Microbiology in Water-Miscible Metalworking Fluids

Abstract

Water-miscible metalworking fluids (MWFs) are used in metal removal and forming operations. For end use, formulation concentrates are diluted in water, providing conditions conducive to microbial growth and metabolism and potentially causing fluid biodeterioration and microbiologically influenced corrosion. Microorganisms in the environment are highly diverse, and bacteria and fungi have been recovered from these fluids at population densities of >10⁶ CFU mL⁻¹. Thus, to control microbial bioburdens in MWFs, microbicides are often incorporated into MWF formulations, used as tankside additives, or both. Some microbicides are suspected as being responsible for adverse health effects. Consequently, their usage has been restricted in recently adopted regulations. Given the limited number of microbicides currently approved for use in MWFs, alternative microbial contamination control strategies are needed. Some of these strategies have already been employed in the market. Studies on microorganisms in MWFs are often hindered by the complexity of the media, especially when trying to examine in-use samples. Historically, microbiological analyses were primarily based on cultivation assays using nutrient-rich media. These analyses have severe disadvantages, and the adoption of more reliable test methods is of utmost importance. Here, we review MWF microbiology, discussing possible consequences and options for both control and condition monitoring testing.

Keywords:

• Bacteria
• bioaerosol
• biodeterioration
• fungi
• microbes
• microbiology
• metalworking fluids

Metalworking fluid categories

Water-miscible metalworking fluids (MWFs) used in metal removal and forming operations are formulated as concentrates that contain 5 to >15 organic ingredients. For end use, water-miscible MWF concentrates are diluted with water on-site to give working concentrations typically ranging from 3 to 15% (v/v). There are four basic classes of MWFs, three of which are water miscible, as classified in ASTM D2881 (1):

1. Straight oils

2. Emulsifiable oils (also known as soluble oils)

3. Semisynthetics

4. Synthetics.

All MWFs are formulated from base stocks and functional additives (2, 3). Base stocks can be mineral oils (naphthenic and paraffinic), vegetable oils (mainly esters), polyalphaolefins, and water. Vegetable, naphthenic, and paraffinic mineral oils are used as straight oil, emulsifiable oil, and semisynthetic MWF base stocks. Vegetable oils, water, and polyalphaolefins are used in semisynthetic and synthetic MWF formulations. Functional additives are ingredients that influence MWF performance properties such as lubricity, foaming tendency, emulsion stability, biostability, corrosion protection, and others.

Straight (or neat) oils are formulated from base oils and functional additives. These products can be formulated from any of the base stocks listed above. They neither contain water nor are they intended to be mixed with water. Their usage is limited to applications requiring high lubricity and little cooling.

Emulsifiable oil formulations are sold as concentrates and include base oils and functional additives that are dispersed in water by means of emulsifiers and coupling agents, because water’s heat transfer properties are much greater than those of oil (2). Emulsifiable oil MWFs are often referred to as soluble oils, which is a misnomer, given that oil is not soluble in water. Emulsifiable oil concentrates contain ≥30% (v/v) oil and may include water at <20% (v/v). When diluted for end use, emulsifiable oil MWFs typically form oil-in-water droplets that are <1 to 10 µm in diameter, giving them anything from a slightly opaque to a milky white appearance. As metalworking cutting speeds and workpiece feed rates accelerated during the mid-20th century, water-miscible MWFs displaced straight oils in many applications. In 2018, water-miscible MWFs accounted for 90% of the global MWF demand. Emulsifiable oils account for an estimated 40% of the global volume of water-miscible MWFs used (4).

Semisynthetic MWF concentrates contain ≥20% water and <50% oil. In semisynthetic concentrates, oil droplets are well dispersed in the water. Functional additives are either water soluble or co-dispersed with the oil. They are usually further diluted by the end user and mainly provide cooling and excellent chip removal properties. Typically, the emulsion droplet size in end-use diluted semisynthetic MWFs is <1.0 µm. As of this writing, semisynthetic MWFs account for an estimated 30% of the global water-miscible MWF demand.

The third category of water-miscible MWFs is synthetics. These products are formulated with water as their primary ingredient and water-soluble performance additives to provide lubricity, corrosion inhibition, and other functionalities. Synthetic MWFs are used in applications where excellent cooling properties are of primary importance. For more information about the various classes of MWFs, consult ASTM classification D2881 (1).

Microbiology in water-miscible MWFs

The high ratio of water and organic compounds, aerated by recirculation at velocities ≥400 L min⁻¹, provides conditions conducive to microbial growth and metabolism. The consequent MWF microbial infections create a risk of fluid biodeterioration and production of allergenic molecules. Thus, uncontrolled microbiological contamination can reduce MWF performance. Bioaerosols can increase the incidence of allergenic diseases ranging from mild allergic rhinitis to debilitating hypersensitivity pneumonitis.

Microorganisms (Greek µικρoζ [mikrós, “small”] and oργανισµoζ [organismos, “organism”]) comprise single cells, cell clusters, and multicellular organisms and have colonized any place imaginable (5, 6). For example, microbes have been recovered from deep ocean thermal vents where temperatures approach 130 °C and pressures are ∼20 MPa (200 atm) (7). Viable microbes have also been recovered from ice up to 20,000 years old (8). In fact, microbiologists have yet to find a natural environment from which they have not been able to recover microbes (9). They are highly diverse organisms originating from all domains of life: Bacteria, Archaea, and Eukarya (10, 11), as well as viruses (12). The three domains differ in cellular construction, biochemistry, and genetics as discussed below. However, it remains unclear when and how these three domains separated, and there is also growing evidence for a two-domain tree of life with Eukarya as a sister lineage to archaeal superphylum (13, 14) (Fig. 1).

Figure 1. Microorganisms are highly diverse organisms originating from all domains of life—Archaea, Eukarya, and Bacteria—plus viruses (not shown). These three domains differ in cellular construction, biochemistry, and genetics. (A)The three-domain model postulates that Eukarya and Archaea shared a common ancestor, whereas (B) the two-domain model claims that Eukarya actually descends from Archaea. It remains unclear which model is correct.

Historically, knowledge about microorganisms was acquired by observation of the reaction of microbes to various stains and subsequent analysis of the physiological properties of individual microbes growing in pure cultures on solid media (15). Initially, the majority of microbiological studies were performed in support of medical science. Investigators did not understand biases caused by test method limitations that strongly influenced our thinking about microorganisms (16). Those that were not culturable were ignored, and unjustified extrapolation of knowledge gained from the study of medically important microbes has resulted in numerous misconceptions about microbial ecology in natural and industrial systems. With the advent of genetic (genomic) testing, microbiologists have discovered that microbes that seemed to be similar—based on microscopy and physiological tests—often belong to different and genetically distant taxonomic groups. Consequently, microbial taxonomy is in the midst of a revolution (17). Still, most genomic databases primarily archive genetic profiles of medically significant microbes. But this is rapidly changing, and genomic testing is now used more broadly for environmental and industrial microbiology research (18). As a consequence, databases are becoming increasingly reliable and useful.

Bacteria

Bacteria, historically classified as eubacteria (“true bacteria”), are a domain comprising unicellular microorganisms, first described in 1676 by Antoni van Leeuwenhoek, who built the first sufficiently functional microscope (19). Prior to the discovery of the archaea, the primary distinguishing characteristic of bacteria was the absence of visible, membrane-bound, internal structures. Bacteria appear in a plethora of shapes (20), but arguably the most common bacterial forms are spheres (cocci, in either strands or clusters), rods (bacilli), comma-shaped (vibrios), and spirals (spirochetes). During the first century of microbiology, the concept of polymorphism (the ability of bacterial cells to change their shape) was hotly debated. However, by the mid-1970s, researchers had confirmed, unequivocally, that polymorphism was a common capability among bacteria. Microbes that appear to be rod shaped under one set of conditions can transform into spherical or comma shapes when conditions change (20). In several biofilm studies, genetically identical Pseudomonas aeruginosa cells differed in both their appearance and their physiological characteristics, depending on the cells’ location within the biofilm. This is analogous to the differences among cells from different tissues of the human body—genetically identical but otherwise quite distinct.

The typical size range for bacteria is between 0.5 to 2 µm in diameter and between 1 to 5 µm long. However, cells as small as 200 nm (Mycoplasma genitalium) and as large as 750 µm (Thiomargarita namibiensis) have been described (21). In the past, bacteria were characterized by their reaction to staining (gram-negative and gram-positive) (22), later discovered to be based on bacterial cell wall chemistry, their shapes, and the substances they were able to metabolize. Increasingly, genomic data (23) are being used to characterize bacteria (as well as all other domains).

At first, most bacteria appear as simple structures surrounded by a distinct cell membrane and cell wall structures that add rigidness and stability while lacking a membrane-bound nucleus (→ prokaryotes). But a closer look reveals that most bacterial cells are sophisticated, containing functional analogues to eukaryotic membrane-bound organelles called bacterial microcompartments encapsulated by protein shells (24, 25) and cytoskeletons (26).

Although no individual bacterial or archaeal species can thrive in all environments overall, these two branches of life exhibit temperature tolerance and metabolic ranges that substantially exceed both the environmental tolerance and metabolic diversity scope of all other organisms. As a domain, bacteria are present in most habitats on Earth, in the atmosphere, and in space (27–29). Some species (aerobes) require oxygen; others (anaerobes) can only thrive when oxygen is absent. Facultative anaerobes—bacteria that behave like aerobes when sufficient oxygen is present and like anaerobes when the amount of oxygen present is not sufficient to support aerobic metabolism—are especially important biodeterioration agents. Particularly within biofilm communities, they scavenge oxygen, creating conditions suitable for obligate anaerobes. Some bacterial species use carbon dioxide as their sole source of carbon, whereas others require organic carbon as food. Bacteria thrive in temperatures low or high (−5 °C to well over 100 °C), classically divided into psychrophiles (<20 °C), mesophiles (20–40 °C), thermophiles (40–60 °C), and extreme thermophiles (>60 °C). Bacteria may withstand various concentrations of pollutants and salts and feel right at acidic, neutral, or alkaline pH ranges (30).

