GB2517441A - Novel Biofilm reactor, and novel methods for assessing biofilms - Google Patents

Abstract

A biofilm reactor is disclosed which is particularly useful in assessing the incidence and the growth of biofilms above, at, and below an air/liquid interface region. The biofilm reactor comprises at least one substrate 10 (for growing the biofilm), a vessel 52 having an internal cavity of sufficient size and dimensions to retain a quantity of liquid therein, such that at least part of the substrate may remain immersed within the liquid and any remaining parts of the substrate may be retained in a headspace above the top surface of the liquid but within the internal cavity of the vessel, and a suspending means for suspending the at least one substrate. The reactor preferably comprises a lid 71. In a further aspect, the present invention relates to a novel method for assessing biofilms present upon a substrate at, and below an air/liquid interface region.

Description

NOVEL BIOFILM REACTOR, AND

NOVEL METHODS FOR ASSESSING BIOFILMS

The present invention relates to a biofilm reactor, believed to be of a novel design, which is particularly useffil in assessing the incidence and the growth of biofilms above, at, and below an air/liquid interface region. In a further aspect, the present invention relates to a novel method for assessing biofilms present upon a substrate, in the region of the substrate which is above, at, and below an air/liquid interface region.

Biofilms may be defined as being an assemblage of microbial cells which are associated with a surface, in which microbial cells are enclosed in a matrix. In a further definition, biofllms are defined to be conglomerates of microbial organisms embedded in highly hydrated matricies of exopolymers, frequently primarily polysaccharides, and/or other macromolecules. Further materials, depending upon the nature of the biofilm, may also be present including but not limited to: mineral crystals, corrosion particles, clays, silt particles. Such a biofllm adheres to surfaces that are regularly, or intermittently in contact with the liquid, such as water, and include colonies of bacteria and or other microorganisms, some of which may express a matrix which provides a further barrier between the biofilm and its ambient environment. Biofllms thus typically form amacrocellular mass of microorganisms, which are generally strongly adhered to substrates and surfaces, which can vary widely in their own respect. In industrial applications, such as water treatment, chemical processing, the incidence and presence of biofilms is typically scrupulously to be avoided as such reduces the overall operating efficiency of any such industrial process, e.g., pipe fouling, blockage of filtration media, and the like. In medical applications, biofilms are also scrupulously avoided as providing a source of one or more undesired microorganisms which may undesirably come into contact with a living organism, and/or reducing the efficiency of one or more biological processes, e.g., filtration processes such as dialysis, blood filtration, and the like. Therefore, means for understanding the characteristics of bioflims, which may lead to further methods for their control and/or eradication, as well as apparatus useful in such methods are of considerable technical interest, and for which there exists a continuing need.

Various apparatus are known to the relevant technical art, each which may be useful in the study of various biofilms. A first such apparatus is described to be a "CDC Biofilm Reactor" which is described to be apparatus, which includes an approximately I litre glass vessel within which is contained a support device which comprises eight "coupon" holders. Each of the coupons are formed of a polymer (e.g., polyethylene) and arc substrates upon which a biofilm can be grown. The support device further incorporates a stir bar which is used to mix a growth medium broth liquid within which the coupons are fully immersed. An inlet to the glass vessel allows for the controlled delivery of a growth medium to the interior of the CDC Biofllm Reactor, and an output positioned above the level of the coupons within the reactor, allows for the withdrawal of the growth medium from within the reactor. A second such apparatus is described to be a "Drip Flow Biofllm Reactor", which is a container having a plurality of chambers, each of the chambers being generally horizontal (viz. may be angled by up to about 15 degrees of arc from the horizontal), and which provide a support base for a "coupon" (e.g, a glass microscopy slide) over which can be continuously, or periodically dosed a liquid material, such as a growth medium. The design of this reactor provides for a low shear, high gas transfer environment to permit for the growth of biofIlms on the test coupons. Each of the chambers includes an inlet, and an outlet to permit for the admission ot and the removal of the liquid material. The design of this reactor however is not directed towards containing any larger quantity of a liquid in contact with the surface of a coupon within an individual chamber, but rather his models to assess the characteristics of biofllm to a "drip flow" of the liquid. A third apparatus is a "Rotating Disc Biofllm Reactor" which includes a Teflon® and Viton® disc containing recesses for one or more coupons. The design of this reactor such that the disc is positioned at the base of a vessel, such that the coupons are totally immersed within a liquid, such as a growth medium. The disc is rotatable, and being rotatable allows for the assessment of moderate surface shear of the biofilm upon the test coupon when immersed within the liquid. A fourth apparatus is a "Biofllm Annular Reactor", which includes a stationary outer cylinder, rotating inner cylinder which define an annulus therebetween. Coupons having upon their surfaces a bioflim(s) arc flush surface mounted within this reactor, and a liquid is provided within this annuhis. The inner cylinder may then be rotated at a variety of speeds, in order to provide specific liquid/surface shear conditions at which the biofllm may be evaluated. While each of the foregoing do provide useful apparatus, in each case whcrein the coupons are maintained in a vertical orientation, they are fully immersed within a liquid, while those apparatus for the coupons are maintained in a horizontal orientation, there are either fully immersed within a liquid, or only subjected to a "drip flow" of the liquid which drains down the surface of the coupon upon which a biofilm is present. Each of the foregoing are presently commercially available from the Biosurface Technologies Corp. (Bozeman, MT, USA).

In a paper titled "A repeatable laboratory method for testing the efficacy of bioeides against toilet bowl ofbiofilms" published in the Journal of Applied Microbiology (2001) Vol. 91, 110-117, the authors of that paper utilized as reactors, 1 L glass beakers fitted with drain spouts, each having a magnetically driven base placed at the bottom of each vessel in a horizontal configuration (and parallel to the base of the glass beaker) wherein each base was constructed from a Teflon and silicone rubber disc, each base also incorporating a magnetic stir bar. Each of the bases also included six removable porcelain ceramic discs, each of which were support substrates for biofllms. When positioned at the bottom of the glass beakers, each of the bases could be rotated by placing the beakers upon a magnetic stirrer base which when activated, magnetically coupled with the magnetic stir bar causing rotation of the bases and the porcelain ceramic discs. According to the method described, the discs are rotated at 500 revolutions per minute ("rpm"). The glass vessels were described as containing a 400 ml. working volume, which based upon the size of the vessel and of the rotating base would ensure that the base, and any of the porcelain ceramic discs would be thlly immersed within the 400 ml. working volume of liquid during the testing as described in that paper. The reactors of this paper, were thus very similar to the "Rotating Disc Biofllm Reactor" (cx. Biosurfaee Technologies Corp.) Notwithstanding the availability of the foregoing to the relevant art, there remains a real and continuing need in the art for providing new apparatus, and new methods for understanding the characteristics of a biofilms, which may lead to further methods for their control and/or eradication. More particularly there is a real and continuing need to provide new apparatus, and new methods for the growth and analysis of biofilms which grow at an air/liquidwater interface. It is to these aspects that the current invention is directed.

in a first aspect the present invention provides a novel biofllm reactor apparatus useful in the evaluation of biofilms upon a surface, wherein the biofilm is simultaneously exposed to an airspace, a liquid and the air/liquid interface. The liquid may be a growth medium, a treatment composition, or any other liquid with which the biotilm maybe contacted. The airspace maybe a sealed annulus or may be an airspace open to the ambient environment of the apparatus.

