Microbial Influenced Corrosion: Role of Bacterial Attachment
and Biofilm
Honor’s Project 4200:497
Sponsor: Dr. Bi-min Newby
By: David M. Vaughn-Thomas
Executive Summary
The project consists of a literature review of the subject microbial influenced corrosion. In
addition, a preliminary study performed by Hua Wang of the University of Akron was reviewed.
The study analyzed bacterial attachment in which the surface affinity for Escherichia coli and
Pseudomonas aeruginosa (PAO1) was observed using a low shear flow chamber enabling in situ
monitoring of cell attachment and biofilm formation. In addition, the effects on corrosion
behaviors of carbon steel and aluminum were assessed when exposed to E coli and PAO1. The
purpose of the monitoring and similar future monitoring was to individualize the affinity of
specific microorganisms to specific surfaces. From performing a literature review I have learned
about the pre-requisites for biofilm formation as well as mechanisms on the molecular scale. In
addition, surface roughness was found to positively correlate with bacterial attachment for
most cases but not all situations. In these instances very smooth and very rough surfaces
showed the highest level of bacterial attachment (Medilanski et al. 2002). Sulfur reducing
bacteria was found to be a major contributor to MIC and its mechanisms were documented
(Suen 1994). Lastly the formation of pits on corroded surfaces was found to be autocatalytic.
Bacterial cell counts were limited to estimations based on averaging five counts over an area of
100mm2. Lastly, carbon steel and aluminum coupons were placed in the chamber containing
both microorganisms for the purpose of comparing the corrosion induced by microorganisms to
the corrosion in a cell-free aqueous environment (control).
Both the cell chamber and cell-free control chamber received an organic nutrient solution
constantly flowing over the coupons and the glass slides. By first characterizing such relative
Vaughn-Thomas 1
affinities, the structural properties of the surface along with the specific metabolic processes of
the organisms can be studied to pinpoint more specific causes of cellular attachment. By
identifying these specific properties, future experimenters may tailor protective coatings and
materials to thwart the attachment of common microorganisms to a specific locale. By
completing this project I have learned to acquire valid academic references from a variety of
credible sources. The hands on aspects of the project has increased my skills with various
techniques such as using a microscope, utilizing recording software, safe handling of
microorganisms, record keeping, and various laboratory skills. This has increased my
independence. My future recommendations for the project would be to attain a list of the
various cellular byproducts that contribute to corrosion. The researcher could then study the
effects of these byproducts on different surfaces. Lastly, by attaining statistical averages of the
various marine life in certain regions a manufacturer could development methods of corrosion
prevention that consider the specific marine life the equipment will be exposed to.
Vaughn-Thomas 2
Table of Contents
Introduction ............................................................................................................................................ 4
Part I: Literature Survey ........................................................................................................................... 4
Biofilms and its Role in MIC.................................................................................................................. 4
Carbon Source in Biofilm ...................................................................................................................... 6
Bacterial Attachment and Formation of Biofilms .................................................................................. 7
The Role of Iron in Biofilm Formation ................................................................................................. 10
Cell Signaling and Genetic Factors for Biofilm Formation.................................................................... 10
Detection of Biofilms.......................................................................................................................... 11
Sulfur Reducing Bacteria .................................................................................................................... 11
Pitting ............................................................................................................................................... 12
Part II: Bacterial Attachment Experiment ............................................................................................... 13
Procedure .......................................................................................................................................... 14
Results............................................................................................................................................... 14
Conclusions........................................................................................................................................ 17
References ............................................................................................................................................ 19
Appendix ............................................................................................................................................... 21
Vaughn-Thomas 3
Introduction
Microbial Influenced Corrosion (MIC) is a mechanism referring to the corrosion of surfaces
influenced by the physiological processes of microorganisms. Each specific physiological process
can exacerbate existing corrosion sites or create new sites entirely. Microbial influenced
corrosion affects numerous industries, plant facilities, and mainly marine structures. Corrosion
costs the US and estimated 3% of the gross domestic product. (Punckt et al 2004). Microbial
influenced corrosion is estimated to account for 20 to 30% of all corrosion (Gu 2011). The
methods by which MIC takes place include the effects of cellular byproducts on surfaces and
the ability to maintain high concentrations of cellular byproducts on a surface. The ability to
maintain such high concentrations will be explained later within the topic of biofilms. The
objective of this report is to gain a broad understanding of the conditions by which MIC takes
place through a literature survey. In addition, the attachment behavior of two bacterial species
will be observed in order to gain an understanding of bacterial attachment.