Arguably, bacteria are the form of life most frequently recovered from MWFs and MWF systems. Commonly recovered bacteria include aerobic and facultative anaerobic genera such as Acinetobacter spp., Alcaligenes spp., Bacillus spp., Blastomonas spp., Citrobacter spp., Comamonas spp., Empedobacter spp., Micrococcus spp., Morganella spp., Mycobacterium spp., Pseudomonas spp., Shewanella spp., Sphingomonas spp., and Wautersiella spp. (31–34). Pseudomonas spp. is the most frequently recovered genus in MWFs and is characterized as gram-negative, facultative anaerobic Gammaproteobacteria, occurring ubiquitously in environmental sources such as soil and water. As noted above, bacterial taxonomic classification is in flux, and numerous current genera (for example, Acinetobacter, Citrobacter, and Comamonas) were historically all members of the genus Pseudomonas. Consequently, although recent papers reporting MWF taxonomic profiles appear to indicate that the common taxa recovered from MWFs have changed since the 1960s and 1970s, in reality, most of the change reflects differences in taxonomic classification rather than actual differences in bacterial populations. Additionally, genomic profiling has identified microbes not previously detected due to the limitations of historical culture test methods.

It is important to recognize that even if bacteria may exist as free-living (planktonic) cells, they are much more likely to form aggregates on surfaces, building 3D structures called biofilms (35, 36).

Archaea

At first thought to be just a subdomain of Bacteria dubbed archaebacteria, in 1977 the Archaea were recognized as a unique domain (37–39) of single-celled microorganisms. Like bacteria, archaea have no nucleus, their cell membranes are usually bounded by a cell wall, and they are most abundant in biofilms (36). Archaea and bacteria are similar in size and shape, but archaea possess genes and many metabolic pathways that are much more closely related to the domain of Eukarya. Other aspects are truly unique, such as the composition of the cell membrane, demonstrating the ancestral distance from bacteria and eukaryotes (40).

Originally thought to inhabit extreme and harsh environments only—extreme temperatures and highly saline, acidic, or alkaline media (41)—archaea have now been acknowledged to be ubiquitous, including mesophiles that live in mild conditions in marshes, sewage, the oceans, and soils. However, our knowledge here is predominantly based on genomic data (42).

So far, evidence of archaea in MWFs is scarce (34), but there is no apparent reason why they should not populate MWFs or MWF systems.

Eukarya

The term eukarya describes organisms whose cells contain a nucleus (eukaryote from the Greek εὖ [true] and κάρυον [kernel]) and intracellular, membrane-bound structures (organelles). They evolved from prokaryotic ancestors involving mergers between bacteria and archaea in multiple symbiotic steps (43). Three main groups were described: animals, plants, and fungi. Most microbial eukaryotes (such as protozoa and algae) are of no concern in MWFs. Fungi are the notable exception. Fungi, ubiquitous in nature (44), are typically subdivided into multicellular, filamentous molds or unicellular yeast and routinely detected in MWFs or MWF systems (31), though not as frequently as bacteria.

Yeasts are unicellular, typically measuring 3 to 5 µm in diameter and often living planktonically. Most yeasts reproduce asexually by mitosis (45) or by budding (46). Using mainly sugars as an energy source, they do not require sunlight to grow and prefer moderately acidic environments (pH 5 to 6.5). Thus, the presence of yeast in MWFs was unusual, often associated with pH drops, and their presence was an indicator of general biostatic imbalance. However, some yeasts, such as Candida rugosa, do survive and multiply in MWFs even at pH of 9 or higher (Küenzi, unpublished).

The term mold is applied to large number of fungal species that play an immensely important role in the breakdown of organic matter. Generally, molds can be found in soil and other shady, damp areas (47) as well as on moist building materials (44), growing as multicellular filaments called hyphae (48). Molds can reproduce asexually or sexually, producing spores as reproductive bodies at the head of special aerial hyphae. Spores are highly resistant to heat and desiccation, are easily transported by air and fluids, and capable of growing into a new organism, comparable to plant seeds (49).

Mold taxa commonly recovered from MWFs include members of the genus Fusarium spp. (50), Exophiala spp., Trichoderma spp., and Penicillium spp. (31–33).

However, switching from hyphae to yeast and vice versa, known as dimorphism, has been described for many fungi, including Candida, which makes the classification into mold and yeasts increasingly difficult. Up to now, switching from one form to the other was implicated with virulence and was not described in the industrial environment (51, 52).

Viruses

Viruses are obligate intracellular, acellular microorganisms that infect living cells of all domains (referred to as hosts) (53). Most viruses feature a nucleic acid core (DNA or RNA), often surrounded by an outer protein capsid, a lipid envelope, or both. They are only able to replicate within a host. There is an extraordinary diversity of viruses, and they have remarkable host specificity (54). Viruses that infect bacteria are called bacteriophages or phages (55). Analogously, mycoviruses infect fungi (56). There is no consensus on what viruses that infect Archaea should be called. Both archaeal phages and archaeal viruses were suggested (57).

There have been few studies of either the types of viruses in MWFs or their frequency of distribution. Recently, some investigators have begun to explore the potential use of phages and mycoviruses to control prokaryotic and eukaryotic microbial contamination in various industrial systems (58, 59). However, successful transmission of viruses depends on having large numbers of host cells present in dense populations. Although bacterial and fungal population density can be sufficient in isolated zones within MWF systems, densities are insufficient in recirculating MWF. Viruses are most effective when there are ≥10⁹ potential host cells per milliliter. The challenge of using viruses as antimicrobials is exacerbated by the specificity between virus and host cell. Each type of virus can infect, at most, a few closely related bacterial species. Consequently, although phages have worked well under laboratory conditions, they are unlikely to be successful bacterial control agents in MWF systems.

Two basic cycles of reproduction can be distinguished for bacteriophages: lytic (or virulent) and lysogenic (or temperate). Whereas lytic reproduction cycles lead to production of virus particles and eventually death of the host, the lysogenic cycle leads to integration of bacteriophage DNA into the host’s genome, also known as prophage. This integration offers new ways for evolutionary and ecological adaptations for the host (60) as new genetic material is incorporated into the bacterial genome.

Biofilms

Although microorganisms can spend some part or all of their lives as planktonic organisms, most prefer to exist in dense, multicellular communities called biofilms (35, 36, 61). Such aggregates appear as slimy films on wetted surfaces or as flocs in liquids and are easily formed on inert and living surfaces that are in contact with liquid or vaporous water (Fig. 2). Biofilms can consist of single or multiple species. Consortia comprised of members of all three domains have also been described (62). Biofilms are certainly the dominant lifestyle of prokaryotes and possibly also the dominant lifestyle of eukaryotic microorganisms.

Figure 2. Some bacteria live as solitary and planktonic organisms, at least for a while. However, (A) this lifestyle has disadvantages and (B) the majority seeks refuge on surfaces, forming biofilms. This lifestyle confers many advantages on their inhabitants protecting them from the environment. Biofilms represent a much higher level of organization than single cells do.

Depending on the physicochemical properties of a given surface, microbes might directly attach to it, or prior adsorption of (macro) molecules (proteins, carbohydrates, lipids, minerals, and/or calcium soaps) on the substrate is necessary to facilitate attachment. Subsequently, microorganisms attach reversibly to the substrate before they use their own cell adhesion structures to promote a strong connection to the colonized surface (63, 64). Subsequent production and excretion of compounds—called extracellular polymeric substances (EPS)—lead to the formation of an extracellular matrix that provides protection and reinforcement (65). As the biofilm matures, it adheres more strongly to the surface on which it has developed. Biofilms not only provide shelter from unfavorable conditions; they also enable their inhabitants to perform in a way that would be impossible for the individual cells. Biofilms have a complex structure that includes channels for fluid and nutrient transport, zones that are nearly cell free, and zones that are densely populated. In many respects, biofilm consortia look and behave like primitive, multicellular organisms, rather than accidental assemblages of different microbial cells. Depending on their location within the biofilm matrix, even genetically identical microbes can exhibit different morphologies and metabolic capabilities, much like different types of cells in multicellular organisms.

This complex biofilm architecture creates a number of chemical gradients. In particular, oxygen and organic nutrient concentrations and pH gradients develop, allowing co-habitation of anaerobic and aerobic, acidophilic, and acidophobic microorganisms. All of these cells “talk” to each other to coordinate adhesion, biofilm maturation, exploitation of extracellular public goods, and swarming in a process known as quorum sensing (QS) (66–68). This communication among biofilm cells involves the production and detection of signaling molecules and the horizontal transfer of genetic material (cell-to-cell transfer of extrachromosomal DNA). Additionally, heterogenic biofilms consisting of multiple species may support each other in gaining the necessary organic molecules for growth and survival (69) in a way that comes close to symbiosis.

The combined effects of the EPS matrix and metabolic processes to sequester and neutralize microbicides make biofilm communities up to a 1,000 times more resistant than planktonic microbes to microbicide treatment (70–74).

Biofilms are also a source of dormant cells that are able to survive most unfavorable conditions and to evade killing by potentially lethal doses of antimicrobial agents (antibiotics, biocides, disinfectants) without genetic change (75). These cells are termed persisters (76–78) and may remain dormant for an undefined period until environmental conditions are deemed suitable enough for their return to vegetative growth. In MWFs, persister cells are able to repopulate their environment as soon as the pressure of antimicrobial agents, the primary factor preventing microbial activity, is relieved.

