CA1206057A - Siderophoric compositions - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:

The present invention relates to a composi-tion useful for the removal of metal, in particular iron, from a liquid medium. Thus, there is provided an insoluble composition comprising:
(1) one or more organic chelating compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said organic compounds possessing one or more coordinating sites. The organic chelating compounds can be selected from the class consisting of catechol and microbial siderophores.

Description

The present invention relates to a composi-tion useful for the removal of metals, in particular iron, from liquid media. The composition, can for example, be used to lower the iron concentration of a liquid medium to less than 0.1 ~M.
Iron is an essential nutrient for all living thinys;
a large number of cellular enz~nes and other proteins require iron in order to function properly. Although iron is amongst the most plentiful of metals, it is difficult for biological systems to acquire; in aerobic environments of substantially neutral pH, iron exists as its oxidized Fe3~ form which readily hydrates to highly insoluble Fe(OH)3 polymeric forms. To ensure accessability of iron in their environment, aerobic and facul-tative micro-organisms synthesize and release into their environ-ment highly selective iron chelating agents called siderophores, the function o which is to provide the microbes with this vital nutrient. The siderophores release~d by the microbes solubilize iron, putting it into a orm readily usable by them. Thus, a free, microbial siderophore is a growth promoting substance for those or~anisms which can utilise the particular siderophore in ~uestion.
In accordance with the present invention it has been determined that removal of iron (e.~ Fe3+) from a liquid nutrient medium will substantiall~ restrict the prolieration of microbes provided that the residual iron concentratlon in ~5 the medium is below 0.1 ~M; and this notwithstanding that the other required nutrients may be present in amounts suEicient for the support of microbial growth.
~hus, it would be advantageous to have compositions able to remove iron rom a liquid, nutrient medium since the absence or limited presence of iron will inhibit rnicrobial growth in such a medium.
For example, the speciEic removal of iron from an ophthalmic solution will inhi.bit microbial growth and spoilage o such a solution. l'he removal of iron from SUC}l a solu-tion would obviate the addition of conventional microbial grow-th ~f~V~

inhibitors which can create their own problems such as toxicity, etc.
Removal o iron from liquid media prior -to the present invention did present problems (see Neilands, J. sacteriology 149: 830, 1982). Commercially available products (e.g. Chelex 100 sold by BioRad) have a low selec-tivi-ty for iron. Addition-ally, other important cations (Mn2~, Mg2+) removed by such commercial products are often desirable components of a licluid medium. Commercial ion exchange products can also liberate iO sodium or potassim ions into the liquid medium being treated which may not be desirable.
Thus, it would be advantageous to be able to remove iron from a liquid medium while at the same time Awoid:ing liberating into the liquid medium undesirable ions.
lS In general, it would also be advantageous to be able to remove iron ~rom a liquid medium when the presence of iron is undesirable, e.g~ when iron is considered a contaminant at concentrations greater than 0.1 ~M.
Free microbial siderophores cannot be used to remove iron from a liquid medium; the nat:ural purpose of such side-rophores is to make iron soluble and available to micro-organisms. The addition of ree siderophores to a liquid medium would therefore enhance the growth of microorganisms which could utilise iron solubilized ~hereby. Additionall~
it would be e~tremely difficult at the very least, to recover such siderophores loaded with iron.
Nevertheless, it would be advantageous to be able to make use of the properties of siderophoric compounds such as microbial siderophores and other organic compounds which can provide co-ordinating groups or ligands for the chelating of iron (ie Fe3 ).
The present inven-tion in general relates to inso-luble compositions, which are capable oE removing metal (e.~.
selective:ly) Erom solution (e.g. Fe3 from a liquicl nu-trient `Tr~6lt ~ 2 -.~ ,.

medium so as to lower the Fe3 content to less than 0.1 ~M), the insoluble composi-tions comprise:
(i) a sui-table insoluble carrier and (ii~ organic co-ordinating sites covalently fixed to the surface of said carrier.
In accordance with the present invention the necessary co-ordinating sites may, for example, be provided by fixing an organic chelating cornpound such as a microbial siderophore to a suitable carrier (infra).
Thus in accordance with one aspect of the present invention, there is, in particular, provided a method for inhibiting mlcrobial growth in a liquid nutrient medium containing Fe3~ by lowering the Fe3~ content thereof to less than 0.1 ~M characterized in that said medium is contacted with an insoluble siderophoric composition and thereafter said insoluble siderophoric composition loaded with Fe3~ is separated from said medium, said insoluble siderophoric composition comprising:
(1) one or more organic siderophoric compounds, covalently fixed to the surface of

(2) a suitable insolublle carrier, said oryanic siderophoric compounds possessing one or more co-ordinating sites capable of chelating Fe3+.
An insoluble siderophoric composition , for the purposes of this aspect of the invention, is a composition having a chelating activity with respect to iron, and in particular a selective chelating activity.
The organic co-ordinating sites of suitable organic siderohporic compounds may, for example, be provided by groups selected from the class consisting of (a) N-substituted hydroxamate groups of formula O OH
Il l I
- C - N ~ C -

3 -(b) phenolate groups of formula OH X

X being an atom of O or N-(c) catecholate groups of formula OH OH
~C~-X' being an atom of O or N- and m being 1, and (d) mixtures of two or more of the above groups;
see below.
If desired, catechol (1,2 dihydroxybenzene) may be used as a siderophoric compouncl to provide catecholate type co-ordinating sites.
The insoluble siderophoric composition can for example be used to speciically remove iron from liquid media such as water, juices, wine, beer, cider, chemical so]utions, microbial and tissue culture media, pharma-ceutical media , etc.
In accordance with another aspPct of the present invention, tnere is provided an insoluble composition comprising a member selected from the class consisting of (A) an insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface oE
(2) a suitable insoluble carrier, said organic chelating compounds possessing one or more co-ordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores and (B) an insoluble composition comprising ~! , .

(1) catechol covalently fi~ed to the surface of (2) a sui-table insoluble carrier, the catechol being covalently fixed ko the surface of said carrier at the benzene ring thereo.
The above compositions, in accordance with -this other aspec-t of the present invention can be used to remove Fe3 from solu-tion.
Thus this other aspect of the present inven-tion also provides a method for removing Fe3 from solution characterized in that the solution is contacted with an insoluble composition as defined above. Thereafter, the compos.ition loaded with metal may be separated from the treated solution~ For example, the iron content of a liquid medium may in this way be lowered to less -than 0.1 ~M. Thus an insoluble composition in accordance with this aspect of the present invention may advantageously be used asa siderophoric composition to remove Fe3~ from li~uid nutrient medium .
In paxticular, this aspect of the present invention also provides a method for inhibiting microbial growth in a liquid nutrient medium containing Fe3+, by lowering the Ee3 content thereof to less than 0.1 ~M characterized in that said medium is contacted with an insoluble compo-sition as defined above and thereaf-ter said composition loaded with Fe3 is separated from the medium. The iron loaded composition can, for example be recovered by filtration.
Compositions as defined above, loaded with Fe3~
may possibly be regenerated by chemical means sui-table for the removal o the chelated metal; the so regenerated compo-sition can thereafter be recycled for further use.
The pres~n-t invention thus provides not only a mechanism for the removal of Fe3~ from liquid media but also for the preservation of various liquid media through the removal of iron thereErom, i.e. rendering liquid nu-trien-t 3~ media highl~ resistall-t to microbial grow-th since any micro-~1 ~2V~

