Automated active system for optimal HVAC hygiene and efficiencies

Abstract

An automated production and distribution system for optimal HVAC hygiene is designed for installation in HVAC plant rooms of buildings. The method, apparatus and system use beneficial bacterial strains, which are safe for humans, animals, and the environment, as an antimicrobial agent to facilitate HVAC hygiene. Intermittently misting the HVAC with beneficial bacteria at least partly inhibits pathogenic microorganisms, disrupts biofilms, and reduces allergens and organic debris. The hygiene solution comprising beneficial bacteria is produced in a bioreactor having sensors for real-time monitoring, a central control unit for automated adjustments, and a high-pressure distribution network for hygiene solution delivery to one or a plurality of HVAC units. This system improves air quality, system efficiency, and longevity while offering a cost-effective, eco-friendly, and compliant solution for maintaining hygienic HVAC systems in commercial buildings and hospitals.

Description

[0001] TITLE

[0002] AUTOMATED ACTIVE SYSTEM FOR OPTIMAL HVAC HYGIENE AND EFFICIENCIES TECHNICAL FIELD

[0003] THIS INVENTION relates to the field of Heating, Ventilation, and Air Conditioning (HVAC) systems, and more particularly to a hygiene method, apparatus and / or system for maintaining optimal hygiene in HVAC systems through the delivery of beneficial bacteria to the HVAC system.

[0004] BACKGROUND

[0005] Heating, Ventilation, and Air Conditioning (HVAC) systems are critical components in commercial buildings, hospitals, and other facilities, as they ensure indoor air quality and comfort and consume approximately 50% of a buildings total energy usage. However, maintaining these systems in a clean and efficient state presents numerous challenges. The following outlines the existing problems and inadequacies of current solutions.

[0006] HVAC systems are prone to the accumulation of contaminants such as dust, mould spores, bacteria, and viruses. These contaminants can proliferate within various components of the HVAC system, including air handling units (AHUs), ducts, filters, coils, blowers and cooling towers. Over time, this accumulation can lead to significant degradation of air quality, posing health risks to building occupants.

[0007] Contaminated HVAC systems can circulate harmful microorganisms throughout the building, leading to respiratory problems, infections, and allergic reactions. Specific pathogens such as Legionella pneumophila, Staphylococcus aureus (including MRSA), and various fungi and viruses can thrive in HVAC environments, exacerbating health risks, especially in hospitals and healthcare facilities where vulnerable populations are present.

[0008] Biofilms, which are complex communities of microorganisms adhering to surfaces, are particularly problematic in HVAC systems. They are resistant to conventional cleaning methods and can harbor pathogens, making them a persistent source of contamination. Biofilms can form on coils, ducts, and other moist areas within the HVAC system, reducing efficiency and increasing maintenance challenges. Dirty HVAC components such as filters, coils, and ducts restrict airflow, forcing the system to work harder and consume more energy. This increased energy consumption leads to higher operational costs and a greater environmental impact. Furthermore, the accumulation of contaminants accelerates the wear and tear of mechanical components, shortening the lifespan of the system and increasing the frequency of repairs.

[0009] Current HVAC cleaning methods are often inadequate for addressing the full spectrum of contaminants. Manual cleaning is labour-intensive, costly, and often fails to reach all areas of the system, especially in complex and inaccessible components. Chemical treatments, while effective to some extent, pose risks to building occupants and the environment and may not completely eradicate biofilms and other persistent contaminants.

[0010] Regular maintenance of HVAC systems is essential to ensure their efficiency and longevity. However, many facilities struggle with maintaining consistent cleaning schedules due to high costs, limited resources, and the complexity of the systems. Irregular and insufficient maintenance exacerbates the problems of contaminant build-up and system inefficiency, which leads to high cleaning costs.

[0011] Cleaning and maintaining HVAC systems often require partial or complete shutdowns, disrupting normal operations in commercial buildings and critical healthcare services in hospitals. This disruption can be particularly challenging in environments where continuous operation is essential.

[0012] Commercial buildings and hospitals must comply with stringent indoor air quality regulations set by organizations such as OSHA, ASHRAE, AIRAH, and the CDC and EPA. Failure to maintain clean HVAC systems can result in non-compliance, leading to legal and financial consequences.

[0013] SUMMARY

[0014] The present invention addresses at least some of the hereinbefore described problems by providing a method, apparatus and / or system for delivery of one or more bacterial strains to HVAC systems as a natural, safe and eco-friendly antimicrobial agent. The method, apparatus and / or system enhances at least one of HVAC air quality, reduces health risks, improves energy efficiency, extends system longevity, and offers a cost-effective and compliant solution for maintaining hygienic HVAC systems.

[0015] In a preferred form, the method, apparatus and / or system is an automated, integrated method, apparatus and / or system.

[0016] An aspect of the invention provides a method for maintaining microbial hygiene in an HVAC environment including the steps of: producing a biologically active hygiene solution that comprises one or more microorganisms and / or one or more bioactive molecules; and cyclically or continuously delivering the biologically active hygiene solution into one or a plurality of HVAC systems and / or environments; wherein producing the biologically active hygiene solution includes: providing purified water and one or more nutrients at a target pH; measuring at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and regulating microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

[0017] In an embodiment, a distribution system cyclically or continuously delivers the biologically active hygiene solution into the HVAC system(s) and / or environment(s).

[0018] In an embodiment, the method is at least partly automated to cyclically or continuously produce, measure, regulate and / or deliver the hygiene solution to the HVAC system(s) and / or environment(s).

[0019] In an embodiment, the one or more microorganisms are bacteria.

[0020] In an embodiment, the bacteria are spore-forming bacteria.

[0021] In particular embodiments, said one or more microorganisms comprise one or more Bacillus species. Preferably, the hygiene solution comprises at least one strain of Bacillus subtilis and / or Bacillus amyloliquefaciens. Suitably, these bacteria degrade organic contaminants and reduce airborne pathogens within the HVAC system.

[0022] In a particular embodiment, the bacteria comprise one or more probiotic bacteria or a formulation comprising probiotic bacteria.

[0023] Suitably, the one or more microorganisms, such as bacteria, are cultivated preferably in a bioreactor. Suitably, the bioreactor is in fluid communication with the distribution system, thereby providing the hygiene solution to the distribution system.

[0024] Suitably, the concentration or bacteria during and / or after cultivation is about 1O4-1O10cfu / mL.

[0025] Preferably, the biologically active hygiene solution is at a temperature of about 30-37°C.

[0026] Preferably, the pH is about 6.5 to 7.5.

[0027] Preferably, dissolved oxygen levels are in the range of about 5-8 mg / L.

[0028] Preferably, turbidity is about 0.8 - 1.5 at OD600.

[0029] Preferably, total dissolved solids is in the range 500-1500 ppm.

[0030] In an embodiment, hygiene solution droplets are delivered to the HVAC system(s) and / or environment(s) in the range of about 1-50 microns. Preferably, the hygiene solution is delivered to the HVAC system(s) and / or environment(s) at critical airflow convergence points, including at least one of: air handling unit (AHU) cabinets, supply air ducts, return air ducts, cooling coils, evaporators, or exhaust pathways.

[0031] In use, the method disrupts biofilms, degrades organic matter, competes with other microbes on HVAC surfaces, reduces airborne contaminants and / or substantially improves HVAC airstream quality.

[0032] Another aspect of the invention provides an HVAC hygiene apparatus or system comprising: a bioreactor configured to produce a biologically active hygiene solution that comprises one or more microorganisms and / or one or more bioactive molecules; a fluid supply system configured to provide purified water and one or more nutrients at a target pH to the bioreactor;. a distribution system configured to cyclically or continuously deliver the hygiene solution from the bioreactor into one or a plurality of HVAC systems and / or environments; a sensor array configured to measure at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and a control unit operatively connected to the sensor array and adapted to regulate: microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

[0033] Suitably, the bioreactor is in fluid communication with the distribution system, thereby providing the hygiene solution to the distribution system.