Dormancy

Minimizing metabolic activity over a long period of time is one way to conserve energy and is closely associated with environmental changes in an organism’s life cycle known to many plants and animals: seed dormancy, for example, prevents germination during unsuitable conditions (79), and hibernation (80) is a common survival strategy to save precious metabolic energy.

In the microbial world, dormancy is also well known: spore-forming bacteria produce endospores under stressful environmental conditions such as lack of nutrients. Endospores are tough, desiccated, nonreproductive structures that help these bacteria to survive for perhaps millions of years (81). Fungi also produce spores as part of their reproduction that—like plant seeds—are able to persist for a long time without germinating. The common term spore for both fungal spores and bacterial endospores can be confusing. The structure and chemical composition of fungal spores are quite different from those of bacterial endospores.

Until recently, microbiologists believed that only endospore-forming bacteria were able to persist in a dormant state. Over the past several decades it has become increasingly apparent that many different types of bacteria can transition into a dormant state and persist there for a long time (82). The state of dormancy has also been designated as the viable but not cultivable (VBNC) strategy of non-spore-forming cells (83). This term describes the fact that a majority of bacterial species from the environment cannot be induced to grow in the laboratory under various conditions despite adequate nutrient supply and being alive. A VBNC state has also been reported for yeast (84). In brief, dormancy has been considered a principal tool for microbial survival, because processes are more difficult to corrupt when they do not occur.

However, the concept of VBNC has created considerable confusion within the microbiology community and those who routinely rely on microbiological data. Few MWF stakeholders realized that the two types of growth media most commonly used for MWF condition monitoring were from a catalogue of more than 5,000 growth medium recipes. Nor were they aware that any given recipe is unlikely to detect more than 1% of all microbes present in environmental samples. Many growth media detect taxa that will not grow on any other nutrient medium. Not only were different nutrient recipes used but growth conditions such as temperature and gas mixture (oxygen free, carbon dioxide, carbon monoxide, and numerous other gas mixtures) were varied. Industry consensus was that the time and effort required testing MWF samples on numerous different types of growth was not feasible. As culture-independent genetic testing became commonplace (85), it was increasingly apparent that traditional culture test methods typically detected less than 1%.

Two examples of the major impact that small culture method changes had on our understanding of microbial ecology illustrate the VBNC challenge. The first example pertains to our understanding of the gut microbiome. Before the early 1970s, researchers relied on either fecal samples or samples taken from gut sections exposed to air, before the samples were incubated under oxygen-free (anoxic) conditions. Once investigators started to first place gut sections into anoxic chambers and then collect samples, we discovered that the microbes once thought to be the dominant gut bacteria were actually an infinitesimally small percentage of the total. All of the other gut bacteria were so sensitive to oxygen that a few seconds’ exposure was sufficient to kill them. Consequently, they had been undetectable. A similarly simple methodological change transformed the field of marine microbiology. Initially, marine microbiologists used the same nutrient medium as the one used to test drinking water, except seawater or seawater salts replaced the tap or distilled water component. This medium selected for gram-negative rods. Thus, conventional wisdom was that gram-negative rods were the dominant operational taxonomic unit (OTU) in marine environments. By simply reducing the nutrient concentration tenfold, marine microbiologists were able to detect OTU that previously had been killed by the high nutrient concentrations provided in the traditional growth media. As with the gut microbiome, our understanding of the marine microbiome changed dramatically, almost overnight. These discoveries, along with those made using genomic test methods, have changed the meaning of VBNC. The term now refers to microbes that we do not yet know how to culture but that are viable in their natural environments. Thus, the term VBNC reflects two substantially different concepts: VBNC is used to refer to microbes that have been injured (generally by microbicide treatment) but not killed—they are viable but do not proliferate to form visible colonies on growth media. In addition, it refers to microbes that might be robust and healthy in their natural environment but do not form colonies on the media used. To add another layer of confusion, when we use culture methods to monitor microbial bioburdens in MWFs, both concepts are concurrently relevant.

Sources for microbes

Water

Water is a compound with the formula H₂O, one of the most common molecules in the universe after H₂. It occurs in form of liquid, ice, and vapor. However, water used in everyday life and for industrial purposes is a complex mixture of H₂O, salts, organic substances, ions, microbes, and other components (86). Depending on its source and subsequent exposure to treatment, water often contains diverse molecules that serve as organic or inorganic macro- or micronutrients. Macronutrients are used as primary sources of energy, molecular building block materials, or both. Micronutrients (such as copper, iron, manganese, magnesium, nickel, and zinc) are essential to cell metabolism in trace quantities but often toxic at higher concentrations.

Organic and inorganic properties characterize water quality. Total organic carbon is a measure of all organic material dissolved or suspended in a water sample. Water’s chemical oxygen demand is a measure of the amount of oxygen required under specified test conditions for the oxidation of water-borne organic and inorganic matter (87). The biochemical oxygen demand tells how much dissolved oxygen is needed for aerobic organisms to decompose the organic matter present (88).

The sum of divalent metallic ions dissolved in water defines the water hardness that has a profound effect on the performance and stability of the mixed-in MWFs. The main elements contributing to hardness are Mg²⁺ and Ca²⁺, whereas Fe²⁺, Sr²⁺, Zn²⁺, and Mn²⁺ play a lesser role. When water hardness varies, pretreatment is an option to provide a supply of water with a constant water hardness. Water softening systems exchange sodium (Na⁺, a monovalent cation) for divalent cations. Consequently, softening reduces water hardness but does not reduce the total dissolved solids. To control total suspended solids, deionization technologies such as distillation, reverse osmosis, or nanofiltration must be used (57). By eliminating mineral nutrients, treatment strategies that pass water through ion-exchange resin beds or filters can lead to a notable reduction in microbes initially but eventually lead to higher numbers due to the increased surfaces on which microbes can form dense biofilm communities.

In essence, (drinking) water provides a home for a plethora of microorganisms (89, 90) and provides the first and arguably the most important point of entry. Commonly, tanks used to store deionized water support biofilm populations of billions of cells per square centimeter. These populations can develop within a few weeks after tank surfaces have been disinfected. That implies that water storage before use is not the best of ideas. The quality of water drawn from deionized water storage tanks can be a significant source of microbial contamination in end-use diluted MWFs.

In studies dating back to the early 1960s, it was repeatedly demonstrated that the taxonomic profiles of MWF microbiomes tend to mimic those of the makeup water. Consequently, improving the microbiological quality of the makeup water with which MWFs are diluted for end use can substantially reduce microbial contamination in in-service MWFs.

(Indoor) air

Microorganisms are transported easily as airborne particles (aerosols), across large distances. Fungal spores and bacteria can be transported as individual particles. Bacteria, on the other hand, are usually transported within water droplets or adsorbed onto other particles such as dust. Depending on air currents (direction and speed), viruses, bacteria, archaea, fungi, bacterial endospores, and fungal spores can be transported over large distances (91–94).

The microbial community in outdoor air varies geographically (95) and directly affects the microbiome of the indoor air (96, 97), which is of particular interest. In a genomic survey of microbial contaminants in jet fuel tanks around the globe, U.S. Air Force researchers found that the nearby soil microbiome was the primary factor contributing to fuel system contamination (98).

In addition to outdoor air, human occupancy contributes to bioaerosols and affects total number and microbial communities in indoor air by respiration and the shedding of skin cells, especially in poorly ventilated environments (99, 100). Moreover, cooling tower aerosols and plumbing system faucets (including showerheads) are important bioaerosol reservoirs (sources): Sink faucets and shower heads are major reservoirs for Fusarium spp. (101), arguably the most common fungal microorganism present in MWFs and/or MWF systems (50).

Indoor air quality investigations have suggested that the bacterial flora of indoor air and dust is dominated by Proteobacteria, Firmicutes, and Actinobacteria, including Pseudomonas spp. (99), the most commonly recovered genus in MWFs (34).

In metalworking facilities, bioaerosols may contain microbes, microbial metabolites, and cell fragments such as endotoxins (see Endotoxins).

Materials, tools

Microorganisms generally cover work materials, tools, and the work environment, and some microbes clearly interact with metals in both natural and synthetic environments (102). However, studies in the industrial environment are lacking in this regard, and it remains unclear whether this entry point is of importance or not.

MWF systems

The systems in which water-miscible MWFs recirculate vary in size from 0.1 to 350 m³. Consequently, MWF systems provide a large surface area on which biofilm communities may develop, depending on sump and piping system design. Biofilms do not coat MWF system surfaces uniformly. Quiescent zones, reverse-flow (eddy) zones, the MWF system surface–air interface (i.e., system surfaces that are periodically but not continuously immersed in MWF), and splash zones (surfaces routinely moistened by MWF mist) are the sites where biofilm accumulation tend to be greatest. These biofilm zones serve as reservoirs for MWF microbes, re-inoculating the bulk recirculating fluid whenever preservative concentrations fall below effective levels. Additionally, in piping dead legs, MWF biodegrades, thereby supporting a tremendous bioburden. The partial vacuum created by the Venturi effect as MWF flows through a header, past the dead leg–header connection, draws contaminated MWF into the recirculating stream.

Incomplete, poorly executed, or inadequate cleaning before filling thus presents a substantial risk of recontamination of the new MWF. To optimize system cleaning, dead legs should be eliminated. Cleaners should be used to disperse and flush out biofilm communities. The process of draining, cleaning, and recharging (D, C, and R) should be repeated until the fluid flushed out during draining is clean. Too often, operators perform a single D, C, and R cycle and assume that the system is pristine. If the process has been inadequate, the time saved by quickly returning the MWF to production will result in shortened MWF performance life and production quality and increased costs.

