WO2001066471A2

WO2001066471A2 - Control of spore forming bacteria in aqueous systems

Info

Publication number: WO2001066471A2
Application number: PCT/US2001/006132
Authority: WO
Inventors: Alexander W. Breen; Charles Taylor; Kelly S. Smith
Original assignee: Hercules Incorporated
Priority date: 2000-03-08
Filing date: 2001-02-23
Publication date: 2001-09-13
Also published as: WO2001066471A3; AU2001241774A1

Abstract

Methods for controlling spores in aqueous systems, especially closed systems. These methods include contacting germinating agent with an aqueous closed system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells, and subjecting the germinated vegetative cells to biocidal treatment. These methods also include contacting germinating agent with the aqueous system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells, subjecting the germinated vegetative cells to biocidal treatment in a hostile environment; and cycling between hostile and non-hostile environments.

Description

CONTROL OF SPORE FORMING BACTERIA

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to germination of spore forming bacteria, and especially relates to the sensitization of spore forming bacteria to agents which can kill or eradicate the spores. The invention further relates to the control of organisms, such as microorganisms, and in particular the inhibition of such organisms in aqueous systems, such as papermaking systems and process water systems, in particular systems that involve circulating streams, and especially recirculating water systems, such as closed aqueous systems including closed paper making systems.

2. Background and Material Information

Spores are known to form from aerobic Bacilli, anaerobic Clostridia, selected sarcinae and a few actinomycetes. Spores resemble certain plant seeds in that they do not carry out any metabolic reactions. In this regard they are especially suited to withstand severe environmental stress and are known to survive prolonged exposures to heat, drying, radiatforT ihdToxic chemicals. In p articular ,^{" ~}spores^~are Bacterial survival structures capable of surviving for thousands of years in an inert state. These properties make spores especially difficult to kill in environments, like living tissue or objects which come in contact with living tissue, or production processes, such as paper production processes, which would be adversely effected by extreme conditions.

Fungi, viruses and vegetative cells of pathogenic bacteria are sterilized within minutes at 70°C; many spores are sterilized at 100°C. However, the spores of some saprophytes can survive boiling for hours. Heat is presently the most commonly used means to insure sterilization of spores. The outer coat of spores is made of a keratin-like protein which comprises as much as 80 wt% of the total protein of the spore. It is this protein coat which is responsible for the resistance of spores to chemical sterilizing agents. A variety of compounds have been used to insure sterilization of spores and have found acceptance depending upon constraints imposed by environment and the required efficacy of action. Acids, alkali, phenols, iodophors, salts, heavy metals, chlorine, hypochlorite, alcohols, glutaraldehyde, formaldehyde, ethylene oxide, organic solvents and surfactants all have been shown to have some action as a sterilant. Of these compounds aldehydes, ethylene oxide, hypochlorites, and Alcide (EPA Reg. No. 456310-03) are commonly used to kill spores.

Expanding upon the above, it is noted that, as spores, bacteria are resistant to stresses, such as dessication, heat and many antimicrobial compounds. However, when environmental conditions become favorable, spores will germinate into vegetative cells and proliferate. At this point, these organisms can cause spoilage or be a human health concern.

The food and paper industries have long taken measures to attempt to control these organisms in their products and production facilities. For example, heat or oxidizing compounds are often used to control spores. More recently, these organisms have received increased attention from the military. In particular, the resilience of these organisms to stress has made them a major concern in the area of biological warfare. It is generally accepted that the most effective biological warfare agent is the spore forming bacterium Bacillus anthracis, the causative agent of anthrax. This organism, as with other spores, can be easily prepared as a dry powder which can be disseminated in a number of ways including crude spraying devices or explosive shells.

The mechanisms of choice for controlling these organisms are harsh. Extreme heat, formaldehyde or bleach are the most frequently used means to control spore forming populations. These methods are often incompatible with keeping equipment in working order, or are too disruptive of manufacturing processes, or pose a safety hazard, to be practical.

In contrast to spores, which are very difficult to kill, vegetative bacteria are sensitive to many gentler forms of microbial control. Moreover, it has long been known that certain compounds can cause dormant bacterial spores to germinate and convert into vegetative cells. In this regard, attention is directed to Wax et al., J. Bacteriol. 95:433- 438 (1968), "Initiation of the Germination of Bacillus subtilus Spores by a Combination of Compounds in Place of L-Alanine"; Keynan et al, J. Bacteriol. 83: 100-105 (1962), "Calcium Dipicolinic Acid-Induced Germination of Bacterial spores"; and Keynan et al.„ Nature, 192: 1211-1212 (1961), "Dipicolonic Acid Content, Heat Activation and the Concept of Dormancy in the Bacterial Endospore."

