AU2015337804A1

AU2015337804A1 - Method and compositions for preventing bacterial corrosion of a substrate

Info

Publication number: AU2015337804A1
Application number: AU2015337804A
Authority: AU
Inventors: Garth Brown; Brenton HAMDORF; Stuart JESSOP; David Mcminn; Brendan Murray; Scott Rice
Original assignee: IXOM OPERATIONS Pty Ltd
Current assignee: IXOM OPERATIONS Pty Ltd
Priority date: 2014-10-31
Filing date: 2015-10-29
Publication date: 2017-05-25
Also published as: WO2016065423A1

Abstract

A method of preventing bacterial corrosion of a substrate and of preventing formation of a microbial biofilm on a substrate, which method comprises applying to the substrate a composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of the alkaline inhibitor and wherein nitric oxide is released from the nitric oxide donor compound when the pH of the alkaline inhibitor is reduced.

Description

WO 2016/065423 PCT/AU2015/050678 - 1 -

METHOD AND COMPOSITIONS FOR PREVENTING BACTERIAL CORROSION OF A SUBSTRATE

Field of the invention 5 The present invention relates to the treatment of substrates to prevent bacterial corrosion and formation of biofilms. The present invention is particularly concerned with providing corrosion protection to, and preventing biofilm formation on, concrete structures, such as sewers and associated structures. 10 Background of the invention

Bacterial corrosion of substrates represents a significant problem, especially in relation to sewers and associated structures, such as manholes. 15 A variety of techniques have been employed to inhibit corrosion of concrete in this context. One approach involves pre-treating sewage in order to increase pH or to precipitate or oxidise sulfides that are present thereby inhibiting the activity of sulfur oxidising bacteria. However, such approaches tend to have only a temporary effect, and repeated and continuous treatment is necessary for prolonged corrosion protection. This 20 can be labour intensive and expensive.

Another approach involves providing a layer of magnesium hydroxide onto a concrete surface susceptible to bacterial corrosion. This may be done by application to the surface of a magnesium hydroxide slurry or gel product. The aim of the layer is to provide a 25 highly alkaline environment that is acid neutralising and that substantially reduces the density and activity of corrosion causing bacteria. Once applied the magnesium hydroxide layer can maintain a suitably high pH at the concrete surface for a period of time, after which re-application is necessary in order to maintain corrosion resistance. PCT/AU2015/050678 WO 2016/065423 -2- A problem with this approach is that the efficacy of the applied layer diminishes as bacteria colonise the layer and the pH is reduced. This can curtail the effective life of the layer. 5 A further issue relates to the formation of biofilms on a substrate. Biofilms are three dimensional microbial growth forms comprising bacterial communities and the extracellular matrix they produce. Biofilms are extremely common and generally form on surfaces where water is available or in suspension. Biofilms are problematic as they can cause significant industrial damage, for example, fouling and corrosion in fluid processes 10 such as water distribution and treatment systems, pulp and paper manufacturing systems, heat exchange systems and cooling towers, and contributing to the souring of oil in pipelines and reservoirs. Biofilms may also present health concerns as they are reservoirs of pathogens and may be associated with a number of chronic infections in humans. 15 Efforts to control biofilms normally involve the use of antimicrobial agents, but it would be desirable to avoid their use as they can find their way into the environment.

Against this background it would be desirable to provide means for preventing bacterial corrosion of a substrate, and controlling biofilms on a substrate, that avoids the drawbacks 20 of conventional approaches. In particular, it would be desirable to provide on a substrate a coating/layer that provides enhanced duration of effectiveness and that is less prone to being nullified by colonisation of bacteria over time.