Operators’ skin

The skin is a complex barrier organ densely covered by up to 10¹² microorganisms from as early as birth—a microbiome that changes frequently until a final state of equilibrium is acquired by adulthood. The skin forms a symbiotic relationship between microbial communities and host tissue and includes two groups: (i) the resident microorganisms that are a routinely found in the skin and (ii) transient microorganisms that do not establish a permanent residency. Under normal conditions, neither group is pathogenic (103, 104). Four main phyla were characterized: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroides, whereas Corynebacteria, Propionibacteria, and Staphylococcus were the most common genera (105). The main interaction point is the skin on hands and forearms that mainly contains Betaproteobacteria, Staphylococcus, and Flavobacteriales (106–108). None of these taxa are dominant in the MWF microbiome. Thus, it can be assumed that operators’ skin is not a significant source of microbial contamination in MWFs.

Saliva

Saliva, a clear liquid secreted by several glands within the oral cavity, consists of proteins and minerals as well as planktonic bacteria shed from the surrounding oral cavity. This cavity is home for dense populations (often >10⁸ CFU mL⁻¹) of diverse bacteria. These bacteria live as biofilm communities on intraoral surfaces and dental plaque that shed bacteria into saliva. Spitting—most commonly introduced into MWFs by workers using the recirculating system as a spittoon—can inoculate MWFs with saliva-borne bacteria. Bioaerosols generated from sneezes are secondary sources of saliva-borne bacteria introduced into MWFs. There are no data available on the survival of saliva-borne bacteria in MWFs, but of the many bacterial taxa found in saliva (109, 110), only Streptococcus spp. have been reported to dwell in MWFs (32). Given that Streptococcus spp. are commonly found in potable water, their recovery from MWFs is not an unequivocal link between saliva contamination and Streptococcus spp. in MWFs. There have been no reported studies of the survival of blood-borne pathogens in MWFs. Moreover, there have been no reports of clusters of communicable diseases in metalworking facilities.

Biodeterioration

Natural and industrial aquatic environments are highly diverse and provide habitats suitable for microbial proliferation and metabolism. Biodeterioration—in the form of biofouling, biodegradation, or both—is the natural consequence of microbial proliferation and metabolism (62). Universally, microorganisms tend to organize themselves into biofilm consortia (see Biofilms) and use QS for organization, differentiation, and maturation (Fig. 3).

Figure 3. Biofilms in MWF systems may vary in size and maturity. (A) Very young biofilms are easily removable, whereas (B) mature ones offer a lot of resistance to cleaning agents. (C), (D) Molds like to settle in places outside the waterline, and the surfaces covered may be very small at the beginning but offer them a chance to distribute all over, using spores as transportation devices.

Metabolism refers to all chemical reactions needed to support life, including conversion of organic or inorganic compounds into energy or into structural components that may end up as waste (111), subsequently supplying energy and building blocks for other organisms (112). As discussed regarding biofilm EPS, biosurfactants, and QS, excreted metabolites can also serve essential ecological functions for the microbes producing them. In the MWF environment, the costs associated with microbial-derived biodeterioration are mainly due to decreased MWF service life. Decreased service life translates into decreased tool life and shortened intervals between D, C, and R events. In turn, D, C, and R costs include the following:

• MWF disposal and replacement

• MWF system cleaning (labor, lost production during downtime, and waste disposal).

Biodegradability depends on the type of microorganisms present as well as physicochemical parameters. The most important parameters contributing to MWF biodeterioration include

• MWF chemistry

• Organic molecule accessibility

• MWF system surface area

• MWF cleanliness

• Oxygen availability

• Water availability

• Temperature

• pH

• Biocide availability

• Surface size.

In recirculating systems using water-miscible MWFs, neither oxygen nor water availability is a limiting factor (more on oxygen availability below). Recognizing that there are microbes that thrive at temperatures <0 °C and others that thrive at >120 °C and that MWF temperatures typically range from 15 to 40 °C, temperature is not a limiting factor for microbial activity in water-miscible MWFs. However, it is very clear that the environment of each MWF system selects for specific organisms .

MWF characteristics

Formulated MWFs are cocktails of diverse organic compounds. Typically, MWF formulations include one or more functional additives in each of these categories:

• Buffering (neutralizing) agents

• Corrosion inhibitors

• Coupling agents (to help keep polar and nonpolar molecules in solution)

• Emulsifiers (to keep micelles in stable, uniform suspension)

• Performance additives to enhance lubricity

• Foam control agents.

• One or more microbicides.

In each of the listed categories, some alternatives are more bioresistant or biostatic than others. A bioresistant substance is not readily degraded, even when the MWF hosts a substantial microbial population. A biostatic substance restricts the metabolic activity and/or proliferation. It is important to consider the fluid’s waste treatability when formulating bioresistant or biostatic MWFs. This creates the paradox to develop eternally stable MWFs in-service that are readily biodegradable upon disposal.

Although the majority of organic molecules used in MWFs are nominally biodegradable, actual degradation rates vary with conditions. Microbes tends to preferentially attack simple, high-energy molecules such as alkanes in base stocks and primary amine neutralizing agents such as monoethanolamine and triethanolamine. When microbes use specific MWF components as nutrients, the concentrations of those molecules become depleted relative to the total MWF concentration. Consequently, the primary symptom of selective component depletion is typically loss of the functional property the component was designed to provide. For example, as amine additives are consumed, the MWF’s alkalinity will decrease. As noted above, molecules excreted or secreted by microbes that directly attack MWF components (primary degraders) can serve as nutrients for microbes that cannot use MWF components as food (secondary degraders), whereas organic acids produced as metabolites can cause emulsions to split. Conversely, biosurfactants can tighten emulsions and contribute to increased foaming and mist levels (113, 114).

One research group described Chryseomonas luteola, Ochrobactrum anthropi, Pseudomonas vesicularis, and Alcaligenes faecalis as the most efficient MWF-degrading taxa (115). Another group demonstrated that Clavibacter michiganensis, Methylobacterium mesophilicum, Rhodococcus erythropolis, and Pseudomonas putida readily degraded MWFs (116). Yet another consortium consisting of Agrobacterium tumefaciens, Comamonas testosteroni, Microbacterium saperdae, and Ochrobactrum anthropi was shown to efficiently degrade MWF compounds (117).

Microbiologically influenced corrosion

The physical presence of biofilm is sufficient to create conditions that promote electrochemical gradients—galvanic cells—in which electrons flow from an anodic region under the biofilm toward the cathode where the metal surface is not covered (118). As electron flow forms anodes, metals dissolve. Consequently, the most common morphology for microbially induced corrosion (MIC) damage is pitting. As noted above, microorganisms produce and excrete acidic metabolites as byproducts of their metabolism (119), primarily as C₁ to C₆ carboxylic acids. These weak organic acids are not particularly aggressive but can react with inorganic chloride salts (most commonly sodium chloride, calcium chloride, and magnesium chloride) to form weak organic bases and strong inorganic acids, particularly hydrochloric acid: $$\mathrm{R}-\text{COOH}\mathrm{ }+\mathrm{ }\text{NaCl}\to \mathrm{R}-{\text{COO}}^{-}·{\mathrm{ }\text{Na}}^{+}+\mathrm{ }\text{HCl}.$$

Other strong inorganic acids commonly produced by reactions with weak organic acids include sulfuric, nitric, and nitrous acids. MIC effects are usually in the context of biofilm formation that unavoidably starts after prolonged contact of metals with aqueous liquids. Although water plays an important electrolytic role in MIC, surfaces do not need to be immersed. MIC also often affects splash zones and surfaces on which mist droplets condense.

In MWF nearly all MIC is due to galvanic cell formation. Although galvanic cell electrochemistry is universally identical, corrosion rates vary substantially based on local physicochemical conditions like ion concentration, pH, and oxygen levels in the overlying fluid and their respective gradients between the bulk fluid–biofilm–metal surface interfaces (120). In addition to their role as MIC agents, some bacteria have been described as degrading commercial corrosion inhibitors (121, 122), which may render MWFs corrosive.

In some cases, however, biofilm consortia were described as actually inhibiting MIC (123).

Odor

Microorganisms produce a wide range of small (<300 Da) microbial volatile organic compounds (MVOCs) that are released as chemical signals to other organisms in the environment, known as olfaction (124), and also play an important role in QS in biofilms or simply as waste products. Some of the bacterial MVOCs have also been described to fight fungi and to diminish fungal contamination (125).

In well-aerated, recirculating MWF systems, MVOCs are oxidized by abiotic chemical reactions with dissolved oxygen in the MWF. However, during shutdown periods the remaining oxygen is rapidly used up, creating anoxic conditions. This forces facultative anaerobic bacteria to switch from oxidative metabolism to fermentation, allowing obligate anaerobic bacteria to become metabolically active. Although MVOCs are produced during aerobic and anaerobic metabolism, the diversity of malodorous MVOC molecules produced during fermentation and anaerobic respiration is higher because in anaerobic respiration, the inorganic terminal electron acceptor oxygen is replaced by sulfate, nitrate, and nitrite (126, 127). Metabolically active microbes in MWFs thus produce volatile organic and inorganic metabolites. Hydrogen sulfide (H₂S) and ammonia (NH₃) are the most common examples of volatile, inorganic metabolites (128, 129). In quiescent systems, this may lead to an accumulation of MVOCs in the MWF and major emission of these during recommission of the systems. Typically, it takes several hours for the facility’s ventilation system to replace high-MVOC air with fresh air and for the MVOCs to dissipate to below detectible concentrations.

Hydrogen sulfide’s characteristic rotten egg odor is detectable at ≥0.5 ppb. At 2 to 5 ppm, H₂S becomes toxic, and exposures to ≥700 ppm can cause sudden death (130, 131). In inadequately ventilated facilities, where airflow is insufficient from floor to ceiling, H₂S can accumulate around machines and other stagnant air zones, because it is heavier than air. Ammonia (NH₃) can be a byproduct of amine metabolism (132). At ≥2.6 ppm, NH₃ can be detected by its strong, acrid odor (133). Due to its pungent smell, significant exposure without the operator’s knowledge is almost impossible, and even concentrations of up to 100 ppm in the air are well tolerated for several hours (134).