Additionally, there is a need to avoid the proliferation of microorganisms in papermaking environments. Numerous locations in paper mills are suitable for growth of microorganisms including the growth of anaerobic and facultatively anaerobic bacteria. As discussed in Schwingel et al., "The Implication of the Growth of Anaerobic Bacteria in the Papermaking Process", Biological Sciences Symposium, 1997, pages 139- 143, which is incorporated by reference herein in its entirety, the growth of facultative anaerobes may be of particular importance in these systems due to alternating aerobic and anaerobic conditions found in many papermaking systems. Bacterial growth under anaerobic conditions causes specific problems not associate with bacterial growth under aerobic conditions. Anaerobic bacteria produce H₂, H₂S and volatile fatty acids (NFA) as part of their metabolism. Along with the health and safety issues related to toxic and explosive gas production, the volatile fatty acids generated cause significant odor problems in both the mill and the products being produced. While anaerobic bacterial growth in paper mills is not new, the problems associated with growth of anaerobes has become more prevalent in recent years. The major contributor to anaerobic growth appears to be the closure of mill water systems, such as closed systems (zero-effluent systems). Accordingly, there is a need to control microorganisms, and especially anaerobic microorganisms in papermaking environments, without the requirements of utilizing large amounts of biocides with their prohibitive costs and effects. While the use of certain compounds is known to cause spores to germinate and thereby be converted into vegetative cells, there is still a need for improvement. For example, there is a need to be able to control spores even under milder conditions, such as not to interfere with production processes and/or without creating harmful or toxic environments. SUMMARY OF THE INVENTION

The present invention relates to methods for controlling spores in an aqueous closed system, comprising contacting germinating agent with the aqueous closed system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells; and subjecting the germinated vegetative cells to biocidal treatment.

The present invention also relates to methods for controlling spores in an aqueous system, comprising contacting germinating agent with the aqueous system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells; subjecting the germinated vegetative cells to biocidal treatment in a hostile environment; and cycling between hostile and non-hostile environments.

The germinating agent can comprise at least one of L-alanine, L-arginine, L- phenylalanine, glutamic acid, inosine, sucrose, lact s^, ^d nosine dL-valine, dL-cysteine, tyrosine, lactate, malate, guanine, methione, priopionate, formate, CO₂ , xanthosine, mannose, adenine, fumarate, oxaloacetate, quaternary ammonium compounds, dipicolinic acid, phosphate, and cobalt, preferably L-alanine.

The closed aqueous system preferably comprises a pulping or papermaking system. The contacting can comprise contacting the germinating agent with the closed aqueous system for at least about 10 minutes, more preferably at least about 20 minutes, and even more preferably at least about 30 minutes.

The contacting can comprise contacting the germinating agent with the closed aqueous system to obtain at least about 50% germination of the total number of spore formers that are capable of germinating into vegetative cells, more preferably at least about 60% germination of the total number of spore formers that are capable of germinating into vegetative cells, even more preferably at least about 70% germination of the total number of spore formers that are capable of germinating into vegetative cells, even more preferably at least about 80% geπnination of the total number of spore formers that are capable of germinating into vegetative cells, and even more preferably at least about 90% germination of the total number of spore formers that are capable of germinating into vegetative cells.

Prior to the contacting under the non-hostile conditions, the closed aqueous system can be contacted with the germinating agent under hostile conditions. The spores are preferably activated prior to the contacting with the germinating agent.

The closed aqueous system can cycled between hostile and non-hostile environments. Thus, for example, the biocidal treatment can comprise heat treatment of the aqueous system, and cycling between hostile and non-hostile environments can comprise heat cycling between lower temperature non-hostile conditions and higher temperature hostile conditions. Moreover, the biocidal treatment can comprise contacting the aqueous system with chemical biocidal agent, and cycling between hostile and non- hostile environments can comprise chemical biocidal agent cycling. The chemical biocidal agent cycling can comprise consumption of the chemical biocidal agent whereby concentration of the chemical biocidal is reduced to obtain a non-hostile environment, and additional chemical biocidal agent is added to cycle to a hostile environment.

The biocidal treatment can comprise heat treatment or contacting the closed aqueous system with chemical biocidal agent. The chemical biocidal agent can preferably comprise at least one of hydrogen peroxide, chlorine, hypochlorite, e.g., sodium hypochlorite, dithiol and glutaraldehyde.

The spores can comprise spores capable of forming bacteria that form volatile fatty acids. For example, the spores can comprise spores of at least one of the genera Bacillus Clostridia and Paenbacillus.

The geπriinating agent can be added with additives to the pulping or papermaking system. For example, the additive can comprise at least one of coating and filler material, such as starches or proteins.

The contacting can comprise adding the germinating agent to a consistency chest, a headbox, fiber stock storage tank and/or to furnish. DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise stated, all percentages, parts, ratios, etc., are by weight. Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.

Further, when an amount, concentration, or other value or parameter, is given as a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of an upper preferred value and a lower preferred value, regardless whether ranges are separately disclosed. The present invention is directed to germinating agents, such as L-alanine, which are utilizable to sensitize spores to lethal agents, such as heat and/or biocides. Moreover, the present invention is directed to germinating agents which sensitize spores so that the spores are formed into vegetative cells which are susceptible to lethal agents at less harsh conditions that presently available. In such manner, by being able to utilize lethal agents that have reduced harshness as compared to what is conventionally used, it is possible to reduce the amount of bacteria or kill the bacteria while minimizing or avoiding adverse effects on processes, such as manufacturing processes for producing paper.

Various germinating agents can be utilized to effect the formation of vegetative cells from the spores, such as germinating agents disclosed by U.S. Patent No. 3,687,815 to Scharpf, Jr. and JP-A-61-015672, which disclosures are incorporated by reference in their entireties. Preferred germinating agents include, but are not limited to, L-alanine, L-arginine, L-phenylalanine, glutamic acid, inosine, sucrose, lactose, adenosine dL- valine, dL-cysteine, tyrosine, lactate, malate, guanine, methione, priopionate, formate, CO₂ , xanthosine, mannose, adenine, fumarate, oxaloacetate, quaternary ammonium compounds, dipicolinic acid, phosphate, and cobalt. Most preferably, the germinating agent comprises L-alanine, especially because L-alanine has a broad range. It causes permeation in virtually every spore former tested.