In a second aspect the present invention provides a novel method for the evaluation of the characteristics of biofilms (e.g., growth, retention, control, removal and/or eradication of the biofilm) upon a surface, wherein the biotilm is simultaneously exposed to an airspace, a liquid and the air/liquid interface. The liquid may be a growth medium, a treatment composition, or any other liquid with which the biofilm may be contacted. The airspace may be a sealed anmilus or may be an airspace open to the ambient environment of the apparatus. The air/liquid interface is at the interface of the airspace and the uppermost level of the (bulk) liquid within the apparatus.

These and further aspects of the invention are set forth in the following further

description of the invention.

Figure 1 illustrates a preferred substrate useful in the biofilm reactor apparatus according to the invention.

Figure 2 illustrates a first embodiment of a biofilm reactor according to the invention.

Figure 3 illustrates second embodiment of the biofllm reactor according to the invention Figure 4 illustrates a third embodiments of a biofilm reactor according to the invention.

Figure 5 illustrates a fourth embodiment of a biofilm reactor according to the invention.

Figure 6 illustrates a fifth embodiment of a biofllm reactor according to the invention.

Figure 7 illustrates an embodiment of a biofilm growth system.

Figure 8 provides a photograph of a test substrate upon which is present a biofilm.

Figure 9 is a bar chart representing the observed bioflim level, or alternately, the level of growth incident upon test substrates resulting from the evaluation performed according to Example 2.

These and further aspects of the invention will be better understood from a

further consideration following specification.

Biotilm reactors of the invention necessarily comprise one or more substrates upon which biofllms are grown. The substrates are advantageously generally rcctangular in form, although thcy may assume any geometrical configuration which is suitable and upon which biofilms may be successfully grown. Nonlimiting examples include substrates which have one or more planar surfaces, or one or more curved surfaces. Such may be referred to as "face regions". With regard to the former such include articles having two or three sides, e.g. generally square or rectangular plate-shaped or strip shaped articles, while with regard to the lafter, such include articles having curved, oblate, hemispherical, or spherical surfaces, e.g. ball shaped articles. The materials of the construction of the substrate can be essentially any material desired, and suitable for use with the biofilm reactor. Coming into consideration are porous and nonporous materials. Examples of porous materials include textiles, fabrics, felts, sponges, membranes, and the like. Examples of nonporous materials include polymers, vitreous materials such as ceramics, stone, metals, metallizcd surfaces, coated surfaces, and the like. The selection of the material of construction, and its surface characteristics are primarily dictated by the intended use of the biofilm reactor. Generally, such are dictated by the ultimate surface sought to be studied. For example, where the characteristics of biofilms upon glazed ceramic or the vitreous surfaces are subject to investigation, then glazed and fired ceramic tiles may be used as a substrate. The substrates are generally preferably formed of nonporous materials.

Optionally the substrates may include one or more cavities extending inwardly from a face region into the substrate (e.g., concavities), or one or more passages (e.g., perforations) extending or passing through the substrate. Further optionally, the substrates may include portions or regions extending outwardly from one or more face regions, e.g., convex portions or other elements extending from a face region of a substrate. The substrates need not be solid articles, such as plates, but portions

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thereof may be hollow. Advantageously, the configuration and/or geometry of the substrates providc physical support for biofllms grown thcrcupon, and particularly at the air-liquid interface present within the biofilm reactor.

Further, optionally but preferably, the substrates may include one or more retaining means, e.g, at an end thereof, particularly if the substrate is generally square or rectangular plate-shaped or strip shaped article, e.g., a curled or helical shaped substrate. Alternately the retaining means may extend outwardly from the substrate, such as may be a hook, hanger, pin, rod, or strip by which the substrate may be suspended within the interior of a biofllm reactor. An alternative, the retaining means of the substrates may be one or more elements which cooperatively attach with a further element, viz, a suspending means, forming part of the biofilm reactor and via which the substrate(s) maybe suspended within the interior of the biofllm reactor. For example, a portion of either the biofllm reactor or the substrate may have a "hook part" or a "loop part" of a hook-and-loop type fastener system (e.g., VELCRO) while the other part of the corresponding hook-and-loop type fastener system is present on either the biofllm reactor of the substrate. In a further and preferred example, a part of the substrate includes a hole (e.g., perforation) extending through part of the substrate, or includes one or more cavities extending into the substrate, and the hole and/or cavity (cavities) may be removably engaged with a further cooperating suspending means of the biofilm reactor, such as a clamp or hook which engages the substrate, preferably a hole or cavity (cavities) present. According to a still further example, a part of the biofllm reactor, e.g, a lid, may include one or more suitably sized cavities into which a part of the substrate may be movably attached by insertion, and removed by withdrawal. Such may be a friction fit or close tolerance fit between a part of the substrate, and said cavity. In all aspects of the invention, the biofilm reactors configured to receive, and to retain one or more substrates within the interior of the biofilm reactor.

In certain embodiments the biofllm reactors of the invention may include one or more suspending means, which may be separate from the lid of the biofllm reactor, and from which the one or more substrates may be suspended within the internal cavity of the vessel. As noted, the suspending means may cooperate with the retaining means of a substrate. Non-limiting examples of a suspending means include a rod, or a rack insertable within the internal cavity, or may be a hook which may be removably affixed to a part of the vessel, such as to a part of the rim. Alternately the suspending means may be a part or element which spans across a part of the open end of thc vcsscl, or still altcrnativcly may be one or more parts or clcmcnts rcmovably affixed to the lid, e.g. to a part of the underside of the lid. Preferably, and advantageously the supporting means which are used to engage the substrate(s) are formed as part of, or are elements forming part of or are otherwise affixed to the lid such as to the underside of the lid, or to a peripheral rim of the lid.

