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In earths crust Iron is the fourth abundant metal but the concentration of soluble ferric ions in solution at physiological pH is less than 10-17 M due to oxidation of ferrous to ferric oxyhydroxide polymers [4, 6]. Microorganisms require iron for various metabolic activities like electron transport chain, synthesis of heme, deoxyribonucleotide synthesis, methanogenesis, TCA cycle etc. [1, 2]. Microbes need minimum of 1 µM of Iron for their optimum growth and development [4, 6]. Cellular iron is very low in humans and is sequestered by ferritin, transferrin and lactoferrin as a primary defense mechanism at onset of infection . In order to sustain in the ubiquitous iron limiting environment, microbes synthesize and secrete selective and strong Fe (III) chelating low molecular weight compounds (500-1500 Da) that chelate iron from ferric complexes. These components are called siderophores and these uptake iron and other metals and transport them into the cell.
Siderophores have been divided into 3 classes based on the functional groups: a) Catecholate, b) Hydroxamate and c) Carboxylate [9, 10]. Siderophores are highly valuable molecules which possess potential application in environmental, agricultural and clinical aspects [8, 11]. Siderophores have shown antimalarial, ant parasitic, antiviral properties [12, 13, 14 ]. Albomycin and Ferrimycin (sideromycins) has shown ant parasitic activity employing Trojan horse strategy [17, 18]. Marine ecology which houses for many undiscovered species can be a potential source for new siderophores which might provide solution for many present and future challenges.
The present work is to study the effect of environmental factors on production of siderophore from Kocuria spp which was isolated from marine sponge Tedania anhelans collected from Arabian sea. The aim is to study the influence of the parameters such as media, NaCl concentration, carbon source and its concentration, Iron Fe(III) concentration , media pH and organic salts on production of siderophore and to optimize the maximum siderophore production.
Materials and Methods
Microorganism and growth conditions
Kocuria spp used for this study was maintained by frequent transfer on Nutrient Agar (pH 6.6 ± 0.2). Glycerol stocks were made for preserving the cultures and for further use. Defined Nutrient Broth (DNB) consisting (gl-1) peptic digest of animal tissue-5, beef extract- 1.50, yeast extract- 1.50, NaCl- 90, Glucose- 15, FeC l3- 0.003, K2HPO4 -2.70 .
Inoculum preparation and culture conditions
A loopful of cells was introduced into 30 mL of autoclaved Nutrient broth and incubated for 48 h. This was used as inoculum for further purposes.
The culture conditions were 37°C for 15 days, 1% inoculum volume. To remove any residual iron present, the glassware used for the experiments were prior washed with 6N HCl and rinsed with distilled water.
Factors affecting production of siderophore Growth medium
To study the production of siderophore, different media were used DNB [(g l-1) peptic digest of animal tissue-5, beef extract- 1.50, yeast extract- 1.50, NaCl- 90, Glucose- 15, FeCl3- 0.003, K2HPO4 -2.70 ]. Potato Dextrose Broth [(g l-1) potatoes infusion- 200, Dextrose- 20]. Actinomycetes broth [(g l-1) beef heart infusion-10, Tryptone-10, Casein enzyme hydrolysate-4, yeast extract, Dextrose-5, L-cyseteine hydrochloride-1, Starch-1, NaCl-1, KPO4 -15, NH4SO4-1, MgSO4 - 0.2, CaCl2 -0.02]. Production of siderophore was checked after 15 days incubation at 37°C.
Since Kocuria spp is a marine organism, NaCl concentration plays a crucial role in growth and development and its influence on production of siderophore was also studied. NaCl concentration varied from 0% - 15% w/v in steps of 3% w/v.
The influence of various carbon sources such as glucose, fructose, maltose, sucrose and glycerol was studied on production of siderophore by Kocuria spp. All carbon sources were added at 1.5 % w/v. also experiments were performed varying the concentration of glucose and sucrose in the range of 1 % to 2 % w/v.
