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MICROBIOLOGY FOR THE UNITINTIATED – PART 6: BACTERIA


A rod-shaped, polarly flagellated bacterium.
Source: https://factrepublic.com/facts/33765/

Introduction

Now that I’ve provided an overview of the characteristics of all life, I’ll describe the most common types of organisms that contaminate industrial process fluid systems – i.e., oilfield injected and produced waters, fuel-associated waters, heat exchange systems, and countless other systems in which fluids are contained of through which they flow. Historically, the two primary types of microbes recovered from systems not exposed to light are bacteria and fungi. More recently a third group – archaea – have been detected too. When light is present, algae are common contaminants. In this article I’ll discuss bacteria. In future articles I’ll focus on each of the other types of microbes.

The Tree of Life

Taxonomy is the science of classifying living things. In 1735, Carl Linnaeus published Systema Naturae in which he described a binomial system for identifying plants (i.e., originally plant and subsequently all organism based on whether they shared or didn’t share a characteristic). Under Linnaean taxonomy, there are eight categorical levels (Figure 1). The highest – most inclusive – level is domain. As illustrated in Figure 3a, there are three domains – Bacteria, Archaea, and Eucarya. Each domain is divided into Phyla. Thus far, the domain Bacteria has approximately 1,300 phyla – with the number still increasing. Phyla are further divided among classes. Again, using Bacteria as our example, the phylum Proteobacteria is comprised of five Classes – Alphaproteobacteria (α-proteobacteria), Betaproteobacteria (β-proteobacteria), Gammaproteobacteria (γ-proteobacteria), Deltaproteobacteria (δ-proteobacteria) and Epsilonproteobacteria ( ε-proteobacteria). The γ-proteobacteria includes 14 Orders which are further divided into 404 Families. For example, the Order Pseudomonadales includes two Families – Moraxellaceae and Pseudomonadaceae – both of which include bacteria commonly recovered from industrial process water systems. Families are comprised of genera (singular – genus). Thus, the genus Pseudomonas is a member of the family Pseudomonadaceae. Traditionally, species was the smallest taxonomic unit (for example Pseudomonas aeruginosa). However, current taxonomic schemes typically include two additional levels – strains and biovariants (abbreviated as biovar.).


Fig 1. Linnaean taxonomy’s eight taxonomic levels.


Fig 2. Phylogenic trees – a) simplified tree showing genetic distances between Bacteria, Archaea, and Fungi; b) more complex genomic map illustrating Bacteria’s emergence approximately 3.2 billion years ago, Archaea’s emergence approximately 1.5 billion years ago, and fungi arriving at approximately the same time as vascular plants – approximately 1 billion years ago. Sources: a) Carreón, Gustavo & Hernández-Zavaleta, Jesús Enrique & Miramontes, Pedro. (2005). DNA Circular Game of Chaos. 757. 10.1063/1.1900503, b) https://i.pinimg.com/originals/46/28/92/46289232f53138d4134ea0e829589d09.gif, c) https://organismalbio.biosci.gatech.edu/biodiversity/eukaryotes-and-their-origins/.

In traditional taxonomy – including microbial taxonomy – organisms were clustered based on their observable similarities. Thus, members of a genus share more characteristics than members of a family. Diversity increases with the classification level. Currently, microbial taxonomists estimate there are at least a billion bacterial species, of which fewer than 10,000 have been characterized (that’s <0.001 % in case you are wondering). Figure 1b offers a visual perspective on the diversity of bacterial life. The black, web-like region on the bottom left reflects the genetic branching since bacteria first existed – an estimated 3.2 billion years ago (the Earth’s estimated age is 4 billion years). I don’t expect readers to use Figure 2b as anything more than an illustration of just how divers the kingdom Bacteria is. Figure 1c provides a simplified tree showing first the evolution of eukaryotes as hosts for bacteria and second the similar age of the Archaea and Eukaryote domains. Figure 3c also illustrates how genetic material (ribonucleic acid – RNA, deoxyribonucleic acid – RNA, and proteins were probably components of self-replicating proto-microbes before the last universal common ancestor (LUCA). Based on genomic analyses, the LUCA is estimated to have existed between 3.48 and 4.1 billion years ago – soon after Earth formed. Although the term LUCA suggests that there was a single genetic entity from which all subsequent life evolved, it is more likely that numerous types of protomicrobes developed and the most successful continued to evolve.

What are Bacteria?

ASTM provides a good working definition. A bacterium is any member “of a class of microscopic single-celled organisms reproducing by fission or by spores. Characterized by round, rod-like, spiral, or filamentous bodies, often aggregated into colonies or mobile by means of flagella. Widely dispersed in soil, water, organic matter, and the bodies of plants and animals. Either autotrophic (self-sustaining, self-generative), saprophytic (derives nutrition from nonliving organic material already present in the environment), or parasitic (deriving nutrition from another living organism).” I discussed bacterial reproduction in July 2023.

By current estimates, Bacteria are the most diverse organisms. To paraphrase Donald Rumsfeld’s “Known knowns” comment, bacterial diversity and total biomass estimates fall into the category of known unknowns. Figure 3 illustrates a typical dendogram created from DNA data from a soil sample. The dendrogram’s branches represent genetic similarities, with similarities between two DNA molecules increasing as the dendogram branches from left to right. Most commonly, the individual organism type – operational taxonomic units (OTU) are identified only by alphanumeric codes. This is because only a small percentage of bacterial OTU that have been detected by genomic profiling have been identified at the Family, Genus, or Species levels. During the past decade, some researchers have suggested that apparent similarities among DNA molecules based on their migration through an electric field gel (denaturing gradient gel electrophoresis – DGGE – see the black bands in the center of Figure 3) is misleading. DNA molecules with different genes can appear to be common OTUs. Some have argued the amplicon sequence variant (ASV) is more precise. Repeating my mantra, I make this point only to illustrate my point about bacterial diversity. As I explained in my July 1018 What’s New article, unlike antibiotics which are designed to kill specific microbes without damaging surrounding human cells (or microbes that are essential for good health), industrial microbicides typically target a broad spectrum of microbes. My favorite analogy is Bambi v. Godzilla (Figure 4). Full taxonomic profiles can be useful to understand the microbial ecology of the system under investigation, but is unlikely to inform contamination control decisions.


Fig 3. Dendogram of genomic profile of bacteria recovered form water samples. Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261970


Fig 4. Bambi versus Godzilla (Source: 1969 animated feature – https://www.indiewire.com/2015/01/a-bambi-meets-godzilla-live-action-remake-123687/#!)

Classifying Bacteria

Shape

Recognizing that the microscope was the first tool use to detect and identify bacteria, the earliest taxonomic schemes were based on their appearance (morphology). With variations within each group, the primary forms are rod, spheres, commas, and spirals (Figure 5). Once the Gram stain – developed by Hans Christian Gram – was able to differentiate between bacteria with cell walls thet retained an iodine-based stain and those that didn’t – bacteria in each of the morphological groups were classified as being either Gram positive (Figure 6a) or Gram negative (Figure 6b).


Fig 5. Primary bacterial morphologies.


Fig 6. Primary bacterial morphologies after Gram staining – a) Gram positive rods; b) Gram negative rods; c) Gram positive spheres; d) gram negative spheres; e) Gram negative commas; f) Gram positive spirals; and g) Gram negative spirals.

It would be decades before the biochemical basis for Gram positive and Gram negative reactions were understood. As illustrated in Figure 7, the Gram positive cell walls are structurally less complex from those of Gram negative bacteria. The largely carbohydrate Gram positive cell wall retains the iodine stain. The peptidoglycan structure of Gram negative cells do not. Other groups of microbes have subsequently been classified based on their reactions to specific stains (acid fast stain, spore stain, etc.).


Fig 7. Cell wall structure – a) Gram positive cell (mostly peptidoglycan); b) Gram negative cell (the cell wall is substantially more complex; the peptidoglycan layer is covered by a lipopolysaccharide layer).

Although morphology can be a useful tool, it is limited due to bacterial pleomorphism – the ability of a specific microbe’s shape to change in response to its environment. More that 20 years ago, researchers at University of Montana’s Center for Biofilm Engineering demonstrated that during biofilm development both the morphology and physiology of genetically identical cells varied, depending on where cells were located within the biofilm matrix. I’m not sure why this was such a startling discovery. Just consider the morphological and physiological diversity of human cells, depending on where they are in (or on our bodies – Figure 8).


Fig 8. Human tissue cells – a) epidermis (skin); b) hair follicle cells; c) lung cells; d) nerve cell; e) retinal cells; f) stomach cells; g) tongue cells.

Physiology

Before genetic testing became affordable and readily accessible, physiological tests were the primary tool – after morphology – to identify microbes. Physiological testing profiles a microbe’s ability to utilize specific nutrients (e.g., glucose or other sugars, various amino acids, hydrocarbons, etc.), produce characteristic metabolites (e.g., ethanol, acetic acid, various gases, etc.), or grow under particular conditions (e.g., oxic or anoxic; acidic, neutral, or alkaline; cold, temperate or hot; etc.). A full battery of physiological tests can include more than 400 different conditions. Traditional microbiological taxonomy standards (for example, Bergey’s Manual of Determinative Bacteriology – nine editions published between 1923 and 1994 – was long considered to be the bible for bacterial identification).

One underappreciated limitation of physiological testing is that these tests could only be run on microbes that could be cultivated in the first place. Moreover, the physiological capabilities of a pure culture can vary with the culture’s age. Consequently, cultures are subject to misidentification due to the variations in their physiological properties as a function of age. I one project, I sent specimens from a single colony to several different labs. Although all the labs used the same automated physiological testing equipment, each lab reported different identities for the culture. When specimens of the same culture were sent to several labs for genetic testing, all identified the microbe correctly.

That said, grouping microbes into physiological classes to assess biodeterioration risk can be useful. I routinely run BART™ (BART – Biological Activity Reaction Test – is a trademark of Droycon Bioconcepts, Inc., Regina, Canada) tests on fuel or fuel-associated water samples that have high ATP-bioburdens. BART vials each contain one of nine different dried media. I typically test for acid producing bacteria (APB), iron related bacteria (IRB), denitrifying bacteria (DN), nitrifying bacteria (NB), slime-forming bacteria (SLYM), and sulfate reducing bacteria (SRB) – microbes commonly associated with microbiologically influenced corrosion. Figure 9 illustrates the reactions in APB, IRB, SLYM, and SRB BART vials inoculated from fuel-associated water.


Fig 9. BART (left to right) APB, IRB, SLYM, and SRB vials inoculated with fuel-associated water – a) Immediately after inoculation; b) after 4-days incubation. The sample was positive for all four metabolic groups.

Figure 9a shows the vials’ appearances immediately after inoculation. Figure 9b shows how each vial changed color and became turbid after four-days’ incubation. The microbes growing in each vial represented diverse types of individual taxa that shared a physiological property – i.e., acid production, etc.