[0034] Suitably, the one or microorganisms are cultivated in the bioreactor.

[0035] Suitably, the apparatus and / or system disrupts biofilms, degrades organic matter, competes with other microbes on HVAC surfaces, reduces airborne contaminants and / or substantially improves HVAC airstream quality.

[0036] In an embodiment, the one or more micro-organisms are bacteria.

[0037] In an embodiment, the bacteria are spore-forming bacteria.

[0038] In particular embodiments, said one or more microorganisms comprise one or more Bacillus species. Preferably, the hygiene solution comprises at least one strain of Bacillus subtilis and / or Bacillus amyloliquefaciens. Suitably, these bacteria degrade organic contaminants and reduce airborne pathogens within the HVAC system.

[0039] In a particular embodiment, the bacteria comprise one or more probiotic bacteria or a formulation comprising probiotic bacteria.

[0040] Suitably, the one or more microorganisms, such as bacteria, are cultivated in the bioreactor. Suitably, the concentration or bacteria during and / or after cultivation in the bioreactor is about 1O4-1O10cfu / mL.

[0041] In an embodiment, the bioreactor is thermally insulated. Suitably, the bioreactor comprises one or more of: a thermostatically controlled heater; and an aeration system. Suitably, the bioreactor maintains a temperature of about 30-37°C and dissolved oxygen levels in the range of about 5-8 mg / L.

[0042] In an embodiment, the fluid supply system comprises a water purification unit. In an embodiment the fluid supply system comprises one or more of: a reverse osmosis (RO) unit; a water purification unit and a pH control mechanism to facilitate maintaining water entering the bioreactor within a target pH range conducive to microbial growth. A preferred target pH range is about pH 6.5-7.5.

[0043] Preferably, turbidity is about 0.8 - 1.5 at OD600.

[0044] Preferably, total dissolved solids is in the range 500-1500 ppm.

[0045] In an embodiment, the distribution system comprises at least one misting device or aerosol sprayer or other fluid dispersion mechanism. In an embodiment, the distribution system comprises a high-pressure, low-volume pump adapted to facilitate producing and delivering droplets in the range of about 1-50 microns, thereby facilitating improved HVAC surface coverage and airborne dispersion. In a particular embodiment, the misting devices are positioned at critical airflow convergence points in the HVAC, including at least one of: air handling unit (AHU) cabinets, supply air ducts, return air ducts, cooling coils, evaporators, or exhaust pathways.

[0046] In an embodiment, the sensor array is integrated with the bioreactor.

[0047] In an embodiment, the control unit comprises an automated feedback loop configured to adjust microbial dosing and / or nutrient levels. In particular embodiments, this is based on real-time sensor data, historical HVAC contamination records and / or user-defined thresholds.

[0048] Suitably, the control unit maintains operational logs of dosing intervals, resource consumption, and contamination data for predictive maintenance, compliance reporting, and trend analysis.

[0049] In an embodiment, the system further comprises a remote monitoring module. The remote monitoring module may be operable by way of a cloud-based platform to gather sensor data, track compliance with indoor air quality standards, and / or issue automated maintenance or contamination alerts.

[0050] Suitably, the control unit is configured to implement advanced data analytics or an intelligent control algorithm to predict contamination trends and optimize microbial solution parameters, optionally incorporating machine learning or Al-based models without requiring continuous human oversight.

[0051] It will be appreciated that in a preferred form, the bioreactor, sensor array, control unit and distribution system are integrated into an automated apparatus or system that can cyclically or continuously produce, measure, regulate and / or deliver the hygiene solution to the HVAC system(s) and / or environment(s).

[0052] A further aspect of the invention provides a liquid culture medium comprising one or more polyols, yeast extract, one or more magnesium, iron, manganese and / or calcium compounds, a pH buffer, one or more organic acids, one or more antimicrobial preservatives, one or more antioxidants, one or more antifoaming agents and optionally one or more surfactants.

[0053] A preferred embodiment of the liquid culture medium is shown in Table 4.

[0054] Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

[0055] It will also be appreciated that the indefinite articles "a" and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers.

[0056] The term "about" is used herein to refer to a tolerance or variation in a stated amount. The tolerance or variation may be no more than ± 10%, ± 9%, ± 8%, ± 7%, ± 6%,± 5%, ± 4%, ± 3%, ± 2%, ± 1% of a stated amount.

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Reference is made to the drawings in describing non-limiting embodiments of the invention wherein: FIG. 1 provides an overview of an embodiment of the apparatus and / or system;

[0059] FIG. 2 provides an embodiment of a distribution system; and

[0060] FIG. 3 provides an embodiment of a distribution system configured for a plurality of HVAC systems.

[0061] DETAILED DESCRIPTION

[0062] The invention is a method, apparatus and / or system that maintains optimal hygiene in HVAC systems by utilizing cultivation of beneficial bacterial strains to produce a hygiene solution deliverable to the HVAC system. This system is typically installed in HVAC plant rooms of buildings and operates cyclically or continuously to enhance air quality, disrupt biofilms, and reduce allergens and organic contaminants in the HVAC system. The method, apparatus and / or system may be performed with manual or automated features that include sensing and control of bacterial cultivation, temperature, pH, dissolved oxygen levels and turbidity, although without limitation thereto. A distribution system facilitates optimal delivery of the hygiene solution to the HVAC system by way of manually or automatically controlled misting.

[0063] An aspect of the invention provides a method for maintaining microbial hygiene in an HVAC environment including the steps of: producing a biologically active hygiene solution, wherein the hygiene solution comprises one or more microorganisms and / or one or more bioactive molecules; and cyclically or continuously delivering the biologically active hygiene solution into one or a plurality of the HVAC system(s) and / or environment(s)s; wherein producing the biologically active hygiene solution includes: providing purified water and one or more nutrients at a target pH; measuring at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and regulating microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

[0064] Another aspect of the invention provides an HVAC hygiene apparatus or system comprising: a bioreactor configured to produce a biologically active hygiene solution, wherein the hygiene solution comprises one or more microorganisms and / or one or more bioactive molecules; a fluid supply system configured to provide purified water and one or more nutrients at a target pH to the bioreactor; a distribution system configured to cyclically or continuously deliver the hygiene solution from the bioreactor into one or a plurality of the HVAC system(s) and / or environment(s)s; a sensor array configured to measure at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and a control unit operatively connected to the sensor array and adapted to regulate: microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution

[0065] Suitably, the method, apparatus and / or system disrupts biofilms, reduces airborne contaminants and / or substantially improves HVAC airstream quality.

[0066] As generally used herein, the term "HVAC environment" refers to one or more components, regions or other features of a HVAC system including but not limited to a HVAC airstream, air handling unit (AHU) cabinets, supply air ducts, return air ducts, cooling coils, evaporators and / or exhaust pathways. At least some of the aforementioned may be referred to as "critical airflow convergence points” in the HVAC system(s) and / or environment(s). The hygiene solution disclosed herein may comprise one or more microorganisms and / or one or more bioactive molecules.

[0067] The one or more microorganisms are preferably bacteria. As generally used herein, "bacteria" may include Gram positive and Gram negative bacteria, aerobic anaerobic and facultative anaerobic bacteria, probiotic bacteria, rods, bacilli and spore-forming bacteria which may include naturally occurring bacterial species, isolates and strains, bacterial species and strains produced by laboratory culturing and selection and also genetically-modified or enhanced bacterial species, isolates and strains.

