Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility

The metalworking and machining industry utilizes recirculating metalworking fluids for integral aspects of the fabrication process. Despite the use of biocides, these fluids sustain substantial biological growth. Subsequently, the high-shear forces incurred during metalworking processing aerosolize bacterial cells and may cause dermatologic and respiratory effects in exposed workers. We quantified and identified the bacterial load for metalworking fluid and aerosol samples of a machining facility in the US Midwest during two seasons. To investigate the presence of potentially pathogenic bacteria in fluid and air, we performed 16S rRNA gene surveys. The concentration of total bacterial cells (including culturable and nonculturable cells) was relatively constant throughout the study, averaging 5.1 × 10? cells mL?1 in the fluids and 4.8 × 10? cells m?3 in the aerosols. We observed bacteria of potential epidemiologic significance from several different bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was identified through both direct sequencing and culturing in every sample collected. Elucidating the bacterial community with gene surveys showed that metalworking fluids were the source of the aerosolized bacteria in this facility.     

Use of MTT assay for determination of the biofilm formation capacity of microorganisms in metalworking fluids

Biofilm formation is a well-known problem in management of metalworking fluid systems. Due to persistence of microorganisms within biofilms, the reappearance of various species of bacteria, including nontuberculous mycobacteria is often observed after the use of biocides and/or cleaning of delivery systems and replacement of cooling fluid. The aim of this study was to determine the usefulness of the tetrazolium salt assay (MTT assay) for assessing the viability of bacteria in biofilms formed in vitro in fresh and used cutting oils, as well as their susceptibility to antimicrobial biocides. Biofilms were established in the microtiter plate format. The results showed that quantification of formazan, a product of the tetrazolium salt reduction by electron transport system could be used for determination of the propensity of bacteria to form biofilms in these complex media. The use of the assay allows also determination of antimicrobial activity of biocides against biofilms in fresh and used metalworking fluids. Biofilms produced by Gram-negative isolates recovered from field metalworking fluids as well as the wild bacterial communities differed in metabolic activity depending on the type of fresh coolants. The MTT assay has high-throughput potential and can be efficiently used for determination of biofilm-forming capacity of microorganisms from individual machines in metalworking industry. The use of the assay may also guide the selection of the most appropriate biocide to fight these microorganisms.     

Land-use patterns and cultural change in the Middle to Late Bronze Age in Ireland: inferences from pollen records

In Ireland, the Middle to Late Bronze Age (1500–600 cal b.c.) is characterised by alternating phases of prolific metalwork production (the Bishopsland and Dowris Phases) and apparent recessions (the Roscommon Phase and the Late Bronze Age-Iron Age transition). In this paper, these changes in material culture are placed in a socio-economic context by examining contemporary settlement and land-use patterns reconstructed from the pollen record. The vegetation histories of six tephrochronologically linked sites are presented, which provide high-resolution and chronologically well-resolved insights into changes in landscape use over the Middle to Late Bronze Age. The records are compared with published pollen records in an attempt to discern if there are trends in woodland clearance and abandonment from which changes in settlement patterns can be inferred. The results suggest that prolific metalworking industries correlate chronologically with expansion of farming activity, which indicates that they were supported by a productive subsistence economy. Conversely, declines in metalwork production occur during periods when farming activity was generally less extensive and perhaps more centralised, and it is proposed that disparate socio-economic or political factors, rather than a collapse of the subsistence economy, lie behind the demise of metalworking industries.     

Modification of a French pressure cell to improve microbial cell disruption

A procedure for modification of the valve stem of a 40 K French pressure cell is described. The modification should be done by a machinist and requires a metalworking lathe. After modification of the valve stem, a torlon 4203 plastic ball is used between the valve stem and valve seat to control the pressure within the cell. The torlon plastic ball is a key component needed to obtain the high pressures required for efficient disruption of microbial cells.     