Colonization by molds is easily perceptible by a musty odor (reminiscent of sweat socks) and their visible mycelial growth (44). Most of the MVOCs produced by molds are not unique to one species and include alcohols, amines, ketones, and terpenes (135). Still, some MVOCs are allergenic, others are toxic, and some are both allergenic and toxic (136). As discussed earlier, molds tend to stick to wet surfaces (splash zones). Once they are no longer in direct contact with recirculating MWF, fungi growing in splash zones are no longer exposed to fungicides incorporated into or added to MWFs. Mechanical removal followed by appropriate cleaning and decontamination procedures are the main way to get rid of molds and related odors (137).

Nominally, machine shop air turnover rates range from 10 to 15 times per hour (138). However, it is not uncommon for the airflow pattern to bypass the breathing zone (Fig. 4). Under these circumstances, fresh air is not supplied where it is needed, or at least not within the required time.

Figure 4. Machine shop ventilation. Air change rate is 10 h⁻¹ in both facilities. (A) Air enters low and exits high so that the facility is well ventilated. (B) Air intakes are above the breathing zone. Consequently, the net air exchange rate belies stagnation in the breathing zone.

Health effects

Skin problems

The skin is the first mechanical and biological barrier to any environmental exposures and protects the inner body from all sorts of attacks, including chemical, physical, and microbial insults. The physiological pH of the stratum corneum is 4.1 to 5.8, which is beneficial for the plethora of commensal microorganisms (139, 140) known as the skin microbiota, or the skin microbiome, that is crucial to support the skin’s immune system (107). Anatomically, the skin comprises two distinct compartments: the epidermis, mainly composed of keratinocytes, and the dermis, a network of collagen and elastin that provides elasticity as well as strength. The skin is also home to immune cells such as phagocytes, mast cells, antigen presenting cells, and T cells. These immune cells directly react to environmental challenges by inflammation and removal of the infective agents while ensuring tolerance against and communication with commensal microbes (141).

The alkaline nature of MWFs—typically buffered in the pH 8.8 to 9.2 range—can be irritating to the skin by removing the skin’s protective fats and oils. This leaves the dermis vulnerable to microinjuries and allergic reactions due to exposure to MWFs (142). Metal fines suspended in recirculating MWFs can cause physical injuries, thereby contributing to oil acne, folliculitis, and irritant and allergic dermatitis.

Dermatitis

Irritant dermatitis occurs when alkaline solutions such as MWFs remove protective oils in the skin and subsequently damage proteins in the skin’s outer layer. This results in dry, thickened, fissured, and inflamed skin.

Allergic dermatitis can develop when antigens (or allergens) such as biocides (143), preservatives, corrosion inhibitors, amines, metal abrasion (nickel, silver, titanium, chromium, copper, cobalt, gold (144)) pass through the damaged skin and cause an immune system response.

In contrast to allergic dermatitis, irritation is caused by either chemical or physical damage to the skin. The primary method to prevent skin problems is to avoid direct contact with MWFs by applying skin cream and wearing protective clothing.

Microorganisms can also contribute to dermatitis, if the commensal skin microbiota is destroyed or imbalanced by environmental or behavioral factors such as poor or excessive hygiene regimes, antimicrobials, climate, or age (145, 146). ASTM E2693 (147) provides detailed guidance on best practices for minimizing dermatitis risk in the metalworking environment.

Infections

Infections occur when pathogenic microorganisms gain entry into the body, proliferate, and cause an immune system response. To date, there are no indications that infections are common when working with MWFs, because “there is no evidence that the incidence of infectious disease at metalworking plants differs from that of the general population” (31), p 256. Passman hypothesized that machinists are similar to sewage treatment workers and schoolteachers. Because they are exposed to novel bioaerosols and microbes, new workers experience a period of frequent colds, gastroenteritis attacks, and other minor types of infectious disease. Within a short period—3 to 6 months—these new workers are no longer susceptible to the diseases that discomfited them initially. Once workers have become acclimated to the microbes to which they are routinely exposed, disease rates of metalworking facility personnel become indistinguishable from those among unexposed populations.

Oil acne and folliculitis

Folliculitis (inflammation of the hair follicles) and oil acne are mostly due to extended contact to neat oils (148) and not related to microbes.

Respiratory diseases due to microorganisms and bioaerosols

Exposure to MWF aerosols can result in respiratory problems such as nose and throat irritation, shortness of breath, asthma, or hypersensitivity pneumonitis (HP). Because most MWF components are antigenic, respiratory systems reflect individual workers’ allergic responses to the components of MWF mist. The severity of an individual’s response depends largely on that individual’s sensitivity to the allergen. The phenomenon is the same as that for food allergies. Most exposed people do not have allergic reactions to common food. However, foods that provide essential nutrition to >99% of the population can cause fatal anaphylactic allergenic reactions in susceptible individuals. Individuals who experience an allergic reaction to MWF or MWF mists must be reassigned to jobs where they are no longer exposed to the allergen.

Aerosols are defined as colloids of microscopic to very fine solid particles or liquid droplets in a gas (e.g., air), such as dust, smoke, or sea salt. Aerosol droplets that are ≤1.0 µm in diameter are called vapor. Larger aerosol particles are called mist. Due to their small size (typically between 30 nm and 100 µm), aerosols can enter the upper respiratory tract unopposed, leading to deposits in the bronchial airways. Very small aerosols may even force entry into the blood circulation, a mechanism that is also used in drug delivery. Respirable aerosols ≤10 µm in diameter can reach the lungs’ alveoli, whereas inhaled aerosol droplets in the >10to 40 µm diameter range (149) are trapped in the thoracic zone (from the entry into the nasal passages to the thorax). Mist droplets in this size range typically cause milder symptoms than do smaller particles. These particles are then attacked by immune cells harboring in lung tissues such as macrophages. Continuous exposure to such particles may inflict phenotypic changes in these macrophages that are thought to contribute to respiratory diseases (150).

Bioaerosols are defined as suspensions of airborne particles that contain living organisms or metabolic products. These particles can be allergenic, toxic, or both. Toxic bioaerosol molecules are classified as endotoxins or exotoxins (Figure 5). Exotoxins are secreted, whereas endotoxins are structural components of living organisms and are among the most potent inducers of inflammatory cytokines (151).

Asthma

Asthma is conventionally defined as a type I allergic airway disease mediated by T-helper cells and immunoglobulin E (152) and occurs with sufficient exposure to airborne irritants. These irritants, known as antigens or allergens, trigger an immunoglobulin E-mediated release of molecules that cause diverse symptoms ranging from sneezing to chronic inflammation (153). Viruses are the predominant cause for asthma and other respiratory illnesses (154), and it was suggested that disruption of the commensal microflora might lead to immune dysfunction (155). There are no indications that airborne microorganisms from MWFs have a specific function in these diseases. It is more likely that chemical components of MWFs are responsible for occupational asthma (156, 157).

Hypersensitivity pneumonitis

Also known as allergic alveolitis and numerous other industry-specific names (farmer’s lung, pigeon breeder’s lung, machinist’s lung, etc.), HP is an immunologically mediated inflammatory disease, featuring a neutrophilic inflammation of the respiratory bronchioles, alveoli, and interstitial tissue of the lungs (152). Like asthma, HP is characterized by an inappropriate immune response to an antigen that not only eliminates the antigen but also extensively damages the host’s tissues. Although the word hypersensitivity implies that the response is heightened, this is not necessarily the case.

HP has been linked to microbial exposures (158), including fungi (Thermoactinomyces vulgaris, Aspergillus niger, Aspergillus fumigatus, and others) and bacteria (Mycobacterium abscessus, Mycobacterium immunogenum, and others). Occurrence of M. abscessus (159), M. immunogenum (160), and Mycobacterium avium (161) has been reported in MWFs, but M. immunogenum was identified as causative agent at several metalworking facilities where HP clusters occurred. Additionally, it was shown that M. immunogenum activates JNK and p38 mitogen-activated protein kinase in alveolar macrophages. This comes as no surprise due to the fact that mitogen-activated protein kinases are primary agents activated during various stimulations (162). It was reported that M. immunogenum causes HP in mice (163, 164), and M. immunogenum was recovered from MWFs at some facilities where HP clusters had occurred. However, it was not detected at others (165–169). Although it was initially speculated (H. W. Rossmoore, Wayne State University, unpublished) that biocides used to suppress rapidly growing MWF microbes selected for M. immunogenum, Passman et al. (167) subsequently demonstrated that there was no relationship between the abundance of M. immunogenum and that of other MWF microbes. Moreover, Aspergillus spp., Fusarium spp., and yeasts (170) are common MWF fungal contaminants known to cause HP.

Still, further studies are needed to understand a possible link between HP and M. immunogenum and other mycobacteria in general. It is important to note that between the 1992 sentinel HP cluster at an automotive manufacturing facility in the United States and 2002, approximately 250 cases of HP were reported for personnel who were routinely exposed to MWFs. Since then, there have been approximately 50 additional cases reported within the industry. In the United States, HP morbidity data indicate that the disease affects 2 of every 100,000 members of the general population. Among machinists the rate is <1 per 100,000 person-years exposure—approximately half of the general population’s incidence rate. Among pigeon breeders the rate is approximately 25 per 100 breeders (i.e., 25%).

Exotoxins

Bacterial exotoxins are toxic substances released by bacteria and damage host cells by disrupting cellular mechanisms or forming pores (Fig. 5) (171). Some bacteria routinely found in MWFs such as Pseudomonas aeruginosa (172) have been reported to produce exotoxins, but most recognized exotoxin-producing bacteria (173) are not commonly recovered from MWFs.