organism present cannot prolifera-te due to -the insuf~icient amount of iron present.
Organic chelating compounds, e.g. siderophoric compounds, useful in accordance with -the present invention may also ~orm complexes with chromium ions and alpha-emi-tting actinide ions due to the struc-tural (a-tomic~
similarities with iron; however, the complexPs are formed at lower aEfinities than iron . Al-though the compositions o~ the present invention can possibly be used Eor the removal of these other metals from liquid media, the following discussion will be directed to the removal of Fe3+ from liquid media.
In accordance with the present invention, a general process for the preparation of an insoluble composition as de~ined above can be characterized in that organic co-ordinating sites capable of chelating metal are covalently fixed to the surface of a suitable carrier. Any suitable means of covalently Eixing organic co-ordinatlng sites to a carrier can be used provided that the composition obtained has the necessary chelating activity.
If it is desired to procluce a side:rophoric compo-sition comprising one or more organic siderophoric compounds fixed to a suitable carrier, then the process o~ its pre-paration may be characterized in that a sui-table carrier is reacted with one or more organic siderophoric compounds possessing co-ordinating sites capable of chelatin~ Fe3~
so as to covalently bond said siderophoric compounds to -the surEace of said carrier, while maintaining the Fe3~ chela-ting activity o~ said siderophoric compounds.
The siderophoric compounds as indicated above can be microbial siderophores. In this particular case, the process of preparation can be characterized in that a sui-table carrier is reac-ted ~i-th one or more microbial sidero-phores so as to bond said microbial siderophores -to the surEace oE said carrier, said carrier and said microbial ",~", "

~;~(36~i7 siderophores possessing fu~c-tional groups reac-tl~e one with the other so as to covalently bond said microbial sidero-phores to said carrier while main-taininy the Fe3 chelating ac-tivi-ty of said microbial siderophores.
Turning now to the chela-ting compounds, sufficient metal coordination sites to chelate the metal ions (e.g.
Fe3~) may be provlded by a single organic chelating (e.g.
siderophoric) compound or alternatively hy two or more such compounds; the number of compounds participa-ting in the chelation of the metal ions being dependent upon the number of coordinating sites which are available from a particular compound fixed to a carrier.
The organic chelating compound used can as indi-cated above be a microbial siderophore. ~ microbial side-rophore may have a molecular weight of less than 2500 Daltons, e.g. a molecular weight in the range of 500 to 2500 Daltons. A microbial siderophore useful in accor-dance with the present invention ~an also possess one or more types of metal coordinating sites within i-ts structure.
The si-tes can be provided by groups selected from the class of groups referred to earlier, e.y. N-substituted hydroxamate groups, catecholate groups, etc. Siderop~ores possessing these groups display high selec-tivity and very high affi-nities for Fe3 A r~presen-tative list o~ microorganisms and their siderophores is given in ~ollowing table 1: -- 6 a -~2C~

Table 1 COMMON NAMES OF SIDEROPHORE
ORGANISM NAME OBTAINED THEREFROM
Prokaryotes Enteric spec.ies Enterobactin ~enterochelin), Aerobactin Agrobacterium tumefaciens Agrobactin Pseudomonas species Pyochelin, Pyoverdine, Pseudobactins, Ferribactin Bacillus megaterium Schizokinen Anaboena species Schizokinen Art _obaoter species Arthrobactin Azotobacter vinelandii a,~-bis-2,3,-dihydroxyben-zoyllysine Actinomy~e_ species Ferrioxamines Mycobacterium species Mycobactins Eukar~otes (Funqi) Penicillium species, Ferrichromes, Copragen A~E~ lus species/
Neur_spora, Ustillago Rhodotorula species Rhodotorulic acids ctomycorrhizal species Hydroxamate type The basic chemical structure, trivial names and possible sources oE some types of microbial ~iderophores are listed below; the term microbial siderophore o course includes any suitable functional de.rivatives, analogs or enantioforms of these molecules:
a) Fe oxamlne H-N ~ ~CONH/ CONH
('CH2)s ~CH2)2 ~C\2)5 ~CH2)2 ( \2 n R~
o d o ~ ~ - o o Linear ferrioxamine Q~7 ,~ , H--~ ~ CONH\ / CONH\ ~=
2 5 ; 12)2 (C\2)5 jCH2)2 (C ~ (t N - N - Cl -N --- 0_ _ O~ ~ -- O ~ ' ,, A

C~lic ferrioxamine wherein: ~
R = H or -COCH3; R' = CH3- or HOOC-(CH2)2-; n = 4 or 5-For ferr:ioxamine B, R=H and R' - CH3-. The mesylate salt of deEerrioxamine is marketed by Ciba-Geigy as Desferal (U.S. pat. 1964 3,118,823 and 3,153,621; Can. pat. 1962 548981 and 715051.

b ) F~rr ichrome s H Hl ~ ~C ' C ~
Q~ .. c~/ \ N /H

Fe3 ¦ I
'~~` ~C~O I -C N~

\ W--~ CH ~C H

H~N ~ C l / C
ll C ~ -H
R~
wherein:
R', R" , ~"' = H Ferrichrome R = -CH (prototype) ~L2~

c) Ci.trate Hydrox-dmate Derlvatives wherein for:
fH3 ICH3 R n f=O O=C schi~okinen H 2 N-OH HO-N aerobactin COOH 4 (CH2)n (CH2)n arthrobactin H 4 ¦ 1l CIOOH 1l H-C-N-C~CH - -CH - -N-C-H

R H OH H R
d) Rhodotorulic Acids 1l NH ~ N ~ C~-CH3 rhodotorulic acid H3C-C- ~ ~ HO H
H H ~ r~CH20H
!l I H O ~ N ~ ~ N ~ ~ CH dim~mic CH ~ C - N ~ ~ ~ Hl O 3 aci.d ~ H

(d) Rhodotorulic Acids (cont.) -CO~3 ¦¦ 2~C3 (CH2) 3 ~ / \
CO ~0 0 ) 03 copragen CH~ / ~ \N

CH2 ,~ I
C/3C ~C~2)2 ~2 - ~ NH

~Z~ )5~

(e3 Mycobactins ~ IIO)~ HR5 N~ ~ 1HP4 1l IOH C\O R3 j ~N NH
R1 ~1H 2 ) 4 --CO

wherein:
R1 = methyl ethyl or alkyl or alkenyl of 11 to 20 carbon atoms R2 = H or methyl R3 = H or methyl 20R~ = methyl, ~thyl, or alkyl of 15 to 18 carbon atoms R5 - H or methyl (f) Fusarinines O NH2 H0 O wherein:
HO ~ ~ ~ C (CH2) 3 N C ~H2-CH2-0 ~ H, n - 1 to 3 H

