A METHOD FOR CONSTRUCTING A DIVERSITY INDEX AND VIABILITY INDEX
OF MICROORGANISMS IN PROCESS SAMPLES
Cross-Reference to Related Applications
This Application is a continuation in part of US Patent Application 13/360,238
filed on January 24, 2012.
Statement Regarding Federally Sponsored Research or Development
Not Applicable.
Background of the Invention
The present invention relates generally to compositions of matter, apparatuses and
methods useful in detecting, identifying, and addressing microorganisms present in commercial
process systems.
The presence and growth of certain microorganism in commercial process systems
is an ongoing challenge. Many of the various stages of commercial process systems contain a
variety of conditions having different amounts of water, nutrients, heat, shelter, anchoring
substrates, chemical conditions, and/or an absence of predators which often function as
environmental niches suitable for colonization by all sorts of microorganisms. Population
growth by these microorganisms often pose a number of problems including degrading process
functions and fouling the end products.
One such problem is microorganism induced crust deposit formation. Crust is the
accumulation on a surface of an item present in a commercial process system of a rigid solid
composition comprising deposited organic and/or inorganic material. The crust can be secretions
and/or colonies of microorganisms themselves. In particular crust can include or consist of the
accumulation of one or more kinds of hard shelled and/or chitin bearing and/or coral organisms.
Crust can have many negative impacts on systems such as decreased operational efficiency,
premature equipment failure, loss in productivity, loss in product quality, and increased healthrelated
risks. Worst of all crust must often be physically removed by scraping or other physical
means and this requires expensive shut downs or disassembly of part or all of the process system.
Another problem microorganisms pose is through the formation of biofilms.
Biofilms are layers of organic materials comprising microorganisms or exopolymeric substance
secreted by microorganisms which aid in the formation microbial communities. Biofilms can
grow on the surfaces of process equipment as well as in pools of fluid. These biofilms are
complex ecosystems that establish a means for concentrating nutrients and offer protection for
growth. Biofilms can accelerate crust, corrosion, and other fouling processes. Not only do
biofilms contribute to reduction of system efficiencies, but they also provide an excellent
environment for microbial proliferation of other microorganisms including pathogenic
organisms. It is therefore important that biofilms and other fouling processes be reduced to the
greatest extent possible to maximize process efficiency and minimize the health-related risks
from such pathogens.
Several factors contribute to the extent of biological contamination and govern the
appropriate response. Water temperature; water pH; organic and inorganic nutrients, growth
conditions such as aerobic or anaerobic conditions, and in some cases the presence or absence of
sunlight, etc. can play an important role. These factors also help in deciding what types of
microorganisms might be present in the water system and how best to control those
microorganisms. Proper identification of the microorganism is also crucial to responding
appropriately. Differences regarding whether the microorganisms are plants, animals, or fungi,
or if they are planktonic or sessile determines how effective various biocontrols will be. Because
different microorganisms induce different problems, proper identification is crucial to properly
remediating unwanted microbial effects. Finally because chemically caused problems cannot be
remediated with biocides, it is also necessary to identify which problems have non-biologically
based origins.
Standard techniques typically used to monitor process systems include standard
plate count techniques. These techniques require lengthy incubation periods and do not provide
adequate information for pro-active control and prevention of problems related to microbial
growth. More recently, adenosine triphoshphate (ATP) measurements have been used as a
means of pro-active control. However, the reagents are costly and small volumes are sampled
from large water systems. While it is possible to quantify microbial activity in a sample with the
use of the ATP assay, the reaction is unable to discriminate between ATP that is produced by one
type of microorganism compared to another and it does not detect organisms that are viable but
inhibited. Another disadvantage is that this method cannot be used to determine microbial
contribution to sheet defects because most organisms are not viable following exposure to the
heat of the dryer section. Data collection is also infrequent, leading to significant gaps in data.
Therefore, this approach provides limited information on the status of microorganisms in the
system of interest. In addition, these approaches are typically used to monitor planktonic
bacteria. Although in some cases, surfaces might be swabbed and analyzed in order to quantify
biofilm bacteria. These approaches are very tedious and time-consuming.
Dissolved oxygen (DO) probes have been used to measure microbial activity in
fluids, as it is well known that microbial activity and aerobic metabolism leads to a decrease in
dissolved oxygen concentrations. U.S. Patents 5,190,728 and 5,282,537, disclose a method and
apparatus for monitoring fouling in commercial waters utilizing DO measurements. However,
the approach requires the use of nutrient additions to differentiate biological from non-biological
fouling and there is no mention of how the probe is refreshed for further measurements after the
probe surface has fouled. In addition, the approach disclosed requires a means of continuously
supplying oxygen.