The biofilm reactors of the invention optionally but most desirably comprise a lid, and necessarily comprise a vessel having an internal cavity adapted to contain a quantity of a liquid, sometimes referred to as a "bulk liquid" haying a composition with which a biofilm present upon the one or more substrates is in contact. The internal cavity of the by from reactor is of sufficient size and a suitable dimensions to retain a quantity of the liquid, such that at least a part of the substrate(s) may remain immersed within the liquid, and any remaining parts of the substrate(s) may be retained in a hcadspacc abovc thc top surface of thc liquid, yct within the internal cavity of the vessel. The internal cavity preferably includes one or more sidewalls, e.g., when the vessel is of a cylindrical configuration, then a single sidewall is considered to be present, whereas other vessel geometries may provide two or more sidewalls which may intersect or join at corners, wherein the sidewall(s) extend upwardly from a base. Preferably, and from a cost perspective, the general configuration of the vessel is generally cylindrical, and has a circular base. However it is to be understood that biofllm reactors according to the invention also and can pass other geomctrical configurations, such as hcmisphcrical or spherical shapcd vcsscls which have only one continuous sidewall/base. The vessel includes at least one open end thereof which permits for the insertion of, and withdrawal of the one or more substratcs into tim intcrnal cavity. Thc opcn end can bc closcd by a suitably sizcd the lid, which may bc looscly fitted which may thus provide for the passage of ambicnt air into an out of the internal cavity, or alternately may include a seal element (e.g., band, gaskct, 0-ring), or mating thrcads which engagc corrcsponding thrcads on portions of the sidewall(s), or which lid may otherwise be configured to provide a close tolerance type fit with the vessel and thus reduce or deny the exchange of air between the ambient environment and the headspace of the internal cavity. The biotilm reactor includes at least one fluid port which proyides for the fluid communication of the liquid (bulk liquid) for my supply source into the interior cavity. While the fluid port may bc provided anywhere within or upon the by from reactor, such as passing through a sidewall thereof; preferably and in preferred embodiments the lid further includes at least one fluid port through which the liquid can be introduced and/or removed (but preferably only removed) from the cavity. The at least one fluid port may be connected to a supply tube which provides a fluid conduit through which the liquid (bulk liquid) may be supplied continuously, or on a periodic basis, into the interior cavity. Optionally but less desirably, the lid may include at least one further fluid port through which the liquid within the internal cavity may be withdrawn. Preferably however, such at least one further fluid port forms part of the vessel and preferably at a lower part thereof to thereby facilitate the withdrawal or drainage of the liquid from the internal cavity of the vesscl. Such a further fluid port is to be connected to a drain tube, which allows for the introduction of; but preferably (only) the withdrawal of fluid or liquid from the internal cavity, preferably via a drain tube. The supply of liquid into, and out from the internal cavity of the vessel is advantageously accomplished by the use of a suitable pumper pumps, valve or valves, or any other conventional fluid control means which can provide such functions. In a particularly preferred embodiments, liquid provided via the supply tube and/or liquid being withdrawn via the drain tube is metered by the use of one or more peristaltic pumps which are configured or othetwise suitably controlled to provide the bulk liquid entering and/or exiting the biofilm reactor at a desired and controlled rate (e.g., volumetric rate). Of course, in an alternative, a like function may be supplied such as by providing controllable valves inline with on one or both of the supply tube(s) and/or drain tube(s), and/or at any of the fluid ports of the vessel and operating said valves in a desired and controlled manner in order to provide a desired and controlled rate (e.g., volumetric rate) of liquid entering and/or exiting the by from reactor.

With regard to the further fluid port via which liquid may be withdrawn from the internal cavity, advantageously this fluid port is present at a portion of the sidewall near the base of the vessel, and/or forms part o extends through the base of the vessel. In this manner, this further fluid port, particularly when fluid communication with the drain tube, may advantageously take advantage of the hydrostatic head of the liquid present in the internal cavity, which facilitates drainage (e.g, gravity drainage) of liquid from the biofllm reactor.

Any of the fluid ports may be formed as integral parts of the lid and/or the vessel, or both. Alternately any of the fluid ports may be formed as one or discrete

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elements or parts which are inserted into and/or through parts of the lid and/or the vessel, or both. For example, a fluid port may be supplied by a portion of a tube extending through a suitably sized hole through a part of the lid, sidewall or base of the biofllm reactor, and a liquid-tight (and preferably further also, a gas-tight) sealing element such as a suitably sized grommet is provided to provide such seal. In a further example, fluid port may be a tube, or other fluid conduit provided in a tight, close tolerance or friction type fit in a suitably sized orifice present within the a part of the lid, sidewall or base of the biofllm reactor without requiring a sealing element.

Optionally, the case of the latter, a cement, or adhesivc may bc applied.

It may, in certain embodiments, bc advantageous to have a "self-lcveling" feature provided by the vessel of the biofilm reactor. In such an instance, the configuration of the fluid ports can be established or modified in order to provide such a feature. For example, the length and the direction of the fluid port via which the liquid may be drained from the internal cavity can be configured, such as by extending upwardly from a point at or near the base of the vessel, and upwardly and away their from to provide a standing column, which may terminate at a point above the base but below the open end or the lid of the biotilm reactor. The length of this fluid port also establishes the vertical height of the liquid that can be contained within the internal cavity of the vessel, before it exits via this fluid port.

When the biofllm reactor is at least partially filled with bulk liquid, the upper surface of the bulk liquid defines an air/liquid interface, with the headspaee within the vessel being above this air/liquid interface. The headspace, when the lid is present, may be defined to be the volume of the intemal cavity of the vessel above the upper surface the bulk liquid.

Preferably most or all parts of the biofilm reactor and/or the substrates are formed of sterilizable, or autoelavable materials such that prior to any use, these elements of the biofilm reactor may be suitably treated, e.g., cleaned, sanitized, or autoelaved. However, such is not an essential requirement and advantageously parts of the biofilm reactor are formed of synthetic thermoplastic materials, including but not limited to: polyolefins such as polyethylene, polypropylene, polybutylene; polyamides including various grades of nylons, especially Nylon 6 and Nylon 66; polyalkylene terephalates such as polyethylene terephalates and polybutylene terephalates, polysulfones, as well as mixtures or blends of such polymers. The use of ceramics, glasses and vitreous materials may also be use in the construction of the biofilm reactors 50 and/or the substrates 10 of the invention.

In a preferred embodiment, the vessel and lid of the biolilm reactor maybe provided by a Nalgene® jar and lid, through which each of is bored a suitably sized hole into which is inserted an end of a supply tube, which end functions as the fluid port, and the drain tube which extends through a hole adjacent to the base via a tightly lifting rubber grommet which forms a liquid seal with the sidewall, which functions as the fluid port. A plurality of substrates formed from white, glazed ceramic lavatory tiles which are cut into pieces having a length of about 4 inches (approx. 10cm) and a width of about 1 inch (approx. 2.5 cm) are suspended on bent metal hangers within the Nalgene® jar and thereafter the lid is was rested upon the rim of the Nalgene® jar but not sealed by engagement of corresponding mating threads thereby providing for passage of ambient air from the environment of the biofi lm reactor to be exchanged with the air present within the headspaee of the vessel.