Part I: Literature Survey
Biofilms and its Role in MIC
Biofilms play a substantial role in MIC. Cellular byproducts are capable of being contained
within biofilms. As a result, hydrogen ions, salt, and oxygen are contained in much higher
concentrations within the biofilm than in the normal environment. The metabolic processes
that create said byproducts can continue as long as the microorganism or colony is provided
conditions that facilitate or maintain life. In order for this to occur the microorganisms must
have access to the following: (1) water, (2) an energy source, (3) a carbon source, (4) electron
donors, (5) and electron acceptors (See Appendix).
Vaughn-Thomas 4
(1) Water provides the media for which corrosive byproducts can be dissolved. Such byproducts
include certain acids and salts. In addition, water is the key requirement for the colony to exist.
Without water, a microorganism cannot survive and thus cannot contribute to MIC. (2) An
energy source is needed to ensure the colony can live, grow, and reproduce. For phototrophs
the energy source is light. Therefore, it is not possible to attribute MIC to phototrophs in
environments that are cut off from light sources such as underground structures, pipes, and
very deep underwater structures. For chemotrophs the energy source is organic matter such as
other organisms, dead food matter, and waste. Other uncommon organisms such as
methylotrophs can obtain energy from single carbon molecules such as methanol, methyl
amines, formaldehyde, and formate. (3) A carbon source is required to manufacturer the
appropriate cellular machinery that enables the organism to grow and continue the metabolic
processes contributing to MIC and the formation of biofilms. For heterotrpohs the carbon
source can include a diverse array of organic matter. For autotrophs the carbon source is
created via the fixation of carbon dioxide, a process by which gaseous carbon dioxide is
converted to a solid organic compound. Naturally dissolved carbon dioxide in water may play
an increasing role in MIC from which the culprits include autotrophs. For cellular processes to
occur, the microorgansim must have access to the appropriate (4) electron donor. Depending
on the specific metabolic pathway an organism takes, a specific electron donor is required to
maintain a hydrogen gradient for the production of adenosine triphosphate (ATP), the prime
molecule for energy transfer to take place. For lithotrophs, the electron donors include
inorganic molecules such as ferrous iron (Fe 2+), sulfur, hydrogen, ammonia, and phosphate.
Such organisms such as Acidthiobacillus ferrooxidans (iron bacteria) can directly contribute to
Vaughn-Thomas 5
MIC by oxidizing iron made structures. Organotrophs rely on organic molecules for their
electron donors. Lastly the organism requires (5) an electron acceptor. Depending on the
respiratory pathway, many different types of molecules can be used such as oxygen, water,
nitrates, sulfates, and carbon dioxide. Thus the diverse requirements for countless
microorganisms can account for the various substrates, and media in which MIC takes place.
These diverse requirements can even account for some conflicting data behind current MIC
theory.
Carbon Source in Biofilm
Certain microorganisms possess the ability to use inorganic substances as their electron donors
during respiration. Lithoheterotrophs possess such ability but unlike lithoautotrophs, they do
not possess the ability to fix carbon dioxide into biomass. Thus without a source of organic
carbon, a lithoautotroph cannot grow. Since corrosion is caused by biofilms it may be intuitive
to assume that limited food (organic carbon) would result in reduced biofilm and reduced
corrosion. Earlier experiments confirmed that suspicion but they were somewhat misleading.
Professor Tingyue Gu of Ohio University performed an experiment using previously established
biofilms and found that limiting the organic carbon source actually resulted in increased
corrosion. Dr. Gu concluded that the microorganisms are replacing the energy provided by
organic carbon with energy provided via the oxidation of inorganic substances such as iron. The
latter option contributes to corrosion. However, if the organic carbon source was completely
removed corrosion would decrease because a small amount of carbon is needed to form the
enzymes necessary to oxidize iron. The result was that the highest amount of corrosion resulted
from limiting the organic carbon by 99%.
