DESC:Complete Specification

Siderophore based method for sequestration of arsenic and phosphorous from aqueous bodies

Cross-reference to related applications: This specification is being filed pursuant to patent application No. 202121016710 dated 09/10/2021 with provisional specification the contents of which are incorporated herein in their entirety by reference.

Field of the invention
The present invention relates generally to biomolecules and applications thereof for remediating heavy metal pollution of water bodies. More particularly, the invention subject hereof narrates a novel siderophore, compositions based on the same, and furthermore methods to prepare and to apply said siderophore composition for effective sequestration of arsenic, phosphorous, nickel and cobalt from water bodies.

Background of the invention and description of related art
The past few decades have seen exponential rise in use of chemical fertilizers, soil conditioning agents, as well as efflux of industrial effluents into water bodies or soil (which ultimately finds its way into the ground table and the water bodies such as streams, rivers, lakes, bays and coastal waters they eventually link). These occurrences, over time, have resulted in large-scale pollution of water bodies / reservoirs which are inherently the natural habitat of myriad flora and fauna besides being essential for supporting life of higher organisms including humankind itself. There is hence a pressing need in the art to have some means of remediating such manmade wanton pollution of water bodies.
Among various pollutants observed to be introduced into water bodies, heavy metal pollutants deserve grave alarm being highly toxic in nature. On a global front, arsenic is observed to be naturally present at high levels in the groundwater of a number of countries, including Argentina, Bangladesh, Chile, China, India, Mexico, and the United States of America.
Use of waters contaminated with heavy metal pollutants, especially arsenic, for drinking, food preparation and irrigation has been proven to cause cancer, skin lesions, cardiovascular disease, hyperkeratosis, heart attacks, kidney failure, pulmonary disease, adverse pregnancy outcomes and infant mortality, negative impacts on cognitive development in young children as well as increased deaths in young adults. (Ref: (1) Association of arsenic with adverse pregnancy outcomes/infant mortality: a systematic review and meta-analysis. Quansah R, Armah FA, Essumang DK, Luginaah I, Clarke E, Marfoh K, et al. Environ Health Perspect. 2015; 123(5):412-21; (2) In utero and early life arsenic exposure in relation to long-term health and disease. Toxicol Appl Pharmacol. Farzan SF, Karagas MR, Chen Y. 2013; 272(2):384-90; (3) The developmental neurotoxicity of arsenic: cognitive and behavioural consequences of early life exposure. Tolins M, Ruchirawat M, Landrigan P. Ann Glob Health. 2014;80(4):303-14).
The International Agency for Research on Cancer (IARC) has classified arsenic and arsenic compounds as carcinogenic to humans, and has also stated that arsenic in drinking-water is carcinogenic to humans. It is now recognized that at least 140 million people in 50 countries have been drinking water containing arsenic at levels above the WHO-prescribed limit of 10 µg/L (Ref: Arsenic Pollution: A Global Synthesis. Ravenscroft P, Brammer H, Richards K. Wiley-Blackwell; 2009).
Similarly, pollution due to man-made excessive application of phosphorus-based fertilizers and other chemicals is reaching dangerously high levels in freshwater bodies, evident from the ill-effects including eutrophication that causes algal blooms, which in turn leads to mortality of fish and plants due to lack of oxygen and light besides reducing utility of such water for purposes such as consumption and swimming. On a global scale, the Huang-He (Yellow) river in China, the Indus and Ganges rivers in India and the Danube river in Europe are widely regarded as highly polluted by phosphorus. (Ref: Mesfin M. Mekonnen et al, Global Anthropogenic Phosphorus Loads to Freshwater and Associated Grey Water Footprints and Water Pollution Levels: A High-Resolution Global Study, Water Resources Research, 2017).
Due to mining, the phosphorus reservoirs have been depleted to meet the global demands of phosphate fertilizers (Gilbert, 2009). An efficient method for phosphate recovery can help to solve this problem. There are several ways to removes the phosphorus from the environment, such as struvite precipitation from wastewater treatment, drainages, and wastewater lagoons (Le Corre et al., 2009). This is one of the proven methodologies to be used for the recovery of phosphorus from the wastewater effluents, where the phosphate concentration is high (Le Corre et al., 2009).
Across the world, provision of a safe arsenic and / or phosphorus -free water supply has been proposed either via a) substitution of water in use by a low- arsenic and / or phosphorus, microbiologically-safe source such as rain water and treated surface water; b) Blending of low- arsenic and / or phosphorus water with higher- arsenic and / or phosphorus water to achieve an acceptable arsenic concentration level; and c) employment of arsenic and / or phosphorus removal methodologies either on centralized or local deployment scales for removing arsenic from water intended for utilization.
Various physical and chemical procedures are recognized today for abatement of water pollution attributable to introduction and residency of arsenic and / or phosphorus contaminants in water bodies. These procedures are based on oxidation, coagulation-precipitation, absorption, ion exchange, and membrane techniques. However, their additive nature, implementation costs, and less-than perfect efficacies have kept said procedures from mass-application. It would be hence extremely beneficial to have some means of ameliorating water pollution attributable to introduction and residency of arsenic and / or phosphorus which is qualified in that it is competent, inexpensive, and environmentally friendly besides involving little or no technical complexities and amenable to skill levels of even a layman to inculcate.
Phosphate recovery in the form of struvite crystals from wastewater treatment plant or livestock waste is of major concern. It needs to be carried out by controlling the pH by alkali dosing and CO2 stripping, which increases the cost of struvite production and also hampers the reaction rate of the struvite formation (Le Corre, et al., 2009). The Ca2+, K+ and other ions present in the waste samples also interfere and easily get introduced and co-precipitated with struvite, which leads to the formation of struvite variants (Hao et al., 2008). The rapid precipitation may cause the formation of smaller particle size as a result it reduces the potentials of crystal growth and also increase the cost of the process for large scales (Hao et al., 2013; Mayer et al., 2016; Pastor et al., 2010).
Struvite crystallization occurs naturally in the diverse biological ecosystems. Biochemical and physiological changes occur in these environments are through microbial metabolism that induces the combination of ions such as ammonia, phosphate and magnesium (Rivadneyera, 1985). There are number of different environments studied for the struvite crystallization, especially from anaerobic digester (Maqueda et al., 1994; Pastor et al., 2010), waste of animal farming liquid (Huang et al., 2015), and municipal treatment plants (Parsons, et al., 2008; Le Corre et al., 2009). Another approach used for struvite crystallization was using urine and synthetic urine of humans as well as animals as a source of phosphorus (Ronteltap et al., 2010). Chemically synthesized struvite could also be obtained by mixing the equimolar concentrations of ammonia, phosphorus and magnesium salts (Kurtulus and Tas, 2011). Microbial activities in various ecosystems, plays a key role in the formation of various minerals including struvite (Frankel and Bazylinski, 2003). There are number of microbial isolates viz., Trypanosoma cruzi (Adroher et al., 1988), Arthrobacter sp., Pseudomonas sp., (Perez-Garcia et al., 1990), Proteus mirabilis (Prywer and Torzewska, 2010), Myxococcus sp., (Omar et al., 1998), Enterobacter sp. EMB19 (Sinha et al., 2014), and Acinetobacter calcoaceticus SRB4 (Han et al., 2015) reported in the lab scale studies for struvite biomineralization.
Microorganisms are known to synthesize low molecular weight, ferric ion-specific chelating agents, known as siderophores, under iron starvation conditions. These siderophores are released for acquisition of iron, after binding to iron molecule it transports back to organism via high affinity and receptor-dependent transport system. The regulation of the production of siderophores depends on the availability of iron (Crosa and Walsh, 2002). There are three major functions of siderophores, as a Trojan horse strategy for drug delivery, plant growth promotion, controlling the noxious species in the soil, and for solubilization and mobilization of wide range of metal ions (Moll et al., 2009; Schalk et?al., 2011). This forms one of the foundational aspects of the present invention.
Prior art, to the limited extent presently surveyed, does not list a single effective solution embracing all considerations and issues mentioned hereinabove, thus preserving an acute necessity-to-invent for the present inventors who, as result of their focused research, have come up with novel solutions for resolving all needs of the art once and for all. Work of the presently named inventors, specifically directed against the technical problems recited hereinabove and currently part of the public domain including earlier filed patent applications and other publications, is neither expressly nor impliedly admitted as prior art against the present disclosures.
A better understanding of underlying principles of the present invention will be obtained from the following narration which sets forth an illustrative yet-preferred embodiment.