[0068] In some embodiments, preferred bacteria may be referred to herein as "beneficial bacteria". Typically, the beneficial bacteria have antimicrobial properties, environmental resilience and are non-pathogenic and / or otherwise safe to use in the apparatus, system and method. Suitably, these beneficial bacteria provide effective pathogen control, environmental safety, promotion of beneficial microbial balance, versatility, stability, cost-effectiveness and broad application potential. Pathogenic microbes may include bacteria, fungi (e.g yeasts), viruses (e.g airborne respiratory viruses) and protozoa including but not limited to Pseudomonas, Staphylococcus Enterococcus, Escherichia, Legionella, Candida, Blastomyces, Histoplasma and Aspergillus species.

[0069] Preferably, beneficial bacteria produce one or more antimicrobial compounds (e.g bacteriocins, lipopeptides, and antibiotics) that inhibit the growth of a wide range of pathogenic bacteria and fungi. Further to this, beneficial bacteria preferably produce enzymes and compounds that break down biofilms, enhancing the effectiveness of antimicrobial treatments. The antimicrobial compounds produced by beneficial bacteria may be natural and / or biodegradable, thereby reducing environmental impact compared to synthetic chemicals. The beneficial bacteria and their byproducts are typically non-toxic.

[0070] In some embodiments, beneficial bacteria may be typically used in agriculture (e.g as plant growth promotion), food industry (e.g in food preservation, extending shelf life) and / or healthcare (e.g as probiotics, topical antimicrobial treatments). In this regard, the beneficial bacteria may act as probiotics which normally colonize various environments such as the gut, skin and plants and outcompete pathogenic microorganisms to promote a healthy microbiome. In some embodiments, beneficial bacteria may stimulate a host immune system, enhancing its ability to fight off infections. In some embodiments, the beneficial bacteria form resilient spores and are stable in various environmental conditions, thus are long-lasting and suitable for different applications.

[0071] Suitably, the beneficial bacteria are readily cultured on a cost-effective large scale, whereby antimicrobial compounds by the beneficial bacteria reduce the need for conventional antibiotics and pesticides, lowering overall treatment costs.

[0072] A non-limiting list of bacteria contemplated by the present invention and examples of sources for these is provided in Table 1.

[0073] Suitably, said one or more microorganisms comprise one or more Bacillus species, Paenibacillus species, Lysinibacillus species, Aneurinibacillus species, one or more Priestia species, one or more Heyndrickxia species, one or more Brevibacillus species and / or combinations thereof.

[0074] Preferably, the hygiene solution comprises at least one strain of Bacillus subtilis and / or Bacillus amyloliquefaciens.

[0075] B. subtilis has broad spectrum antimicrobial activity such as through production of subtilin, bacitracin, and other compounds. B. subtilis also produces resilient spores that can withstand extreme conditions. In a particular embodiment, Bacillus subtilis is strain qst713

[0076] B. amyloliquefaciens has broad spectrum antimicrobial activity such as by producing iturins, bacillomycin, and fengycins which assists in reducing antibioticresistant bacteria. B. amyloliquefaciens can be produced cost-effectively production on a large scale.

[0077] Suitably, these bacteria degrade organic contaminants and reduce airborne pathogens within the HVAC system.

[0078] In an embodiment, the beneficial bacterium is, or comprises, Bacillus licheniformis. B. licheniformis has broad spectrum antimicrobial activity such as by producing lichenysin and bacitracin. B. licheniformis also forms spores which are highly stable and long-lasting.

[0079] In an embodiment, the beneficial bacterium is, or comprises, Bacillus pumilus. This bacterium has broad spectrum antimicrobial activity such as by producing pumilacidins and subtilisin and therefore provides an effective alternative to conventional antibiotics.

[0080] In further embodiments, bacteria may include one or more of:

[0081] Bacillus velezensis, which is closely related to B. amyloliquefaciens but genetically distinct and has a strong biosurfactant profile;

[0082] Bacillus clausii, a probiotic known for antimicrobial metabolite production and stress resilience;

[0083] Bacillus safensis which is isolated from extreme environments and is a resilient surface colonizer with biosurfactant traits;

[0084] Paenibacillus polymyxa, a spore-forming, nitrogen-fixing bacterium with strong biofilm disruption capabilities and antimicrobial peptides;

[0085] Lysinibacillus sphaericus, which produces biosurfactants and enzymes environmentally used for microbial hygiene and pest control;

[0086] Bacillus mojavensis, an antifungal surfactin producer that is genetically close to B. subtilis but independently viable;

[0087] Bacillus sonorensis, a thermotolerant, protease-rich strain similar to B. licheniformis with distinct taxonomy; and / or

[0088] Aneurinibacillus migulanus, which produces antimicrobial peptides and biosurfactants used in agricultural biocontrol.

[0089] Priestia megaterium has fungicidal and antiviral activity and produces penicillin amidase used to make synthetic penicillin and several enzymes, such as amylases and glucose dehydrogenase, enzymes for modifying corticosteroids, amino acid dehydrogenases and bioactive molecules such as cyclic lipopeptides, belonging to the surfactin, iturin, and fengycin lipopeptide families;

[0090] Heyndrickxia coagulans, which is a spore-forming probiotic bacterium that produces lactic acid; and / or

[0091] Brevibacillus laterosporus, which is a probiotic facultative anaerobe which produces mall peptides known as AMPs, which exhibit a broad spectrum of inhibitory efficacy against bacteria, fungi, and viruses.

[0092] Suitably, the one or more microorganisms, such as bacteria, are cultivated in the bioreactor. Preferably, the concentration or bacteria during and / or after cultivation is about 1O4-1O10cfu / mL. This includes about 105-109cfu / mL, about 106-108cfu / mL, about 107cfu / mL, about 5x 105-109cfu / mL, about 5 x 106- 5 x 108cfu / mL and about 5 x 107cfu / mL

[0093] The microorganisms disclosed herein are typically cultivated after inoculation into a culture medium that is prepared with specific concentrations of nutrients at a target pH required for optimal microorganism (e.g bacteria) growth and viability. Typically, the culture medium is sterilized to eliminate any unwanted contaminating microorganisms.

[0094] The culture is maintained at an optimal temperature range of 30-37°C, preferably using a thermostatically controlled heater. This temperature range may include about 31°, 32°, 33°, 34°, 35° and 36°C. Preferably, temperature sensors continuously monitor the culture medium to ensure consistent conditions.

[0095] The pH is maintained within a target range of pH about pH 6.5-7.5. This includes about pH 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 and 7.4.

[0096] Maintenance of pH may be achieved by way of a pH controller that automatically doses pH buffering agents as needed. In an embodiment, pH sensors provide real-time monitoring and feedback to the control system. Non-limiting examples of pH buffering agents include: sodium bicarbonate (NaHCO3), potassium bicarbonate (KHCO3), sodium carbonate (Na2CO3), potassium carbonate (K2CO3), calcium carbonate (CaCO3), magnesium carbonate (MgCO3), sodium citrate (trisodium salt), potassium citrate, disodium phosphate (Na2HPO4), dipotassium phosphate (K2HPO4), TRIS (Tris(hydroxymethyl)aminomethane), MOPS buffer and HEPES buffer, although without limitation thereto.

[0097] Oxygen levels are preferably maintained using an aerator (e.g an air pump) such as with fine bubble diffusers. Dissolved oxygen sensors monitor oxygen levels may be monitored continuously or periodically, whereby the control system adjusts aeration as necessary.

[0098] Preferred oxygen levels are about 5-8 mg / L, which includes about 5.5, 6.0, 6.5, 7.0 and 7.5 mg / L.

[0099] The concentration of total dissolved solids (e.g minerals, salts and organic matter) is preferably maintained within an optimal range of 500-1500 ppm. This includes about 600, 700, 800, 900, 1000, 1100, 1200, 1300 and 1400 ppm. Suitably, total dissolved solids (TDS) sensors continuously monitor the medium, and an automated dosing system adds water or nutrients based on readings.