Moreover, it is preferable to utilize certain germinating agents in certain environments. For example, when activating spores in food products and/or materials that may come into contact with food products, it is preferable to utilize edible and/or non-toxic germinating agents, such as L-alanine.

In order to effect germination of spores, it is necessary that conditions be favorable for germination of the spores into vegetative bacteria. In particular, it is noted that when spores are in a hostile environment, the spores will not germinate. Therefore, in order to effect germination of spores, the spore containing environment should be adjusted to render the environment non-hostile. In other words, the germinating agent can be added to the spores in a non-hostile environment to cause the spores to germinate. Alternatively, the germinating agent can be added to a hostile environment, which environment is subsequently rendered non-hostile, whereby the spores will germinate upon the environment becoming non-hostile. In such an instance where the environment is initially hostile, a bacteriostatic condition will be maintained, because the spores cannot germinate to proliferate. However, in such an instance, if the environment is not harsh enough, the spore will not be killed, but will remain active until such time when, and if, the environment is rendered non-hostile.

Thus, when the terminology "hostile environment" is utilized herein, it refers to an environment wherein spores will not germinate, or at most will have minimal germination. In contrast, when the terminology "non-hostile environment" is utilized herein, it refers to an environment wherein spores will germinate. It is noted that non-hostile and hostile environments will be varied depending upon the spore formed involved. In general, an example of a non-hostile environment included, but is not limited to, environments having a temperature of about 10°C to 60°C, or higher, aqueous metachromatic dye compositions a pH of about 5 to 9, more preferably about 6 to 8, and even more preferably about 7 to 8. Expanding upon the above, it is noted that depending upon the spore formed, higher and/or lower temperatures and pH's can provide a non-hostile environment. For example, in the paper industry, non-hostile environments usually include areas of process water and storage pulp stocks.

The germinating agent can be added to an environment in a number of different manners. For example, the germinating agent can be solid, such as powder, and/or could be in a solution or dispersion, such as an aqueous solution or dispersion. The germinating agent could be added to a solution, such as an aqueous solution containing the spores, or could be sprayed onto surface to be treated.

Without limiting the environments to which a germinating agent can be utilized, it is noted that in a papermaking process the germinating agent can be added, such as in powder or solution form to process water, an/or locations where additives, such as coatings and fillers, including proteins, starches, clays and/or latex, are added to the process. Without wishing to be bound by theory, addition of the germinating agents to areas where additives are utilized would be beneficial, because such additives can themselves be a source of contamination.

In particular, and without wishing to limit the invention, the germinating agent can be added to any papermaking system at any point in the system, such as in any manner where additives are employed in papermaking processes, and in any manner that additives used in the paper industry are employed. Non- limiting examples of suitable aspects for addition include:

Refined or unrefined furnish stock in stock tanks.

Pulp refining conducted in a refiner.

Refined pulp stored, ready for use in stock chests.

Paper making furnish drawn from stock chests. The flow box, the point at which the paper making furnish flows at a controlled rate onto the fourdrinier wire.

Or any combination of the foregoing, upstream of any of the foregoing l cc-lous, or any point inbetween such locations, or any other suitable location, or combination of locations, concurrently or at different times. Expanding upon the above, the germinating agent can be added in the process, such as in the papermaking process, at any location where biocides are routinely added and/or at other locations. For example, the germinating agent can be added at the feed points in a paper mill which include the pulper, broke, headbox, consistency chest, clarifiers, fillers, additives, fiber stocks. Moreover, germinating addition to non-hostile environments in the paper mill, such as fiber stock storage tanks and furnish, allows germination to be initiated. This renders the spore forming bacteria sensitive to heat killing in the dryer section of the paper formation process, thereby reducing bacterial load in food grade packaging material.

As noted above, anaerobic spore forming bacteria, including those of the genus Clostridium and B. subtilus, can be responsible for the production of volatile fatty acids (VFA's) in paper mills. These products, such as acetate, butyrate and propionate cause a rancid odor in both the mill and the paper produced. Paper contaminated with VFA's cannot be sold. This problem is particularly bad in mills that recycle process water. Recycling of water in closed systems (also known as zero-effluent systems or complete recycle systems) is an industry trend, hence abatement of odor problems is of paramount importance. Continuous treatment of mills experiencing VFA problems has not been economically or environmentally feasible prior to the present invention, both at the mill and in the resulting product. However, the present invention enables the addition of germinating agent, preferably L-alanine, to sites in ^•- pul ing and papermaking systems, such as stock storage chests, the headbox and/or consistency chest to help control odor producing bacteria.

Still further, when added to an aqueous system, the germinating agent and biocide utilized therewith can become a component of resultant product formed in the process.