The standard Clark style electrochemical DO probe has many limitations such as:
chemical interferences (H2S, pH, C02, NH3, S04, C1-, C12, C102, MeOH, EtOH and various
ionic species), frequent calibration and membrane replacement, slow response and drifting
readings, thermal shock, and high flow requirements across membranes. Anew type of
dissolved oxygen probe, which has recently been made commercially available by a number of
companies (e.g., HACH, Loveland, CO), overcomes nearly all of these limitations so that DO can
be measured on-line in process waters. This new DO probe (LDO) is based on lifetime
fluorescence decay where the presence of oxygen shortens the fluorescence lifetime of an excited
fluorophore. The fluorophore is immobilized in a film at the sensor surface and the excitation is
provided with a blue LED. U.S. Patents 5,698,412 and 5,856,119 disclose a method for
monitoring and controlling biological activity in fluids in which DO is measured in combination
with pH and/or ORP (oxidation-reduction potential) to measure transitions in metabolic behavior,
specifically related to nutrient/substrate depletion.
Conventional plating techniques and oxidant residuals may indicate adequate
biocide dosing and control of microbial growth, while deposition, defects and breaks remain
prevalent. There is a clear need to provide more accurate information regarding microbial
growth and biofilm formation in industrial systems. Quantitative PCR techniques allow for rapid
analysis of sheet defects, felts, process water samples, etc. to determine the contribution of
microorganisms to quality issues. This new approach has been demonstrated to allow for a more
proactive diagnosis of problems leading to improved machine efficiency and product quality.
Thus it is clear that there is clear utility in novel methods and compositions for the
proper identification of microorganisms present on in commercial process systems. 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. In addition, this section should not be construed 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
At least one embodiment of the invention is directed towards a method of
addressing a microorganism infestation in an industrial process system. The method comprises
the steps of: 1) taking at least one first measurement which identifies the relative concentration of
two or more organisms present in at least one portion of the industrial process system, the
identifications at least partially defining a baseline diversity index, 2) taking at least one second
measurement which identifies the relative concentration of two or more organisms present in the
at least one portion of the industrial process system, the identifications at least partially defining a
subsequent diversity index, the at least one second measurement taken later than the first
measurement(s), 3) noting any relative change in concentration of the two organisms, 4)if the
second measurement differs from the measurement by an amount greater than a pre-determined
threshold amount, determining if the change is associated with an unwanted effect on the
industrial process system, and 5) implementing a remedy to remediate the unwanted effect.
The first and second measurement may be performed by at least one item selected
from the list consisting of DNA analysis, PCR analysis, qPCR analysis, and any combination
thereof. The threshold amount may be 100 cells per ml of fluid taken from the system or 100
cells per gram of an end product of the industrial process, or other solid samples taken from the
process including but not limited to felts. The method may further comprise the step of
identifying if one of the organisms is a pioneer and if one is an adaptor, if one is a pioneer and its
concentration increases by more than the threshold in the subsequent index, the remediation
includes applying a biocide regimen targeting the pioneer, if no biofilm formers are detected the
remediation includes identifying and eliminating a non-biological vector which facilitates the
settlement of the microorganisms.
Regardless of the identity of the at least one organisms, if their relative
concentrations increase relative to the prior measurement by an amount more than the threshold
even if the overall biological population remains the same, a biocide treatment may be added to
the system.
The method may further comprise the step of correlating the change in diversity
index to another event that occurred in the industrial system, the other event selected from the list
consisting of: changing the source of at least one feed material, changing the kind of at least one
feed material, changing the rate of operating at least a portion of the system, and any combination
thereof, and reversing the event. The overall concentration of cells in the sample may remain
unchanged between the first and second measurements. The measurements may be taken in a
portion of system that a deposit has formed on and the deposit does not contain any significant
biological component. The measurements may be taken in over a plurality of locations
throughout the system and the indices compare overall system populations.
At least one third diversity index measurement may be taken subsequent to the
second measurement and subsequent to the remediation and the efficacy of the remediation is
evaluated by the change in the relative concentrations of the at least two organisms as measured
in the third diversity index measurement. The overall concentration of cells in the sample may
remain unchanged between the first and second measurements, the identity of the first and
second organisms are not known to cause any unwanted effects on the process equipment or end
product, and an effective biocide may be added to the system to kill the first and second
organisms when a threshold change is detected. One of the organisms may be capable of
forming spores which are resistant to biocides and when the relative amount of that organism
grows in excess of the threshold, the treatment may be targeted to the area of the process with
vegetative cells to prevent sporulation.
Brief Description of the Drawings
A detailed description of the invention is hereafter described with specific
reference being made to the drawings in which:
FIG. 1 contains three graphs illustrating the application of the invention for rapid
detection of total bacteria (A), primary (B) and adaptive (C) biofilm-forming bacteria in headbox
deposits collected at a coated free sheet mill.
FIG. 2 illustrates a graph of the total bacterial load of sheet defects from a coated
free sheet mill (1-5), a tissue mill (6), and an uncoated free sheet mill (7) to which the invention
was applied to.
FIG. 3 is a graph of the total bacterial load of sheet defect samples the invention
was applied to.
FIG. 4 illustrates pie charts denoting microbial diversity in D samples
collected from machine felts from three different paper mills.