Summary of the invention 25

Accordingly, in one embodiment the present invention provides a method of preventing bacterial corrosion of a substrate, which method comprises applying to the substrate a composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of the 30 alkaline inhibitor and wherein nitric oxide is released from the nitric oxide donor compound when the pH of the alkaline inhibitor is reduced. PCT/AU2015/050678 WO 2016/065423 -3-

The present invention also provides a method of preventing formation of a microbial biofilm on a substrate which method comprises applying to the substrate a composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of the alkaline inhibitor 5 and wherein nitric oxide is released when the pH of the alkaline inhibitor is reduced.

In another embodiment, the present invention provides a composition suitable for use in the methods of the invention, the composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is 10 stable at the initial pH of in the alkaline inhibitor and wherein nitric oxide is released from the nitric oxide donor compound when the pH of the alkaline inhibitor is reduced.

The present invention further provides a substrate treated in accordance with the methods of the invention. 15

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 20

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to 25 which this specification relates.

Detailed discussion of the invention

In the following reference may be made to treating a concrete substrate as this is believed 30 to be an area of particular applicability of the invention. However, the invention should not be read as limited to treatment of concrete substrates, as noted above. WO 2016/065423 PCT/AU2015/050678 -4-

Central to the present invention is the combination of components used in the composition, and the operative relationship between the components. The composition comprises as one essential component an alkaline inhibitor of bacterial corrosion. In accordance with the 5 invention it is intended that this alkaline inhibitor provides protection against bacteria (induced acid) corrosion due to the alkaline environment provided by it. However, over time the pH environment of the composition may reduce, for example as acid-producing bacteria colonise and produce a biofilm. As the pH decreases, it is intended that the nitric oxide donor compound will release nitric oxide thereby dispersing biofilm and thus 10 providing prolonged corrosion protection. By combining the alkaline inhibitor and nitric oxide donor compound in accordance with the invention it is believed that extended protection may be achieved when compared with the use of either component alone.

Herein the term "bacterial corrosion" means acid corrosion due to the activity of bacteria. 15 One example mechanism that may be involved relates to sulfur oxidising bacteria typically present in a substrate biofilm surface. These bacteria oxidise hydrogen sulphide produced by anaerobic bacteria, to sulfuric acid. Depending upon the substrate, the sulfuric acid may be corrosive. This form of corrosion is a significant problem in concrete sewerage systems where hydrogen sulfide is produced by the action of bacteria on untreated sewage 20 and sulfuric acid produced is corrosive to the concrete. Millions of dollars are spent worldwide on the repair and maintenance of affected structures.

The alkaline inhibitor used in the invention functions by providing a highly alkaline environment that is acid neutralising and retards proliferation of acid-forming bacteria. As 25 such, this type of reagent is known in the art. To be useful in the invention, especially when it is desired to treat sewers and associated structures such as manholes, the reagent should be thixotropic and easily pumped. On application to substrate the reagent should also adhere strongly. Typically, the alkaline inhibitor will be magnesium hydroxide based, and such inhibitors are generally available and used in the form of a slurry. Magnesium 30 hydroxide slurries useful in the invention are known in the art. They are usually a slurry comprising at least 50-60 wt% of magnesium hydroxide. By way of example, useful PCT/AU2015/050678 WO 2016/065423 -5- magnesium hydroxide slurries are disclosed in International Patent Publication No. WO 96/003346, the contents of which are hereby incorporated by reference. The rheological properties of a magnesium hydroxide slurry ( and / or other alkaline inhibitor) may need to be modified to provide useful properties. The invention embraces the use of 5 such (modified) alkaline inhibitors.

Generally, the initial pH of the alkaline inhibitor, i.e., the pH of the alkaline inhibitor prior to use, is in the range 8 to 14. At the alkaline pH of the alkaline inhibitor, the nitric oxide donor compound should be stable in that there is no, or no appreciable release, of nitric 10 oxide. However, as the pH environment of the donor compound becomes less alkaline, as would be the case when acid-producing bacteria proliferate and colonise, nitric oxide is released. This is desirable since the nitric oxide will inhibit and/or reverse formation of microbial-produced biofilm. Advantageously, this will contribute to control of microbial growth when the effectiveness of the alkaline inhibitor has diminished. The alkaline 15 inhibitor and nitric oxide donor compound may thus provide enhanced duration of corrosion protection. Typically, the nitric oxide donor compound is stable from alkaline to neutral pH conditions. At about neutral pH nitric oxide will start to be released.