Accordingly, one of the advantages of the present invention is that lower amounts of biocides can be utilized due to the use of the germinating agent to render the spores more susceptible to biocidal activity. For example, in a papermaking composition, such as a papermaking pulp or slurry, lower amounts of biocide can be utilized therewith, whereby products formed from the papermaking composition will contain lower levels of biocide therein. Thus, the present invention provides cellulosic products, such as paper and paper products, which include the germinating agent and lower levels of biocide than ordinarily may appear in the paper and paper products. Thus, the presence of non-toxic germinating agents and lower amounts of biocide in products, including cellulosic products such as paper and paper products, provides advantages of effectively controlling organisms in the aqueous systems associated with the production of such products, while lowering safety, toxicity and/or related concerns, and/or adverse physiological reactions. This is of particular significance with respect to cellulosic products comprising members selected from stationery paper, paper towel and tissue paper.

For sterilization of surfaces, the germinating agent, such as in a solution or a dispersion, can be sprayed onto the surface, such as a metallic or plastic surface. Thus, for example, heating and air-conditioning ducts can be sprayed with germinating agent After the germinating agent is added to the environment, the spores should be permitted to remain in contact with the germinating agent for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells prior to biocidal treatment. In this regard, it is noted that not all of the spore formers form vegetative cells when the spore formers are germinated. The contact conditions preferably provide at least about 50% germination of the total number of spore formers that are capable of germinating into vegetative cells, more preferably at least about 60%), more preferably at least about 70%>, even more preferably at.least about 80%, even more preferably at least about 90%>, and even more preferably substantially all or all of the spore formers that are capable of germinating into vegetative cells are germinated.

With the above in mind, the germinating agent is preferably maintained in contact with the spores for at least about 10 minutes, more preferably at least about 20 minutes, and even more preferably at least about 30 minutes prior to biocidal treatment. The contact time between the germinating agent and the spores need not be limited, and can be as long as desired. However, when contact is maintained under non-hostile conditions, a bacterial environment may proliferate, and therefore biocidal treatment should preferably be initiated at less than about 20 hours, more preferably less than about 30 minutes after contact of the germinating agent and spores under non-hostile conditions.

The contacting of the germinating agent and the spores need not be made under non-hostile conditions, but can be made under hostile conditions, whereby the spores will not germinate and form vegetative cells. In such an instance, it can be stated that a bacteriostatic condition is present, because the bacteria cannot proliferate. Such bacteriostatic condition can be maintained until it is desired to activate the spores for any purpose, and especially for forming vegetative cells that can be killed by biocidal treatment. For example, a germinating agent can be incorporated into a paper product, which paper product has a hostile environment. In such an instance, the spores cannot germinate into vegetative cells until the paper product is treated to remove the hostile environment. Thus, for example, the paper product can have a hostile environment due to heating and/or dessication, and the formation of a non-hostile environment can be achieved by cooling or adding water to the paper product. Similarly, a solution containing germinating agent and spores can be maintained at a hostile environment by adding biocide, which can be changed to a non-hostile environment by dilution the solution, removing the biocide and/or reducing the amount of biocide in he solution.

The germinating agent is added to the spores at a sufficient concentration to permit the spores to form vegetative cells within the above-disclosed contact period of the spores with the germinating agent under non-hostile conditions. It is noted that the amount of germinating agent will vary with the spore former. As an example, preferably, the germinating agent is at a concentration of about 0.5 to 10 mM, more preferably about 1 to 10 mM, and even more preferably about 2.5 to 10 mM. In the case of L-alanine, preferred concentrations are about 0.1 to 10 mM, more preferably about 0.3 to 5 mM, and can range from about 0.5 to 3 mM, about 0.7 mM to 2.5 mM, and about 1 to 2 mM, with one value being about 1. With regard to the above, it is noted that there is a balance between the amount of germinating agent used and expense. Usually, it is desirable to use higher amounts of germinating agent; however, the amount of germinating agent used is balanced against higher expense. Thus, amounts of, for example, 1 mM, 2.5 mM or 5 mM can be utilized depending of the desired balance. Preferably, the spores are activated so that the formation of vegetative cells is more efficient. The activation of the spores can be performed prior to contact with the germinating agent, at the same time as contact with the germinating agent, or subsequent to contact with the germinating agent, or any combination thereof. Preferably, the activation of the spores is initiated prior to contacting of the spores with germinating agent. Activating conditions will vary with the spore former. In general, activating can be achieved, in the case of 5. cereus and B. subtilus, by raising the temperature of the spores to one or more temperatures in the range of about 50°C to 70°C, more preferably about 55°C to 65°C, and even more preferably about 63°C to 65°C, for about 20 minutes to 180 minutes, more preferably about 30 minutes to 90 minutes, and even more preferably about 35 minutes to 45 minutes, with preferred combinations of time and temperature being about 65°C for 45 minutes for B. cereus and B. subtilus.

Moreover, the activating can be achieved by adding materials to the spores that enable the spores to form vegetative bacteria more efficiently. For example, the materials can comprise, but are not limited to, activating agents including elemental calcium; dipicolinic acid and/or inosine.

The biocidal treatment can be any treatment that will reduce or kill the bacteria. For example, high temperatures, such as temperatures of at least 50°C, and preferably within the range of about 80°C to 200°C, more preferably about 100°C to 200°C, and even more preferably about 150°C to 200°C can be utilized. Moreover, chemical agents, including but not limited to conventional chemical agents, can be utilized. For example, the chemical agents can comprise one or more of acids, such as benzoic acid; alkalis, such as sodium hydroxide; phenols, such as dichlorophenol; iodophors, such as betadine; salts, such as sodium chloride; heavy metals, such as copper; chlorine; hypochlorite, such as sodium hypochlorite; alcohols, such as methanol, ethanol, isopropanol; aldehydes, such as glutaraldehyde and formaldehyde; alkylating agents, such as ethylene oxide; organic solvents, such as chloroform; hydrogen peroxide; quaternary ammonium compounds, such as N-alkyl dimethylbenzylammonium chloride and alkyl trimethylammonium salts; and surfactants, such as alkyl sulfosuccinates, such as dioctyl and dinonyl sulfosuccinates.

