AU2015365575A1

AU2015365575A1 - Compositions and methods of use

Info

Publication number: AU2015365575A1
Application number: AU2015365575A
Authority: AU
Inventors: Michael BRALKOWSKI; Graeme George Paine; Paul Geoffrey SMYTH
Original assignee: Global Bioprotect Ip Pty Ltd
Current assignee: Global Bioprotect Ip Pty Ltd
Priority date: 2014-12-15
Filing date: 2015-12-15
Publication date: 2017-07-27
Also published as: WO2016097857A1

Abstract

This invention relates to methods of treatment of a sewage transport system, including reducing odour, reducing hydrogen sulfide production, reducing and/or preventing corrosion, reducing and/or preventing biofilm formation and/or adhesion and disrupting biofilms. Compositions suitable for use in such methods, comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof, preferably with at least one microorganism of the genus Bacillus, are also described.

Description

PCMB2015/002477 WO 2016/097857 - 1 -

COMPOSITIONS AND METHODS OF USE

This application claims the benefit of Australian Provisional Patent Application No. 2014905067, filed on 15 December 2014, which is hereby incorporated by reference in its 5 entirety.

Field of the Invention

This invention relates to methods of treatment of a sewage transport system, including reducing odour, reducing hydrogen sulfide production, reducing and/or preventing 10 corrosion, reducing and/or preventing biofilm formation and/or adhesion and disrupting biofilms. Compositions suitable for use in such methods, comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof, preferably with at least one microorganism of 15 the genus Bacillus, are also described.

Background of the Invention

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 20 admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Although the general population does not commonly contemplate the journey taken by 25 their waste to its final destination at a sewage treatment plant, the effective management and processing of sewage is essential for a functional modern society. The increasing population and trend towards urbanisation has resulted in problems for the sewage transport system. Sewage treatment plants are commonly located away from urban areas, which, with expanding urbanisation, results in a longer residence time of the sewage in the 30 sewage transport system prior to treatment. Long residence times may cause the sewage to turn septic, which may produce problems with sewage treatment and result in an increase PCT/IB2015/002477 WO 2016/097857 -2- in malodour around the pipelines and pump stations and corrosion in the sewage transport system.

Septic sewage produces offensive odours such as the odour associated with hydrogen 5 sulfide gas (rotten egg gas) and other malodourous gaseous substances. Such odours can be toxic at specific concentrations, which presents a significant health risk, particularly for people working in and around the sewage transport system. Hydrogen sulfide gas at 1000 ppm will cause instant death, at 500 ppm carries a significant mortality risk, at 100 ppm causes a loss of a sense of smell and at 10 ppm will react with mercury in teeth fillings and 10 present other health risks.

Furthermore, high levels of dissolved sulfides (such as HS“ or H2S) within the sewage arriving at the sewage treatment plant must be oxidised to sulfates (such as SO42' or H2SO4) increasing the energy requirements for oxidation. High levels of dissolved sulfides 15 also inhibit sewage digestion by aerobic and anaerobic methane-forming bacteria and, therefore, may inhibit a significant component of the sewage decomposition process. Inhibition of the sewage decomposition process may result in high sludge volumes.

Hydrogen sulfide gas is a resulting product of the microbial activity of sulfur reducing 20 bacteria in environments such as a sewage transport system (equations 1 and 2). Activity of other microorganisms may also contribute to the production of hydrogen sulfide gas through the reduction of the redox potential of the sewage, thereby creating optimal conditions for sulfate reduction (Neethling et al. 1989, Causes and Control of Concrete

Pipe Corrosion <http://www.seas.ucla.edu/stenstro/r/r29>). 95

Anaerobic bacteria SO42" + organic matter —> S2" + H20 + C02 (1) S2" + 2H+ —> H2S (2)

The sulfides may then be converted to sulfates, such as sulfuric acid, by another group of 30 bacteria (equation 3). WO 2016/097857 PCT/IB2015/002477 -3 - H2S + 2O2 —> H2SO4 (3)

Sulfuric acid causes significant corrosion within the sewage transport system, particularly of concrete and metal. Corrosion of the sewage transport system can be a source of 5 significant cost through the continual maintenance and replacement of infrastructure. It is estimated that the cost of sewage infrastructure corrosion in the United States is US$13.75 billion/year.

There are several treatments which are used to control odour and corrosion in sewerage 10 systems, for example, biofilters, activated carbon filters, vent filters and chemical treatments such as acid or alkali for pH control, disinfectants, biocides, antibiotics, surfactants, deodorants, fragrances, chelating agents, oxidising compounds and oxygen gas. Many of the filtration systems are only targeted at reducing malodour and do not address corrosion issues. Large doses of the chemical treatments are typically required to 15 observe an effect, particularly in systems with a high sewage flow rate, which significantly increases the salt content of the sewage effluent and cost of treatment.

Conventional chemical treatments easily disperse within the sewage and are rapidly diluted and removed from the system with sewage flow. A treatment with a longer residence time 20 is desired to enable a lower dosage to be applied and result in a more cost effective treatment.

There is a need for active, cost effective, environmentally friendly treatments that avoid one or more of the above problems. 25

Summary of the Invention

The present invention is predicated in part on the discovery that compositions comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl 30 substituents, alkyl ester sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w and at least one microorganism of the genus Bacillus PCT/IB2015/002477 WO 2016/097857 -4- reduces biofilm formation or adhesion, disrupts biofilms, reduces odour and reduces or prevents corrosion in a sewage transport system.

In one aspect of the invention, there is provided a method of treatment of a sewage 5 transport system comprising addition of a composition comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w and at least one microorganism of the genus Bacillus to the sewage transport system. 10

In another aspect of the invention, there is provided a composition comprising at least one microorganism of the genus Bacillus in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL and one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with 15 alkyl or aryl substituents, alkyl ether sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w.

Figures

Figure 1: Average effect of composition 1 on the growth of Desulfovibrio desulfuricans (n 20 = 2).

Figure 2: Average effect of composition 1 on the growth of Enterobacter cloacae (n = 2).

Figure 3: Average effect of composition 1 on the growth of Rahnella aquatilis (n = 2).

Figure 4: Average effect of composition 1 on the growth of Pseudomonas baetica (n = 2).

Figure 5: Average effect of composition 1 on the growth of Enterobacteriaceae bacterium 25 (n = 2).