Figure 5. Exotoxins and endotoxins. (A) Exotoxins are diffusible proteins secreted into the surrounding medium by mostly gram-positive bacteria. (B) Classic endotoxins are part of the outer membrane of the cell wall of gram-negative bacteria. They are liberated upon lysis.

Fungal exotoxins are called mycotoxins (174) and are produced by molds, such as Fusarium and Aspergillus, known to dwell in MWFs. Still, poisoning by mycotoxins while working with MWFs has never been credibly reported (166).

Endotoxins

The most common endotoxin to which machinists are exposed is lipopolysaccharide, a component of the gram-negative bacteria outer cell membrane (Fig. 5) (175). Structure and bioactivity are heterogenous and vary between and even within species (176). In addition, components of the gram-positive cell wall have been suggested to function as endotoxins as well (177, 178). Endotoxins are not only toxic but are also allergenic, suspected to cause anything from mild fever to toxic shock as well as obesity, chronic fatigue, and atherosclerosis, and are released from both damaged and intact cells.

Endotoxins from planktonic bacteria in the MWF itself are of no real concern, because uptake of MWF as well as infections due to contact with MWFs are rare events (if they occur at all). Airborne endotoxins from either aerosolized bacteria or bacterial components are another story, because they are apparently easily absorbed. They are measured in units per cubic meter (EU/m³), typically ranging from 2 to 183 EU/m³ in the metalworking environment (179). Currently, no occupational exposure limits associated with endotoxins exist, and risks caused by exposure remain unidentified. However, in 2010 the Dutch Expert Committee on Occupational Safety recommended a health-based exposure limit for airborne endotoxin of 90 EU/m³ as an 8-h time-weighted average (180), although lower levels may already cause inflammation in some workers. This may be attributed to different methods to measure endotoxins that may or may not reflect the presence of endotoxin capable of stimulating host cells (176). Of course, the impact of endotoxins is strongly influenced by the host’s own inactivation mechanisms, which themselves are shaped by stress responses and state of health (176, 181).

One bioaerosol survey of automotive part manufacturing plants in the United Kingdom reported endotoxin concentrations ranging from 1 to 7,600 EU m⁻³ (182). However, neither Crook and Swan (182) nor any other authors reported any possible relationship between endotoxin bioaerosol concentration or connected adverse health effects among exposed workers. Consequently, a relationship between MWF bioburden and endotoxin bioaerosol concentration, as well as between endotoxin bioaerosol concentration and respiratory disease in the metalworking environment, remains speculative (183, 184).

Minimizing exposure to aerosols by sufficient area ventilation and effective housekeeping is likely the best, most convenient, and most efficient way to protect MWF end users from respiratory diseases (185).

Microbial contamination control

All MWFs, regardless of type, require good working conditions and management, which includes the monitoring of pH and concentration. In addition, effective microbial contamination control in MWF systems depends on engineering controls, condition monitoring, and contamination control treatments.

Engineering controls

Maintaining a clean, safe working environment heavily contributes to reducing the risk of uncontrolled microbial contamination. Although keeping machines clean and tidy is important, provisions should be made to minimize introduction of contamination into recirculating MWFs. Historically, wastes generated from power-washing machine surfaces flowed directly into MWF sluices. This practice often introduced substantial microbial contamination into the MWFs.

Although personnel responsible for MWF management rarely have any say in system design, particularly for systems that are already in operation, they might be able to influence metalworking facility owners and operators to consider the impact of system design for future installations. Minimizing the existence of stagnant zones is important for both central and individual sump systems. In central systems, MWF flow from clean fluid reservoirs to machines should be laminar to minimize biofilm development. In contrast, flow through return sluices should be turbulent to optimize chip transport to a filtration system. Machine surfaces should be accessible for cleaning and sample collection. All MWF systems, regardless of size, should include a means for removing contamination. Typically, this includes the ability to remove chips, swarf, and tramp oil. Best practice also includes installation of equipment to ensure that the water used for MWF dilution has consistent, high-quality characteristics, particularly uniform hardness, appropriate for the chosen MWF.

Installation of mist collectors and other local exhaust systems on any machines improves the overall air quality of a given facility and efficiently remove aerosols. However, mist collectors must be properly maintained to work at maximum efficiency and to prevent them from becoming bioaerosol sources.

Condition monitoring

The number of parameters tested and the frequency with which they are tested will vary with the system size and type of metalworking operation. Minimally, MWF concentration should be checked daily. Other commonly monitored parameters included pH, alkalinity, and tramp oil concentration. For critical machining operations, specific component concentrations of biocides, corrosion inhibitors, defoamers, and dirt load are monitored. Because MWFs are formulated to include chemicals that help to resist pH change (i.e., buffers), alkalinity testing provides a much earlier indication of MWF biodeterioration than pH testing does. The pH drop only occurs after buffer concentration has become depleted. Studies have shown that microbial contamination levels increase with tramp oil and dirt loads (166); consequently, it is important to monitor and control these parameters. Monitoring for microbial contamination is also critical but extensively complicated despite the fact that the vast availability of microbial techniques should make it easy to assess the populations in MWFs. However, the composition of the fluid itself often—if not always—demands additional preparation steps such as filtration or centrifugation that may blur the overall picture by changing cellular or community properties. This is the obvious reasons for the persistent popularity of culture-dependent tests that hark back to beginning of human civilization when microorganisms were first used to produce a wide variety of food, including alcoholic beverages and cheese.

Culture-dependent tests

The most commonly used technique in both the laboratory and in the field is culture testing, by heterotrophic plate counts (HPCs) or use of dip slides (paddles). A plethora of different nutrient media is available, with each growth medium recipe optimized to support the cultivation of one or more microbial taxa. No individual nutrient medium is likely to detect >1% of all bacteria or fungi present. Microbes that produce visible colonies on nutrient media are called culturable. Microbes that are viable in the sample but do not produce colonies are termed VBNC (see Dormancy).

The concept underlying culture-dependent techniques is that each individual cell placed onto solid or semisolid media will proliferate to form a visible colony. Typically, the individual cell (or floc of cells) must proliferate to approximately a billion cells before colonies are sufficiently large to be recognized by the naked eye. That translates into 30 generations (one generation is the time required for the population to double; i.e., for the majority of cells to replicate). Microbial generation times vary among taxa and growth conditions. Most commercial culture tests assume that all bacteria have generation times in the 0.5 to 1.5 h range (i.e., will produce visible colonies within 48 h). However, it is common for MWF microbes to have generation times ranging from 2 to 8 h or even longer. Consequently, culture testing terminated after 48 h is likely to underestimate the culturable population density in a given sample. The standard plate count method (ASTM D5465 (186)) is typically performed by a qualified laboratory technician. For HPC, tryptic soy agar plates are conventionally applied with subsequent specification based on morphology (shape, elevation, smell, and color). Additional identification of colony-forming microbes is possible by determination of biochemical properties, analysis of protein patterns by matrix-assisted laser desorption/ionization time-of-flight (187), or genetic testing by polymerase chain reaction (PCR).

Commercially available dip slides (nutrient agar–coated plastic paddles) can be used tankside and do not require technical training to use. They are imprecise, providing an approximation with results that typically vary by an order of magnitude (i.e., ±1 Log₁₀ CFU mL⁻¹ so that an observed value of 10³ CFU mL⁻¹ actually indicates a population density of 10² to 10⁴ CFU mL⁻¹) of culturable population density. Typically dip slides include bacterial growth medium on one side and fungal growth medium on the other, making it possible to quantify bacteria, molds, and yeast (molds and yeasts are differentiated by their respective colony morphologies on fungal growth media). Dip slide culture data provide adequate microbial contamination measurements for most users. Two caveats affect culture test interpretation. First, unless antibiotics are formulated into the fungal medium, bacteria can grow on it and be misidentified as fungi. Second, test kit instructions typically recommend observing dip slides for colonies after 24, 48, and 72 h. Slower growing microbes can require a week or longer to produce visible colonies. Unless dip slides are observed for 10 to 14 days before being scored as negative, there is a substantial risk that negative test results will be artifacts of inadequate incubation periods.

That said, both methods are widely applied in the industry due to their apparent simplicity in estimating microbial population, and both methods do not require additional preparation steps. Still, because the majority of microorganisms do not grow on artificial media (188), or at least not within a reasonable time, the disadvantages are immense, leading to underestimation of the real population density and diversity (34).

Culture-independent tests

Microscopy

Microscopy can be used to directly visualize microorganisms in a sample. However, enumeration is difficult (due to the size of bacteria), and differentiation between live and dead cells requires additional staining and more sophisticated instruments. Direct counting after staining with microbe-specific fluorochromes is a suitable method for the enumeration of live microorganisms in environmental samples (189) and theoretically could also be applied to MWFs. Traditional staining procedures such as the acid-fast stain to detect mycobacteria (190) and the Gram stain have been used. However, interferences due to bacteria-sized emulsion droplets and swarf particles can make direct counting in MWFs impractical. Speciation remains challenging beyond differentiation between gram-negative and gram-positive bacteria and other morphological features.

Fluorescent in situ hybridization

An option for specification may be fluorescent in situ hybridization (FISH). FISH is used to detect specific nucleic acid sequences (conventionally targeting 16s rRNA) in fixed and permeabilized samples by a fluorescently labeled probe. The specimen is subsequently analyzed by fluorescence microscopy, and by combining two or more differently labeled probes, this technique can be used to simultaneously detect different species in a specimen. FISH is mainly used in diagnostic microbiology to detect human pathogens in a given sample, and we refer to the review by Frickmann et al. (191) for a concise treatment of the subject.

Catalase activity

Catalase is an enzyme found in almost all aerobic cells, including fungi and bacteria. Catalase (and other scavengers) protect cells from oxidative damage by reactive oxygen species (192).