Similarly, representatives from diverse taxa are included in each respiration and fermentation category (see August 2023). Classifying microbes based on their ecologically important physiological properties is like classifying trades people based on their skills – carpenters, electricians, plumbers, welders, etc. It provides no indication of their family lineages.

Optimal and Tolerated Growth Conditions

Just as genetically diverse microbes fall into different physiological categories, they also fall into different growth condition categories. Here, I’m using growth condition to include environmental factors such as temperature, pH, and salinity.

Temperature

Psychrophilic bacteria grow optimally at temperatures between 10 °C and 15 °C. Most will not grow at temperatures >20 °C. Mesophiles – the microbes most commonly associated with disease – require moderate temperatures in the 20 °C to 40 °C range. Most bacteria isolated from fuel and industrial process fluid systems where temperatures are typically <45 °C are mesophilic. Thermophiles require hotter temperatures (≥45 °C). Thermophiles have been recovered from environments where the temperature is >100 °C. Figure 10 illustrates how microbes are classified based on the temperature range in which they will grow.


Fig 10. Microbe classification by optimal and tolerated temperatures.

pH

The Mirriam-Webster online dictionary defines pH as “a measure of acidity and alkalinity of a solution that is a number on a scale on which a value of 7 represents neutrality and lower numbers indicate increasing acidity and higher numbers increasing alkalinity and on which each unit of change represents a tenfold change in acidity or alkalinity and that is the negative logarithm of the effective hydrogen-ion concentration or hydrogen-ion activity in gram equivalents per liter of the solution.” Mathematically,

pH = -Log10 [H+]

Where “p” is a term with an interesting history (which I won’t share here). In water, the fractional sum of hydrogen (H+) and hydroxide (OH) ions is always 1. As shown in Figure 11, pH decreases as Log10 [H+] increases. At neutral pH, Log10 [H+] = Log10 [OH]. Neutrophiles only grow when the in the pH range 7 ± 1.5. Acidophiles thrive at lower pHs. Some (extreme acidophiles) grow in water with pH = 1. Conversely, alkalinophiles require pH ≥ 9 (Figure 12).


Fig 11. Relationships between hydrogen ([H+] and hydroxyl ([OH]) ions and pH.


Fig 12. Microbe classification by optimal and tolerated pH ranges.

Other environmental conditions

Microbes can also be classified based on their preference (i.e., condition for optimal or maximal growth) for atmospheric pressure (barophiles only grow in environments with pressures ≥ 10,000 kPa (100x atmospheric pressure, and vacuumphiles grow in environments with pressures in the 5 kPa to 15 kPa range – 0.05x to 0.14x atmospheric pressure), osmotic pressure, salinity, radiation, or other factors that span ranges that select for different microbes at different requins of the parameter’s range. Organisms that prefer life at the high or low ends of each factor are called extremophiles. Some types of microbes enjoy environments that are extreme by more than one criterion. For example, microbes thriving around deep-sea ocean vents are thermophilic barophiles. They require both high temperatures and pressures.

Most higher life forms can tolerate only a narrow (in Goldilocks terms – just right) range of each of these factors. Bacteria and archaea are unique in the range of environmental conditions they enjoy as kingdoms. However, individual taxa are limited in the range of environments in which they can thrive. Still each time a group of scientists report they have discovered an environment on Earth that is completely devoid of life, they are proven wrong. The development of genetic testing has made it possible to detect bacteria and archaea (more on archaea in my next article) that had previously been undetectable. Regarding the ability of one or more life forms to exist in Earth’s most hostile environments, Carl Sagan’s adage – “The absence of evidence is not evidence of absence” – has been proven true. Of course, Professor Sagan was referring to life beyond earth. We will have to wait and see on that front.

Summary

The domain Bacteria is genetically the most ancient and diverse of the three domains into which all life on earth is divided. To date, microbiologists estimate that we have detected <0.001 % of the different types of bacteria thought to exist in nature. Although the tools used to traditionally identify bacteria are still of some value, genomic test methods have changed the game. Microbes that once seemed to be closely related, based on their appearance and nutritional characteristics (physiology) have been shown to be genetically distant. Conversely, other microbes, once thought to be dissimilar are now known to be close genetic relatives. Because of their ecological and physiological diversity, bacteria play major roles in biotransformation processes (I’ll write about nutrient cycles in future What’s New articles). When biotransformation causes damage, we call it biodeterioration. When it is used to produce useful products, we call it biotechnology, and when it facilitates remediation, we call it bioremediation. We need to remember that microbes live their lives unaware of the labels we humans apply to their activities.

In this article I have barely scratched bacteriology’s surface. Quite often clients ask me to provide them with a list of microbes present in a contaminated system. I invariably ask what they will do with that information. In my opinion, the art and science of microbial contamination control depends on understanding what the population is doing rather than identifying taxonomic names. What do you think? As always, please share your comments and questions with me at fredp@biodeterioration-control.com.

STLE’S TRIBOLOGY & LUBRICATION TECHNOLOGY JUNE 2023 ISSUE SOUNDING BOARD


Bacteria
Source: BACTERIA – Bing images

June TLT Sounding Board Question 1: “Do you think that biolubricants are more sensitive to microbiological attacks?”

In Talmudic style, my response to that question is another question: is biolubricant susceptibility to microbial attack – i.e., biodeterioration – a matter of conjecture?! Most of the respondents apparently thought so. However, one responded: “Depends on the composition and overall resistance to microbial attack. I would imagine testing on microbial resistance would be part of the testing for new products.” This individual chose not to take the bait. They recognized that “sensitivity to microbial attack” depended on the finished stock’s chemistry rather than its source.

Having it both ways?

As ASTM D8324 Standard Guide for Selection of Environmentally Acceptable Lubricants for the U.S. Environmental Protection Agency (EPA) Vessel General Permit points out, the concept of an environmentally acceptable lubricant (EAL) aligns with Humpty Dumpty’s comment: “When I use a word,’ Humpty Dumpty said in rather a scornful tone, ‘it means just what I choose it to mean — neither more nor less.’” Figure 1’s exchange between Humpty Dumpty and Alice is relevant for several reasons.

1. There are numerous EAL labels and the criteria for each label is unique. Germany’s Blue Angel, EU’s Ecolabel US EPA’s EAL, respective criteria are similar but different criteria. Thus, the concept of what defines an environmentally friendly lubricant is a matter of debate.

2. Alice’s response is a question all too commonly asked by marketers. One common trope among companies selling water-miscible metalworking fluids (MWF) is the claim that their product is “biocide-free” when it most likely contains an unregistered component that’s more toxic than most registered biocides (see What’s New, February 2022).


Fig 1. Humpty Dumpty’s perspective on definitions.

3. Humpty Dumpty’s retort is again relevant to the extent that folks can use meaningless generalizations to support any point of view. The fact is that biolubricant biodegradability can and has been tested.

Lubricant marketers want to be able to claim that their products are environmentally acceptable, but are considerably less enthusiastic about also claiming that readily biodegradable products are – well – also readily susceptible to biodeterioration.

Biodegradability testing

All of the environmental label issuers require biodegradability testing. Typically testing is performed in accordance with Organisation for Economic Co-operation and Development (OECD) Test 301 OECD Guideline for Testing of Chemicals – Ready Biodegradability. OECD 301 has a number of variants – 301A through 301F. As illustrated in Figure 2, the first step is to determine a product’s partition coefficient (OECD 117). If the product’s n-octanol/water partition coefficient – POW (POW = ; Log POW = KOW) – is in the ≤3 KOW ≤ 7 range, then the substance is potentially bioaccumulative. Bioaccumulative substances are those that can be captured irreversibly in the tissues of various organisms. When KOW is in the 3 to 7 range, the next step is to test for biodegradability. Lubricants are biodegradable if > 95 % (w/w) of the formulation is biodegradable (>70 % of each molecular species is mineralized within 28 days). If >5 % of the formulation is not biodegradable, the product is then tested in accordance with OECD 305 Bioaccumulation in Fish. The key points here are:

1. Biobased feed stocks and lubricants can readily be tested for their partition coefficient, and

2. If KOW is in the 3 to 7 range, the lubricant’s water accommodated fraction can be further tested for biodegradability.

There is no need to speculate about biobased lubricant biodegradability (i.e., susceptibility to microbiological attack). The OECD test methods are standardized, making it possible to compare how readily any finished lubricant or lubricant stock might be.


Fig 2. A very simplified flow chart for assessing lubricant or lubricant base stock biodegradability and bioaccumulative properties.

Keep in mind that if there is no water present, then neither lubricants nor base stocks – regardless of their chemistry – will be susceptible to microbiological attack. Similarly, if none of the lubricant or base stock partitions into the aqueous phase when water is present, it is not going to be susceptible to microbiological attack.

Here’s the having it both ways conundrum: by definition, the more biodegradable a substance by OECD 301, the more susceptible it is to microbiological attack. Ecological good news is necessarily operational bad news unless you keep water out of the lubricating system!

Are all biobased stocks equal?

Fatty acid methyl ester (FAME) is used to blend biodiesel fuels. Consequently, FAME biodegradation susceptibility has been studied quite extensively. It is not unreasonable to use FAME biodegradation knowledge as a starting point to speculate about biolubricant basestock resistance to microbial attack.

Generally speaking, biodegradability inversely related to oxidative stability. The primary factors affecting both oxidative stability and bioresistance are carbon chain length and saturation. FAMEs with shorter chain length (i.e., <C 15) tend to be more biologically and oxidatively stable than those with chain lengths > C15. Also, saturated molecules (i.e., those with no carbon-carbon double bonds – C=C) are more stable than mono- or polyunsaturated carbon chains (Figure 3). Note, for example that coconut FAME (C12-C14; >85 % saturated) is quite stable. It is even used as a topical antimicrobial agent. Conversely soy and canola FAME (are composed of primarily C16-C18 and C24-C26 mono- and polyunsaturated acids – soy is ~14 % saturated and ~canola is 5 % saturated). Both are readily biodegradable.


Fig 3. Relative biodegradability of selected biobased oils and fatty acid methyl esters.

Summary

Biobased lubricants and lubricant stock susceptibility to biodegradation should not be assessed by polling. As my friend and STLE Metalworking Fluid Education Committee colleague John Burke frequently observes, “without data, you are just another person with an opinion.” Everybody has an opinion, but objective data are relatively easy to develop. The question should have been the basis of a research effort rather than an opinion poll.

What do you think? As always, please share your comments and questions with me at fredp@biodeterioration-control.com.

MICROBIOLOGY FOR THE UNITINTIATED – PART 5: MICROBIAL RESPIRATION


Tree of life

All Organisms Share Common Characteristics

As I explained in April’s column, all life forms share at least six common properties (variations of this list that include additional properties):

  • Order
  • Growth
  • Homeostasis
  • Metabolism
  • Reproduction
  • Respiration

In April I focused on order and growth. In May I covered homeostasis and metabolism and in July, reproduction. In this month’s article I will focus on respiration.