Vaughn-Thomas 6
Bacterial Attachment and Formation of Biofilms
In order for a biofilm to form a surface must be exposed to a flowing fluid containing
microorganisms. The surface must then be conditioned via the adsorption of nutrients and
organic molecules to the surface (Trulear & Characklis 1982). This conditioning is necessary not
only to sustain initial biofilms but also to provide incentive for motile microorganisms to move
toward the surface. Numerous researchers have demonstrated that materials with diverse
properties are rapidly conditioned by absorbing such organics when exposed to natural water
(Little 2007). Once initially attached the formation of a polysaccharide material is necessary for
binding and cellular reproduction is necessary for growth and continued maintenance of the
biofilm. Trulear et. al. divided biofilm development into three phases: Induction, Accumulation,
and Plateau. The induction phase was described as the conditioning of the surface along with
the initial attachment of microorganisms. The accumulation stage was described as an initial
logarithmic increase in biomass followed by a constant rate. Eventually the biofilm reaches a
mass in which the biofilm growth rate and the biofilm loss rate due to shear stress reaches
equilibrium and thus plateaus.
Figure 1: Initial steps and formation of biofilm (Taken from Todar’s Online Textbook of Bacteriology Todar 2008).
Vaughn-Thomas 7
The initial cell attachment can be attributed to mechanical adhesion among other forces. In
mechanical adhesion, folds of the plasma membrane, flagella, or any extendable structure of
the cell interlocks with crevices or grooves inherent in the substrate. Roughness is a unit used
to quantify such surface irregularities. A rougher substrate can provide greater surface area for
cell attachment as demonstrated by Dr. Chai A. Korber in a study on the substratum influences
on biofilms formed by Salmonella enteritidis (Korber et al. 1997). In addition, Nickels et. al.
showed a reduction in biomass retained on surfaces of silica containing equal size grains but
different surface topography. The conclusion was a reduced biomass results from the absence
of cracks and crevices (Nickels et al. 1981). However, some studies have shown smoother
surfaces resulting in increased cell adhesion. Medilanski et. al. showed that 4 separate bacterial
species on surfaces of varying roughness show increased adhesion on either extremes i.e. a
minimum adhesion not correlated to minimum roughness see figure 2 (Medilanski et al. 2002).
Figure 2:Normalized levels of adhesion of Pseudomonas putidamt2( )
Pseudomonas aeruginoas PA01 ( )Rhodococcus( ) and Desulfovibriodesuluricans(
) on UNS S30400 stainless-steel surfaces of different roughness. (Reprinted from
Medilanski et al., 2002, without permission)
Vaughn-Thomas 8
The type of substrate plays a role in biofilm attachment. It has been demonstrated through vast
amounts of literature that a metal substratum greatly influences the formation rate and cell
distribution of biofilms during the few first hours of formation. Gerchakov et. al. performed a
study involving four surfaces: glass, stainless-steel, brass, and a copper-nickel alloy. His findings
showed a significant increase in colony formation for stainless steel (see figure 3).
Figure 3:Number of marine heterotrophic bacteria cultured from various
substrates in relation to exposure time on surfaces including glass, 304 stainless
steel (UNS S30400), 60/40 copper-zinc brass (UNS C28000), and 90/10 coppernickel (UNS C70600) (Gerchakov et al., 1977 reprinted without permission).
Vaughn-Thomas 9
The Role of Iron in Biofilm Formation
Certain bacteria such as Acidithiobacillus ferrooxidans rely on iron as a source of electrons (see
appendix) which contribute to the formation of pits via pitting. In addition, iron plays an
important role by serving as a signal for biofilm synthesis. In a study in which the iron was
sequestered from PAO1, the bacterial species failed to form biofilms from previously
established thin layers of cells (Banin, Vasil, & Greenberg, 2005). Banin et. al. concluded that by
sequestering iron, inhibitory concentrations of the chelator lactoferrin block the ability of PAO1
biofilms to mature. This is thought to be a result of the motile structures of PAO1 persistently
twitching in response to low iron concentrations.
Cell Signaling and Genetic Factors for Biofilm Formation
Biofilms, such as those created by pseudomonas aeruginosa (PAO1) consist of an extracellular
polysaccharide matrix containing embedded bacterial cells. Certain bacteria can communicate
with one another via cell signals which trigger the formation of macroscopic groups or biofilms.