Catalase activity is easily measured by mixing MWF samples with 30% hydrogen peroxide (H₂O₂) in a sealed reaction tube. The amount of liberated oxygen gas is then directly proportional to the catalase concentration and the pressure increase is set in relation to the microbial activity. However, the limit of detection is rather high (≥10⁵ mL⁻¹), and anaerobic and other bacteria that do not have a complete catalase enzyme go undetected (193). It also presents a further disadvantage: MWF ingredients or chemicals incorporated during use interact with the assay, leading to false-negative as well as false-positive results (Küenzi, unpublished). For example, dissolved iron reacts with H₂O₂ quantitatively so that the catalase reaction is proportional to the iron concentration. In MWFs, the most common cause of false-negative results is the dominant presence of catalase-negative bacteria.

Genomic testing

Amplification and subsequent detection of nucleic sequences (DNA, mRNA, rRNA) are often done by application of PCR, a molecular technique that uses the ability of DNA polymerase to synthesize new strands of DNA complementary to the offered template strand. This technique can be used for microbial enumeration as well as for specification. Originally developed in the 1970s and 1980s, it has experienced many alterations and modifications since (194). Frequently, the invariable 16S rRNA gene (18S rRNA in eukaryotes) is targeted for identification and enumeration of OTUs. However, the technique is also useful for the detection of specific genes or the determination of transcriptional activity (enzyme-mediated process by which DNA gene codes are copied into messenger RNA) and is particularly useful for the detection of slow-growing microorganisms such as mycobacteria (195) in MWFs.

Quantitative PCR (qPCR), also known as real-time PCR, is a DNA amplification technique using fluorescent reporter dyes that bind to DNA. Whereas traditional PCR measures the accumulation of the product only at the end of all amplification cycles, qPCR quantifies the amplification as it occurs by measuring the increase in fluorescent signal that is dependent and directly proportional to the amount of double-stranded DNA produced during each cycle. qPCR can conveniently be used for enumeration and specification, if the appropriate sequence information of the microorganism in question is available.

Reverse transcriptase PCR allows the amplification of RNA that, subsequently, is reversibly transcribed into complementary DNA (cDNA), made possible by the enzyme reverse transcriptase. cDNA is then used as template for any of the PCR methods available.

Propidium monoazide-PCR (PMA-PCR or viability PCR) is a newer technique (196). PMA-PCR is in fact an effective tool to discriminate between intact and physically damaged cells in microorganisms. Therefore, analyzing PMA-treated DNA from populations reflects the structure of metabolically active/viable cells to a great extent.

Metagenomics, also known as next-generation sequencing, is the study of genetic material from environmental samples and enables population analysis of microbial DNA of both culturable and unculturable microbes. This newer technique allows examination of thousands of organisms in parallel and has been widely applied in recent microbiological studies (197). Di Maiuta et al. (34) used this technique to analyze in-use MWFs. However, this technique also has drawbacks, because contamination in the laboratory and of DNA extraction kits is widespread and does impact microbiome studies, especially analysis of low microbial biomass samples (198, 199).

Flow cytometry

Originally developed for the counting of red blood cells and subsequently adapted to other eukaryotic cells, flow cytometry (as well as fluorescence-activated cell sorting) has recently been updated to analyze bacteria (200–202). This technique allows the simultaneous analysis of physical and/or chemical parameters by counting up to thousands of particles per microliter and is currently replacing HPC for routine microbiological monitoring of drinking water (89, 203).

The cell surface or cell components may be labeled with fluorescent dyes (modern machines are able to analyze multiple fluorescent dyes simultaneously) and are then aligned to pass individually through a laser beam by hydrodynamic focusing, mainly limiting this technique to single, nonclumped cells. However, flow cytometry has also been applied to cell pellets (204), using specially designed instruments. In general, flow cytometry allows the examination of a large number of cells per sample, recording (for each single cell) parameters such as cell membrane integrity, viability, and activity, even allowing later identification.

Our own unpublished experiences clearly demonstrate the usefulness of this technique, especially concerning the determination of quantity and activity. Even the identification of single species or genera might be possible using specific dyes. However, MWF samples need sophisticated isolation steps such as buoyant density centrifugation because emulsion droplets or MWF ingredients and other chemicals present in in-use samples greatly interfere with staining and subsequent analysis.

Quantification of adenosine triphosphate

Adenosine triphosphate (ATP) is the most important transporter of chemical energy in all known cells for metabolism (205), and an ASTM standard test method to measure intracellular ATP associated with microorganisms found in water-miscible MWFs exists (206). In principal, this method does allow the quantification of the ATP content and an assessment of the population size within 5 min or so but no conclusion on the type of microorganism present (207). Recently a new method was developed that allows a qualitative differentiation between bacterial and fungal cells (208). ATP measurement is most suitable for recurrent measurement, allowing rapid recording of the on-site contamination in respect to earlier readings.

Testing for endotoxins

For an extensive overview, please consult Munford (176). In general, none of the assays available, including the Limulus amebocyte lysate assay and endotoxin activity assay is foolproof, but all may be useful under specific conditions. ASTM practice E2657 (209) describes a consensus protocol for endotoxin testing in MWFs, and ASTM practice E2144 (210) provides a protocol for monitoring endotoxin concentrations in MWF aerosols.

Contamination control treatments

Physical treatments

Sound (sonication systems), ultraviolet light, photocatalysis, ozonation heat, radiation, and filtration are physical means to reduce or eliminate microorganisms. All of these methods were applied to in-use MWFs with little or no success. Application of these technologies may even lead to the degradation of the MWF (31, 211). A major challenge to these single-point treatments is that even those that are 99.9% effective (i.e., 99.9% kill of a 10⁶ cells mL⁻¹ population leaves 10³ surviving cells per milliliter), the surviving microbes are likely to colonize downstream surfaces and never again be exposed to the treatment. Moreover, the treatments select for resistant microbes. They could partially be of use to treat spent MWFs before disposal or to reduce bioburdens in makeup water.

Antimicrobial activity of metals

Metals as antimicrobials have been used since antiquity, especially silver (Ag) and copper (Cu). Whereas silver had not been described to have any function in biochemical processes (nonessential metals), some others such as copper are indispensable in all organisms (essential metals). However, if present in excess, all metals are toxic (212). Still, “excess” is difficult, if not impossible, to define: for example, Enterococcus faecalis is able to survive and dwell in high iron concentrations that are toxic to most other bacteria (213). Toxicity of metals is more pronounced when used as nanoparticles (214). However, incorporation of metals makes most sense on regularly cleaned surfaces—for example, in the health care setting (215)—and is largely worthless in MWFs. To date, tests using antimicrobial surfaces on MWF systems or formulation of MWF with metal nanoparticles (silver) have all been unsuccessful. All in-use MWFs already have high concentrations of the metals contained in the workpiece alloys (iron, nickel, lead, vanadium, tungsten, etc.). Given the substantial bioburdens supported by MWFs that have high concentrations of toxic heavy metals, additional use of heavy metals as biocidal substances is unlikely to be of benefit in MWFs. Moreover, heavy metals are categorized as priority pollutants. Their intentional use in MWFs could increase waste disposal costs, make it more difficult for site operators to comply with discharge permit requirements, or both. ASTM Subcommittee E34.50 on health and safety of MWFs has recently chartered a task force to develop a guide on the human health risks associated with metal ion accumulation in MWF systems. These risks provide an important argument against the use of heavy metals as microbicides in these systems.

Chemical treatments

Microbicides (also called biocides or antimicrobial pesticides) are chemical products designed to kill bacteria (bactericides), fungi (fungicides), or both (broad-spectrum microbicides). In contrast, bioresistant chemicals are functional additives (e.g., corrosion inhibitors, buffers, emulsifiers, etc.) that are recalcitrant to microbial attack (i.e., not readily used as food) but do not directly influence MWF bioburden. In Europe, biocides are all registered under the European Union’s (EU) biocidal products regulation (BPR) (216) and cognizant national regulatory agencies. Although the amount of toxicological information required for a pesticide registration is substantially greater than that needed for other chemicals registered under REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), biocidal products are not necessarily more toxic than other chemicals used to formulate MWFs.

Each registered biocidal product is approved for use in one or more specific applications. At present, there are 27 biocidal products either approved or in the review process for use in MWFs under BPR Product Type 13. Until final biocides panel decisions are made, all 27 products may be used. The number of biocidal products available is a small fraction of the number products sold prior to the EU’s adoption of BPR.

In the United States, all pesticides must be approved by the U.S. Environmental Protection Agency’s Office of Pesticides Programs. In accordance with the U.S. Code of Federal Regulations (40 CFR 152 et seq.) pesticides are approved for use against specific target pests in specific end-use applications (217). Products that target microbes are designated as antimicrobial pesticides. At present there are approximately 50 active substances approved by Office of Pesticides Programs as antimicrobial pesticides for use in MWFs (218). Within the United States, antimicrobial pesticides must also be approved by the environmental protection agency of each state in which they are sold or used. Similarly, within the EU, each nation approves the use of biocides within their country. In addition to the EU and United States, other nations have their own biocide registration processes and requirements. Manufacturers, marketers, and users all share responsibility for ensuring that biocides have the required regulatory approvals and are use in accordance with local regulations.

Biocides can be classified by target organism or by chemical class. The most common chemical classes of MWF microbicides are formaldehyde-condensate (F-C) chemistries and isothiazolinones (all isothiazolinone biocides have a five-molecule ring that includes a nitrogen, a sulfur atom, and a keto (R′(C = O)R″) group. Formaldehyde acts on proteins by denaturation and on nucleic acids by alkylation, isothiazolinones act on the membrane and proteins, whereas pyrithiones are general inhibitors of membrane transport processes in fungi (219–221).