Respiration

Respiration is the three-step (note: here I am using step to indicate a group of biochemical reactions, not a single reaction) each metabolic process by which non-photosynthetic cells obtain (conserve) energy. In oxic (oxic – an environment in which oxygen is present) environments aerobic respiration is the primary energy producing process. In anoxic (anoxic – in environment in which oxygen is absent or at a concentration too low to support aerobic metabolism) environments anaerobic respiration or fermentation occur. Fermentation is a form of energy metabolism that does not involve the electron transport chain (ETC). Figure 1 illustrates these three forms of energy metabolism. For aerobic respiration, oxygen (O2) serves as the electron transport chain’s (ETC’s) terminal electron acceptor. For anaerobic respiration, alternative inorganic molecules (for example, carbon dioxide (CO2), nitrate (NO3), nitrite (NO2), iron (Fe3+), manganese (Mn4+), sulfate (SO42-), sulfur (S0), etc.) serve as the ETC’s terminal electron acceptor.


Fig 1. Respriation – all respiration pathways begin with glycolysis.

Respiration Pathways

Step 1Glycolysis is the metabolism of glucose to pyruvate (Figure 2). Each 6-carbon (C6) glucose molecule is catabolized (broken down) to two C3 pyruvate molecules. This pathway generates 8 adenosine triphosphate (ATP) molecules (remember that ATP is the primary energy molecule in all cells).


Fig 2. Glycolysis – Note the role of ATP and the net generation of 2 ATP molecules per glucose molecule.

Nicotinamide adenine dinucleotide (NAD+ and NADH) plays a key role in electron transfer. The reversable electron transfer between NAD+ and NADH (NAD+ + e ↔ NADH) drives oxidative phosphorylation (ADP + PO4 → ATP). As we will see below, flavin adenosine dinucleotide (FAD+ and FADH2) play a similar role to NAD+ and NADH in the ETC.

Step 2 – As illustrated in Figure 3, pyruvate is metabolized via the Krebs Cycle (also known as the Citric Acid Cycle or Tricarboxylic Acid Cycle). The Krebs Cycle can theoretically generate an additional 24 ATP molecules.


Fig 3. The Krebs Cycle.

Step 3 – The final sequence of respiration reactions occurs in the ETC (Figure 4). The ETC is a membrane-bound system through which electrons flow down a redox gradient. As noted above, O2 is the terminal electron acceptor for aerobic respiration and other inorganic molecules can function as terminal electron acceptors for non-fermenting anaerobic bacteria. Sulfate reducing bacteria (SRB) use SO4=, nitrate reducers use NO3, and other, specific metabolic groups use the other anions listed above. Note that organisms assigned to a group based on their terminal electron acceptor can be genetically diverse.


Fig 4. Electron Transport Chain – ETC.

Aerobic and anaerobic respiration generate a net of 30 to 32 ATP molecules per molecule of glucose. In contrast, fermentation yields only two ATP molecules per molecule of glucose.

Fermentation Pathways

Figure 5 shows two fermentation pathways – homolactic and heterolactic. Homolactic fermentation produces two molecules of lactate per glucose molecule, Heterolactic fermentation produces one lactate and one ethanol molecule (note – 6 carbon atoms in and 6 carbon atoms out). As shown in Figure 6, alcohol fermentation produces two ethanol and two CO2 molecules (again 1 C6 sugar &rarr 2 C2 alcohol + 2 CO2 molecules – six carbon atoms in and six caron atoms out).


Fig 5. Homolactic and heterolactic acid fermentation pathways.


Fig 6. Ethanol and acetic acid fermentation pathways.

From an energy generation perspective, fermentation generates only 2 ATP per glucose molecule. Thus, respiration produces energy (i.e., ATP) much more effectively than fermentation does.

Summary

As with the other characteristics common to all living organisms, energy generation is universal. The two primary energy production processes used are respiration and fermentation. Anerobic or anaerobic respiration produces 32 to 38 ATP molecules per glucose molecule catabolized. Fermentation produces only two. The figures I’ve presented in this overview are simplified versions of the actual pathways. I invite those who are interested in a more detailed explanation of any of these metabolic pathways to search the internet or pick up a biochemistry textbook.

As always, please share your comments and questions with me at fredp@biodeterioration-control.com.

MICROBIOLOGY FOR THE UNITINTIATED – PART 4: MICROBIAL REPRODUCTION


Tree of life

All Organisms Share Common Characteristics

As I explained in April’s column, all life forms share at least six common properties (variations of this list that include additional properties):

  • Order
  • Growth
  • Homeostasis
  • Metabolism
  • Reproduction
  • Respiration

In April I focused on order and growth. I followed with a discussion of homeostasis and metabolism in May. In this month’s article I will focus on reproduction.

Reproduction

The online Mirriam-Webster dictionary defines reproduction as “the process by which plants and animals give rise to offspring and which fundamentally consists of the segregation of a portion of the parental body by a sexual or an asexual process and its subsequent growth and differentiation into a new individual.” Were it not for reproduction, the first generation of a given type of microbe (or any other organism) would never be followed by a second generation.

In previous posts, I’ve distinguished between growth (increasing mass) and proliferation (increasing cell numbers). However, in microbiology, it is common to refer to proliferation as population growth (or simply growth). The time required for the number of cells to double is called the generation time (G). As I discussed in my August 2021 What’s New article. In that article, I made the point that bacterial colonies typically become visible once they contain approximately 1 billion cells and that it takes 30 generations for one cell to proliferate into 1 billion cells (Figure 1; 230 = 1.0 x 109 – 1 billion). Figure 2 illustrates the slopes and 9.0 Log10 cells mL-1 endpoints for microbes that have generation times ranging from 0.5 h to 4 h. Reiterating the take home lesson from August 2021, if you are depending on culture test data, continue to record colony counts until they no longer change after an additional 48 h observation period. It is not uncommon for ecologically important microbes to have generation times that are 10 h or longer. Such microbes will require a week or longer to form visible colonies.


Fig 1. Bacterial proliferation – bacteria reproduce by binary fission. The time required for cells to divide is the Generation time (G). Once approximately 1 billion cells accumulate (30 generations) their aggregated mass is visible as a colony.


Fig 2. Exponential growth – impact of generation time (G) on time lapsed before a single cell multiplies through 30 generations. Bacteria with 30 min generation times can produce visible colonies within 15 h. As G increases, so does the delay between inoculation and colony visibility.

Bacteria – Bacteria reproduce by binary fission (Figure 3). As binary fission begins, the cell’s chromosome becomes visible when observed through a microscope (Figure 3a). Next, the cell wall and plasma membrane grow, and the chromosome begins to replicate itself (Figure 3b). Once the chromosome has duplicated itself and the two daughter chromosomes separate, a septum begins to form between the two cells (Figure 3c). The two daughter cells typically separate once the septation process is complete (Figure 3d). However, with some types of bacteria the cells do not separate. These microbes form chains or filaments (Figure 4).


Fig 3. Binary fission – a) chromosome condenses; b) chromosome replicates as cell wall and plasma membrane grow; c) newly formed replicate chromosomes separate while cell wall and plasma membrane begin to form a septum; d) once septum has formed, daughter cells separate. Adapted from Bacterial reproduction medical images for power point (slideshare.net).


Fig 4. Bacterial chains and filaments – a) Bacillus megaterium (rod-shaped bacterium) chain (source: https://www.flickr.com/photos/occbio/6414377511/); b) Streptococcus sp. (spherical – coccoidal – bacterium) chain (source: https://www.wikidoc.org/index.php/Streptococcus); Beggiatoa sp. sheathed filament (source: https://alchetron.com/Beggiatoa).

Fungi – Fungi reproduce either sexually or asexually. As discussed for bacteria, during asexual reproduction a cell replicates its entire complement of genes so that daughter cells each receive their genes from a single parent. In contrast, during sexual reproduction, the parents’ cell’s chromosomes separate (see below) with half of the genes going into each of two gametes. For some fungal species, gametes from different hyphae can combine. For others, gametes must come from two different colonies.

Unlike bacteria and archaea, fungi are eukaryotes – their internal structures, including the nucleus, are membrane bound. Consequently, fungal reproduction begins with either mitosis or myosis. After mitosis, the cell’s DNA is duplicated to that each daughter cell’s nucleus has a complete copy of the parent cell’s DNA. In this regard, mitosis is similar to bacterial binary fission.

There are three asexual fungal reproduction mechanisms:

  • Budding – as illustrated in Figure 5, yeast cells most commonly reproduce by budding. The process begins as a yeast cell produces a bud. As the bud continues to emerge, the nucleus migrates towards the bud as DNA replicates by mitosis. The original (parent) nucleus divides and the two daughter nuclei migrate apart as one nucleus enters the bud and the other returns to the nucleus’ original location within the parent cell. As the bud matures, the cell membrane and wall develops until there are two separate cells. This process is called cytokinesis. The daughter cells can separate or remain attached to one another. If the latter occurs, the yeast cells can form chains or clusters.
  • Fragmentation – cells in fugal filaments (hyphae) divide by mitosis. As shown in Figure 6, hyphae can elongate, form branches, or both as cells divide.
  • Sporulation – filamentous fungi form specialized hyphae called aerial hyphae. Spores then form at or near the top of these areal hyphae (Figure 7). Spores formed by fungi such as Hormoconus resinae or Penicillium sp. that commonly grow in fuels or metalworking fluids are called conidia. These spores are pigmented and give fungal colonies their characteristic colors (Figure 8).


Fig 5. Yeast reproduction by budding – parent cell forms bud, cell’s nucleus begins DNA replication, once daughter DNA has been produced, the nucleus divides and the two nuclei migrate to the bud and parent cell respectively, the cell ultimately divides into two daughter cells which then continue to grow to maturity, after which each daughter cell will form one or more buds.


Fig 6. Filamentous fungus with fragmenting hyphae – note that as cells within filaments divide, filaments can elongate, branch, or both. Source: https://images.fineartamerica.com/images-medium-large-5/fungal-hyphae-dennis-kunkel-microscopyscience-photo-library.jpg


Fig 7. Penicillium colony with close up images of aerial hyphae and conidia.


Fig 8. Fungal colonies on growth medium. Different colors reflect characteristic pigmentation of each species’ spores. Source hold-breath-fungus-goes-airborne-easier-than-we-thought.w1456.jpg (1456×1092) (wonderhowto.com)

To reproduce sexually, gametes are formed in special hyphae. As I noted above, each gamete is haploid (i.e., contains a single set of chromosomes). Depending on the species, either two of these specialized hyphae from a single mycelium fuse or hyphae from two different mycelial masses (colonies) fuse. A pair of gametes then fuses to form a cell that contains two haploid nuclei. The two nuclei then fuse to form a diploid (i.e., paired chromosomes) zygote. Each zygote then divides to produce ascospores. When these ascospores disperse and settle onto suitable surfaces, they germinate and form new mycelia (Figure 9). This is a superficial explanation of fungal sexual reproduction. The details vary substantially among fungal species. The key point to remember is that sexual reproduction is the primary means by which genetic diversity occurs among fungi of a given species.