PA01 was shown to contain two such signals that encourage the formation of biofilms. (Davies
et. al.). Davies et. al. performed a study in which the biofilms produced by a mutant PAO1
(incapable of expressing the signaling genes) was compared to that of a typical species of PAO1.
The results concluded that the biofilm produced by the mutant was a single sheet 20% thinner
than the control. Furthermore, the biofilm formed by the control contained complex layers of
biofilms in which water channels permeated between each layer. They concluded that while
cell to cell signaling may not play a crucial role in biofilm formation, it is highly involved in the
maintenance of thicker and more complex biofilms
Vaughn-Thomas 10
Detection of Biofilms
The presence of corrosion products and corrosion inhibitors can sometimes mask the normal
signs of biofilms formation such as its characteristic slimy texture along with its distinct organic
odor. A simple method for verifying the presence of a biofilm has been developed by detecting
the presence of organics in a sample (Beech et al. 2000). The sample is centrifuged at 3000rpm.
The pellet is transferred to a small amount of distilled water and placed in a pre-weighted
melting-pot where it is heated at 105 degrees Celsius until dry. The dry weight is estimated
from the difference in weights. The sample is then heated to 500 degrees Celsius upon which
the difference in weight yields an approximation of the amount of organic matter in the
sample. In mature biofilms, the organic matter can account for 30 to 40 percent of the dry
weight. The contribution of the organic matter from microorganisms can be detected by
analyzing the protein and carbohydrate content via spectrophotometry.
Sulfur Reducing Bacteria
Sulfate reducing bacteria (SBR) has been recognized as one of the most important
microorganisms relating to the anaerobic corrosion of metals (Suen 1994). SBR are bacteria that
obtain energy by oxidizing organic compounds while reducing sulfates into hydrogen sulfide
and other sulfides. In conjunction with a biofilm, the presence of SBR is typified by the
formation of a hydrogen layer over the surface for which the bacteria can oxidize to create
hydrogen sulfide. SBR causes corrosion under anaerobic conditions by removing hydrogen from
the layer via the enzyme hydrogenase. This depolarization of the system, often referred to as
the Cathodic Depolarization Theory, is characterized by the following equations (Nace 1982).
Vaughn-Thomas 11
Pitting
Pitting is a form of corrosion resulting in the creation of small holes in the substrate. In nonbiologically induced corrosion pitting is the result of interactions between an anode and a
cathode. A media containing electrolytes enables ions to move from the anode to the cathode
causing losses in the anode leading to the formation of small pits. In microbial influenced
corrosion the anode or substratum is losing ions to the microorganism so it can maintain its
respiratory pathways. In addition, the formation of biofilms enables high concentration of
hydrogen ions leading to a highly acidic environment. This environment is thus more
susceptible to continued corrosion and pitting. Pitting is said to be autocatalytic for two main
reasons. (1) As the size of the pit increases, the rate of material loss increases. This is due to the
ratio of diameter to surface area i.e. as diameter doubles, the surface area quadruples. (2)
Stainless-steel which is naturally protected from corrosion via an oxide layer can undergo
pitting. A previously formed pit is the site for reactions that produce species that move laterally
on the surface and weaken the protective oxide layer (Punckt et al. 2004). Therefore, the
Vaughn-Thomas 12
presence of pits increases the probability of further pit formation analogous to a chain reaction.
Professor Tingyue Gu of Ohio University spoke in a lecture at the University of Akron regarding
the complications of numerous short pits vs. few but deep pits. While numerous short pits may
be the most readily visible, a single long pit can be the most devastating in terms of evaluating
material failure.
Part II: Bacterial Attachment Experiment
Because of the diversity of biofilms and their major contribution towards MIC it is important to
study the individual organisms that make up the biofilms (micro-scale). The easiest way to limit
biofilm formation is to prevent the initial attachment of cells. Initially a study involving bacterial
attachment was performed over winter break initiated by Hua Wang for which I assisted by
photographing results, and cutting the glass slides used in the apparatus. Due to unforeseen
circumstances the results were unable to be used. An additional study was performed at the
University of Akron by Hua Wang along with other faculty and students at the University of
Akron with the purpose of monitoring the attachment behaviors of Escherichia coli and
Pseudomonas aeruginosa (PAO1). The purpose of the monitoring and similar future monitoring
is to individualize the affinity of specific microorganisms to specific surfaces. By first
characterizing such relative affinities the structural properties of the surface along with the
specific metabolic processes of the organisms can be studied to pin point more specific causes
of cellular attachment. By identifying these specific properties future experimenters may
enable the production of tailored coatings and materials that can thwart the attachment of
common microorganisms to a specific locale.