Formaldehyde is a ubiquitous chemical found in the environment, albeit in extremely low concentrations, because it is rapidly broken down by sunlight or bacterial activity. It is also formed endogenously by any living cell as a normal physiologic chemical, which may be involved in metabolism (222). It has been used in medical applications as a sterilizer and is often found in consumer products such as food, cosmetics, and cleaning agents. However, it has been suspected to cause contact allergy (223, 224) and to have toxic effects on the nervous system (225). Additionally, it was published that chronic exposure of rats to formaldehyde vapor led to formation of nasopharyngeal cancer, and other studies found increased numbers of nasopharyngeal carcinoma and leukemia in humans (226). Formaldehyde is currently listed by the International Agency for Research as a probable human carcinogen (227). This topic is controversially discussed, and further epidemiological studies using a more effective risk assessment are needed to support or falsify the presented evidence (228). In addition, it must be kept in mind that C¹³-nuclear magnetic resonance studies (unpublished but submitted to regulatory agencies) have demonstrated that at MWF pH (i.e., >8.0) the concentration of free formaldehyde released into MWFs is below detection limits (i.e., <0.1 mg L⁻¹). Even more important, formaldehyde aerosol studies have shown that formaldehyde concentrations in aerosols above F-C microbicide-treated, versus untreated, MWFs are not significantly different (229). Moreover, only F-C microbicides have been shown to denature endotoxins. A major advantage of F-C microbicides is that they are compatible with a great variety of MWF formulations (>160 of 200 tested formulations). No other active substance has demonstrated efficacy and chemical compatibility in nearly as many different MWFs as F-C biocides have.

The isothiazolinone biocides include bactericidal, fungicidal, and broad-spectrum active substances. Some isothiazolinone chemistries are stable in MWFs; others can only be used as tankside additives. Isothiazolinones have been implicated with contact dermatitis (230) and are known to be skin sensitizers. In addition, they exhibit an unusually high acute aquatic toxicity (231). Other chemical classes include pyrithiones (most commonly the sodium salt of a molecule with the chemical formula C₅H₅NOS, a six-atom ring structure) and phenolics (six-atom aromatic ring with one or more hydroxyl groups attached).

Efficacy of biocides depends on the concentration and on the formulation: emulsifiers, sequestrates, and other compounds have an influence on the efficacy for good or bad (232). As fear of health risks increases and regulations on the use of biocides are tightened, it will also become increasingly difficult to use effective concentrations in MWFs. Passman et al. (233) have demonstrated that underdosing with microbicides can exacerbate microbial contamination in MWFs. Sublethal doses of biocides are likely to select for resistant strains rather than having the desired effect (234). This phenomenon has also been well documented for nonlethal concentrations of antibiotics (235, 236). One major, often underappreciated challenge to formulating MWFs with microbicides is the ranges of biocide concentrations likely to be present in the end-use diluted MWF. Most water-miscible MWFs are used at end-use concentrations ranging from 3 to 10% (v/v), depending on the application. If an MWF is formulated to deliver the maximum permissible concentration when an MWF is used at 10%, then it is unlikely to be present at an effective concentration when that same MWF is used at 3%. Conversely, when the microbicide is formulated to deliver the maximum permissible concentration when the MWF is used at 3%, it is quite likely to exceed the maximum permissible concentration when that MWF is used at 10%. Many formulators assume that on average their products will be used at 5% (v/v). Consequently, depending on the actual MWF end-use dilution, microbicides formulated into the concentrate are likely to be present at inappropriate concentrations in MWF systems.

Concerns have also been raised about the use of biocides as a contributing factor to the development of antimicrobial resistance in humans (221, 237). Thus, effective usage of biocides in concentrates will become less and less feasible. This may be different when used as tankside additive or system cleaners, where higher doses are practicable in a short and clearly defined period. Thus, manufacturers may often resort to toxic substances, which, not registered as biocides, can pose further, yet unknown health risks.

Toxic substances—from the viewpoint of the microorganisms—may also lead to more diverse populations as degrading toxic substances is facilitated in presence of multiple species (117, 238) as predicted by the stress gradient hypothesis (239).

As previously stated, microorganisms usually grow in dense, multicellular communities called biofilms that offer extensive protection from attack by antimicrobials: Microorganisms in biofilms are considered up to a thousand times more resistant, thus surviving most onslaughts (70–74). As a consequence, biocides and other toxic substances in MWFs are not able to diminish biofilms, repress their formation, or achieve their removal. On the contrary, they might select for persister cells (240).

Biocides can still be very effective when used per appropriate guidelines and in combination with effective hygiene practices and frequent monitoring.

Phages

The use of phages in MWFs as a tool to prevent or limit bacterial contamination is worth a discussion. Several research teams (58, 240, 241) have proposed using phages as alternatives to antibiotics and biocides against planktonic organisms and biofilms. To date, several limitations have impeded the adoption of phase treatment for microbial contamination control in MWFs. First, the density of planktonic cells in these fluids is rarely sufficient to ensure efficient transmission from one host to the other. Phages are not mobile. Consequently, they rely on cell-to-cell contact for transmission from one host cell to another. In laboratory studies, phages are used against populations in the 10⁹ cells mL⁻¹ range. This is a 100–1,000 times the population density in even heavily contaminated MWF. The distance between planktonic cells can be too great to sustain a phage infection. Second, phages are host specific. This means that in order to achieve effective control, the phage cocktail would have to include phages effective against all of the microbial taxa present in the MWF. If only some taxa are killed, those that remain could potentially be more biodeteriogenic than the original population was. Given that the taxonomic profile of bacteria in MWFs is almost system unique (and often evolving within any individual MWF system), a given phage cocktail is unlikely to be effective for long. Third, many phage infections are lysogenic. Rather than producing a new phage and then lysing, infected bacteria integrate phage DNA into the host cell genome. This can change the host’s physiology but not kill the cell. As a bonus (for the bacteria), lysogenically infected cells are immune to subsequent phage attacks. Fourth, both bacteria and phages mutate at a rate of one successful mutation per billion cells or phages. Finally, bacteria have several efficient defense mechanisms that enable portions of the target population to become immune, further limiting the use of phages in MWFs.

Bio-concept

Bio-concept products do not contain listed bactericides and are designed, through their constituents, to become populated by a substantial number of waterborne bacteria, mainly Pseudomonas oleovorans. Generally, more than 50 different genera have been detected in MWFs in the planktonic phase, but bio-concept fluids exert a selection pressure chiefly allowing the growth of this bacterial species in the range of 10³ to 10⁸ CFU mL⁻¹ (34). Additionally, bio-concept fluids are less toxic to microorganisms, which may lead to competition among diverse species, favoring single-taxon populations (117). P. oleovorans is globally ubiquitous and primarily originates from the water source used.

What exactly exerts the selection pressure is unknown, and diverse biologically active molecules have been suspected to be indispensable, such as low concentrations of fungicides that may confer a selective advantage to P. oleovorans. Similar effects have been shown in other settings by using sublethal doses of antibiotics (242). Pseudomonas spp. have also been reported to produce antibiotics that attack other microorganisms (243–248), which remains a further possibility, as well as other (volatile) compounds such as long-chain alkyl cyanides (249). Another option is the presence of fatty acids in some MWFs that may have a role in signaling, which could lead to selection for Pseudomonas spp. (250). On the other hand, P. oleovorans may simply be most efficient at consuming organic substances that either originate from the water source or are introduced into the coolant, thereby effectively preventing the establishment of other bacterial species in turn; a comparable behavior has already been shown for bacteria on plant leaves (251).

As a positive side effect, large planktonic populations of P. oleovorans might weaken biofilm populations by forming transient pores that help to either prevent biofilm establishment or (partially) dissolute them by providing access of high-pH MWFs into the biofilms themselves. Such behavior was shown for bacilli and other flagellated bacteria in early stage and mature biofilms (252).

Another positive aspect may be the transformation of nitrate and nitrite to di-nitrogen gas by Pseudomonas spp. (253) and thus removal of precursors of the potentially carcinogenic N-nitrosamines.

Conclusions

Recent innovations in water-miscible MWF formulations have resulted in improved performance and performance life. However, regulatory pressures affecting the availability of active substances (microbicides) that have been used traditionally to control microbial contamination in MWFs are increasing the contamination control challenge. The dynamics of water-miscible MWF use are conducive to microbial growth. Formulation chemistry provides all of the required organic nutrients. Makeup water—whose content usually varies between 85 and 98% in the end-use diluted MWF—provides inorganic nutrients and a suitable growth habitat. Turbulent flow created by MWF recirculation at velocities of ≥0.8 m³ min⁻¹ (≥200 gpm) creates an oxygenated environment for aerobic microbes.

If left uncontrolled, microbial contaminants can cause both direct (using MWF chemistry as food) and indirect (producing metabolites that react with MWF components) MWF biodeterioration. Moreover, microbes in MWFs and biofilms that have developed on system surfaces act as bioaerosol reservoirs. Bioaerosol inhalation has been linked to a range of allergenic respiratory diseases, and prolonged contact with antigenic metabolites in MWF has been associated with dermatitis. Consequently, effective microbial contamination control is important in terms of both MWF performance and worker health and safety.

As regulatory pressures impact microbicide use, alternative microbial control strategies are being developed. Improved engineering controls have reduced worker exposure risks. Increased use of biostable additives and adjuvants has contributed to MWF bioresistance and decreased microbicide demand. Formulation of bio-concept MWFs that select for non-biodeteriogenic microbes have also been proven as an effective means of preventing MWF biodeterioration and reducing worker health risks.

Recent developments in microbiology test methods are providing tools for better understanding the microbial ecology of MWFs. As these tools gain broader use, it is likely that innovative microbial contamination control strategies will be developed. The marriage of industrial hygiene, non-culture-based microbiology testing, and MWF formulation should result in improved MWF stability and decreased allergenic disease risk among machinists and other workers in the metalworking environment.