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)