Effects of Growth Conditions on Siderophore Producing Bacteria and Siderophore Production from Indian Ocean Sector of Southern Ocean
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Received: 28 September 2018 | Revised: 20 December 2018 | Accepted: 29 December 2018 DOI: 10.1002/jobm.201800537 RESEARCH PAPER Effects of growth conditions on siderophore producing bacteria and siderophore production from Indian Ocean sector of Southern Ocean Alok K. Sinha1 | Bhaskar Parli Venkateswaran1 | Sarat C. Tripathy1 | Amit Sarkar2 | Sabu Prabhakaran1 1 National Centre for Polar and Ocean Research, Vasco da Gama, Goa, India Iron is an important element for growth and metabolism of all marine organisms, 2 Kuwait Institute for Scientific Research, including bacteria. Most (99.9%) of iron in oceans are bound to organic ligands like Safat, Kuwait siderophores and siderophore-like compounds. Distribution of bioavailable iron Correspondence mainly depends on pH and temperature of the ocean. Due to global warming and Parli V. Bhaskar, National Centre for Polar ocean acidification, bioavailability of iron may alter and in turn effect the response of and Ocean Research, Vasco da Gama marine bacteria. In this study, we investigated the effect of growth conditions like pH, 403804, Goa, India. Email: [email protected] temperature, and iron (III) concentrations on growth and siderophore production in selected heterotrophic bacteria isolated from waters around Kerguelen Islands (KW) Funding information and Prydz Bay (PB). Microcosm experiments were carried out on two KW-isolates Ministry of Earth Sciences, Govt. of India (Enterococcus casseliflavus and Psychrobacter piscatorii) and five PB-isolates (Pseudoalteromonas tetraodonis, Bacillus cereus, Psychrobacter pocilloporae, Micrococcus aloeverae, and Pseudomonas weihenstephanensis) which produced either hydroxamate-type or catecholate-type siderophores. Increasing iron concen- trations (10 nM to 50 μM) increased the growth rate of all isolates while siderophore production (% siderophore) generally reduced at higher iron concentration. Siderophore production peaked at early log phase, probably in response to higher iron-demand. Temperature and pH experiments showed that most isolates produced more siderophore at 15 and 25 °C temperature and pH 8.5. These results reveal that in future ocean conditions (warmer and acidified waters), bacterial growth and siderophore production may get affected and thereby influencing iron uptake and associated biogeochemical processes. KEYWORDS iron, microcosm, siderophore, Southern Ocean, warmer and acidified water 1 | INTRODUCTION essential metabolic activities of microorganisms [2]. Unlike terrestrial environment, concentrations of iron in open ocean Iron is one of the most important trace elements required for is extremely low and its distribution is patchy [3]. Dissolved growth of many terrestrial and marine microorganisms [1]. It iron concentration is higher in marine waters closer to land is required in micro-molar concentrations for growth and and/or surrounded by land mass as compared to open ocean. J Basic Microbiol. 2019;1–13.www.jbm-journal.com © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim | 1 2 | SINHA ET AL. For example, dissolved iron concentration in Arabian Sea is these waters can play an influential role in regulating greater and ranges from 1.25 to 60 nM [2] compared to open primary production and therefore affect the biological draw- ocean waters of Indian Ocean sector of Southern Ocean down of atmospheric carbon [3,18]. Siderophore production (IOSO) (0.046–0.38 nM) [3]. is influenced by bioavailability of iron and growth In recent decades, studies based on high sensitive conditions (temperature, nutrients, carbon source, etc.). electrochemical methods suggests that 99.9% bio-available Increasing atmospheric CO2 levels are reported to cause an iron in ocean are attached to organic ligands [4]. These are increase in the dissolution of CO2 in surface oceans, differentiated into two classes viz., organic compounds especially in the cold waters of Southern Ocean (SO), strongly bound to iron (L1) and is present in photic layers of altering the ocean pH [19]. Such decrease in pH may alter the water column, while second group of organic compounds the concentrations of bio-available iron for bacteria in (L2) which are weakly associated with iron and abundant in the marine waters [20]. However, none of the previous studies entire water column [5,6]. Although the structure of these in IOSO have highlighted the presence of siderophore organic ligands is not clear, these compounds are considered to producing bacteria or investigated the effect of growth be siderophores or siderophore-like compounds due to their conditions including pH, temperature, and iron concen- high affinity for iron [7]. Siderophores are low molecular trations