Figure 6: Average effect of composition 1 on the growth of Pantoea agglomerans (n = 2). Figure 7: Average effect of composition 1 on the growth of Shewanella oneidensis (n = 2). 30

Detailed Description of the Invention WO 2016/097857 PCT/IB2015/002477 -5 - 1. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the 5 invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. 10 The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term “about” is used herein to refer to conditions (e.g. amounts, concentrations, time, 15 etc.) that vary by as much as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a specified condition.

As used herein, the term “alkyl” refers to a straight chain or branched saturated hydrocarbon group having 1 to 20 carbon atoms. Where appropriate, the alkyl group may 20 have a specified number of carbon atoms, for example, Cs-Cix alkyl which includes alkyl groups having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms in a linear or branched arrangement. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl and octadecyl. Optionally, one or more -CFU- in an alkyl chain may be 25 substituted with one or more substituents selected from hydroxyl, acyl (C(=0)R), acyloxy (OC(=0)R) and oxo (=0), wherein R is Ci-Cio alkyl or aryl.

The term “aryl” is used herein to refer to any stable monocyclic or bicyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl 30 elements include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, biphenyl and binaphthyl. WO 2016/097857 PCMB2015/002477 -6-

As used herein, the term "biosurfactant" refers to a surfactant that is produced by bacteria, yeast or fungi. For example, the biosurfactant may be an antimicrobial peptide such as a cyclic lipopeptide biosurfactant including surfactin, lichenysin, iturin, fengycin, 5 bacillomycin or mycosubtilin.

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 10 but not the exclusion of any other integer or step or group of integers or steps.

As used herein, the term “sewage transport system” refers to the system of sewerage pipeline or sewage mains, man holes, hydraulic systems such as pumps, wet wells, pumping stations and lift stations that transport the sewage from its origin to the sewage 15 treatment plant. This term refers to both gravity transport systems and pressure transport systems.

As used herein, the term “wet well” refers to an underground pit for sewage storage, typically at a pumping station. 20 2. Methods of the Invention

The present invention relates to the use of a composition comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester 25 sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w to reduce odour, reduce hydrogen sulfide production, reduce and/or prevent corrosion, reduce and/or prevent biofilm formation and/or adhesion and/or disrupt existing biofilms in a sewage transport system. In preferred embodiments, the composition useful in the invention further comprises at least one microorganism of the genus Bacillus. 30

In one aspect of the present invention, there is provided a method of treatment of a sewage PCT/IB2015/002477 WO 2016/097857 -7 - transport system comprising addition of a composition comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% 5 w/w to the sewage transport system.

In another aspect of the present invention, there is provided a method of treatment of a sewage transport system comprising addition of a composition comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or 10 sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w and at least one microorganism of the genus Bacillus to the sewage transport system. 15 The surfactant is an anionic surfactant selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts or mixtures thereof. Suitable alkyl sulfates include, but are not limited to, Cg-Cig alkyl sulfates such as sodium dodecyl sulfate, lithium dodecyl sulfate, ammonium dodecyl sulfate, sodium tetradecyl 20 sulfate, sodium 7-ethyl-2-methyl-4-undecyl sulfate and sodium 2-ethylhexyl sulfate, especially sodium dodecyl sulfate. Suitable alkyl ether sulfates include, but are not limited to, Cs-Cis alkyl ether sulfates such as sodium laureth sulfate and sodium myreth sulfate. Suitable alkyl sulfonates or sulfonic acids include, but are not limited to, Cs-Cis alkyl sulfonates such as sodium tetradecyl sulfonate, sodium octadecyl sulfonate, sodium 25 dodecyl sulfonate, sodium hexadecyl sulfonate and the corresponding sulfonic acids. Suitable aryl sulfonates or sulfonic acids, optionally substituted with alkyl or aryl substituents include, but are not limited to, Cs-CiS alkylbenzene sulfonates or sulfonic acids such as sodium dodecylbenzene sulfonate, dodecylbenzene sulfonic acid and dodecylbenzene sulfonic acid isopropylamine salt; C4-C18 alkylnaphthalene sulfonates or 30 sulfonic acids such as sodium butylnaphthalene sulfonate and sodium hexylnaphthalene sulfonate; and C6-C16 alkyl diphenyloxide disulfonates or disulfonic acids such as sodium PCT/IB2015/002477 WO 2016/097857 -8- dodecyl diphenyloxide disulfonate; especially sodium dodecylbenzene sulfonate or dodecylbenzene sulfonic acid. If optionally substituted with an alkyl substituent, the aryl sulfonate or sulfonic acid may be positioned at any point along the alkyl chain, for example on a primary, secondary or tertiary carbon. Suitable alkyl ester sulfonates or 5 sulfonic acids include, but are not limited to, Cs-Cis alkyl methyl ester sulfonates or sulfonic acids such as methyl ester sulfonate, sodium dodecyl methyl ester a-sulfonate, sodium tetradecyl methyl ester α-sulfonate and sodium hexadecyl methyl ester a-sulfonate. The sulfate, sulfonate or sulfonic acid groups may be positioned at any point along the alkyl chain or aryl ring, for example on a primary, secondary or tertiary carbon. Each of 10 the anionic surfactants may contain a mixture of compounds varying in the length of the alkyl chain.

In some embodiments, one surfactant is used. In other embodiments, more than one surfactant is used, for example, two or three surfactants may be used together. 15

Suitable optional substituents include, but are not limited to, alkyl groups such as C4-C18 alkyl and aryl groups such as phenyl, naphthyl, tetrahydronaphthyl, biphenyl and binaphthyl. 20 Suitable salts include, but are not limited to, those formed with cations such as sodium, potassium, lithium, ammonium and isopropylamine; especially sodium.