Fig 9. Fungal (Ascomycete) sexual reproduction cycle – specialized mycelial cells fuse to form a heterokaryotic cell – unmerged, haploid chromosomes from two gametes. The two chromosomes then merge to form a zygote. Zygote divides to produce spores. Spores germinate to initiate new mycelia.

Gene Transfer Beyond Reproduction

I often talk about promiscuity among microbes. This reflects the ease with which microorganisms transfer genetic material among cells within a microbiome. There are three primary mechanisms by which horizontal gene transfer occurs:

  • Transformation
  • Transduction
  • Transfection

Transformation – a DNA fragment from a dead or damaged cell is released into the environment, enters another cell, and replaces a fragment of the receiving cell’s DNA. Typically, DNA fragments contain approximately 10 genes. Figure 10 illustrates the transformation process.

Transduction – a bacteriophage (type of virus that infects bacteria) transfers DNA from one cell to another. The process is illustrated in Figure 11.

Conjugation – genetic recombination occurs when two cells attach to one another (in Gram negative bacteria, a sex pilus (tubular cell structure) links the cells and a DNA fragment (plasmid) transfers from one cell to the other. The plasmid can remain as extra chromosomal (i.e., not integrated into the recipient’s chromosome) DNA or be integrated into the recipient’s chromosome. One common plasmid that is transferred via conjugation carries genes responsible for microbicide (or antibiotic) resistance. Figure 12 illustrates conjugation.


Fig 10. Transformation: Step 1: A donor bacterium dies and is degraded. Step 2: DNA fragments, typically around 10 genes long, from the dead donor bacterium bind to transformasomes on the cell wall of a competent, living recipient bacterium. Step 3: In this example, a nuclease degrades one strand of the donor fragment and the remaining DNA strand enters the recipient. Competence-specific single-stranded DNA-binding proteins bind to the donor DNA strand to prevent it from being degraded in the cytoplasm. Step 4: RecA proteins promotes genetic exchange between a fragment of the donor’s DNA and the recipient’s DNA for the functions of RecA proteins). This involves breakage and reunion of paired DNA segments. Step 5: Transformation is complete. Source: Horizontal Gene Transfer in Bacteria


Fig 11. Transfection – a) A bacteriophage adsorbs to a susceptible bacterium; b) The bacteriophage genome enters the bacterium. The genome directs the bacterium’s metabolic machinery to manufacture bacteriophage components and enzymes. Bacteriophage-coded enzymes will also breakup the bacterial chromosome; c) Occasionally, a bacteriophage capsid mistakenly assembles around either a fragment of the donor bacterium’s chromosome or around a plasmid instead of around a phage genome.; d) The bacteriophages are released as the bacterium is lysed. Note that one bacteriophage is carrying a fragment of the donor bacterium’s DNA rather than a bacteriophage genome; e) The bacteriophage carrying the donor bacterium’s DNA adsorbs to a recipient bacterium; f) The bacteriophage inserts the donor bacterium’s DNA it is carrying into the recipient bacterium; g) Homologous recombination occurs and the donor bacterium’s DNA is exchanged for some of the recipient’s DNA; h) Specialized Transduction by Temperate Bacteriophage. Source: Horizontal Gene Transfer in Bacteria


Fig 12. Conjugation – Mobilizable plasmids, that lack the tra genes for self-transmissibility but possess the oriT sequences for initiation of DNA transfer, can also be transferred by conjugation if the bacterium containing them also possesses a conjugative plasmid. The tra genes of the conjugative plasmid enable a mating pair to form while the oriT sequences of the mobilizable plasmid enable the DNA to move through the conjugative bridge. Source: Horizontal Gene Transfer in Bacteria

Summary

Reproduction is the means by which all organisms proliferate. Without reproduction, even a theoretically immortal cell would not have much impact on the environment in which it lives. The time required for a population to double is called the generation time. Colonies in or on solid nutrient media become visible after approximately 30 generations (one cell proliferates to approximately one billion – 109 – cells). Broth media typically becomes turbid when the population density is approximately one million (106 cells mL-1). This takes 20 generations (220 = 1.0 x 106). Bacteria reproduce asexually. Fungi can reproduce either asexually or sexually. An organism’s characteristics evolve when a successful mutation occurs, when DNA fragments are transferred among cells, or via sexual reproduction. But this is a topic for a future article.

For more information, contact me at fredp@biodeterioration-control.com.

MICROBIOLOGY FOR THE UNITINTIATED – PART 3: WHAT IS LIFE – CONTINUED


Tree of life

All Organisms Share Common Characteristics

As I explained in last month’s column, all life forms share at least six common properties (variations of this list that include additional properties):

  • Order
  • Growth
  • Homeostasis
  • Metabolism
  • Reproduction
  • Respiration

In April I focused on order and growth. This article will focus on homeostasis and metabolism.

Homeostasis

To survive, cells must be able to regulate their internal environment. Collectively, the numerous processes by which cells maintain a stable internal environment are called homeostasis. For example, cells function optimally when the sodium (Na+) and potassium (K+) concentrations are in balance and at the right levels. Similarly, although some microbes can thrive in environments with pH <2 and others can thrive in environments with pH >12, most microbes maintain an internal pH of approximately 7.2 ± 0.2. Although there are exceptions, conditions inside cells differ from those of their environment. Cell membranes control the movement of solvents (i.e., water) and solutes (dissolved molecules such as Na+, K+, organic molecules used as food, etc.). The membranes function as semipermeable barriers but also contain various molecular structures that actively control the movement of molecules across the barrier. The key point for homeostasis is that, as illustrated in Figure 1, the composition of a cell’s cytoplasm is substantially different from that of its environment.


Fig 1. Homeostasis illustrated – solute concentrations inside the cell (e.g., [Na+]I) are maintained at levels needed for healthy metabolism regardless of their concentrations in the external environment (e.g., [Na+]o). Osmol is osmolarity – the total number of moles of solute per L in a solvent (dissolved organic chemicals + cations + anions).

Metabolism

Metabolism includes all the biochemical reactions by which cells obtain energy and convert nutrients into new cell mass, waste products, and heat. Catabolism includes the biochemical reactions that convert large, food molecules (e.g., carbohydrates, proteins, and lipids) into their individual building blocks (e.g., sugars, amino acids, and fatty acids) as illustrated in Figure 2. Anabolism includes the biochemical reactions that assemble building block molecules into new cell components (i.e., carbohydrates, proteins, nucleic acids, lipids, etc.) as shown in Figure 3. Catabolic metabolic pathways typically capture energy and anabolic pathways consume energy. Both types of metabolism generate heat that is lost to the environment in which microbes are growing. Commonly, microbes are classified on how they obtain energy and how they use carbon-based molecules for anabolism.


Fig 2. Catabolism – macromolecules are broken down into their building blocks. In this figure, proteins are catabolized to amino acids (top) and carbohydrates are catabolized to individual sugar molecules (bottom).


Fig 3. Anabolism – building block molecules such as amino acids (top) and sugars (bottom) are assembled into macromolecules.

Energy metabolism

Phototrophic microorganisms – bacteria and algae – obtain their energy from sunlight. Phototrophic microbes are only likely to cause biodeterioration problems in systems exposed to sunlight.

All other microorganisms are chemotrophs – they conserve energy from chemicals. If the microbe conserves energy by obtaining electrons from inorganic molecules such as ammonium (NH4), hydrogen (H2), iron (Fe2+), nitrite (NO2), phosphite (HPO3), sulfide (HS), sulfur (S0), among others, it is a lithotroph. If the microbe conserves energy from organic molecules it is a fermenter.

Carbon metabolism

Autotrophic microorganisms use inorganic carbon – i.e., either carbon dioxide (CO2), carbon monoxide (CO), or methane (CH4) – to build organic molecules. Strictly speaking CH4 is a C1 organic compound. However, in the world of microbiology, all three C1 compounds listed here are considered to be inorganic. Thus, photoautotrophs conserve energy from light and use inorganic carbon (CO2). Chemoautotrophs conserve energy from inorganic compounds and use inorganic carbon (CO2, CO, or CH4). Chemoautotrophs are also called chemolithoautotrophs.

Heterotrophic microorganisms use organic molecules (molecules built from carbon and hydrogen, either with or without other atoms such as nitrogen – N, oxygen – O, phosphorus – P, or sulfur – S) as their carbon sources. As noted in the previous paragraph, in the context of microbial metabolism, compounds with two or more carbon atoms are organic. Just like autotrophs, heterotrophs are divided in to two groups, based on their energy source. Photoheterotrophs obtain their energy from light and chemoheterotrophs obtain their energy from organic compounds.

Nutritional requirements vary tremendously among microbes that obtain carbon from organic compounds. Some microbes can only use C2 or C3 molecules such as acetate. Others can attack complex, high molecular weight molecules (macromolecules). Some microbes are fastidious – their diets are severely limited. Others can utilize a variety of organics.

Table 1 summarizes the types of energy and carbon utilization modes used by microorganisms. The possible combinations of energy sources x terminal electron acceptors x carbon sources provide the basis for the microbial world’s tremendous diversity. No single type of microbe has all of the different tools for obtaining energy or food. However, as I explained in May 2022’s What’s New article, microbes most commonly live within biofilm communities.

Table 1. How microorganisms conserve energy and obtain carbon.



Consortia

Acting in community – consortia – the metabolic capabilities of individual types of microbes create a net capability that far exceeds that of a single type. This is a critical point to understand. Throughout much of the history of environmental microbiology – particularly diagnosis of biodeterioration damage to industrial systems – investigators typically relied on culture test methods to recover and identify the microbes present. Investigators would then attempt to reproduce the biodeterioration process in jars that contained the process fluid (cooling tower water, fuel over water, water-miscible metalworking fluids, etc.). Invariably, it was rarely possible to reproduce the biodeterioration phenomenon. Stakeholders would then speculate that the damage had been caused by “superbugs.” Only after investigators started to use fluids and materials from the systems in which damage was observed did they begin to recognize the power of consortia. The loose analogy here is the difference between one individual and team of tradespeople – carpenters, electricians, plumbers, building a house. It’s the rare individual that can build a modern house. Even a person with all of the required skills will need months or years to complete the work that a crew can finish in a few weeks. Similarly, in industrial systems, biodeterioration damage is caused by different types of microbes – each which a limited number of capabilities working in synergy with the others.

One common dynamic in consortia is the successive degradation of high molecular weight molecules. Consider Figure 4. Some microbes are able to attack fuel molecules. In a closed system, the waste products (metabolites) produced by these microbes accumulates to toxic concentrations Figures 4a. The traditional bacterial growth curve (Figure 4b) reflects the combined effects of nutrient consumption and metabolite toxicity. In consortia, microbes that are unable to eat large molecules eat the metabolites of those that can (Figure 4 c). Additionally, within biofilm environments, aerobic and facultatively anaerobic microbes consume oxygen to create anoxic zones in which obligate anaerobes can thrive (Figure 4d).


