Material method and apparatus for inhibiting microbial growth in an aqueous medium

A biocidal material comprises a biocide immobilized in a porous inorganic polymer network such as a sol-gel matrix. The polymer may be coated on an inorganic support. The material can be used for inhibiting microbial growth in an aqueous medium, e.g., the wash solution of a photoprocessing system. The material can be housed in a flow-through container.

CROSS REFERENCE TO RELATED APPLICATION 
Reference is made to and priority claimed from Great Britain Application 
No. 961S994.7, filed Jul. 30, 1996. 
FIELD OF THE INVENTION 
The invention relates to a material, method and apparatus for inhibiting 
microbial growth in an aqueous medium. 
BACKGROUND OF THE INVENTION 
Microbial growth occurs in many systems in which aqueous media such as 
water, aqueous solutions and aqueous dispersions are employed. 
For example, significant biofouling can occur in many areas of 
photoprocessing systems and, in particular, where low-flow rate washes and 
water recycling is used. The problem may be overcome by adding biocides to 
the wash water tanks when bacterial biofilm formation becomes evident 
visually. However at this point the biocides may not work and even at 
quite high concentrations are not particularly effective because the 
bacteria have attached to surfaces to form colonies that have built up in 
layers. Hence, any biocide in solution can only reach the outer biofilm 
layer and not the inner layers of the biofilm that are protected. 
Furthermore, widespread use of such biocides is not desirable because they 
are relatively expensive and require specialized disposal to protect the 
environment. 
Alternative methods of inhibiting bacterial growth in aqueous media involve 
the gradual release of a biocide through interaction with water, e.g., by 
leaching. 
U.S. Pat. No. 4,552,591 describes a biocidal composition for inhibiting 
microbial growth in oil field waters that comprises a biocide and a solid, 
particulate adsorbent therefor. The biocides are conventional water 
soluble compounds traditionally used in the treatment of oil field waters, 
e.g., 2-methyl-4-isothiazolin-3-one which are adhered to a known adsorbent 
e.g., diatomaceous earth. The compositions avoid the personal and 
environmental contamination which can result from spillage of the biocide 
used previously in liquid form. After addition to oil field waters, such 
compositions release the biocide through leaching. 
A problem associated with the prior art methods and materials for 
inhibiting bacterial growth in aqueous media using biocides is that 
biocide is released in the media. 
Furthermore, there is a need for a method and materials in which the 
biocide is only used on demand when the bacteria are present. Methods and 
materials that reduce the exposure of operators to toxic biocides are also 
sought. 
SUMMARY OF THE INVENTION 
The invention provides a biocidal material comprising a biocide and an 
inorganic carrier that is a porous inorganic polymer network in which the 
biocide is immobilized. 
The invention also provides a method for inhibiting bacterial growth in an 
aqueous medium comprising contacting the aqueous medium with a biocidal 
material comprising a biocide and an inorganic carrier that is a porous 
inorganic polymer network in which the biocide is immobilized. 
The invention also provides apparatus for inhibiting bacterial growth in an 
aqueous medium comprising a container having fluid inlet means and fluid 
outlet means, the fluid inlet and fluid outlet means communicating with an 
inner chamber such that, when the apparatus is in use, fluid entering the 
inner chamber through the fluid inlet means flows through the inner 
chamber and leaves the container through the fluid outlet means, 
characterized in that-the inner chamber holds a biocidal material 
comprising a biocide and an inorganic carrier that is a porous inorganic 
polymer network in which the biocide is immobilized. 
The invention removes the need for conventional dosing of biocides in 
solution, either directly or by gradual release, which has many drawbacks. 
The biocide is only used on demand when the bacteria are present. The 
direct exposure of operators to toxic biocides is minimized. The invention 
is able to utilize inexpensive, readily available inorganic supports.

DETAILED DESCRIPTION OF THE INVENTION 
Suitable inorganic polymer carrier materials include those derived from 
sol-gel materials, silicon nitride, metal esters, e.g., acetates, 
nitrates, phosphates and ormosils (organically modified silicates). 
