BR112017021943B1 - METHOD FOR ANTICIPATING A PROBLEM CAUSED BY MICROORGANISM IN A WATER PROCESS SYSTEM - Google Patents

Abstract

METHOD FOR ANTICIPATING A PROBLEM CAUSED BY MICROORGANISM IN A WATER PROCESS SYSTEM. The invention is directed to methods and compositions for identifying the specific microorganisms present in a particular portion of a water process system. The method involves obtaining a sample from the process and determining whether a problematic organism exceeds an acceptable threshold. If exceeded, a remediation biocontrol procedure can be applied well before any defects or unwanted problems occur.

Description

CROSS REFERENCE TO RELATED ORDERS

[001] This application is filed as an international PCT application on April 14, 2016, claiming the benefit of US Application No. 14/687,017, filed on April 15, 2015, the disclosure of which is incorporated in its entirety.

DECLARATION REGARDING RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

[002] Not Applicable.

BASICS OF THE INVENTION

[003] The present invention generally relates to compositions of matter, apparatus and methods useful in determining diversity and viability thresholds. The threshold can be used to determine whether microorganisms are contributing to process efficiency problems or product quality problems. The threshold is also useful in identifying where a particular microorganism entered a process stream and/or where it proliferated.

[004] The presence of unwanted microorganisms in an industrial process stream can lead to a series of problems, such as deposition on equipment surfaces, impaired machine performance, loss of production efficiency and out-of-specification final products. Having the ability to accurately identify which organisms are proliferating and how they are doing so can greatly help in dealing with these problems.

[005] For example, in papermaking systems microbial growth can be very harmful and costly. The growth of microorganisms on equipment surfaces can lead to the formation of deposits that flake off and contribute to sheet defects and pinholes. Shower water treatments or contaminated process water can lead to the growth of microbes on felts that commonly result in the formation of felt plugs. These plugs, in turn, cause a series of problems most notably in compromising water removal from the paper web. As a result microbial growth can result in an excessive and costly need for multiple pre-cleanings and cleanings of felts or other papermaking equipment. These problems can be compounded when an incorrect determination of which microorganisms occur due to the fact that this can result in a requirement for aggressive chemical treatment which can lead to a reduction in the life of the felt, further degrade the quality of the paper, additional impact on process equipment and/or may not even control the underlying microbial infestation. Furthermore, incorrectly distinguishing between biologically caused problems and mechanically or chemically caused problems can still result in inappropriate, wasteful, and possibly counterproductive efforts.

[006] A number of prior art methods are known to identify which microorganisms are present in a papermaking system. These methods, however, are particularly deficient when applied to paper sheets or felts. Some of the prior art methods such as US Patents 8,012,758, 7,981,679, 7,949,432 detect various effects on papermaking system fluids produced by living microbiological organisms. Other methods such as US 5,281,537 rely on obtaining a sample of contaminant from a living microorganism and growing more of it in order to perform various analyses. In the context of paper sheets and felt, however, these methods are particularly inadequate, because by the time samples of the felt or paper are taken they no longer contain enough living organisms (or any of them) to culture or any of the chemicals they produce. Additionally, items in the papermaking system (such as paper sheets and felts) that are downstream of the heating or drying sections will have all defect-causing microorganisms killed after they have already caused the defects. Alternative methods that do not depend on the presence of living organisms also tend to be deficient due to the fact that they often produce false positives. For example, ninhydrin (which is used to detect primary or secondary amines) and IR spectroscopy often produce false positives or negatives due to the fact that they detect materials that may have non-biological origins (such as chemical additives or contamination). ).

[007] Thus, it is clear that there is clear utility in new methods and compositions for the adequate identification of microorganisms present in industrial process streams and equipment. The art described in this section is not intended to constitute an admission that any patent, publication, or other information referred to herein is “Prior Art" with respect to this invention unless specifically designated as such. Furthermore, this section should not be interpreted to mean that a search has been made or that no other pertinent information, as defined in 37 CFR § 1.56(a), exists.

BRIEF SUMMARY OF THE INVENTION

[008] To satisfy the long-existing but unresolved needs identified above, at least one embodiment of the invention is directed to a method for anticipating a problem caused by microorganisms in a water process system. The method comprises the steps of: measuring the total microorganism population in at least a portion of the system; measuring the amount of at least one subgroup of the microorganism population in relation to the total microorganism population; determining whether the amount of at least one subgroup of microorganisms exceeds a threshold, the threshold being at least one of a predetermined: absolute amount of the subgroup of microorganisms, a relative amount of the subgroup of microorganisms and any combination thereof; and, optionally, establishing a biocontrol procedure to remedy the presence of at least one subgroup of microorganisms.

[009] The subgroup of microorganisms may comprise filamentous bacteria. The subgroup of microorganisms may comprise Spirogyra, Cladophora, Pithophora, O. Siphonocladales pithophora pithophora, Ulvibacter littoralis, Vetellibacter vladivostokensis, Weeksella virosa, Fucobacter, Gelidbacter, Ornithobacterium rhinotracheale, Riemerella cohambina, Flavobacterium psychrofilum, Ctyophaga succinicans, Vladibacter, Pfeiffer ella, Bacillus aquatillis, Flexibacter marinus, Flavobacterium odoratum, Microscilla, Flexithrix, Capnocytophaga, Taxeobacter, Sporocytophaga, Saprospira, Chryseobacterium, Hymenobacter and any combination thereof. The threshold may be that the subgroup of microorganisms is measured to be in an amount of at least 5 to 90% of the total microorganism population.