Objectives of the present invention
The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:
It is a primary objective to establish a green and sustainable process for phosphate removal / recovery through struvite formation by using bacterial siderophore for efficient removal of even low phosphate concentrations with high recovery efficiency.
It is another objective of the present invention to establish a mass-utilizable process for removal / recovery of phosphates from phosphate-rich industrial wastes.
It is another objective of the present invention to establish a nutrient medium for up-scaled production of siderophores.
The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claims. Other advantageous embodiments of the invention are specified in the dependent claims.

Brief description of drawings
The present invention is explained herein under with reference to the following drawings, in which:
Figure 1 includes pyoverdine structures reported by (a) Chen et al. and (b) Baune et al.
Figure 2 represents pyoverdine structure reported in the present invention.
Figure 3 is a liquid chromatography profile of siderophore solution after partial purification according to the disclosures hereof.
Figure 4 is a liquid chromatography coupled mass spectroscopy profile of prominent peak at 1356 m/z value indicative of the siderophore species according to the disclosures hereof.
Figure 5 is a schematic to illustrate siderophore mediated struvite formation according to the disclosures hereof.
Figure 6 is a schematic to show mechanism of struvite formation induced by siderophore in presence of different media ions like Mg2+ + PO43- + NH4+.
The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. The drawings are not to scale (unless so stated) and are intended for use solely in conjunction with their explanations in the following detailed description.
Attention of the reader is now requested to the brief description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

Statement of the invention
This invention particularly establishes a green as well as sustainable process for repeatable phosphate recovery for up to twenty cycles through struvite formation by using bacterial siderophore produced by Pseudomonas taiwanensis R-12-2 for removal of phosphate as low as 1.3 mM with 99% recovery efficiency.

Detailed description
Principally, general purpose of the present invention is to assess disabilities and shortcomings inherent to known systems comprising state of the art and develop new systems incorporating all available advantages of known art and none of its disadvantages.
Biologically induced mineralization leads to the formation of minerals of different elements like Ca, Mo, Zn, Cu, Ni, and Co (Lowenstam, 1981) and finally contributes to the biogeochemical cycles. Metal speciation occurs due to microbial mediated mobilization and immobilization, which is a complex and interdisciplinary process contributes to the biomineralization (Gadd, 2001; 2004).
Also as will be elaborated further in this document, siderophore plays an important role in struvite formation by trapping the phosphate ions. Besides, struvite (magnesium ammonium phosphate hexa hydrate; MgNH4PO4.6H2O) is one of the best fertilizers that provide three important macro elements such as N, P and Mg in a single unique combination.
Accordingly, the disclosures herein are directed towards a novel siderophore, compositions based on the same, and furthermore methods to prepare and to apply said siderophore composition for effective sequestration of arsenic and phosphorous from water bodies.
According to one aspect of the present invention, the siderophore of concern was obtained from Pseudomonas taiwanensis R-12-2 (Accession No. MCC 4695) general deposit in National Centre for Microbial Resource, Pune). The culture was routinely maintained at 4 °C on 0.1 x tryptic soya agar. Said strain, in liquid culture, expresses a mixed siderophore output comprising pyoverdine and pyochelin. Among said output, pyoverdine was observed to be the predominant species. Broth for microbial enrichment and siderophore expression was derived from standard succinate media (“SSM”) compositions reported by various researchers, as noted from Table 1 below.
Media components SSM
(g/L) A
(g/L) B
(g/L) C
(g/L) Present invention (g/L)
1) Succinate 4.0 4.0 4.0 4.0 5.0
2) (NH4)2SO4 1.0 1.0 1.0 1.0 -
3) NH4Cl - - - - 2.5
4) KH2PO4 6.0 12.9 3.0 3.0 0.1
5) K2HPO4 3.0 - 3.0 6.0 0.1
6) MgSO4.7H2O 0.2 0.2 0.2 0.1 0.75

Legend: A=Kilz et al., 1999; B= Hannauer et al., 2012; C= Yin et al., 2014
Table 1

Media optimization for siderophore
The reader shall appreciate that contribution of the present inventors in reaching the media composition favouring yields high siderophore lied in replacement of (NH4)2SO4 by NH4Cl, use of minimal amount of phosphate (10 mg % compared to 600 mg% or 300 mg % reported elsewhere), and use of higher amount of MgSO4.7H2O (75 mg %) compared to equivalent media reported elsewhere. Consequently, the media composition used by the present inventors is unique, being not identified with equivalent media reported elsewhere. Significance as such, of this contribution is evident from that industrially viable of siderophore was possible using dilute media, that is, around 4.0 to 5.0 g/L of siderophore powder was obtained, via lyophilization of 1 L broth.
The siderophore production ability of the P. taiwanensis R-12-2 (a Gram-negative, short rod, motile and non-spore forming bacterium) was evaluated by growing it initially in standard SM broth (Meyer and Abdallah, 1978) composed of (g/l): succinate, 4.0; NH4Cl, 1.0; K2HPO4, 3.0; KH2PO4, 6.0; and MgSO4.7H2O, 0.2 and pH 7.0. Four different combinations of modified succinate broth medium (mSM) were prepared to observe simultaneous formation of siderophore and struvite (Table 1). All the media components were dissolved in Milli Q water. The P. taiwanensis R-12-2 was inoculated (0.05 %, 48 h grown culture) in respective sterilized mSM mediums. In order to increase the production of siderophore, 0.05 % sterilized deferrated tryptone (3 % stock) broth was added and flasks were incubated at 30°C, with 150 rpm shaking for 48 h. The intense greenish-yellow fluorescent coloration of the media was observed as an indicator for the production of siderophore.
In order to increase siderophore production ability of P. taiwanensis R-12-2, the media was designed by lowering the phosphate content. To induce siderophore production with less ionic interference in standard SM medium (Meyer and Abdallah, 1978), four different combinations of mSM (Table A below) were studied. Different media compositions of modified SM media (mSM) for siderophore and struvite formation, as under-