The degradation of sodium O,O-diethyl dithiophosphate by bacteria from metalworking fluids

The breakdown of sodium O,O-diethyl dithiophosphate (O,O-diethyl phosphorodithioate) by four bacterial strains (tentatively identified as strains of Aeromonas, Pseudomonas, Flavobacterium and Bacillus) isolated from contaminated metalworking fluids was shown to involve the successive formation of ethanol, aldehyde and orthophosphate. An acid phosphodiesterase was identified in cell-free extracts that was five- to sevenfold enhanced in specific activity in bacteria grown on O,O-diethyl dithiophosphate as sole phosphorus source, compared with bacteria grown on orthophosphate. This is thought to initiate the breakdown process.     

The potentiation of industrial biocide activity with Cu2+. I. Synergistic effect of Cu2+ with formaldehyde

Earlier studies suggested that copper enhances the antimicrobial activity of some formaldehyde (FA)-condensate biocides in metalworking fluids as well as FA in laboratory media. The possible synergistic interaction between FA and Cu²⁺ in combination were tested against Pseudomonas aeruginosa in trypic soy broth, mineral salt base-glucose medium, and 0·9% NaCl solution. In all cases, Cu²⁺ enhanced the FA bactericidal activity. A sequential treatment of bacterial cultures was employed to study the increased effectiveness of the Cu²⁺ and FA combination. The cells were exposed to FA or Cu²⁺ and subsequently exposed to the alternate compound with centrifugation and washing between exposures. Results varied depending on the medium. Synergistic activity of FA and Cu²⁺ was established based on the interpretation of the results.     

Biocidal Activity of Formaldehyde and Nonformaldehyde Biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in Pure and Mixed Suspensions in Synthetic Metalworking Fluid and Saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

The potentiation of industrial biocide activity with Cu2+. II. Synergistic effects with 5-chloro-2-methyl-4-isothiazolin-3-one

The role of Cu²⁺ in enhancing 5-chloro-2-methyl-4-isothiazolin-3-one (IT) activity was investigated. This study was carried out in two directions. Firstly, the level of enhanced activity from a Cu²⁺ and IT mixture where environmental destruction of IT is minimal was examined. Secondly, the level of protection from Cu²⁺ in environments known to irreversibly reduce IT activity was studied. The bactericidal activities were determined in tryptic soy broth medium, mineral salt base-glucose medium and 0·9 NaCl solution. Under certain conditions, Cu²⁺ also stabilizes and/or protects the sensitive chlorinated IT molecule. Both synergistic activity and stabilization or protection by Cu²⁺ enhance the antimicrobial activity of IT under test conditions. Alternative sequential treatment with IT and Cu²⁺ was used to further characterize enhanced activity. The results suggest synergism. The utility of all the findings was investigated in metalworking fluid.     

Changes in Pyrenean woodlands as a result of the intensity of human exploitation: 2,000 years of metallurgy in Vallferrera, northeast Iberian Peninsula

This research is based on the discovery of a large number of charcoal kiln sites and abandoned iron mines in Vallferrera (Axial Pyrenees, northeastern Spain). The study reveals that this region has been affected by the metal mining and smelting industry for at least 2,000 years, with maximum intensity in the eighteenth and nineteenth centuries, followed by abandonment of the activity. The region’s woodland dendrochronology and historical records indicate that exploitation of wood charcoal for metalworking affected the past vegetation in the area and impeded the development of mature woodland. Our findings suggest that the greatest changes in vegetation and landscape history occurred at times of particular specialization in socioeconomic activities.

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.

     

Does the Emperor Have Any Clothes: Using Mechanistic Information or Doing Houdini Risk Assessments?

Risk assessment research rarely quells controversy. Mega-mouse, and mega-rat, experiments contradicted a threshold for carcinogenesis, yet thresholds are still argued. High to low dose continuity of response from cigarette smoking to environmental tobacco smoke, and from occupational asbestos exposure to take-home asbestos, contradict thresholds in people. Nevertheless, mechanistic hypotheses allege “Houdini Risk Assessments”, which make risks disappear or allow industries to escape from protecting workers. Despite concerns for animal-to-human extrapolations, priority occupational exposures with sufficient or substantial evidence of carcinogenicity in people not addressed by new exposure limits include silica, sulfuric acid mist, chromates, diesel particulate matter, particulate matter generally, metalworking fluids, welding fume, and formaldehyde. “Houdini Risk Assessments” are exercises in “anti-hypothesis generation”: ignore selected tumor sites and types; ignore data from people (as with formaldehyde and diesel); choose the most resistant species in laboratory tests; select biochemical parameters in which the most resistant species resembles people; assume a mechanism that gives threshold or steep exposure response for carcinogenic effect; and reduce estimated people risk by the parameter ratio to the most resistant species. NORA research should focus on quantitative reconciliation of laboratory and epidemiology studies, and develop a counter “anti-hypothesis” generation research agenda for key exposure circumstances.     