Preferably, the biocidal agent comprises an agent that is non-hazardous and/or not detrimental to the environment that is being treated, and/or can be utilized in concentrations less than would be expected to be utilized. For example, in the case of utilizing temperature as a biocidal agent, the present invention enables the use of lower temperatures. Still further, in the case of use of chemicals, such as glutaraldehyde, as a biocidal agent, the present invention enables the use of lower concentrations of glutaraldehyde. For example, phenolics and quaternary ammonium compounds are common biocides that control bacterial populations at typical concentrations from about 5 mg/1 to 30 mg/1. These concentrations do not affect the viability of spores, and would need to be applied at such high concentrations that they would be environmentally or economically unacceptable.

Expanding upon the biocidal treatments that can be utilized with the germinating agents of the present invention, it is noted that the biocidal treatments can create a hostile environment which can remain hostile for an extended period of time. However, the biocidal treatment is preferably a treatment in which cycling between a non-hostile and hostile environment can be achieved. Such cycling in biocidal treatment is especially preferred in closed systems wherein the aqueous environment is recycled. In this manner, a non-hostile environment can be established so that the spores will germinate and form vegetative cells in the presence of the germinating agent. Once the spores are germinated, the environment can be modified to form an environment in which the more easily killed vegetative cells can be subject to a less harsh treatment than would be needed to kill the spores. The biocidal treatment can then be modified or permitted to change so that the environment will become non-hostile in order that any further spores in the aqueous system can be converted to vegetative cells for biocidal treatment. Cyclic biocidal treatments include, but are not limited, to heat cycling, and the use of rapidly consumed biocidal agents and/or biocidal agents that can be removed from the aqueous system. For example, with regard to heat cycling, the temperature cf the aqueous system can be at a temperature or modified to be at a temperature at which the spores can form vegetative cells in a non-hostile environment, such as temperatures, depending on the spore former, of about 10°C to 55°C, more preferably about 20°C to 45°C, and even more preferably about 25°C to 35°C. The temperature can then be raised in any manner to a temperature at which the vegetative cells can be killed, which temperature would be expected to be lower than the temperature required to kill the spores. Thus, depending upon the vegetative cells, the vegetative cells can be killed by raising the temperature to preferably at least about 65°C, with ranges of temperatures including 65°C to 75°C, about 75°C to 80°C, about 80°C to 85°C, and even higher. In a papermaking system, the raised temperature for killing the vegetative cells can comprise the temperature in the dryer section, such as a temperature of about 95°C to 100°C. With regard to the use of rapidly consumed biocidal agents, it is noted that chemical biocidal agents, such as hydrogen peroxide and chlorine are rapidly consumed, and can vary between a hostile and a non-hostile environment in a matter of minutes. Accordingly, when using these chemical agents, the environment can be cycled between a non-hostile environment and a hostile environment by adding the chemical agent to form the hostile environment, and then removing the chemical agent or permitting the chemical agent to be consumed to return to a non-hostile environment.

Practice of the invention will become further apparent from the following non-limiting examples.

EXAMPLES

The invention is illustrated in the following non-limiting examples, which are provided for the purpose of representation, and are not to be construed as limiting the scope of the invention. All parts and percentages in the examples are by weight unless indicated otherwise.

Preparation of Spores

The following procedure is utilized to prepare spore stocks for the examples. A colony of spore forming bacterium, i.e., B. cereus ATCC 14579, or B. subtilus is taken from an agar plate comprised of trypticase soy agar (TSA) and grown in trypticase soy broth. The culture is grown overnight at 37°C, and then placed at 80°C for 15 minutes to induce sporulation. Spores are harvested in a centrifuge and resuspended in sterile water at 4°C for at least 48 hours before use in germination experiments. In each example, prior to L- alanine exposure the spore forming bacteria are heat activated by incubation of the culture at 65°C for 20 minutes to obtain optimal germination efficacy Examples 1-10

Spores of 5. cereus ATCC 14579 are added to obtain a concentration of about 10⁴ to 10⁵ spores/ml to sterile water and paper mill process water in the from of headbox material from the International Paper Mill at Augusta, GA. The sterile water and paper mill process water containing activated spores were divided into separate solutions to which no L-alanine germinating agent or varying concentrations (0, 0.5, 1.0, 2.5 and 5.0 mM) of L-alanine were added. After 2 hours, the L-alanine dosed cultures and undosed cultures were heated to 80°C for 20 minutes, and then the cultures were plated on tryptic soy medium (obtained from Difco Laboratories, Detroit, Michigan), the cultures were placed at 37°C overnight, and colonies appearing on the medium were counted. The results are show in Table 1, and it is noted that results were only measured for Examples 2, 3, 6 and 8.