The effect of nitric oxide on antimicrobial biofilm formation is known and is believed to be 20 linked to the accumulation of reactive oxygen and nitrogen species within organisms in a biofilm. More specifically, it is believed that nitric oxide promotes programmed cell death and dispersal of cells within the biofilm. This effect is described in more detail, for example in International Patent Publication No. WO 06/125262, the contents of which are hereby incorporated by reference. 25

Nitric oxide donor compounds useful in the present invention must be compatible with the alkaline inhibitor. The donor compound must be stable in the alkaline inhibitor and it must not compromise the rheological properties that the inhibitor is required to possess. If the donor compound does impact on rheological properties of the alkaline inhibitor to the 30 extent that the resultant composition is unsuitable for an intended application, it may be possible to use one or more additives to remediate properties of the composition. WO 2016/065423 PCT/AU2015/050678 -6- A variety of known nitric oxide donor compounds may be used in the present invention. The efficacy and compatibility of a given combination of donor compound and alkaline inhibitor may be assessed by experiment. Examples of nitric oxide donor compounds for 5 use in the present invention include sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO), GSNO monoethyl ester, S-nitroso-N-acetylpenicillamine (SNAP), glycol-SNAP, L-arginine, N,N'-dinitroso-N,N'-dimethylphenylenediamine (BNN3), N,N'-dinitrosophenylenediamine-N,N'-diacetic acid (BNN5), BNN5-Na, BNN5 methyl ester, 2-hydroxybenzoic acid 3-nitrooxymethylphenyl ester (B-NOD), dephostatin, 10 3,4-dephostatin, diethylamine NONOate, diethylamine NONOate/AM, /i S,S'-dinitrosodithiol, S-nitrosocaptopril, N -hydroxy-L-arginine monoacetate salt, Angeli's salt, l-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-l-triazene (NOC-5), l-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-l-triazene (NOC-7), 6-(2-hydroxy-l-methyl-2-nitrisohydrazino)-N-methyl- 1-hyxanamine (NOC-9), l-hydroxy-2-oxo-3-(N-ethyl-2-15 aminoethyl)-3-ethyl-l-triazene (NOC-12), 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18), (±)-(E)-Methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1), (±)-(E)-4-ethyl-2-[(E)-hydroxyiminol-5-nitro-3-hexenamide (NOR-3), (±)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-l-yl]-3-pyridine carboxamide (NOR-4), 4-phenyl-3-furoxancarbonitrile, PROLI/NO (L-proline in methanolic sodium 20 methoxide), 3-morphorlinosydnonimine (SIN-1), S-nitroso-N-valerylpenicillamine (SNVP), spermine NONOate, ethyl nitrite, streptozotocin and DETA-NO and DETA-NONOate and combinations thereof.

Examples of microorganisms within a biofilm or capable of forming a biofilm include 25 bacterial or fungal species or both, and may comprise one or more species selected from, for example, Thiobaccilus spp. Candida spp., Hormoconis spp., Pseudomonas spp., Pseudoalteromonas spp., Staphylococcus spp., Streptococcus spp., Shigella spp., Mycobacterium spp., Enterococcus spp., Escheirichia spp., Salmonella spp., Legionella spp., Haemophilus spp., Bacillus spp., Desulfovibrio spp., Shewanella spp., Geobacter 30 spp., Klebsiella spp., Proteus spp., Aeromonas spp., Arthrobacter spp., Micrococcus spp.,