Influence of a triazine derivative-based biocide on microbial biofilms of cutting fluids in contact with different substrates

Although biofilms are often associated with hospital infection problems owing to their high resistance to antimicrobial agents, in recent years biofilms have also been studied in the industrial sector, mainly because they are a major cause of contamination outbreaks in facilities and products. The aim of this study was to investigate whether different materials commonly found in the metalworking industries have different biofilm formation characteristics when in contact with contaminated cutting fluid as well as to establish an optimal concentration of a triazine-based antimicrobial agent to protect the oil/water emulsion and also to delay or interrupt the development of biofilms. Biofilms grown on the surface of carbon steel, stainless steel, aluminum, polyvinyl chloride, and glass were analyzed in terms of cell growth and susceptibility to the tested biocide. The results showed that the type of material used had little influence on cell adhesion or on the microbicide concentration required to control and eradicate microorganisms suspended in the emulsion and in the biofilms.     

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.     

Harmonisation of chemical and biological process in development of a hybrid technology for treatment of recalcitrant metalworking fluid

Disposal of operationally exhausted metalworking fluids (MWFs) is enormously challenging. In this study the feasibility of employing a sequential Fenton-biological oxidation for the treatment of recalcitrant components of MWF wastewater was investigated. A statistical experimental design was employed to address Fenton reagent (H?O?, Fe2?) dose optimisation which ensured minimal concentrations of the reagents, thus making the treatment environmentally less toxic to subsequent biological steps and economically viable. This was achieved by employing a five-level-two-variable central composite experimental design. The results demonstrated that Fenton pre-treatment of the MWF effluent greatly improved biodegradability index (BOD?)/COD increased from 0.160 to 0.538) with a synchronous lowering in the toxicity of the wastewater, making the recalcitrant component more amenable to subsequent biological treatment. An overall decrease of 92% and 86% in chemical oxygen demand (COD) and total organic carbon (TOC), respectively, was achieved by the two-step treatment method developed.     

Influence of biocide treatment regimen on resistance development to methylchloro-/methylisothiazolone in Pseudomonas aeruginosa

Resistance development among microbial populations exposed to industrial biocides intending to control microbial levels has received increasing attention in the last few years. Usually studies dealing with resistance development are done after the process has taken place. Characterization of resistant organisms by isolation of dominant populations reveals some details, but the steps leading to resistance usually remain unclear. In this study, development of resistance to methylchloro-/methylisothiazolone (IT) biocide under laboratory conditions is described. Results with experimental setups relevant to field dosing conditions demonstrated how the pattern of biocide treatment influenced the degree of resistance development. The induction of higher resistance in the laboratory populations exposed to different dosing patterns varied. Sequential treatment of culture with a constant increase in concentrations of isothiazolone (5–10 μl l⁻¹ of commercial product) resulted in increasing resistance, exceeding ten-fold. However, additional increases of 25–50 μl l⁻¹ in each step were lethal, suggesting threshold levels of resistance in populations tested. Extrapolation of laboratory data to field conditions appeared more relevant after Pseudomonas species, highly resistant to IT, were isolated from metalworking fluids. In these fluids the biocide treatment regimen in the field was similar to the one used in the laboratory. An understanding of the factors contributing to resistance development and selection in the field is emphasized.     