The amount of surfactant in the composition may depend on the treatment site and volume of sewage to be treated. The amount of surfactant is optimised to prevent over foaming or 25 loss of activity. In particular embodiments, the surfactant in the composition is in an amount in the range of from 0.1% to 2% w/w, 0.3% to 1.7% w/w, 0.5% to 1.5% w/w, especially 0.8% to 1.2% w/w, more especially 0.9% to 1.1% w/w, most especially about 1% w/w. 30 In some embodiments, the composition for use in the methods of the invention comprises at least one microorganism of the genus Bacillus. WO 2016/097857 PCT/IB2015/002477 -9-

In some embodiments, the microorganism is in a vegetative state. In other embodiments, the microorganism is in a dormant state, for example, an endospore. In further embodiments, the microorganism is present in a mixture of dormant and vegetative states. 5

The microorganism of the genus Bacillus may be selected from Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus popilliae, Bacillus circulans and combinations thereof; especially Bacillus subtilis and Bacillus licheniformis·, more especially Bacillus subtilis. In some embodiments, the at least one microorganism of 10 the genus Bacillus is a specific strain of Bacillus subtilis, especially Bacillus subtilis NRRL B-3383 (US Department of Agriculture, Agricultural Research Service, ARS Culture Collection NRRL), B. subtilis ATCC 21331, B. subtilis ATCC 21332, B. subtilis SD901 (FERM BP.7666), B. subtilis ATCC 6051, B. subtilis RSA-203 or combinations thereof; especially B. subtilis RSA-203. 15 B. subtilis RSA-203 is a rod-shaped, aerobic, Gram positive, β-haemolytic microbe capable of forming endospores. Nucleic acid sequence analysis confirms that it is a strain of B. subtilis. A sample of this microorganism was deposited at the ATCC depository, 10801 University Boulevard, Manassas, Virginia 20110-2209, United States of America on 9 20 January 2013 and has been allocated Accession No. PTA-13451.

In some embodiments, the composition comprises more than one microorganism from the genus Bacillus. 25 The amount of microorganism of the genus Bacillus is an amount sufficient to achieve an effective population at the site of application. In some embodiments, the microorganism of the genus Bacillus is present in the composition in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL, 5 x 104 cfu/mL to 7.5 x 106 cfu/mL, 1 x 105 cfu/mL to 5 x 106 cfu/mL, 5 x 105 cfu/mL to 2.5 x 106 cfu/mL, especially 7.5 x 105 cfu/mL to 2 x 106 30 cfu/mL, most especially 1 x 106 cfu/mL or 1.2 x 106 cfu/mL. PCT/IB2015/002477 WO 2016/097857 - 10-

In some embodiments, the composition useful in the invention may be applied to a wet well, including, but not limited to, the surface of the sewage in the wet well or to the wet well wall. In particular embodiments, the composition useful in the invention may be applied as a foam. 5

The composition may be applied directly to the surface of the sewage, may be applied directly to the surface of the sewage in the wet well or may be applied to the wet well wall, especially as a foam. The composition may be added as a bolus. In other embodiments, the composition may be added continuously at a rate suitable to maintain contact with the 10 sewage and/or surface of the sewage transport system. In particular embodiments, the composition is added as a bolus. In some embodiments, the sewage is not further mixed after addition of the composition to maintain a high local concentration of the composition. In some embodiments, the composition is applied at the commencement of the fill cycle in the wet well. 15

In particular embodiments, the composition of the invention is applied as a foam. In some embodiments, the foam has a long residence time at the surface of the sewage or on surfaces in the sewage transport system. For example, the foam may be present for greater than 15 minutes, especially greater than 30 minutes, most especially greater than one hour. 20

Without wishing to be bound by theory, the formulation of the composition as a foam is thought to maintain a high local concentration of the composition at the point of application, for example on the surface of the sewage or on the surfaces of the sewage transport system, particularly on the surfaces of the wet well, such that the composition is 25 added in a phase specific manner. This is thought to enable a prolonged contact time between the composition of the invention and the odour and corrosion causing microorganisms and, therefore, a lower effective dosage rate. A prolonged foam residence time is advantageous for such activity. 30 The foam may be generated prior to application to the sewage transport system or may be generated during the application process. For example, the foam may be generated by PCT/IB2015/002477 WO 2016/097857 - 11 - spraying, misting, infusing with gas or air, stirring, using a spray or jet head or a foam dispenser.

In still other embodiments, the composition of the invention is applied to the sewage 5 transport system by spraying, pouring, hosing, misting or dripping; especially spraying or misting.

Sewage has a variable composition which depends on the substances being discharged into the sewage transport system. For example, sewage comprises carbonaceous and 10 nitrogenous waste matter and populations of many different microbes. The flow rate of sewage in the sewage transport system also varies, which determines the retention time of the sewage in the sewage transport system. For example, the flow rate of sewage is slower at night time compared with daytime and is faster in the wet weather compared with the dry weather. 15

Treatment regimes may be established for different sites of application by a person skilled in the art. Optimal concentrations of the composition will be required to maintain activity and maintain sufficient foaming whilst avoiding over foaming. The dosage rate of the composition will be a dose sufficient to maintain a suitable local concentration of the 20 composition at the site of application. The sewage flow rate, the sewage volume to be treated and environmental factors will also be taken into account when determining the dosage rate of the composition.

Suitable dosage rates of the compositions of the invention may include 1:1000 to 25 1:1000000 parts relative to sewage flow, 1:5000 to 1:500000 parts relative to sewage flow, 1:7500 to 1:250000 parts relative to sewage flow, especially 1:10000 to 1:100000 parts relative to sewage flow or 1:25000 to 1:75000 parts relative to sewage flow, most especially 1:50000 parts relative to sewage flow. 30 In some embodiments, biofilm formation and/or adhesion on surfaces of the sewage transport system is reduced and/or prevented. In some embodiments, biofilms on surfaces PCT/IB2015/002477 WO 2016/097857 - 12- of the sewage transport system are disrupted. Said biofilms may have been formed prior to treatment with a composition of the invention.

Sulfur and/or sulfate reducing bacteria may be present in biofilms on surfaces in the 5 sewage transport system. The use of compositions of the present invention to prevent or reduce biofilm formation and/or adhesion and/or to disrupt existing biofilms will assist in the reduction of malodourous hydrogen sulfide and other malodourous gases in the sewage transport system. 10 The reduction of hydrogen sulfide in the sewage transport system may also reduce the amount of sulfuric acids and sulfates, therefore reducing the corrosion of surfaces in the sewage transport system. Thus, it is contemplated that the reduction of the number of bacteria or the inhibition of the growth of bacteria, particularly sulfur and/or sulfate reducing bacteria, in a sewerage transport system resulting from application of the 15 compositions of the invention will consequently result in the reduction of odour and/or corrosion in the sewerage transport system.

Microbial populations that may be affected by the methods of the invention may include Gram positive and Gram negative bacteria, autotrophic bacteria, heterotrophic bacteria, 20 nitrogen-fixing bacteria and methanogenic archaea that are present in sewage. The methods of the invention may also affect populations of aerobic, facultative anaerobic and anaerobic bacteria, and are particularly useful against strains of bacteria that produce malodourous gas such as hydrogen sulfide gas, and bacteria that convert sulfide to sulfate. The methods of the invention are also useful against ammonia forming, nitrite forming, 25 nitrate forming, denitrifying, methane forming, metal reducing such as iron reducing and acid producing bacteria. In particular embodiments, the methods of the invention are active against sulfur and/or sulfate reducing bacteria.