~2C~

( g ~ Er~terobact in OH

~OH
C--O
Hl~

C^' H-- C~
/ El '`~

O--C E~CH

~ ~

O / / \ /C\N

2 5 ~ cH O ~ \\
HO
OH

(h) Agrobactirls .N~

3 ~0~1 ~ N ~ H
N

where in:
R = H,OH

-- :L3 --s~

(i) Pseudobactin o o C~-C~ -CH -C
2 2 ~H H
H2N ~/

OH HO ~ \C~ ) f ~33 OH

CH~ \ / C113 C~C~ f A detailed description of the above siderophores is given by Neilands (Annu. Rev. Biochem. 50: 715-731, 1981), and their coordination chemistry has been reviewed by Raymond & Carrano (Account of Chemical Research, vol. 12, No. 5, 1979 at pages 183-190).
Microbial siderophores can be extracted for example from spent microbial culture media with organic solvents.
Examples of such methods are given in United States Patent Nos. 3,118,823 and 3,153,621 as well as Canadian Patent Nos.
648,981 and 715,051. For example, siderophores possessing hydroxamate ligands may be obtained in this fashion. Hydro-xamate microbial siderophores are distributed widely through-out the prokaryotic and eukaryotic microbial world, but to date, only bacteria are known which produce typical mono- and dihydroxybenzoic acid-bearing sidexophores.
Some microbial siderophores, their analogs and/or their enantioforms have been chemically synthesized in the laboratory:
(i) enterobactin, its enantioform and carboxylic, methyl, and aromatic analogs;
(ii) Nl, N8-bis-2,3-dihydroxybenzoylspermidine;
(iii~ ferrichrome and enantio-ferrichxome.
Other processes for the preparation o~ various siderophores are described in Canadian Patent Nos. 742,670, 746,~73, 773,540 and 775~539O
A discussion of the preparation of siderophores by denovo synthesis can be Eound in Neilands Review 1981 and Neilands et al J. Biol. Chem. 256 ; 3831 - 3238, 1381.
The ferrioxamine B is sold commercially under the designation Desferal which is a trademark of Ciba-Geigy~
The microbial siderophores, ferrioxamine and enterohactin, referred to above are prototypical natural micro-bial siderophores and each represents the general structure and properties of hydroxamate and catecholate-bearing sidero-phores respectively. These particular siderophores will be referred to below (e.g. m the exa~ les). For the purposes o ~is speciica-tion-the expression des as it appears before ferrioxamine e-tc is to be under-s-tood to refer to errioxamine etc wl~erein co-ordinating sites are unoccupied e.g.
-they are not iron loaded.
Turning now to carriers suitable in accordance with the present invention, they must of course be insoluble in the liquid medium of intended use; for example, the carrier can be water insoluble. Desirably, the carrler is also inert in the liquid medium of intended use. The carriers can be in particulate or solid form.
The carrier can be an organlc or inorganic compound.
For example, the carrier may be a natural or modiflecl natural polymer (e.g. lignin, agar, alignate, glucan, ce]lulose, dex-tran, cellulose aceta-te, humic acid, e-tc.) a synthetic organic polymer (e.g. a polyamide, a polyamine, a polyacrylamide, a polyester, a polyurethane, a polyethylene, a polystyrene, a polypropylene, a polycarbonate, a silicone, nylon, latex, a poly1uroolefin, etc.) or an inorganic material (a ceramic, a glass, carbon, etcO~.
As indicated above the present invention provides a (siderophoric) composition comprising one or more microbial siderophores which are covalen-tly immobilized or fixed on a suitable insoluble carrier in such a way ~ha~ the microbial siclerophores re-t~in the:ir high chela-ting affinity for metal ions, i.e. iron.
A number of known processes are suitable for the binding of microbial siderophores to carriers so as to preserve the iron chelating or cornplexing properties thereof. Forexample, the commonly used methods Eor covalently binding enzymes to insoluble carriers can be adapted Eor the immobilization of microbial siderophores. See, for example ~Me-thods of Enzy-mology, XXXIV s:30 (Jakoby W.l3. Ed.) Academlc Press, New York (197~).
Carriers which are su:Ltable for the process oE
prepc3ring slderophoric compositions using microbial siderophorcs are ~.hose which have active surfaces; the active surfaces have 6~

functional groups which can bond to a cornpatib]e functional group`
of the chosen siderophoric compound. The Eunctional group can, for example, be selected from the class consisting of O O
2~ (CH2)n-NI12, n being 0, 1, 2 3 etc OH
~-H~-CH2-X, X being a halogen atom, for example, Br, O-C - NH, -OH ~ -C-XI X bein~, as defined above~-C-N3-SO3H,and ~ N2 .
Howevex, any functional group can be used which will react with a functional group on the microbial siderophore in question to bind it to the carrier, the microbial siderophore retaining its iron chelating capacity.
It is possible to put some distance between a microbial siderophore and the surface oE the carrier, e.y.
in order to limit the effect on the microbial siderophore of a surface characteristic of the carrier. For example, teflon may be used as a carrier. However, teflon has a highly hydrophobic surface which is non-wetting. There-fore, it is desirable to put some distance between ths sur-face of the teflon and the microbial siderophore to allow the siderophore to extend well into an aqueous liquid medium.
A spacer compound may be used to provide a spacer group to space apart a carrier and a sideroph~re.
A suitable spacer compound is bifunctional; i.e.
it has a functional group which can react with a functional group of the carrier to bind it thereto; and it has also a second functional group which can react with a compatible functional group on the chosen microbial siderophore to bind it thereto: see the above groups. The spacer ~roup may alternatively have a second functional group which while not reactive with a compatible functional group on the siderophore, may be convertible into such a group.
spacer compound can,for example, tn addition to ~- 17 -}~7 the above referred to functional groups, include a hydrocarbon chain, the length of which is chosen in accordance with the distance which it is desired to place between the carrier and the siderophore. The spacer compound used may be l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride salt or glutaraldehyde. However any compound can be used which will space the microbial siderophore from the carrier, the necessary or desired distance provided of course that it is bifunctional.
The spacer compound may be bound, to a carrier by making use of conventional reac-tions involving the formation of ester groups, amide groups, amino groups, diazo groups, ether groups, sulphonamide groups, amidino groups; the reaction may be a carbon-carbon condensation.
Thus a carrier suitable for the process of the present invention may be represented generally by the formula r back t R - ~cn J

wherein n is an integer, "back" is a carrier backbone, i'R"is a single bond or a suitable spacer group and " ~cn" is a functional group as defined above. For example " ~cn" may be a carboxyl group and R may be a group such as ( 2)2 C NH - (CH2)2 - NH - ~ - (CH2)n -A useful carrier may need to have its surEace treated in order to provide the surf ace with a suitable Eunctional group which can bond to a microbial siderophore.
Nylon, for example, is a carrier which requires a pretreatment to provide it with suitable Eunctional groups.
Since the nylon eontains the amide group, its surEace may be sub~ected to partial hydrolysis uslng known techniques -to glve Eree amino and carboxyl Eunctional groups. The aqueous metho~l may proceed ~s below:

~z~c~æ~

O H O H
R - C - N-R HCl ~E~ - C - OH HN - R
, 1 Mylon (R being Modified Nylon the rest of the chain~
Alternatively, nylon can be reacted in a non-aqueous~edium with, for example, thionylchloride to give rise to the functional group - C = N. If desired, an Cl appropriate spacer group can be readily attached through~
for example, the use of ethylene diamine or another func-tionally equivalent species such as N02 ~ NH2 followed by a reduction of -N02 to -NH2 suitable for generation of diazonium salts which are then suitable for coupling to a mucrobial siderophore,e.g. enterobactin. Succinic ~nh~dride can thereafter be used as a further extension of the spacer group; i.e. to form an amide linkage.
Teflon is another useful carrier which must be pretreated in order to provide it with a suitable functional -,~ group which can bond to a microbial siderophore.
Teflon~ a tradename or pol~vtetrafluoroethylene from DuPont, is highly inert and is not readily attacked by acids and bases. No easy displacement of the fluorine atomes is known. Fluorine atom can,however,be displaced by ion-radicals such as sodium or potassium napthalenP of formula:
Ell NO
Na+
~ ~ El or On reaction between te10n and such sodium or potassiumnaphthalene, a sodio or potassio species of teflon is formed of formula:
E;' E~`
--C -- C --Na F