Fig 4. Microbial consortia – a) Microbe A eats C17-hydrocarbons but as metabolites accumulate they become toxic to the microbe; b) traditional closed system growth curve – initially, microbes acclimate to the environment (lag phase); once acclimated the population doubles logarithmically as a function of time (log phase); eventually, as metabolites accumulate, cells die at the same rate new cells are created (stationary phase); ultimately, the die-off rate exceeds the growth rate (death phase); c) Microbe A eats C17-hydrocarbons and Microbe B eats the metabolites – preventing them from accumulating in the closed system; d) oxygen concentration gradient ([O2]) through a biofilm – aerobes at the biofilm-bulk fluid interface (yellow-green) consume oxygen to create an anoxic (red) environment in which anaerobes can thrive.

Summary

Physical and chemical conditions inside microbial cells differ from those of the surrounding environment. Consequently, cells expend substantial energy maintaining constant internal conditions. Collectively, all of the metabolic processes that contribute to maintaining stable internal conditions are called hormonesis. Metabolic processes are either catabolic – when larger molecules are broken down into smaller molecules that are then used as building blocks – or anabolic – the means by which building block molecules are converted into cell components. Additionally, metabolic activity can be divided into energy and carbon metabolism. Energy metabolism involves the use of an energy source and terminal electron donor. Although some microbes can use single carbon (C1) molecules, the vast majority of microbe types that have been identified to date obtain their carbon from organic molecules. Microbes working in consortia do things that no single microbe can.

If you have any questions about this article or microbial contamination-related issues, please contact me at fredp@biodeterioration-control.com.

MICROBIOLOGY FOR THE UNITINTIATED – PART 2: WHAT IS LIFE?


Tree of life

All Organisms Share Common Characteristics

In last month’s column, I wrote about what microbes are, where they are found, and why everyone should have some understanding of the microbial world. In this article, I will provide a superficial overview of how microbes live. I’ll start with a summary of the common characteristics of all living things. As illustrated in Figure 1, all life forms share at least six common properties (variations of this list that include additional properties):

  • Order
  • Growth
  • Homeostasis
  • Metabolism
  • Reproduction
  • Respiration

I’ll spend this and the next two articles discussing each of these properties. This article will focus on order and growth.

Order

All organisms are ordered. Each cell is bound by a membrane, wall, or both. The fluid (cytoplasm) within each cell contains the ingredients the cell needs to function. Bacteria and Archaea (prokaryotes) have no visible, membrane-bound internal structures. All protozoan, plant, animal, and fungal cells have membrane-bound, internal organelles and are classified as eukaryotes. Figure 2 compares prokaryotic (Figure 2a) and eukaryotic (Figure 2b) cell structures.


Fig 1. Common properties of all living things.


Fig 2. Typical cell structures – a) prokaryote (bacterial cell); b) eukaryote (yeast cell).

Growth

Growth is an organism’s increase in size, mass, or both. Often growth is conflated with proliferation. As I’ll discuss in a future article, proliferation is the increase in cell numbers as a result of reproduction. Thus, growth and proliferation (reproduction) are two different properties. Figure 3 illustrates growth. The size of the cell increases to a specific size, after which it begins to divide.


Fig 3. Cell growth – a) cell’s initial size; b) cell’s size as it begins to divide.

Summary

All living things – organisms – share a set of properties. The list of common properties varies among authors, but the list I provided at the beginning of this article is uncontroversial. The first two universal properties that define living beings are order and growth.

Order means that there is a consistent structure. Bacteria and archaea are protists – they have cell membranes and walls but no membrane-bound internal structures. Algae, fungi, and protozoans (like all plant and animal cells) are eukaryotes – their cells all have membrane-bound internal structures. I’ll return to these properties in a future article.

Growth means that the organism’s mass increases – cells become larger – to a certain point – as they mature. I’ll explain the difference between growth and reproduction in my June What’s New article.

Some have argued that viruses meet these two criteria. They are indeed ordered – i.e., have genetic and structural components – but individual virions do not grow.

If you have any questions about this article or microbial contamination-related issues, please contact me at fredp@biodeterioration-control.com.

MICROBIOLOGY FOR THE UNINITIATED – PART 1: WHAT ARE MICROORGANISMS AND WHY SHOULD WE CARE?


Microorganisms – a) virus; b) bacteria; c) archaea; d) yeast; d) mold.

Each year, on the first morning of STLE’s Metalworking Fluid Management Certificate Training Course, I ask attendees about their backgrounds. Invariably, the majority have at least undergraduate degrees in chemistry, engineering, or chemical engineering. However, of the more than 600 people who have taken the course since 2004, fewer than 50 have had a microbiology course.

Globally, biodeterioration – damage that organisms cause – has been estimated to range between $100 billion and $500 billion annually. Of this, 70 % to 80 % of biodeterioration damage is caused by microorganisms. Add to this an estimated $1 trillion cost due to infectious disease. On the flipside, biotechnology is a $500 billion industry that is based on microbiology. Just considering the economic impact microbe have on our lives, one might reasonably ask why so few people have received the most rudimentary education about microbes. Perhaps it’s the six syllables in microorganism (mic-ro-org-an-is-m) and microbiology that make the topic so intimidating. Still, given the role of microbes in disease, nature’s primary cycles, and biotechnology, one might think that every high school graduate would have learned something about microbiology. In this post and the next several to follow, I will do my best to demystify microbiology.

In this month’s article and those that follow, I’ll offer a very superficial overview of microbiology from an ecological and industrial perspective. There are numerous, excellent introductory microbiology textbooks. I don’t intend to provide any level of detail approaching that of a microbiology textbook. My intention is to help non-technical readers gain a fundamental appreciation for how microbes can affect their lives and their businesses.

Definition

Microorganisms (micro – meaning: small + organism – meaning: something having many related parts that function together as a whole) are living things that are too small to be seen by the naked eye. Microbiologists agree that bacteria, archaea, and some fungi (yeasts and molds) are microorganisms.

The status of virus is still a matter of debate. Viruses are essentially genetic material packaged in a protein coat. They don’t perform most of the functions that are used to define living beings. Thus most microbiologists do not consider viruses to be life forms. However, viruses reproduce by infecting cells and hijacking their victim’s (host’s) metabolic machinery to reproduce. Consequently, some microbiologists believe viruses should be classified as living things. Image (a) in the title figure shows a tobacco mosaic virus (TMV). The TMV virus is structurally complex, regardless of how we classify it. I included a virus in the in the figure because there are research groups investigating the use of viruses to control microbial contamination in industrial systems. I’ll return to this topic in a future post.

Abundance

One recent study, illustrated in Figure 1, estimated that microorganisms represent ~17% of Earth’s total biomass (estimated total biomass 550 gigatons of carbon – Gt C; 1Gt = 109 tons – and bacterial biomass ≈70 Gt)1. The contribution of bacteria to the Earth’s biomass is second only to that of plants. Microorganism biomass – including that of archaea, bacteria, fungi, protists, and viruses – account for ~93Gt.

A microbiome is the population of all microbes living in a specific ecosystem (fuel tank, cooling tower, human gut, etc.). Researchers investigating the human microbiome have estimated that the average human body has between 1x to 10x as many microbial cells as human cells. Moreover, it has been discovered that tissues, long thought to be microbe-free, have specific microbiomes that are likely to be essential for healthy tissue function (we have known about skin and gut microbes for nearly 170 years, but finding microbiomes specific to nearly every human tissue type (organs, muscle, etc.) was a surprise. Investigators have only scratched the surface of human microbiome research. We have little idea of how microbes interact with human cells and what roles they play in maintaining good health.


Fig 1. Relative abundance of Earth’s lifeforms, in Gt.

Diversity

As I will explain in future articles, the microbial world is remarkably diverse. The number of different types of bacteria has been estimated to range from hundreds of thousands to tens of millions, of which only a fraction of a percent has been identified.

I’ll discuss why below. Despite their central importance to life as we know it, even the most rudimentary discussion of microorganisms or microbiology (i.e., the study of microorganisms) is rarely included in high school or university curricula.

A Brief History

Tree of Life

Microbes were the first organisms to exist on earth. Our current understanding is that Earth formed 4.6 billion (4,600,000,000) years ago. There is evidence that the first microbes came into existence approximately 3.5 to 3.8 billion years ago (the fossil record indicates that mammals appeared 65 million years ago, and humans showed up a mere 315,000 years ago). Figure 3 illustrates the life on Earth timeline. The bar illustrating Earth’s age is 5 inches (in) long and the one for microorganisms is 4.1 in long. By comparison, the one for plants is 0.2 in and the one for humans is 0.000003 in – too thin to see! Thus, for more than 3 billion years, microbes were the only life forms on Earth.

If we visualize life as a tree, the organisms began to diversify genetically approximately 3.2 to 3.5 billion years ago (Figure 3). This was the time of the last universal common ancestor – LUCA – of all cellular organisms, starting with the bacteria and (thus far) culminating in humans. Before the microbes now classified as members of the kingdom Archaea were discovered near ocean floor, thermal vents in the Marianas Trench, in 1960, the tree of life was thought to have three Kingdoms: Monera (Prokaryota – all single cell organisms that do not have a nucleus or other membrane-bound internal bodies, Protista – all single cell organisms with a nucleus and other membrane-bound bodies, and Eukaryota – all multicellular organisms). As depicted in Figure 3a, in the 1960s through 1990s, Archaea were classified as Archaebacteria. When viewed through a microscope, they appeared to be bacteria. Believing that conditions around Marianas Trench thermal vents was similar to those of primordial Earth, microbiologists initially assumed that Archaea were more ancient than true bacteria – Eubacteriales. As genetic tools became available in the early 1990s, it became apparent that a) Eubacteriales are more ancient than Archaea (Figure 3b), and b) the Archaea are sufficiently distinct genetically to be its own phylogenic Kingdom (phylogenics – the study of evolutionary development and diversification of a species or group of organisms). When Figure 3a was created, fungi were thought to be much more ancient than plants or animals. As Figure 3b illustrates, the phylogenetic tree of life branched off to fungi, plants, and animals a mere half-billion years ago. Thus, fungi are more closely related to us than they are to bacteria.


Fig 2. Timelines – microorganisms appeared approximately 1 billion years after Earth was formed and more than 3 billion years before the first plants appeared. Brown bar – Earth’s age; blue bar microorganisms’ age; green bar – time since plants first appeared; yellow bar – time since first animals appeared; purple bar (invisibly narrow line) – time since humans appeared.


Fig 3. Phylogenic trees – a) Tree from mid-1960s depicting Archaebacteria as being more ancient than Eubacteria; b) Woese et al. (19902) phylogenic tree showing three Kingdoms – Bacteria, Archaea, and Eucarya. Line segment lengths are based on genetic differences (longer lines indicate greater differences). The initial split at bottom center is LUCA.

Genomic testing has been a cottage industry since the late 1990s. A recent diagram (Figure 4) is based on the genomics of 2.3 million different species, from bacteria to humans, illustrates how complex the Tree has become (or as some authors note, the Tree now looks more like a Bush).