For example, affected bacteria may include, but is not limited to, sulfur and/or sulfate 30 reducing bacteria such as Desulfovibrio species, particularly Desulfovibrio desulfuricans, Desulfovibrio salixigens, Desulfovibrio africanus and Desulfovibrio vulgaris', PCT/IB2015/002477 WO 2016/097857 - 13-

Desulfotomaculum species particularly Desulfotomaculum nigrificans and Desulfotomaculum halophilum\ Desulfosporosinus species such as Desulfosporosinus orientis; Desulfosporomusa species such as Desulfosporomusa polytropa\ sulfide oxidising bacteria such as Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Thiobacillus 5 concretivorous, Thiobacillus intermedins and Thiobacillus thioparus', bacteria from the Enterobacteriaceae family such as Pantoea agglomerans, Enterobacter cloacae and Rahnella aquatilis and Pseudomonas species such as Pseudomonas baetica\ metal reducing bacteria such as Shewanella oneidensis and Shewanella putrefaciens; Clostridium species such as Clostridium perfringens, Clostridium butyricum, Clostridium tetani and 10 Clostridium difficile·, and Listeria species such as Listeria monocytogenes.

In some embodiments, there is provided a method of reducing odour in a sewage transport system, comprising addition of a composition of the invention to the sewage transport system. Without wishing to be bound by theory, it is thought that odour is reduced through 15 microbial control, mainly through the direct reduction of biological activity and the resultant increase in dissolved oxygen levels, which further suppresses anaerobic microbial activity.

In particular embodiments, hydrogen sulfide production in the sewage transport system is 20 reduced by application of a composition of the invention. Without wishing to be bound by theory, it is thought that hydrogen sulfide production is reduced through the control of sulfur or sulfate reducing bacteria in the sewage transport system. The amount of hydrogen sulfide in the sewage transport system may be reduced to an amount below 10 ppm, 7 ppm, 5 ppm, 3 ppm or 1 ppm. 25

In some embodiments, corrosion of surfaces in the sewage transport system is reduced and/or prevented. Any surface in the sewage transport system may be susceptible to corrosion, especially either concrete or metal surfaces. The corrosion may occur at any point in the sewage transport system, particularly in the pipes, wet wells and/or hydraulic 30 sections such as the pumps. PCT/IB2015/002477 WO 2016/097857 - 14-

Although the anionic surfactant in the composition may have some foaming activity, in some embodiments, the composition for use in the methods of the invention further comprises a foaming agent. Suitable foaming agents may include non-ionic surfactants, such as alkyl and polyalkyl esters of poly(ethylene oxide), alkyl and polyalkyl ethers of 5 poly(ethylene oxide), alkyl and polyalkyl glycosides or polyglycosides in particular alkyl and polyalkyl glucosides or polyglucosides and alkylalkanolamides; zwitterionic surfactants such as alkylamidopropyl betaines, for example cocamidopropyl betaine and lauramidopropyl betaine; and combinations thereof. In particular embodiments, the foaming agent is a non-ionic surfactant selected from an alkylmonoethanolamide or an 10 alkyldiethanolamide, for example a Cg-Cig alkylmonoethanolamide such as cocomonoethanolamide or a Cg-Cig alkyldiethanolamide such as cocodiethanolamide; most especially cocomonoethanolamide. In some embodiments, each of these foaming agents may contain a mixture of compounds varying in the length of the alkyl chain. 15 The amount of foaming agent in the composition depends on the treatment site and volume of sewage to be treated. The amount of foaming agent is optimised to prevent over foaming and leakage of the foam from the sewage transport system, but still maintain an effective amount of foam at the surface of the application site of the sewage transport system, such as a wet well. In some embodiments, the foam formed is a stable foam and 20 may maintain structural integrity for an extended period of time, for example for greater than 15 minutes, especially greater than 30 minutes, most especially greater than one hour. In particular embodiments, the foaming agent in the composition is in an amount in the range of from 0.1% to 1% w/w, especially about 0.5% w/w. 25 In some embodiments, the composition for use in the methods of the invention further comprises a thickening agent. Suitable thickening agents include, but are not limited to, natural gums such as xanthan gum, guar gum, gum arabic, carrageenan, locust bean gum, alginate, karaya gum, tragacanth, curdlan, scleroglucan, gellan gum, gum ghatti, beta-glucans, chicle gum, dammar gum, glucomannan, mastic gum, tara gum and agar; 30 carboxymethyl cellulose; hydroxypropylmethyl cellulose; hydroxyethyl cellulose; hydroxypropyl cellulose; ethyl cellulose; methyl cellulose; carboxymethylhydroxyethyl PCT/IB2015/002477 WO 2016/097857 - 15- cellulose; pectin; starch; gelatin; carbomers; fumed silica; hydroxypropyl guar gum; polyethylene glycols; and combinations thereof. The thickening agent may be present in the composition in an amount in the range of from 0.1% to 1% w/w, especially about 0.3% w/w. 5

In some embodiments, the composition for use in the methods of the invention further comprises a biosurfactant. Suitable biosurfactants may include, but are not limited to, antimicrobial cyclic lipopeptide biosurfactants such as surfactin, lichenysin, iturin, fengycin, bacillomycin, mycosubtilin and combinations thereof; especially surfactin. Each 10 of these biosurfactants may contain mixtures of compounds, varying in chain length of the fatty acid moiety of the lipopeptide.

In some embodiments, the biosurfactant is produced by the microorganism of the genus Bacillus that is in the composition. In these embodiments, the biosurfactant may be 15 produced by the microorganism during production of the microorganism for inclusion in the composition. In some embodiments, the microorganism and biosurfactant are produced together and included in the composition without isolation from one another. Alternatively, in some embodiments, the biosurfactant is added to the composition during manufacture. 20

In some embodiments, the biosurfactant is not produced by the microorganism of the genus Bacillus that is in the composition. In these embodiments, the biosurfactant is added to the composition during manufacture. 25 The biosurfactant may be present in the composition in an amount in the range of from 0.001% to 0.1% w/w, 0.003% to 0.05% w/w, 0.004% to 0.04% w/w, 0.005% to 0.03% w/w, 0.006% to 0.02%, especially about 0.01% w/w or about 0.0067% w/w.