[0010] At least one measurement may be taken by a process selected from the list consisting of: Chain Termination Sequencing, Denaturation Gradient Gel Electrophoresis (DGGE), Digital Polymerase Chain Reaction, Baseline Analysis of DNA, Ion Torrent Semiconductor Sequencing, MALDI, MALDI-TOF, Mass Spectrometry, PCR Analysis, Pyrosequencing, qPCR Analysis, Sequencing by Ligation and Oligonucleotide Detection, Sequencing by Synthesis, measurement of antibodies associated with one or more microorganisms, measuring hormones associated with one or more microorganisms, measuring secretions associated with one or more microorganisms, measuring proteins associated with one or more microorganisms, measuring organic molecules associated with one or more microorganisms, measuring molecules associated with one or more plus microorganisms, fluorescent phages, cell separators and any combination thereof.

[0011] The method may further comprise identifying an input source that is the cause of at least a subset of the microorganism population. The input source may be feed water, raw materials, fiber or dyes from a source selected from the group consisting of: municipal water, tap water, lake water, sea water, lake water, filtered water , desalinated water, recirculated water, waste water, distilled water, condensed boiler water, cooling water, titanium dioxide, clay, process additives, polymers, dyes, glossing agents and any combination thereof.

[0012] The method may further comprise the step of identifying the water source from which the subgroup of microorganisms comes and establishing a biocontrol procedure to remedy the presence of the at least one subgroup of microorganisms only subsequent to and no later than 1 week from the introduction of water from the water source from which the subgroup of microorganisms comes.

[0013] The water process system may be one or more stages of an item selected from the group consisting of: papermaking, cooling water, boiler water, food manufacturing, beverage manufacturing, ore processing , alumina processing, biodiesel manufacturing, distillation, petrochemical refining, petrochemical synthesis, polymer synthesis, plastic manufacturing, wastewater processing, laundry, dishwashing, water treatment, solid-liquid separation and any combination thereof. The method may further comprise the step of recording the identified organism in a format that can be stored and/or transmitted.

[0014] The method may further comprise the step of conducting a biocidal program associated with the remediation of the identified organism. The method may further comprise taking at least one measurement of a sample product from the process system. The sample may be so desiccated that there are few or no living organisms in it.

[0015] Additional features and advantages are described here and will be evident from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] A detailed description of the invention is hereinafter described with specific reference being made to the drawings, in which:

[0017] Figure 1 illustrates the pie charts of qPCR analysis of felts from a paper machine, without felt permeability problems (PM2 capture felt) and felt samples from the machine with felt permeability problems (PM2 felt). PM1 capture).

[0018] Figure 2 illustrates pie charts of relative abundance of bacteria in felt fabrics over time as determined by qPCR. The numbers on the pie charts represent the total bacterial load of the sample.

[0019] Figure 3 is a graph illustrating the life (in days) of felt fabric in the machine before (Month 1) and during (Months 2 to 7) program optimization.

[0020] For the purposes of this disclosure, similar reference numerals in the figures will refer to similar features unless otherwise indicated. The drawings are only an exemplification of the principles of the invention and are not intended to limit the invention to the particular embodiments illustrated.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0021] The following definitions are provided to determine how the terms used in this application and, in particular, how the claims will be interpreted. The organization of the definitions is for convenience only and is not intended to limit any of the definitions to any particular category.

[0022] “Chain Termination Sequencing” means a method that requires a single-stranded DNA template, a DNA primer, a DNA polymerase, normal deoxynucleoside triphosphates (dNTPs) and modified nucleotides (dideoxyNTPs) that terminate strand elongation. DNA. These chain-terminating nucleotides lack a 3'-OH group necessary for the formation of a phosphodiester bond between two nucleotides, causing DNA polymerase to cease DNA extension when a ddNTP is incorporated. ddNTPs can be radioactively or fluorescently labeled for detection on automated sequencing machines. In some cases chain termination sequencing may involve the synthesis of new DNA strands complementary to a single-stranded template (step I). The DNA template is provided with a mixture of all four deoxynucleotides, four dideoxynucleotides--each labeled with a different colored fluorescent marker, and DNA polymerase (step II). Since all four deoxynucleotides are present, chain elongation continues until, by chance, DNA polymerase inserts a dideoxynucleotide. The result is a new set of DNA chains of all different lengths (step III). The fragments are then separated by size using gel electrophoresis (step IV). When each labeled DNA fragment passes through a detector at the bottom of the gel, the color is recorded. The DNA sequence is then reconstructed from the color pattern representing each nucleotide sequence (step V).

[0023] “Defect” means an undesired attribute of an item associated with an industrial process or process stream, in the context of a papermaking process that includes, but is not limited to, one or more plugs in a felt, and these attributes of paper such as holes, discoloration, scratches, stains, translucent spots and any combination thereof.

[0024] “Denaturating Gradient Gel Electrophoresis (DGGE)” means a form of electrophoresis that uses a chemical gradient to denature the sample as it moves through an acrylamide gel. DGGE can be applied to nucleic acids such as DNA and RNA, thus allowing the user to evaluate the number of different DNA/RNA sequences present in a sample. A representative example can be found in the Scientific Article: Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA, by G. Muyzer et al., Applied and Environmental Microbiology, Vol. 59, No.3, pp. 695-700, March (1993).