Fig 4. 2015 Tree of Life based on genetic data from 2.3 species.3

Human Awareness

Humans have been using microbes from time immemorial. We have been fermenting grains and grapes, and making cheeses, for as long as we have been cultivating plants or maintaining livestock. Modern microbiology dates from 1665, when Roert Hooke published Micrographia: or some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries Thereupon. A decade later, Antonie van Leeuwenhoek published his observations. Hooke and van Leeuwenhoek had each constructed microscopes through which they observed and created sketches of microorganisms (Figures 5a and 5b). However, it took another 200 years before Louis Pasteur and Robert Koch demonstrated that microbes were living beings and were responsible for fermentation, disease, and spoilage.


Fig 5. First microscopes – a) replica of Robert Hook’s microscope; b) replica of Antonie van Leeuwenhoek’s microscope.

Louis Pasteur conducted experiments to disprove the theory of spontaneous generation (belief that microbes developed from inanimate components of the materials which they caused to rot) and prove the germ theory of disease (i.e., that microorganisms did not form spontaneously and that they caused disease, biodeterioration, and were the agents responsible for fermentation). Pasture used goosenecked flasks (Figure 6) to demonstrate that microbes did not proliferate (multiply) in broth boiled in the flasks but did in identical flasks that contained unboiled broth. Proliferation also occurred when Pasteur intentionally permitted boiled broth to be exposed to microbially contaminated air (i.e., either by breaking off the gooseneck or tipping the flask). The gooseneck shape allowed air but not microbes to enter the flasks. These experiments led to Pasteur’s development of the pasteurization – the process of heating substances at temperatures sufficient to disinfect but not degrade them.


Fig 6. Drawing of Louis Pasteur’s gooseneck flask used to disprove spontaneous generation theory.

While Pasteur was focusing on fermentation microbiology, Robert Koch demonstrated the unequivocal relationship between microbes and disease. Koch demonstrated that the disease, anthrax was caused by the spore forming bacterium, Bacillus anthracis. He also developed the first solid growth media so that he could isolate pure cultures from colonies (Figure 7, zone 5). Pasteur’s and Koch’s research launched the modern age of microbiological research.


Fig 7. Obtaining a pure (single type of bacterium) culture by the streak plate method – a) specimen is collected using a sterilized inoculating loop; b) successively, initial specimen is deposited onto solid nutrient medium using a back-and forth motion (1), inoculating loop is heat sterilized, cooled, and oved across initial inoculation zone (2). This dilutes the sample. The final iteration (5) typically produces individual colonies after the inoculated plate has been incubated.

As now, much of the research performed during the late 19th century was focused on the relationship between microbes and disease. However, Sergei Winogradsky became the father of microbial ecology. Based on his pioneering research on sulfur metabolism in the late 1880s, Winogradsky developed the theory of biogeochemical cycles. This theory states that elements like sulfur, carbon, nitrogen, and phosphorous cycle through nature (Figure 8). These cycles are primarily mediated by microbial activity. The first paper describing gasoline deterioration by microbes was published in 1895. Starting in the 1920s, considerable effort was focused on oilfield damage caused by microorganisms. This research included the first studies on what was originally called microbially induced corrosion (MIC – now microbiologically influenced corrosion – see MICROBIOLOGICALLY INFLUENCED CORROSION – Biodeterioration Control Associations, Inc. (biodeterioration-control.com)). However, the term biodeterioration was not coined until 1965, when H. J. Hueck offered the definition: “any undesirable change in the properties of a material caused by the vital activities of organisms.”4


Fig 8. Biogeochemical cycles – this illustration provided a simplified depiction of how carbon (C), nitrogen (N), phosphorous (P), and sulfur (S) cycle through nature.

In my seminars on the topic I explain that biodeterioration and bioremediation are two sides of the same biodegradation coin (Figure 9). As Winogradsky observed, microbes drive biogeochemical cycles. These cycles occur regardless of human intent. When we want biodegradation to occur, we call it bioremediation. When microbial activity causes changes, we’d prefer to prevent, we call it biodeterioration (see https://biodeterioration-control.com/2018/03/).


Fig 9. Like the obverse (front) and reverse (back) sides of this 2000 Sacagawea U.S. dollar coin, bioremediation and biodeterioration are flips sides of biodegradation.

Summary

Microbes play invaluable roles in our lives. Our bodies would not function without the microbes that make up he human microbiome. Fewer than 2,000 pathogenic microbes have been identified among the tens of thousands that have been identified and the millions of different types of microbes that exist in nature. Microbes mediate nutrient cycles. This cycling conserves essential nutrients and prevents wastes from accumulating. Biodegradation includes all processes that breakdown organic substances. On one hand, biodegradation is the foundational element of a $0.5 trillion biotechnology industry. On the other, biodeterioration and infectious disease cost $1.5 trillion per year. We are part of the microbial world. To me, that seems like an excellent reason why everyone should have a basic understanding of microbiology.

For more information about fuel system condition monitoring and predictive maintenance, contact me at fredp@biodeterioration-control.com.


1 Bar-On, Y.M, Phillips, R., Milo, R., 2018. The biomass distribution on Earth. Proc Natl Acad Sci U S A., 115(25):6506-6511. https://pubmed.ncbi.nlm.nih.gov/29784790/.
2 Woese, C. R., Kandler, O., Wheelis M.L., 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proceedings of the National Academy of Sciences of the United States of America. 87 (12): 4576–9. https://doi.org/10.1073/pnas.87.12.4576.
3 Hinchliff, C. et al., 2015. Synthesis of Phylogeny and Taxonomy Into a Comprehensive Tree of Life. Proceedings of the National Academy of Sciences https://www.pnas.org/doi/full/10.1073/pnas.1423041112.
4 Hueck, H.J., 1965. The Biodeterioration of Materials as a Part of Hylobiology. Material und Organismen, 1, 5-34.

MICROBIOLOGICALLY INFLUENCED CORROSION


Leaking underground storage tank. Microbiologically influenced corrosion (MIC) created numerous holes in this home heating oil fuel tank.

Definition

AMPP (formerly NACE) and ASTM define corrosion as “the deterioration of a material, usually a metal, that results from a chemical or electrochemical reaction with its environment” (ASTM Terminology G193). AMPP defines microbiologically influenced corrosion (MIC) as “corrosion affected by the presence or activity, or both, of microorganisms.” The AMPP definition of MIC adds: “The microorganisms that are responsible for MIC are typically found in biofilms on the surface of the corroding material.”

Background

A 2016 NACE study estimated that globally the cost of corrosion was $2.5 trillion U.S. ($2,500,000,000,000).

A 2022 Ohio University study suggested that damage attributed to MIC represented 20 % of the total, or $500 billion U.S.

Historically, MIC was the acronym for microbially induced corrosion, and later, microbiologically induced corrosion. Well into the 1990s, the prevailing theory was that MIC was primarily caused by a process called cathodic depolarization (more on that below). By the end of the 1990s, most investigators recognized that MIC was more complicated and that is was more common for microbes to influence rather than cause MIC. Conveniently, the MIC acronym worked as well for microbiologically influenced corrosion as it did for microbially induced corrosion and microbiologically induced corrosion.

AMPP TM0166 Detection, Testing, and Evaluation of Microbiologically Influenced Corrosion (MIC) on External Surfaces of Buried Pipelines provides an excellent overview of MIC. Although the document’s focus is pipeline corrosion, the general principles it describes are generally applicable. TM0166 is a consensus standard. There are also several excellent books that cover MIC in detail. A few of my favorites include:

  • Manual of Biocorrosion, H. A. Videla, CRC Press, Boca Raton, 273 pp, 1996, ISBN 0-87371-726-0
  • Microbiologically Influenced Corrosion, B. J. Little and J. S. Lee, John Wiley & Sons, Inc. Hoboken, 279 pp, 2007, ISBN 978-0-471-77276-7
  • CorrCompilations: Introduction to Corrosion Management of Microbiologically Influenced Corrosion, R. Eckert, AMPP, Houston, 489 pp, 2015, ISBN 978-1-575-90285-2

Additionally, a substantial body of MIC literature has been published in the past decade. In January 2023 alone, 180 peer-reviewed papers were published. A citation service (ScienceDirect) search of papers published since 2010 listed more than 8,000 publications. Consequently, in today’s article, I’ll share only a bit of history and a offer superficial overview of MIC.

Cathodic Depolarization Theory

A relationship between microbial contamination and corrosion has been recognized since the late 19th century. By the mid-20th century, researchers were in general consensus about the relationship between biofilms and MIC (for a refresher on biofilms, see my May and August 2022 What’s New articles). However, the mechanisms are still being investigated. One the earliest models – cathodic depolarization – was proposed in the 1930s. According to this model, when a metal surface is exposed to water, metal dissolution can occur. At the anode, positively charged metal ions (Me2+) form (in the case of iron – Fe – ferrous – Fe2+ – ions form), releasing two electrons (e) per metal ion (Table 1, Reaction 1). The electrons migrate to the cathode where they bond with hydrogen (H+) ions (Table 1, Reaction 3) dissociated from water (Table 1, Reaction 2) to passivate the cathode. Sulfate reducing bacteria enzymes utilize the hydrogen ions from the cathodic surface and catalyze sulfate reduction to sulfide (Table 1, reaction 4). The dissolved Fe2+ ions react with sulfide (S2-) and hydroxide (OH-1) to form ferrous sulfide (FeS) and ferrous hydroxide (Fe(OH)2) deposits (Table 1, Reactions 5 and 6, respectively). The mechanism is illustrated schematically in Figure 1.

Table 1. MIC SRB-mediated cathodic depolarization.




Fig 1. Cathodic depolarization. Numbers in circles refer to reaction numbers listed in Table 1. Hydrogen coating at cathode passivates surface – inhibiting electron flow. SRB-mediated hydrogen utilization depassivates the cathode and promotes electron flow.

The early discovery of the relationship between SRB and MIC continues to influence how investigators think about MIC to this day. However, it is now recognized that SRB-mediated cathodic depolarization is only one MIC mechanism.

Common Denominator

The definition I quoted in the second sentence of this article is quite broad. Microbiologists and corrosion engineers now recognized that microbes influence corrosion through a variety of mechanisms in addition to cathodic depolarization. However, MIC is invariably associated with biofilms. The presence of biofilm creates an electropotential gradient between biofilm-free surfaces and those beneath biofilm polymer – extracellular polymeric substances (EPS). Figure 2 is from one of my fuel microbiology course modules. It illustrates the oxygen concentration ([O2]) as a function of distance from the bulk fluid to deep within a biofilm matrix. Aerobic (bacteria that require O2) and facultatively anaerobic bacteria (bacteria that use O2 for respiration when it is available then switch to fermentation when the [O2] is insufficient to support aerobic respiration) that are part of the biofilm microbiome scavenge and thereby deplete O2 (a microbiome is all the microorganisms present in a specific environment) from the biofilm matrix. Near the biofilm surface that is in contact with the bulk fluid (e.g., water, fuel, water-miscible metalworking fluids, etc.), diffusion is sufficient to keep the [O2] close to saturation (O2 saturation concentrations are temperature dependent). At 20&deg C 100 % saturation in water = 8.77 mg L-1 . Deep within a biofilm, [O2] can be <0.4 mg L-1anoxic.