In some embodiments, the composition does not contain an added biosurfactant. 30

The compositions useful in the invention may also comprise additional components PCT/IB2015/002477 WO 2016/097857 - 16- produced by the microorganism of the genus Bacillus that is in the composition. These components may be produced by the microorganism during production of the microorganism for inclusion in the composition. For example, the composition may further comprise one or more isocoumarins such as amicoumacin A, B and C, and/or other 5 low molecular weight precursors for biosurfactants (i.e. components with a molecular weight less than 700 Da). These components may be added to the composition without isolation from the microorganism. In other embodiments, these components are not produced by the microorganism of the genus Bacillus that is in the composition and may be added to the composition during manufacture. 10

In some embodiments, the composition for use in the methods of the invention comprises a preservative. Suitable preservatives include, but are not limited to, sodium benzoate, benzoic acid, sorbic acid, lactic acid, nitrites such as sodium nitrite, nitrates such as sodium nitrate, propionic acid, sodium propionate, sodium sorbate, ascorbic acid and combinations 15 thereof; especially sodium benzoate. The preservative may be present in the composition in an amount in the range of from 0.03% to 0.6% w/w or 0.1% to 0.4% w/w, especially about 0.3% w/w or about 0.2% w/w.

In some embodiments, the composition for use in the methods of the invention comprises a 20 liquid medium. Suitable liquid mediums include, but are not limited to, aqueous carriers such as water, buffer, culture medium, culture medium used to culture the microorganism and combinations thereof. In particular embodiments, the composition comprises the culture medium containing the microorganism in a sufficient quantity produced directly from the culture process. 25

Suitable buffers include, but are not limited to, citric acid and phosphate such as monosodium phosphate, monopotassium phosphate, disodium phosphate and dipotassium phosphate. In some embodiments, the pH of the composition is in the range of from pH 4 to 10, especially pH 5 to 9, most especially pH 6 to 8. 30

Exemplary culture medium may comprise aqueous monopotassium phosphate, disodium PCT/IB2015/002477 WO 2016/097857 - 17- phosphate, magnesium sulfate heptahydrate, calcium chloride hexahydrate, disodium EDTA dehydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, ammonium chloride, sodium nitrate, glucose monohydrate and yeast extract. 5 3. Compositions of the Invention

In another aspect of the invention, there is provided a composition comprising at least one microorganism of the genus Bacillus in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL and one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with 10 alkyl or aryl substituents, alkyl ether sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w.

The amount of microorganism of the genus Bacillus is an amount sufficient to achieve an effective population at the site of application. In some embodiments, the microorganism of 15 the genus Bacillus is in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL, 5 x 104 cfu/mL to 7.5 x 106 cfu/mL, 1 x 105 cfu/mL to 5 x 106 cfu/mL, 5 x 105 cfu/mL to 2.5 x 106 cfu/mL, especially 7.5 x 105 cfu/mL to 2 x 106 cfu/mL, most especially 1 x 106 cfu/mL or 1.2 x 106 cfu/mL. 20 In some embodiments, the microorganism is in a vegetative state. In other embodiments, the microorganism is in a dormant state, for example, an endospore. In further embodiments, the microorganism is present in a mixture of dormant and vegetative states.

The microorganism of the genus Bacillus may be selected from Bacillus subtilis, Bacillus 25 licheniformis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus popilliae, Bacillus circulans and combinations thereof; especially Bacillus subtilis and Bacillus licheniformis', more especially Bacillus subtilis. In some embodiments, the at least one microorganism of the genus Bacillus is a specific strain of Bacillus subtilis, especially Bacillus subtilis NRRL B-3383 (US Department of Agriculture, Agricultural Research Service, ARS 30 Culture Collection NRRL), B. subtilis ATCC 21331, B. subtilis ATCC 21332, B. subtilis SD901 (FERM BP.7666), B. subtilis ATCC 6051, B. subtilis RSA-203 or combinations WO 2016/097857 PCT/IB2015/002477 - 18- thereof; especially B. subtilis RSA-203. B. subtilis RSA-203 is a rod-shaped, aerobic, Gram positive, β-haemolytic microbe capable of forming endospores. Nucleic acid sequence analysis confirms that it is a strain of B. 5 subtilis. A sample of this microorganism was deposited at the ATCC depository, 10801 University Boulevard, Manassas, Virginia 20110-2209, United States of America on 9 January 2013 and has been allocated Accession No. PTA-13451.

In some embodiments, the composition comprises more than one microorganism from the 10 genus Bacillus.

The surfactant is an anionic surfactant selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts and mixtures thereof. 15 Suitable alkyl sulfates include, but are not limited to, Cg-Cig alkyl sulfates such as sodium dodecyl sulfate, lithium dodecyl sulfate, ammonium dodecyl sulfate, sodium tetradecyl sulfate, sodium 7-ethyl-2-methyl-4-undecyl sulfate and sodium 2-ethylhexyl sulfate, especially sodium dodecyl sulfate. Suitable alkyl ether sulfates include, but are not limited to, C8-Ci8 alkyl ether sulfates such as sodium laureth sulfate and sodium myreth sulfate. 20 Suitable alkyl sulfonates or sulfonic acids include, but are not limited to, Cg-Cig alkyl sulfonates such as sodium tetradecyl sulfonate, sodium octadecyl sulfonate, sodium dodecyl sulfonate, sodium hexadecyl sulfonate and the corresponding sulfonic acids. Suitable aryl sulfonates or sulfonic acids, optionally substituted with alkyl or aryl substituents include, but are not limited to, Cg-Cig alkylbenzene sulfonates or sulfonic 25 acids such as sodium dodecylbenzene sulfonate, dodecylbenzene sulfonic acid and dodecylbenzene sulfonic acid isopropylamine salt; C4-C18 alkylnaphthalene sulfonates or sulfonic acids such as sodium butylnaphthalene sulfonate and sodium hexylnaphthalene sulfonate; and C6-C16 alkyl diphenyloxide disulfonates or disulfonic acids such as dodecyl diphenyloxide disulfonate; especially sodium dodecylbenzene sulfonate or dodecylbenzene 30 sulfonic acid. If optionally substituted with an alkyl substituent, the aryl sulfonate or sulfonic acid may be positioned at any point along the alkyl chain, for example on a PCT/IB2015/002477 WO 2016/097857 - 19- primary, secondary or tertiary carbon. Suitable alkyl ester sulfonates or sulfonic acids include, but are not limited to, Cg-Qg alkyl methyl ester sulfonates or sulfonic acids such as methyl ester sulfonate, sodium dodecyl methyl ester α-sulfonate, sodium tetradecyl methyl ester α-sulfonate and sodium hexadecyl methyl ester α-sulfonate. The sulfate, 5 sulfonate or sulfonic acid groups may be positioned at any point along the alkyl chain or aryl ring, for example on a primary, secondary or tertiary carbon. Each of the anionic surfactants may contain a mixture of compounds varying in the length of the alkyl chain.