[0025] “Digital Polymerase Chain Reaction” or (digital PCR, DigitalPCR, dPCR or dePCR) means a refinement of conventional polymerase chain reaction methods that can be used to directly quantify and clonally amplify nucleic acids including DNA, cDNA or RNA. The key difference between dPCR and traditional PCR lies in the method of measuring quantities of nucleic acids with the former being a more accurate method than PCR. PCR performs a single sample reaction. dPCR also performs a single reaction within a sample, however, the sample is separated into a large number of partitions and the reaction is performed on each partition individually. This separation allows for more reliable collection and sensitive measurement of nucleic acid quantities. Representative examples are described in Patent 6,143,496 and in the scientific article Digital PCR, by B. Vogelstein et al., Proc. Natl. Academic. Sci. USA, Genetics, Vol. 96, pp. 9236-9241, August (1999).

[0026] “DNA-Based Analysis” means a method of DNA analysis including, but not limited to, qPCR, PCR, digital PCR, ion semiconductor sequencing, pyrosequencing, sequencing by synthesis, sequencing by litigation, termination sequencing. chain and any combination thereof.

[0027] “Felt” means a belt made of interwoven wool or any other fiber used in a papermaking process which functions as a material conveyor in which the interwoven fibers define a plurality of lumens through which water or other fluids pass. can pass. Felts can also provide cushioning between press rolls and can also be a means used to remove water from papermaking materials. Felts include, but are not limited to, bottom felts, bottom plate felts, cylinder cloth wet felts, dryer felts, endless felts, pickup felts, suction pickup felts, Harper top felts, and top felts .

[0028] “Ion Torrent Semiconductor Sequencing” means a DNA sequencing method based on the detection of hydrogen ions that are released during DNA polymerization. This is a “sequencing by synthesis” method, during which a complementary strand is constructed based on the sequence of a template strand, a representative example of this is described on the website: http://www.lifetechnologies.com/us/ en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing- technology.html (as accessed December 23, 2013).

[0029] “MALDI” stands for Matrix Assisted Laser Desorption/Ionization, a form of Mass Spectrometry used to identify biomolecules (biopolymers such as DNA, proteins, peptides and sugars) and large organic molecules (such as polymers, dendrimers and other macromolecules), it typically involves ablation to a gaseous ionic state from a sample by pulsing a laser against it and then analyzing the sample by mass spectrometry. Representative examples of using MALDI to identify organisms are described in the scientific articles:

[0030] Phyloproteomics: Species Identification of Enterobacteriaceae using Matrix-Assisted Laser Desorption/ Ionization Time-of Flight Mass Spectrometry, by G. C. Conway, et al., J. Mol. Microbiol. Biotechnol. 3: 103-112, (2001),

[0031] Real-time identification of bacteria and Candida species in positive blood culture broths by matrix-assisted laser desorption ionizationtime of flight mass spectrometry, by A. Ferroni, et al., J Clin Microbiol., 48(5), 1542- 1548, (2010 May), and

[0032] Application of MALDI-TOF MS for the Identification of Food Borne Bacteria, by M. Pavlovic, et al., Open Microbiol J.; 7: 135-141 (2013).

[0033] “Mass Spectrometry” or (MS) means an analytical chemistry technique used to identify the amount and type of chemicals present in a sample by measuring the mass to charge ratio and the abundance of ions in the gas phase, the same is more fully described in the textbook: Mass Spectrometry - A Foundation Course, by K. Downard, Cambridge UK: Royal Society of Chemistry (2004).

[0034] “Microorganisms” means any organism small enough to insinuate itself into, adjacent to, on top of, or attached to equipment used in an industrial process or stream, such as, but not limited to, a papermaking process, the same includes, but is not limited to, those organisms so small that they cannot be seen without the aid of a microscope, collections or colonies of such small organisms that can be seen with the naked eye, but which comprise a series of individual organisms that are too small to be seen with the naked eye, as well as one or more organisms that can be seen with the naked eye, they include, but are not limited to, any organism whose presence in any way hinders a water process system, the The same also includes, but is not limited to, those so-identified microorganisms described in the textbooks: Brock Biology of Microorganisms (14th Edition) Hardcover, by Michael T. Madigan et al., Benjamin Cummings Publisher, (2014) and The Prokaryotes: Vol 7: Proteobacteria: Delta and Epsilon Subclasses. Deeply Rooting Bacteria, by Dworkin, M., Falkow, S. Springer Science and Business Media, (2006).

[0035] “Paper Product or Paper Sheet” means any final product with a fibrous structure formed from a traditional papermaking process, but not necessarily comprising cellulose fibers. Examples of such end products include, but are not limited to, facial tissue, bath tissue, tablecloths, copy paper, printing paper, writing paper, notebook paper, newspaper, cardboard, printing paper, bond paper, paperboard and the like.

[0036] “Papermaking Process” means any portion of a method of manufacturing paper products from pulp comprising forming an aqueous cellulosic papermaking supply, draining the supply to form a sheet, and drying the sheet. The steps of forming the papermaking supply, draining and drying can be carried out in any conventional manner generally known to those skilled in the art. The papermaking process may also include a pulping stage, i.e. making pulp from a lignocellulosic raw material and bleaching stage, i.e. chemical treatment of the pulp to improve gloss, the same may also include , but without limitation, one or more of such steps as pulping, digestion, refining, drying, calendering, pressing, crepeing, dewatering, and bleaching, papermaking is further described in reference Handbook for Pulp and Paper Technologists, 3rd Edition , by Gary A. Smook, Angus Wilde Publications Inc., (2002) and The Nalco Water Handbook (3rd Edition), by Daniel Flynn, McGraw Hill (2009) in general and in particular, pp. 32.1 to 32.44.

[0037] “PCR analysis” means polymerase chain reaction analysis.