Media Media components (g %)
Succinate NH4Cl K2HPO4 KH2PO4 MgSO4.7H2O
mSM-A 0.5 0.25 0.01 0.01 0.075
mSM-B 0.5 0.25 0.05 0.05 0.09
mSM-C 0.5 0.1 0.005 0.005 0.035
mSM-D 0.5 0.075 0.001 0.001 0.025
Table A
It was found that both the characteristics of simultaneous siderophore and struvite formation were demonstrated in mSM-A medium only. While no struvite crystals were found in either of these controls at a low concentration, this could be due to the fact that, the ideal concentration of media ions necessary for the struvite production (Kofina and Koutsoukos, 2005). The standard SM was only used for the production of siderophores and not for struvite formation. However, in the present study, it was observed that during media optimization P. taiwanensis R-12-2 was able to simultaneously precipitate struvite in the medium along with the siderophore production.
In various iron-free media such as MM9 (Schwyn and Neilands, 1987), Cas-amino acid medium (Meyer and Abdallah, 1978), Barbhaiyya and Rao medium (Barbhaiyya and Rao, 1985) and SM medium (Meyer and Abdallah, 1978) there were different species of Pseudomonas and other bacterial genera were reported for siderophore synthesis. While struvite formation has been studied separately in various media such as B-17, B-26, B-27, B-28, B-29, B-30 and B-411 (Rivadeneyra et al., 1983; 1992) by using different Pseudomonas sp. and other bacterial genera.
Out of four different media, mSM-A medium was found to be suitable candidate for both siderophore and struvite formation, because it showed enhanced production of siderophore compared to mSM-B, where the media components are at higher concentrations. At low concentration of phosphate (~1.3 mM) in mSM-A bacterial growth and crystals of struvite were obtained after incubation for 24 h. However, below this concentration of phosphate, like mSM-C and mSM-D there was no synthesis of siderophore and struvite. Moreover, there is no struvite crystallization occurred in two controls used. The struvite formation was not observed in media devoid of siderophore and in water control having pH 9.0, signifies the role of siderophore in the struvite formation. Thus, from these studies, we reiterate that optimal concentration of phosphate is required for the bacterial growth and siderophore. In this study we reports for the first time the formation of struvite at a low phosphate concentration (1.3 mM) by inorganic media salts compared to the earlier reports where 1.63 mM of phosphate concentration was used from organic media like yeast extract (Sinha et al., 2014). In the current investigation, simultaneous siderophore synthesis and struvite formation were seen in the same medium, mSM-A, which has not been previously reported.

Screening of siderophore production and characterization
The ability of P. taiwanensis R-12-2 to synthesize the siderophore was tested on standard universal chrome azurol sulphonate (CAS) agar plate by spot inoculation. The universal CAS plate was prepared as reported previously (Schwyn and Neilands, 1987). The CAS plate was incubated at 30 °C for 48 h, to check for the formation of orange halo zone around the colony. The siderophore production was carried out by growing P. taiwanensis R-12-2 in standard succinate broth medium (SM) (Meyer and Abdallah, 1978). After 48 h incubation, the media was centrifuged at 8000 × g at 4 ºC and the cell-free supernatant was examined for formation of siderophore types by using different chemical tests such as tetrazolium test (Snow, 1954), Arnow’s test (Arnow, 1937), and carboxylate test (Vogel, 1992). The produced siderophore was diluted in 100 mM Tris-HCl (pH-7.0) and qualitatively analysed by fluorescence spectra (Jasco FP-8300, Japan) in the range of 350 to 500 nm wavelength by UV-visible absorption spectra (Baysse et al., 2000).
The formation of struvite crystals is driven by surrounding pH (9.0) and presence of Mg2+ and NH4+ ions along with PO43- and siderophore which was further validated by computational studies. The morphology of struvite was characterized by scanning electron microscopy, followed by elemental analysis. Accordingly, siderophore obtained from the process described above was characterized using state-of-art procedures like LC-MS/MS equipment, and on the basis of LC-MS/MS profile we identified final structure of siderophore (Fig A and B) so determined to comprise pyoverdine as a predominant species. Prior art refers two pyoverdine structures being reported (Ref: Chen et al., 2016; Baune et al., 2017). Attention of the reader is requested to the accompanying Figures 1 and Figure 2, in which Figure 1 includes pyoverdine structures reported by (a) Chen et al. and (b) Baune et al. On the other hand, Figure 2 represents pyoverdine structure reported in the present invention, which differs notably from that a total 10 amino acids are present with characteristic cysteine group, which imparts new function, hence novel biomolecule for use in the overall scheme of the present invention.
Figure 3 is a liquid chromatography profile of siderophore solution after partial purification, showing a single peak of siderophore produced according to the foregoing narrative. Furthermore, Figure 4 is a liquid chromatography coupled mass spectroscopy profile of prominent peak at 1356 m/z value indicative of this siderophore species was selected for siderophore production on the basis of its ability to produce an orange halo zone around the colony on the universal CAS agar plate and the formation of green colour pigment in mSM-A medium. Siderophore scavenges selectively Fe from the Fe-dye complex, and releases Fe from the dye, as a result media colour changes from blue to green and orange halo zone around the colonies in the petriplates (Payne, 1994). The siderophores produced by microorganisms are categorized by different types based on their principal chelating groups. These are phenol-catecholates, hydroxamates, carboxylates, peptide based, mycobactins, citrate-hydroxamates and keto hydroxy bidentates (Cornelis and Matthijs, 2002; Winkelmann and Drechsel, 1997). Initially, siderophore formation was confirmed by CAS assay and followed by the tetrazolium test. The tetrazolium salt turned dark red after adding the siderophore, suggesting that P. taiwanensis R-12-2 synthesized the hydroxymate form of siderophore. The linear and cyclic backbone configurations of the hydroxamate siderophores contain peptide chains in their core which is related to the form of pyoverdine siderophore. Pyoverdine, however, is a mixed kind of siderophore mostly produced by Pseudomonas sp. The pigment is yellowish green, water-soluble and has high ferric chelation ability (Holsberg and Artis 1983; Meyer, 2000). In addition, an intense peak was observed at 400 nm in cell-free supernatant suggesting that pyoverdine type siderophore was secreted by P. taiwanensis R-12-2. Moreover, presence of relative abundance of pyoverdine was measured by fluorescence (excitation at 405 nm and emission at 450 nm), which was functionally equivalent to an established method of pyoverdine abundance determination (Meyer and Abdallah, 1978; Cox and Adams, 1985; Visca et al., 1992).

In-vitro struvite formation by siderophore
Out of four different mSM broths (Table 1), mSM-A was selected for both siderophore production as well as in vitro struvite formation. The media mSM-A (deferrated, without Fe) was used as test and mSM-A (ferrated, with 50 µM Fe) as a biotic control, both the flasks were inoculated with P. taiwanensis R-12-2. All the flasks were incubated at 30 °C for 48 h at 150 rpm. The pH of the test flask reached to pH 9.0 after siderophore production from the initial pH 7.0, whereas in biotic control the pH ranged between pH 7.0-8.0 after the growth. The flasks were centrifuged at 4°C, at 6000 × g for 20 min to collect the supernatant. The supernatant of test, biotic and abiotic control (sterilized Milli Q) were adjusted to pH 9.0, biotic and abiotic control served as siderophore-free controls which were further used to check for struvite crystallization. All the flasks were autoclaved at 15 lbs pressure for 30 min, cooled and centrifuged to eliminate any role of proteins in the struvite formation. The supernatants were collected in different separating funnels. All the media components of mSM-A (g %) were added into the respective separating funnels and mixed thoroughly and kept at static condition. The precipitate formation was started after 5 min in the test funnel and it is settled down at the bottom of funnel within 10-15 min, whereas there was no precipitation observed in the control flasks. After 15 min, the amount of phosphate was estimated from supernatant by Fiske-Subbarow method (1925). The settled precipitate from test flask was collected by filtration, washed for 3-4 times with Milli-Q water, and dried at 50 °C for 12 h. After drying the crystals were further characterized and analyzed. The above procedure of struvite formation was repeated for twenty times and the phosphate concentration was estimated before and after every cycles. The amount of phosphate sequestration (phosphate recovery) by siderophore was determined.

