Modified chrome azurol S method for detection and ...

Environmental Sustainability https://doi.org/10.1007/s42398-018-0005-3

ORIGINAL ARTICLE

Modified chrome azurol S method for detection and estimation of siderophores having affinity for metal ions other than iron P. R. Patel1 · S. S. Shaikh1 · R. Z. Sayyed1  Received: 13 March 2018 / Revised: 24 April 2018 / Accepted: 25 April 2018 © Society for Environmental Sustainability 2018

Abstract Siderophores are small molecular weight (generally 1 kDa) ferric specific ligands produced by variety of organisms to chelate iron under iron limiting conditions. Various assays have been in use to detect and estimate different phenotypes of siderophores. Though there are various methods available for detection of iron specific siderophore or modified method for Cu specific siderophore [chalkophore], reports on modified methods for detection of siderophore having affinity for various other metal ions are scarce. In present study, a modified method was designed for screening siderophores that can bind to heavy metal ions such as C ­ u2+, ­Ni2+, ­Mn2+, ­Co2+, ­Zn2+, ­Hg2+ and A ­ g2+. Each of the modified chrome azurol S (CAS) solution when used to screen siderophore resulted in instant colour change similar to the traditionally used CAS solution for screening ­Fe3+ specific siderophores. Two bacterial cultures isolated from local rhizospheric soil produced hydroxamate and catecholate siderophores that could remove C ­ u2+, ­Ni2+, ­Mn2+, ­Co2+, ­Zn2+, ­Hg2+ and A ­ g2+ metal ions from CAS solution resulting in instant colour change. Similar observations were recorded on CAS agar amended with modified CAS solution. The order of complexation of metals with siderophore produced by Alcaligenes sp. RZS2 in CAS assay was as follows; ­Cu2+ > Ni2+ > Mn2+ > Fe3+ > Co2+ > Zn2+ > Hg2+ > Ag2+. The order of complexation of metals with siderophore produced by Pseudomonas aeruginosa RZS3 in CAS assay was as follows; N ­ i2+ > Co2+ > Mn2+ > Zn2+ > Fe3+ > Cu2+ > Hg2+ > Ag2+. Keywords  Siderophore · Heavy metals · Chrome azurol sulphonate · CAS agar · CAS shuttle assay

Introduction Siderophore are defined as small molecules (often 1030 M−1) to coordinate with the ferric ion (Neilands 1981; Neilands 1995; Miethke and Marahiel 2007; Sonawane et al. 2016; Wani et al. 2016; Niehus et al. 2017; Ellermann and Arthur 2017). However, microorganisms also need metal ions other than ­Fe3+ and in order to chelate these metal ions, they produce various other types of siderophores. For detection of ferric specific siderophores variety of methods/assays have been developed (Rane et  al. 2005; Louden et al. 2011; Passari et al. 2015). These assays are * R. Z. Sayyed [email protected] 1



Department of Microbiology, PSGVPM’s Arts, Science and Commerce College, Shahada, Maharashtra 425409, India

based on chemical properties of siderophores and include ferric perchlorate assay (Atkin et  al. 1970), Csaky test (Csaky 1984), Arnow test (Arnow 1937), as well as on biological or functional properties for example CAS universal assay (Schwyn and Neilands 1987), modified by AmesGottfred et al. (1989), Milagres et al. (1999), and Machuca and Milagres (2003) and bioassays (Sung et al. 2001). Variations to the CAS assay have been developed together with the CAS agar plate technique; however, traditional methods can only show the chelation between siderophore and ferric ion (Louden et al. 2011; Gusain et al. 2015; Saha et al. 2016; Abbamondi et al. 2016). This warrants the need to develop assays for detection of various other phenotypes of siderophore that possess affinity for metal ions other than iron. Therefore, modified methods for detection and estimation of siderophores need to be searched. Thus the present study was aimed to develop an assay capable of surpassing previous methodological limitations for qualitatively and quantitatively estimating siderophores capable of binding variety of metal ions other than ­Fe3+.