[0038] “Buffer” means a solid, semisolid, viscous deposit and/or other deposit of material positioned within the lumens of a felt. Plugs can inhibit the flow of material through the lumens and/or can damage any other functionality of a felt.

[0039] “Primer” means a composition of matter, typically a short strand of nucleotides, known to be complementary to specific sections of DNA and serve as a starting point for synthesis of a nucleotide chain complementary to DNA adjacent to the specific section of DNA.

[0040] “Probe” means a composition of matter constructed and arranged to bind to a target DNA section and which can be readily detected when so bound and thereby be used to indicate the presence or absence of the target DNA section.

[0041] “Pyrosequencing” means a method of DNA sequencing (determining the order of nucleotides in DNA), based on the principle of “sequencing biosynthesis”. It differs from Sanger sequencing, in that it depends on detecting the release of pyrophosphate upon nucleotide incorporation, rather than chain termination with dideoxynucleotides. The desired DNA sequence has the ability to be determined by light emitted upon incorporation of the next complementary nucleotide by the fact that only one of four of the possible A/T nucleotides /C/G be added and available at a time, so that only one letter can be incorporated into the single-stranded template (which is the sequence to be determined.) A representative example can be found in the Article Pyrosequencing Sheds Light on DNA Sequencing, by Mostafa Ronaghi, Genome Research, 11:3-11 (2001) which can be found at http://genome.cshlp.Org/content/11/1/3.full.html#ref-list-1 (as accessed December 23, 2013).

[0042] “qPCR analysis” means quantitative and/or qualitative polymerase chain reaction analysis.

[0043] “Sequencing by Ligation and Oligonucleotide Detection” means a way to generate hundreds of millions to billions of short sequence reads at a time. This is achieved by attaching small fragments of an unknown sequence to magnetic beads which then undergo immersion PCR. Representative examples can be found on the website: http://gtc.soe.ucsc.edu/content/solid-technology-overview (as accessed December 23, 2013). And the sales brochure: See the Difference Discover the Quality Genome by Life Technologies Corporation (2010).

[0044] “Sequencing by Synthesis” means a technique used to determine the series of base pairs in DNA, also known as DNA sequencing. This sequencing method is based on reversible dye terminators that allow the identification of simple bases when they are introduced into DNA strands. Representative examples can be found on the website: http://nxseq.bitesizebio.com/articles/sequencing-by-synthesis-explaining-the- illumina-sequencing-technology/ and http://www.illumina.com/ sequencing technology/ technology.ilmn (as accessed December 23, 2013).

[0045] In the event that the above definitions or a description indicated elsewhere in this application are inconsistent with a meaning (explicit or implied) that is commonly used in a dictionary or indicated in a source incorporated by reference in this application, the Terms of the particular application and claims are understood to be construed in accordance with the definition or description of this application and not in accordance with the common definition, dictionary definition, or the definition that is incorporated by reference. In view of the above, in the case where a term can only be understood if it is interpreted by a dictionary, if the term is defined by the Kirk-Othmer Encyclopedia of Chemical Technology, 5th Edition, (2005), (Published by Wiley, John & Sons, Inc.) this definition will control how the term is defined in the claims. All chemical structures illustrated also include all possible alternative stereoisomers.

[0046] At least one embodiment of the invention is directed to a method for determining the threshold at which a change in the diversity index guarantees a change in biocontrols of a process system. As described, for example, in US Patent Applications 13/374,949, 13/550,748, and 14/138,526 a diversity index is a useful tool for determining the potential for a defect or problem caused by a microorganism. Furthermore, identifying infestations of specific microorganisms can help indicate what types of defects or problems may result. Diversity indices depend on identifying a threshold of change in the index that is a precursor to such a defect or problem. Thus, there is great utility and value in methods for determining the threshold at which a microorganism is considered to be the main cause of the problem.

[0047] In at least one embodiment of the method of determining whether a change in the diversity index indicates a potential future or present problem or defect comprises: determining the microbial diversity present in a water process system, determining the abundance of one/some /each microorganism detected in relation to the total population, determine whether the abundance of the total population is indicative of which microorganisms are likely to cause a defect or problem, determine whether the relative or absolute abundance of any one or some microorganisms exceeds the predetermined threshold, and, optionally, establish a biocontrol protocol or procedure to eliminate/treat the microorganism(s).

[0048] In at least one embodiment, threshold value is identified using historical microbial and process efficiency and quality data that correlate the level of abundance associated with production issues and not merely variability in the analysis. This value may vary from one process application to another, as some systems may be able to tolerate higher levels of microbial contamination before the microbes negatively impact efficiency or product quality. In at least one embodiment further analysis is used to confirm that the treatment has reduced the population below the predetermined threshold to provide assurance that production or quality problems related to this growth of the microorganism will cease to occur.

[0049] In at least one embodiment for carrying out one or more of the above process steps, one or more methods, compositions and apparatus from any, any or all of the following references are used: US Patent Applications 13/289,547, 13/ 374,949, 13/550,748, 14/138,526, US Patents: 8,613,837, 7,018,793, 6,849,395, 6,054,267, US Published Patent Application: 2002/0031771, 2013/0186582, and Patent Documents International Patent: WO 2008/061193 A2, WO 2007/024295 A2, WO 2004/046375 A2, and WO 2004/042082 A1.