7-f~ r1~ ~ 19 ~3LZ~t~

These organo metallic species of teflon are highly reactive towards many organic functional groups and their general behaviour is similar to the well known Grignard reagents.
Thus, they can be reacted with a dimethyl carbonate to give rise to an alkoxy carbonyl substituted polytetrafluoroethylene.
This substituted ethylene can subsequently be subjected to hydrolysis to provide a polytetrafluoroethylene with carboxyl substituents. The carboxylated teflon thus generated, can then be used for direct coupling to microbial siderophores (or chelators) such as Desferal . As indicated above, it may be desired to space the siderophore from the surface of the teflon. If so, ethylenediamine and similar compounds can be readily attached through the carboxyl group by standard procedures. Since the teflon's backbone is very inert to many organic and inorganic reagents, very vigorous reaction conditions can be employed in further derivatization using the carboxylic functional group. See, for example, ^-Methods in Enæymology- Supra.
As indicated above, the microbial siderophore must also possess compatible unctional groups which will react with those of the carriers without interfering with the chelating activity thereof. For example, suitable functional group in the siderophore enterobactin is the 2, 3-dihydroxy benzoic group which is susceptible to dia onium coupling under neutral to almost neutral conditions. The acylamine xequired in the generation of diazonium salts can be prepared from aminopropylsilylated glass.
Examples of suitable functional groups on -the microbial siderophores axe tha amino group, the carboxyl group, the phenolate group and the cathecolate group, etc.
The previous comments relating to carriers, spacer groups etc for microbial siderophores apply to the use of other s:iderophoric compounds, ~or example catechol.
II cakechol i5 used ~s the ~iderophoric compound it may be ~ Tr~de ~

bonded to a carrier by diazo coupling or through reaction with a functional group on the carrier such as o - C - H . In these latter cases -the functional yroup on the carrier is directly reactive with -the benzene ring of the catechol.
When using a composition in accordance with the present in~ention, the conditions of use should of course be such as to avoid the break--down or decomposition of the composition; i.e. conditions such as pI-I, temperature, pressure, etc. should be chosen so as to avoid the break-down of the composition.
As indicated above, an insoluble (siderophoric) composition, in accordance with the present invention, can be used to remove iron from a liquid medium. In use, the (sideorphoric) composition is intermixed with a desired liquid medium for a suitable time, whieh will of course depend upon the amount of (siderophorie) eomposi-tion used, the initial iron eoncentrat on, the desired final iron eoncentration, etc. The Fe in the medium combines with the Isiderophoric) composition and can thus ba physically sepa~ated from the medium. The affinity of (siderophoric) eompounds for iron can be so great that even small amounts of iron can ~e removed from a liquid medium. The ~irst concentration of iron in a treated nutrient medium can for example be far below that req~lired to support mierobial growth.
In drawings which illustrate the present invention Figure l is a yraph illustrating the inhibition oE microbial growth due to the removal of iron from liquid medium, re 2 illustrates regeneration o-f a siderophoric com-posi-tion by pH manipulation; and FicJure 3 illustrates regerleration of a siderophoric compo-sition by a reducing agent.

Figure 1 as indicated above is a graph illustra-tive of the inhibition of microbial growth in even the most nu-tritional - - -- 21 a -~Z~ ;7 solutions (e.g. bacteriological broth medla~ on removal oE
iron therefrom.
In particular, fi~ure 1 illustrates the inability of the bacterium Neisseria menin~itidis to grow in a complex -highly nutritional medium (neisseria defined medium ~ NDM) from which only iron has been extracted with ferrioxamine immobilized OllagarOSe~ the agarose having previously been activated by cyanogen bromide for coupling to ferrioxamine.
Thus, one gram of the above siderophoric composition was contacted with 200 ml NDM at 22C for a time period of 20 min. before recovering the siderophoric composition.
Prior to treatment, the NDM contained about 3.6 ~M of iron;
after treatment, it contained less than 0.1 ~M iron. The treated medium was then divided in-to two portions and FeC13 was added to one of them~ A control consisting of untreated NDM and -the two portions were then inoculated with microbes and maintained at a pH of 7.4 and a temperature of 37C.
As can be se~n in figure 1, unhibited growth occurs in the control ~0)~ However, in the treated medium, (~), cells are unable to undergo anymore than one or two divisions due to the absence of the vital nutrient iron. On the other hand if exogenous iron is added back to the tr~ated medium full growth is again realized (~).
~iquid media to be treated to remove Fe3~ can 2S have, for example, a p~I in the range of 4.5 to 9. During the contact with the siderophoric composition, the temperature of the mixture can for example range from 1C to 50C and the contact can occur under atmospheric pressure. Fxamples of different media which can be treated with the composition are listed in table 2 which follows:

~2~ 7 Table 2 Classes of liquid ~edia Specific example thereof Liquid foods - fruit and vegetable ~uices, clear meat broth (e.g. consommé), culture media for microbial, plant and animal cells Breveragec - wine, beer, natural and synthetic juices, cider, drinking water ~harmaceutica] - buffer solutions for lavage ie.g.
ophthalmic solution, peritoneal lavage), water used in the manu-0 facture OI various solutions and 1 preparationS, antibiotic solutions, Cosmetics (li~uid) - those susceptible to microbial degradation, contamination, or spoilage Indus-trial water and - cooling tower, process and waste waste water water Natural water - removal of actinides (e.g. plutonium) and chromium The sidephoric composition can, as indlcated above, for example, be used to remove iron from microbial fermentation cu]tures to stop further growth o~ microbes in the fermenter.
Thus, the siderophoric compositio~ in accordan~e with the present invention may be used to treat wine in order to inhibit microbial growth therein.
The composition of the present invention may also be used to remove iron from cosmetic solutions to prevent contamina-tion by the growth of microbes. Components for cosmetic solu-tions are often obtained from natural sources and are susceptible to microbial degradation.
The siderophoric composition oE the present invention may also be used for the removal of iron from drinking water, pharmaceutical and biological solutions, and industrial water.

Al-though -the microbial siderophores are selective ~or iron, they can also bind me-tals that are classified as ac-tinides e.y. plutonium. Thus, the present inven-tion addi-tionally provides means for removing such hazardous metals as plutonium ~rom contam~ated wa-ter; and a rapid means to collect (concentrate) the radioactive metals (e.g. pluto-nium) to determine the concentration thereof in standard water volumes. Such metals are selectively removed from water due to their structural similarity (i.e. atomic) to iron.
As indicated previously, compositions in accor-dance with the present invention~ can be regenerated for further use by the removal of the iron therefrom by suitable chemical means. In this way, the composi-tion can be econo-mically used since it can be recycled ~or repeated use.
The regeneration, for example, of a composition loaded with iron, can be carried out either through -the manipulation of the pH of a medium surrounding -the iron-loaded ~siderophoric) composition and/or by treating the iron-loaded composition with a suitable reducing agent.
In either case, appropriate conditions should be chosen which will not decompose the composition or destroy -the iron binding capacity -thereof.
If regeneration is affected by manipulation of the pH, the pH must be brought to or beyon~ a point at which the iron is released.
In general, when making use of a microbial sidero-phore, a pH oE 1 or lower should be avoided. The use o~
mineral acids should also be avoided. The pH can be manipu-lated through the use of organic acids (for example, acetic acid, s~ccinic acid, citric acid, isoci-tric acid, ketomalonic acid, malic acid, oxalic acid or pyruvic acid).
Figure 2 illustra-tes the regenera-tion oE a siderophoric composi-tion by the manipula-tion of pH. The 2~ -i7 designation (~) represents en-terobactin immobiliæed on a polyacrylamide carrier whereas the designation (O) repre-sents ferrioxamine immobilized to the same type of carrier.
The pH was lowered in the presence of 15 mM ci-tra-te and 0.05 M ~ - _ - 24 a -0~