Examples 11-14 A suspension of spores of B. cereus was centrifuged and resuspended to obtain a concentration of about 10⁴ to 10⁵ spores/ml in headbox material from the International Paper Mill at Augusta, GA, which contains high levels of fiber and an indigenous bacterial population of 10⁴ bacteria per ml, in order to test the efficacy of L-alanine induced germination in paper mill headbox material. Following heat activation at 65°C for 20 minutes, L-alanine was added to half the suspensions to a concentration of 5 mM for 30 minutes. Exposure to L-alanine was followed by exposure to RX- 1,000, a biocide comprising as active ingredients 5.3 wt%> sulfone, and a 16 wt% quaternary ammonium compound (N-alkyl dimethylbenzylammonium chloride) obtained from Hercules Incorporated, Wilmington, Delaware. The results illustrated in Table 2 show that L- alanine can induce sensitivity in the presence of fiber and an indigenous bacterial population as compared to suspensions not containing L-alanine.

Examples 15-20

Suspensions of B. cereus spores were added to sterile water to obtain a concentration of about 10⁴ to 10⁵ spores/ml. L-alanine was added to the half of the activated suspensions to a concentration of 1 mM for 30 minutes. Each of the suspensions was then exposed to heat (80°C), dithiol obtained from Hercules Incorporated, Wilmington, Delaware, or RX- 1,000 obtained from Hercules Incorporated, Wilmington, Delaware. The results in Table 3 show that L-alanine can greatly increase the sensitivity to biocidal agents as compared to compositions containing the same spores and initial concentration thereof in the absence of L-alanine.

Examples 21-26

As discussed previously, anaerobic spore forming bacteria of the genus Clostridium can be responsible for the production of volatile fatty acids (VFA's) in paper mills. These products, such as acetate, butyrate and propionate cause a rancid odor in both the mill and the paper produced. Paper contaminated with VFA's cannot be sol This problem is particularly bad in mills that recycle process water. Recycling of water is an industry trend, hence abatement of odor problems is of paramount importance. Continuous treatment of a mill experiencing VTA problems has not been economically or environmentally feasible prior to the present invention.

The present examples were performed to ascertain whether a germinating agent, such as L-alanine, can be used in a closed (zero effluent) paper mill environment to germinate spore former in that environment. In the examples, B. subtilus is utilized as the spore former. Therefore, the instant examples are directed to the use of L-alanine in mill process water from a recycle mill expeπencing VFA problems from the bacteπa B subtilus. In particular, concentrations of L-alanme of 2 5 mM and 1 mM were sufficient to sensitize the B subtilus when subjected to heat of 80°C for 30 minutes

These examples include the spore suspension of B subtilus which was centrifuged and then resuspended in stenle water samples and samples of process water from a recycle mill (Green Bay Packaging) Either no L-alanme, or amounts of 2 5 or 1 mM of L-alamne for an exposure time of 1 hour were added The results illustrated in Table 4 show that L-alanme reduces the load of spore forming bacteria in a paper mill experiencing VFA problems. It should therefore be possible to apply L-alanine to sites such as stock storage chests, the headbox or consistency chest to help control odor producing bacteπa

Examples 27-32 Suspensions of B cereus were added to stenle water to obtain a concentration of about 10⁴ to 10⁵ spores/ml. L-alamne was added to the half the activated suspensions to a concentration of 1 mM for 1 hour. To each of the suspensions was added varying concentrations of glutaraldehyde (obtained from Umon Carbide) for 15 minutes, with no glutaraldehyde being added to some sample. The results in Table 5 show that L-alamne can greatly increase the sensitivity to glutaraldehyde, which is often employed as a biocide to control spore forming bacteria in paper mills producing food grade packaging material. Thus, treatment of process water or stock chests with L-alanine prior to the addition of glutaraldehyde will be expected to decrease the load of spore forming bacteria in a paper manufacturing system, such as by adding the L-alanine at feed points as for glutaraldehyde application, or anywhere in the system in advance of application of the glutaraldehyde.

Examples 33-40

Suspensions of B. cereus were added to sterile water to obtain a concentration of about 10⁴ to 10⁵ spores/ml. L-alanine was added at a concentration of 1 mM. Replicate cultures were not exposed to L-alanine. RX- 1,000 obtained from Hercules Incorporated. Wilmington, Delaware, was added at a concentration of 10 ppm to treated and untreated spore cultures at: less than 1 minute, 5 minutes, 30 minutes and 60 minutes of L-alanine exposure time. The results are illustrated in Table 6 with the numbers being determined by plate counts on tryptic soy medium. In Table 6, %Kill is equal to the number of bacteria growing in the absence of L- alanine minus the number of bacteria growing in the presence of L-alanine divided by the number of bacteria growing in the absence of L-alanine x 100.

Examples 41-49

Bacillus cereus spores treated in the manner described earlier were added to obtain a concentration of about 10⁴ to 10⁵ spores/ml to paper mill white water obtained from a paper mill in Finland. The heat sensitivity of the spores in Whitewater was tested by dosing the Whitewater spiked with the B. cereus spores with L-alanine concentrations of 1 or 2.5 mM for 45 minutes at 37°C, and then placed at 80°C for 15 minutes. A control sample that did not receive L-alanine was also placed at 80°C. After heat exposure the samples were diluted and plated onto tryptic soy agar. After 24 hours colonies appearing on the plates were counted. The results are illustrated in Table 7.

The effect of L-alanine on spores in Whitewater exposed to H₂O₂ was also tested. Three concentrations of H₂O₂, 0.02 wt%, 0.01 wt% and 0.0015 wt%, were tested in samples that had been exposed to 2.5 mM L-alanine for 30 minutes. Samples were exposed to H,O₂ for 20 minutes. Following exposure the samples were diluted and plated onto tryptic soy agar. After 24 hours, colonies appearing on the plates were counted. The results are illustrated in Table 8.