Examples of suitable materials are referred to in "Sol-gel Science," by C. 
J. Brinker and G. W. Scherer, Academic Press, 1990, Chapters 2-3. 
The preparation of inorganic, e.g., silica glasses through the low 
temperature "sol-gel" synthesis is known. For example, see Chem. Rev. 
1990, 90, 33-72 "The Sol-Gel Process" by L. L. Hench and J. K. West. 
An amorphous matrix of the glassy material may be prepared by the room 
temperature polymerization of suitable monomers, usually metal alkoxides. 
The polymerization of metal alkoxide mixtures results in a transparent 
porous solid (xerogel) with surface areas of up to hundreds of square 
meters per gram and having small pores, e.g., from 0.5 to 500 nm. The low 
temperature glass synthesis allows doping of the inorganic glass with 
organic molecules, e.g., a chemically sensitive dye. 
The sol-gel glass has a cage-like porous molecular structure in which a 
single doping molecule can be isolated in an individual cage, even at high 
concentrations of additive. Molecules trapped in sol-gel glasses can 
interact with diffusible solutes or components in an adjacent liquid or 
gas phase in the pore space. 
The sol-gel matrix may comprise one or more of SiO.sub.2, TiO.sub.2 and 
ZrO.sub.2. In a preferred embodiment of the invention, a SiO.sub.2 
/TiO.sub.2 sol-gel matrix is used. Preferably, the mole ratio of Si:Ti in 
the sol-gel glass is from 90:10 to 50:50, more preferably from 80:20 to 
60:40. 
Suitable types of biocide include those described in "Microbiocides for the 
Protection of Materials", W. Paulus, published by Chapman Hall, 1993. They 
are agents capable of killing or inhibiting the multiplication of 
microorganisms such as bacteria, yeasts, fungi, algae and lichens. 
Examples include heterocyclic N,S compounds, compounds with activated 
halogen groups and quaternary ammonium salts. 
Preferred biocides include those currently employed in the treatment of 
photoprocessing systems, e.g., isothiazolinones. 
Examples of isothiazolinone biocides are those having the structure 
##STR1## 
wherein 
R represents hydrogen, alkyl, aryl, alkaryl and aralkyl; and, 
R.sup.1 and R.sup.2 independently represent hydrogen, halogen, alkyl, or 
R.sup.1 and R.sup.2 taken together represent the atoms necessary to 
complete a fused carbocyclic ring, preferably a 5- or 6-membered ring, 
e.g., a benzene ring. 
Preferred biocides include those having the following structures: 
##STR2## 
wherein R.sup.3 is an alkyl group having from 4 to 20 carbon atoms or an 
aryl group having from 6 to 20 carbon atoms; 
##STR3## 
wherein R.sup.5 and R.sup.6 are selected from hydrogen and halogen, and 
R.sup.4 is an alkyl group having from 5 to 20 carbon atoms; and, 
##STR4## 
wherein each of R.sup.7, R.sup.8 and R.sup.9 is hydrogen or an alkyl group 
providing a total of from 2 to 20 carbon atoms; R.sup.10 is substituted or 
unsubstituted alkyl or aryl, e.g., phenoxyethyl; and, Y is any suitable 
counter anion, e.g., halide. 