on production of siderophores. weight organic ligands produced by bacteria (including some In the present study, we investigate the influence of cyanobacteria), fungi, and some higher plants [8]. Bacteria different concentrations of iron (III), temperature, and pH on produce siderophore for iron sequestration to support it's the growth and siderophore production of selected bacterial growth under iron limiting conditions (concentrations isolates obtained from marine waters near Kerguelen islands <10−5 mol L−1) in environment [8,9]. Some siderophore (KW) and Prydz Bay (PB). producing marine bacteria are also reported to stimulate the growth of other bacteria in iron deficient conditions by a 2 | MATERIALS AND METHODS process called siderophore signaling [10]. Apart from supporting the bacterial iron-demand, siderophore producing 2.1 | Sampling area bacteria in iron deficient marine environment may synergisti- cally support primary productivity by providing iron in In Indian Ocean sector of Southern Ocean (IOSO), exchange for organic carbon, thereby fueling the indirect Kerguelen basin has a surface area of 7000 km2 swept nutrient requirement to support microbial loop [11]. In by Antarctic Circumpolar Current (ACC), that is rich in contrast, siderophore producing marine bacteria were found nutrients [3] and naturally fertilized with iron from the to compete with phytoplankton for available iron and vitamins, islands, resulting in sporadic phytoplankton blooms while resulting in suppression of phytoplankton growth [12]. PB is the third most productive bay around Antarctica [21]. Bacterial siderophores are either hydroxamate or cat- Sampling locations were selected based on the proximity echolate-type siderophores; some bacteria are also reported to to land, nutrient rich waters, possible higher iron inputs produce carboxylate siderophore [13]. Two broad categories due to land mass effect (KW), and sea ice melting (PB). of siderophore viz. α-hydroxyaspartic acid or “citric acid” and Moreover, both KW and PB stations reflect different amphiphilic siderophores have been abundantly reported regimes based on differences in ambient temperature, from ocean regimes [11]. The α-hydroxyaspartic acid-type salinity, and nutrients. siderophores are photoreactive siderophores, which are prominent in surface ocean e.g., aerobactin, pertrobac- 2.2 | Sampling strategy tins [14], while amphiphillic types contain peptide heads and fatty acid tails e.g., aquachelins [15]. Seawater samples were collected from two stations each from IOSO waters encompass diverse oceanic regimes warm, PB (68°S 72°E and 68°S 76° E) and KW (47°S 64°E and 51°S nutrient limited frontal waters forming its northern limits to 64°E) during the 9th Indian Expedition to SO (ISOE-9) cold nutrient replete polar waters in the south. Nevertheless, onboard research vessel SA Agulhas in austral summer 2016– these waters are considered to be high-nutrient low- 2017 (Fig. 1). Sea water samples were collected using clean chlorophyll (HNLC) waters wherein iron is considered to 10 l Niskin bottles attached to CTD rosette equipped with be a major growth limiting nutrient of phytoplankton [16]. Sea-Bird CTD system (SBE911 plus, Sea-Bird Electronics, The IOSO extends from 40°S to the coast of Antarctica and USA) from three different depths (0 m, Deep Chlorophyll includes various frontal regimes with strong gradients in Maxima (DCM) and 200 m). Aliquots of water samples were hydrography and nutrients [17]. Presence of islands like collected for total cultivable bacteria (measured as colony Kerguelen islands and Crozet islands are known to naturally forming units, CFU), isolation of siderophore producing fertilize these waters, resulting in sporadic blooms of bacteria, total bacterial abundance, chlorophyll a, and phytoplankton [3]. Sequestration of iron by siderophores in nutrients. SINHA ET AL. | 3 2.3.3 | Nutrient analyses of KW and PB waters Aliquots (250 ml) of nutrient samples were collected in acid- cleaned bottle. Sample were stored at −20 °C and brought to the laboratory for analyses. The samples were thawed, brought to room temperature, and analyzed for dissolved nutrients (nitrate, nitrite, phosphate, silicate) using automated segmented nutrient analyzer (Seal UK; Model AA3), following standard colorimetric methods [24]. Standards were used to calibrate the auto-analyzer and frequent baseline checks were made. The standard deviation for all nutrients analyzed was ±1% and linearity of calibration curve yielded a correlation coefficient of R2 > 0.9990. 2.4 | Culturable bacteria from KW and PB waters Total heterotrophic culturable bacterial load in the sample was determined by serially diluting 100 μl sample into FIGURE 1 Sampling locations in Kerguelen waters (KW) and in different dilution (103,105, and 107) and spread on Zobell Prydz Bay (PB) during 9th Indian