Fig 2. Oxygen gradient between bulk fluid and depths of biofilm matrix.

Although biofilms can develop from a single microbe, more commonly, biofilm microbiomes include a variety of microbes. As I discussed in May 2022, both the types of microbes and their respective physiologies vary depending on their location within a biofilm community. Consequently, although MIC encompasses several different types of corrosion processes, biofilm presence is the common denominator.

MIC Mechanisms

Sulfate reduction

As discussed under Cathodic depolarization, sulfate reducing bacteria (SRB) and archaea (SRA) – collectively referred to as sulfate reducing prokaryotes (SRP) utilize SO42- instead of O2 for respiration. The process – called dissimilatory sulfate reduction – generates H2S (Table 1, Reaction 4). When ferrous iron (Fe2+) is present, the H2S react with Fe2+ to produce ferrous sulfide (FeS – Table 1, Reaction 5). Deposition of FeS one ferrous metal surfaces stimulate the galvanic cell’s (Figure 1) cathodic reaction – accelerating the corrosion rate.

Acid production

Nominally, acid producing bacteria (APB) defines a category of bacteria that produce organic or inorganic acids. Classifying microbes as APB is arbitrary. The metabolic pathways by which all organisms generate energy produce low molecular weight (C1 to C6) organic acids (LMWOA) – mono-, di-, and tricarboxylic acids (Table 2). Thus, all microorganisms are acid producers. However, there are some classes of bacteria that generate greater yields (i.e., mg acid excreted per cell) than others. For example, bacteria in the Family Acetobacteraceae ferment sugars and ethanol to acetic acid (the metabolic processes the convert wine into vinegar).

Table 2. Low molecular weight organic acids produced as energy metabolism metabolites.



These LMWOA can attack metals directly. When chloride, sulfate, nitrate, or nitrite salts are present, they can react with LMWOA to form strong inorganic acids (hydrochloric, sulfuric, nitric, and nitrous, respectively) and weak organic bases. The strong inorganic acids are aggressively corrosive.

Metal deposition

Iron oxidizing bacteria and manganese oxidizing bacteria form metal oxides and hydroxides that typically take the form of tubercles (Figure 3). Oxygen becomes depleted under the deposits. This creates the anode terminal of a galvanic corrosion cell.


Fig. 3. Iron oxide corrosion tubercles on interior surface of a firemain line. Source: Scott McNamara, Liberty Corrosion Solutions https://www.flickr.com/photos/59127492@N07/5416316387/

Metal reduction

Respiration is the process by which organisms obtain energy. The last step in respiration is the release of an electron which transfers to a terminal electron acceptor. For all aerobic organisms O2 is the terminal electron acceptor. In anaerobic respiration a different molecule serves this role. SRP use SO42- as a terminal electron acceptor. Other microbes can use nitrate (NO3), nitrite (NO2), ferric iron (Fe3+), or manganese (Mn) as terminal electron acceptors. Microbes that use either iron o manganese for respiration are called metal reducing bacteria (MRB). Ferric iron is reduced to ferrous (Fe2+) and Mn4+ is reduced to Mn2+ both of which are water soluble. Thus, MRB dissolve metal oxides, thereby accelerating localized corrosion.

Hydrogen embrittlement

Hydrogen embrittlement occurs when hydrogen atoms permeate metals. Figure 4 shows an intergranular crack cause by hydrogen embrittlement in steel. Hydrogen accumulates at the cathodic end of galvanic reaction cells. For example, it is a biproduct of dissimilatory sulfate reduction and other metabolic processes. Thus, the hydrogen that accumulates around the cathode can infiltrate steel’s intergranular spaces. This most commonly occurs in systems using cathodic protection too aggressively.


Fig 4. Hydrogen embrittlement stress cracks formed between metal grains of a steel specimen. Source: https://uscorrosion.com/index.php/astm-f1940-testing-services-for-hydrogen-embrittlement-of-fasteners/

MIC Morphology:

Historically, it was a believed that pitting corrosion was diagnostic for MIC. However, we now know that abiotic mechanisms can cause MIC and that MIC can cause both localized (e.g., pitting) and more general corrosion. This means that visual (with or without microscopy) observation of corrosion patterns is insufficient to diagnose. Correct diagnosis is made even more challenging when the corrosion is inside tanks. Health and safety-focused regulations typically require that tanks been cleaned and rendered gas-free before inspectors can enter them. The processes that render the space safe for entry also disrupts or destroys much of the evidence. As illustrated in Figure 5, regions of heavy biofilm accumulation are apparent in this UST. In the 1980s, when this photo was taken, an inspector could enter the tank (wearing appropriate personal protective equipment) and collect surface swab, scrape, and fluid samples. Those samples could then be tested microbiologically and chemically (for organic chemical composition and minerology) to facilitate diagnosis. Confined space entry practices used today, remove most of the residue visible in Figure 5 before an inspector is permitted to enter the tank. Thus, although we know considerably more about MIC today than we did half a century ago, there are still considerable challenges to timely and accurate diagnoses in systems likely to be affected by MIC.


Fig 5. UST interior view, after product removal but before tank cleaning. Note bands of heavy slime accumulation 15° either side of bottom dead center. Numerous corrosion pits were observed under these slime-covered regions.

Summary

Our understanding of MIC continues to evolve. Analytical tools that are currently available did not exist in the 1930s when the seminal papers describing MIC were published. There are several well documented MIC mechanisms. All are associated with the presence of biofilms. Moreover, observing pitting corrosion is no longer recognized as a sufficient basis for diagnosing MIC.

For more information about fuel system condition monitoring and predictive maintenance, contact me at fredp@biodeterioration-control.com.

RETAIL AND COMMERICAL FUEL SYSTEM CLEANLINESS – VALUE PROPOSITION ONE MORE TIME


Bottom sample from ultra-low-sulfur diesel tank showing hazy fuel over bottoms-water loaded with suspended solids.

Opportunity Cost

In two previous articles, I’ve discussed the opportunity costs associated with premature filter plugging (see November 2016 and February 2019). My November 2016 article focused on the disconnect between operator awareness and the actual incidence of substantial contamination in underground storage tanks (UST) and fuel transfer equipment between UST and dispensers. In February 2019, I presented an opportunity cost calculation model that I had first suggested to fuel retailers in 1992. My 2019 calculations were based on unleaded gasoline sold at $2.30 U.S. per gal. Today, as I passed several forecourts gasoline prices ranged from $2.60 to $2.80 per gal and ultra-low-sulfur diesel (ULSD) prices were more than $6.00 U.S. per gal. In my February 2019 article I defined opportunity cost as follows (note: the maximum permitted flow rate at commercial dispensers is 40 gpm):

Opportunity cost is the difference between the economic value of the theoretically optimal use of an assist and the value realized by its actual use. At fuel retail sites that experience rush hour peaks, where the dispenser flowrate is the primary factor controlling fuel sales revenues, the opportunity cost is the difference between sales generated while dispensing at 10 gpm (U.S. EPA’s maximum permissible flowrate at retail dispensers) and sales generated while dispensing at slower flowrates.

If you assume that dispensers can operate for a maximum of 30 min per hour – allowing 30 min per hour for payment and vehicle movement), a commercial dispenser can deliver 1,200 gph. At $6.00 gal-1 , that generates $7,200 gross revenue. For each 1 % of flow rate reduction, the opportunity cost is $72 h-1 . That doesn’t seem like much but, if flow rate is the limiting factor for 4h day-1 , the opportunity cost per dispenser per year is $105,000 for each 1 % of flow rate below the 40 gpm maximum.

Case Study

Since I first started providing fuel retailers with my opportunity cost model in 1992, few have been willing to check their flow rates and test my calculations. However, my cost model was validated in an article the that appeared in the 3rd quarter issue of PEI Journal (Vol 16, Issue 3). The title of the article was Preventing Fuel Contamination Issues by Bill Jones and Jessica Montgomery of Warren Rogers and Clean Fuels National, respectively. The article is a case study that illustrated the impact of maintaining clean fuel systems on opportunity cost.

Predictive Maintenance

In their PEI article, Jones and Montgomery advocated for a predictive maintenance approach (see What’s New December 2016, and January 2017), using flow rate testing as a fast and easily performed routine check. As shown in Figure 1a, five-weeks after new filters were installed, flow rates began to decrease. Jones and Montgomery noted that it is important to run flow rate tests during quiet periods. At sites with submerged turbine pumps (STP) that were under-capacity, flow rates were affected by the number of dispensers operating simultaneously (Figure 1b). At this site, flow rate testing at each dispenser needed to be performed when no other dispensers were operating.


Fig 1. Retail gasoline dispenser flow rate testing – a) flow rate as a function of time in service since last filter change; b) flow rate as a function of the number of dispensers operating simultaneously (from Jones and Montgomery, PEI Journal, 16(3): pp 26-32).

Corrective Action Impact

Jones and Montgomery reported the impact of contaminant removal (UST system cleaning) on daily fuel sales volumes (Figure 2 – Figure 1-4 in the PEI article). The figure did not identify which dispensers handled unleaded gasoline and which handled ULSD. Nor did Jones and Montgomery report the impact of tank cleaning on the time lapse between new filter replacement and flow rate degradation. I always recommend recording dispenser totalizer readings when changing filters. Premature filter plugging (e.g., flow-rate reduction) relates to how many gallons have been filtered before flow rate (or pressure differential) is affected. When the fuel is clean, a 10 µm dispenser filter should be able to process at least 500,000 gal before the flow rate decreases by 10 %. In the case of dispensers H18 and H19 – each delivering approximately 4,000 GPD – that translates to approximately 4 months (125 days) of at least 90 % maximum flow rate. For the lower volume dispensers (H9, H10, H11, H12, and H17) that handled approximately 200 GPD, filter performance life should be years. Here, I’ll focus on the impact of tank cleaning on dispenser H18T to illustrate the opportunity cost associated with premature filter plugging. After tank cleaning and fuel polishing:

  • Average daily sales increased by approximately 2,400 gallons.
  • If the product was ULSD @ $6 gal-1 , after cleaning, H18 generated an additional $14,400 gross revenue per day, or $5.3 million per year.
  • If the product was regular unleaded gasoline @ $4 gal-1 , after cleaning, H18 generated an additional $9,600 gross revenue per day, or $3.5 million per year.

Table 1 summarizes the net additional revenue for all the dispensers at which substantial fuel sales volumes were observed. Although sales at several dispensers decreased (the article made no mention of other contributing factors), the net increase was 1,200 GPD (438,000 gal per year). I used current fuel pricing here in the Princeton New Jersey area to compute the sales $s impact.


Fig 2. Effect of tank cleaning on fuel sales volumes (from Jones and Montgomery, PEI Journal, 16(3): pp 26-32).

Table 1. Effect of tank cleaning on revenues (data taken from Figure 2).