[0100] Preferably, turbidity is about 0.8 - 1.5 at OD600. This includes about 0.9, 1.0, 1.1, 1.2, 1.3 and 1.4 at OD600.

[0101] The culture medium is suitably a liquid medium or "broth". Non-limiting examples include Luria Bertani (LB) broth, nutrient broth, glucose broth, brain-heart infusion (BHI) broth, alkaline peptone water (APW), tryptic soy broth (TSB), and selenite F broth. However, the present invention also provides a liquid culture medium that is preferred to the aforementioned media for bacterial growth.

[0102] Accordingly, in a further aspect the invention provides a liquid culture medium comprising one or more polyols (e.g glycerol), yeast extract, one or more magnesium, iron, manganese and / or calcium compounds (e.g mineral salts), a pH buffer, one or more organic acids (e.g citric acid monohydrate, lactic acid, tartaric acid), one or more antimicrobial preservatives, one or more antioxidants, one or more antifoaming agents and optionally one or more surfactants.

[0103] The pH buffer may comprise one or more of the pH buffering agents hereinbefore described.

[0104] A preferred embodiment of the liquid culture medium is shown in Table 4, including preferred components, concentrations and concentrations ranges. Data in the Examples demonstrate the effectiveness of the liquid culture medium disclosed herein compared to LB and TSB nutrient media.

[0105] A non-limiting example of preparation of the liquid culture medium as a 10X concentrate includes the following steps:

[0106] 1. Start with 6-7 L of RO or deionized water, preheated to 40-50 °C.

[0107] 2. Add and mix ingredients in the following order: polyol (e.g glycerol), yeast extract, mineral salts, pH buffers, citric acid, antioxidants, preservatives, surfactant (if used), antifoam.

[0108] 3. Top up to exactly 10 L with RO water after full dissolution (~15 minutes mixing).

[0109] 4. Pasteurize by heating to 65-70 °C for 30-40 minutes. 5. Cool to below 40 °C and store in sterile, light-blocking food-grade containers.

[0110] The liquid culture medium of this aspect is a nutrient medium or broth for HVAC bioreactor applications by inclusion of sporulation triggers (e.g. Mn, Ca), surfactant and enzyme enhancement (Fe, Mg, glycerol), pH buffering and stability during extended cycles, mist-compatible behaviour, controlled foaming under aeration and / or preserved shelf stability for automated systems.

[0111] As previously described, the hygiene solution may comprise one or more bioactive molecules, alone or in combination with the one or more beneficial bacteria. Generally, the one or more bioactive molecules facilitate biofilm disruption, organic matter degradation, allergen reduction, and competitive exclusion of pathogens within HVAC systems and / or environments.

[0112] In one embodiment, the bioactive molecule is a compound normally produced by beneficial bacteria, such as hereinbefore described. The bioactive molecule of this embodiment may be purified from bacterial cultures, chemically synthesized or produced in recombinant form.

[0113] In another embodiment, the bioactive molecule may comprise one or more biosurfactants. The biosurfactants may be naturally produced by the cultured bacteria within the system or dosed from an external source in purified, chemical synthetic or recombinant form. Table 2 provides some non-limiting examples of biosurfactants.

[0114] In yet another embodiment, the bioactive molecules may comprise one or more enzymes.

[0115] The enzymes may be naturally produced by the cultured bacteria within the system or dosed from an external source in purified, chemical synthetic or recombinant form. Enzymes may act synergistically to digest organic matter embedded on HVAC surfaces, expose and destabilize pathogenic microbial colonies and spores and / or facilitate deeper penetration of the hygiene solution, although without limitation thereto.

[0116] Table 3 provides non-limiting examples of enzymes.

[0117] As hereinbefore described, the hygiene solution comprising the one or more microorganisms and / or one or more bioactive molecules is delivered to the HVAC system(s) and / or environment(s). In an embodiment, delivery is by way of a distribution system.

[0118] Suitably, the distribution system is configured to cyclically or continuously deliver the hygiene solution from the bioreactor into one or a plurality of HVAC systems and / or environments. Suitably, the distribution system is in fluid communication with the bioreactor, whereby the bioreactor provides hygiene solution to the distribution system.

[0119] It will be appreciated that the distribution system may be configured to deliver the hygiene solution comprising the one or more microorganisms and / or one or more bioactive molecules to each of a plurality of HVAC systems and / or environments. Suitably, delivery to each of the plurality of HVAC systems and / or environments is independently controllable.

[0120] In an embodiment, the distribution system comprises at least one misting device or aerosol sprayer or other fluid dispersion mechanism. In an embodiment, the distribution system comprises a high-pressure, low-volume pump adapted to facilitate producing and delivering droplets in the range of about 1-50 microns, thereby facilitating improved HVAC surface coverage and airborne dispersion. In some embodiments, the droplets may be about 2-40, 3-30, 4-20 or 5-10 microns. In a particular embodiment, the misting devices are positioned at critical airflow convergence points in the HVAC, including at least one of: air handling unit (AHU) cabinets, supply air ducts, return air ducts, cooling coils, evaporators and / or exhaust pathways.

[0121] The distribution system may deliver hygiene solution to the HVAC system(s) and / or environment(s) cyclically or continuously. By this is meant that the hygiene solution is delivered to the HVAC system(s) and / or environment(s) periodically, such as at regular time intervals or for regular periods, or continuously while the HVAC system is in operation.

[0122] As previously described, a sensor array is configured to measure at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s). A control unit is operatively connected to the sensor array and adapted to regulate microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

[0123] Optimal pH, temperature, dissolved oxygen, total dissolved solids (TDS) and turbidity parameters are as hereinbefore described.

[0124] In an embodiment, the control unit comprises an automated feedback loop configured to adjust microbial dosing and / or nutrient levels. In particular embodiments, this is based on real-time sensor data, historical HVAC contamination records and / or user-defined thresholds.

[0125] Suitably, the control unit maintains operational logs of dosing intervals, resource consumption, and contamination data for predictive maintenance, compliance reporting, and trend analysis.

[0126] In an embodiment, the system further comprises a remote monitoring module. The remote monitoring module may be operable by way of a cloud-based platform to gather sensor data, track compliance with indoor air quality standards, and / or issue automated maintenance or contamination alerts.

[0127] Suitably, the control unit is configured to implement advanced data analytics or an intelligent control algorithm to predict contamination trends and optimize microbial solution parameters, optionally incorporating machine learning or Al-based models without requiring continuous human oversight.

[0128] It will be appreciated in light of the foregoing that in a preferred form, the bioreactor, sensor array, control unit and distribution system are integrated into an automated apparatus or system that can cyclically or continuously produce, measure, regulate and / or deliver the hygiene solution to the HVAC system(s) and / or environment(s).

[0129] By this is meant that the aforementioned components of the apparatus and system are integrated into a single unit. Further to this, production of the hygiene solution may include automated measurement and regulation of microbial cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution. Preferably, this measurement and regulation are performed in "real-time", although periodic or "end-point" measurement and regulation are also contemplated.

[0130] Referring to FIG. 1 apparatus or system 10 comprises bioreactor 11 which is preferably thermally insulated and is used to produce a hygiene solution comprising beneficial bacteria. Bioreactor 11 comprises a thermostatically controlled heater 12 to maintain the optimal temperature range of about 30-37°C for bacterial growth. Aerator 13 bubbles oxygen into bioreactor 11 by way of fine bubble diffuser 14 to achieve a preferred oxygen concentration of about 5-8 mg / L and also to facilitate mixing of the hygiene solution. Reverse osmosis (RO) system 15 is in fluid communication with mains water supply 16, backflow check valve 17 and 10-40 micron filter 18 to supply filtered, purified water to bioreactor 11. The RO system 15 may also provide regular maintenance alerts to ensure consistent water quality. Nutrient tank 19 is in fluid communication with bioreactor 11 and provides nutrients to the hygiene solution by way of dosing pump 25 and conduit 20. Culture tank 21 is in fluid communication with bioreactor 11 and provides beneficial bacteria by way of dosing pump 26 and conduit 22. pH balancing tank 23 is in fluid communication with bioreactor 11 and by way of dosing pump 27 and conduit 24. There is also a drain port 29 in bioreactor 11 to remove wastewater, excess beneficial microbes and / or enzymes.