Detailed Description of the Invention
The following definitions are provided to determine how terms used in this
application, and in particular how the claims, are to be construed. The organization of the
definitions is for convenience only and is not intended to limit any of the definitions to any
particular category.
"Adaptor" means an organism that exhibits some level of tolerance to the
biocontrol program. When the adaptor's microbial competition is reduced by a biocide, this
adaptive organism is able to flourish and may form a biofilm.
"Biological" means a composition of matter in which at least 10% of the
composition (by volume or mass) comprises cells from an organism.
"Defect" means an unwanted attribute of an item associated with an industrial
process. It includes but is not limited to one or more plugs on a felt, and such attributes of paper
sheet as holes, discoloration, streaks, spots, translucent spots, and any combination thereof.
"Felt" means a belt made of interweaved wool or any other fiber used in a
papermaking process which functions as a conveyer of materials wherein the interweaved fibers
define a plurality of lumens through which water or other fluids may pass. Felts may also
provide cushioning between press rolls and may also be a medium used to remove water from
papermaking materials. Felts include but are not limited to bottom felts, bottom board felts,
cylinder tissue wet felts, drier felts, endless felts, pickup felts, suction pickup felts, Harper top
felts, and top felts.
"Opportunist" means an organism that thrives by settling into pre-established
biofilms, crusts, deposits, or other colonies of organisms, and tends to supplant, displace, or
coexist alongside pioneer organisms and/or previous opportunist organisms.
"Paper Product or Paper Sheet" means any formed fibrous structure end
product of a papermaking process traditionally, but not necessarily, comprising cellulose fibers.
Examples of such end products include but are not limited to facial tissue, bath tissue, table
napkins, copy paper, printer paper, writing paper, notebook paper, newspaper, paper board,
poster paper, bond paper, cardboard, and the like.
"Papermaking Process" means one or more processes for converting raw
materials into paper products and which includes but is not limited one or more of such steps as
pulping, digesting, refining, drying, calandering, pressing, crepeing, dewatering, and bleaching.
"PCR Analysis" means polymerase chain reaction analysis.
"Pioneer or Primary" means an organism which attaches to a clean surface or
region, thereby initiating biofilm, crust, or deposit formation at that surface.
"Plug" means a solid, semisolid, viscous, and/or other deposit of material
positioned within the lumens of a felt. Plugs may inhibit the flow of material through the
lumens, and/or may impair any other functionality of a felt.
"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.
"Probe" means a composition of matter constructed and arranged to bind to a
targeted section of DNA and which can be readily detected when so bound and thereby be used
to indicate the presence or absence of the targeted section of DNA.
"qPCR Analysis" means quantitative polymerase chain reaction analysis.
"Microorganisms" means any organism small enough to insinuate itself within,
adjacent to, on top of, or attached to equipment used in an industrial process (including
papermaking), it 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
by the naked eye but which comprise a number of individual organisms that are too small to be
seen by the naked eye, as well as one or more organisms that can be seen by the naked eye, it
includes but is not limited to any organism whose presence, in some way impairs the industrial
process such as forming plugs within felts and/or causing defects within paper sheets.
In the event that the above definitions or a description stated elsewhere in this
application is inconsistent with a meaning (explicit or implicit) which is commonly used, in a
dictionary, or stated in a source incorporated by reference into this application, the application
and the claim terms in particular are understood to be construed according to the definition or
description in this application, and not according to the common definition, dictionary definition,
or the definition that was incorporated by reference. In light of the above, in the event that a term
can only be understood if it is construed by a dictionary, if the term is defined by the KirkOthmer
Encyclopedia of Chemical Technology, 5th Edition, (2005), (Published by Wiley, John
& Sons, Inc.) this definition shall control how the term is to be defined in the claims.
At least one embodiment of the invention is directed to a method of identifying a
microbiological infestation by comparing the current diversity index of at least a portion of the
system to a baseline index. Virtually no commercial process system is 100% free of
microbiological organisms. Process system facilities often encompass huge volumes with many
inputs through which organisms can enter and contain numerous different niches for them to
colonize so it always has some sort of biological population. From a commercial standpoint
however it is far preferable for a system to be populated with benign organisms than to be
populated with harmful organisms such as those which impair the process, damage the product,
or pose dangers to people. As a result using a diversity index is a useful diagnostic approach
which correlates changes in population with changes from benign effects to harmful effects. A
method which correctly identifies which organisms are present and where they are, can aid in
selecting the proper remedy and in deploying it in the optimal location.
A diversity index is a snapshot of the biological diversity of the organisms present
in a commercial process system. Diversity indices can be system wide or can be limited to
certain portions of a process system. For example because it is the convergence point of many
rich fluid inputs, the headbox of a papermaking process is often highly populated with
microorganisms and may be expected to have a diversity index which varies widely over time.
In contrast the treated fresh water that is used in the papermaking process is nearly organism free
so a change in diversity and abundance there from a few organisms to an array of bacteria would
indicate a problem. As a result sometimes noting the diversity index of a particular section
affords insights that a system wide diversity index would not provide. Noting the kinds of
changes in diversity and where they are located influences where the feed points for biocide
should be located and how the population should be addressed.
