Module 1-4 - MWF Microbiology

7/25/2019 Module 1-4 - MWF Microbiology

1/492014 STLENot to be distributed, copied, or replicated without express written consent of STLE.


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Upon completion of this module, students will:

Understand the adverse cost impact of uncontrolled microbial contamination.

Understand:

-how microbes contaminates metalworking fluid systems,

-why microbes thrive in them, and-where they are most likely to be found.

Recognize the primary types of contaminant microbes.

2014 STLENot to be distributed, copied, or replicated without express written consent of STLE.


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As we shall illustrate repeatedly through the course of the day, many of the factors that

affect production efficiencies also affect H&S. The mirror image of this statement is: a

safe MW environment is a more productive one.

Clean MWF that is in good condition: Permits faster feed-rates

More parts per hour

More parts/tool

Reduced # of rejected parts

Fewer drain, clean & recharge events

Less waste generated

Healthy workers are more productive:

Greater focus

Less illness-related lost time

A fuller discussion of the relationship between profitability is beyond the scope of

todays course, but a strong business case can be made for the investment in good

industrial hygiene practices, including mist and microbial contamination control.



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Uncontrolled microbial contamination in MWF systems is expensive.

Uncontrolled microbial contamination adds to the operations Cost of Qualityby

increasing:

-Operational costs-increased additive use costs

-increased tool costs (decreased tool life)

-decreased productivityfewer good pieces per work shift

-Waste treatment

-Actual labor and disposal costs

-System down-time during cleaning (loss production time)

-Waste handling coststreatment & disposal

-Employee health

-Sick employees are less productive than healthy ones-Increased change of making mistakes (inattentiveness)

-Sluggish movements

-Inability to focus on job

-Very sick employees = lost time and can infect co-workers; more on this in

Microbiology Potential Health Effectsmodule, later this morning.



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As we will see in this session, microbes can contribute directly, indirectly or both ways

to many of the coolant failure mechanisms that John Burke discussed yesterday

afternoon.



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The same microbial processes that degrade coolant in your system, continue to

degrade the coolant when it goes to waste treatment. The microbes in the coolant are

already adapted to thrive in the coolant.

Biofilm development (well discuss biofilms in more detail during this presentation) canplug the filter surfaces, inhibiting fluid flow and causing the need fro frequent, costly

element replacement.



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This topic will be discussed in module: Microbiology Potential Health Effects, later this

morning.



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The major lesson here is that by controlling microbial contamination in MWF systems

you can increase your profitability!!



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As Ill explain over the next several slides, MWF have all of the nutrients and conditions

that favor microbial growth.

When microbes grow in MWF systems, they use coolant components as food and they

produce wastes that react with MWF. Since they degrade the economic value of theMWF, we call these processes biodeterioration. Biodeterioration is any process in

which organisms degrade materials, causing economic harm.

When microbes use a MWF chemical as food, thereby removing it from the fluid, the

effect is direct.

When microbes produce wastes, such as biosurfactants (biomolecules that act like

detergents) that then react with the fluid (in this case causing excessive foaming) the

effect is indirect.



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Left photo: destabilized emulsionacidic byproducts of microbial metabolism

(biological activity) spit emulsion.

Center photo: rotting fish (typical amine odor)

Right photo: rotten eggs (sulfide odor)



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Note how reserve alkalinity starts falling off before productivity does. (No control limits

set for alkalinity). The correlation coefficient between productivity and alkalinity is

0.59. Thats a pretty strong correlation.

These data dont tell us why alkalinity is falling off, but they do suggest that alkalinity isa critical factor in maintaining production rates.



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Bacteria produce a wide variety of volatile compounds, a few of which are illustrated

here.

Hydrogen sulfide, characterized by its rotten egg odoris very toxic.

Ammonia pungent odor is severely irritating , but not toxic.

Mercaptans (thiols) are noxious but not toxic except at very highconcentrations. Methylthiol is responsible for the characteristic odor of urine

after one has eaten asparagus. Thiols typically smell like garlic.

Skatole (3-methylindole) is a toxic VOC that smells like feces.

Bacterially generated MVOC also include a variety of mono- and dicarboxylic acids,

including acetic, lactic, pyruvic, butyric, citric, succinic, and malic acids, among others.

This complex bouquet of MVOC gives MWF a characteristic cesspool/swampy odor.



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MWF systems with heavy fungal contamination have a characteristic early, musty odor.

The primary MVOC contributing to this earthy odor is geomsin (4,8 -dimethyldecalin-

4 -ol), although a broad range of alcohols, ketones, terpenes, furans, and ethers

contribute to this scent.