Serratia spp., Porphyromonas spp., Fusobacterium spp. and Vibrio spp., representative PCT/AU2015/050678 WO 2016/065423 -7- examples of such species being Candida albicans, P. aeruginosa, Staphylococcus epidermidis, Escherichia coli, Bacillus licheniformis, Serratia marcescens, Fusobacterium nucleatum, and Vibrio Cholerae. The efficacy of nitric oxide on these may be assessed by experiment. 5

The nitric oxide donor compound is present in the alkaline inhibitor in an amount to provide the required efficacy. The amount may be varied and this will of course impact on the effectiveness in controlling biofilm formation and on the duration of this functionality. Generally, the nitric oxide donor compound will be present in an amount of 0.016 mg / L 10 to 32 g/ L based on the total weight of the composition. Usually, the nitric oxide donor compound will be used at a concentration of up to about 200 mM, for example from 0.1 μΜ to 200 mM. The most suitable concentration to achieve the desired effect will depend on a number of factors and may be determined by routine experiment. Such factors include the reagents used, how the composition is applied and the prevailing 15 microbes and/or biofilm.

In accordance with an embodiment of the invention the composition may include one or more antimicrobial agents, noting here that the action of nitric oxide may increase the sensitivity of microbial cells to antimicrobial agents. Suitable antimicrobial agents 20 include, for example antibiotics, detergents, biocides (e.g., chloramine) surfactants, agents that induce oxidative stress, bacteriocins and antimicrobial enzymes, peptides and phage. The antimicrobial agents may be natural or synthetic. A composition in accordance with the invention may be produced by mixing of the 25 individual ingredients. The extent of mixing (intensity, duration and speed) may be optimised by experimentation. As noted, the composition of the invention may also include other functional additives, provided that this does not compromise the efficacy of the slurry in terms of bacterial corrosion resistance and crack resistance. PCT/AU2015/050678 WO 2016/065423 -8-

After formation, the composition of the invention should maintain sufficient fluidity for application to a chosen substrate. After application the composition should form an adherent layer (usually a gel) on a substrate. 5 Once formulated the composition of the present invention must exhibit suitable rheological properties for application to a concrete substrate. Typically, the substrate will be the internal surfaces of a pipe. The composition will typically be used in the form of a slurry. This means that the slurry in accordance with the invention must be pumpable for application by spraying as is usually done for coating such substrates. After pumping 10 desirably the slurry develops a yield stress (i.e., a gel) that prevents the slurry from falling under gravity.

The composition of the invention may be applied to any concrete substrate that is susceptible to bacterial corrosion and biofilm formation. Typically, the substrate is a 15 structure, such as a pipe, that comes into direct contact with sewage. The substrate may also be (the internal surface of) a manhole. Depending upon the rheological properties of the composition, it may be applied by spraying on the substrate. This would be done in conventional fashion. For an in situ structure, the substrate surface to be treated is usually cleaned with water to remove any slime layer and/or loosely bound corrosion products. 20 For substrate surfaces that have not been in service, cleaning may not be required prior to coating with slurry.

Embodiments of the invention are illustrated with reference to the following non-limiting examples. 25

Examples Example 1 30 This example explores the incorporation of a NO donor into commercial magnesium hydroxide based corrosion inhibitor for concrete sewer pipe applications. The example PCT/AU2015/050678 WO 2016/065423 -9- involves compatibility and NO release studies of a coating with the NO donor and the effect of the modified coating on laboratory bio films. The coating with NO is compared with an unmodified coating to determine if NO can extend the protective effect of the coating. Samples were monitored for changes in the microbial community as well as 5 indicators of corrosion.

The corrosion inhibitor is highly alkaline and has a pH of between 9.5 and 10. The high pH should make the corrosion inhibitor compatible with the NO donor which is stable at high pH and releases NO from the donor as the pH approaches neutral. Thus, in theory NO 10 should only be released when acid producing bacteria colonise the coating and reduce the pH by acid production.