tris-s~di.um acetate. The lowering of the pH was accomplished by an addi.tion of appropriate amounts of acetic acid.
Alternatively, the iron loaded siderophoric com-position may be treated with a suitabl.e reducing agent to release the iron. In accordance with the presen-t invention, it is possible-to llse suitable di-thionites or ascorbates as the reducing agents, e.g. sodium or potassium dithionite and sodium or potassium ascorbate. The dithionites can be used for the reduction of compositions which include hydroxamate ligands whereas the ascorbates can be used for the reduc-tion of siderophoric compositions containing phenolate/catecholate group ligands. Other useful reducing agents include hydro-xylamine and hydroquinone.
The siderophoric compositions wherein the sidero-phore includes catecholate ligands (e.g. siderophoric compo-sitions consisting o enterobactin fixed to glass) and especially the siderophoric compositions containing diazo linkages must be subjected to mild reduction conditions such as provided using ascorbic acid. The siderophoric compositions which include hydroxamate groups ~Desferal), can be reduced with 1.0 molar sodium dithionite.
Other reduciny agents may possibly be used to regenerate the sidephoric composition; however, the reducing agent used must be chosen on the basis that it ~ill no-t 25 destroy the integrity or iron-bindi.ng capacity of the sidero-phoric compositio~. Sodium dithionite (Na2S2O4),hydroxylamine (including its acid addition salts) and hydroquinone are as indicated above examples of useful reducing agents. In par-ticular the reducing agent can be hydroxyl amine chloride.
rrhe regeneration of the composition may take place in the presence of a suitable organic acid that will complex with the iron that is rcleased. Suitable acids are di or tricarboxylic organic acids that will chelate the Iibera-ted iron ions.

~z~

Figure 3 illustrates the repeated regeneration of sidero-phorlc compositions consis-ting of enterobactin ( ~ ) or ferrox~mine ( ~ ) bound to polyacrylamide carriers, the reducing agent consisting of sodium ascorbate. The regeneration solution had a pH of 7 in the presence of 15 mM sodium citrate and 0.05 M tris-sodlum acetate.
The insoluble composi-tions in accordance with the present invention thus provides for -the advantageous removal of metals (e.g. iron) from liquid media. Such media remain essentially unchanged except for the absence of iron.
The liquid media reEerred to herein may be aqueous, organic or mixtures thereoE.
Reference will now be made to a number of examples which deal with embodiments of the present invention.
Example 1~ Activation of silica gel (glass) The activation methods were analogous to those as described by H. Weetal & A.M. Filbert, Methods of Enzymology XXXIV B:59~72 1974.

(a) Pretreatment -lOO grams of silica gel designated Sigma S-4133 (sold by Si~ma Chemical Company) of 100 to 200 mesh (70 to 140 microns) chromatographic grade and of pore diameter of about 25 angstrom was suspended in a .., :, .
"~{ ." f ~ ~

mixture of 50 ml of 70% HNO3 and 300 ml of distilled water. The suspension was re1uxed ~ith mixing for about 1 hour.
The gel was allowed to se-ttle and the liquid layer drawn ofE. The gel was then washed repeatedly with distilled water until the wash water was about neutral pH.

(b) Amine activated silica gel:

The above pretreated silica gel (wet) was used for the following amination without drying. A 10%
solution of ~-aminopropyltriethoxysilane in distilled water 1500 ml) was added to the above obtained silica gel. The pH was then adjusted to 3O45 with 6N hydrochloric acid. The suspension was then maintained under stirring at a temperature o 75C for about 3 hours. The gel was then filtered and washed with 500 ml of distilled water and dried in an oven at 100 to 110~C.
The above amination can be described graphicall~
as follows:
Et ~ OH O
glass - f ~ i-OH ~ Et-o-si-cH2-cH2-c~2-NH2 aqueO~15 O o 75C.
Et I-l Et ~ I O , if desired, glass--~ Si-O-Si-CH2-CH2-CH2-NH2 ~ >
~ b O treated to add group H Et -Si-OH
-Si- OH
H O to block reactive group r , I of Et.
glass ~ Si-O-Si-CH2-CH2-CH2-NH2 El -Si-I Et = ethyl The above obtained amineactivated silica gel will hereinafter be ref~rred to as glass fNH2 (c) Aminoarylcarbonyl activated silica gel:

50 grams of glass JNH2 wa5 suspended in 300 ml of ethanol Eree chloroEorm. 2~5 grams p~nitrobenzoyl chloride ~Eastman Kodak~ was subsequently admixed therewith.

- 2~ ~

:

~2~6~

Thereafter, 30 ml of dry trieth~lamine was added and the resultant mixture was refluxed for 20 hours. The beads were allowed to settle and the liquid phase drawn off. The beads were then wash repeatedly with chloroform then repeatedly with ethanol and finally with distilled water with ethanol.
The wet water-washed gel was subjected to a treatment for the reduction of the nitroaryl group to an aminoaryl group by adding the gel to a solution of 50 gm of sodi~ dithionite in 250 ml. of distilled water. The whole suspension there-ater being refluxed with mixing for 45 min. The suspensionwas then filtered while still hot and washed repeatedly with dilute hydrochloric acid and then washed with distilled water.
After thorough washing with distilled water, the gel was dried in an oven at 70 to 80C; this t~pe of activated~carrier can be used for subsequent diazotization and coupling.
The above reaction can be represented graphically as follows ~2 / ~ Chloroform H O
glass ~ NH2 + l J CHCl~glass J N-e- ~ No2 J ~ Triethylamine C=O Et3N
Cl A

Reduction , R ~
2S2O4 ~ glass ~ N-C - ~ NH2 S (d) Carboxyl activated s.ilica gel:

50 gm of glass ~ H2 (see above), were suspended in 250 ml of water cold in an ice bath. 50 ml of 1 N sodium hydroxide was added followed by 15 gm.of solid succinic anhydride. After mixing or 2 hours, the pH was adjusted with the addition of 1 N sodium hydroxide to a pH of 5-6. This adjustment of pH with sodium hydroxide was repeated hourly three additional times and the mixture was left overnight. The suspension was thexeafter filtered and washed thoroughly with distilled water and dried at 100 to 1~0C
The above reaction can be represen~ed graphicall~
.20 as follows:

H
glass f N-H ~ CH2-CH2 NaOH _ ~ f 1 ¦ aqueous HO-C=O

(e) Aldehyde activated silica gel:

20 gm of glass f NH2 ( see above) were suspended in 50 ml of 0~1 M sodium phosphate at a p~ of 7 followed by the addition of 10 ml purified 8% glutaraldehyde.
The mixture was subjected to vacuum and mixed occasionally.
The reaction was allowed to proceed for 3 hours and the mixture was thereafter filtered, washed thoroughly with distilled water and dried under vacuum.
The reaction outlined above can be represented gra~hically as follows:

H H H
glass ~ N-H ~ o=C-cH2-c~2-cH2-c=
H H
f l I
glass ~ N=C-CH2-CH2-CH2-C=O

~v~

Example 2: Activation of polyacrylamide (azide coupling) 60 ml of ethylene diamine activated polyacrylamide gel (per Inman, Methods in Enzymology XXXIV
B:35 1974) was diluted with water to make up to 100 ml volume. About 1~2 y of p-nitrobenzoyl azide was dissolved in 100 ml of tetrahydrofuran and the obtained solution was subjected to filtration.
I'he obtained filtrate was added immediately to the aqueous gel suspension referred to above.
1.5 ml of triethylamine was then added to the suspension. The gel mixture was then stirred gently for 30 min. An additional portion of 1.2 grams of p-nitrobenzoyl azide in 50 ml of tetrahydrofuran was added -Eollowed by another 1 ml of triethylamine. Gentle stirring was continued Eor an additional hour. The obtained gel was filtered and washed-tho~ughly with 1:1 tetrahydrofuran: 0.2 M sodium chloride and then resuspended in 0.2 M sodium chloride. 3 ml of acetic anhydride was added to the suspension which was then mixed for 1 hour, the gel being thereafter washed withO.l M NaCl.
The obtained gel can be used directly for diazo coupling.
The reaction can be represented graphically as ~ollows:

l3 C=O

- N-CII -CH2-~H2 + ~ - 3 ~ B f N--CH -CH2-~-C ~ NO
2 ~ triethyla~ine 2 2 ~ I-I H O
_ ~ B ~ N-cH2-cH2-N-c- ~ NH2 wherein:B = polyacrylamide backbone (ie carrier) Example 3: Activati.on of agarose with cyanogen bromide.