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Upper right: Photo (courtesy of J. Byers, Milacron, Inc., Cincinnati OH) shows MWF

headers the >70% blockage due to swarf particles having been glued together by

biopolymer.

Lower left: MWF header showing MIC holes



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Biofouling is the accumulation of biomass on surfaces.

BUT!

Not all fouling is readily visible.

Photos:

Upper left: Slime on sluice-way and filtration-unit structuresfrom STLE MWF Training

Course Module 2.6, courtesy of J. Burke, Houghton International, Valley Forge, PA

Lower left: Similar to upper leftfrom STLE MWF Training Course Module 2.6,

courtesy of J. Burke, Houghton International, Valley Forge, PA

Upper right: Deck plates appear to be clean, but undersides (Lower right) are heavilyfouledcourtesy of F. Passman.



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Its normal for filters to plug overtime.

Its NOT normal for them to become coated with black, slimy residue.

Refer back to Module 1.3 for the good, bad and ugly of filter performance.



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As we learned in Module 1.3, filtration is not absolute. Filter efficiency is measure as

the -ratio : particles/mL of fluid entering filter particles/mL of fluid downstream of

filter. Regardless of filter efficiency, more particles in the fluid upstream more

particles downstream.

This is only true for inert particles. Microbes trapped by the medium can grow on and

within the medium. Eventually, microbes will colonize the downstream side of the

medium. Fluid passing through the medium will cause flocs of microbes to slough off

of the filters downstream face.

If the fluid is MWF, then these microbes will reinoculate the system downstream of the

filter.

If the fluid is air (think mist collectors) then these microbes will be aerosolized. Moreon that during the discussion of health issues.



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Decreased intervals between filter indexing cycles can be an early symptom of

biofouling. In this example, a paper medium filter indexes (rolls clean media into place)

when the pressure differential between the upstream and downstream sides of the

filter is > 20 psig. The upper and lower control limits are 11.5h and 8.5h, respectively.

Indexing cycles >11.5h can be symptomatic of lose of filter integrity (fluid is bypassingthe filter medium, the filter is torn, etc.). Shorter cycle intervals indicate increased

burdens (for example: particle loadings, grease clumps, biomass).

In this illustration, the indexing interval fell below the LCL on 21 March. The first step is

to examine the filter. Based on the appearance of the medium, additional testing will

be required to complete root cause analysis on the problem.



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Left photo: MWF demulsification.

Right photo: water line that has been attacked by acid corrosion.



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Antimicrobials are also known as biocides.

RP additives are used to prevent rust (corrosion); RP = rust preventative.

High microbe population densities in the recirculating MWF will cause filter media toplug more frequently. When paper filtration media are used, the annual cost of media

increases as the indexing rate increases. The filter media indexes(rolls forward to fresh

paper) each time the back pressure increases to a specified control limit.



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Note that heavy accumulation may be found on surfaces beneath the fluid level

where recirculating fluid is in constant contact with the surface,

Or

In splash zones, where microbe-laden MWF droplets hit zones not normally in contact

with the recirculating fluid. Microbes growing on splash zone surfaces wont be treatedby microbicide addition to the recirculated fluid. More on this in the module:

Controlling Microbial Contamination.

The microbial population density (cells per cm3) within surface films may be 100,000 to

1,000,000 time greater than in the recirculating MWF.



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For every CFU/mL in the recirculating MWF, there are probably 106CFU/cm2on the

system surfaces

For every CFU there are probably > 100 viable but not culturable (VNBC) cells.

Biofilm structures are not uniform. In fact they are quite complex. Actual cells

comprise only a small percentage of total biofilm mass. Secreted peptidoglycans,

entrained metal ions/particles and water/MWF make up the bulk of the biofilm. The

also act as a protective barrier between the biofilm community and the bulk-fluid

environment.

Jerry Byers' photo in Slide 5 illustrates an extreme case. Metal fines (Swarf) have been

cemented together by biofilm to reduce the cross-sectional area of this pipe by > 80%!

Photo credits:

Top left: Staphylococcus - www.dartmouth.edu/~gotoole/heparin.htm

Bottom left: www.technet.pnl.gov/.../projects/ES4FBioFilm.stm

http://www.dartmouth.edu/~gotoole/heparin.htmhttp://www.technet.pnl.gov/sensors/biological/projects/ES4FBioFilm.stmhttp://www.technet.pnl.gov/sensors/biological/projects/ES4FBioFilm.stmhttp://www.dartmouth.edu/~gotoole/heparin.htm


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The distribution of cells can be quite complex. Moreover, theproteomics(gene

expression as reflected in by the production of different types of enzymes) is amazingly

complex. Much like undifferentiated (all identical) somatic cells in a developing

mammalian embryo differentiate into different types of tissues, genetically identical

microbes within biofilms can differentiate so much that by traditional microbiologicaltest methods they appear to be different types of microbes.