Formulation of Proli-NO into commercial product and verification of NO release 15 The commercial product was mixed with NO donor Proli to a final concentration of: 1) 10 mM, 2) 1 mM or 3) 0.1 mM and no Proli controls (4-5), and was cast on round glass coupons.

Prior to quantification of the NO release, the pH of the commercial product in PBS was 20 determined. The 200 pi commercial product coupon was added to 10 ml of PBS (pH 7.4). This resulted in the coupon completely dissolving the PBS and the pH changed to approximately 10. Since lower pH is known to promote NO release from the Proli donor, the commercial product coupons, 500 μΐ, were also added to 10 ml of either 0.1 M or 1 M Tris buffer (pH 7.4). The pH changed to 8.81 and 8.12 respectively. This is not 25 unexpected since the commercial product is meant to mediate its long term effects partly through maintaining an elevated pH.

The release of NO from the coatings was quantified in 10 ml of PBS using the Apollo Free Radical analyser. The results obtained show a concentration dependent increase in the 30 ampomeric response to the presence of the NO donor in the commercial product. No response was observed for the lowest concentration of Proli tested, 0.1 mM, while NO was PCT/AU2015/050678 WO 2016/065423 - 10- detected at increasing concentrations of the donor from 1 mM to 10 mM. These results indicate that Proli can still release NO after having been formulated in the commercial product for 24 h at room temperature. The pH was not measured here, but would be expected to be elevated in line with the results presented above for the commercial product 5 added to PBS (pH 10). Thus, even at the elevated pH, some NO release can be observed. As a background control, the commercial product without added NO donor was tested and no NO response was detected.

To test long term stability of NO donors in the commercial product, coupons of the 10 commercial product were cast with 1 mM and 10 mM Proli-NO and held at room temperature. Coupons were collected on different days, 1, 4, and 7, dissolved in 1 M Tris as above and the NO release was quantified. Visual inspection of the coupons on these days indicated that there was no visible cracking for any of the coupons as they were kept in a humidified condition. At day 1, the coating still looked a bit shiny. At days 4 and 7, 15 although the coating looked of matted gray and set, visible mark left by the forceps was observed when pick up the coupon.

At day 1, NO was detected at 1 and 10 mM Proli, while no response was observed for Proli at 0.01 mM or 0.1 mM which was similar to the results referred to above for Proli at 1 mM 20 after 24 h in PBS (pH 7.4). At day 4, a slight response was detected for the 1 mM sample and a larger NO response for the 10 mM sample. At day 7, NO production was not readily apparent at 1 mM, but could be observed at 10 mM. It was observed for the Day 4 samples that the coupons did not completely dissolve in the PBS buffer, which may have affected the NO release. 25

To determine if the effect of the commercial product was strictly mediated by the elevated pH, samples were acidified by adding HC1 to the commercial product. Nitric oxide was not detected after 24 h of incubation in these experiments for the Proli-NO. This would suggest that the NO was released rapidly as a consequence of the acidification and this was 30 in-line with previous data on the rates of NO release under different pH regimens. WO 2016/065423 PCT/AU2015/050678 - 11 -

Example 2

It is known that biofilms that have been dispersed using NO donors are more sensitive to biocides than non-dispersed biofilms. Therefore, one strategy for controlling biofilms may 5 be to combine the dispersal effects of NO with a biocide to control the biofilm. To determine if the NO donors were compatible with a biocide, chloramine, in the commercial product formulation, 1 mM Proli-NO were added to the commercial product with either 10 or 100 ppm chloramine. The NO release was quantified after 24 h. Results showed that NO could be detected for Proli 1 mM with chloramine at 100 ppm. 10

Example 3

Dispersal of model biofilms from NO donor containing coatings of a commercial product. 15

This example is aimed at determining if NO donors will disperse biofilms that form on the commercial product.

