Module 1-4 - MWF Microbiology

2014 STLE Not to be distributed, copied, or replicated without express written consent of STLE.
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.
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.
Uncontrolled microbial contamination in MWF systems is expensive.

Uncontrolled microbial contamination adds to the operations Cost of Quality by
increasing:
-Operational costs
-increased additive use costs
-increased tool costs (decreased tool life)
-decreased productivity fewer good pieces per work shift
-Waste treatment
-Actual labor and disposal costs
-System down-time during cleaning (loss production time)
-Waste handling costs treatment & 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 Effects module, later this morning.
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.
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) can
plug the filter surfaces, inhibiting fluid flow and causing the need fro frequent, costly
element replacement.
This topic will be discussed in module: Microbiology Potential Health Effects, later this
morning.
The major lesson here is that by controlling microbial contamination in MWF systems
you can increase your profitability!!
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 the
MWF, 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.
Left photo: destabilized emulsion acidic byproducts of microbial metabolism

(biological activity) spit emulsion.
Center photo: rotting fish (typical amine odor)
Right photo: rotten eggs (sulfide odor)
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 is
a critical factor in maintaining production rates.
Bacteria produce a wide variety of volatile compounds, a few of which are illustrated
here.
Hydrogen sulfide, characterized by its rotten egg odor is very toxic.
Ammonia pungent odor is severely irritating , but not toxic.
Mercaptans (thiols) are noxious but not toxic except at very high
concentrations. 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.
MWF systems with heavy fungal contamination have a characteristic early, musty odor.
The primary MVOC contributing to this earthy odor is geomsin (4,8 -dimethyldecalin4 -ol), although a broad range of alcohols, ketones, terpenes, furans, and ethers
contribute to this scent.
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
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 structures from STLE MWF Training
Course Module 2.6, courtesy of J. Burke, Houghton International, Valley Forge, PA
Lower left: Similar to upper left from 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 heavily
fouled courtesy of F. Passman.
Its normal for filters to plug over time.

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.
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. More
on that during the discussion of health issues.
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 bypassing
the 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.
Left photo: MWF demulsification.

Right photo: water line that has been attacked by acid corrosion.
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 to
plug 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.
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 treated
by 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.
For every CFU/mL in the recirculating MWF, there are probably 10 6 CFU/cm2 on 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
The distribution of cells can be quite complex. Moreover, the proteomics (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 microbiological
test methods they appear to be different types of microbes.
Images are from Center for Biofilm Engineering, Bozeman, MT.
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 geometry of a typical biofilm. The
illustration in this slide shows oxygen (O2) concentration gradients in a cross section of
biofilm. Compare the [O2] shown on this slide with sulfide (S=) concentrations shown
on the next slide.
Note that the [S=] increases as the [O2] decreases and vice versa.
Different [O2] zones select for different types of microbes.
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.
Mist droplets generated inside the plant can transport microbes from one sump to
another.
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.
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.
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
or flumes).
Good news: Older, open-grate decking has been replaced with solid plates at most
facilities its 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.
Left Photo: Sump with substantial tramp oil contamination emulsion has been split by
microbial attack.
Right Photo: Mixed bacterial and fungal population forming a slime mat on the surface
of a MWF sump.
Now that we understand the impact of microbial contamination, lets turn to the
subjects of that science the microbes.
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 trunk
reflect 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.
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 smallest
bacteria are too small to be seen with a conventional light microscope (maximum
magnification = 1,200x).
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.
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.
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.
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 as
thick 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 nimbly
than 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."
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 Gram cells. 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.
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.
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.
Regardless of their general preferred growth temperature, most microbes follow a

similar 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
k is the rate coefficient of the reaction.

T is the absolute temperature.
A is the prefactor. The units of the pre-exponential factor are identical to those of the
rate constant and will vary depending on the order of the reaction. If the reaction is
first order it has the units s-1, and for that reason it is often called the frequency factor
or attempt frequency of the reaction.
Ea is called the activation energy of the reaction R is the gas constant When the
activation energy is given in molecular units, instead of molar units, e.g. joules per
molecule instead of joules per mol, the Boltzmann constant is used instead of the gas
constant.
Above the optimum T, biomolecules start to denature and growth-rate tends to
plummet as temperatures continue to increase. If you heat fuel to a sufficient
temperature to kill microbes, you stand a considerable risk of starting a fire or causing
an explosion.
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 photograph

on right) weighs <10-9 g and, if it burst open while plating, would produce > 100
colonies.
This is why visual, microscopic examination of MWF suspected of having fungal
contamination is important.
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 medium typically in liquid media fungal
colonies will appear as spheres. If the areal hyphae the specialized filaments that carry
spores) 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.

Module 1-4 - MWF Microbiology

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2014 STLE Not to be distributed, copied, or replicated without express written consent of STLE.

Upon completion of this module, students will:

Clean MWF that is in good condition:

Uncontrolled microbial contamination in MWF systems is expensive.

Left photo: destabilized emulsion acidic byproducts of microbial metabolism

Lower left: MWF header showing MIC holes

Biofouling is the accumulation of biomass on surfaces.

Its normal for filters to plug over time.

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

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

Left photo: MWF demulsification.

Antimicrobials are also known as biocides.

Many machinists also tend gardens and raise livestock.

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

Bacteria often from smooth or glistening colonies on growth media.

In MWF systems mesophilic bacteria seem to predominate.

Regardless of their general preferred growth temperature, most microbes follow a

k is the rate coefficient of the reaction.

In contrast the spore-bearing portion of a fungus aerial hypha (shown in photograph

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