In some embodiments, the composition comprises one surfactant. In other embodiments, 10 the composition comprises more than one surfactant, for example, two or three surfactants.

Suitable optional substituents include, but are not limited to, alkyl groups such as C4-C18 alkyl and aryl groups such as phenyl, naphthyl, tetrahydronaphthyl, biphenyl and binaphthyl. 15

Suitable salts include, but are not limited to, those formed with cations such as sodium, potassium, lithium, ammonium and isopropylamine; especially sodium.

The amount of surfactant in the composition may depend on the treatment site and volume 20 of sewage to be treated. In particular embodiments, the surfactant in the composition is in an amount in the range of from 0.1% to 2% w/w, 0.3% to 1.7% w/w, 0.5% to 1.5% w/w, especially 0.8% to 1.2% w/w, more especially 0.9% to 1.1% w/w, most especially about 1% w/w. 25 Although the anionic surfactant in the composition may have some foaming activity, in some embodiments, the composition further comprises a foaming agent. Suitable foaming agents may include, but are not limited to, non-ionic surfactants, such as alkyl and polyalkyl esters of poly(ethylene oxide), alkyl and polyalkyl ethers of poly(ethylene oxide), alkyl and polyalkyl glycosides or polyglycosides in particular alkyl and polyalkyl 30 glucosides or polyglucosides and alkylalkanolamides; zwitterionic surfactants such as alkylamidopropyl betaines, for example cocamidopropyl betaine and lauramidopropyl PCT/IB2015/002477 WO 2016/097857 -20- betaine; and combinations thereof. In particular embodiments, the non-ionic surfactant is an alkylmonoethanolamide or an alkyldiethanolamide, for example a Cs-Cis alkylmonoethanolamide such as cocomonoethanolamide or a Cs-Cis alkyldiethanolamide such as cocodiethanolamide; most especially cocomonoethanolamide. In some 5 embodiments, each of these foaming agents may contain a mixture of compounds varying in the length of the alkyl chain.

The amount of foaming agent in the composition depends on the treatment site and volume of sewage to be treated. In some embodiments, the composition forms a stable foam and 10 may maintain structural integrity for an extended period of time, for example for greater than 15 minutes, especially greater than 30 minutes, most especially greater than one hour. In particular embodiments, the foaming agent in the composition is in an amount in the range of from 0.1% to 1% w/w, especially about 0.5% w/w. 15 In some embodiments, the composition further comprises a thickening agent. Suitable thickening agents include, but are not limited to, natural gums such as xanthan gum, guar gum, gum arabic, carrageenan, locust bean gum, alginate, karaya gum, tragacanth, curdlan, scleroglucan, gellan gum, gum ghatti, beta-glucans, chicle gum, dammar gum, glucomannan, mastic gum, tara gum and agar; carboxymethyl cellulose; 20 hydroxypropylmethyl cellulose; hydroxyethyl cellulose; hydroxypropyl cellulose; ethyl cellulose; methyl cellulose; carboxymethylhydroxyethyl cellulose; pectin; starch; gelatin; carbomers; fumed silica; hydroxypropyl guar gum; polyethylene glycols; and combinations thereof. The thickening agent may be present in the composition in an amount in the range of from 0.1% to 1% w/w, especially about 0.3% w/w. 25

In some embodiments, the composition further comprises a biosurfactant. Suitable biosurfactants may include, but are not limited to, antimicrobial cyclic lipopeptide biosurfactants such as surfactin, lichenysin, iturin, fengycin, bacillomycin, mycosubtilin and combinations thereof; especially surfactin. Each of these biosurfactants may contain 30 mixtures of compounds, varying in chain length of the fatty acid moiety of the lipopeptide. PCT/IB2015/002477 WO 2016/097857 -21 -

In some embodiments, the biosurfactant is produced by the microorganism of the genus Bacillus that is in the composition. In these embodiments, the biosurfactant may be produced by the microorganism during production of the microorganism for inclusion in the composition. In some embodiments, the microorganism and biosurfactant are 5 produced together and included in the composition without isolation from one another. Alternatively, in some embodiments, the biosurfactant is added to the composition during manufacture.

In some embodiments, the biosurfactant is not produced by the microorganism of the 10 genus Bacillus that is in the composition. In these embodiments, the biosurfactant is added to the composition during manufacture.

The biosurfactant may be present in the composition in an amount in the range of from 0.001% to 0.1% w/w, 0.003% to 0.05% w/w, 0.004% to 0.04% w/w, 0.005% to 0.03% 15 w/w, 0.006% to 0.02%, especially about 0.01% w/w or about 0.0067% w/w.

In some embodiments, the composition does not contain an added biosurfactant.

The compositions of the invention may also comprise additional components produced by 20 the microorganism of the genus Bacillus that is in the composition. These components may be produced by the microorganism during production of the microorganism for inclusion in the composition. For example, the composition may further comprise one or more isocoumarins such as amicoumacin A, B and C, and/or other low molecular weight precursors for biosurfactants (i.e. components with a molecular weight less than 700 Da). 25 These components may be added to the composition without isolation from the microorganism. In other embodiments, these components are not produced by the microorganism of the genus Bacillus that is in the composition and may be added to the composition during manufacture. 30 In some embodiments, the composition further comprises a preservative. Suitable preservatives include, but are not limited to, sodium benzoate, benzoic acid, sorbic acid, PCT/IB2015/002477 WO 2016/097857 -22- lactic acid, nitrites such as sodium nitrite, nitrates such as sodium nitrate, propionic acid, sodium propionate, sodium sorbate, ascorbic acid and combinations thereof; especially sodium benzoate. The preservative may be present in the composition in an amount in the range of from 0.03% to 0.6% w/w or 0.1% to 0.4% w/w, especially about 0.3% w/w or 5 about 0.2% w/w.