Agarose swollen in water was mixed with an equal volume of water. Finally divided CNsR
~50~300 m~ per ml of agarose~ was added atonce to the stirred suspension. The pH of the suspension was in~ediately adjustec~ and maintainad at pH 11 with sodium hydroxide. The temperature oE the suspension was maintained at 20C and the reaction allowed to proceed ior about 30 mi.n. Thereafter t the gel was then washed rapidly with a large amount of ice cold water followed by washing with an appropriate buffer. The obtained activated gel should be used as soon as possible.
The chemical reactions involved in the agarose activation can be indicated ~raphically as ~ollows:

agarose agarose _ ~-OH e ~
+ CN Br ~ /C=N-H + H2O
_ I OH ~ O
_~ J

Cyanogen bromide, by analogous procedures, can be used to activate other polysaccharides;
for example, alginate, glucans, cellulose/ agar, ~; dextrans, etc.

Example 4: Immobilization of ferrioxamine or enterobactin 1 mM HCl-washsd, water swollen CNBR acti~ated carrier, obtained in accordance with Example 3,was mixed with ferrioxamine or enterobactin (20 mg/mlP in an NaHCO3 buffer (O.1 M, pH 8.3) cc)ntaining 0.5 M NaCl.
The mixture was agitated overnight at 4C. The gel was then ~7ashed w.ith 0.1 M acetate buE-Eer pH

4.0 containing 0.5 M NaC:L to remove excess uncoupled ferrioxamine or enterobactin.

Example 5: Immobilization oE Des.~exal to polyacrylamide gel -ta) Biogel P~150 polyacrylamide from ~ioRad was linked through ethyLenediamine and then succinic anhydride followin~ published procedures as described by 6~.3 Inman (John K. Inman, Covalen-t Linlcage of Functional Groups, Ligands, and Proteins to polyacrylamide beads in -Methods of Enzymology , XXXIV B, 30 (Jakobyr W.B. ed.) Academic Press, New York (1974) and Biochemistry 8:4074 (1969)S (see examples 16 and 17) infra. Unreac-ted free amino groups in ethylene-diamine were blocked by reaction of acetic anhydride at the end of the reaction period.
Test for presence of any free amino group was through the TNBS ~trinitrobenzene sulfonic acid) testn The addition of acetic anhydride was repeated twice until TNBS ~est were negative.

This activated, extended polyacrylamide was immediately used for coupling.

(b) Coupling of Desferal to activated polyacrylamide gel 700 mg Desferal was dissolved in 5 ml oi deionized water and followed by addition of163 mg ferric chloride. A deep red solution was formed. 20 ml activated gel, obtained above, was washed twice wi-th ethanol by centrifigation and decantation.
The total solid shr~nk to a very small volume after the second volume of ethanol (20 ml) was added.
The 5 ml of ferric complexed Desferal was added at 20C -to this shrun~cen gel and the mixture was mixed by swirling. Thè gel gradually swelled to give a solid mass. 5 ml of H2O was added to help disperse the gel and -the pH of the suspension ~2~

was adjusted to 4.2 with NaOH llN). 200 mg EDC
(l-ethyl-3-(3-dimethylamino propyl) carbodiimide), 200 mg was added in one portion and the pH of the suspension monitored and kept at 4.3 to 4~6 by addition of HCl (1 N), for 3 hrs. A further portion of 200 mg EDC was added and the mixture, allowed to stand overnight at room temperatuxe. The gel was filtered on a sintered flass funnel and washed with 0~2 M NaCl.
The reaction can be graphically represented as follows:

PA

I ~ 2 C~2 N l-C~I2~CH2-C-OH + H2N-CH2-Def ~ C2~5N=C=N
PA ~ CE13~ C~H2 ~N CH2 CH2 ~N3~
cle H-C-C-N-CH -CH N-C-CH2-CH -C-N-CH -De~ t C2E12-N-C;O
1l t 2 2 1 11 2 2 ~3 ~AO H H O ~N-CH -CH~-CH2 cle wherein: PA = polyacrylamide ~ackbone or carrier and Def = desferal.

~z~

Example 6: Immobilization of ferrioxamine or enterobactin to polyacrylamide carrier -A swollen polyacrylamide gel acyl azide derivative freshly prepared as in accordance with example 2 was suspended in a solu-tion containing the Eollowing: 0.1 M CaC12, 0.001 N HCl acid, ferrioxamine or enterobactin at 0.3 mg/ml (pH 4.0).
The pH was immediately adjusted to 9.0-and the mixture was stirred for 60 min. at 0C. In the case of enterobactin, the solutions are S0% ethanol.
The coupled gel waswashed with large volumes of Q.05 M Tris-acetate-0.15 M citrate, pH 7Ø

Example 7: Immobiliæation of Desferal to silica yel (glass beads) -About 700 mg (or approximately one mMole) of Desferal was dissolved in 25 ml of water followed by the addition of 170 mg of ferric chloride.
The pH of the solution was then adjusted to 4.3 with one normal hydrochloric acid and to thls was added 25 ym oE succinylated silica gel characterized by the Eormula:
, R
ylAss ~ _ N - C - CH2 - CH2 - C-NH2 s~

200 mg of ~DC: HCl was added to this mixture which was thereafter agitated for 3 houro at room temperature. The pH of the solution was then adjusted to 4.3 and the mixture allowed to stand overnight. Aftex filtration, the obtained ~mpo~lte was washed thoroughly with distilled water until the washing water was colorless. Thereafter, the obtained composite was dried under vacuum.

Example 8: Immobilization of enterobactin to aminoarylcarbonyl activated silica gel-

5 gm of aminoarylcarbonyl activated silica gel obtained as in step (c) of example 1, was mixed with 10 ml of 1 M sodium nitrite and cooled in an ice bath. 1 ml of concentrated hydrochloric acid was added dropwise to the cooled solution. The mixture was maintained in the ice bath for an additional 45 min. to allow for complete diazoti-zation. The mixture was then filtered in the cold, washed with cold distilled water and, while still cold, the gel was added to a solution of about 30 mg of enterobactin in 10 ml of ethanol including about 2 ml of saturated borax solution. The reaction was allowed to proceed for 1 hr in an ice bath. The composite was then recovered by flltration and wash~d with an ethanol water mixture containing 0.1 normal hydrochloric acid and thereafter with ethanol until the filtra-te obtained ~L2~

was colorless. The obtained composite was then dried under vacuum.