Specific examples of commercially available isothiazolinone biocides 
include Proxel.TM. (manufactured by Zeneca): 
##STR5## 
Promexal.TM. (manufactured by Zeneca): 
##STR6## 
Kathon.TM. (manufactured by Rohm and Haas): 
##STR7## 
Other commercially available biocides are: Bronopol.TM. (manufactured by 
Boots): 
##STR8## 
Domiphen.TM. bromide (manufactured by Ciba-Geigy): 
##STR9## 
Vantocil.TM. (manufactured by Zeneca): 
##STR10## 
Densil S.TM. (manufactured by Zeneca): 
##STR11## 
Biocides which are hydrophobically modified Proxel.TM. and Kathon.TM. have 
been prepared having the following structures: 
##STR12## 
R.sup.3 =--(CH.sub.2).sub.7 CH.sub.3 (Compound 1) R.sup.3 
=--(CH.sub.2).sub.15 CH.sub.3 (Compound 2) 
##STR13## 
R.sup.4 =--(CH.sub.2).sub.7 CH.sub.3, R.sup.5 =H, R.sup.6 =Cl (Compound 3) 
R.sup.4 =--(CH.sub.2).sub.17 CH.sub.3, R.sup.5 =H, R.sup.6 =Cl (Compound 
4) 
R.sup.4 =--(CH.sub.2).sub.7 CH.sub.3, R.sup.5 =H, R.sup.6 =H (Compound 5) 
R.sup.4 =--(CH.sub.2).sub.7 CH.sub.3, R.sup.5 =Cl, R.sup.6 =Cl (Compound 6) 
Many commercially available biocides are soluble in aqueous media and an 
increase in their hydrophobicity is required to convert them into the 
preferred hydrophobic compounds for use in the invention. 
It is preferred that the biocides having a log P of at least 1.5 wherein P 
represents the partition coefficient between n-octanol and water defined 
as follows 
##EQU1## 
Log P is a well-known term used in literature on biocides. As used herein, 
it provides a measure of the hydrophobicity of the biocide. 
A variety of commercial and hydrophobically-modified biocides have been 
studied. Partition coefficients between octanol and water have been 
determined at 25.degree. C. by UV/visible absorption. First, the 
calibration curve of each biocide was determined as optical density 
(OD.sub.abs) versus concentration of biocide in .mu.g/g (ppm) of water for 
the predominantly water-soluble materials and .mu.g/g of octanol for the 
predominantly oil-soluble biocides. 
A known amount of biocide was placed in a glass vessel containing either 10 
ml of water or 10 ml of octanol depending on the solubility of the 
biocide. An equal volume of the other solvent was added and the glass 
vessel sealed. The vessel was shaken vigorously for a few minutes and then 
every few hours for more than 48 hours. Each mixture was placed in a 
sealed separating funnel and left for a further 24 hours. The water phase 
of each mixture was removed and the UV/visible spectra run against water 
with appropriate dilutions to bring absorbance between 0 and 1.5 for the 
commercial biocides and the octanol fractions were examined for the 
hydrophobically modified biocides. 
The following partition coefficients shown in Table 1 were determined. 
TABLE 1 
______________________________________ 
Biocide P 
______________________________________ 
Promexal .TM. .about.4.5 
Vantocil .TM. .about.0.3 
Domiphen .TM. .about.50 
Kathon .TM. .about.1 
Proxel .TM. .about.0* 
Compound 1 &gt;330 
Compound 3 &gt;560 
Compound 2 &gt;130 
Compound 4 &gt;480 
______________________________________ 
*i.e. there was almost no biocide in the oil phase. 
The log P value of the biocides that are used in the invention is 
preferably at least 1.5, more preferably at least 2.0. 
Preferably, the amount of biocide used is from 5 to 35 mole percent, more 
preferably from 20 to 30 mole percent based on the metal alkoxide or other 
precursor used to construct the inorganic polymer network. 
Preferably, the inorganic polymer carrier is coated on a support. Preferred 
support materials are those to which the inorganic polymer readily 
adheres. 
Inorganic support materials are advantageous. Many provide the additional 
benefits of low cost and physical robustness. Suitable materials include 
pumice, clay, sand, gravel, chalk, zeolites and glass. Such materials give 
the further advantage of easy disposal and are potentially more stable 
over wide pH ranges than organic polymer-based systems. 
Polymers suitable for use as support materials include any inert, water 
insoluble polymers having appropriate surface properties. Preferably, such 
polymer supports have a non-crystalline surface. Preferably, the polymer 
supports have a hydrophilic surface comprising groups such as --OH and 
--COOH. 