As part of the biofilm assay development, various methods to quantify colonization of the 20 commercial product have been considered, including crystal violet staining, protein quantification and CFU counts. Crystal violet staining was not compatible with the commercial product as it was not possible to add the CV without solubilizing the gel and the excess stain could not be washed away (data not shown). To determine if protein quantification could be used, a standard curve of bovine serum albumin (BSA) was made 25 in PBS and formulated with the commercial product. Results showed that the commercial product interfered with the protein quantification at the concentration range used and therefore was not suitable to measure biofilm formation (data not shown).

The formation of Colony forming units (CFUs) is a standard method for quantifying 30 bacteria in biofilms and therefore, it was investigated whether the commercial product would alter bacterial viability or quantification. Here, dilutions of P. aeruginosa were PCT/AU2015/050678 WO 2016/065423 - 12- added to commercial product and determined CFUs (Table 1.1). When equal amounts of the bacterial culture quantified in the absence or presence of the commercial product, it was observed that the presence of the commercial product resulted in a 75% reduction (1.89E +09 vs 4.4E +08) in CFU counts. This could suggest that the commercial product 5 has some antimicrobial activity or that the bacteria are sequestered, e.g. through adhesion, by the commercial product. However, when dilutions of P. aeruginosa were added to the commercial product, the CFUs obtained were consistent with the 10 fold dilutions. Therefore, while there is a loss of CFUs when comparing bacterial numbers with and without the commercial product, the CFU method can still be used measure CFUs but 10 those numbers can not be compared to a no-commercial product control.

Table 1.1. CFU counts (spread plate on LB agar)

Commercial product 500 μΐ only No growth PAOl 500 pi only 1.89E+09 Commercial product 500 μΐ + 4.40E+08 Commercial product 500 μΐ + 1.60E+08 Commercial product 500 μΐ + 3.94E+07

Conclusions 15

It has been shown that NO can be formulated into and released from a commercial magnesium hydroxide-based product. The high pH of the product may improve the life-time of the NO donor in the formulation due to the pH mediated release characteristics of the NO donor. 20

Longer term experiments, over 7 days, showed that NO could be detected in the samples using the NO donor Proli-NO at 10 mM for up to 7 days.