[0131] Sensor array 28 in bioreactor 11 is configured to monitor and maintain optimal growing conditions for the beneficial bacteria: a pH sensor monitors the pH level of the liquid culture; a temperature sensor monitors the temperature of the hygiene solution; a total dissolved solids (TDS) sensor monitors the concentration of dissolved substances; a turbidity sensor monitors the turbidity levels for microbial growth; a dissolved oxygen sensor monitors oxygen levels; one or more nutrient sensors monitor the concentration of substances such as nitrogen, phosphorus, and / or potassium, although without limitation to these; and water level sensors monitor and facilitate maintaining appropriate water levels in the bioreactor. A central control unit (CCU) 40 comprises programmable logic controller 41 that processes data from sensor array 28 and controls heating, aeration, nutrient delivery, and water levels. Programmable logic controller 41 may be connected to a touchscreen display for local monitoring and manual control. A Wi-Fi / Ethernet module may facilitate remote monitoring and control.

[0132] Before provision to distribution system 50, hygiene solution is filtered by sediment filter 30 and enters holding tank 32 by way of respective conduits 31 and 33. Sediment filter 30 removes particles in the range 40-150 microns from hygiene solution exiting bioreactor 11 by way of conduit 31 before entering holding tank 32 by way of conduit 32. With reference to FIG. 1 and FIG. 2, pump 51 is preferably a high-pressure, low-volume pump that distributes the hygiene solution from holding tank 32 by way of conduit 34 and delivers the hygiene solution to HVAC system 60 by way of conduit 52 which delivers the hygiene solution to a plurality of small-diameter, high-pressure conduits 54. The direction of fluid flow is indicated by the solid arrow in FIG. 2. Micro-droplet misting heads 55 create a fine mist of the hygiene solution delivered to HVAC 60, preferably at set intervals. A preferred droplet size delivered by misting heads 55 is about 1-50 microns. Distribution points for the hygiene solution via misting heads 55 are strategically located within the HVAC system 60, including one or more of return air ducting, air handling units (AHUs), cooling and heating coils, air filters, supply air ducting and cooling towers (not shown).

[0133] As shown in FIG. 3, the distribution system 150 comprising pump 151 may be configured to deliver hygiene solution to each of a plurality of HVAC systems 161, 162, 163 by way of misting heads 155A, 155B and 155C.

[0134] In a typical HVAC plant room, bioreactor 11 with sensor array 28, aerator 13 and fine bubble diffuser 14, RO system 15, nutrient tank 19, culture tank 21, pH balancing tank 23, sensor array 28, central control unit (CCU) 40, sediment filter 30, holding tank 32, pump 50 and their associated conduits are located together in a cabinet or other closeable space (not shown), whereby the hygiene solution exits by way of conduit 52 for delivery to one or a plurality of HVAC systems or environments 60,160.

[0135] In use, the bioreactor 11 is filled with water, nutrients, and inoculated with beneficial bacteria. Sensor array 28 that measures pH, temperature, dissolved oxygen, TDS, and nutrient levels is calibrated and the programmable logic controller 40 is programmed with the target operating parameters. Real-time monitoring is conducted by way of sensor array 28 continuously monitoring the conditions in the bioreactor 11, ensuring optimal temperature, pH, dissolved oxygen, TDS, and nutrient levels. Automated adjustments are made by programmable logic controller 41 processing sensor array 28 data and making real-time adjustments to maintain optimal conditions.

[0136] Typical operational parameters are as follows: optimal pH range of 6.5-7.5, wherein the sensor data is sent to the programmable logic controller 40 for real-time adjustments; optimal temperature range of 30-37°C wherein the programmable logic controller 40 adjusts the thermostatically controlled heater based on sensor readings; optimal dissolved oxygen levels in bioreactor 11 in the range of 5-8 mg / L, wherein the programmable logic controller 41 controls the aerator 13 and fine bubble diffuser 14 to ensure adequate aeration; the concentration of total dissolved solids in the culture medium is optimally within 500-1500 ppm, wherein automated dosing systems adjust water and nutrient levels; and optimal turbidity levels of the culture medium are within 0.8 - 1.5 OD600, wherein automated dosing systems adjust microbe dosing.

[0137] The apparatus / system 10 is typically installed in a cabinet or other closeable space in a HVAC plant room and connected to a water supply, single-phase power, and water drains. All sensors, pumps, and distribution lines are connected and tested. Safety checks are conducted to ensure safe operation during the initial setup. This includes verifying all electrical connections and ensuring that there are no leaks in the water supply and drainage systems.

[0138] As previously described, the apparatus / system 10 periodically mists the hygiene solution into the HVAC 60, targeting critical airflow convergence points to ensure thorough coverage. The frequency of misting can be adjusted based on the HVAC system's usage, typically once a day, but this may be extended to once a day, week, or month. Periodic manual checks and maintenance is performed such as by checking spray tips for correct operation, replacing reverse osmosis filters (e.g annually), dosing fresh bacteria (e.g every three months and replacing the nutrient bottle as needed. Remote monitoring and control can include monitoring system performance remotely via a web interface or mobile app, featuring user-friendly dashboards and customizable alerts. Real-time data from sensor array 30 and operational logs may be accessible for analysis and predictive maintenance. The apparatus or system 10 generates automated alerts to notify of any deviations from optimal conditions or required maintenance tasks.

[0139] The apparatus or system 10 may also be manually controlled and operated ("manual mode"). A touchscreen display connected to the programmable logic controller 41 assists local monitoring and manual control of the system by providing real-time sensor data and operational status on the display. In manual mode, there can be manual adjustment of temperature, pH, dissolved oxygen levels and nutrient concentrations using the touchscreen interface. Manual controls allow for fine-tuning and immediate response to any specific requirements or unexpected conditions. In manual mode, apparatus or system 10 can be set to mist the hygiene solution on demand, based on immediate needs or specific cleaning schedules.

[0140] The method, apparatus and system disclosed herein provides a HVAC hygiene solution comprising beneficial bacteria that reduces bacterial and fungal pathogens, disrupts biofilms, controls allergens and break down of organic matter, leading to improved respiratory health for building occupants. The method, apparatus and system prevents clogging and blockages, reduces mechanical strain, and improves airflow, resulting in lower energy consumption and extended HVAC component lifespan. Automation reduces manual cleaning requirements, lowers energy bills, and minimizes maintenance costs and helps maintain compliance with air quality standards and provides a safe, eco-friendly alternative to chemical disinfectants.

[0141] The use of beneficial bacteria in the HVAC hygiene system offers significant environmental advantages over traditional chemical disinfectants. The beneficial bacteria strains employed in the method, apparatus and system produce natural antimicrobial compounds that effectively reduce bacterial and fungal pathogens without the need for harsh chemicals. This natural approach minimizes the environmental impact associated with chemical production, usage, and disposal. By replacing chemical disinfectants with a biological solution, the system eliminates the release of potentially harmful substances into the environment. Chemical disinfectants can contribute to water and soil pollution and may pose risks to wildlife and ecosystems. Beneficial bacteria are renewable resources that can be cultured and maintained with minimal environmental impact. The system uses a sustainable approach to HVAC hygiene, promoting long-term environmental health. The antimicrobial compounds produced by beneficial bacteria are biodegradable, ensuring that they break down into harmless substances after use. This reduces the risk of long-term environmental contamination. Maintaining clean HVAC systems through biological means enhances energy efficiency by preventing clogging and ensuring optimal airflow. Improved energy efficiency reduces the overall carbon footprint of the HVAC system.