In some embodiments, the composition comprises a liquid medium. Suitable liquid mediums include, but are not limited to, aqueous carriers such as water, buffer, culture medium, culture medium used to culture the microorganism and combinations thereof. In 10 particular embodiments, the composition comprises the culture medium containing the microorganism in a sufficient quantity produced directly from the culture process.

Suitable buffers include, but are not limited to, citric acid and phosphate such as monosodium phosphate, monopotassium phosphate, disodium phosphate and dipotassium 15 phosphate. In some embodiments, the pH of the composition is in the range of from pH 4 to 10, especially pH 5 to 9, most especially pH 6 to 8.

Exemplary culture medium may comprise aqueous monopotassium phosphate, disodium phosphate, magnesium sulfate heptahydrate, calcium chloride hexahydrate, disodium 20 ethylenediaminetetraacetic acid (EDTA) dehydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, ammonium chloride, sodium nitrate, glucose monohydrate and yeast extract.

In some embodiments the composition may be formulated as a concentrate which may be 25 diluted before use. In other embodiments, the composition does not require dilution before use.

In some embodiments, the composition is formulated as a foam. The foam may be formed from the diluted or concentrated composition. 30

The composition of the invention may be prepared by combining the one or more anionic PCT/IB2015/002477 WO 2016/097857 -23- surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts or mixtures thereof with the liquid medium. Foaming agents, thickening agents, biosurfactants and/or preservatives are also added to the liquid 5 medium, if required. The cultured microorganism of the genus Bacillus is then added to the composition, particularly in endospore form.

In other embodiments, the composition of the invention may be prepared by combining culture medium comprising the cultured microorganism of the genus Bacillus, particularly 10 in endospore form, with an aqueous carrier comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof, and, if required, foaming agents, thickening agents, biosurfactants and/or preservatives. In some embodiments, the culture medium may be the 15 medium used to culture the microorganism of the genus Bacillus. WO 2016/097857 PCT/IB2015/002477 -24-

EXAMPLES

Materials

Materials used in the preparation of the composition of the Examples are readily available 5 from commercial sources, such as Sigma-Aldrich Co. LLC. Cocomonoethanolamide is available from, for example, Alpha Chemicals Pvt Ltd. Bacterial species are commercially available from commercial sources, such as American Type Culture Collection (ATCC). Pseudomonas baetica is available from, for example, National Collection of Industrial, Food and Marine Bacteria (NCIMB) Culture Collection. 10

Example 1: Method of producing compositions A composition of the invention was prepared by combining 1 % w/w (final concentration) sodium dodecylbenzene sulfonate, 0.3% w/w (final concentration) xanthan gum, 0.5% w/w (final concentration) cocomonoethanolamide, 0.01% w/w (final concentration) surfactin 15 and spent culture medium (aqueous monopotassium phosphate, disodium phosphate, magnesium sulfate heptahydrate, calcium chloride hexahydrate, disodium EDTA dehydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, ammonium chloride, sodium nitrate and traces of glucose monohydrate and yeast extract) comprising B. subtilis endospores (final concentration of 1 x 106 cfu/mL) in a 1000 gallon stainless 20 steel dilution reactor with slight agitation.

Example 2: Method of producing compositions A composition of the invention was prepared by combining 1 % w/w (final concentration) sodium dodecylbenzene sulfonate, 0.01% w/w (final concentration) surfactin and spent 25 culture medium (aqueous monopotassium phosphate, disodium phosphate, magnesium sulfate heptahydrate, calcium chloride hexahydrate, disodium EDTA dehydrate, ferrous sulfate heptahydrate, manganese sulfate monohydrate, ammonium chloride, sodium nitrate and traces of glucose monohydrate and yeast extract) comprising B. subtilis endospores (final concentration of 1 x 106 cfu/mL) in a 1000 gallon stainless steel dilution reactor with 30 slight agitation. PCT/IB2015/002477 WO 2016/097857 -25-

Example 3: Activity of compositions against bacteria

The bactericidal effect of compositions of the invention was determined using a bacterial growth inhibition assay. The bacterial species tested are representative of bacterial species typically present in a sewage transport system. 5

Composition 1(1% w/w dodecylbenzene sulfonic acid, 0.01% surfactin, 1 x 106 B. subtilis endospores in spent culture medium, pH 4.5-5.5; prepared by the method of Example 2 using dodecylbenzene sulfonic acid) was added (in duplicate) to cultures containing 1 x 105 cells of the test bacterium (Desulfovibrio desulfuricans, Enterobacter cloacae, 10 Rahnella aquatilis, Pseudomonas baetica, Enterobacteriaceae bacterium, Pantoea agglomerans and Shewanella oneidensis) in culture medium. The final concentration of Composition 1 in the culture medium was 250 ppm, 500 ppm and 1000 ppm. The cultures were incubated at 30-37° C. At predetermined time points, the optical density at 600 nm (OD 600 nm) of the cultures were measured to determine the growth of the bacteria 15 (Figures 1 to 7).

Composition 1 inhibited the growth of D. desulfuricans for up to 170 hours at a concentration of 1000 ppm, 78 hours at a concentration of 500 ppm and 46 hours at a concentration of 250 ppm (Figure 1). 20

The growth of E. cloacae was inhibited for up to 145 hours at all concentrations of Composition 1 tested (Figure 2).

Composition 1 inhibited the growth of R. aquatilis for up to 145 hours at all concentrations 25 tested (Figure 3).

The growth of P. baetica was inhibited for up to 145 hours at all concentrations of Composition 1 tested (Figure 4). 30 The growth of Enterobacteriaceae bacterium was inhibited for up to 121 hours in the presence of 500 and 1000 ppm of Composition 1. 250 ppm of Composition 1 slightly PCT/IB2015/002477 WO 2016/097857 -26- inhibited the growth of Enterobacteriaceae bacterium (Figure 5).

Composition 1 inhibited the growth of P. agglomerans for up to 121 hours at a concentration of 1000 ppm. At 500 ppm of Composition 1, growth was inhibited for 50 5 hours. A slight inhibition of P. agglomerans growth was observed in the presence of 250 ppm of Composition 1 (Figure 6).