Vaughn-Thomas 13
Procedure
A flow chamber system was developed by Dr. Newby’s team at the University of Akron’s
Department of Chemical Engineering that allows reproducible in situ monitoring of biofilm
development under low-shear flow conditions. Such conditions are often encountered in
environments subject to MIC. Metal coated glass slides where placed in a glass chamber (see
figure 4).
The chamber slides were conditioned with a nutrient solution. Microorganisms w ere pumped
into the chamber and the initial cell attachment was recorded over time.
Results
The initial cell attachment over the first 3 hours was counted and averaged. In addition, the
slides were rinsed lightly with a saline solution and the remaining cells were counted. The data
for both microorganisms is tabulated below.
Vaughn-Thomas 14
(100cells/mm^2)
Escherichia coli Cell Attachment
40.00
35.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
1
2
3
Rinsed
Time (Hours)
Figure 5: Escherichia coli cell attachment on glass surface (Data taken by Hua Wang, University of Akron).
(100cells/mm^2)
PAO1 Cell Attachment
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
1
2
3
Rinsed
Time (Hours)
Figure 6: PA01 cell attachment on glass surface (Data taken by Hua Wang, University of Akron).
The PAO1 showed less affinity for the glass substrate than the E coli. In addition, the PAO1
showed a smaller retention rate of cells after rinsing. After rinsing, PAO1 lost 31.1% of its
surface cells whereas E coli lost 13.0%.
Vaughn-Thomas 15
In addition to the glass slides, carbon steel and aluminum coupons were placed within the
chambers containing the cells along with a control chamber that contained the nutrient
solution without cells. The chambers were photographed, sonicated, and then photographed
again. The purpose was to obtain a visual on the corrosion caused and subjectively compare the
results to the control.
Figure 7: Escherichia coli after sonification. Coupons of control (left) and experimental (right). Aluminum (right)
and Carbon steel (left) after cleaning. (Photograph taken by Hua Wang, University of Akron).
After cleaning, the control and experimental aluminum coupons for E coli showed little
difference in visible surface corrosion whereas the aluminum deposited on the glass slide
showed some slight discoloration for the experimental sample. The experimental carbon steel
coupon did show a more dark color when compared to the control.
Vaughn-Thomas 16
Figure 8: PAO1 After sonification. Coupons of control (left) and experimental (right). Aluminum (left) and Carbon
steel (right). (Photograph taken by Hua Wang, University of Akron).
Both the control and experimental for PAO1 showed no visible signs of corrosion for the
aluminum coupon and aluminum coated slide. After cleaning, the carbon steel coupon showed
more surface discoloration and irregularities for the experimental when compared to the
control.
Conclusions
Since the glass surface area was void of iron, it is possible to attribute the lower surface affinity
expressed by PAO1 to twitching in response to low iron concentrations as reported by Banin et.
al. The results for both the E coli and PAO1 study showed that the introduction of
microorganisms to a metalic surface increases visible corrosion when compared to a metalic
surface receiving only an aqueous solution of organics. The literature view served to broaden
the understanding of MIC. However, the more answers we receive from knowledge regarding
Vaughn-Thomas 17
MIC the more questions they create. MIC is an extremely complex topic that relies not only on
the many properties of the surface, but also on the numerous metabolic process from many
different microbial species.
Vaughn-Thomas 18
References
Banin, Ehud, Michael L. Vasil, and E. P. Greenberg."Iron and Pseudomonas Aeruginosa Biofilm
Formation." National Academy of Sciences 102.31, 2 Aug. 2005. Web. 3 Mar. 2011.
<http://www.jstor.org/stable/pdfplus/3376219.pdf>.