In at least one embodiment the diversity index is used to preemptively avoid a
harmful microbiological effect before it occurs. Because there are so many different sorts of
organisms that correspond to specific problems in specific in commercial process systems it is
sometimes efficient to focus on the presence or absence or the relative ratio of specific targeted
organisms. For example some organisms are pioneers and some are adaptive biofilm-formers. A
pioneer creates a biofilm or crustdeposit where there previously was none, while an adaptive
biofilm-former exhibits resistance to a treatment program. If a review of the diversity index
shows first the film or crust predominantly comprised one organism then later its composition
changed to a different organism it could indicate the transition from a pioneer to an opportunistic
adaptor and the biocide regimen can be modified to appropriately address this situation.
Similarly if a primary film former tends to gain access to the system from one mechanism and
the adaptive one from another mechanism properly identifying what kind of organism is present
helps to identify the vector sources of the microbial contamination.
In at least one embodiment the diversity analysis can be used to focus quality
control review of the end products. For example some organisms such as some fungi do not
cause significantly impair the process itself but they form masses which tend to become
embedded in end products or machine components and thereby cause unwanted defects, reduced
felt dewatering and reduced mechanical efficiency. A rise in the concentration of fungi in the
diversity index would suggest especially close scrutiny of the end product for defects is
appropriate.
In at least one embodiment the nature of the change in the index is not as
significant as the rate of the change in diversity. For example if a given diversity index over time
tends to show a relatively static population diversity but it suddenly changes, this indicates that
something significant has changed in the system. This could mean a material input may have a
defect which stimulates population change, or a piece of equipment may be damaged or
malfunctioning which opens up new niches for different organisms. As a result, diversity index
analysis can be used to detect non-biological problems in process systems.
In at least one embodiment the change in diversity index can be used to detect a
looming problem before it actually manifests. As previously mentioned a change in diversity
index may indicate a defective material or damaged or malfunctioning equipment. Sometimes
the change in diversity can be detected before other unwanted effects occur (such as loss of
operational efficiency or defective end products) and identification of the cause of the change in
diversity can moot a potential problem before its effects manifest in a significant or expensive
manner. Similarly a change in diversity may indicate that crustdeposit or a biofilm or another
organism induced problem will occur, but the index allows for the problematic microorganism to
be removed before it causes its associated problems. Sometimes the rapid change indicates that a
benign species which previously blocked the colonization efforts of a harmful organism is no
longer potent and the harmful organism is now free to colonize that niche.
In at least one embodiment, the analysis of the diversity index occurs in a situation
where the total cell count within the region analyzed remains unchanged but the composition of
the microorganisms changes. In at least one embodiment, the change in diversity corresponds to
a situation in which the total cell count increases or decreases.
In at least one embodiment, one or more portions of a process system are regularly
sampled for their diversity index. The samples may be time indexed and may be correlated with
other events at the facility such as the activation, deactivation, operating status, rate of
production, and or temperature, of certain equipment, and/or the use of different materials,
additives, or chemicals. This allows for the use of biological diversity as another means of
quality control at the facility. A significant change in diversity that corresponds to some other
event indicates that the other event may have some unexpected positive or negative impact on the
process.
In at least one embodiment some microorganism induced effects are known to
occur after a specific amount of time has elapsed from the moment of contamination. As a result
a change in diversity index can be used to determine how long it takes for the organism to cause
its associated problems. This method can be used both as a diagnostic (to find out how the
organism functions) as well as a cost optimization tool. Cost optimization can be achieved by
receiving advanced warning from the diversity change that a problem will occur within a given
timeframe using the advanced warning to purchase or use of a remedy at a time when it has a
lower cost or higher availability than it would if it was purchased as a sudden response to an
unexpected emergency.
In at least one embodiment the diversity index can be used to detect spore-forming
organisms. When these organisms are in spore form they have little or no metabolic activity and
are highly resistant to biocides. It takes a large amount of biocide to control organisms once they
are in the spore-state and the likelihood of spores making it into the finished product becomes
very high. Dairyman's and liquid packaging standards are likely not to be met in a situation
where spores are present. In contrast when these organisms are in a vegetative state they are
susceptible to biocides and are much easier to control. .. Detection of spore-forming organisms
by the diversity index method shifts the focus of the biocontrol program to prevention of the
formation of spores.
In at least one embodiment the results of the diversity index analysis are used to
augment the biocontrol program by determining how much, what kind, and how often, one or
more biocide compositions are added to one or more locations within a commercial process
system. In at least one embodiment any and all of the above and below embodiments are applied
to a commercial system such as an industrial system including but not limited to a process water
system, papermaking process, pulping process, food processing process, chemical refining
process, wood processing process, water filtration process, water purification process, chemical
synthesis process, coating processes, organic chemistry using processes, and any combination
thereof. In at least one embodiment the diversity index is used to assess problematic
microorganisms found in machine deposits, sheet defects, finished products, felts, etc. The
method is based on analysis of nucleic acids in sample extracts.