In DNB, the source of phosphate is K2HPO4 . Concentration of K2HPO4 varied in the range of 0.0 % - 0.1 % and production of siderophore was estimated to study the influence of phosphate on production of siderophore.
Ferric chloride was added to DNB at various concentrations so that iron content in the media varies. Effect of iron in the range of 0 - 50 µM was studied.
pH of the DNB was varied from pH 5-10 and its influence on production of siderophore was studied.
Production of siderophore was quantitatively detected by Chrome Azural S Liquid Assay of Payne . After 15 days incubation, centrifuged (12000 rpm for 10 min) cell free supernatant was subjected to siderophore estimation in terms of % siderophore units. CAS assay solution and culture supernatant was mixed in 1:1 ratio. Uninoculated medium was used as blank and unicoluated media plus CAS assay solution was used as reference. The reduction of blue color to orange due presence of siderophore was measured at 630 nm. Production of siderophore was calculated using the formula,
% siderophore units = [(Ar-As)/Ar] * 100 Where, As- Absorbance of sample Ar- Absorbance of reference at 630nm
Cell free supernatant was subjected to Arnow's assay, Csaky's assay and to confirm the results, Neilands assay was also performed.
Results and Discussion
Kocuria spp was cultivated in DNB and checked for production of siderophore using Chrome Azurol S Liquid Assay . Effect of carbon source, NaCl, inorganic phosphate, iron, pH and inorganic salts on production of siderophore were studied.
Detection of Siderophore
Wine red color formation in Csaky's Assay  determined presence of Hydroxamate type and confirmed by Neiland's assay with appearance of peak at around 420 nm. Arnow's assay didn't give any red color formation and hence Kocuria spp is not producing any catecholate.
The Chrome Azurol S liquid assay detects siderophore presence based on the mobilization of iron molecules. In this method, a dye linked with iron molecules is used as a source for iron. When the iron molecules are trapped by siderophore, it releases the free dye resulting in the color change.
Influence of media on production of siderophore
Media influence on production of siderophore was studied in 3 different media such as DNB, PDB, A.B. Medium choice, level of iron requirement, producer organism performance and iron content availability in the medium decides level of siderophore production.
DNB favourd maximum production of siderophores ( % siderophore units) (Fig) followed by Potato Dextrose Broth ( % siderophore units) and Actinomycetes Broth ( % siderophore units). Hence for further experiments DNB was used.
Influence of NaCl Concentration
Siderophore production and organism's growth can be influenced by NaCl concentration since Koccuria spp is a marine microorganisms. NaCl concentration in the media varied from 0% - 15% w/v and NaCl concentration at 9 % w/v induced maximum siderophore production - 80.55 % siderophore units (Fig). NaCl concentration above 9% showed negative effect on growth and production of siderophore.
Influence of different carbon sources
Actinomycetes were classified based on their utilization of carbon sources by Pridham et al . Different carbon sources were fortified with DNB for 15 days at 37°C. Glucose and sucrose induced maximum siderophore production i.e., % siderophore units (Fig ).
Influence of Iron concentration on production of siderophore
Iron concentration is the most important factor that influences production and secretion of siderophore . Considering this, influence of Fe (III) added externally to DNB in increasing order on the production of siderophore was observed. Cell growth (cell dry weight) has increased with added Fe but siderophore production decreased. Production of siderophore has gone to maximum in absence of Fe (III) but in its presence, the production of siderophore decreased proportionally with externally added iron.
Iron concentration above µM couldn't induce production of siderophore which implies that this concentration of Fe (III) was sufficient for the Kocuria spp which lead to the production of negligible amount of siderophore production.
Influence of pH on production of siderophore
Dissolved iron influences production of siderophore, its solubility is determined by pH of the media. Iron is soluble at acidic pH and insoluble at neutral to alkaline pH. Hence production of siderophore is less at acidic conditions as Fe (III) solubilizes in the medium .
For Kocuria spp siderophore production the optimum pH of the media came to 8. Maximum siderophore production was at pH 8 ( %siderophore units) while very sharp decrease was shown at pH ( %siderophore units).