Beech, Iwona, Alain Bergel, AlfonsaMollica, Hans-Curt Flemming, Vittoria Scotto, and Wolfgang
Sand."Simple Methods for the Investigation of the Role of Biofilms in
Corrosion."BiocorrossionNetworok (2000). Print.
Brant, Jonathan A., and Amy E. Childress. "Membrane–Colloid Interactions: Comparison of Extended."
Environmental Engineering Science 19.6 (2002). Print.
Davies, David G., Mattew R. Parsek, James P. Pearson, Barbara H. I, J. W. Costerton, and E. P.
Greenberg."The Involvement of Cell-to-Cell Signals in the Deelopment of a Bacterial
Biofilm."Science ns 280.5361 (1998): 295-98. Science.Web. 13 Mar. 2011.
<http://www.jstor.org/stable/pdfplus/2895685.pdf?acceptTC=true>.
Gerchakov, S. M., F. J. Roth, B. Sallman, L. R. Udey, and D. S. Marszalek."Observation on Microfouling
Applicable to OTEC Systems." OTEC Biofouling and Corrosion Symposium (1977): 63-75. Print.
Gu, Tingyue. "Understanding Microbiologically Influenced Corrosion (MIC) Mechanisms." The
University of Akron, Akron. 10 Feb. 2011. Lecture.
Korber DR, Chai A., G. M. Woofaardt, and S. C. Cadwell."Substratum Topography Influences
Susceptibility of Salmonella Enteritidis Biofilms to Sodium Phosphate." Environ Microbiology 63
(1997): 3352-358. PubMed.gov. Environ Microbiology. Web. 2 Feb. 2011.
<http://www.ncbi.nlm.nih.gov/pubmed/9292984>.
Little, Brenda J., and Jason S. Lee.Microbiologically Influenced Corrosion. Hoboken, NJ: WileyInterscience, 2007. 9-10. Print.
M. Van Loosdrecht, Mark C., Johannes Lyklema, Willem Norde, and Alexander J. Zehnder. "Bacterial
Adhesion: A Physicochemical Approach." Microbial Ecology, 17.1 (1989): 1-15. JSTOR.Web. 3
Mar. 2011. <http://www.jstor.org/stable/pdfplus/4251031.pdf?acceptTC=true>.
Vaughn-Thomas 19
Medilanski, E., K. Kaufmann, L. Y. Wick, O. Wanner, and H. Harms."Influence of the Surface Topography
of Stainless Steel on Bacterial Adhesion."Biofouling 41 (2002): 193-203. Print.
NACE (1982) TPC Publication 3.“The Role of Bacteria in the Corrosion of Oil Field Equipment.” Houston,
TX
Nickels, J. S., Ronald J. Bobbie, Robert F. Martz, Glen A. Smith, David C. White, and Norman L.
Richards."Effects of Silicate Grain Shape, Structure, and Location on the Biomass and
Community Structure of Colonizing Marine Microbiota." Applied and Environmental
Microbiology 41.5 (1981): 1262-268. PubMed.gov. Web. 10 Mar. 2011.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC243899/>.
Peng, Ching-Gang, Shing-Yi Suen, and J. K. Park."Modeling of Anaerobic Corrosion Influenced by
Sulfate-Reducing Bacteria."Water Environment Federation 66.5 (1994): 707-15. Print.
Punckt, C., M. Bolscher, H. H. Rotermund, A. S. Mikhailov, L. Organ, N. Budiansky, J. R. Scully, and J. L.
Hudson. "Sudden Onset of Pitting Corrsoin on Stainless Steel as a Critical Phenomenon."
Science ns 305.5687 (2004): 1133-136. JSTOR.Web. 6 Feb. 2011.
<http://www.jstor.org/stable/3837614>.
Todar, Kenneth. "The Normal Bacterial Flora of Humans."Online Textbook of Bacteriology. 2008. Web.
15 Mar. 2011. <http://www.textbookofbacteriology.net/normalflora_2.html>.
Trulear, Michael G., and William G. Characklis."Dynamics of Biofilm Processes." Water Environment
Federation 54.9 (1982): 1288-301. JSTOR.Web. 28 Jan. 2011.
<http://www.jstor.org/stable/25041684>.
Vaughn-Thomas 20
Appendix
Organism Classification
General Organism Requirements
Specific Organism Requirement
Vaughn-Thomas 21