Recovery of phosphate from industrial solid P rich waste sample by using siderophore and its electrochemical applications
To study the application of siderophore based in vitro struvite formation on real waste samples; we have collected the solid waste sample from phosphate fertilizer industry situated near Ranjangaon industrial area (Pune, India). In order to precipitate phosphate in the form of struvite, industrial solid P rich waste sample (1.0g) was dissolved in 100 ml sterilized siderophore solution, and 0.25 g of NH4Cl and 0.09 g of MgSO4.7H2O were added. After 15 min the precipitate was collected and characterized while the supernatant was analyzed for phosphate content by Fiske Subbarow method. The abiotic control (water) was adjusted to pH 9.0 and performed the same above experiment. All the experiments were performed in triplicate.
The electrochemical properties of the formed struvite from industrial waste sample were characterized by a conventional three electrode system in 2 M KOH electrolyte at 25 °C. The struvite (8.0 mg) active material along with 1.5 mg of activated carbon and 0.5 mg of polyvinylidene fluoride binder were mixed in N-methyl-2-pyrrolidene and sonicated to prepare the ink. Typical working electrode was prepared by drop-casting 1.0 mg of ink on the carbon electrode (1.0 x1.0 cm2). Platinum foil was used as a counter electrode and Hg/HgO as a reference electrode. To perform cyclic voltammetric studies, the potential window of 0-1.0 V was used at different scan rates. The galvanostatic charge/discharge tests were carried out in the potential range of 0-0.8 V at different current densities (1.87, 2.5, 3.75 and 5 A g-1).
It was observed that 99 % of phosphate (4.0 mg) was sequestered after each cycle of siderophore mediated struvite formation by using mSM-A media components. The phosphate sequestration was performed for 20 cycles in the same siderophore solution. After every cycle, over 99 % of phosphate was sequestered in to struvite crystals while about 0.5 to 1 % of the phosphate remained after each cycle in the siderophore broth. While in case of industrial waste sample, the amount of phosphate present was 176 mg/g of solid waste sample. Where more than 90 % of phosphate was recovered from industrial phosphate rich waste sample compared to 40 % recovery in control. In both these cases phosphate sequestration occurred within 10 to 20 minutes. In control, amorphous particles were observed with less quantity, whereas fine crystalline nature of particles were observed with high quantity at the bottom of siderophore solution.
Asymmetrical curves can be seen in struvite cyclic voltammograms, which can be attributed by the combined double-layer and pseudo-capacitive contributions to the overall capacitance. Discharging times were found to be much longer than charging times with a small IR-drop, which was due to the higher capacitance values. The specific capacitance (Cs) of the sample was estimated using the following equation,

Where,
i = current; ?t = time of discharge; m = mass of active material (8 mg)
?V = voltage drop upon discharging
The specific capacitance value for the sample was found to be 320 F g-1 at 1.87 A g-1, 96 F g-1 at 2.5 A g-1, 23.4 F g-1 at 3.75 A g-1and 18.75 F g-1 at 5 A g-1 respectively. We did not observe much capacitance different at higher applied current densities ca. 3.75 - 5 A g-1 which attributed to the faster discharging profiles at higher applied current densities. Furthermore, charge retention is an important parameter for the commercial application of any material. Consequently, the cycling stability of struvite sample has been carried out at 2 A g-1 for 250 cycles. Specific capacitance and its coulombic efficiency retained was 92 % even after 250 cycles which illustrates its good super-capacitance behaviour for electrochemical applications.
In the present study the siderophore produced is identified to be pyoverdine (hydroxymate) type of iron chelator. Pyoverdines are chemically composed of three parts a) fluorescent chromophoric group, dihydroquinoline; b) six to twelve different amino acid attached as peptide group; and c) various dicarboxylic acids or their amide derivative group attached to chromophoric group at N-3 position. More than 60 different pyoverdines have been isolated and chemically identified from different Pseudomonas species (Schalk and Guillon, 2013). However; there are no reports till date on siderophore mediated struvite mineral formation.
Siderophore is popularly known for its chelating ability for iron and different metal ions. There is a possibility of chelation of media ions at particular concentration and further may act as a template for struvite nucleation (Dupraz and Visscher, 2005). The chemical groups in siderophore which are mostly negatively charged may interact with media ions like Mg2+, NH4+ and PO43-. Additionally, the alkaline pH of siderophore solution favours the struvite crystallization and helps in enhancing the struvite formation (Sinha et al., 2014). This interaction leads to crystal nucleation of ions to form struvite within 10 to 15 min of incubation at static condition. This method of struvite formation is rapid, compared to 7 days of incubation reported by Sinha et al. (2014).
In vitro struvite crystallization was reported from different biological fluids like human urine (Ronteltap et al., 2010), animal urine (Matsumoto and Funaba, 2008), bacterial extracellular products like proteins (Sinha et. al., 2014), extracellular polysaccharide (Lin, et al., 2012), glycosaminoglycan and glycoproteins like Tamm-Horsfall glycoprotein (McLean and Nickel, 1994; Buffington et al., 1994), and homopolypeptide (Li et al., 2015). The struvite formation was found to occur in heterogeneous ion conditions by extracellular proteins or microbial cells (Sinha et. al., 2014 and Li, et al., 2015). In view of this background we propose the mechanism of siderophore mediated struvite crystallization (Fig. 6) and we corroborate the struvite formation in medium may occur due to siderophore.
A number of studies suggests that, parameters such as pH of the solution, temperature, presence of foreign ions, and Mg:N:P ratio influences the struvite precipitation and properties (Rahman et al., 2014; Kumar and Pal, 2015; Li, et al., 2019). It was also reported that the precipitation is spontaneous at higher equimolar concentrations of Mg:N:P ratio (Kofina and Koutsoukos, 2005). Thus, the 40 % of struvite yield was obtained in control due to natural propensity of these three metal ions to agglomerate. Furthermore, the presence of siderophore increases struvite yield, possibly due to their high chelating action, which may facilitate the fast precipitation of media ions into struvite. The siderophore contains small peptide chain which may act as a template for the nucleation of ions and which further develops the struvite crystals (Dickerson, et al., 2008). The cavity like structure present in pyoverdine and the free -OH, -COOH, and -NH groups, surrounding pH (pH 9.0) may be responsible for initiation of nucleation of media ions. In order to eliminate the involvement of extracellular secreted proteins and glycoproteins for struvite nucleation the siderophore solution was sterilized. The sterilized siderophore solution showed struvite formation which is in contrast to the involvement of bacterial secretory proteins in ion nucleation (Sinha et al., 2014). The sterile siderophore was used multiple times for struvite formation, by adjusting the pH to alkaline after 3-4 cycles of reuse, without any decrease in potential of struvite formation.
Thus, an in vitro crystal growth of struvite biomineral in medium was induced by siderophore, compared to earlier reports of bacterial struvite bio-mineralization (Sinha et al., 2014). In addition, the struvite was subjected to electrochemical applications, and it was found that the siderophore mediated struvite demonstrated high capacitance activity. In future, this research will help to open up new horizons for siderophore-mediated struvite or struvite variant applications and explore more siderophore usability.