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Materials and methods Isolation of heavy metal resistant bacteria Two bacterial isolates obtained from local soil and previously identified as Alcaligenes sp. RZS2 and Pseudomonas aeruginosa RZS3 based on 16S rRNA gene sequencing (Sayyed and Patel 2011) were tested for their resistance to various heavy metals ions by dilution method (Cervantes et al. 1986; Sheng et al. 2008). Log phase cultures of these isolates were inoculated in freshly prepared nutrient agar and nutrient broth amended independently with various heavy metal salts namely ­MnCl2·4H2O, ­NiCl2·6H2O, ­ZnSO4·7H2O, ­ZnCl2, ­FeSO4, ­CuSO4, ­CuCl2, ­HgCl2, ­FeCl3·6H2O, ­AgNO3 and ­CoCl2; 10 mM stock solution of each heavy metal were used and further diluted at various concentrations ranging from 100 to 2000 µM (Sambrook et al. 1989) and incubated at 28 °C for 24 h.

Screening, production, detection and estimation of siderophore Growth and siderophore production was carried out in 500 mL Erlenmeyer flask containing 100 mL of modified succinate medium (SM) (Meyer and Abdallah 1978). For this purpose, Alcaligenes sp. RZS2 and P. aeruginosa RZS3 (6 × 106 cells mL−1) were grown independently in iron free SM consisting of (g L−1) ­K2HPO4; 3.0 or, ­KH2PO4; 2.0, ­MgSO4 ­7H2O; 0.2, ­NH4SO4; 1.0; succinic acid 4.0, and on CAS agar (Schwyn and Neilands 1987; Milagres et al. 1999) at 28 ± 2 °C at 120 rpm for 24–48 h. Following the incubation, cell density of SM broth was measured at 620 nm by using double beam UV–visible spectrophotometer (1240, Shimadzu, Japan) and CAS agar plates were observed for color change. For detection and estimation of siderophore, SM broth was centrifuged at 5000×g for 15 min at 4 °C to obtain cell free supernatant. This supernatant was assayed for qualitative test by using CAS assay and quantitative test by CAS shuttle assay (Schwyn and Neilands 1987; Payne 1994). Percent siderophore units (% SU) produced by isolates were calculated by measuring optical density of sample (As) and reference (Ar) at 630 nm by UV–visible spectrophotometer (1240, Shimadzu, Japan) and using following formula:

% SU =

As − Ar × 100. Ar

Type determination of siderophore Microorganisms produce two types of siderophores such as hydroxamates and catecholates (Miethke and Marahiel

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2007). Hydroxamate nature of siderophore was detected by Csaky test (Csaky 1984) and catecholate nature of siderophores was detected by Arnow test (Arnow 1937).

Screening for binding of siderophore to other metal ions For screening for binding of siderophores capable of conjugating to heavy metal ions other than F ­ e3+, various CAS solutions and CAS agar were prepared by replacing F ­ e3+ 2+ 2+ 2+ 2+ 2+ 2+ separately with C ­ u , ­Ni , ­Mn , ­Co , ­Zn , ­Hg and ­Ag2+. Each of this modified CAS solution was used to screen siderophore. For this purpose different sets containing CAS-metal-HDTMA dye agar were used, where Fe (III) was replaced with other metals (­ MnCl2·4H2O, ­NiCl2·6H2O, ­ZnCl2, ­CuCl2, ­CoCl2, ­HgCl2 and ­AgNO3) to be screened. The concentration of all metal ions was kept constant to 10 mM metal stock in 10 mM HCl. Simultaneously the log phase culture of Alcaligenes sp. RZS2 and P. aeruginosa RZS3 were individually inoculated on modified CAS agar plates where modified CAS solution for each metal ion was used. Plates were incubated at 28 °C for 24 h and observed for color change from blue to orange red.

