Factors influencing the microbial composition of metalworking fluids and potential implications for machine operator's lung

Hypersensitivity pneumonitis, also known as "machine operator's lung" (MOL), has been related to microorganisms growing in metalworking fluids (MWFs), especially Mycobacterium immunogenum. We aimed to (i) describe the microbiological contamination of MWFs and (ii) look for chemical, physical, and environmental parameters associated with variations in microbiological profiles. We microbiologically analyzed 180 MWF samples from nonautomotive plants (e.g., screw-machining or metal-cutting plants) in the Franche-Comté region in eastern France and 165 samples from three French automotive plants in which cases of MOL had been proven. Our results revealed two types of microbial biomes: the first was from the nonautomotive industry, showed predominantly Gram-negative rods (GNR), and was associated with a low risk of MOL, and the second came from the automotive industry that was affected by cases of MOL and showed predominantly Gram-positive rods (GPR). Traces of M. immunogenum were sporadically detected in the first type, while it was highly prevalent in the automotive sector, with up to 38% of samples testing positive. The use of chromium, nickel, or iron was associated with growth of Gram-negative rods; conversely, growth of Gram-positive rods was associated with the absence of these metals. Synthetic MWFs were more frequently sterile than emulsions. Vegetable oil-based emulsions were associated with GNR, while mineral ones were associated with GPR. Our results suggest that metal types and the nature of MWF play a part in MWF contamination, and this work shall be followed by further in vitro simulation experiments on the kinetics of microbial populations, focusing on the phenomena of inhibition and synergy.     

Rapid detection of rRNA group I pseudomonads in contaminated metalworking fluids and biofilm formation by fluorescent in situ hybridization

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial degradation. Microbial communities present in MWFs lead to biofilm formation in the MWF systems, which act as a continuous source of contamination. Species of rRNA group I Pseudomonas dominate in contaminated MWFs. However, their actual distribution is typically underestimated when using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar. To overcome this, fluorescent in situ hybridization (FISH) was used to study their abundance along with biofilm formation by two species recovered from MWFs, Pseudomonas fluorescens MWF-1 and the newly described Pseudomonas oleovorans subsp. lubricantis. Based on 16S rRNA sequences, a unique fluorescent molecular probe (Pseudo120) was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas. The specificity of the probe was evaluated using hybridization experiments with whole cells of different Pseudomonas species. The probe's sensitivity was determined to be 10(3) cells/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (± 1.1) × 10(6) and 5.0 (± 2.3) × 10(6) cells/ml in four different industrial MWF samples collected from three different locations. Biofilm formation was visualized under stagnant conditions using high and low concentrations of cells for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120. On the basis of these observations, this molecular probe can be successfully be used in the management of MWF systems to monitor the levels and biofilm formation of rRNA group I pseudomonads.     

Quantitative real-time PCR detection of Pseudomonas oleovorans subsp. lubricantis using TaqMan-MGB assay in contaminated metalworking fluids

Metalworking fluids (MWFs) are highly prone to microbial contamination, which leads to their degradation and biofouling. Pseudomonas oleovorans subsp. lubricantis, a newly described subspecies, was found to be important to MWF fouling. However, the actual distribution of P. oleovorans subsp. lubricantis in MWF is difficult to study using standard culturing techniques. To overcome this, a study was conducted to design a specific quantitative real-time PCR (qPCR) assay using TaqMan^®MGB (minor groove binding) probe for its identification and estimated quantification in contaminated MWFs. The gyrB housekeeping gene sequence was selected for designing a TaqMan^® MGB primer-probe pair using the Allele ID^® 5.0 probe design software for the assay. Whole-cell qPCR was performed with MWF spiked directly with P. oleovorans subsp. lubricantis (eliminating DNA extractions using commercial kit); the primer-probe pair’s sensitivity was 10¹ colony forming units (CFU) ml⁻¹. The assay provided no amplification with other closely related Pseudomonas species found in MWFs indicating its specificity. It was successful in identifying and enumerating P. oleovorans subsp. lubricantis from several used MWFs having between 10⁴ and 10⁶ CFU ml⁻¹. The designed TaqMan^® MGB probe thus can be successfully used for the subspecies-specific identification of P. oleovorans subsp. lubricantis and facilitates the study of its impact on MWFs.     

Microbial degradation of water miscible metal working fluids

Water miscible metal working fluids (MWF) are prone to contamination by bacteria and fungi. In the present work it was investigated which components of a model MWF emulsion were most readily degraded by microorganisms and which are relatively resistant to biodegradation. The microbial community colonising an MWF emulsion, during its lifetime under in-use conditions was analysed up to species level. Shifts in the composition of microbial community were related to chemical changes in the MWF emulsion over time.     