Examples of suitable types of polymer from which suitable supports can be 
derived include ethenic polymers including polyolefins, polystyrene, 
polyvinyl chloride, polyvinyl acetate and acrylic polymers; and polymers 
formed by condensation reactions including polyesters, polyamides, 
polyurethanes, polyethers, epoxy resins, amino resins and phenol-aldehyde 
resins. 
The support may take a variety of forms, e.g., particulate, sheet or fiber. 
It may be porous or non-porous. 
The thickness of the inorganic polymer carrier coating on the support may 
be from 10 nm to 10 .mu.m, preferably from 50 nm to 5 .mu.m. 
In accordance with one method of preparing a material of the invention, a 
solution of the biocide is made in an organic solvent, e.g., 
tetrahydrofuran or alcohol. The biocide solution is mixed with an alkoxide 
sol-gel forming pre-cursor. The pre-cursor containing the biocide may be 
coated on a support if required by any conventional coating means, e.g., 
dipping, spinning and spraying. The pre-cursor containing the biocide is 
left for some hours, e.g., from 4 to 6 hours, before removing the solvent, 
preferably under reduced pressure. Drying is preferably carried out in a 
vacuum oven at a temperature from 60.degree. to 100.degree. C. 
In use, the aqueous medium is brought into contact with the biocidal 
material. Different ways of achieving contact include passing the aqueous 
medium through a container, e.g., a column containing the material in 
particulate form, passing the aqueous medium through a filter of the 
material and passing the aqueous medium over the material in the form of a 
surface coating. 
The invention is of particular use in photoprocessing systems. Such systems 
comprise stages for developing, fixing, bleaching and washing an exposed 
photographic material. Each stage requires apparatus for applying the 
appropriate aqueous processing solution to the photographic material. The 
apparatus may comprise means for supplying, removing and, possibly, 
recirculating such solutions. 
Particularly, the method of the invention may be used to inhibit microbial 
growth in the wash solution or other solutions used in a photoprocessor. 
FIG. 6 is a schematic representation of apparatus for use in performing the 
method of the invention. The apparatus comprises a container 10 having 
fluid inlet means 11 and fluid outlet means 12 said inlet and outlet means 
11, 12 communicating with an inner chamber 13 of the container. When the 
apparatus is in use, fluid entering the inner chamber through the inlet 
means 11 flows through the chamber 13 and leaves the container through the 
outlet means 12. The inner chamber 13 holds a biocidal material in 
accordance with the invention in the form of particles 14. A filter 15 to 
retain the particles is positioned at the top of the inner chamber to 
prevent loss of the particles from the device. The top of the container 10 
is provided with plugs 16 for venting any gas that accumulates in the 
device. 
Fluid entering the device flows down a central tube and subsequently flows 
up through the particles. The arrows indicate the direction of the flow of 
fluid through the device. 
FIG. 7 is a schematic representation of the use of the apparatus shown in 
FIG. 6. A tank 20 containing water 21 is shown, e.g., the wash water tank 
of a photoprocessor. Tubing 22 has an open end in the water 21 at the 
bottom of tank 20, the other end being connected to the inlet of a pump 23 
outside the tank 20. Tubing 24 connects the outlet of the pump 23 to the 
inlet of a device 25 of the type shown in FIG. 6. One end of tubing 26 is 
connected to the outlet of device 25 and the other end opens into the top 
of tank 20. 
In use, water is pumped from the bottom of tank 20 through device 25 and 
back into tank 20 in a recirculation loop. The arrows indicate the 
direction of the flow of water around the loop. 
The invention is further illustrated by way of example as follows. 
Preparation of Biocide 
A Proxel.TM. analogue (Compound 2) was prepared in three steps from 
commercially available starting materials as outlined in Scheme 1. 