Characterization of struvite crystals
The produced struvite crystals were analysed for their morphological characteristics and elemental composition by Field Emission Scanning Electron Microscopy (FE-SEM) (FEI, NOVA, NanoSem 450) equipped with energy dispersive X-ray (EDAX) spectroscopy (Bruker, XFlash 6|30). The X-ray powder diffraction (PXRD) pattern of struvite crystals were characterized by using an Ultima IV diffractometer (Rigaku, Japan) over the 2? range of 10-80 degrees. The sample was prepared as per the standard procedure and the measurements were carried out with the help of Goniometer using CuKa radiation (Ka=0.1540562 nm) monochromatized with a graphite crystal. The produced struvite was confirmed by comparing values with the standard JCPDS card number 71-2086. Further, the FTIR spectra of dried powder of struvite crystals were recorded in the scanning range of 400-4000 cm-1 on Tensor 37 FTIR (Bruker) using KBr. Struvite crystals of 6.0 mg were subjected to thermogravimetric analysis (TGA) at a atmospheric pressure (DTA-60H, Shimadzu). Thermal gradation was increased at the rate of 10°C per min from 10-1000 °C and differential scanning calorimetry (DSC) (DSC-60, Shimadzu) was performed in the range of 10-300 °C at the same conditions.
To essential to know the exact chemical nature and composition of struvite crystals formed at low phosphate containing medium. The struvite crystals were subjected to FESEM, EDAX, PXRD, FTIR, and TGA analysis.

FESEM and EDAX spectroscopy analysis: The morphological features of struvite crystals were recorded by FESEM. The variations in size of crystals were observed (Fig. 1a). The SEM images of the crystals at higher magnification showed typical prismatic crystal pattern. Bacterial metabolic activities may affect the crystal homogeneity because the bacterial cells synthesize different extracellular components e.g., secondary metabolite or enzymes, etc. in the medium. The variation in size and shape of struvite might be due to different stages of crystal growth, presence of impurities, other ions, and slight changes in pH during struvite synthesis. (Mclean, et al., 1990; Prywer and Torzewska, 2009; Prywer, et al., 2012). The EDAX spectral pattern showed the presence of Mg, P, N, and O in the crystals and the elemental distribution confirms the struvite composition (Fig. 1b).

FTIR and PXRD: The major bands obtained in FTIR spectra (Fig. 2a) were in good agreement with previously reported band values for struvite (Sinha et al., 2014; Kurtulus and Tas, 2011; Huang et al., 2015). The characteristic band at 2879 cm-1 is due to the symmetric stretching vibration of N-H in NH4+ group, a small broad band of water-phosphate hydrogen bonding was observed at 2336 cm-1, while deformation of H-O-H mode of water and H-N-H mode of NH4+ can be seen at 1663 and 1425 cm-1, respectively. The bands at 752 and 678 cm-1 were assigned for water-water hydrogen bonding, although NH4+-water hydrogen bonding can be seen at 881 cm-1. The different vibrational band frequencies of PO43- groups were observed at 1167, 977, 562, 467 and 433 cm-1. The H-O-H bending mode of vibration was observed at 1574 cm-1. Thus, FTIR spectrum of struvite evidenced the presence of different functional constituents in the crystals like N-H bond, P-O bond, and NH4+ and PO43- ions. These observed results can be correlated with previously reported struvite (Sinha et. al., 2014).
The powder XRD pattern of struvite synthesized by siderophore is shown in Fig. 2b. The values of diffraction pattern of the peaks obtained at 2? displayed as consecutive increasing intensity from the 2? range of 10 to 40 values such as 15.74, 16.50, 20.90, 27.10, 31.94, 33.32, and 38.32. These obtained values correlated with (hkl) indices such as (002), (011), (111), (103), (004), (022), (213) of the struvite reported previously from the JCPDS No. 71-2089 (Huang et al., 2015). The synthesised struvite from siderophore is reported to be monophasic in nature.

TGA profile of struvite: The TGA spectra (Fig. 3a) showed that dehydration and decomposition of the struvite started at above 25 °C, whereas continuous weight loss of the product occurred from 70 °C and complete loss at about 250 °C and remained constant up to 1000 °C. This indicates that continuous removal of struvite elementary moieties occurs for e.g., H2O, NH4+, Mg2+, PO43-, etc. At 140 °C, there was a 54.4 % weight loss occurred which is attributed to liberation of water from the struvite crystals (Sinha et al., 2014). The single peak around 125.7 °C of DSC profile (Fig. 3b) showed that the major loss occurred during the heating process. This single peak indicates the simultaneous loss of ammonia and water from struvite. The endothermic heat flow was observed during the heat change as 131.87 kcal/g (Sinha et al., 2014).