In at least one embodiment the identification of the constituents of the diversity
index is achieved through DNA based analysis involving the use of PCR primers to detect the
presence, absence and quantity 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 targeted towards a part of a DNA strand which is highly conserved
among a group of organisms. As a result, detecting the presence of that particular part of DNA is
definitive proof of the presence a specific organism. PCR analysis is of particular use in
analyzing felts and paper sheets due to the difficultly of correctly identifying its contaminating
microorganisms because they lack viable organisms for traditional plating methods or ATP
measurements.
In at least one embodiment the PCR analysis involves utilizing one or more of the
methods described in the Article Primer 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 the PCR analysis involves utilizing 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).
In at least one embodiment the PCR analysis is a qPCR analysis as described in
Trade Brochure qPCR guide, prefaced by Jo Vandesompele, (as downloaded from website
http://www.eurogentec.com/file-browser.html on 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.
In at least one embodiment, the polymerase chain reaction (PCR) is a method for
targeting sequences of nucleic acid (DNA or RNA) and increasing the copy number of the target
sequence to obtain useful quantities of nucleic acid for down-stream analysis. This method can
be applied to the detection of microorganisms in a variety of samples that include, but are not
limited to, machine felts, sheet defects, machine deposits, etc.
In at least one embodiment, once DNA is extracted from the sample, using any of
the DNA extraction kits available commercially, it can be analyzed in real-time using a PCR
approach such as a Quantitative PCR approach. Quantitative PCR utilizes 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 function of a specific
gene. Some form of detection such as fluorescence may be used to detect the resulting DNA or
'DNA amplicon'. The change in fluorescence is directly proportional to the quantity of target
DNA. The number of cycles required to reach the pre-determined fluorescence threshold is
compared to a standard that corresponds to the specific DNA target. A standard is typically the
target gene that is pure and of known quantity at concentrations that span several logs. The
number of copies of target DNA present in the sample is calculated using the standard curve.
The copy number per sample is then used to determine the number of cells per sample.
In at least one embodiment a primer set is used which targets DNA sequences
from bacteria using a conservative approach to quantify total bacteria. In at least one
embodiment a primer set is used which targets primary biofilm-forming bacteria, including, but
not limited to, Meiothermus, Pseudoxanthomonas, and Deinococcus. In at least one embodiment
a primer set is used to target an adaptive biofilm-former which belongs to the Sphingomonadacea
family of bacteria. In at least one embodiment the adaptive biofilm-former 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.
In at least one embodiment the method involves distinguishing between DNA at
the biological domain level. In at least one embodiment the method involves distinguishing
between DNA of Bacteria, Archaea, and Eukaryota. These organisms have hugely differing
DNA and a protocol which focuses on identifying the organism' s DNA at the domain level is
vastly simpler than more specific determinations. Because with felts, the organisms from
different domains are often best treated differently, such a simple form of identification can be
used to accurately identify the specific regimen best targeted to the particular contaminant. In at
least one embodiment the test used is such that it would not distinguish between organisms of the
same domain or between different kinds of Bacteria, or between different kinds of Archaea, or
between different kinds of Eukaryota.
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
the PCR analysis is used to detect genome sequences associated with enzymes unique to or
nearly unique to specific organisms.
In at least one embodiment the method involves detecting a defect and then
utilizing the PCR analysis to properly analyze the diversity index of the defect. In at least one
embodiment the method determines if the defect is totally biologically based, totally nonbiologically
chemical based, or resulting from a combination of non-biologically chemical,
mechanical, and biologically based sources. In at least one embodiment the defect is one or more
plugs on a felt. In at least one embodiment the defect is a paper sheet having at least one or more
of: a hole, a hole with a discolored halo around at least a portion of it, a streak of discoloration, a
spot, a translucent spot, and any combination thereof.
In at least one embodiment a threshold level is methodology used to discount false
positives. Sometimes PCR analysis detects traces of organisms that while 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 pre-determined level known for one or
more particular organisms. In at least one embodiment the method involves discounting the
presence of any organism detected at 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
level lower than 104 cells per ml.
In at least one embodiment the method is able to detect microoganisms that would
not otherwise be detected by prior art methods. For example in cases where foulant is caused by
an infestation of anaerobic or sulfate reducing organisms, methods such as ATP detection would
not correctly identify the foulant source as biological as the amount of ATP produced by a
microorganism under anaerobic conditions is significantly less than under aerobic conditions.
Therefore the foulant source will be identified incorrectly and n chemical not an anti-biological
approach would be used to attempt to resolve the problem. In another example, differentiation of
microbial from chemical contamination in felts using traditional approaches such as plating, ATP
detection, etc. is virtually impossible due to the fact that these samples dry out during transport
and all viable organisms die. Utilizing the DNA approach would always correctly indicate a
biological infestation because all life contains DNA.