Results Isolation of heavy metal resistant bacteria Both isolates grew well on nutrient agar amended with varying concentrations of heavy metal ions. This indicated their ability to resist or tolerate high concentrations of variety of heavy metal ions. Alcaligenes sp. RZS2 showed luxurious growth at 2000 μM concentration of M ­ nCl2 but less growth at 1000 μM of N ­ iCl2, ­FeSO4 and C ­ uSO4, 1400 μM of ­ZnSO4, 1600 μM of Z ­ nCl2 and 1200 μM of F ­ eCl3, indicating the inability of Alcaligenes sp RZS2 to tolerate such concentration of these metal ions. Low concentra­ oCl2 supported the tion (600 and 400 μM) of ­CuCl2 and C growth of Alcaligenes sp RZS2. P. aeruginosa RZS3 tolerated high (1800 μM) concentration of Z ­ nCl2, 1600 μM ­FeSO4, 1200 μM of ­MnCl2, 1400 μM of ­ZnSO4 and ­FeCl3, 800 μM of N ­ iCl2 and C ­ uCl2, Growth of isolate was affected at 400 μM ­CuSO4 and 200 μM of ­CoCl2. Both isolates failed to grow in presence of ­HgCl2 and ­AgNO3.

Screening, production, detection and estimation of siderophore In the shake flask studies, change in the color of succinate medium from colorless to fluorescent green after 24 h and change in the color of CAS agar from blue to orange indicated the production of siderophore. It was further confirmed

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Fig. 1  CAS agar assay for Alcaligenes sp. on a copper CAS agar, b nickel CAS agar, c manganese CAS agar, d iron CAS agar, e cobalt CAS agar, f zinc CAS agar

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Table 1  Chelation of heavy metal ions from modified CAS solution by soil isolates Metal ions

Cu2+ Ni2+ Mn2+ Fe3+ Co2+ Zn2+ Hg2+ Ag2+

Results of CAS test Alcaligenes sp. RZS2

P. aeruginosa RZS3

− ++ + + ++ + − −

+++ ++ ++ ++ ++ + − −

+, positive; ++, strong positive; +++, very strong positive; −, negative

by CAS test, where addition of CAS to cell free supernatant changed the blue color of CAS to orange color while no color change occurred in control (un-inoculated) medium. Change in the color of CAS reagent is due to the fact that siderophore (strong Fe chelator) present in the supernatant chelates the iron from HDTMA (weak chelator) in CAS reagent and results in color change from blue to orange red. Amount of siderophore produced by Alcaligenes sp. RZS2 was higher than the siderophore yield by P. aeruginosa RZS3.

Type determination of siderophore Both isolates produced mixture of siderophores namely hydroxamate and catecholate. However, hydroxamate siderophores were produced in major quantity while catecholate siderophores were produced in minor quantity. Alcaligenes sp RZS2 produced 73.00% hydroxamate SU and 24.61% catecholate SU while P. aeruginosa produced 62.22% hydroxamate SU and 11% catecholate SU.

Screening of metals for complexation with siderophore After 24 h growth of isolates on modified CAS agar, change in the color of CAS agar from blue to orange was observed. This indicated the production of siderophore and chelation of ­Mn2+, ­Ni2+, ­Zn2+, ­Cu2+, ­Co2+, ­Hg2+ and ­Ag2+. Similar observations were observed with modified CAS solution with varying degree of color intensity. Intensity of color change from blue to sunset yellow with CAS solutions having ­Hg2+ and ­Ag2+ was comparatively less. This indicated the weak chelation or binding of siderophore with ­Hg2+ and A ­ g2+. The ability of siderophores of Alcaligenes

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sp RZS2 and P. aeruginosa RZS3 to chelate metal ions was highest with Ni and ­Cu2+ respectively and lowest for ­Ag2+. Amongst all the CAS agar plates Ni-CAS agar plate and CuCAS agar showed superior results with Alcaligenes sp RZS2 (Figs. 1, 2) and P. aeruginosa RZS3 respectively. Intense colour change in this condition indicated the higher affinity of siderophore of isolates with Ni and ­Cu2+. In CAS assay degree of complexation of metals and siderophore of Alcaligenes sp RZS2 and P. aeruginosa RZS3 was also highest with Ni and C ­ u2+ respectively (Table 1). Although siderophores are ­Fe3+ specific they have been reported to bind other metal ions.

