterial isolates
showed that these isolates were Escherichia vulneris (Mangrove-1, ac-
cession number- MN044881), Enterobacter cancerogenus (Mangrove-2,
accession number- MN044882), Pantoea agglomerans (Mangrove-3, ac-
cession number- MN044883) and Enterobacter bugandensis (Mangrove-
4, accession number- MN044884) (Fig. 2). Based on sequence simila-
rities search using EzBioCloud and BLASTn, our isolates showed > 99%
Fig. 2. Neighbour-joining 16S- rRNA gene sequence tree showing the phylo-
genetic relationship of mangrove strain, Mangrove-1 (MN044881), Mangrove-2
(MN044882), Mangrove-3 (MN044883), Mangrove-4 (MN044884). Bootstrap
replications were analysed with uniform rate using MEGA-7.0.9.
similarities with pathogenic strains (Supplementary Table 1).
3.2. Effect of growth conditions on bacterial growth and siderophore
production
Siderophore positive bacterial isolates were grown under five dif-
ferent iron concentrations (Fig. 3). Growth of Enterobacter bugandensis
was characterised by a short lag phase (24 h) followed by 24 h - 72 h log
growth phase, and prolonged stationary phase decrease with time at
different iron concentrations. Other isolates namely Enterobacter can-
cerogenus, Escherichia vulneris, and Pantoea agglomerans have prolonged
lag phase (120 h to 144 h), decreased with increasing iron concentra-
tion followed by short log phase of 48 h to 72 h. The cell density of
Enterobacter bugandensis, Escherichia vulneris, and Pantoea agglomerans
increased with iron concentration while Enterobacter cancerogenus cell
density decreased with iron concentration. In the presence of different
iron concentrations, siderophore production increased with an increase
in cell density after 16–20 h of incubation time (Fig. 3). Production
started at the early lag phase and maximum accumulation of side-
rophores occurred during the late log phase. Enterobacter cancerogenus
and Enterobacter bugandensis produced higher percentage (> 50%) of
siderophores at > 10 nM iron concentration, while Escherichia vulneris
and Pantoea agglomerans produced moderate amounts (30–40%) of
siderophore during lag phase, which increased during exponential
phase after 150 h and decreased after 200 h. Growth and siderophore
production of four isolates were observed in different temperatures
after 120 h (Fig. 4). Selected isolates were grown at three different
temperatures (5 °C, 15 °C and 25 °C). The growth and siderophore
production of all selected isolates were low at 5 °C. The growth of all
isolates increased at 15 °C and was maximum at 25 °C. Siderophore
production also increased with temperature for all isolates except En-
terobacter cancerogenus.
The effect of pH on growth and siderophore production after 120 h

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A.K. Sinha and B.V. Parli Journal of Experimental Marine Biology and Ecology 524 (2020) 151290

Fig. 3. Line graph of bacteria Escherichia vulneris (MN044881), Enterobacter cancerogenus (MN044882), Pantoea agglomerans (MN044883) and, Enterobacter bu-
gandensis (MN044884) represent growth curve (A, C, E, G and I) and siderophore production (B, D, F, H, J) at five increasing iron concentrations of 10 nM, 100 nM,
1 μM, 10 μM, and 50 μM. Abscissa shown at each point is standard deviation of duplicate samples.