The diversity index can use PCR such as but not limited to qPCR for the detection
of total organisms such as bacteria; Sphingomonas species; Erythrobacter species; Pseudomonas
species; Burkholderia species; Haliscomenobacter species; Saprospira species; Schlegelella
species; Leptothrix species; Sphaerotilus natans; Bacillus species; Anoxybacillus species;
members of the Cytophaga-Flavobacterium-Bacteroides phylum; green nonsulfur bacteria,
including Herpetosiphon, members of the Deinococcus-Thermus phylum, including Meiothermus
species; catalase-producing bacteria, amylase-producing bacteria, urease-producing bacteria,
nitrifying bacteria, fungi, etc. These techniques utilize primers and standards pairs that allow for
detection and quantification of target organisms based on conserved sequences. The 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.
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. The diversity index can also be expressed quantitatively as the relative abundance of
several target organisms. The diversity index for any part of a process can be measured at times
when machines or processes are running 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 among
sample locations or relative to a baseline. Treatment strategies and feed points can then be
altered accordingly to combat the problem.
A diversity index based on quantification of DNA measures the presence and
diversity of organisms in a process, independent 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 for assessing the efficacy of treatment
strategies of process waters. This can be accomplished by comparison of the diversity index to
the viability index.
This method would quantify the amount and type of viable bacteria present in
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 blueprint for protein synthesis in a process known as translation.
mRNA is produced only by living cells. RNA from living cells can be isolated with the use of
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
mRNA into its complementary DNA (cDNA). Two sets of primers are required for this
experiment. The first targets specific mRNA, while the second is used to amplify the resulting
cDNA produced by the reverse transcriptase reaction.
EXAMPLES
The foregoing may be better understood by reference to the following examples,
which are presented for purposes of illustration and are not intended to limit the scope of the
invention.
EXAMPLE #1
A coated free sheet mill experienced persistent deposition in one of the machine
headboxes, which was believed to be the cause of defects in the final product. The headbox itself
suffered from an accumulation of chemical deposits and fibrous growths. Microscopic and
chemical analysis showed little to no bacterial presence within the accumulation.
Microorganisms were assumed to be the underlying cause of the problem. However, traditional
monitoring techniques (e.g. standard plate counts and ATP levels) used to analyze process
samples did not indicate elevated levels of microbial activity. Specifically the results indicated
no more than 100 CFU/ml and no more than 100 RLU (ATP).
Deposit samples from the headbox were analyzed over the course of several
months using qPCR techniques to develop a diversity index. Initial qPCR results from the
analysis of headbox deposits exhibited high levels of microbial loading, as well as elevated
densities of pioneering and adaptor biofilm-formers (Figure 1). The treatment strategy was
augmented with the addition of biocides to both the pulper and the broke silo. The feed rate of
the oxidant-based biocontrol program was also increased. Analysis of deposits collected one
month later detected little change in the total bacterial load of the headbox deposits (Figure 1A).
The number of pioneering biofilm-formers decreased one-log, while the density of adaptive
biofilm-formers decreased four-logs (Figure IB and 1C). The amount of headbox deposits and
frequency of sheet defects continued to remain unchanged. Traditional plating and ATP analysis
of the stock and process water system indicated little biological activity. The ATP and plate
count values were averaging less than 100 RLU and 100 colony-forming units per gram (CFU/g),
respectively.
The treatment strategy was further optimized through the addition of unstabilized
chlorine and biocides to the broke silo and the pulper. After implementation of the last set of
changes, additional samples were collected and analyzed. The total bacterial load of the deposit
showed a decrease of nearly one-log (Figure 1A). The final set of deposit samples showed a
decrease of nearly two-logs in the density of primary biofilm-formers (Figure IB). Adaptive
biofilm-formers remained at near-background levels (Figure 1C). Again, despite improved
control of the microbial population, the defect frequency, the nature of the defects, and headbox
deposition remained unchanged.
Sheet defects from this mill were analyzed using the same qPCR-based approach.
It is impossible to determine bacterial content in defects using traditional plating and ATP
methods because many of the bacteria that may have been present in the defect are killed by the
high temperatures of the dryer section. Chemical analysis does not provide a definitive answer
about bacteria present in the sheet as it relies on ninhydrin staining. This approach is non¬
specific and prone to false positive and false negative results. DNA analysis of holes and sheet
defects from this mill detected very low bacterial density (Figure 2, Samples 1-5). Primary and
adaptive biofilm-formers were not detected in the sheet defects analyzed from this mill.
Therefore, bacterial slime was not likely contributing to defects and quality issues at this mill.