Selection and assembly of indigenous bacteria and methanogens from spent metalworking fluids and their potential as a starting culture in a fluidized bed reactor

Waste metalworking fluids (MWFs) are highly biocidal resulting in real difficulties in the, otherwise favoured, bioremediation of these high chemical oxygen deman (COD) wastes anaerobically in bioreactors. We have shown, as a proof of concept, that it is possible to establish an anaerobic starter culture using strains isolated from spent MWFs which are capable of reducing COD or, most significantly, methanogenesis in this biocidal waste stream. Bacterial strains (n = 99) and archaeal methanogens (n = 28) were isolated from spent MWFs. The most common bacterial strains were Clostridium species (n = 45). All methanogens were identified as Methanosarcina mazei. Using a random partitions design (RPD) mesocosm experiment, we found that bacterial diversity and species–species interactions had significant effects on COD reduction but that bacterial composition did not. The RPD study showed similar effects on methanogenesis, except that composition was also significant. We identified bacterial species with positive and negative effects on methane production. A consortium of 16 bacterial species and three methanogens was used to initiate a fluidized bed bioreactor (FBR), in batch mode. COD reduction and methane production were variable, and the reactor was oscillated between continuous and batch feeds. In both microcosm and FBR experiments, periodic inconsistencies in bacterial reduction in fermentative products to formic and acetic acids were identified as a key issue.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

Presence of a Single Genotype of the Newly Described Species Mycobacterium immunogenum in Industrial Metalworking Fluids Associated with Hypersensitivity Pneumonitis

Outbreaks of hypersensitivity pneumonitis (HP) among industrial metal-grinding machinists working with water-based metalworking fluids (MWF) have frequently been associated with high levels of mycobacteria in the MWF, but little is known about these organisms. We collected 107 MWF isolates of mycobacteria from multiple industrial sites where HP had been diagnosed and identified them to the species level by a molecular method (PCR restriction enzyme analysis [PRA]). Their genomic DNA restriction fragment length polymorphism (RFLP) patterns, as determined by pulsed-field gel electrophoresis (PFGE), were compared to those of 15 clinical (patient) isolates of the recently described rapidly growing mycobacterial species Mycobacterium immunogenum. A total of 102 of 107 (95%) MWF isolates (from 10 industrial sites within the United States and Canada) were identified as M. immunogenum and gave PRA patterns identical to those of the clinical isolates. Using genomic DNA, PFGE was performed on 80 of these isolates. According to RFLP analysis using the restriction enzymes DraI and XbaI, 78 of 80 (98%) of the MWF isolates represented a single clone. In contrast, none of the 15 clinical isolates had genetic patterns the same as or closely related to those of any of the others. Given the genomic heterogeneity of clinical isolates of M. immunogenum, the finding that a single genotype was present at all industrial sites is remarkable. This suggests that this genotype possesses unusual features that may relate to its virulence and its potential etiologic role in HP and/or to its resistance to biocides frequently used in MWF.     

Differential biocide susceptibility of the multiple genotypes of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis>

The non-tuberculous mycobacterium Mycobacterium immunogenum colonizes industrial metalworking fluids (MWFs) presumably due to its relative resistance to the currently practiced biocides and has been implicated in occupational respiratory hazards, particularly hypersensitivity pneumonitis. With an aim to understand its inherent biocide susceptibility profile and survival potential in MWF, five different genotypes of this organism, including a reference genotype (700506) and four novel test genotypes (MJY-3, MJY-4, MJY-10 and MJY-12) isolated in our recent study from diverse MWF operations were evaluated. For this, two commercial biocide formulations, Grotan (Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine) and Kathon (5-chloro-2-methyl-4-isothiazolin-3-one) currently practiced for the control of microorganisms, including mycobacteria, in MWF operations were tested. Effect of the fluid matrix on the biocide susceptibility was investigated for the synthetic (S) and semi-synthetic (SS) MWF matrices. In general, the minimum inhibitory concentration values were higher for the HCHO-releasing biocide Grotan than the isothiazolone biocide Kathon. All genotypes (except the reference genotype) showed lower susceptibility in SS as compared to S fluid matrix for Grotan. However, in case of Kathon, a greater susceptibility was observed in SS fluid for majority of the test genotypes (MJY-3, 4 and 10). The test genotypes were more resistant than the reference genotype to either biocide in both fluid types. Furthermore, the individual genotypes showed differential biocidal susceptibility, with MJY-10 being the most resistant. These observations emphasize the importance of using the resistant genotypes of M. immunogenum as the test strains for formulation or development and evaluation of existing and novel biocides, for industrial applications.