[0142] To ensure the system is safe for human use and complies with regulatory standards, several safety measures are implemented. The bacterial strains used in the system are carefully selected for their safety and efficacy. Strains such as Bacillus subtilis, Bacillus amyloliquefaciens, and Lactobacillus species are generally recognized as safe (GRAS) by regulatory agencies. These strains do not pose health risks to humans or animals. The system delivers the hygiene solution in controlled amounts through a misting process, ensuring even distribution without over-application. This prevents excessive exposure to the bacteria and maintains a balanced microbial environment. Continuous monitoring of environmental conditions (temperature, pH, dissolved oxygen) ensures that the bacterial culture remains within safe operational parameters. The central control unit (CCU) adjusts conditions in real-time to prevent the growth of unintended or harmful microorganisms. The system is designed to comply with relevant regulatory standards for indoor air quality and safety, including guidelines from OSHA, ASHRAE, AIRAH, and the CDC. Regular testing and validation ensure that the system meets these standards and provides a safe indoor environment.

[0143] EXAMPLES

[0144] INTRODUCTION

[0145] This invention is directed to hygiene treatment of HVAC systems by delivering a hygiene solution comprising one or more microorganisms and / or bioactive molecules to disrupt biofilms, degrade organic matter, compete with other microbes on HVAC surfaces, reduce airborne contaminants and / or substantially improve HVAC airstream quality. Experiments were performed to determine which bacterial microorganisms are suitable for this purpose by measuring activity against pathogenic microbes including Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae bacteria and Candida albicans and Aspergillus brasiliensis fungi. The bacterial microorganisms tested were B. subtilis, B. licheniformis, B.amyloliguefaciens and B. lateralis.

[0146] Also tested was the efficacy of a nutrient medium as set forth in Table A, which is referred to in these Examples as "PBV nutrient" medium.

[0147] EXAMPLE 1

[0148] Anti-bacterial activity test of B. subtilis, B. licheniformis and B. lateralis

[0149] A sample of each of the test samples (Table 5) was diluted in synthetic hard water for samples diluted at point of use (or distilled water in the case of ready to use samples). A test suspension of each bacterial strain and interfering substance is then added to the dilutions and maintained at 20°C for 1-60 minutes (general purpose disinfection) or 30-60 seconds (hand hygiene samples). At the end of the contact time an aliquot is taken, and the bacterial / bacteriostatic activity is immediately neutralized or suppressed by the method. The numbers of surviving bacteria in each sample are determined and the reduction is calculated.

[0150] The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae as bacterial strains.

[0151] Samples can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance.

[0152] At least a 5 logic reduction in viable bacterial counts is considered to be indicative of efficacy of the test sample. The test is deemed valid where all control requirements are met.

[0153] Testing showed failure to fully neutralize the sample. Neutralization was achieved using a 1:100 dilution of the sample. In order to account for the addition serial dilution from 1:10 to 1:100, 10 plates per duplicate have been sampled for the

[0154] -1 dilution. These were then summed to give the equivalent count at neat.

[0155] The data are shown in Tables 5-13. Test samples 001.04, 002.02, 003.02 and 004.02 did not achieve a >5 logic reduction.

[0156] Test samples 005.06, 006.04 and 007.05 achieved a >5 logw reduction.

[0157] This suggests that B. subtilis alone or together with B. licheniformis and B. lateralis when grown in PBV nutrient medium (see Table 4 for this formulation) provide superior anti-bacterial activity.

[0158] EXAMPLE 2

[0159] Anti-fungal activity test of B. subtilis, B. licheniformis and B. lateralis

[0160] This European Standard test specifies a test method and the minimum requirements for fungal / yeasticidal activity of chemical disinfectant and antiseptic samples that form a homogeneous, physically stable preparation when diluted with hard water, or - in the case of ready-to-use samples - with water. Samples can only be tested at a concentration of 80% or less (97% with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to samples that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means.

[0161] A sample of the test sample (Table 14) as delivered and / or diluted with hard water (or water for ready to use samples) was added to a test suspension of fungi in a solution of an interfering substance. The mixture was maintained at 20°C for the contact times specified for the relevant sample. At the end of this contact time, an aliquot was taken; the biocidal action in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is calculated.

[0162] The test is performed using Candida albicans and Aspergillus brasiliensis fungal strains.

[0163] To be considered "anti-fungal", at least one concentration per test sample should demonstrate a 4 logw or more reduction (for hygienic handwash samples 2 logw or more) and at least one concentration should demonstrate a logw reduction of less than 4 (for hygienic handwash samples less than 2).

[0164] The data are shown in Tables 14-21.

[0165] Test samples 001.04, 002.02, 003.02 and 004.02 did not achieve a >4 logic reduction.

[0166] Test samples 005.06, 006.04 and 007.05 achieved a >4 logw reduction.

[0167] This suggests that B. subtilis alone or together with B. licheniformis when grown in PBV nutrient medium (see Table 4 for this formulation) provide superior anti-fungal activity.

[0168] EXAMPLE 3

[0169] Anti-bacterial test of B. subtilis, B.amyloliquefaciens , B. licheniformis and B. lateralis

[0170] This standard method BS EN 1276:2019 describes a suspension test method for establishing whether a test sample has or does not have bactericidal activity.

[0171] The test takes into account practical conditions of application of the sample, including contact time, temperature, test organisms and interfering substance, ( / .e. conditions which may influence its action in practical situations).

[0172] The test sample is diluted in synthetic hard water for samples diluted at point of use (or distilled water in the case of ready to use samples). A test suspension of bacteria and interfering substance is then added to the dilutions and maintained at 20°C for 1-60 minutes (general purpose disinfection) or 30-60 seconds (hand hygiene samples) At the end of the contact time an aliquot is taken, and the antibacterial / bacteriostatic activity is immediately neutralized or suppressed by the validated method. The numbers of surviving bacteria in each sample are determined and the reduction is calculated.

[0173] The test is performed using Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae bacteria.

[0174] Samples can only be tested at a concentration of 80 % or less, as some dilution is always produced by adding the test organisms and interfering substance.

[0175] Anti-bacterial activity is confirmed when the sample tested as above demonstrates at least a 5 logic reduction in viable bacterial counts. The test is deemed valid where all control requirements are met.

[0176] This method has been audited by UKAS to the ISO 17025 standard, for tests where no deviations from the standard method are stipulated.

[0177] Testing showed failure to fully neutralize the sample. Neutralization was achieved using a 1:100 dilution of the sample. In order to account for the addition serial dilution from 1:10 to 1:100, 10 plates per duplicate have been sampled for the -1 dilution. These were then summed to give the equivalent count at neat.

[0178] The data are shown in Tables 23-27.

[0179] Test samples 008.04, 009.04 and 010.03 achieved a >5 logic reduction when tested as above. Thus, B. subtilis and B. amyloliquefaciens showed anti-bacterial activity together, whether in the presence or absence of B. lateralis and / or B. licheniformis.

[0180] EXAMPLE 4

[0181] Anti-fungal activity test of B. subtilis, B.amyloliquefaciens, B. licheniformis and B. lateralis

[0182] This European Standard test specifies the minimum requirements for fungal / yeasticidal activity of test samples that form a homogeneous, physically stable preparation when diluted with hard water, or - in the case of ready-to-use samples - with water. Samples can only be tested at a concentration of 80% or less (97% with a modified method for special cases) as some dilution is always produced by adding the test organisms and interfering substance. This European Standard applies to samples that are used in the medical area in the fields of hygienic handrub, hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and surface disinfection by wiping, spraying, flooding or other means

[0183] Test sample neat and / or diluted with hard water (or water for ready to use samples) is added to a test suspension of bacteria in a solution of an interfering substance. The mixture is maintained at 20°C for the contact times specified for the relevant sample. At the end of this contact time, an aliquot is taken; the biocidal action in this portion is immediately neutralized or suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane filtration is used. The numbers of surviving bacteria in each sample are determined and the reduction is calculated.