In comparison, a mill suffering from significant biological deposition had defects containing
much higher microbial loading (Figure 2, Sample 6). Furthermore, similar bacterial species were
identified in the deposits and defects. Ninhydrin staining of these defects did result in a positive
reaction indicating the presence of microorganisms. In another case, bacteria were detected in
sheet defects at levels just above the minimum density required to be considered a biological
defect. However, the ninhydrin reaction was negative indicating the defect did not contain
microorganisms (Figure 2, Sample 7). Quantitative qPCR examination of headbox deposits
demonstrated reductions in both primary and adaptive biofilm-formers following each
modification to the treatment strategy. The fact that there was a drastic decrease in these target
organisms and no decrease in the amount of deposition or defect frequency, indicates that
bacteria are likely not responsible for defect problems in this machine system. Primary biofilmformers
colonize machine surfaces and create a favorable environment for attachment and
proliferation of other organism types. Without the presence of these organisms, bacteria may
attach to machine surfaces following the deposition of chemical debris that can serve as a good
growth medium. It is likely that chemical additives and fiber were depositing inside the headbox,
resulting in a microenvironment suitable for microbial colonization. Since the analysis of sheet
defects revealed negligent microbial presence, microorganisms were ruled out as the primary
source of deposition in the headbox and adverse effects on product quality. Efforts to improve
machine performance were focused away from biocontrol and toward better mechanical control
of the system allowing for improved operational conditions and product quality.
EXAMPLE #2
A coated free sheet mill utilized a competitive oxidant-based biocontrol program
for several years. Control of microbial growth was perceived as adequate; however, there was an
opportunity to further reduce sheet breaks for improved process efficiency. The program was
implemented and optimized in several phases. Bacterial density throughout the process remained
low and a reduction in sheet breaks was documented. The average number of breaks per day
decreased from an average of 1.2 breaks per day to 0.42 breaks per day.
Approximately two-years after the implementation of the optimized program, it
was observed that process conditions had become more variable and increasing concentrations of
biocontrol products were required to maintain the same level of control. A system survey using
traditional monitoring tools such as plate counts and ATP measurements, indicated that bacterial
density in the process water system remained low and no or little increase was observed in the
headbox and broke system. However, the mill was suffering a severe outbreak of holes and
defects. While traditional monitoring techniques indicated no change in the performance of the
biocontrol program, the on-line activity monitor detected increasing microbial activity (Figure 3).
A diversity index analysis utilizing qPCR analysis of the machine deposits and
sheet defects all confirmed the presence of pioneering and adaptive biofilm-formers. The density
of total bacteria in the defects was approximately 1.8xl0 7 cells per gram (Figure 3). This
evidence indicates the role of microorganisms in the defect and quality issues. The machine
underwent a caustic boilout after which, the online activity monitor demonstrated a reduction in
microbial activity and a more stable ORP value indicating improved program performance and
resilience. The amount of microorganisms in sheet defects decreased from 107 to 105 cells/g
following the boilout (Figure 3). This confirms that qPCR can detect microbial contribution to
sheet defects which cannot be detected using traditional techniques. In addition, qPCR can be
used to assess the efficacy of the biocontrol program on the final product.
EXAMPLE #3
Felt samples from two paper mills that were experiencing performance issues,
which manifested themselves as on-machine deposits and sheet defects, were analyzed using
qPCR. Each sample was tested for the presence of microorganisms. Once it was determined that
each sample contained high amounts of bacteria, the samples were then analyzed for the presence
of adaptive and primary biofilm-formers, which included Sphingomonadaceaefin., Meiothermus,
Geothermus, and Pseudoxanthomonas which have been known to contribute to problems with
machine efficiency and product quality. Both mills contained normal levels of adaptive biofilmformers,
however, Mill 1had twice as many primary biofilm formers as Mill 2 (Figure 4). The
level of adaptive biofilm formers was determined to be normal as its levels were in the range that
indicated it is likely not contributing to the problem. Diversity index showed that the level of
pioneer biofilm-formers at Mill 2 was at a near-background level. High levels of pioneer
biofilm-formers at Mill 2 suggested biofilm formation in felts which leads to felt plugging and
reduced water removal from the sheet. The presence of biofilm on the felts can lead to increased
deposition of other matter which can then redeposit onto the sheet. Elevated levels of pioneer
biofilm-formers at Mill 1 suggested that additional analysis of other parts of the process such as
shower water, additives, storage chests, etc. were needed to determine where these organisms
were originating.
The result of these examples demonstrates that conventional plating techniques
and oxidant residuals may indicate adequate biocide dosing and control of microbial growth,
while deposition, defects and breaks remain prevalent. Utilizing a diversity index comprising
PCR and qPCR methods provides more accurate information regarding microbial growth and
biofilm formation in industrial water systems. These strategies allow for rapid analysis of the
contribution of microorganisms to deposit formation and can be used to rapidly determine
whether or not deposits containing microorganisms are contributing to defects.
A qPCR based diversity index allows for rapid analysis of sheet defects to
determine the contribution of microorganisms to quality issues. This new approach has been
demonstrated to allow for a more proactive diagnosis of problems leading to improved machine
efficiency and product quality.
While this invention may be embodied in many different forms, there described in
detail herein specific preferred embodiments of the invention. 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 papers, and any
other referenced 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 described herein and/or incorporated herein. In addition the invention
encompasses any possible combination that also specifically excludes any one or some of the
various embodiments described herein and/or incorporated herein.