The coupling reaction can be represented graphically as below:

H OlNaNO2~ 1 1l f ~ 2 HCl f ~ N2 1~ 0 + Entexobactin~ glass-~ N-C- ~ N=
C=O ~
1 HO ~
10 i ~ OH ~f=o OHEnterobactin xample 9: Immobilization of cathechol to polyacrylamide gel:
diazo coupling -20 ml of the gel obtained in accordance withExample 2, was suspended in 20 ml of 0.l normal HCl and cooled in an ice bath. While the suspension was maintained in contact with the ice bath, 2 ml of l M sodium nitrite was added dropwise with agitation ~f the suspension. The resultant i7 mixture was kept in the ice bath for an additional 30 min. and centrifuged to remove the liquid phase.
The gel was then washed twice with ice-cold distilled water and then placed in contact with 20 ml of an ice-cold solution of 10~ catechol in saturated horax.
The reaction was allowed to proceed overnight at 4C~ The composite was recovered by filtration and ik was washed repeatedly with water containing 0.1 normal HCl. The reaction can be indicated schematically as Eollows:

PA f N- tCE12) 2-N-C-~ NH2 + NaN02 ~ HCl --~.

PA ~ ~l-(CH2)2-~-cl- ~ N~ + ~ OH

OH

PA f ~- (CH2 ) 2-N-t~ N--N~

t)l~ t)~

wherein PA is polyacrylamide carri.er or backbone.

0 EXample 10:Immobil:Lzation of catechol to aminoaryl carbonyl activated silica gel-20 gm of aminoarylcarbonyl activated silica gel ob-tained in accordance with example 1 (c) was .suspended in 20 ml of ice-cold water. 10 ml of 1 M scdium nitrite was added to the suspension while maintaining the suspension in an ice bath.
10 ml of 2 normal hydxochloric acid (ice-cold) was slowly added dropwise to the suspension.
The reaction was allowed to proceed at between 0 and 4C for about 1 hour and it was then washed with ice~cold distilled water. The solid was then added to 10 ml of 10% cathecol in saturated borax solution (ice-cold) with mixing.
20 ml of water was added to facilitate mixing and the reaction was allowed to proceed in an ice bath for an additional one hour. The composition was then allowed to settle and the li~uid siphoned off. The composition was washed with distilled water containing 0.1 normal hydrochloric acid and ethanol. The composition was rapeatedly washed until the washing water was colorle6s. The composition was then recovered - and dried under a vacuum.
The reactions involved with the catechol can be represen-ted generally as below:

25 glass-~ N-C ~ NH2 2 glass ~ ~ N2 ~ OH H 1i ~N- ~

OH OH

-- ~1 --;7 Example ll: I~nobilization of catechol to aldehyde activated silica gel -5 gm of aldehyde activated silica gel (activated as in example 1 ~e) abo~e) was suspended in 10 ml of 10~ cathecol in saturated borax solution for l hour. The mixture was then subjected to vacuum evaporation and then heated while still under vacuum at 70C or one hourO The mixture was ~hen cooled to room temperature and water was added thereto, the mixture then being heated at 70C for an additional hour. The mixture was then ccoled to room temperature and 500 mg of sodium borohydride was added and the mixture was maintained at 70~C for a further hour.
The resultant reaction mixture was then cooled in an ice bath and 5 ml of glacial acetic acid was added dropwise with mixing. The reaction was allowed to proceed for an additional 30 min.
and the mixture was then filtered. The recovered composition was then washed repeatedly with distilled water and ethanol alternately and then dried under vacuum.
The general chemical reactions are believed to be as follows:

- ~2 -~2~

g f 2 CH2 CH2 C o + ~ Bora OH

g f 2 CH2 CH2 1 ~ NaBH4 OH OH Sodium Borohydride g f 2 CE~2 CH2 CH2 1 ~
H

OH OH

Example 12: Removal of iron from wine The inability of wine to spoil after extraction of iron usin~ the composition of the present invention is illustrate!d in table 3 below~ This protectlon from spoilage is retained even when the wine is vigourousl~ aerated (agitated continu-ally in open flasks at 25C). The treatment of the commercial wine samples consisted of vigou-rously aerating the sample after inoculating with ac.etobacter xylinum, aeration continuing for a period of about 30 days at 25~C.

Table3 . . .
TRE~TM~NT OBSERVATIONS
... ...
None By three weeks, wine was foul smelling ~including açetic acid smell); turbid from bacterial growth Filter sterilized~no bacteriaNo spoilage (0.45 ~m pore size) added) Iron extraction with No spoilage siderophoric composition of the present invention Addition of iron ions By three weeks, wine was foul to wine subjected to smelling (including acetic acid iron removal by treat- smell); turbid from bacterial ment with the sidero- growth phoric composition of the present invention The following examples (i.e. 13&14) illustrate the procedure which may be used to recycle siderophoric compositions in accordance with the present invention i.e. recycling after removal of bound iron. The reuse of the siderophoric composition of the present invention makes it economically attractive.

Example 13: Regeneration of an active iron-free siderophoric composition comprising enterobactin fixed to silica gel -The siderophoric composition subjected to regeneration can in general be represented bv the following graphic formula:
H O
glass - ~ ~ C ~ N-N-enterobactin The above siderophoric composition loaded with iron, was subjected to treatment with an e~ual volume of 0.1 M sodium ci-trate and 0.1 M ascorbic ~2~

acid, the treatment lasting for a period of about 12 hours. The treatment was repeated twice Eor a recovery of loaded iron in the range of 95~.

Repeated loading and unloading of the siderophoric composition with Fe3+ results in retention of up to 95% of the original iron-binding capacity.

On average, each grarn of iron-loaded siderophoric composition included about 212 to 232 micrograms of iron per gram of composition. In the above procedure, about 80-100mg. of siderophoric composition were subjected to regeneration.

Example 14: Regeneration of a siderophoric composition comprising a catechol fixed to a silica gel -~a~ The siderophoric comp~osition can be xepresented generally by the foll,owing graphic formula:

f ~ ~0 OH H

The siderophoric composition was subjected to the same treatment as in Example 13. The iron-loaded siderophoric composition contained from 110 micxograms to 163 micrograms of Fe per gram of the composition. ~etention of iron-bindlng caDacity after xegeneration was up to 9S%.

- ~5 -~2~ i7 (b) The siderophoric composition having the following general structure was subjected to a reductive regenera-tion:

glass f N-CH2-CH2-CH2-b~I ~

OH OH

The iron loaded siderophoric composition contained from 43 micrograms to 56 micrograms or iron per gram of siderophoric composition. The sidero-phoric composition was regenerated using 5 ml of 0.1 M sodium dithiorlite. Treatment in this way resulted in the recovery of 85 to 93~ of the iron-blnding càpacity ofthe siderophoric composition.

Example 15: A siderophoric composition comprising ferrioxa-mine fix~d to silica gel -0The siderophoric composition was obtained in accordance with the procedure outlined in Example 7. The siderophoric composition had an iron binding capacity of about 740 micro-grams of iron per gram of siderophoric compo-sition. 12 gm of the above composition when exposed to 5 ml of a 500 ~M Fe solution was able to remove or recover 79.1~ of the iron;
on being exposed to about 5 ml of a 5 ~M
Fe3 solution about 98.8% of this iron was removed or recovered from the solution by the siderophoric composition.

'1~2~6~5~l' Example 16: Activation of Biogel P 150 with Ethylenediamine:

PA

fH2 PA
1~ 1 H-C-C-NH2 + H2N-CE~2 CH2 NH2 ~ CH2 PA H~~ --CH --OEl +NH

PA = polyacrylamide back bone:

All operations were carried out in a well ventilated hood. 200 ml. anhydrous ethylene diamine in a 500 ml. 3 necked round bottom flask was heated with a heating mantle and the final temper ture was reached and ad~usted and maintained at 90~C ~ 2~C. The glass was also equipped with a conden~er with outlet protected by a dryi~g tube, a mechanical stirrer and the third neck was used for addition of materials and temperature monitoring.
10 gm of Biogel - P 150 was added through the thermometer neck in one portion and the mixture was stirred and heated at 90~C ~ 2~C for a period of 3 to 4 hours. The solid gel swelled to a great vol~e and the evolution of ammonia can be a~certained by wetted pH paper at the drying-tube outlet. At the end of the reaction, the mixture was poured with mechanical stirring on to a mixture oE 400 ml, ice and water (1:1).
Any gel aclhering to the flask can be washed down by jets of watex. The gel was filtered while the mixture was still cold. The gel was promptly washed repeatedly ~ith 0.2 M NaCl and 0.00 1 N HCl until the filtrate gave a negative TNBS test (Trinitroben~enesulfonic Acid). The total gel volume was about 170 ml. i.e. wet gel.