[0184] The test is performed using Candida albicans and Aspergillus brasiliensis fungi.

[0185] Anti-fungal activity is considered to be shown where at least one concentration per test s a 4 logic or more reduction (for hygienic handwash samples 2 logw or more) and at least one concentration demonstrating a logw reduction of less than 4 (for hygienic handwash samples less than 2).

[0186] The data are shown in Tables 28-32.

[0187] Test samples 008.04, 009.04 and 010.03 achieved a >4 logw reduction when tested as above.

[0188] Thus, B. subtilis and B. amyloliquefaciens showed antifungal activity together, whether in the presence or absence of B. lateralis and / or B. licheniformis.

[0189] Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

[0190] All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

[0191]

[0192] Table 2: Biosurfartants

[0193] Biosurfactant Example Compounds Function in HVAC Hygiene

[0194] Class

[0195] Cyclic Biofilm disruption, anti-microbial

[0196] Surfactin, Iturin A, Fengycin lipopeptides membrane lysis, surface wetting

[0197] Wetting, detergent-like action, surface

[0198] Glycolipids Rhamnolipids, sophorolipids tension reduction

[0199] Mild surfactant effect, biocompatibility

[0200] Phospholipids Lysophosphatidylcholine enhancer

[0201] Table 3 : Enzymes

[0202] Enzyme Class Example Enzymes Function in HVAC Hygiene

[0203] Break down protein-based organic debris, skin

[0204] Proteases Subtilisin, alkaline protease cells, dust-bound allergens

[0205] Degrade starch-based residues (e.g. paper dust,

[0206] Amylases Alpha-amylase textile fibres)

[0207] Hydrolyse oils, skin sebum, fatty acid residues on

[0208] Lipases Lipase A, phospholipase coils and ducts

[0209] Endoglucanase, Decompose plant-based cellulose particles or

[0210] Cellulases beta-glucosidase fibre dust

[0211] Break down hemicellulose, aiding in biofilm

[0212] Xylanases Xylanase penetration

[0213] Chitinases Chitinase A Target fungal biofilms and mould structures

[0214] Laccases / Oxidative degradation of biofilm matrices and

[0215] Oxidoreductases peroxidases VOC residues Table 4: Liquid nutrient medium composition

[0216] (1000* concentrate; intended for 1:1000 dilution into a 50 L bioreactor)

[0217] Batch Volume: 10 L

[0218] Target pH (before dilution): ~6.5-6.8

[0219] Shelf life: 12 months unopened under standard storage conditions

[0220] Dilution: 1 mL per 1 L of reactor refill volume

[0221] Ingredient List and Functional Roles:

[0222] Concentration

[0223] Ingredient Function in Formulation (per 10 L batch)

[0224] Primary carbon source. Supports sustained

[0225] 300 g (range 100- microbial metabolism and surfactin biosynthesis.

[0226] Glycerol (99.5%) 600g) Stable and non-sugar-based, reducing contamination risk.

[0227] Complex nitrogen source. Provides peptides, amino

[0228] Yeast Extract (low 200 g (range 50- acids, and vitamins essential for vegetative growth ash) 500g) and enzyme production. Low ash content minimizes residue.

[0229] Enzyme cofactor. Supports metabolic enzyme

[0230] Magnesium Sulfate

[0231] 20 g (range 5-100g) systems and contributes to stability of (MgSO4-7H2O) biosurfactant output.

[0232] Calcium source. Triggers sporulation pathways and

[0233] Calcium Gluconate 10 g (range 2-50g) aids in buffering against pH shifts. More soluble and stable than carbonate forms.

[0234] Manganese Sulfate Essential trace element. Induces sporulation in

[0235] 0.5 g (range 0.1-2g) (MnSO4-H2O) Bacillus species and activates proteolytic enzymes.

[0236] Iron source. Supports biosurfactant

[0237] Ferric Ammonium

[0238] 2 g (range 0.2-5g) (surfactin / fengycin) synthesis. Highly soluble and Citrate chelated to reduce precipitation risk.

[0239] Potassium Part of dual buffer system. Maintains stable pH

[0240] Phosphate 10 g (range 2-50g) during microbial metabolism and enhances

[0241] Monobasic (KH2PO4) phosphorus availability.

[0242] Sodium Phosphate Complementary buffer component. Stabilizes pH

[0243] 5 g (range 1-25g)

[0244] Dibasic (Na2HPO4) and prevents acidification under high respiration.

[0245] Citric Acid Mild acidulant and chelator. Helps maintain pH and

[0246] 1 g (range 0.2-5g) (monohydrate) solubilize trace minerals. Concentration

[0247] Ingredient Function in Formulation

[0248] (per 10 L batch)

[0249] Ascorbic Acid Antioxidant. Prevents oxidative degradation of the

[0250] 1 g (range 0.2-5g)

[0251] (Vitamin C) formula and stabilizes iron species in solution.

[0252] GRAS antimicrobial preservative. Extends shelf life

[0253] Sodium Benzoate 2 g (range 0.2-5g) and prevents contamination of concentrate.

[0254] GRAS antifungal preservative. Complements

[0255] Potassium Sorbate 2 g (range 0.2-5g) sodium benzoate for broad-spectrum preservation.

[0256] Non-ionic surfactant. Enhances droplet dispersion

[0257] Tween-80 during misting, improving coverage on HVAC (Polysorbate 80) 1 g (range 0.2-5g) surfaces. Optional depending on misting system

[0258] (optional) specs.

[0259] Food-grade silicone emulsion. Suppresses foam

[0260] Antifoam FG-10 0.5-1 .0 g (range formation during aeration in the bioreactor. Critical (PDMS) 0.1 -5g) for stable reactor operation under continuous airflow.

[0261] Table 5

[0262] Table 7

[0263] Table 8 Table 9

[0264] Table 10 Table 11 Table 13

[0265] KEY

[0266] No Logion umber of cfu / ml at the beginning of the contact time = A / / 10

[0267] Nvo is the number of cfu / ml in the validation test suspension at the beginning of the contact time

[0268] A is the verification of experimental conditions control

[0269] B is the neutraliser toxicity control

[0270] C is method validation

[0271] Vc is the colony forming units counted per 1 ml of sample x~ is the average of Vfci & I / C2 x" wm is the weighted mean of A /

[0272] Na Logio number of surviving cfu / ml in the test mixture

[0273] R (Ig No- Ig Na = Ig R) is the calculation for reduction in viability

[0274] Bolded values indicate target log or higher reduction in viability b.sub = B. subtil is b.lich = B. licheniformis b.lat = B. lateralis Table 14

[0275]

[0276] Table 16

[0277] Table 17

[0278] Table 18 Table 19

[0279] Table 20

[0280] Table 21 Table 22

[0281] KEY

[0282] NO LoglO number of cfu / ml at the beginning of the contact time = A / / 10

[0283] NvO is the number of cfu / ml in the validation test suspension at the beginning of the contact time

[0284] A is the verification of experimental conditions control

[0285] B is the neutraliser toxicity control

[0286] C is method validation

[0287] Vc is the colony forming units counted per 1ml of sample x~ is the average of Vc] & Vc2 x" wm is the weighted mean of A /

[0288] Na LoglO number of surviving cfu / ml in the test mixture

[0289] R (Ig NO - Ig Na = Ig R) is the calculation for reduction in viability

[0290] Bolded values indicate target log or higher reduction in viability b.sub = B. subtil is b.lich = B. licheniformis

[0291] Table 23

[0292]

