Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria

3 Biotech, Oct 2017

In this study, siderophore production by various bacteria amongst the plant-growth-promoting rhizobacteria was quantified by a rapid and efficient method. In total, 23 siderophore-producing bacterial isolates/strains were taken to estimate their siderophore-producing ability by the standard method (chrome azurol sulphonate assay) as well as 96 well microplate method. Production of siderophore was estimated in percent siderophore unit by both the methods. It was observed that data obtained by both methods correlated positively with each other proving the correctness of microplate method. By the modified microplate method, siderophore production by several bacterial strains can be estimated both qualitatively and quantitatively at one go, saving time, chemicals, making it very less tedious, and also being cheaper in comparison with the method currently in use. The modified microtiter plate method as proposed here makes it far easier to screen the plant-growth-promoting character of plant-associated bacteria.

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Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria

Biotech Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria Naveen Kumar Arora 0 1 Maya Verma 0 1 0 Rhizosphere Microbiology Laboratory, Department of Environmental Microbiology, BBA University , Lucknow, UP 226025 , India 1 & Naveen Kumar Arora In this study, siderophore production by various bacteria amongst the plant-growth-promoting rhizobacteria was quantified by a rapid and efficient method. In total, 23 siderophore-producing bacterial isolates/strains were taken to estimate their siderophore-producing ability by the standard method (chrome azurol sulphonate assay) as well as 96 well microplate method. Production of siderophore was estimated in percent siderophore unit by both the methods. It was observed that data obtained by both methods correlated positively with each other proving the correctness of microplate method. By the modified microplate method, siderophore production by several bacterial strains can be estimated both qualitatively and quantitatively at one go, saving time, chemicals, making it very less tedious, and also being cheaper in comparison with the method currently in use. The modified microtiter plate method as proposed here makes it far easier to screen the plant-growth-promoting character of plant-associated bacteria. Siderophore; PGPR; CAS assay; Microplate; Plate reader Introduction Siderophores are low-molecular weight secondary metabolites with iron-chelating potential. These are compounds with small peptidic molecules having side chains and functional groups which have high-affinity ligand to bind ferric ions and transport them through the cell membrane (Raymond et al. 2015; Niehus et al. 2017) . Siderophores are produced by various microorganisms and are classified into four main classes (carboxylate, hydroxamates, catecholates, and mixed type) on the basis of their structural features, functional groups, and types of ligands (Table 1) (Ali and Vidhale 2013; Kumar et al. 2017; Miethke and Marahiel 2007; Aznar et al. 2015) . Diverse bacterial and fungal genera ranging from human pathogens to environmental microbes such as plant-growth-promoting rhizobacteria (PGPR) are reported to produce siderophores. One of the key mechanisms of PGPR in promoting plant growth involves the production of secondary metabolites such as siderophores (Verma et al. 2011; Ghavami et al. 2017) . Although iron is abundantly available in soil, most of it is unavailable to the plant or other organisms, because it forms insoluble complexes. Hence, iron deficiency is a major global issue. Siderophores produced by PGPR help in fulfilment of the iron requirement of plants by causing its solubilisation and chelation from organic or inorganic complexes present in soil (Wandersman and Delepelaire 2004; Arora et al. 2013; Singh et al. 2017) . Microbial siderophores strongly chelate iron and enhance iron uptake by forming a ferric–siderophore complex even at very low concentrations (Dimkpa et al. 2009; Ferna´ndez-Scavino and Pedraza 2013; Boiteau et al. 2016) . Siderophores thus not only help in enhancing plant growth, but also play a very important role in providing iron to other organisms including humans. Siderophores produced by PGPR also S. Siderophore no. type 1 2 3 4 Hydroxamate Esters or acid chlorides or carboxylic Ferrioxamine B–Pseudomonas acids fluorescence Catecholates Phenolate or 2,3-dihydroxy benzoate Enterobactin–Escherichia coli (DHB) binding groups Carboxylates Hydroxyl carboxylate and carboxylates Mixed type Mixture of above mentioned Pyoverdine–Pseudomonas functional groups aeruginosa Rhizobactin–Rhizobium meliloti (Maurer et al. 1968) ; Radhakrishnan et al. (2014) Dave et al. (2006) ; Grobelak and Hiller (2017) Smith and Neilands (1984) ; Ghavami et al. (2017) Leong and Neilands (1982) ; Behnsen and Raffatellu (2016) help in protection of plant from phytopathogens (Arora 2015; Saha et al. 2016) . Phytopathogens are inhibited in rhizosphere by siderophore-producing PGPR because of iron starvation or due to competitive exclusion in irondeficient conditions (Beneduzi et al. 2012; Parmar and Chakraborty 2016; Dalvi and Rakh 2017) . Besides plant growth promotion, siderophores also play an important role in bioremediation of heavy metals from contaminated sites by binding to the toxic metals such as Cr3?, Al3?, Pb2?, Cd2?, Hg2?, etc. (Saha et al. 2015) . Siderophore-producing microorganisms can thus be used to detoxify heavy metal contamination by mobilization of insoluble heavy metals (Dimkpa et al. 2008; Rajkumar et al. 2010; Hao et al. 2014; Mishra et al. 2017) . Siderophore-producing microbes can thus be used in a variety of ways including bioremediation, sustainable agriculture as biosensors, and even in medicine. Siderophore production ability of microorganisms is commonly detected by the chrome azurol sulphonate (CAS) assay as given by Schwyn and Neilands (1987) . For quantitative estimation of si (...truncated)


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Naveen Kumar Arora, Maya Verma. Modified microplate method for rapid and efficient estimation of siderophore produced by bacteria, 3 Biotech, 2017, pp. 381, Volume 7, Issue 6, DOI: 10.1007/s13205-017-1008-y