In use, the biofilm reactor may be used to evaluate various characteristics of biofilms present upon the surface of the substrate, particularly at the air/liquid interface within the vessel. A sufficient quantity of a bulk liquid can be retained in the internal cavity for a suitable amount of time to thereby provide for (potential) interaction and physical contact between the bull liquid, and the substrate(s) bearing the biofllm. The biofilm reactor may form part of a biofi lm growth system which further comprises one or more further pumps, valves, or other fluid control means whereby the supply of liquid (bulk liquid) entering the reactor, and the withdrawal or drainage of liquid (bulk liquid) from the internal cavity of the biofilm reactor may be controlled according to any desired protocol. The liquid (bulk liquid) may be provided from one or more suitable reservoirs or vessels which contain a supply or supplies of one or more different liquids, and the liquid (bulk liquid) withdrawn from the biofilm reactor may be provided to a drain vessel or reservoir in which the spent liquid may be collected, if desired for later analysis. For example, it is contemplated that two or more different liquids may be supplied via one or more fluid ports into the internal cavity of the vessel. Such may be, for example distilled or deionized water as a first liquid, and a nutrient broth and/or a "test additive" material as a second liquid (or, third liquid) supplied to the biofllm reactor. By "test additive material" is to be understood any other composition of matter which may be supplied to the biofIlm reactor 50. Nonlimiting examples of "test additive material" include one or more of: surfactants, organic solvents, inorganic solvents, water, aqueous dispersions, emulsions or suspensions or of one or more of organic or inorganic materials, e.g. metals, metal salts, oxidizing agents, bleach, acids, antimicrobially effective compounds, as well as biologically active materials, e.g., bacteria, spores and phages.

The provision of any of the foregoing test additive materials via the liquid supply system provides for a convenient and controlled method for evaluating the effect of such materials provided to the biofllm reactor 50, and the effect of such materials upon biofllms present upon the substrates 10. It is to be understood however that the test additive materials may be provided directly to the biofllm reactor directly if desired.

Such a system may optionally, per preferably further also includes one or more controller means, which may be automatic, or semiautomatic, or for that matter may be fully manual. With regard to the manual option, such can be simply a human operator opening andior closing suitably positioned valves upstream, and downstream of the biofilm reactor at a suitable time or under other suitable conditions. With regard to automatic or semiautomatic controllers, such can be dedicated, or general-purpose digital devices, including but not limited to digital pump controllers, general-purpose computers, logic circuits, and the like. In one preferred embodiment, the system comprises one or more peristaltic pumps through which liquid may be supplied and/or withdrawn, but preferably both supplied, and withdrawn from the biofilm reactor in response to a preprogrammed sequence over a number of hours or days. Such a system providcs for a highly effective, prccisc control over the delivery of, and withdrawal of liquid (bulk liquid) from the biofilm reactor without requiring constant human oversight or control. Such a preprogrammed sequence may for example, include the delivery of, and withdrawal of similar or equal volumetric amounts of the bulk liquid into and out of the biofllm reactor, at one or more time intervals within every 24 hour time interval.. Such a preprogrammed sequence may also for example provide a variable delivery rate, and or withdrawal rate of the liquid (bulk liquid) into and/or out of the biofllm reactor at one or more time periods or time intervals.

Optionally but preferably, the control system may also ffirther include a recorder means, which may be one or more of: a memory storage unit (e.g, RAIVI memory), a visual display device, a printer, or other device whereby a record of the operating characteristics of the biofilm reactor may be recorded for later review by a human operator, and if desired retained. When present, as in preferred embodiments, the controller means may provide additional, ancillary functions including, but not limited to: temperature control, temperature sensing of the ambient environment, the liquid contained anywhere within the bioflim growth system, light levels, as well as any other environmental conditions of the ambient environment, within the bio film growth system. Further ancillary functions include data logging, of any of the elements of the system, the biofllm reactor, or of any of the substrates, liquids, or test additive materials found within the biofilm growth system. Such frirther functions are easily provided by the use of conventional probes or sensor suited for their specific purpose, whose outputs can be provided as inputs to the control means. Is also contemplated that further devices, such as heating or cooling devices, light sources, may also be operated and controlled by the control means in order to provide specific conditions to the system (sometimes referred to as a "biofllm growth system") during any testing protocol.

Any of the biofilm reactors disclosed and described herein and/or the biofllm growth systems disclosed and described herein may be used in a novel method for the evaluation of the characteristics of biofilms (e.g., growth, retention, control, removal and/or eradication of the biofilm) upon a surface, wherein the biofilm is simultaneously exposed to an airspace, a liquid (e.g., bulk liquid) and the air/liquid interface. Generally, the characteristics of biofilm growth on partially immersed substrates within a biofllm reactor, which forms part of a biofilm growth system, may be evaluated over a period of time. During this time period, the conditions of the biofilm reactor can be maintained in a single state or condition, or alternately can be varied over an interval of time. For example, in one embodiment, during an evaluation ofbiofilm growth a quantity of a liquid is charged to biofilm reactor, (which can be used independently of the further elements of a biofllm growth system) within which is present a suitable substrate. The characteristics of a biofllm present upon the surface of the substrate can be assessed and evaluated under such conditions, and no supply of or withdrawal of liquid contained within the biofilm reactors or need occur, although such can take place. In a further embodiment, during an evaluation of biofilm growth, a biofilm growth system comprising a biofilm reactor is supplied. The biofilm reactor contains a suitable substrate, and a quantity of liquid is charged to the biotilm reactor. Over a period of time, additional liquid can be charged the biofilm reactor and/or liquid can be withdrawn from the biofilm reactor. The characteristics of the biofllm present upon the surface of the substrate can be assessed and evaluated under such conditions.

The bulk liquid may be a growth medium, a treatment composition, or any other liquid with which the biofllm may be contacted. The bullc liquid may be water, such as common tap water from a municipal supply source is contemplated, and such would contain naturally present microbiological organisms. The liquid may also be distilled, deionized and/or demineralized water. The liquid may be water which has been specifically balanced to a specific hardness, wherein said water is either common tap water, or a distilled or deionized water. The liquid may be treatment composition, which can include any of a numbcr of chemicals compositions which arc intended to provide a cleaning, antimicrobial, or other technical benefits to the substrates. Non-limiting examples of such chemical constituents include; surfactants, organic acids, inorganic acids, bases, organic solvents, water, chelating agents, thickeners, antimicrobial agents including non-phenolic and phenolic compounds, dyes, colorants, fragrances, inorganic materials which may be provided in a powdered or comminuted form, oxidizing agents including bleaches, hydantoins as well as precursors thereof, as well as further constituents know to the relevant technical art.