[0050] In at least one embodiment the method includes the step of measuring an increase in the amount of filamentous bacteria/microorganisms in relation to the total microbial population between up to 5% and up to 90% or more. In at least one embodiment the method includes the step of measuring a rise in the amount of at least one organism relative to the total microbial population between 5 to 90% selected from the list consisting of: Spirogyra, Cladophora, Pithophora, O. Siphonocladales pithophora pithophora , Ulvibacter littoralis, Vetellibacter vladivostokensis, Weeksella virosa, Fucobacter, Gelidbacter, Ornithobacterium rhinotracheale, Riemerella cohambina, Flavobacterium psychrofilum, Ctyophaga succinicans, Vladibacter, Pfeifferella, Bacillus aquatilllis, Flexibacter marinus, Flavobacterium odoratum, Microscilla, Flexithrix, Capno cytophaga, Taxeobacter, Sporocytophaga, Saprospira , Chryseobacterium, Hymenobacter and any combination thereof. In at least one embodiment the method includes the step of measuring an increase in the amount of at least one organism in relation to the total microbial population between 5 and 90%, the organism being one of those described or mentioned in the textbook The Prokaryotes: Vol 7: Proteobacteria: Delta and Epsilon Subclasses. Deeply Rooting Bacteria, by Dworkin, M., Falkow, S. Springer Science and Business Media, (2006).

[0051] In at least one embodiment the change in the diversity index is used to identify which process input is the potential or actual cause of a problem or defect. Such process inputs include, but are not limited to, water sources, material sources, and specific equipment. For example, a process water system includes a series of new and/or recirculating water sources that may include, but is not limited to, one or more types of: pond water, sea water, lake water, filtered water , desalinated water, recirculated water, waste water, distilled water, condensed water, condensed boiler water, cooling water and any combination thereof. Furthermore, the process system may have one or more raw materials or partially processed materials introduced into it. Furthermore, sometimes one or more pieces of equipment or pipes/flow lines are or are not used in a given operation or particular surfaces thereof do not come into contact with the process object. If a particular problem is the result of an organism originating from only one or a few of these process inputs, it would be effective to use only one biocontrol process when these process inputs are being used or target treatment and/or add additional treatment to the contaminated input. . Furthermore, it is more efficient to establish biocontrol when a threshold is crossed as opposed to waiting until too much of the problem organism accumulates/grows to actually cause a defect or problem.

[0052] In at least one embodiment the method of measuring some or all of the diversity indices using data utilizes one or more of: Chain Termination Sequencing, Denaturation Gradient Gel Electrophoresis (DGGE), Digital Polymerase Chain Reaction , DNA-Based Analysis, Ion Torrent Semiconductor Sequencing, MALDI, MALDI-TOF, Mass Spectrometry, PCR Analysis, Pyrosequencing, qPCR Analysis, Sequencing by Ligation and Oligonucleotide Detection, Sequencing by Synthesis, measurement of associated antibodies to one or more microorganisms, measurement of hormones associated with one or more microorganisms, measurement of secretions associated with one or more microorganisms, measurement of proteins associated with one or more microorganisms, measurement of organic molecules associated with one or more microorganisms, measurement of molecules associated with one or more microorganisms, fluorescent phages, cell separators and any combination thereof.

[0053] In at least one embodiment the process water system may be, but is not limited to, one or more process stages of one or more of: papermaking, cooling water, boiler water, food manufacturing, manufacturing beverage industry, ore processing, alumina processing, biodiesel manufacturing, distillation, petrochemical refining, petrochemical synthesis, polymer synthesis, plastic manufacturing, wastewater processing, laundry, dishwashing, water treatment, solid-liquid separation and any combination thereof.

[0054] In at least one embodiment the method can be applied to the detection of microorganisms in a variety of samples taken from a manufacturing system. These samples include deposits on equipment surfaces, water or fluid samples, and/or whole pieces or partial fragments of intermediate or final stage products. It can analyze defects in the product sample or in the product sample as a whole.

[0055] In at least one embodiment a highly sensitive and rapid detection method is provided for microorganisms located in intermediate or final products of a manufacturing process. The method includes analysis of materials present in sample extracts. These samples can be highly desiccated and may contain little or no live samples of the contaminating microorganisms. Some prior art methods of using DNA analysis include WO 2005/042082 which describes an in situ method using probes to determine the presence or absence of a microorganism. In situ methods, however, are not applicable to paper sheets or felts, as they are dry when sampled. Furthermore, the in situ method involves applying the probes during cell division of the microorganisms, which is not possible on paper sheets or felts with little or no living organisms on them. In at least one embodiment, DNA-based analysis involves the use of probes.

[0056] In at least one embodiment, DNA-based analysis involves the use of PCR primers to detect the presence or absence of microorganisms. US Patent 5,928,875 describes the use of PCR primers to detect the presence or absence of spore-forming bacteria. In at least one embodiment the primer is directed to a part of a DNA strand that is highly conserved among a group of organisms. As a result, detection of the presence of this particular piece of DNA is definitive proof of the presence of a specific organism. PCR analysis is of particular use in the analysis of samples due to the difficulty of correctly identifying their contaminating microorganisms, due to the fact that they lack viable organisms for traditional plating methods or ATP measurements.

[0057] In at least one embodiment, PCR analysis involves the use of one or more of the methods described in the Primer Article Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase, by Randall Saiki et al., Science, Volume 239, pp. 487-491 (1988). In at least one embodiment, PCR analysis involves the use of one or more of the methods described in the Article Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction, by Kary Mullis et al., Methods In Enzymology, Volume 155, pp. . 335-350 (1987).