Discussion Growth of isolates at higher concentrations of various metal ions is due to a variety of reasons such as requirement of heavy metal ions for cell growth and metabolism; requirement of these metal ions as cofactors for various enzymes may be due to multi-dimensional heavy metal resistance mechanisms, and bioaccumulation or transformations of metal ions (Straw et al. 2018). ­Zn2+ and ­Cu2+ ions increase fluorescent siderophore production (Dimkpa et al. 2012). Several siderophore-producing bacteria associated with plants, such as Brassica juncea and Aquilegia bertolonii, have been isolated from metal-contaminated soils (Barzanti et al. 2007). Idris et al. (2004) have isolated PGPR and characterized them as Ni hyperaccumulator. Metal-resistant endophytic bacteria that also produce siderophores have also been isolated from various plant species (Hussein and Joo 2014). Patel et al. (2016) have also reported good growth and siderophore production in A. faecalis RZS2 and P. aeruginosa RZS3 strains in presence of high concentration of various heavy metal ions like ­MnCl2·4H2O, ­NiCl2·6H2O, ­ZnCl2, ­CuCl2 and C ­ oCl2 other than ­FeCl3·6H2O. Change in color of CAS agar and CAS solution from blue to orange is attributed to the principal of replacement of iron from iron-HDTMA complex by strong iron chelator such as a siderophore (Schwyn and Neilands 1987; Milagres et al. 1999). Shaikh et al. (2014) and Sayyed and Patel (2011) have also reported good siderophore yield in Alcaligenes sp. and P. aeruginosa. PGPR are known to produce wide range of siderophore, of these hydroxamate siderophore producing PGPR are useful in agriculture as these are more stable and potent iron chelators (Sayyed and Chincholkar 2006; Mazzola 2002; Patel et al. 2016; Saha et al. 2016; Reshma et al. 2018). Although siderophores are ferric specific ligands, but they can also bind various other metal ions such as ­Cu2+, ­Ni2+, ­Mn2+, ­Co2+, ­Zn2+, ­Hg2+ and A ­ g2+ available in the environment. Many microorganisms require different types of metal

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Fig. 2  CAS agar assay for P. aeruginosa on a copper CAS agar, b nickel CAS agar, c manganese CAS agar, d iron CAS agar, e cobalt CAS agar and f zinc CAS agar

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ions for their growth and metabolism and as cofactors for enzymes. Zn is required as cofactor in Pseudomonas sp. for its carbonic unhydrase enzyme (Schijf et al. 2015). Methanotrophs produce siderophore referred as chalkophore (Yoon et al. 2010), that has high affinity for copper ions. Aiken et al. (2003) and Nair et al. (2007) have reported that siderophore bind divalent metals like C ­ d2+, ­Ni2+, ­Zn2+, ­Cu2+, 2+ 2+ 2+ ­Co , ­Sn , ­Pb with varying capacities. A similar test for uranyl complexation was reported by Renshaw et al. (2003). Sayyed et al. (2015) have reported efficient good growth, siderophore of exopolysaccharide producing Enterobacter sp RZS5 in presence of various heavy metal ions. There are various methods to screen ferric specific siderophores producing microbes, but suitable methods to screen microbes producing siderophore that can also bind to other metal ions are scarce. The proposed modified methods can be employed as best to detect and estimate various phenotypes of siderophores.

Conclusion The traditional methodology available for the detection of siderophore-producing microorganisms proposed by Schwyn and Neilands (1987) has limitations of detecting only ­Fe3+ specific siderophore. In the method hereby, we are successfully reporting method for complexation of eight different heavy metal ions; ­Cu2+, ­Ni2+, ­Mn2+, ­Fe3+, ­Co2+, ­Zn2+, ­Hg2+ and ­Ag2+. Thus this modified CAS-agar assay is simple method for observing the chelation between siderophores and heavy metals. This method can be implemented for the detection of siderophores that possess affinity for metal ions other than iron. Acknowledgements  Author RZS is thankful to UGC, New Delhi for providing financial support in this research project.

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