Such a treatment composition is intended to be useful in evaluating the cleaning and/or antimicrobial efficacy of a composition and its effect upon biofilm over both short (several seconds, to several minutes) time intervals, to much longer time intervals (several minutes, preferably at least several hours, several days, several weeks, months, or years). The liquid may also be a nutrient broth, a growth medium, or other liquid which is useful in fostering the growth of biofilm present upon a substrate within the biofllm reactor. During any testing protocol, one or more liquids can be provided to the biofilm reactor. In such manner, the response of a biofilm present upon the substrates, or otherwise present within the interior of the biofllm reactor can be evaluated, over any suitable time interval.

The airspace within the bioffim reactor, (viz., headspaee) may be a se&ed annulus or it may be open to the ambient environment of the biofllm reactor. While testing utilizing a sealed annulus provides greater control, minimizes contamination of the interior of the biofilm reactor from external sources, e.g., inanimate dirt, debris, or microbial contaminants, such as pathogens, yeasts, spores, and the like, testing protocols wherein the biofllm reactor is open to the ambient environment, e.g., such as per the use of biofilm reactor having a loosely fitting lid may provide a more realistic approximation of "real-life" conditions of a biotilm, and its response to the liquid (s) with which it comes in contact.

Bioflims which are grown in, or treated within the bioflim reactors of the invention may be evaluated by any of a number of quantitative or qualitative techniques. By way of non-limiting examples, the biofllms present on substrates may be visually evaluated without the aid of any computer based optical systems, such as by visual evaluation by a person with or without supplementaiy magnification, and/or a computer based optical system may be used to digitally evaluate images of the biofilms present on substrates. Further, biofilms may be extracted (e.g. scraped from) thc surfaces of the substrates, and subjected to further biological testing, e.g, grown in suitable growth media, such as on plates or Petri dishes under controlled conditions, and thereafter evaluated, such as to identify and/or quantify the biofllm, as well as to identify and/or quantify one or more microorganisms comprised in the biofilm.

Preferred embodiments of the invention are discussed with reference to the following drawing figures. It is to be understood that similar or like elements in the various aspects of the invention are referenced by common numerals. Is also to be understood that features according to a specific embodiment of the invention, may be incorporated in, or adapted to other embodiments of the invention, such as may be represented by different drawing figures.

Figure 1 illustrates a preferred embodiment of a substrate 10 useful in the biofilm reactors according to the invention. It is however to be recognized that other substrates other than shown on Fig. 1 may be used in the biofllm reactor, and that such substrates may indeed, take any form, and may have different geometries than those disclosed in the figure. The substrate 10 advantageously is generally rectangular in form, having a length greater than its width, and a width greater than its height (thickness). Such defines a rectangular, plate-like substrate. Face regions 12 of the substrate 10 may be generally flat. Altemately, the face regions 12 may include patterned regions, (not illustrated) such as one or more concave regions, or one or more convex regions extending into, or outwardly from, the generally flat face region 12. Of course, different surface features, including convex and/or concave patterned regions, crosshatched, or mesh shaped patterned regions, although not illustrated in the drawing figures are also expressly contemplated as being of use. When present, such patterned regions may be present on one, or both sides of the substrate 10, viz., thc opposite face regions 12 of substrate 10. The illustrated substrate 10 optionally but preferably includes at least one end 18 which includes retaining means which may aid in rcmovably affixing or removably clamping the substrate within the bioflim reactor. In the depicted embodiment, the retaining means 20 is a rectangular recess or perforation 20 near one end 18 of a substrate 10. It is to be understood that other configurations, e.g, a circular or oval hole, or a part of the substrate 10 which extends outwardly from one or both faces 12 thereof may also supply a suitable retaining means. In this embodiment, as the characteristics of biofilms upon glazed ceramic or the vitreous surfaces are subject to investigation, then glazed and fired ceramic tiles may be used as a substrate 10.

Figures 2, 3, 4, 5 and 6 illustrate in partial cross-sectional views several embodiments of biofilm reactors 50 of the invention. Each biofilm reactor 50 includes a vessel 52 having an internal cavity 54 adapted to contain a quantity of a liquid 100, (e.g. "bulk liquid",). The vessel 52 has sidewalls 56 which in the case of a generally circular biofilm reactor as is depicted in each of Figs. 2, 3, 4, 5 and 6, is a generally circular sidewall (although other geometric configurations are also clearly possible) which extend upwardly from a base 58. The vessel 52 also includes an open end 60 and includes also at least one fluid port 62 through which the bulk liquid 100 can be introduced, and/or removed (but preferably only removed) from the cavity 54 of the biofllm reactor 50. The biofilm reactor 50 further includes a removable lid 71 which provides an easily removable cover which spans the open end 60 of the vessel 52. In the embodiments of Figs. 1, 5 and 6 the lid 71 is merely placed upon and rests upon the top rim 73 of the sidewall 56 but is not otherwise affixed thereto. Such permits for the entry of and egress of ambient air in the environment to enter the vessel 52 and particularly the headspace 105 above the upper surface 102 of the liquid 100. In the embodiment of Fig. 3 the lid 71 includes a body 72, a top 74, and a sealing means 76, here depicted as a lateral sealing element or band, which is advantageously formed of an autoclavable, or otherwise sterilizable elastomeric material which can be friction fitted within a portion of the internal cavity 54. As illustrated, the sealing means 76 forms a physical seal against the inner surface 53 of the sidewall 56, and with the lid 71 a barrier to the ambient external environment.

Not wholly dissimilarly in the embodiment of Fig. 4, the lid 71 includes near its periphery mating threads 71 B which may be coupled with corresponding mating threads 57 which are formed as part of the vessel 56. Also present is a sealing means 76, here in the form of a flat gasket formed of an autoclavable or sterilizable elastomer, is provided on an underside surface 73 of the lid 71.

Depending from the lid 71 are one or more substrates 10, which maybe such as are depicted on Fig. 1, which substrates 10 are suspended within the internal cavity 54 by suitable means. In the embodiment of Fig. 1, such means are S-shaped metal hooks 71A having two curved sections, the first extending through the perforation 20 of each substrate 10, the second curved section extending over and resting upon the top rim 73. In the embodiment of the lid 71 of Fig. 3, these means are not illustrated, while in the embodiments of in Figs. 4, 5 and 6 these means are hangers 75 from which the substrates 10 may be removably suspended. As illustrated, in use, an upper part 13A of each substrate lOis present within the headspaee 105 of the vessel 52, while a lower part I 3B of each substrate 10 is present within the bulk liquid 100. A narrow region I 3C of each substrate 10 is present within the air/liquid interface.