These examples demonstrate the efficacy of L-alanine in the Finnish Whitewater system showing that gemination can be induced in complex systems with an indigenous bacteria population present. Two factors needed for germination were met in that the L- alanine is bioavailable to the target population, and the environment is non-hostile (or does not prevent germination). The heat experiment demonstrates that L-alanine induced heat sensitivity would enable spore load in finished paper to be reduced by causing spore formers to be in a vegetative state when they reach the temperature extremes of the dryer section so that vegetative cells will not survive, and the bacterial levels in the paper will be reduced.

Moreover, oxidizers, such as H₂O₂, which are frequently used to control spore forming populations, are short lived in the system and may react with many non-target components present in process water. The present data shows that lower concentrations of H₂O₂ will reduce bacterial spore levels if germination is induced. Levels of H₂O₂ of 0.02 wt% and 0.01 wt%> can accomplish similar reductions of spore forming bacteria with or without L-alanine exposure. However, when lower levels (0.0015 wt%) of H₂O₂ are present L-alanine exposure results in a spore former reduction. In particular, it is noted that H₂O₂ is an effective biocide by itself, and the present invention enables the use of low concentrations of H,O₂ while obtaining effective biocidal results.

Examples 50-58 - Effect of Hop Acid On Spores.

Tests of the effect of L-alanine and hop acid were conducted as follows. Suspensions of B. cereus spores were added to sterile water to obtain a spore stock having a concentration of about 10⁴ to 10⁵ spores/ml. The spore stock was then divided into 1.0 ml aliquots for testing. L-alanine was supplied at concentrations of 1 or 2.5 mM. After L-alanine addition, spores were placed at 37°C for one hour. After L-alanine treatment, hop acid (HYDROHOPS from Watertown Hops, Wisconsion) was added to the spores. After a 20 minute exposure the spore samples were taken through a serial dilution and plated on nutrient agar medium and placed at 37°C overnight. After the incubation period, colonies were counted. The effect of L-alanine was assessed by comparing the plate counts to control spore preparations that were not exposed to L-alanine. The results are illustrated in Table 9. In particular, Examples 52 and 53 were subjected to Hop Acid treatment and Example 58 was exposed to heat treatment. It is noted that control Examples 50 and 51, which were subjected to biocidal treatment at 80°C for 15 minutes, show that L-alanine did induce germination. Another control sample (Example 58) similar to samples Examples 50 and 51 were subjected to biocidal treatment at 80°C for 15 minutes to show that these examples did, in fact, germinate, and that the germinated cells could be killed. This may be due to the fact that hop acid is a more "gentle" or slower acting killing agent than heat or other of the compounds tested with L-alanine. e.g., dithiol, RX- 1,000, glutaraldehyde.

The examples describe various embodiments of the invention. Other embodiments will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is understood that modifications and variations may be practiced without departing from the spirit and scope of the novel concepts of this invention. It is further understood that the invention is not confined to the particular formulations and examples herein illustrated, but it embraces such modified forms thereof as come within the scope of the following claims.

Claims

What is claimed is:

1. A method for controlling spores in an aqueous closed system, comprising: contacting germinating agent with the aqueous closed system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells; and subjecting the germinated vegetative cells to biocidal treatment.

2. The method according to claim 1, wherein the germinating agent comprises at least one of L-alanine, L-arginine, L-phenylalanine, glutamic acid, inosine, sucrose, lactose, adenosine dL-valine, dL-cysteine, tyrosine, lactate, malate, guanine, methione, priopionate, formate, CO₂ , xanthosine, mannose, adenine, fumarate, oxaloacetate, quaternary ammonium compounds, dipicolinic acid, phosphate, and cobalt.

3. The method according to claim 2, wherein the germinating agent comprises L-alanine.

4. The method according to claim 1, wherein the closed aqueous system comprises a pulping or papermaking system.

5. The method according to claim 4, wherein the germinating agent comprises L-alanine.

6. The method according to claim 4, wherein the contacting comprises contacting the germinating agent with the closed aqueous system for at least about 10 minutes.

7. The method according to claim 6, wherein the contacting comprises contacting the germinating agent with the closed aqueous system for at least about 20 minutes.

8. The method according to claim 7, wherein the contacting comprises contacting the germinating agent with the closed aqueous system for at least about 30 minutes.

9. The method according to claim 4, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 50% germination of the total number of spore formers that are capable of germinating into vegetative cells.

10. The method according to claim 9, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 60% germination of the total number of spore formers that are capable of germinating into vegetative cells.

11. The method according to claim 10, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 70% germination of the total number of spore formers that are capable of germinating into vegetative cells.

12. The method according to claim 11, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 80% germination of the total number of spore formers that are capable of germinating into vegetative cells.

13. The method according to claim 12, wherein contacting comprises contacting the germinating agent with the closeu . .ueous system to obtain at least about 90% germination of the total number of spore formers that are capable of germinating into vegetative cells.

14. The method according to claim 4, wherein, prior to the contacting under the non-hostile conditions, the closed aqueous system is contacted with the germinating agent under hostile conditions.

15. The method according to claim 4, wherein the spores are activated prior to the contacting with the germinating agent.