Formulation of the NO donors into the gel was compatible with chloramine, which may act to increase the microbial biofilm control. 25 CFU counts can be used to quantify bacterial biofilms on the commercial product.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of preventing bacterial corrosion of a substrate, which method comprises applying to the substrate a composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of the alkaline inhibitor and wherein nitric oxide is released from the nitric oxide donor compound when the pH of the alkaline inhibitor is reduced.
2. A method of preventing formation of a microbial biofilm on a substrate, which method comprises applying to the substrate a composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of the alkaline inhibitor and wherein nitric oxide is released when the pH of the alkaline inhibitor is reduced.
3. A method as claimed in claim 1 or 2, wherein the alkaline inhibitor is a magnesium hydroxide slurry.
4. A method as claimed in claim 1 or 2, wherein the alkaline inhibitor has an initial pH in the range 8 to 14.
5. A method as claimed in claim 1 or 2, wherein the nitric oxide donor compound is selected from sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO), GSNO monoethyl ester, S-nitroso-N-acetylpenicillamine (SNAP), glycol-SNAP, L-arginine, N,N'-dinitroso-N,N'-dimethylphenylenediamine (BNN3), N,N'-dinitrosophenylenediamine-N,N'-diacetic acid (BNN5), BNN5-Na, BNN5 methyl ester, 2-hydroxybenzoic acid 3-nitrooxymethylphenyl ester (B-NOD), dephostatin, 3,4-dephostatin, diethylamine NONOate, diethylamine NONOate/AM, S,S'-dinitrosodithiol, S-nitrosocaptopril, N -hydroxy-L-arginine monoacetate salt, Angeli's salt, l-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-l-triazene (NOC-5), 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-l-triazene (NOC-7), 6-(2- hydroxy-l-methyl-2-nitrisohydrazino)-N-methyl-l-hyxanamine (NOC-9), l-hydroxy-2- oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-l-triazene (NOC-12), 2,2'- (hydroxynitrosohydrazono)bis-ethanamine (NOC-18), (±)-(E)-Methyl-2-[(E)- hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1), (±)-(E)-4-ethyl-2-[(E)- hydroxyiminol-5-nitro-3 -hexenamide (N OR-3), (±)-N - [(E)-4-ethyl-2- [(Z)- hydroxyimino]-5-nitro-3-hexene-l-yl]-3-pyridine carboxamide (NOR-4), 4-phenyl-3-furoxancarbonitrile, PROLI/NO (L-proline in methanolic sodium methoxide), 3-morphorlinosydnonimine (SIN-1), S-nitroso-N-valerylpenicillamine (SNVP), spermine NONOate, ethyl nitrite, streptozotocin and DETA-NO and DETA-NONOate and combinations thereof.
6. A method as claimed in claim 1 or 2, wherein the substrate is a concrete substrate. 7 A method as claimed in claim 1 or 2, wherein the substrate is a structure that comes into direct or indirect contact with sewage.
8. A method as claimed in claim 7, wherein the structure is a pipe or manhole.
9. A composition suitable for use in the method according to claim 1 or claim 2, the composition comprising an alkaline inhibitor of bacterial corrosion and a nitric oxide donor compound, wherein the nitric oxide donor compound is stable at the initial pH of in the alkaline inhibitor and wherein nitric oxide is released when the pH of the alkaline inhibitor is reduced.
10. A composition as claimed in claim 9, wherein the alkaline inhibitor is a magnesium hydroxide slurry.
11. A composition as claimed in claim 9, wherein the alkaline inhibitor has an initial pH in the range 8 to 14.
12. A composition as claimed in claim 9, wherein the nitric oxide donor compound is selected from sodium nitroprusside (SNP), S-nitroso-L-glutathione (GSNO), GSNO monoethyl ester, S-nitroso-N-acetylpenicillamine (SNAP), glycol-SNAP, L-arginine, N,N'-dinitroso-N,N'-dimethylphenylenediamine (BNN3), N,N'-dinitrosophenylenediamine-N,N'-diacetic acid (BNN5), BNN5-Na, BNN5 methyl ester, 2-hydroxybenzoic acid 3-nitrooxymethylphenyl ester (B-NOD), dephostatin, 3,4-dephostatin, diethylamine NONOate, diethylamine NONOate/AM, S,S'-dinitrosodithiol, S-nitrosocaptopril, N -hydroxy-L-arginine monoacetate salt, Angeli's salt, l-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-l-triazene (NOC-5), 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-l-triazene (NOC-7), 6-(2- hydroxy-l-methyl-2-nitrisohydrazino)-N-methyl-l-hyxanamine (NOC-9), l-hydroxy-2-oxo-3-(N-ethyl-2-aminoethyl)-3-ethyl-l-triazene (NOC-12), 2,2'- (hydroxynitrosohydrazono)bis-ethanamine (NOC-18), (±)-(E)-Methyl-2-[(E)- hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1), (±)-(E)-4-ethyl-2-[(E)- hydroxyiminol-5-nitro-3 -hexenamide (N OR-3), (±)-N - [(E)-4-ethyl-2- [(Z)- hydroxyimino]-5-nitro-3-hexene-l-yl]-3-pyridine carboxamide (NOR-4), 4-phenyl-3-furoxancarbonitrile, PROLI/NO (L-proline in methanolic sodium methoxide), 3-morphorlinosydnonimine (SIN-1), S-nitroso-N-valerylpenicillamine (SNVP), spermine NONOate, ethyl nitrite, streptozotocin and DETA-NO and DETA-NONOate and combinations thereof.
13. A substrate when treated in accordance with the method according to claim 1 or claim 2.