[0293] Table 25

[0294] Table 26 Table 27

[0295] KEY

[0296] No Logion umber of cfu / ml at the beginning of the contact time = A / / 10

[0297] Nvo is the number of cfu / ml in the validation test suspension at the beginning of the contact time

[0298] A is the verification of experimental conditions control

[0299] B is the neutraliser toxicity control

[0300] C is method validation

[0301] Vc is the colony forming units counted per 1 ml of sample x~ is the average of Vfci & I / C2 x" wm is the weighted mean of A /

[0302] Na Logio number of surviving cfu / ml in the test mixture

[0303] R (Ig No - Ig Na = Ig R) is the calculation for reduction in viability

[0304] Bolded values = Concentration showed target log reduction or greater b.sub = B. subtil is b.lich = B. licheniformis b. a my = B.amyloliquefaciens b.lat = B. lateralis Table 2§

[0305] Table 30

[0306] Table 31

[0307] Table 32

[0308] KEY

[0309] NO Log10 number of cfu / ml at the beginning of the contact time = A / / 10

[0310] NvO is the number of cfu / ml in the validation test suspension at the beginning of the contact time

[0311] A is the verification of experimental conditions control

[0312] B is the neutraliser toxicity control

[0313] C is method validation

[0314] Vc is the colony forming units counted per 1 ml of sample x~ is the average of Vc^ & Vc2 x" wm is the weighted mean of A /

[0315] Na Log10 number of surviving cfu / ml in the test mixture

[0316] R (Ig NO - Ig Na = Ig R) is the calculation for reduction in viability

[0317] Bolded values = Concentration showed at least target log reduction or greater b.sub = B. subtil is b.lich = B. licheniformis b. a my = B.amyloliquefaciens b.lat = B. lateralis

Claims

CLAIMS1. A method for maintaining microbial hygiene in an HVAC environment including the steps of: producing a biologically active hygiene solution, wherein the hygiene solution comprises one or more microorganisms and / or one or more bioactive molecules; and cyclically or continuously delivering the biologically active hygiene solution into one or a plurality of HVAC systems and / or environments; wherein producing the biologically active hygiene solution includes: providing purified water and one or more nutrients at a target pH to the biologically active hygiene solution; measuring at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and regulating microorganism cultivation; at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

2. A HVAC hygiene apparatus or system comprising: a bioreactor configured to produce a biologically active hygiene solution that comprises one or more microorganisms and / or one or more bioactive molecules; a fluid supply system configured to provide purified water and one or more nutrients at a target pH to the bioreactor; a distribution system configured to cyclically or continuously deliver the hygiene solution from the bioreactor into one or a plurality of HVAC systems and / or environments, wherein the bioreactor is in fluidcommunication with the distribution system, thereby providing the hygiene solution to the distribution system; a sensor array configured to measure at least one parameter selected from pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity, the concentration of the one or more bioactive molecules and / or or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and a control unit operatively connected to the sensor array and adapted to regulate: microorganism cultivation; at least one parameter selected from target pH, temperature, dissolved oxygen, total dissolved solids (TDS), turbidity, humidity; the concentration of the one or more bioactive molecules and / or nutrient concentration within the biologically active hygiene solution and / or the HVAC system(s) and / or environment(s); and / or cyclic or continuous delivery of the hygiene solution.

3. The apparatus or system of Claim 2, wherein the one or more microorganisms are cultivated in the bioreactor.

4. The apparatus or system of Claim 2 or Claim 3, wherein the sensor array is integrated into the bioreactor.

5. The apparatus or system of any one of Claims 2-4, wherein the bioreactor is thermally insulated.

6. The apparatus or system of any one of Claims 2-5, wherein the bioreactor comprises one or more of: a thermostatically controlled heater; and an aeration system.

7. The apparatus or system of any one of Claims 2-6, wherein the fluid supply system comprises one or more of: a reverse osmosis (RO) unit; a water purification unit; and a pH control mechanism to facilitate maintaining water entering the bioreactor within a target pH range conducive to microbial growth.

8. The apparatus or system of any one of Claims 2-7, wherein the distribution system comprises one or more misting devices or aerosol sprayers that produce hygiene solution droplets in the range of about 1-50 microns.

9. The apparatus or system of Claim 8, which comprises a plurality of misting devices that deliver hygiene solution droplets in the range of about 1-50 microns.

10. The apparatus or system of Claim 8 or Claim 9 wherein the misting devices are positioned at critical airflow convergence points in the HVAC, including at least one of: air handling unit (AHU) cabinets, supply air ducts, return air ducts, cooling coils, evaporators, or exhaust pathways.

11. The apparatus or system of any one of Claims 8-10, wherein the distribution system comprises a high-pressure, low-volume pump that facilitates producing droplets in the range of about 1-50 microns for delivery by the misting devices.

12. The apparatus or system of any one of Claims 2-11, wherein the bioreactor, sensor array, control unit and distribution system are integrated into an automated apparatus or system that can periodically or continuously produce, measure, regulate and / or deliver the hygiene solution to the HVAC system(s) and / or environment(s).

13. The method of Claim 1, or the apparatus or system of any one of Claims 2-12, wherein the one or more micro-organisms are bacteria.

14. The method, apparatus or system of Claim 11, wherein, said one or more microorganisms are selected from the bacteria listed in Table 1.

15. The method, apparatus or system of Claim 13 or Claim 14, wherein, said one or more microorganisms comprise one or more Bacillus species.

16. The method, apparatus or system of any one of Claims 13-15, wherein, said one or more microorganisms comprise at least one strain of Bacillus subtilis and / or Bacillus amyloliquefaciens.

17. The method, apparatus or system of any one of Claims 13-16, wherein the bacteria comprise one or more probiotic bacteria or a formulation comprising probiotic bacteria.

18. The method of any one of Claims 1 or 13-17, or the apparatus or system of any one of Claims 2-17, wherein the bioactive molecules comprise one or more enzymes.

19. The method, apparatus or system of Claim 18, wherein the one or more enzymes are listed in Table 3.

20. The method of any one of Claims 1 or 13-19, or the apparatus or system of any one of Claims 2-17, wherein the bioactive molecules comprise one or more biosurfactants.

21. The method, apparatus or system of Claim 20, wherein the one or more biosurfactants are listed in Table 2.

22. The method of any one of Claims 1 or 13-21, or the apparatus or system of any one of Claims 2-21, wherein the temperature is about 30-37°C.

23. The method of any one of Claims 1 or 13-22, or the apparatus or system of any one of Claims 2-22, wherein dissolved oxygen levels are in the range of about 5-8 mg / L.

24. The method of any one of Claims 1 or 13-23, or the apparatus or system of any one of Claims 2-23, wherein turbidity is about 0.8 - 1.5 at OD600.

25. The method of any one of Claims 1 or 13-24, or the apparatus or system of any one of Claims 2-24, wherein total dissolved solids is in the range 500-1500 ppm.

26. The method of any one of Claims 1 or 11-25 or the apparatus and / or system of any one of Claims 2-25, which disrupts biofilms, degrades organic matter, competes with other microbes on HVAC surfaces, reduces airborne contaminants and / or substantially improves HVAC airstream quality.

27. A liquid culture medium comprising one or more polyols, yeast extract, one or more magnesium, iron, manganese and / or calcium compounds, a pH buffer, one or more organic acids, one or more antimicrobial preservatives, one or more antioxidants, one or more antifoaming agents and optionally one or more surfactants.

28. The liquid culture medium of Claim 27, which is prepared as a concentrate.

29. The liquid culture medium of Claim 27 or Claim 28 comprising the compounds listed in Table 4.

30. The method of any one of Claims 1 or 13-26, or the apparatus or system of any one of Claims 2-26, wherein the one or more microorganisms are cultured in the liquid culture medium of any one of Claims 27-29.

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