Mechanism of struvite formation: In order to find out the molecular mechanism of struvite formation by siderophore (pyoverdine) we validated the hypothesis by the computational approach. Prior to start of pyoverdine mediated metal complexation reactions, the geometry of pyoverdine was optimized in presence of six H2O molecules using semi-empirical PM6 method and its interactions with water molecules are depicted in figure 4a. In this energetically (-1090.062 kcal/mol) stable complex, the water molecules interact with the hydroxyl groups of Q-chromophore, hydroxyl-ornithine, D-serine, OH-histidine and aD-threonine (Table B below). These interactions were found to be common in all energetically stable pyoverdine-ions complexes. The metal complexation reaction was initiated by addition of Mg2+ ion in the pyoverdine-water complex. This yields higher (-858.84 kcal/mol) energy for pyoverdine + Mg2+ metal ion complex (Fig. 4b), which was stabilized by hexacoordinate bonds between Mg2+ with two water (W2/W3) molecules and four amino acid residues in pyoverdine. The carbonyl oxygen of hydroxyl-ornithine and D-threonine residues were involved in coordination bonds with Mg2+ along with hydroxyl oxygen and ring nitrogen of OH-histidine. The water molecules (W1/W4/W5/W6) established the hydrogen bonding interactions with Q-chromophore, OH-histidine, and cOH-ornithine (Fig. 4b; Table 2). The common H-bond interactions were found between water molecules and pyoverdine similar to figure 4a.
Molecule Name/H-bonds Pyo.6H2O Pyo.6H2O+ Mg2+ Pyo.6H2O +Mg2++PO43- Pyo.6H2O +Mg2++PO43-+NH4+ Pyo.6H2O +Mg2++NH4+ Pyo.6H2O +Mg2++NH4++PO43- 6H2O +Mg2++ PO43-+NH4+ Pyo.6H2O +Mg2++ PO43-+NH4+
Metal co-ordination interactions
Mg2+....O1(PO43-) -
Mg2+.... O2(PO43-) - 2.11 1.98 - 1.73 -
Mg2+....O(W2) 2.05 - - 2.1 2.05 -
Mg2+....O(W3) 2.08 - - 2.11 2.02 2.03 -
Mg2+.... O(W5) - - - - 2.05 1.96 -
Mg2+....O1(OHHis) 2.1 2.01 2.01 2.09 - - -
Mg2+....N1(OHHis) 2.05 - - 2.11 - - -
Mg2+....O2(cOHOrn) 2.06 2 1.98 - - - -
Mg2+....O(aDThr) 2.01 - - 2.06 - - 1.95
Mg2+....O(Lys) - - - - - - 1.93
Mg2+....O5(Q-Chromo) - 1.94 1.93 2.04 1.95 - 1.9
Hydrogen bonding interactions
(PO43-)O2...H-O(W2) - 1.47 149.52 1.61 169.92 - 1.65 147.12 1.70 152.89 1.67 163.41
(PO43-)O1...H-O(W3) - 1.55 154.07 1.68 153.55 - 1.68 149.55 1.69 137.70 -
(PO43-)O2...H-O(W4) - - - - - 1.79 147.28 -
(PO43-)O1...H-O(W5) - - - - - 1.81 142.09 -
(PO43-)O3...H-O1(cOHOrn) - 1.29 166.40 1.55 159.80 - - - -
(PO43-)O3...H1-N(NH4+) - - 1.77 133.33 - 1.87 131.17 1.35 158.49 2.03 129.71
(PO43-)O3...H2-N(NH4+) - - 1.43 151.49 - 2.00 130.58 - 1.83 141.21
(cOHOrn)O2...H1-N(NH4+) - - - 1.87 169.40 - - -
(aDThr)O...H2-N(NH4+) - - - 2.17 158.02 - - -
(W5)O...H3-N(NH4+) - - - 2.11 103.40 1.71 160.42 - 1.70 164.01
(W6)O...H3-N(NH4+) - - - - - 1.60 162.30 -
(PO43-)O3...H-O5(Q-Chromo) - - - - 1.76 166.20 1.73 141.26 -
(PO43-)O3....H-N(aDThr) - - - - 2.22 148.80 - -
(PO43-)O4....H-N(Lys) - - - - - - 1.76 143.91
(Q-Chromo)O1...H-O(W4) 2.35 138.82 1.97 156.30 1.75 155.35 1.70 157.72 2.01 157.63 - - 2.45 120.65
(W6)O...H-N(cOHOrn) 1.87 156.55 1.72 176.01 - 2.10 127.17 1.97 163.21 - - 1.91 159.54
(W4)O...H-O1(aDThr) 1.97 156.00 1.81 158.20 1.90 122.34 1.68 163.49 1.96 164.22 - - 1.48 154.38
(W2)O...HN(OHHis) 1.48 165.90 1.53 147.95 1.38 162.70 1.48 151.20 - - - 1.74 154.52
(Ser)O....H-O(W3) 2.18 140.43 2.02 155.05 1.78 141.36 - - 1.74 160.76 - 1.78 149.52
(W3)O.....H-O(W2) 1.59 160.44 - 1.69 134.71 2.04 120.01 1.83 150.63 - 1.74 145.74 1.93 135.40
Respective figure no. 5a 5b 5c 5d 5c’ 5d’ 5a’’ 5d’’
Energy in kcal/mol -1090.062 -858.84 -1463.642 -1416.687 -674.856 -1413.539 -667.272 -1425.162
Table B - Intramolecular and intermolecular hydrogen bonding interactions between struvite ions complex and pyoverdine (siderophore).
After the addition of PO43- ion in the preceding complex it yields the Pyo + Mg2+ + PO43- the complex with the lowest (-1463.642 kcal/mol) energy stable conformation, depicted in figure 4c. In this complex, the Mg2+ was forming two coordination bonds with the oxygen of PO43- ion. The Mg2+ was also involved in coordination with carbonyl, carboxyl and hydroxyl groups of cOH-ornithine, Q-chromophore and OH-histidine residues of pyoverdine, respectively. The PO43- was forming hydrogen bonding interactions with cOH-ornithine and two water (W2/W3) molecules (Table 2). The interactions between water molecules and pyoverdine residues (Q-chromophore, serine, and lysine) were found to be similar to preceding complexes (Table 2). In the presence of PO43-, the Mg2+ did not participate in a coordination bond with water molecules as compared to figure 4b. The cOH-ornithine was involved in the stabilization of Pyo + Mg2+ + PO43- complex by establishing the coordinate as well as hydrogen bonding interactions with Mg2+ and PO43- ions, respectively. These interactions might be helpful to produce the lowest energy stable complex (Fig. 4c).
Further, the formation of struvite was accomplished by addition of NH4+ ion in the earlier complex (Pyo + Mg2+ + PO43-), this resulted in slightly higher (-1416.687 kcal/mol) energy complex (Fig. 4d) as compared to Pyo + Mg2+ + PO43- (Fig. 4c). Thus, herein we corroborated this result with previous study where ammonia plays an important role in struvite nucleation, high ammonia concentration helps in the enhanced recovery of phosphorus compared to magnesium ion concentration present in the solution (Tansel et al., 2018). In this pyo-struvite complex, the Mg2+ was involved in four coordination bonds with PO43-, Q-chromophore, cOH-ornithine and OH-histidine residues of pyoverdine (Table 2). Similar to the earlier complex (Fig. 4c), the PO43- was involved in hydrogen bonding interactions with cOH-ornithine and two water (W2/W3) molecules. The PO43- was also forming two H-bonds with NH4+. During struvite formation, two different types of interactions were observed, i.e. one is coordinate bond between Mg2+ and PO43- and other is hydrogen bond between PO43- and NH4+ (Fig. 4d). The pyoverdine holds the Mg2+ and PO43- ions more tightly and the addition of NH4+ leads to struvite formation. Once the struvite is formed, its internal interactions binding profile may reduce the affinity towards the pyoverdine, this may help pyoverdine to release the struvite and become easily accessible for the next cycle of struvite complexation reaction.
The second pathway was initiated by the addition of NH4+ in earlier Pyo + Mg2+ complex (Fig. 4b), which again resulted in higher (-674.856 kcal/mol) energy Pyo + Mg2+ + NH4+ complex (Fig. 4c’). In this complex, the Mg2+ was forming hexacoordinate bonds with two water (W2/W3) molecules and three residues (Q-chromophore, D-threonine, and OH-histidine) of pyoverdine (Fig. 4c’, Table 2). The NH4+ ion was positioned ~4.5Å away from the Mg2+ ion within the pyoverdine cavity where it interacts with D-threonine, cOH-ornithine, and water (W5) molecule. The cOH-ornithine was also interacting with the other two water (W1/W6) molecules. The interaction network (two coordinate and four hydrogen bonds) was observed between two ions (Mg2+, NH4+), two water molecules (W1/W6) and two pyoverdine residues (D-threonine and OH-ornithine) in Pyo + Mg2+ + NH4+ complex (Fig. 4c’). This complex is energetically unstable as compared to the Pyo + Mg2+ complex but retains a similar binding pattern of Mg2+ with water molecules and pyoverdine residues (Figs. 4b and 4c’).
Further, the addition of PO43- in the preceding complex leads to production of energetically favourable complex of struvite (Fig. 4d’). In this stable complex, the Mg2+ was forming five coordinate bonds with PO43-, Q-chromophore and three water (W2/W3/W5) molecules (Table 2). The oxygen atoms of PO43- were involved in hydrogen bonding interactions with water (W1/W4/W5/W6) molecules, NH4+ ion, Q-chromophore and D-threonine residues of pyoverdine (Fig. 4d’). The water (W1) molecule was also involved in H-bond with D-serine and NH4+ ion. In this complex, the NH4+ was positioned in the middle of pyoverdine cavity formed by Q-chromophore, OH-histidine, D-serine, and aD-threonine residues. While water molecules interacted with struvite complex through coordinate and H-bonds interactions. The struvite complex (Fig. 4d’) contains one coordinate bond between Mg2+ and PO43- ions and two H-bonds between PO43- and NH4+ ions similar to struvite complex formed in the earlier pathway (Fig.4d). Therefore, the pyo-struvite complexes produced by two complexation reaction pathways (Fig. 4) possesses similar energy content and same internal interactions pattern suggests that struvite formation could occur through different pyoverdine mediated pathways based on sequential addition of ions.
Moreover, to understand the role of pyoverdine in struvite formation, we have performed conformer distribution calculations in the absence and presence (addition of all ions at a time) of pyoverdine (Figs. 4a’’ and 4d’’). In the absence of pyoverdine (water control), the obtained struvite complex has higher (-667.272 kcal/mol) energy content as compared to earlier formed struvite complexes (Figs. 4d, 4d’). In this complex (Fig. 4a’’), the Mg2+ was involved in four coordinate bonds with PO43- and water (W2/W3) molecules. The PO43- was also forming hydrogen bonding interactions with five water molecules and NH4+ ion (Fig. 4a’’). Two coordinate bonds (between Mg2+ and PO43-) and one H-bond (between PO43- and NH4+ ions) were observed in struvite complex which were produced in absence of pyoverdine (Fig. 4a’’). Whereas, one coordinate bond (between Mg2+ and PO43-) and two H-bonds (between PO43- and NH4+ ions) were noticed in struvite complex which were produced in presence of pyoverdine (Figs. 4d, 4d’).
Furthermore, we have performed conformer distribution calculations on pyoverdine where all (Mg2+, PO43- and NH4+) ions were added in a single step. The resulting lowest (-1425.162 kcal/mol) energy stable complex has similar interactions and location of struvite within the cavity of pyoverdine that observed in struvite complex produced in the presence of pyoverdine (Figs. 4d and 4d’). The pyoverdine mediated all struvite complexes (Figs. 4d, 4d’ and 4d’’) are energetically more stable than produced in absence of pyoverdine (Fig. 4a’’) which could be explained by coordinate and hydrogen bonding interactions between the complexes. The interactions of water molecules with struvite and pyoverdine residues were significantly contributed to the proper positioning of struvite within the cavity of pyoverdine. The first pathway (Pyo + 6H2O + Mg2+ + PO43- + NH4+) is energetically stable as compared to second pathway (Pyo + 6H2O + Mg2+ + NH4+ + PO43-) therefore first pathway of complexation reaction could be more favourable for the formation of struvite.
Nonetheless, Li et al., (2015) used a peptide model in previous study to demonstrate in vitro struvite biomineralization in solution for e.g. polyaspartic acid (PASP) as urinary protein for studies of interaction between protein and mineral formation. They suggested that PASP interacts with the ions in solution by its chelating action to form stable complexes. As a result, the activity of free metal ions decreased to limit crystal growth (Wu, et al., 2002; Wang, et al., 2009). Numerous studies have shown that the presence of special acidic proteins or macromolecular matrices plays an important role in the nucleation of metal ions on the surface of the macromolecule in the biomineralization process of different mineral formation (Addadi and Weiner, 1985; Weiner, 1979, 2008; Addadi et al., 1987).
From the theoretical studies, we concluded that magnesium, ammonium, and phosphate ions with water have -667.272 kcal/mol of energy indicating very unstable complex. Whereas in presence of pyoverdine it showed very stable complex with the energy of -1416.687 kcal/mol. This shows that, siderophore plays an important role in struvite formation by trapping the phosphate ions.