##STR14## 
EXAMPLE 1 
Compound 2, dissolved in a small quantity of tetrahydrofuran (THF), was 
added at 25% mol/mol to a 70/30 Si/Ti alkoxide sol-gel forming precursor 
("liquid-coat," Merck ZLI 2132,1857). The mixture was coated onto pumice 
stones (Prolabo 26398293), leaving the sol in contact with the support for 
about 5 hours before removing the solvent under reduced pressure (14 mm 
Hg) and drying in a vacuum oven. Analysis (IR, MS and elemental) of dried 
samples showed the presence of immobilized biocide. 
Control (blank sol-gel) and active (immobilized bioeide in sol-gel) coated 
pumice stones were tested in a nutrient broth containing .about.10.sup.4 
bacteria/ml (Pseudomonas aeruginosa). The control particles and active 
particles were each put separately into a 10 cm glass column with 
screw-tight plastic adapters and polypropylene nozzles. A nylon mesh 
filter was put at the bottom and top of each column in between two rubber 
washers. The columns, all silicone rubber tubing, flasks and nutrient 
broth necessary to complete the flow circuit were sterilized by 
autoclaving at 120.degree. C. for &gt;20 minutes. Each column was placed in 
circuit with 50 ml of nutrient broth as shown in FIG. 1. A shaking water 
bath held at 30.degree. C. was used to keep the 250 ml wide-neck 
round-bottomed flasks containing the culture at this constant temperature. 
A small inoculum of pre-prepared bacterial culture was added to each flask 
to give a known number of bacteria/ml in the flask. At time zero, a small 
aliquot of the bacterial culture was removed from each flask for further 
counting/analysis and the pumps started to give a volume flowrate of 13.5 
ml/min. The flow direction was upwards through the stones. 
Aliquots were removed from the reaction flask at time intervals of 0.5, 8 
and 24 hours and the viable counts performed. These data are summarized in 
FIG. 2. There is the usual lag phase as the bacteria become accustomed to 
the new medium, followed by a growth phase in each system. However, it is 
quite evident that the bacterial population is significantly lower in the 
active system, which shows a bactericidal effect compared to the control. 
Differences could be seen visually between the active and control systems 
since solutions become more cloudy as bacterial populations increase due 
to light-scattering phenomena. Light scattering and perhaps UV absorption 
could be used to detect total number of bacteria; but, unlike plating, 
these techniques would not distinguish between viable and non-viable 
organisms. 
In addition, the solutions were analyzed after the experiment. None of the 
Compound 2 or obvious metabolites were detected by HPLC or mass 
spectrometry. 
EXAMPLE 2 
An identical method to Example 1 was used except that porous clay beads 
(OBI, 8-16 mm) were used in place of the pumice stones. Microbiological 
evaluation was carried out in a similar fashion to Example 1, and the data 
is given in FIG. 3. This demonstrates that bacteriostatic activity is 
obtained for the Compound 2 immobilized in sol-gel coated on clay beads. 
EXAMPLE 3 
An identical method to Example 1 was used except that Compound 3 was used 
in place of Compound 2. Microbiological evaluation was carried out in a 
similar fashion to Example 1, using untreated pumice stones (no sol-gel 
coating) and those having a sol-gel coating only (no biocide); the data 
are given in FIG. 4. 
The data demonstrate that the biocidal activity of the hydrophobic biocide 
is maintained using immobilization in sol-gel coated onto pumice stones. 
EXAMPLE 4 
An identical method to Example 1 was used except that Compound 3 was used 
in place of Compound 2. The resultant active and control pumice stones 
were dried for 1 hour at 90.degree. C. and tested as described previously. 
The microbiological results in FIG. 5 show that the drying treatment has 
removed any contribution from the blank sol-gel coating. In this case 
Compound 3 immobilized in sol-gel coated on pumice stones is strongly 
bactericidal. 
The invention has been described in detail with particular reference to 
certain preferred embodiments thereof, but it will be understood that 
variations and modifications can be effected within the spirit and scope 
of the invention.