Bottom Line

In 1992, when I founded BCA, I shared an opportunity cost calculation tool with prospective fuel retailer clients. Thirty years later, a case study reported in PEI Journal validated the opportunity costs estimated in my model.

There are factors other than filter plugging that affect dispenser flow rates. The most common is pump power insufficient to deliver maximum permissible flow to all dispensers when customers are fueling at multiple dispensers. In my March 2021 What’s New post, I summarized other common causes of fuel system flow rate reduction.

There are factors other than uncontrolled microbial contamination that contribute to premature (i.e., >10 % flow rate reduction before 500,000 gal have been filtered) filter failure. However, uncontrolled microbial contamination is one of the most common causes.

Cost effective condition monitoring relies on tiered testing (see What’s New, February 2017) in which a fast, easily performed test is run most frequently and more diagnostic tests are run when the results from the frequently run test are at or above their control limit (see What’s New, October 2020). The Jones and Montgomery case study article published in PEI Journal (Volume 16, Issue 3, 2022) demonstrates the value of both tiered condition monitoring and linking test results to appropriate actions.

For more information about fuel system condition monitoring and predictive maintenance, contact me at fredp@biodeterioration-control.com.

17TH INTERNATIONAL CONFERENCE ON THE STABILITY, HANDLING AND USE OF LIQUID FUELS


International Association for Stability, Handling and Use of Liquid Fuels (IASH)

IASH was founded in 1983 when Nahum Por, a chemist at Oil Refineries Ltd., Haifa, Israel, obtained support from the Israel Institute of Petroleum & Research to sponsor a conference to discuss issues related to the stability and handling of liquid fuels. The focus was on fuels and crude oil stored as strategic reserves. Between 1983 and 2003, IASH met triennially – venues alternating between Western Europe and North America. Since 2005, the society has been meeting biennially. The association is intimate (approximately 200 members) but include an international group of subject matter experts and people responsible for intermediate to long-term fuel storage. Invariably topics addressed during IASH Conferences range from practical fuel storage and condition monitoring considerations to esoteric explorations of fuel deterioration physicochemical processes. Each conference has included a half-day fuel microbiology session.

I have been attending the IASH conferences since the 1991 Orlando, FL meeting. I have yet to be disappointed by the quality of the presentations and the number of new insights I gain from the speakers. September’s conference in Dresden was no exception.

IASH 2022

The 17th International Conference on the Stability & Handling of Liquid Fuels (IASH 2022) was held in Dresden from 11 through 15 September. In keeping with the precedents set at the previous 16 conferences, the program included excellent papers covering the gamut of fuel quality and stability issues (visit Dresden Agenda (iash.net) to see the conference program). This year’s conference included many papers addressing sustainable fuels – particularly sustainable aviation fuels (SAF) – and fuel quality modelling. I was particularly encouraged to see many younger participants presenting their research. Typically, the conference proceedings are available form the IASH website (Home (iash.net)) three to five months after the meeting. If you are particularly interested in any of the presentations, you can contact the authors directly for copies of their papers or presentations. I won’t attempt to provide a synopsis of every session or paper presented in Dresden. Instead, I’ll focus on the fuel microbiology papers.

IASH 2022 Fuel Microbiology Presentations

Detection Technologies

Dr. Jiri Snaidr of Vermicon AG (Hallbergmoos, Germany) reported on the development of a gene probe system for detecting specific microbes in fuel systems. The prototype flow cytometry system relied on fluorescent dye linked, ribonucleic acid (RNA) to label target microbes. After specimens are stained, they are drawn through a flow cytometer. Each target microbe is stained with a dye that fluoresces at a different wavelength so that the abundance of each organism can be quantified. Over the past decade, a new generation of flow cytometer has been developed that has made the technology an increasingly useful tool for quantifying microbial contamination in liquids. There are several challenges related to the use of flow cytometry for microbial contamination in fuels. As I’ll discuss below, the variety of microbes and types that exist in different fuel systems is considerable. Probes designed to detect specific microbes are likely to miss others that might be present. Additionally, microbial population densities in fuel samples is likely to be <1 cells mL-1 . This means that in order to reliably detect fuel contaminant microbes, specimens will need to be ≥1 L.

Dr. Osman Radwan of University of Dayton Research Institute (Dayton, OH, USA) discussed the use of an electrochemical biosensor to detect biofilm development on fuel system surfaces. Dr. Radwan’s multi-phase, fluidic device is comprised of an array that is pretreated with a protein molecule believed to be universally present among fuel degrading fungi (i.e., a biorecognition element – BRE). When microbes are bound to the protein, they generate an electrochemical signal. To date, Dr. Radwan and his colleagues have performed in-lab tests demonstrating their ability to detect filamentous fungi and single-cell yeasts. One challenge common among devices such as the one that Dr., Radwan described is that the sensor quickly becomes saturated. The sensors detect initial biofilm formation but cannot differentiate between a 10 µm or 1,000 µm thick film. It is generally recognized that fuel and fuel system biodeterioration are primarily due to the activities of biofilm communities. Thus, being able to assess three-dimensional biofilm growth is an important requirement for surface sensor arrays. Both Dr. Snadir and Dr. Radwan presented cutting edge technologies that are likely to take years of further development before they will be ready for commercial deployment, but their work is exciting and points towards the future.

Biodeterioration Mitigation and Contamination Control

Graham Hill, of ECHA Microbiology, Ltd. (Cardiff, Wales, UK) discussed the relationship between salinity and sulfate reducing bacterial (SRB) activity in marine, seawater ballasted, fuel tanks. Microbiologically influenced corrosion (MIC) has been a problem in ships’ fuel tanks since the early 20th century transition from coal to liquid fuels. In order to maintain stability, ships take on seawater as they consume fuel. One of the most serious, unintended consequences of sea water ballasting is that the combination of seawater and fuel create an excellent environment for the proliferation of microbes. Aerobic and facultatively anaerobic microbes scavenge oxygen and produce low molecular weight organic acids on which SRB can thrive. Mr. Hill reported the results of lab-scale tests run at ECHA. His team found that by using freshwater, rather than seawater – i.e., keeping the salinity at <9 g L-1 (the typical salinity of ocean water is 35 g L-1) – reduces SRB activity (sulfate production) substantially.

Dr. Oscar Ruiz, U.S. Air Force research Lab (AFRL, Dayton, OH, USA) reported on successful laboratory tests that demonstrated the efficacy of a graphene oxide (GO), nanoparticle, depth filter to scrub microbes from fuel. Dr. Ruiz report the results of a 227 m3 (60,000 gal) trial. At flow rates ≤ 40 L min-1 (≤10 gpm) the device removed >90 % of the bioburden. At the end of the test the pressure differential across the filter was < 28 kPa (4 psi). Given the increased regulatory pressure against the use of fuel-treatment, antimicrobial pesticides (biocides), filtration is a promising alternative. Traditional filter media develop a cake that improves filtration efficacy to a point. Once the filter has accumulated too much material, pressure differentials increase, and flowrates decrease. If GO media can overcome this limitation, they might change the nature of fuel filtration. Filtration’s primary limitation is that the microbes that pass through the medium can colonize downstream surfaces and form biofilm communities. Filtration does not address biofilm control.

Microbial Ecology

Dr. Gareth Wiliams of ECHA Microbiology, Ltd. (Cardiff, Wales, UK) reported the results of an investigation into the factors contributing to hydrogen sulfide generation in salt caverns in which butane was stored. The ECHA team used quantitative polymerase chain reaction (qPCR) and next generation sequencing (NGS) to identify microbes in salt dome samples. Dr. Williams reported that of three caverns tested, hydrogen sulfide accumulated only in one. However, the microbial population profiles were similar among all three caverns. All three had SRB and Halobacterium species (Halobacterium is a genus within the domain of Archaea). The primary difference among the three caverns was the suction line installation. The cavern in which substantial concentrations of hydrogen sulfide had developed had an irregular floor. The uptake line was above the bottom; allowing brine to accumulate. The uptake lines in the other two caverns were at the bottom. In these caverns there was no bottom brine layer.

I presented NGS genomic data from the CRC-sponsored diesel fuel microcosm study from which I had reported adenosine triphosphate (ATP and adenylate energy charge (AEC) results at ICSHLF 16 in 2019 (The Relationship Between Planktonic and Sessile Mirobial Population Adenosine Triphosphate Bioburdens in Diesel Fuel Microcosms | IASH Online Library of Conference Proceedings and Newsletters (conferencespot.org) and The Relationship Microbial Community Vitality and ATP Bioburden in Bottoms Waters Under Fuel Microcosms | IASH Online Library of Conference Proceedings and Newsletters (conferencespot.org) – both are accessible to IASH members only. If you cannot access the papers, contact me for copies). Although during the original study (DP-07-16-01-FINAL-REPORT-REV-30JUL21-COMPLETE.pdf (crcao.org)) only a few aqueous phase samples were subjected to NGS testing. We were able to obtain the microcosms after 18-months. Marathon Petroleum, LuminUltra Technologies Ltd., and BCA collaborated to collect samples from the fuel-water interface and aqueous phases f 32 microcosms. LuminUltra Technologies Ltd. Performed the 18-month NGS tests and a different lab had performed the 3-month tests. Among the eight microcosms for which there were data from 3 and 18 months, the genomic profiles of four microcosms had remained stable and those of the other four changed substantially. Duplicate samples from several 18-month microcosms demonstrated that variability among duplicate aqueous samples was excellent but that variability among duplicate interface samples was substantial. Genomic test methods continue to evolve rapidly. As the protocols are refined, it will be essential to understand the sources of variation related to sampling and testing. Without this understanding, results from environmental samples will be difficult to interpret. I’ll devote a future What’s new post to genomics, transcriptomics, and metabolomics.

Lifetime Achievement Award

During the conference banquet I was taken totally by surprise when I was called to the stage to receive IASH’s Lifetime Achievement Award. I confess that when 1 st Vice-Chair, Matt DeWitt announced that it was time to present awards, I commented to my tablemates that these awards have never been given to microbiologists we are such a small minority within the Association. As I was making the comment, I heard my name. Did I mention that I was taken totally by surprise?

The award citation reads:

“By the members of IASH in recognition of his significant technical contributions and leadership related to testing, control and mitigation of microbiological contamination in fuels and oils. His notable technical contributions include over 70 technical publications on fuel, lubricant and metalworking fluid microbiology and biodeterioration. He has had numerous Chair and leadership positions in ASTM, IBBS, and IATA, and contributed significantly to the development of best industry practice documents and guidelines for the prevention and control of microbiological contamination in fuels.”



1st Vice-Chair Matt DeWitt presenting IASH Lifetime Achievement Award to Fred Passman

Summary

In summary, the conference was an exceptional opportunity to learn about various aspects of fuel degradability and its control. I most strongly recommend that individuals who are responsible for fuel quality join IASH and make plans to attend future IASH conferences. Again, for more information about IASH, visit Home (iash.net).

To contact me, please send an email to fredp@biodeterioration-control.com.

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