Mechanism of struvite formation by siderophore: A computational approach
In order to understand the mechanism of siderophore mediated struvite (MgNH4PO4 6H2O) formation, further analyzed conformer distribution calculations on different metal ions complexes. From our experimental results, we predicted two different pathways of struvite formation based on the alteration in the sequence of addition of ions into the siderophore containing media. The first pathway is siderophore.6H2O + Mg2+ + PO43- + NH4+ and the second pathway is siderophore.6H2O + Mg2+ + NH4+ + PO43-. These pathways are distinct from each other due to change in the sequential addition of NH4+ and PO43- ions during the complexation reactions. The amino acid sequence of siderophore has been adopted from the previous study (Chen et al., 2016). The initial structure of siderophore was constructed and geometrically optimized by semi-empirical PM6 method using Spartan’14 molecular modeling software (Shao et al., 2006). Each ion was individually optimized by DFT (B3LYP/6-31G** basis set) method to ensure their correct electronic charge during the calculations (Becke et al., 1992). Hundred runs of conformer distribution calculations were performed on each of metal ion complexes in the presence of siderophore. Similar calculations were performed without siderophore and by using all ions and water, this served as control. The lowest energy stable conformations were selected and used as a starting geometry for the next step of complexation reactions. Likewise, eight different siderophore and ions complexes were generated and used for the calculations. The lowest energy stable complex from each complexation reactions was analyzed for hydrogen bonding and metal coordination interactions. These complexes are positioned on the potential energy surface diagram with respect to their energy content.
For ameliorating phosphate pollution in accordance with the present invention, phosphate-rich industrial waste was treated with a suspension of siderophore obtained as per the preceding narration. Particularly, the process was typified by the following sequence of steps-
1) Preparation of mSM-A media broth containing media ions as per the foregoing narration;
2) Inoculating P. taiwanensis culture into the mSM-A media broth, incubating the same for 24 to 48 h, subjecting the resultant medium to centrifugation to remove the broth which contains siderophores at a concentration of about 5U. This siderophore solution, after addition of media salts as per aforesaid description (thus constituting a phosphate-sequestration agent) is ready to use for removal / recovery of phosphates as per the present invention;
3) Phosphate-rich industrial waste (concentrated liquid which contains around more than 200 mg per 100 ml or 200 mg per gram of any industrial sample like phosphate rich fertilizer effluent waste or solid waste, ceramic industrial waste, municipal waste, sewage waste, detergent industrial waste, animal husbandry waste, lavatories waste, and fly ash waste, etc) is added to the siderophore solution prepared in step 2) to thus constitute a process admixture;
4) Minimum 50 mg per 100 ml or 50mg per gram of phosphate is removed by siderophore solution of step 2) above, in a batch process at room temperature with holding time of 20 to 25 min, with chemistry as explained in the foregoing narration. In presence of magnesium and ammonia, phosphate ions get precipitated;
5) The siderophore solution is monitored for pH for successive addition of batches of solid industrial waste to be treated, and can be regenerated if pH falls OR after every 3 batches of removal of phosphate from waste by adding 1M NaOH solution in system.