16. The method according to claim 4, wherein the biocidal treatment comprises heat treatment.

17. The method according to claim 4, wherein the biocidal treatment comprises contacting the closed aqueous system with chemical biocidal agent.

18. The method according to claim 4, wherein the closed aqueous system is cycled between hostile and non-hostile environments.

19. The method according to claim 18, wherein the biocidal treatment comprises heat treatment of the aqueous system, and cycling between hostile and non-hostile environments comprises heat cycling between lower temperature non-hostile conditions and higher temperature hostile conditions.

20. The method according to claim 18, wherein the biocidal treatment comprise contacting the aqueous system with chemical biocidal agent, and cycling between hostile and non-hostile environments comprises chemical biocidal agent cycling.

21. The method according to claim 20, wherein the chemical biocidal agent cycling comprises consumption of the chemical biocidal agent whereby concentration of the chemical biocidal is reduced to obtain a non-hostile environment, and additional chemical biocidal agent is added to cycle to a hostile environment.

22. The method according to claim 20, wherein the chemical biocidal agent comprises at least one of hydrogen peroxide, chlorine, hypochlorite, dithiol and glutaraldehyde.

23. The method according to claim 22, wherein the chemical biocidal agent comprises hydrogen peroxide.

24. The method according to claim 22, wherein the chemical biocidal agent comprises chlorine.

25. The method according to claim 22, wherein the chemical biocidal agent comprises sodium hypochlorite.

26. The method according to claim 4, wherein the spores comprise spores capable of forming bacteria that form volatile fatty acids.

27. The method according to claim 4, wherein the spores comprise spores of at least one the genera Bacillus Clostridia and Paenbacillus.

28. The method according to claim 4, wherein the germinating agent is added with additives to the pulping or papermaking system.

29. The method according to claim 28, wherein the additive comprises at least one of coating and filler material.

30. The method according to claim 28, wherein the additive comprises starch.

31. The method according to claim 28, wherein the additive comprises protein.

32. The method according to claim 4, wherein the contacting comprises adding the germinating agent to a consistency chest.

33. The method according to claim 4, wherein the contacting comprises adding the germinating agent to a headbox.

34. The method according to claim 4, wherein the contacting comprises adding the germinating agent to a fiber stock storage tank.

35. The method according to claim 4, wherein the contacting comprises adding the germinating agent to furnish.

36. A method for controlling spores in an aqueous system, comprising: contacting germinating agent with the aqueous system for a sufficient period of time and under non-hostile conditions so that spores capable of germinating can germinate into vegetative cells; subjecting the germinated vegetative cells to biocidal treatment in a hostile environment; and cycling between hostile and non-hostile environments.

37. The method according to claim 36, wherein the germinating agent comprises at least one of L-alanine, L-arginine, L-phenylalanine, glutamic acid, inosine, sucrose, lactose, adenosine dL-valine, dL-cysteine, tyrosine, lactate, malate, guanine, methione, priopionate, formate, CO₂ , xanthosine, mannose, adenine, fumarate, oxaloacetate, quaternary ammonium compounds, dipicolinic acid, phosphate, and cobalt

38. The method according to claim 37, wherein the germinating agent comprises L-alanine.

39. The method according to claim 36, wherein the closed aqueous system comprises a pulping or papermaking system.

40. The method according to claim 39, wherein the germinating agent comprises L-alanine.

41. The method according to claim 39, wherein the contacting comprises contacting the germinating agent with the closed aqueous system for at least 10 minutes.

42. The method according to claim 39, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 50%> germination of the total number of spore formers that are capable of germinating into vegetative cells.

43. The method according to claim 42, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 60%> germination of the total number of spore formers that are capable of germinating into vegetative cells.

44. The method according to claim 43, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 70% germination of the total number of spore formers that are capable of germinating into vegetative cells.

45. The method according to claim 44, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 80% germination of the total number of spore formers that are capable of germinating into vegetative cells.

46. The method according to claim 45, wherein contacting comprises contacting the germinating agent with the closed aqueous system to obtain at least about 90% germination of the total number of spore formers that are capable of germinating into vegetative cells.

47. The method according to claim 39, wherein, prior to the contacting under the non-hostile conditions, the closed aqueous system is contacted with the germinating agent under hostile conditions.

48. The method according to claim 39, wherein the spores are activated prior to the contacting with the geπninating agent.

49. The method according to claim 39, wherein the biocidal treatment comprises heat treatment of the aqueous system, and cycling between hostile and non-hostile environments comprises heat cycling between lower temperature non-hostile conditions and higher temperature hostile conditions.

50. The method according to claim 39, wherein the biocidal treatment comprise contacting the aqueous system with chemical biocidal agent, and cycling between hostile and non-hostile environments comprises chemical biocidal agent cycling.

51. The method according to claim 50, wherein the chemical biocidal agent comprises at least one of hydrogen peroxide, chlorine, hypochlorite, dithiol and glutaraldehyde.

52. The method according to claim 51, wherein the chemical biocidal agent comprises hydrogen peroxide.

53. The method according to claim 51, wherein the chemical biocidal agent comprises chlorine.

54. The method according to claim 51, wherein the chemical biocidal agent comprises sodium hypochlorite.

55. The method according to claim 39, wherein the spores comprise spores capable of forming bacteria that form volatile fatty acids.

56. The method according to claim 39, wherein the germinating agent is added with additives to the pulping or papermaking system.