The lid 71 optionally but preferably includes at least one fluid port 78 which may form part of, or be connected to (as shown) a supply tube 80. Such is depicted on Figs. 2, 3, 4 and 6, whereas in Fig. 5 a fluid port 78 is omitted. The fluid port 78 may be an angled tube passing through the lid 71 having and inlet end 77 in fluid communication with a supply tube 80 and an outlet end 79 thereof extending into the internal cavity 54, as illustrated on Figs. 3 and 6, or may be a generally sfraight fining passing through the lid 71 as shown on Figs. I and 4. Although not shown, two or more fluid ports may be provided to lids 71. The lid 71 of Fig. 5 omits a fluid port 71, however it is to be understood that a side fluid port 78 is provided extending through an upper part of the sidewall 56 of the vessel 52, and is in fluid communication with a supply tube 80. The lid of 71 further includes a passage 110 which extends through the lid 71 which permits for the exchange of ambient air in the immediate environment of the biofllm reactor 50 with the headspace 105 or inner volume of the internal cavity 54. Such a configuration permits for the effect of ambient air upon the growth ofbiotilm within the biofllm reactor 50 to be studied.

Although not depicted on Fig. 5 is to be understood that the lid 71 can take alternate configurations than as specifically shown. For example the lid 71 may include a plurality of passages 110 passing therethrough. Such would be advantageous to provide increased opportunity for the exchange of air within the headspace 105 with that of the ambient environment. Such a benefit can be ffirther realized if the lid 71 is configured to include a mesh in its construction, or a series of intersecting or non-intersecting elements such as bars or rods from one or more of which may be provided hanger means 75 to which substrates 10 can be suitably and removably attached.

Adjacent to or proximate the base 58 of the vessel 52 is at least one fluid port 62 which may form part of, or is connected to a drain tube 82 which allows for the introduction of or withdrawal of a fluid or liquid into the internal cavity 54 of the vessel 52. In use, downstream of the fluid port 62 and/or the drain tube 82 there may be provided a valve (not illustrated) or other means (e.g., a pump) for controlling/restricting the flow of liquid outwardly from the biofllm reactor 50, which may be used to establish and/or control the level of fluid or liquid in the internal cavity 54. In the embodiments of Figs. 2,3,5 and 6 the fluid port 62 is formed as an integral part of the vessel 52, whereas in the Fig. 4 the fluid port 62 extending through the sidewall 56 is fluid port 62 which in the depicted embodiment is a short tube passing through a portion of the sidewall, and sealingly affixed there to by means of a grommet 59. The grommet 59 operates in a manner similar to that of the sealing means 76, and advantageously is formed of an autoclavable, or otherwise sterilizable elastomeric material. Additionally, the depicted biofilm reactor 50 of Fig. 4 also illustrates, in "phantom lines" and alternative positioning of the fluid port 62', and a grommet 59' which passed through the base 58 of the vessel 52. Such is an alternative configuration of the biotilm reactor 50, and illustrates that the fluid port 62 may be placed within the bottom 58 of the vessel 52. In such a position, more effective and near total drainage of the vessel 52 may be achieved. The foregoing also illustrates that a plurality of fluid ports may be provided and form part of a biofilm reactor 50 according to the invention, e.g. wherein both fluid ports 62, 62' are simultaneously present. The fluid port 62 of Fig. 6 differs from prior illusrated embodiments in that it is an elongated "S-shaped" fluid port 62 which comprises an inlet section 62A which passes through a portion of the sidewall 56 of the vessel, and which extends to an upstanding generally linear intermediate leg section 62B which next extends away from the base 58, which in turn passes through an inverted bend section 62C which redirects the fluid port 62 towards the base. A final end section 62D can be coupled to a drain tube 82 as shown. Thus, as is seen from the drawing, the sections 62A, 62B, 62C and 62D define a continuous fluid conduit, and represent an alternative embodiment of the fluid port 62. An advantage of such configuration is that the volume of the bulk liquid 100, and thereby the height and placement of the upper surface 102 of the bulk liquid, which establishes the air/liquid interface, can be conveniently controlled without the need of an external downstream valve, or other flow-stoppage means, downstream of the fluid port 62 which would be otherwise necessary in order to control the retained the volume of the bullc liquid 100 within the vessel 52. As is recognized and understood to a skilled artisan from the drawing figure, the relative height or distance of the inverted bend section 62C from the base 58 of the vessel 52 conveniently establishes the maximum distance or height of the upper surface 102 of the bulk liquid 100 within the biofilm reactor 50.

When the biofilm reactor 50 is at least partially filled with bulk liquid 100, the upper surface 102 of thc bulk liquid 100 is at the air/liquid interface, with the headspace 105 being the volume of the internal cavity 54 of the vessel 52 above the upper surface 102 the bulk liquid 100.

According to the invention, none, preferably some, but more preferably all parts of the biofilm reactor 50 and/or the substrates 10 may be formed of sterilizable, or autoelavable materials of construction such that prior to any use, these elements of the biofllm reactor 50 may be accordingly treated, e.g., cleaned, sanitized, or autoclaved. However, such is not an essential requirement and advantageously parts of the biofilm reactor are formed of synthetic thermoplastic materials, e.g., including but not limited to: polyolefins such as polyethylene, polypropylene, polybutylene; polyamides including various grades of nylons, especially Nylon 6 and Nylon 66; polyalkylene terephalates such as polyethylene terephalates and polybutylene terephalates, polysulfones, as well as mixtures or blends of such polymers. The use of ceramics, glasses and vitreous materials may also be use in the construction of the biotilm reactors 50 and/or the substrates 10 of the invention.

In a preferred embodiment, the vessel 52 and lid 71 of the biofilm reactor 50 were respectively provided by a Nalgene® jar and lid, through which each of has been bored a suitably sized hole into which was inserted an end of a supply tube 80, which end functioned as the fluid port 78, and the drain tube 82 which extended through a hole adjacent to the base 58 via a tightly fitted rubber grommet which formed a liquid seal with the sidewall 52, which functioned as the fluid port 62, the above biofilm reactor being most similar to the embodiment of Fig. 4. Further, a plurality of substrates 10, formed from white, glazed ceramic lavatory tiles which had been cut into pieces having a length of about 4 inches (approx. 10cm) and a width of about 1 inch (approx. 2.5 cm) were suspended on bent metal hangers within the Nalgenc® jar (vessel 52) as depicted on Fig. 2, and the lid (lid 71) was rested upon the rim 73 of the Nalgene® jar as depicted on Fig.2 but not scaled by engagement of corresponding mating threads as per Fig. 4.

in use, one or more substrates 10 may be provided to the internal cavity 54 of the biofllm reactor 50, and thereafter a desired amount of bulk liquid 100 may be supplied to the vessel 52 via fluid port 78 from a suitable supply source (not shown), and a sufficient quantity of the bulk liquid 100 may be retained in the internal cavity 54 for a suitable amount of time, such as by controlling the egress of the bulk liquid via the fluid port 62. Optionally some or all of the bulk fluid 100 may be removed from thc bioflim rcactor 50 such as by draining via the fluid port 62 and drain tube 80, and further optionally a fresh supply of bulk liquid 100 may be supplied to the biofllm reactor 50 via the fluid port 78 from a suitable supply source. The bulk fluid 100 may be retained in the biofllm reactor 50 fbr any desired interval of time, and periodic draining and refilling of the bioflim reactor on a periodic basis is aLso contemplated and may be perfbrmed with any of the biofllm reactors of the invention.