The above disclosure is intended to be illustrative and not exhaustive. This
description will suggest many variations and alternatives to one of ordinary skill in this art. All
these 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 which equivalents are
also intended to be encompassed by the claims.
All ranges and parameters disclosed herein are understood to encompass any and
all subranges subsumed therein, and every number between the endpoints. For example, a stated
range of " 1to 10" should be considered to include any and all subranges between (and inclusive
of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a
minimum value of 1 or more, (e.g. 1to 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.
This completes the description of the preferred and alternate 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 attached
hereto.
Claims
What is claimed is:
1. A method of addressing a microorganism infestation in an industrial process system, the
method comprising the steps of:
taking at least one first measurement which identifies the relative concentration of two or
more organisms present in at least one portion of the industrial process system,
taking at least one second measurement which identifies the relative concentration of two
or more organisms present in the at least one portion of the industrial process system, the
identifications at least partially defining a subsequent diversity index, the at least one second
measurement taken later than the first measurement(s),
noting any relative change in concentration of the two organisms,
if the relative concentration of one of the measured organisms exceeds a pre-determined
threshold amount, determining if the change is associated with an unwanted effect on the
industrial process system, and
implementing a remedy to remediate the unwanted effect.
2. The method of claim 1 in which the first and second measurement are performed by at
least one item selected from the list consisting of D A analysis, PCR analysis, qPCR analysis,
and any combination thereof.
3. The method of claim 1 in which the threshold amount is 104 cells per ml of fluid taken
from the system or 104 cells per gram of an end product of the industrial process or of a solid
sample from the industrial process.
4. The method of claim 1 further comprising the step of identifying if one of the organisms
is a pioneer and if one is an adaptor, if one is a pioneer and its concentration increases by more
than the threshold in the subsequent index, the remediation includes applying a biocide regimen
targeting the pioneer, if no pioneer formers are detected the remediation includes identifying and
eliminating a non-biological vector which facilitates the settlement of the microorganisms.
5. The method of claim 1 further comprising the step of identifying if one of the organisms
is a pioneer and if one is an opportunist, if one is a pioneer and its concentration increases by
more than the threshold in the subsequent index, the remediation includes applying a biocide
regimen targeting the pioneer, if no pioneers are detected the remediation includes identifying
and eliminating a non-biological vector which facilitates the settlement of the microorganisms.
6. The method of claim 1 in which regardless of the identity of the at least one organisms, if
their relative concentrations increase relative to the prior measurement by an amount more than
the threshold even if the overall biological population remains the same, a biocide treatment is
added to the system.
7. The method of claim 1 further comprising the step of correlating the change in diversity
index to another event that occurred in the industrial system, the other event selected from the list
consisting of: changing the source of at least one feed material, changing the kind of at least one
feed material, changing the rate of operating at least a portion of the system, and any combination
thereof, and reversing the event.
8. The method of claim 1 in which the overall concentration of cells in the sample remains
unchanged between the first and second measurements.
9. The method of claim 1 in which the measurements are taken in a portion of system that a
deposit has formed on and the deposit does not contain any significant biological component.
10. The method of claim 1 in which the measurements are taken in over a plurality of
locations throughout the system and the indices compare overall system populations.
11. The method of claim 1 in which at least one third diversity index measurement is taken
subsequent to the second measurement and subsequent to the remediation and the efficacy of the
remediation is evaluated by the change in the relative concentrations of the at least two
organisms as measured in the third diversity index measurement.
12. The method of claim 1 in which the overall concentration of cells in the sample remains
unchanged between the first and second measurements, the identity of the first and second
organisms are not known to cause any unwanted effects on the process equipment or end product,
and an effective biocide is added to the system to kill the first and second organisms when a
threshold change is detected.
13. The method of claim 1 in which one of the organisms is capable of forming spores which
are resistant to biocides and when the relative amount of that organism grows in excess of the
threshold, treatment is targeted to the area of the process with vegetative cells to prevent
sporulation.
14. A method of addressing a microorganism infestation in an industrial process system, the
method comprising the steps of:
taking at least one first measurement which identifies the relative concentration of at least
one organism present in at least one portion of the industrial process system,
determining if the concentration of the at least one organism exceeds a predetermined
threshold for that organism,
if exceeding, determining if the threshold exceeding organism is an adaptor or is an
pioneer,
if an adaptor implement a remedial strategy which takes into account the organism's
resistance to biocides,
if a pioneer implement a remedial strategy which utilizes a lower dosage of biocide than
if the organism were an adaptor.
15. The method of claim 14 in which a measurement is also taken determining the absolute
population of all microorganisms infesting the industrial process system, and
determining if the concentration of the at least one organism exceeds a predetermined
threshold for that organism relative to the overall population of microorganisms,
if exceeding, determining if the threshold exceeding organism is an adaptor or is an
pioneer,
if an adaptor implement a remedial strategy which takes into account the organism's
resistance to biocides,
if a pioneer implement a remedial strategy which utilizes a lower dosage of biocide than
if the organism were an adaptor.