Recovery of phosphate from industrial waste: In the studies undertaken, the amount of phosphate was 173.29 mg/g solid waste. More than 90 % of P was recovered in the form of struvite in presence of siderophore. Around 40 % of P was recovered by in the form of struvite in water (Control). The amount of phosphate used in the in vitro mSM-A study for struvite formation with siderophore was 40 ppm, whereas the solid P rich waste contained almost 43 times more phosphate than used in the study. By using siderophore more than 90 % of phosphate was recovered compared to 40 % recovery in control. While, in water control, amorphous particles were observed with far less quantity whereas fine crystalline nature of particles was observed with high quantity at the bottom of siderophore solution. In the siderophore solution with the rapid recovery (10 to 20 min) the amount of P entrapment was as high as 90 %, whereas in control it takes more than 1 h for precipitation. Even after further incubation for 48 h, the precipitation did not increase

Reusability of the phosphate-recovery agent: Referring to FIG. 7, it can be seen that Phosphate removal from industrial sample (a, b) industrial waste sample, (c) siderophore mediated struvite formation and with water as control and (d) Phosphate removal from industrial solid waste sample by using siderophore with water as a control. The blue colour bar indicates total amount of phosphate sequestered (<90 %) and the orange colour region indicates the phosphate recovered in control after every cycle in the siderophore broth (> 40 %). The SD ± mean corresponds to 3 independent experiments.
According to another aspect of the present invention, the siderophore-based treatment protocol mentioned in the foregoing narrative was assessed to verify generation of downstream products of value. Here, struvite was observed as a by-product. Further addition of salts of magnesium, phosphate, and ammonia in the broth resulted in their removal via precipitation. The insoluble salts settled at the bottom and could be easily removed by conventional means for separation. Figure 5 is a schematic to illustrate siderophore mediated struvite formation according to the disclosures hereof.
It shall be appreciated that in the water remediation protocol (indirectly, as the industrial wastes originally being dumped into the water bodies is now able to be treated by method of the present invention) established above, a commercially important biomineral is synthesized, namely Struvite (Magnesium ammonium phosphate hexahydrate; MgNH4PO4.6H2O) which is known to be a slow release fertilizer providing three important macroelements such as N, P and Mg in single unique combination. This evidences that siderophore of the present invention was engaged in struvite formation process, that is, cell-free siderophore solution acted as a nucleation matrix for struvite formation which can be led to crystallization by adding salts of magnesium, ammonium, and phosphate in the form of dry powder. (Even though number of processes are reported so far for struvite synthesis, this invention scores above them in that it allows even the lowest concentration of media ions i.e., 0.6 mM being amenable to siderophore-based sequestration into struvite mineral. However till date only 1.3 mM phosphate ions sequestration was only possible reported so far.)
In a parallel study undertaken by the applicant named herein, the electrochemical super-capacitance performance of the struvite was studied. The specific capacitance value for the struvite was found to be 320 F g-1 at 1.87 A g-1 and retained 92 % capacitance after 250 cycles.
The reader shall now appreciate, that sequestration of magnesium, ammonium, and phosphate is possible in the scheme mentioned above, with generation of struvite as an end-product. The siderophore solution is reusable, for at least nineteen times as independently confirmed by the inventors hereof, for sequestration / recovery of said contaminants.
According to yet another aspect of the present invention, the inventors named herein have experimented with different corollary trials including influx of one or more among magnesium, ammonium, cobalt, nickel and phosphate and thus realized different complexes which may accord independent commercial applications ranging from / fuel cell applications. A summary of these trials is represented in Table 2 below.
Components Combinations (gm/100ml siderophore)
(1) (2) (3) (4) (5)
1) K2HPO4 0.150 0.150 0.150 0.150 0.150
2) KH2PO4 0.150 0.150 0.150 0.150 0.150
3) MgSO4.7H2O 0.100 0.100 0.100 0.100 0.100
4) NiCl2 0.050 - 0.050 0.034 0.016
5) CoCl2 - 0.050 0.050 0.016 0.034
Table 2
In line with the preceding paragraph, the inventors named herein have taken waters containing Ni and Co as a major pollutant (for example, from electroplating industries) for sequestration of both these metals. Thus, removal of said two metals was observed in the sample as a insoluble metal complex resulting at the end. On basis of these initial findings, the inventors named herein have designed modified succinate broth for newer synthesis route thereby improving on prior route for struvite synthesis using siderophore. Ni-struvite and Co-struvite synthesized thus using this new approach were found to be useful for fuel cell application in hydrogen evolution reaction.
The standard material used for Hydrogen Evolution Reaction (HER) - platinum - showed 99 % efficiency for HER activity utilized for fuel cell technology. However, Ni-Co struvite material synthesized as per the foregoing narration showed 98% activity which is very near to platinum, proving utility of the present invention for fuel cell technology.
From the foregoing narration, the present invention is identified in having the following salient features-
a) Establishment of a simple, yet able technology for remediating heavy metal pollution of water bodies with improved performance, endurance, and output than any of its closest peers in state-of-art.
b) Establishment of a standardized phosphate removal process, using siderophore which plays an important role in struvite biomineralization.
c) Output of commercially viable by-product, struvite which can be used as slow release phosphate fertilizer / super capacitor / piezoelectric / fuel cell catalyst / other applications.
d) Successful demonstration of phosphate sequestration by using industrial waste samples, as possible application for environmental sustainability and phosphate conservation.
As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention which will be limited only by the appended claims. ,CLAIMS:1) A reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products, the process comprising-
a) Preparing a phosphate-sequestration agent, being a suspension of media ions and siderophores;
b) Adding the solid industrial waste to the phosphate-sequestration agent and subjecting the resulting process admixture at room temperature to a holding time of 20 minutes to 25 minutes to result in chelation of phosphate ions and formation of commercially valuable by-products which settle down and hence sequestered, by separation from said process admixture; and
c) Repeating step b) for successive addition of solid industrial waste to be treated, therein monitoring the pH of the process admixture between each instances of repetition of step b), and in the event pH falls, adjusting the same by adding 1M NaOH to the process admixture to regenerate said process admixture for repeated use in rapid sequestration of phosphates from solid industrial waste.
2) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the step of preparing the phosphate-sequestration agent consists of-
a) Preparing a siderophore-inducement medium;
b) Inoculating the siderophore-inducement medium with a bacterial culture known for extracellular production of siderophores and incubating said culture broth for 24 hours to 48 hours for production of siderophores;
c) Upon completion of incubation, subjecting the culture broth to centrifugation for separation of the supernatant containing inorganic ions and siderophores in suspended format, being the phosphate-sequestration agent ready for use.
3) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the siderophore-inducement medium is modified succinate medium broth, prepared by admixing-
a) 0.5 g % of succinate;
b) 0.25 g % of NH4Cl;
c) 0.01 g % of K2HPO4;
d) 0.01 g % of KH2PO4; and
e) 0.075 g % of MgSO4.7H2O.

4) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the media ions are selected among Mg2+, PO43-, and NH4+.
5) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the bacterial culture known for production of siderophores is Pseudomonas taiwanensis R-12-2 isolated from the tannery soil.
6) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the step of addition of 1M NaOH to the process admixture is undertaken after every three instances of addition of the solid industrial waste, to ensure peak processivity of the phosphate-sequestration agent.
7) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the industrial waste is selected among phosphate rich fertilizer effluent waste, ceramic industrial waste, municipal waste, sewage waste, detergent industrial waste, animal husbandry waste, lavatories waste, and fly ash waste having phosphate concentration at least 0.6 mM.
8) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 1, wherein the commercially valuable by-products are phosphate, struvite and salts of magnesium, phosphate, and ammonia.
9) The reusable method for rapid sequestration of phosphates from solid industrial waste and therein outputting commercially valuable by-products as claimed in claim 8, wherein the struvite formed is typified by the formula MgNH4PO4.6H2O.
10) An aqueous medium for cultivating Pseudomonas taiwanensis R-12-2 for high production of siderophores, the medium comprising-
a) 0.5 g % of succinate;
b) 0.25 g % of NH4Cl;
c) 0.01 g % of K2HPO4;
d) 0.01 g % of KH2PO4; and
e) 0.075 g % of MgSO4.7H2O.