Example 17: Succinylation: Carboxylic arm extension:

~A PA
l~2 ~H2 IH2 ~H2 I~ ~
¦2 CH2 NH2 ~ C~ j O - ~ H f---C-N-CH2-CH2-N-H

~00~1 PA = polyacrylamide bacX-bone.

50 ml. wet gel (Biogel P-150~Ethylenediamine activated) is suspendecl in 50 ml. 0.1 N NaOH
in a 250 ml. beaker. Ext~rnal cooling in an ice bath and gentle mechanical stirring is also provided. 1.~ gm. succinic anhydride (10 mmole) was added in one portion and the mixtur~ stirred in the cold for 2 hrs. A
Eurther 1 gm. portion of succinic anhydride was added with further cooling and stirring for an additional hr. During the addition of the second portion of succinic anhydride, the mixture pH is monitored lntermittently ~ ~8 -ail;~O605i7 with a pH meter and additional amounts of 1 N NaOH were added to maintain a pH of 3.5 to 4Ø A third portion of 1 g. succinic anhydride was added and the monitoring procedure was same as the previous addition. The TNBS Test showed that there were still free amino group on khe gel and these were blocked by addition 10 ml. acetic anhydride and stirred for 30 min. The mixture was eventually washed thoroughly with 0.1 M NaCl. TNBS Testwas negative for the gel.

~ 49 -

Claims (31)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for inhibiting microbial growth in a liquid nutrient medium containing Fe3+ by lowering the Fe3+ content thereof to less than 0.1 µM characterized in that said medium is contacted with an insoluble sidero-phoric composition and thereafter said siderophoric com-position loaded with Fe3+ is separated from said medium, said insoluble siderophoric composition comprising:
(1) one or more organic siderophoric compounds covalently fixed to the surface of (2) a suitable insoluble carrier, said organic siderophoric compounds possessing one or more coordinating sites capable of chelating Fe3+.

2. A method as defined in claim 1, wherein said coordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate group of formula (b) a phenolate group of formula X being an atom of O or N-, and (c) a catecholate group of formula X' being an atom of 0 or N- and m being 1.

3. A method for inhibiting microbial growth in a liquid nutrient medium containing Fe3+ by lowering the Fe3+ content thereof to less than 0.1 µM characte-rized in that said medium is contacted with an insoluble siderophoric composition and thereafter said siderophoric composition loaded with Fe3+ is separated from said medium, said insoluble siderophoric composition comprising (1) one or more organic siderophoric com-pounds covalently fixed to the surface of (2) a suitable insoluble carrier, said organic siderophoric compounds possessing one or more coordinating sites capable of chelating Fe3+, said organic siderophoric compounds being selected from the class consisting of microbial siderophores.

4. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight of less than 2500 daltons.

5. A method as defined in claim 3, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons.

6. A method as defined in any one of claims 3, 4 and 5, wherein said coordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate group of formula (b) a phenolate group of formula X being an atom of 0 or N-, and (c) a catecholate group of formula X' being an atom of 0 or N- and m being 1.

7. A method as defined in claim 5, wherein said coordinating sites are provided by one or more N-substituted hydroxamate groups of formula

8. A method as defined in claim 5, wherein said coordinating sites are provided by one or more catecholate groups of formula

9. A method as defined in claim 3, wherein said organic siderophoric compounds are selected from the class consisting of desferrioxamines.

10. A method as defined in claim 3, wherein the organic siderophoric compound covalently fixed to the carrier is enterobactin.

11. A method for inhibiting microbial growth in a liquid nutrient medium containing Fe3+ by lowering the Fe3+ content thereof to less than 0.1 µM characterized in that said medium is contacted with an insoluble side-rophoric composition and thereafter said siderophoric com-position loaded with Fe3+ is separated from said medium, said insoluble siderophoric composition comprising (1) unsubstituted catechol covalently fixed to the surface of (2) a suitable insoluble carrier, the catechol being covalently fixed to the surface of said carrier at the benzene ring thereof.

12. An insoluble composition comprising a member selected from the class consisting of (A) an insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface of (2) a suitable insoluble carrier, said organic chelating compounds possessing one or more coordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores and (B) an insoluble composition comprising (1) unsubstituted catechol covalently fixed to the surface of (2) a suitable insoluble carrier, the catechol being covalently fixed to the surface of said carrier at the benzene ring thereof.

13. An insoluble composition comprising (1) one or more organic chelating compounds, covalently fixed to the surface of (2) a suitable insoluble carrier, said organic chelating compounds possessing one or more coordinating sites, said organic chelating compounds being selected from the class consisting of microbial siderophores.

14. A composition as defined in claim 13, wherein said microbial siderophores have a molecular weight of less than 2500 daltons.

15. A composition as defined in claim 14, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons.

16. A composition as defined in any one of claims 13, 14 and 15, wherein said coordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate group of formula (b) a phenolate group of formula X being an atom of 0 or N-, and (c) a catecholate group of formula X' being an atom of 0 or N- and m being

17. A composition as defined in claim 15, wherein said coordinating sites are provided by one or more N-substituted hydroxamate groups of formula

18. A composition as defined in claim 15, wherein said coordinating sites are provided by one or more catecholate groups of formula

19. A composition as defined in claim 13, wherein said organic chelating compounds are selected from the class consisting of desferrioxamines.

20. A composition as defined in claim 13, wherein the organic chelating compound fixed to the carrier is enterobactin .

21. An insoluble composition comprising (1) unsubstituted catechol covalently fixed to the surface of (2) a suitable insoluble carrier, the carrier being covalently fixed to the surface of said carrier at the benzene ring thereof.

22. A method for removing Fe3+, from solution characterized in that the solution is contacted with an insoluble composition as defined in claim 12.

23. A method for removing Fe3+ from solution characterized in that the solution is contacted with an insoluble composition as defined in claim 13.

24. A method as defined in claim 23, wherein said microbial siderophores have a molecular weight of less than 2500 daltons.

25. A method as defined in claim 23, wherein said microbial siderophores have a molecular weight in the range of 500 to 2500 daltons.

26. A method as defined in any one of claims 23, 24 and 25, wherein said coordinating sites are provided by one or more members of the class consisting of (a) a N-substituted hydroxamate group of formula (b) a phenolate group of formula X being an atom of 0 or N-, and (c) a catecholate group of formula X' being an atom of 0 or N- and m being 1.

27. A method as defined in claim 25, wherein said coordinating sites are provided by one or more N-substituted hydroxamate groups of formula

28. A method as defined in claim 25, wherein said coordinating sites are provided by one or more cate-cholate groups of formula

29. A method as defined in claim 23, wherein said organic chelating compounds are selected from the class consisting of desferrioxamines.

30. A method as defined in claim 23, wherein the organic chelating compound covalently fixed to the carrier is enterobactin.

31. A method for removing Fe3+ from solution characterized in that the solution is contacted with an insoluble composition as defined in claim 21 .