[0058] In at least one embodiment the PCR analysis is a qPCR analysis as described in the Trade Brochure qPCR guide, prefaced by Jo Vandesompele, (as downloaded from the website http://www.eurogentec.com/file-browser.html at January 19, 2012). In at least one embodiment, the method is a quantitative qPCR analysis. In at least one embodiment, the method is a qualitative qPCR analysis.

[0059] As illustrated in at least one embodiment, once DNA is extracted from the sample using any of the commercially available DNA extraction kits, it can be analyzed in real time using a PCR approach, such as a Quantitative PCR approach. Quantitative PCR uses the same methodology as PCR, but it includes a real-time quantitative component. In this technique, primers are used to target a DNA sequence of interest based on the identity of the organism or the function of a specific gene. Some form of detection, such as fluorescence, can be used to detect the resulting DNA or 'DNA amplicon'. The change in fluorescence is directly proportional to the change in the amount of target DNA. The number of cycles required to reach the predetermined fluorescence threshold is compared to a standard that matches the specific DNA target. A standard is typically the target gene that is pure and of known quantity at concentrations spanning several logs. The number of target DNA copies present in the sample is calculated using the standard curve. The number of copies per sample is then used to determine the number of cells per sample.

[0060] In at least one embodiment a set of primers is used which targets bacterial DNA sequences using a conservative approach to quantify total bacteria. In at least one embodiment a set of primers is used which targets primary biofilm-forming bacteria, including Meiothermus, Pseudoxanthomonas, and Deinococcus. In at least one embodiment a set of primers is used to target an adaptive biofilm former that belongs to the Sphingomonadacea family of bacteria. In at least one embodiment the adaptive biofilm former has exhibited higher tolerance to oxidant-based biocontrol programs compared to other biofilm and planktonic microorganisms. In at least one embodiment, the primer is used to distinguish between fungal and bacterial infestations.

[0061] In at least one embodiment the process system involves materials passing into and out of shower streams and basins of liquids containing various microorganisms containing various microorganisms from which live samples can be readily obtained. Sometimes, however, the process system is a dynamic environment, including but not limited to changes such as transitioning between wet and dry conditions, rapid passage through air and liquids, and drastic changes in pH, temperature, salinity, light, rheology, viscosity, hydrophobicity, hydrophilicity and shape/orientation (such as bending, bending, rolling, etc.) and any combination thereof. As a result, the population of organisms inhabiting the sample at a given point in the process stream may be different from that present within water sources or application devices, such as shower streams and liquid basins. As a result a typical analysis of shower streams and liquid basins will not correctly identify which microorganisms are present within the sample. An analysis of a sample that takes into account the types of organisms that are known to be capable of inhabiting these environments, however, allows for a truly accurate analysis of sample contaminations.

[0062] In at least one embodiment the mode involves distinguishing between microorganisms at a category level such as the Domain Level. Biological life can be categorized according to one of three domains: Archaea, Bacteria, and Eukarya. Likewise the method involves distinguishing between microorganisms at the biological kingdom level category. Biological life can be categorized according to five kingdoms: Monera, Protista, Plant, Animal and Fungi. Organisms in these different categories have extremely different DNA and a protocol that focuses on identifying the organism's DNA in one of these distinctions is much simpler than more specific determinations. As different categories of organisms are often treated differently with samples, such a simple form of identification can be used to accurately identify the specific regimen best targeted at the particular contaminant.

[0063] In at least one embodiment more than one primer is used to identify organisms that have more than one uniquely recognizable nucleotide sequence. In at least one embodiment, PCR analysis is used to detect genome sequences associated with enzymes unique or nearly unique to specific organisms.

[0064] In at least one embodiment the method involves detecting a defect and then using PCR analysis to appropriately associate the source of the defect. In at least one embodiment the method determines whether the defect is entirely biological based, entirely non-biological chemical based, or resulting from a combination of non-biological chemical-based, biological mechanical sources.

[0065] In at least one embodiment the defect in the sample is one or more of: a hole, a hole with a discolored halo around at least a portion thereof, a sequence of discoloration, a stain, a translucent stain and any combination thereof.

[0066] In at least one embodiment a threshold level is the methodology used to discount false positives. Sometimes PCR analysis detects traces of organisms that, although present, are not causes of a particular defect. In at least one embodiment the method involves discounting the presence of any organism detected at a concentration lower than a known predetermined level for one or more particular organisms. In at least one embodiment the method involves discounting the presence of any organism detected at a level lower than 104 cells per gram (of the defect). In at least one embodiment the method involves discounting the presence of any organism detected at a level lower than 104 cells per ml.

[0067] In at least one embodiment, the results of the analysis are used to enhance the biocontrol program by determining how much, what type, and how often, one or more biocidal compositions are added to one or more locations within a process system. water.

[0068] In at least one embodiment, the method is capable of detecting microorganisms that would otherwise not be detected by prior art methods. For example, in cases where the scale is caused by an infestation of anaerobic or sulfate-reducing organisms, methods such as ORP detection would not correctly identify the source of scale as biological and would therefore incorrectly suggest applying a chemical approach rather than an antibiological one. Using the DNA approach, however, would always correctly indicate a biological infestation due to the fact that all life contains DNA.

[0069] In at least one embodiment, the method is used to assess microbial diversity. The method can be based on analysis of nucleic acids in sample extracts. More specifically, it uses PCR such as, but not limited to, qPCR, for the detection of whole organisms, such as bacteria; Sphingomonas species; Erythrobacterium species; Pseudomonas species; Burkholderia species; Haliscomenobacter species; Saprospira species; Schlegelella species; Leptothrix species; Sphaerotilus natans; Bacillus species; Anoxybacillus species; members of the phylum Cytophaga-Flavobacterium-Bacteroides; green non-sulfur bacteria, including Herpetosiphon, members of the phylum Deinococcus-Thermus, including Meiothermus species; catalase-producing bacteria, amylase-producing bacteria, urease-producing bacteria, fungi, etc. These techniques use primers and standard pairs that allow the detection and quantification of target organisms based on conserved sequences. Primers target regions in the microbial genome that are highly conserved through evolution, while primers for specific phyla or genera target more variable regions of the genome.