Fig. 7 illustrates an embodiment ofabiofllm growth system 150. Central to the said system 150 is a biofllm reactor 50 which can be any of the foregoing embodiments, or may be embodiments not specifically described with reference to the foregoing drawings but which nonetheless still operate according to the teachings of the present invention. For sake of illustration, the biofllm reactor 50 as depicted in Fig. 5. A liquid supply system 152 comprising a sterile water supply 152A and a nutrient supply 152B is separately connected by suitable fluid conduits 154 to a (first) fluid controller 156. The depicted fluid controller can be any of a variety of devices which meter the flow of the liquid from the liquid reservoir 152 to the reactor 50.

Such can be a simple "on-off" fluid controller such as a manually operated valve, or may be automatically controlled valve, or maybe a pump, a peristaltic pump, or virtually any other device which fulfils such a function. In the depicted embodiment, it is understood that the fluid controller 156 is a peristaltic pump which provides for both the controlled simultaneous delivery of aliquots of the sterile water and nutrient through supply tube 80, to the bioflim reactor. Not dissimilarly, liquid can be withdrawn from the biofllm reactor via a drain tube 82 which, downstream thereof, it in fluid communication with a further (second) fluid controller 158. Such may be the same as, or different than the fluid controller 156. This further fluid controller 158 is connected to a liquid drain reservoir 160 via an intermediate fluid conduit 159.

It is to be understood that the liquid supply system 152 may be one or more of: a non-sterile water supply, a sterile water supply, a nutrient supply, and/or a test additive material supply. Advantageously both the first fluid controller 156 and the second fluid controller 158 are devices or apparatus which are responsive to a controller 170, which may be any of a number of known-art devices, e.g., analog control device, a programmable digital controller device which may be a programmable computer having appropriate control andlor signal outputs which can communicate, or signal lines 172 to appropriate inputs present on the first fluid controller 156 and/or the second fluid controller 158. Use of a programmable digital controller and/or a general-purpose computer program to provide such a function permits for the control of various operative characteristics of the biofilm growth system 150 including the level of the liquid contained within the biofllm reactor 50, the frequency of, and the quantity of liquid supplied and/or removed from the biofilm reactor 50, as well as the overall duration of any testing protocol which is performed utilizing the biofilm growth system 150. Although not illustrated, it is also contemplated that the controller 170 can provide additional, ancillary functions to the biotilm growth system 150 including, but not limited to; temperature control, temperature sensing of the ambient environment, the liquid contained anywhere within the biotilm growth system, light levels, as well as any other environmental conditions of the ambient environment, within the bioflim growth system. For the ancillary functions include data logging, of any of the elements of the biofilm growth system, the biofilm reactor, or of any of the substrates, liquid, or test additive materials found within the biofilm growth system. Such further functions are easily provided by the use of conventional probes or sensor suited for their specific purpose, whose outputs can be provided as inputs to the controller 170. Isis also contemplated that further devices, such as heating or cooling devices, light sources, may also be operated and controlled by the controller 170 in order to provide specific conditions to the biotllm growth system 150 during any testing protocol.

The following examples demonstrate certain particularly prefened aspects of the invention.

Example I -Control Samples, Biofllm Samples An exemplary embodiment of a biofllm growth evaluation testing protocol was carried out utilizing a biofilm growth system similar to that disclosed with reference to Fig. 7, but wherein that the biofilm reactor was more closely akin to that of the by from reactor described on Fig. 2.

In this test, the effect of biofilm generation and growth was evaluated on a series of test substrates wherein the bulk liquid of the biofllm reactor was an aqueous composition based on tap water, and which additionally included standardized growth medium tTryptic Soy Broth). However, test additive materials as recited above were omitted from this test.

Both the vessel and the lid of the biofllm reactor were formed of a synthetic thermoformed polymer (brand: NALGENE). The total liquid volume of the (empty) vesscl was 1 litre, and the vessels were cylindrically shaped having a closed base, and an open end upon which the lid was fittable. A hole was provided through the sidewall of the vessel at a place adjacent to the base of the vessel. Into this hole was provided an autoclavable grommet, through which was inserted a drain tube, and which extended into a earboy which collected the spent bulk liquid generated during the test and which exited the biofilm reactor. To supply the biofilm reactor during the test, separate supply tubes which extended via a peristaltic pump, extended into the interior of the vessel via an opening in the lid, but the ends of the tubes were kept separate and only extended into the headspace of the biofllm reactor. Such reduced the likelihood of contamination by the bulk liquid whose level within the biotilm reactor was always below the ends of these supply tubes.

A number of commercially available, glazed white ceramic bathroom tiles were used to form the substrates. Each of the length and width of the square tiles was approximately 4 inches (about 10 cm) on each side, and the tiles were divided into four equal pieces being approximately 1 inch (about 2.5 cm) in width, and about 4 inches (about 10 cm) in length. During the end of each, small hole was drilled using a conventional ceramic drill in order to provide a perforation passing thcrcthrough.

Short segments of metal were bent into hooks, with one end thereof passing through the perforation of a substrate, and the other end it was suspended from the rim of the open end of the vessel. In such a manner, the placement of the lid would rest upon the portions of the hooks extending over the rim of the vessel, thus ensuring that the headspace of the vessel was exposed to the ambient environment of the biofilm reactor.

The test was performed at room temperature, (approx. 20°C) and the temperature of the materials supplied to the biofilm reactor was also at room temperature.

For the test, a lx Tryptic Soy Broth was prepared for use as a nutrient broth, and placed in a sterile supply vessel from which led a supply tube, through the peristaltic pump and to the biofllm reactor. A supply of unfiltered tap water (containing residual chlorine, and naturally occurring microorganisms) was also preparcd, and similarly was placed in a sterilized supply vessel from which led a supply tube, through thc peristaltic pump and to the biofilm reactor. During the test, water feed was set to flowrate of I SmI/min and the pump for the nutrient (TSB) feed was set to a flowrate of 1.5m1/min. With each tilling cycle which was set for 40 minutes, such supplied a total fluid volume of 660m1 of liquid (bulk liquid) to the biotilm reactor, which comprised 10% TSB concentration in the biofllm reactor. Thus with each fill cycle, approximately one-half of the available volume of the cylindrically shaped biofilm reactor vessel was filled with the bulk liquid, and the remaining volume above the liquid and below the lid was the headspace.