Images are from Center for Biofilm Engineering, Bozeman, MT.



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This slide and the next show how variable the chemical environment can be within the

biofilm matrix.

If you look back at slide 27, youll see the complex geometryof a typical biofilm. The

illustration in this slide shows oxygen (O2) concentration gradients in a cross section ofbiofilm. Compare the [O2] shown on this slide with sulfide (S

=) concentrations shown

on the next slide.



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Note that the [S=] increases as the [O2] decreases and vice versa.

Different [O2] zones select for different types of microbes.



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Studies performed in the 1950s and 1960s demonstrated that most often, the kinds of

microbes found in MWF reflect the microbial population in the water used to blend

end-use MWF.

On rare occasions, MWF concentrates harbor microbial contamination.



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Mist droplets generated inside the plant can transport microbes from one sump to

another.



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Employees often dont realize that when they dump trash (rags, cigarette butts,

partially eaten food, etc.) or when they sweep chips, etc. into MWF systems, they are

inoculating those systems with microbes that can cause biodeterioration, disease or

both.



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Plant ventilation systems draw in fresh air and microbe-laden dust and water particles

too.

Being located downwind of significant sources of airborne microbes (bioaerosols) can

increase the microbial challenge to the MWF in your plant.



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Many machinists also tend gardens and raise livestock.

Garden and barnyard wastes carried into the plant on the soles of workers boots may

fall into MWF flowing through below-deck coolant return sluices (also called trenches

orflumes).Good news: Older, open-grate decking has been replaced with solid plates at most

facilitiesits now more difficult to track or sweep debris into trenches.

Bad news: The new plates weigh much more that the old grates. Biomass

accumulation on the underside of plates goes unnoticed and becomes a constant

source of re-infection for the MWF.



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Left Photo: Sump with substantial tramp oil contaminationemulsion has been split by

microbial attack.

Right Photo: Mixed bacterial and fungal population forming a slime mat on the surface

of a MWF sump.



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Now that we understand the impact of microbial contamination, lets turn to the

subjects of that sciencethe microbes.



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Genome mapping technology (genomics) has enabled molecular biologists to compare

the DNA (deoxyribonucleic acid) of organisms with increasing ease and sophistication.

The current Tree of Life model identifies three primary Kingdoms: Bacteria, Archaea

and Eukaryota. The intersections between the branches in the tree and the main trunkreflect our best estimate of the evolutionary point at which groups of organisms

diverged genetically.

Historically (before genomics), the theory was that fungi evolved from bacteria. Note

the genetic difference between fungi and bacteria on this tree. Compare it with the

difference between fungi and animals. Also note how short the entire Animals branch

is. When first discovered, the Archaea were thought to be a Family within the Bacteria

kingdom.

The take home lesson here is that similarities (or dissimilarities) in gross form and

function do not necessarily reflect genetic similarities.

The Gram Negative bacteria we find in fuel belong to the Family Proteobacteria.



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These two illustrations provide a sense of the size of bacteria and fungal spores relative

to one another.

Bacterial are >1,000x as large as viruses, and fungi are 100 times as large as bacteria.

If you look closely at the illustration on the right, youll note that some of the smallestbacteria are too small to be seen with a conventional light microscope (maximum

magnification = 1,200x).



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There are many different kinds of bacteria that can grow in MWF, since the chemicals

used to kill bacteria generally work against a great variety of bacteria, it is generally

sufficient to know whether a significant number of bacteria are present. For routine

condition monitoring and contamination control, you dont need to know the names

(taxonomy) of the bacteria present.

Fungi are >100,000 times larger than bacteria. A typical yeast cell is >100 microns dia.

Fungal cells (within the filament) are typically 10 microns x 100+ microns. By

comparison, most MWF bacteria are rods measuring 0.5 microns x 1 to 2 microns.



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Bacteria often from smooth or glistening colonies on growth media.

Upper left: Comparator Chart for the bacterial side of a typical Dipslide or paddle type

test kit. To make it easier to see colonies, the medium contains a dye that causes the

colonies to turn red.

Lower Right: Diverse bacterial colony types of a culture plate that had been inoculated

with tap water. Notice range of sizes, shapes and colors.



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Current research is showing that in biofilms formed by a pure culture (starting with one

cell of one type of bacterium) individual bacteria assume different shapes and

physiological characteristics, very much like cells in different parts of our bodies.

Form (shape): most commonly spheres (cocci), rods (bacilli) , commas (vibrio) or spirals(spirochetes).