4
A.K. Sinha and B.V. Parli
Fig. 4. Bar graph of bacteria Escherichia vulneris (MN044881), Enterobacter cancerogenus (MN044882), Pantoea agglomerans (MN044883) and, Enterobacter bugandensis
(MN044884) representing growth (A, C and E) and siderophore production (B, D and F) after 120 h incubation at three different temperatures of 5 °C, 15 °C, 25 °C.
Abscissa on each bar represent standard deviation of duplicate samples.
incubation is shown in Fig. 5. All bacterial isolates did not show any
significant growth and siderophore production at pH 5.5. Growth in-
creased at pH 6.5 and 7.5, but siderophore production was maximum at
6.5 and decreased at 7.5. Both growth and siderophore production
decreased at pH 8.5. When subjected to three different carbon sources
namely glucose, xylose and cellobiose, growth and siderophore pro-
duction were maximum in the presence of glucose compared to xylose
and cellobiose (Fig. 6).
4. Discussion
Surface mangrove sediments are alluvium types rich in organic
matter (Kathiresan and Bingham, 2001) that act as a sink of organic
carbon and can support a variety of microorganisms (Holguin et al.,
2001). However, recycling of nutrients and detritus and decomposition
of organic matter by microbes, leads to an increase in oxygen demand
creating a reducing environment within mangrove sediments. Bacterial
abundance was high (45 to 48 × 106 CFU/g wet sediment) in the top
3–5 cm at two locations and gradually decreased with depth, which
corroborated with earlier studies from mangroves and sand dunes
(Gaonkar et al., 2012; Gaonkar and Borkar, 2016). A decrease in
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Journal of Experimental Marine Biology and Ecology 524 (2020) 151290
bacterial abundance (CFU) with depth could be attributed to (a) in-
cubation conditions that supported aerobic bacteria and (b) reduced
sediment nutrient and/or organic carbon availability with depth. We
found that 45 to 53% of morphologically different bacterial isolates
were siderophore positive. This was higher than those reported from
sand dunes (16 to 20%) (Gaonkar et al., 2012). Since siderophore
producing bacteria can also induce other bacteria to produce side-
rophores (Champomier-verges et al., 1996), such a likelihood cannot be
ruled out in our samples.
Most of the mangrove based microbial studies have focussed on
microbial diversity (Chen et al., 2016; Ghizelini et al., 2012; Haldar and
Nazareth, 2018), productivity (Alongi, 1988), biotechnological poten-
tial (Yu et al., 2005; Zhou et al., 2008; Dias et al., 2009), nutrient re-
generation and recycling of organic matter (Cao et al., 2011). More-
over, reports on siderophore producing bacteria from mangrove
sediments focused on endophytes, phyto-pathogens, nitrogen fixers etc.
(Gaonkar et al., 2012; Gaonkar and Bhosle, 2013; Eljounaidi et al.,
2016; Naik et al., 2017; Haldar and Nazareth, 2018; Zhu et al., 2018).
In this study, all siderophore positive bacterial isolates were Gram-ne-
gative bacteria belonging to Proteobacteria phylum, and γ- proteobacteria
class which produced hydroxamate type siderophores. Strains of these
A.K. Sinha and B.V. Parli Journal of Experimental Marine Biology and Ecology 524 (2020) 151290

Fig. 5. Bar graph of bacteria Escherichia vulneris (MN044881), Enterobacter cancerogenus (MN044882), Pantoea agglomerans (MN044883) and, Enterobacter bugandensis
(MN044884) representing growth (A, C, E and G) and siderophore production (B, D, F and H) after 120 h incubation at pH 5. 5, 6.5, 7.5 and 8.5. Abscissa on each bar
represents standard deviation of duplicate samples.

isolates are known to be invasive and opportunistic pathogens
(Atanassova et al., 2014; Aydin et al., 1997; Kellogg et al., 2016; Li
et al., 2015) and are widely reported in wound infections (Pati et al.,
2018). Pathogenicity of the isolates in this study was not addressed,
however, they showed high similarities to pathogenic strains (Supple-
mentary Table 1). Therefore, these isolates may be considered as re-
presentative of pathogenic strains.
Since siderophores are produced to sequester extremely low con-
centrations of bioavailable iron, the hypothesis that iron concentration
may regulate the production of siderophores in bacteria was tested. In
the microcosm experiments, acidified iron was added to the media in
five different iron concentrations; although bioavailable iron con-
centration was not estimated, it is assumed that the bulk of the acidified
iron added was bioavailable. The concentrations of iron added were
selected based on an earlier experiment (Cabaj and Kosakowska, 2009)
for comparative purposes. The lower iron concentration (10 nM) mat-
ched the dissolved available iron concentration of the Arabian Sea
(Witter et al., 2000), while 1 μM concentration was close to the dis-
solved iron concentration reported from the overlying waters in the
sampling sites (Mesquita and Kaisary, 2007). Although higher iron

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A.K. Sinha and B.V. Parli Journal of Experimental Marine Biology and Ecology 524 (2020) 151290

Fig. 6. Bar graph of bacteria Escherichia vulneris (MN044881), Enterobacter cancerogenus (MN044882), Pantoea agglomerans (MN044883) and, Enterobacter bugandensis
(MN044884) representing growth (A, C and E) and siderophore production (B, D and F) after 120 h incubation using different carbon sources namely Glucose, xylose,
and cellobiose. Abscissa on each bar represents standard deviation of duplicate samples.