The growth of S. oneidensis was inhibited for up to 130 hours in the presence of all tested concentrations of Composition 1 (Figure 7). 10

It is evident from this data that Composition 1 inhibits the growth of bacterial species typically present in a sewage transport system and, therefore, Composition 1 is expected to be effective in reducing or preventing biofilm formation, reducing hydrogen sulfide production or reducing corrosion in a sewage transport system. 15

Preliminary field trials indicated that compositions of the invention comprising 1.2 x 106 cfu/mL B. subtilis endospores in spent culture medium, 0.0067% w/w surfactin, 1% w/w sodium dodecylbenzene sulfonate and 0.2% w/w sodium benzoate disrupt biofilms and reduce hydrogen sulfide levels in a sewage transport system. 20

Example 4: Activity of compositions against Listeria monocytogenes

The activity of a composition of the invention against L. monocytogenes was compared with a composition comprising sodium dodecylbenzene sulfonate alone. Composition 2 comprised 1.2 x 106 cfu/mL B. subtilis endospores in spent culture medium, 0.0067% w/w 25 surfactin, 1% w/w sodium dodecylbenzene sulfonate and 0.2% w/w sodium benzoate and was produced in accordance with the method of Example 2. Composition 3 comprised aqueous 1% sodium dodecylbenzene sulfonate (pH adjusted to 7 with sodium hydroxide). 100 mL of Agar as Tryptic Soy Broth was added to each sterile plate and allowed to dry. 1 30 mL of L. monocytogenes (5700 cfu/mL) was added to each plate. Once dry, 1 mL of the test compositions at various dilutions were applied to the plates. The plates were incubated PCT/IB2015/002477 WO 2016/097857 -27- for five days at 30°C, checking daily. After incubation, the number of L. monocytogenes colonies on each plate were counted (Table 1).

Table 1: L. monocytogenes growth in the presence of Compositions 2 and 3.

Dilution (composition:water) L. monocytogenes count (cfu) Composition 2 Composition 3 Neat 0 36 1:2 11 44 1:3 27 54 1:4 39 68 1:5 49 79 5

At their highest concentrations, application of Composition 2 caused a log 3.75 reduction in the number of L. monocytogenes colonies, whereas Composition 3 caused a log 2.20 reduction. 10 It is evident from the data above that the composition comprising sodium dodecylbenzene sulfonate alone (Composition 3) is less effective at inhibiting L. monocytogenes growth than the composition comprising B. subtilis endospores in spent culture medium, surfactin, sodium dodecylbenzene sulfonate and sodium benzoate (Composition 2). Composition 2 effectively inhibited L. monocytogenes growth, particularly at higher concentrations. 15

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of treatment of a sewage transport system comprising addition of a composition comprising one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ester sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w and at least one microorganism of the genus Bacillus to the sewage transport system.
2. The method according to claim 1, wherein odour in the sewage transport system is reduced.
3. The method according to claim 2, wherein hydrogen sulfide production in the sewage transport system is reduced.
4. The method according to any one of claims 1 to 3, wherein corrosion in the sewage transport system is reduced and/or prevented.
5. The method according to any one of claims 1 to 4, wherein biofilm formation and/or adhesion on surfaces of the sewage transport system is reduced and/or prevented.
6. The method according to any one of claims 1 to 4, wherein biofilms on surfaces of the sewage transport system are disrupted.
7. The method according to any one of claims 1 to 6, wherein the composition is applied as a foam.
8. The method according to any one of claims 1 to 7, wherein the composition is applied to a wet well.
9. The method according to any one of claims 1 to 8, wherein the composition is applied at a dosage rate in the range of from 1:10000 to 1:100000 parts relative to sewage flow.
10. The method according to any one of claims 1 to 9, wherein the at least one microorganism of the genus Bacillus is present in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL.
11. The method according to any one of claims 1 to 10, wherein the surfactant is a Cs-Ci8 alkylbenzene sulfonate or sulfonic acid or salts thereof.
12. The method according to claim 11, wherein the surfactant is dodecylbenzene sulfonic acid or a salt thereof.
13. The method according to any one of claims 1 to 12, wherein the at least one microorganism of the genus Bacillus is present in the vegetative state, as an endospore or a mixture thereof.
14. The method according to any one of claims 1 to 13, wherein the at least one microorganism is Bacillus subtilis.
15. The method according to any one of claims 1 to 14, wherein the composition further comprises a foaming agent.
16. The method according to claim 15, wherein the foaming agent is an alkylmonoethanolamide or an alkyldiethanolamide non-ionic surfactant.
17. The method according to claim 16, wherein the foaming agent is cocomonoethanolamide.
18. The method according to any one of claims 1 to 17, wherein the composition further comprises a thickening agent.
19. The method according to any one of claims 1 to 18, wherein the composition further comprises a biosurfactant.
20. The method according to claim 19, wherein the biosurfactant is selected from surfactin, lichenysin, fengycin, iturin, bacillomycin, mycosubtilin and mixtures thereof.
21. The method according to claim 20, wherein the biosurfactant is surfactin.
22. The method according to claim 1, wherein the composition comprises dodecylbenzene sulfonic acid or a salt thereof, Bacillus subtilis endospores and surfactin.
23. A composition comprising at least one microorganism of the genus Bacillus in an amount in the range of from 1 x 104 cfu/mL to 1 x 107 cfu/mL and one or more anionic surfactants selected from alkyl sulfates, alkyl ether sulfates, alkyl sulfonates or sulfonic acids, aryl sulfonates or sulfonic acids optionally with alkyl or aryl substituents, alkyl ether sulfonates or sulfonic acids and salts thereof in an amount in the range of from 0.1% to 2% w/w.
24. A composition according to claim 23, wherein the at least one microorganism of the genus Bacillus is present in the vegetative state, as an endospore or a mixture thereof.
25. A composition according to claim 23 or 24, wherein the at least one microorganism is Bacillus subtilis.
26. A composition according to any one of claims 23 to 25, wherein the surfactant is a C8-Ci8 alkylbenzene sulfonate or sulfonic acid or salts thereof.
27. A composition according to claim 26, wherein the surfactant is dodecylbenzene sulfonic acid.
28. A composition according to any one of claims 23 to 27, further comprising a foaming agent.
29. A composition according to claim 28, wherein the foaming agent is an alkylmonoethanolamide or an alkyldiethanolamide non-ionic surfactant.
30. A composition according to claim 29, wherein the foaming agent is cocomonoethanolamide.
31. A composition according to any one of claims 23 to 30, further comprising a thickening agent.
32. A composition according to any one of claims 23 to 31, further comprising a biosurfactant.
33. A composition according to claim 32, wherein the biosurfactant is selected from surfactin, lichenysin, fengycin, iturin, bacillomycin, mycosubtilin and mixtures thereof.
34. A composition according to claim 33, wherein the biosurfactant is surfactin.