Concurrently with the supply of the water feed and nutrient feed, the bulk liquid present in the biofllm reactor was drained by use of a peristaltic pump which required approximately 1 -3 minutes, and immediately after draining a fill cycle was initiated.

Such an operation mimics the flush and refill cycle of a domestic lavatory appliance, particularly a domestic toilet.

The unfiltered tap water used contained naturally occurring water bacteria, molds and fungi that form biofilm on surfaces which have not been cleaned for a while. The water source for the reactors can be non-sterile tap water that still contains these naturally occurring microorganisms (unknown mixed species). A tap water sample may be initially obtained, enriched, split into 0.Sml aliquots and frozen at -70 C to provide an inoculum source. This inoculum source may be used every time reactors arc started and ensured that the biofllm reactor contained a very similar set of natural organisms throughout the duration of the test.

During the test, if and when required, the nutrient and water supply vessels (e.g, carboys) were cleaned, sterilized and refilled whenever needed. Similarly as the waste carboy was nearly filled with spent liquid from the biofllm reactor, it was similarly removed, washed, autoclaved and reconnected when needed.

Utilizing the peristaltie pumps, the reactor was flushed and refilled at set time intervals so that spent bulk liquid was substantially drained and fresh medium along with water and additive is replenished. These cycles imitate the flushing and refilling which mimics the behaviour of domestic toilets. In such a manner the biofllm reactor provides an effective apparatus for studying the incidence of biolfllms and their growth in systems which pcriodieally flush and refill a bulk liquid.

According to the test, at the initiation of the test the peristaltic pumps were initiated to provide a first filling cycle, wherein an initial fluid volume of66Oml of liquid (bulk liquid) was charged to the biofllm reactor, which bulk liquid comprised 10% TSB concentration as described above. Thereafter for the duration of the test, the peristaltie pumps were cycled at 12 hours intervals in order to drain and fill the biotilm reactors as described above. The testing proceeded for a plurality of days, often 14-18 days.

During the test, at each of day 5, 8, 13 and 16, two substrates were removed from the biofilm reactor. Removalwas accomplished by simply lifting the lid, and withdrawing two of the substrates from the interior of the biofilm reactor, and immediately afterwards replacing the lid. The removed substrates were placed in a petri dish (or other container) and dried in incubator at 36°C with airflow.

Each of the thus dried substrates was inserted into an Ortery Photosimile 200 light box with a daylight balanced fluorescent light source using a Canon EOS® 7D digital camera. The images thus obtained were subsequently converted to gray-scale using ImagePro® Plus software (Ver. 7) and part of each substrate at the air/liquid interface region of the tile was analyzed, which resulted in a numerical grey scale value which was graduated into values having a range of 0-255, wherein a totally black surface is represented by a value of "0", and a maximum brightness has a value of "255". (Untreated reference substrates, viz., the glazed white ceramic bathroom tiles registered an averaged value of "160" -"165" on this "0 -255" graduated greyseale.) Reference is made to Fig. 8 which indicates the interface region of a representative test substrate which was subjected to image analysis and assignment to the graduated greyscale. This value was exported to an Excel spreadsheet and used for later data analysis. The results from each of the two test substrates removed on a particular day were numerically averaged.

As a control sample, one or more new substrates were photographed and evaluated as above to represent a "clean" reference test substrate measurement.

Subsequently, at the conclusion of the test (day 16), and following the evaluation of the final set of test substrates, the data exported to the Excel spreadsheet was analyzed to provide a measure of relative brightness of the averaged brightness readings of the two test substrates withdrawn from the biofilm reactor on each of days 5, 8, 13 and 16, as compared to the clean reference test substrate.

Example 2 -Additive Materials Testing on Biofilms The protocols of foregoing Example I repeated this tests with the following variations.

As a control, a first biofllm growth system was configured, and operated as described above with reference to Example 1 for a 16 day test period, wherein the liquid input to the biofllm reactor was the tap water sample and the standardized Trypie Soy Broth growth medium which was present in the bulk liquid at a 10% concentration, as described supra. The test was performed for 16 days, during which two test substrates were withdrawn from the biofilm reactor on each of days 5, 8, 13 and 16, analyzed as discussed above, and as compared to a clean reference test substrate which was also analyzed discussed above.

A second biofilm growth system was configured, and operated as described above (with reference to Example 1) for a 16 day test period, wherein the liquid input to the biofllm reactor was the tap water sample to which was added a sufficient amount of copper sulphate (in order to provide a fmal aqueous concentration of 0.01% CuSO4,). The standardized Trypic Soy Broth growth medium which was present in the bulk liquid at a 10% concentration, as was described supra. The test was performed for 1 6 days, during which two test substrates were withdrawn from the biofilm reactor on each of days 5, 8, 13 and 16, analyzed as discussed above, and as compared to a clean reference test substrate which was also analyzed as discussed above.

A third biofllm growth system was configured, and operated as described above (with reference to Example 1) for a 16 day test period, wherein the liquid input to the biofllm reactor was the tap water sample and the standardized Trypic Soy Broth growth medium which was present in the bulk liquid at a 10% concentration, as described supra. The tap water sample was a 10 liter aliquot of water, to which was added 1 gram of a powdered product (cx. Novozymes) which comprised Bacillus arnyloliquefhciens at a minimum spore count of 50 x io per gram of the powdered product. In this matter, the standardized tap water sample further included a 0.01% concentration of the powdered product, when supplied to the bioflim reactor. The test was performed for 16 days, during which two test substrates were withdrawn from the biotilm reactor on each of days 5, 8, 13 and 16, analyzed as discussed above, and as compared to a clean reference test substrate which was also analyzed discussed above.

The results of the tested substrates from each of the first, second and third biofilm growth systems are disclosed on Fig. 9, which represents the relative graduated grcyscalc readings of these test substrates at each of days 5,8, 13 and 16.

As is understood therefrom the incidence of the biofilm growth on test substrates at thc air/liquid interface was similar for days 5 and 8, but were increasingly pronounced as compared to the control test substrates from the first biofilm growth system at days 13 and 16 of the test.

Assessment of the substrates may also be performed by withdrawing the substrates treated in biofllm reactor according to the invention, and thereafter removing part or all of the biofilm upon the surface of the substrate, and thereafter evaluate the removed biofllm sample according to conventional laboratory practices.

For example the biofilm samples may be visually evaluated, with or without the use of optical magnification apparatus, or the biofilm samples may be later plated or flasked with a suitable nutrient media and permitted to grow for a period of time, after which the sample and any grown microorganisms may be separated from the growth media and evaluated. The biofllm reactors of the invention also permit for the selective assessment of biofllms which arc present growth of biofllms above, at, and below an air/liquid interface region.