Physiology: primarily a profile of what nutrients the organism can use as food, the

presence of characteristic enzymes and the chemicals produced as waste metabolites.



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Since the invention of the compound microscope in ~1595, and Antony van

Leeuwenhoeks discovery of bacteria in 1683, shape has been the first trait used to sort

bacteria taxonomically.

On September 17, 1683, Leeuwenhoek wrote to the Royal Society about his

observations on the plaque between his own teeth, "a little white matter, which is asthick as if 'twere batter." He repeated these observations on two ladies (probably his

own wife and daughter), and on two old men who had never cleaned their teeth in

their lives. Looking at these samples with his microscope, Leeuwenhoek reported how

in his own mouth: "I then most always saw, with great wonder, that in the said matter

there were many very little living animalcules, very prettily a-moving. The biggest sort.

. . had a very strong and swift motion, and shot through the water (or spittle) like a pike

does through the water. The second sort. . . oft-times spun round like a top. . . and

these were far more in number." In the mouth of one of the old men, Leeuwenhoek

found "an unbelievably great company of living animalcules, a-swimming more nimblythan any I had ever seen up to this time. The biggest sort. . . bent their body into curves

in going forwards. . . Moreover, the other animalcules were in such enormous numbers,

that all the water. . . seemed to be alive."



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The Gram stain (named after its inventor, Hans Christian Gram) has been used since

1884 to characterize bacteria based on their cell wall chemistry.

Iodine stains starch purple. Consequently, the peptidoglycan cell wall of Gram +

bacteria retains the iodine stain and appears purple under a light (brightfield)microscope.

In the Gram procedure, a cell preparation is first stained with iodine, next rinsed

(decolorized) with acid-alcohol and final stained with a non-specific safranin solution.

The acid-alcohol rinse washes the iodine stain from Gramcells. The safranin stain

then gives them the characteristic pink color shown in this slide.

The Gram stain was used for nearly 100 years before we understood the details of the

cell wall chemistry that resulted in the positive and negative stain results.



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Lets dispel the myth that maintaining a high pH will kill off fuel microbes. Different

species of microbes are capable of living in environments covering the entire pH range.

Some species (for example Thiobacillus thiooxidans) thrive in acid mine-drainage

streams that are 2N sulfuric acid. Others (for example Bacillus alcalophilus) favor pH 9

to 11.5.

Regardless of whether water is acidic or alkaline, microbes can grow.



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In MWF systems mesophilic bacteria seem to predominate.

Hyperthermophiles have been discovered growing in deep-ocean thermal vents where

pressures are ~200 ATM and temperatures are ~130 C.

The key point here is that the temperatures within fuel systems are optimal for many

microbes.



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Regardless of their general preferred growth temperature, most microbes follow asimilar pattern of growth-rate as a function of temperature.

At temperatures below the optimum, growth-rate v. temperature relationship is

described by the Arrhenius equation:

k =Ae-Ea/RT

kis the rate coefficient of the reaction.Tis the absolute temperature.Ais theprefactor. The units of the pre-exponential factor are identical to those of therate constant and will vary depending on the order of the reaction. If the reaction isfirst order it has the units s-1, and for that reason it is often called thefrequency factoror attempt frequencyof the reaction.Eais called the activation energy of the reaction Ris the gas constant When theactivation energy is given in molecular units, instead of molar units, e.g. joules permolecule instead of joules per mol, the Boltzmann constant is used instead of the gasconstant.

Above the optimum T, biomolecules start to denature and growth-rate tends toplummet as temperatures continue to increase. If you heat fuel to a sufficienttemperature to kill microbes, you stand a considerable risk of starting a fire or causingan explosion.



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In Wisconsin, USA, theres a single fungus colony (started from one cell) that weighs >

20 MT and covers several hectares!! Theoretically, it would form a single colony on a

nutrient growth medium.

In contrast the spore-bearing portion of a fungus aerial hypha (shown in photographon right) weighs 100

colonies.

This is why visual, microscopic examination of MWF suspected of having fungal

contamination is important.



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Top Photo: Comparator Chart for the fungal side of a typical dipslide or paddle type test

kit. Fungal colonies are typically white, dry & fuzzy on this medium.

Bottom Left Photo: Fungal colonies in liquid mediumtypically in liquid media fungal

colonies will appear as spheres. If the areal hyphae the specialized filaments that carryspores) face into the medium, the spheres will be fuzzy. If the areal hyphae face into

the center of the mass, then the outside will be smooth; almost leather like in

toughness. These colonies are very difficult to break apart.

Bottom Right Photo: Plate that had been left open to indoor air. Note variety of colors,

shapes and sizes. Fungal colony coloration is due to color of spores.



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Documents

Module 1-4 - MWF Microbiology