concentrations are not reported in marine waters, total iron con-
centration in mangrove sediments is significantly higher (Mesquita and
Kaisary, 2007), resulting in bioavailable iron concentrations up to
50 μM. Nevertheless, it may be too high for bacterial growth (Cabaj and
Kosakowska, 2009). The influence of iron (III) on the growth and
production of siderophore at lower iron concentration was relatively
slower than at higher iron concentration (Fig. 3). This was similar to
those reported from mangrove sediments and sand dunes (Gaonkar and
Bhosle, 2013).
Under low iron concentration, bacteria tend to produce more side-
rophore (Schalk and Braud, 2011) to chelate iron for metabolic activ-
ities. By contrast, under higher iron concentration, this element may be
taken up using alternate pathways other than the siderophore me-
chanism (Sandy and Butler, 2010). Overall siderophore produced by
bacteria decreased with an increase in iron concentration. Enterobacter
bugandensis produced siderophores during the lag phase (faster pro-
duction rate) and attained the log phase earlier (faster growth rate)
than other bacterial isolates (Fig. 3). The slow siderophore production
(96–120 h) and slow growth (after 120 h) might be due to the need to
accumulate iron for metabolic activities followed by the initiation of
growth (log phase) (Andrews et al., 2003). Growth of Enterobacter
cancerogenus decreased with increasing iron concentration, but side-
rophore production increased with iron concentration. Although bac-
teria produce siderophores primarily to sequester iron at low con-
centrations, siderophores also play an important role in the dissolution
of minerals (Liermann et al., 2000; Schalk and Braud, 2011), uptake of
vitamins/growth factors, virulence in pathogens (Neilands, 1986). The
observed growth and siderophore production may be in response to
physiological needs and may vary with species (Tsolis et al., 1996).
Temperature can affect the bacterial metabolic activity, especially
respiration rate, and protein expression, while pH can affect the side-
rophore structural stability and iron redox chemistry (Borer et al.,
2009). Bacterial density, and siderophore production of all isolates,
increased with temperature (5 °C < 15 °C < 25 °C), which reflected
the ambient temperature (23 °C) of the sampling sites. These isolates
were obtained from inter-tidal sediments which are periodically ex-
posed to high insolation during low tides. The high production of
siderophores at higher temperatures (Fig. 4) indicated that the side-
rophore gene expression increased with incubation temperature, which
may be in response to their ambient environmental conditions.
Stability of siderophore is observed between pH 6 to 8, but it is
negatively affected at lower and higher pH (Pullin and Cabaniss, 2003).

7
A.K. Sinha and B.V. Parli
Mangrove sediments in the sampling sites are constantly flushed by
tidal waters, land run-off and freshwater inputs from near-by human
habitats. This may either directly alter the pH of overlying waters or
indirectly influence the pH due to resuspension of surface sediments
(Lankford and Byers, 1973). Bacterial growth and siderophore pro-
duction (Fig. 5) was influenced by pH and production was higher at
pH 6.5 and 7.5. This is similar to those reported earlier (Pullin and
Cabaniss, 2003). Sinha et al. (2019) reported maximum siderophore
production at pH 8.5 in strains isolated from open oceans. Although the
pH of overlying waters at the sampling site was 8.2, lower siderophore
production at pH 8.5 in this study may be due to the physiological traits
of the isolates.
Carbon sources used in this study were monomers and oligomers of
complex polysaccharides including cellulose and xylan. All the isolates
showed slight variations in growth in glucose, xylose and cellobiose
supplemented medium. Nevertheless, siderophore production was de-
coupled from growth and all isolates showed similar production of
siderophores in all sugars. Mangroves are woody plants and are rich in
cellulose and xylan. Xylose is a monomer of xylan while cellobiose is a
dimer obtained by degradation of cellulose. Both glucose and xylose are
abundantly found in mangrove sediments of the sampling area (Khodse
et al., 2008). In our experiment, all isolates used glucose as the pre-
ferred source of carbon, but also could utilize diverse carbon sources to
support their growth and siderophore production.
Mangroves harbour euryhaline microbial species that are exposed to
diverse organic carbon sources, varying pH and bioavailable iron con-
centrations. Our results show that for a dynamic environment such as
mangroves, iron concentrations and pH influenced both growth and
siderophore production of sediment bacterial isolates, where tempera-
ture affected only growth of the isolates. Moreover, the isolates were
more adapted to utilize different carbon sources and siderophore pro-
duction was not influenced by carbon source. Several strains of isolates
used in this study are pathogenic in nature. Hence our results may serve
as an indicator of possible responses of such organisms to changing
global climatic conditions. In a rapidly changing global climate wherein
ocean temperatures are predicted to be warmer by 1.5 °C by 2030
(Masson-Delmotte et al., 2018), increased siderophore production and
growth at warmer temperatures by such strains should be of concern
and needs to be addressed.
Funding
Ministry of Earth Science (MoES), Government of India (GOI).
Research involving human participants and/or animals
This article does not contain any studies with human participants or
animals performed by any of the authors.
Declaration of Competing Interest
The authors declare that they have no competing interest.
Acknowledgments
We thank the Director, National Centre for Polar and Ocean
Research (NCPOR) for his continued support and Miss Pallavi Bhardwaj
for her contribution during this study. We also thank the Ministry of
Earth Sciences (MoES), Government of India (GOI) for funding of
SOCarP project and fellowship of Alok K. Sinha. We also express our
thanks to the two anonymous reviewers and Prof. Ruben Sommaruga
whose comments and suggestions vastly improved the quality of the
text. This is NCPOR contribution number J-47/2019-20.
8
Journal of Experimental Marine Biology and Ecology 524 (2020) 151290
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jembe.2019.151290.
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