[0070] Being able to accurately quantify an organism of interest present in a sample makes it possible to express that organism as a percentage of the total bacterial load in the sample. Given the large number of organisms that can be detected, a snapshot of the diversity of the microbial population in the sample can be determined. This snapshot is called the diversity index. The diversity index can also be expressed quantitatively as the relative abundance of various target organisms. The diversity index for any part of a process can sometimes be measured when machines or processes are operating well, thus creating a baseline. The diversity index measured at times of poor machine or process performance can then be compared to the baseline to look for fluctuations in microbial populations and to determine which bacterial groups are responsible for problems in the process. The diversity index can also be quantified for ease of comparison using the Shannon Diversity Index calculation to compare monitoring data across sample sites or against a baseline. Treatment strategies and feeding points can then be changed accordingly to combat the problem.

[0071] A diversity index based on DNA quantification measures the presence and diversity of organisms in a process, regardless of their viability. Ribonucleic acid (RNA), specifically messenger RNA (mRNA), is a molecule that is produced only by living organisms and has properties such that, depending on the target, are unique to a specific phylum or genera of bacteria. By amplifying mRNA sequences that are unique to the organisms listed above it becomes possible to determine which bacteria are present in their viable form. Accurate detection of viable organisms can then be used as a tool to evaluate the effectiveness of process water treatment strategies. This can be achieved by comparing the diversity index with the viability index.

[0072] This method would quantify the amount and type of viable bacteria present in the process samples. The quantitative (real-time) polymerase chain reaction method can be applied to detect messenger ribosomal nucleic acids (mRNA). mRNA is transcribed DNA that is sent to the ribosome to serve as a template for protein synthesis in a process known as translation. mRNA is only produced by living cells. RNA from living cells can be isolated using commercially available kits. Detection of mRNA requires an extra step in the quantitative polymerase chain reaction. Reverse transcriptase is added to the reaction cocktail to transcribe the mRNA to its complementary DNA (cDNA). Two sets of primers are needed for this experiment. The first uses specific mRNA as a wash, while the second is used to amplify the resulting cDNA produced by the reverse transcriptase reaction.

EXAMPLES

[0073] The foregoing can be better understood with reference to the following examples which are presented for purposes of illustration and are not intended to limit the scope of the invention. In particular, the examples demonstrate representative examples of principles innate to the invention and these principles are not strictly limited to the specific condition recited in these examples. As a result, it will be understood that the invention encompasses various changes and modifications to the examples described herein and such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its intended advantages. Therefore, it is intended that such changes and modifications are covered by the attached claims.

EXAMPLE OF DETERMINING THRESHOLD VALUE

[0074] An uncoated free sheet mill was suffering from a felt permeability problem on one of its paper machines. The permeability of the felt fabrics on one of the machines in the factory decreased at a significantly greater rate than on the other two machines. All chemical means to solve this problem were exhausted and the factory began to look towards microbial contamination as a possible problem. However, the plant's biocontrol program was perceived to be working well due to low bacterial counts, low ATP measurements, and lack of sludge deposition on machine surfaces.

[0075] In order to resolve this issue, felt samples from the unaffected machine were compared with those from the affected machine using qPCR (Figure 1). Although both felts contained the same amount of total bacteria (~107 cells/g), it was immediately obvious that the problematic felt had a bacterial population that was dominated by a single group of organisms: adaptive biofilm formers. These bacteria were present in small quantities on the other felt.

[0076] After the discovery of a dominant bacterial population growing on the felt, the felt cleaning chemical was changed. The tissue was reanalyzed during the subsequent shutdown period. It was observed that the previous adaptive biofilm population was reduced to 25% of the total population, with the total bacterial load remaining unchanged (Figure 2).

[0077] Based on these data, a second chemical, which has been shown to have good efficacy against adaptive biofilm formers, was added to the felt washing protocol. After this second chemistry adjustment, the felts were reanalyzed during a shutdown period. DNA results showed that the total bacterial load remained unchanged, while adaptive biofilm formers were reduced to roughly 10% of the total population (Figure 2). The new chemical combination was allowed to operate in the machine for the next four months. Over these months, the previous adaptive biofilm population was eliminated from the felt fabrics in this machine. The elimination of this group of bacteria resulted in an improvement in the efficiency of water removal from the felts and in extending the life of the fabrics by 4 days (Figure 3).

[0078] Although this invention can be configured in many different ways, specific preferred embodiments of the invention are described in detail here. The present disclosure is an exemplification of the principles of the invention and is not intended to limit the invention to the particular embodiments illustrated. All patents, patent applications, scientific articles and any other reference materials mentioned herein are incorporated by reference in their entirety. Furthermore, the invention encompasses any possible combination of some or all of the various embodiments mentioned herein, described herein and/or incorporated herein. Furthermore, the invention encompasses any possible combination which also specifically excludes any one or any of the various embodiments mentioned herein, described herein and/or incorporated herein.

[0079] The above disclosure is intended to be illustrative and not exhaustive. This description will suggest many variations and alternatives to someone skilled in the art. All such alternatives and variations are intended to be included within the scope of the claims where the term "comprising" means "including, but not limited to." Those familiar with the art may recognize other equivalents to the specific embodiments described herein whose equivalents are also intended to be encompassed by the claims.

[0080] All ranges and all parameters disclosed herein are understood to encompass any and all subranges included therein and each number between extreme points. For example, a stated range of "1 to 10" shall be considered to include any and all subranges (and including) the minimum value of 1 and the maximum value of 10; that is, all subranges starting with a minimum value of 1 or more, (e.g., 1 to 6.1) and ending with a maximum value of 10 or less, (e.g., 2.3 to 9.4, 3 to 8, 4 to 7) and finally to each number 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 contained within the range. All percentages, ratios and proportions presented herein are by weight unless otherwise specified. All percentages, ratios and proportions in this document are by weight unless otherwise specified.

[0081] This completes the description of the preferred and alternative embodiments of the invention. Those skilled in the art may recognize other equivalents to the specific embodiment described herein which equivalents are intended to be encompassed by the claims appended hereto.

Claims (13)

1. Method for anticipating a problem caused by microorganisms in a water process system, the method being characterized by the fact that it comprises: measuring the total microorganism population in a selected water process system feedwater source from the group consisting of: municipal water, tap water, pond water, sea water, lake water, filtered water, desalinated water, recirculated water, waste water, distilled water, condensed boiler water, cooling water and any combination thereof; measuring the amount of at least one subgroup of the microorganism population in relation to the total microorganism population; determining whether the amount of at least one subgroup of microorganisms exceeds a predetermined threshold, the predetermined threshold being at least 5 to 90% of at least one subgroup of the population of microorganisms in relation to the total population of microorganisms; identifying a source of input that is the cause of at least one subgroup of the microorganism population; and establishing a biocontrol procedure to remedy the presence of at least one subgroup of microorganisms. 2. Method according to claim 1, characterized by the fact that the subgroup of microorganisms comprises filamentous bacteria. 3. Method according to claim 1 or 2, characterized in that the subgroup of microorganisms comprises Spirogyra, Cladophora, Pithophora, O. Siphonocladales pithophora pithophora, Ulvibacter littoralis, Vetellibacter vladivostokensis, Weeksella virosa, Fucobacter, Gelidbacter, Ornithobacterium rhinotracheale, Riemerella cohambina, Flavobacterium psychrofilum, Herpetosiphon, Haliscomenobacter, Sphaerotilus, Ctyophaga succinicans, Vladibacter, Pfeifferella, Bacillus aquatilllis, Flexibacter marinus, Flavobacterium odoratum, Microscilla, Flexithrix, Capnocytophaga, Taxeobacter, Sporocytophaga, Saprospira, Chryseobacterium, Hymenobacter and any combination thereof. 4. Method according to any one of claims 1 to 3, characterized in that at least one measurement is taken by a process selected from the list consisting of: Chain Termination Sequencing, Denaturation Gradient Gel Electrophoresis (DGGE), Digital Polymerase Chain Reaction, DNA-Based Analysis, Ion Torrent Semiconductor Sequencing, MALDI, MALDI-TOF, Mass Spectrometry, PCR Analysis, Pyrosequencing, qPCR Analysis, Ligation Sequencing and Detection Oligonucleotide, Sequencing by Synthesis, measurement of antibodies associated with one or more microorganisms, measurement of hormones associated with one or more microorganisms, measurement of secretions associated with one or more microorganisms, measurement of proteins associated with one or more microorganisms, measurement of organic molecules associated with one or more microorganisms, measurement of molecules associated with one or more microorganisms, fluorescent phages, cell separators and any combination thereof. 5. Method according to any one of claims 1 to 4, characterized in that the input source is selected from the group consisting of: municipal water, tap water, pond water, sea water, water lake, filtered water, desalinated water, recirculated water, waste water, distilled water, condensed boiler water, cooling water and any combination thereof. 6. Method according to claim 1, characterized by the fact that it further comprises establishing a biocontrol procedure to remedy the presence of the at least one subgroup of microorganisms only subsequent to and no later than 1 week of the introduction of the water from the source of feed water from which the subgroup of microorganisms comes. 7. The method of any one of claims 1 to 6, wherein the water process system is one or more stages of an item selected from the group consisting of: papermaking, cooling water, boiler water, food manufacturing, beverage manufacturing, ore processing, alumina processing, biodiesel manufacturing, distillation, petrochemical refining, petrochemical synthesis, polymer synthesis, plastic manufacturing, wastewater processing, laundry, dishwashing , water treatment, solid-liquid separation and any combination thereof. 8. Method according to any one of claims 1 to 7, characterized by the fact that it further comprises recording the identified microorganism in a format that can be stored and/or transmitted. 9. Method according to any one of claims 1 to 8, characterized by the fact that it further comprises conducting a biocidal program associated with the remediation of the identified microorganism. 10. Method according to any one of claims 1 to 9, characterized by the fact that at least one measurement is taken from a sample product of the process system. 11. Method according to claim 10, characterized by the fact that the sample is so desiccated that there are few or no living microorganisms in it. 12. Method according to any one of claims 1 to 11, characterized in that the subgroup of microorganisms comprises biofilm formers. 13. Method according to claim 1, characterized by the fact that it further comprises identifying raw materials, intermediate products, or process additives added to the system from which the subgroup of microorganisms comes and establishing a biocontrol procedure to remedy the presence of the at least one subgroup of microorganisms only subsequent to and no later than 1 week of the introduction of that subgroup of microorganisms water source.