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.     

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.     

Progress in the development of methods used for the abatement of microbial contaminants in ethanol fermentations: a review

Biofuel research and development roadmap is currently underway in several countries and is expected to pave a way for the establishment of a viable renewable energy sector that can compete with petroleum-based fuels. Ethanol fermentation has garnered increasing attention amongst various stakeholders (industries, governments, and academia) due to its economic and environmental merits. However, microbial contamination continues to be one of the major barriers in ethanologenic processes, resulting in low ethanol yields and thereby translating into economic losses. To this end, technological innovations geared towards effective elimination of microbial contamination are constantly being developed. This review explores and discusses the fermentation conditions that facilitate the growth of undesired microorganisms during ethanol fermentation processes. It highlights the methods that are currently used in biorefineries as well as innovative and advanced biotechnological methods currently being evaluated as viable alternative strategies to control or eliminate microbial contaminants in ethanol fermentations. These methods have the potential to minimize or control the contamination problem and could pave a way for the development of an efficient biofuel sector.

     

Bacterial community structure and function in a metal-working fluid

The diversity of bacterial populations colonizing spatially and temporally separated samples of the same metal-working fluid (MWF) formulation was investigated. Analyses were performed with a view to improve strategies for bioaugmentation of waste MWF in bioreactor systems and prevention of in-use MWF biodeterioration in engineering workshops. Significantly, complementary phenotypic, genotypic and in situ methods revealed that the bacterial communities in operationally exhausted MWFs had low diversity and were similar in species composition from different locations and uses. Of the 179 bacterial isolates studied, only 11 genera and 15 species were identified using fatty acid methyl ester (FAME) analysis, with culture independent analyses by 16S rDNA denaturing gradient gel electrophoresis (DGGE) and fluorescent in situ hybridization being congruent with these FAME data. In order to gain some insight into functional role of detected populations, we correlated the MWF chemical composition and potential pollution load with bacterial abundance and community composition detected within samples.     

Selection of microbial consortia for treating metal-working fluids

The aim of this research was to determine the effectiveness of a strategy for constructing microbial consortia for treating chemically mixed industrial effluent, based on a more thorough understanding of communities within waste metal-working fluids (MWFs). Complementary phenotypic and genotypic methods revealed that the microbial communities in spent MWFs had low diversity and were very similar in species composition in samples originating from different locations and uses. Of 65 bacterial isolates studied, only 9 species were identified using fatty acid methyl ester (FAME) analysis. The results of genotypic analysis by denaturing gradient gel electrophoresis (DGGE) were congruent with observations made using FAME analysis. The metabolic potential of the isolates was assessed in terms of assimilation ability and tolerance of co-contaminants. The three isolates, selected (Clavibacter michiganensis, Methylobacterium mesophilicum, and Rhodococcus erythropolis) to form a consortium, were representative of three of the four most abundant populations and when combined could utilise'or tolerate all of the individual MWF components, including the biocide and the recalcitrant compound benzotriazole. Journal of Industrial Microbiology & Biotechnology (2002) 29, 20–27 doi:10.1038/sj.jim.7000271 Received 19 December 2001/ Accepted in revised form 02 May 2002     

Activity assessment of microorganisms eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium chloride: a quantitative comparison of flow cytometry to epifluorescent microscopy

Enhanced natural recovery may be successfully implemented at contaminated sediment sites, which are often characterized by large volumes of sediments with low to moderate levels of contamination to cost-effectively reduce human and ecological risks. In order to evaluate the potential for microbial contribution to remediation strategies, physiological assessment of indigenous microorganisms is essential. We report here a method for rapid and accurate assessment of metabolically (5-cyano-2,3-ditolyl tetrazolium chloride [CTC]) active microorganisms eluted from sediment, based on flow cytometry (FCM). Microorganisms eluted from sediment and suspended in estuarine medium were stained with CTC and counterstained with the DNA stain Picogreen (PG). Optimal stain concentrations and incubation times were employed. FCM quantification of the dual-stained microorganisms was not statistically different (paired t test; alpha=0.05; df=10) from enumeration (total or active numbers) by an established method (fluorescent microscopy) over two orders of magnitude (approximately 10(4)-10(6)/ml). This research suggests that FCM, which allows the collection and analysis of multiple parameters (light scatter and fluorescence emission), is a good candidate for microbial characterization in complex environmental matrices, such as sediments, across a broad range of activity levels (approximately 2% to 84% of total). Potential applications for this FCM-based method include the rapid assessment of changes in sediment microbial activity in response to enhanced bioremediation strategies.     

Microbial Stability of Pharmaceutical and Cosmetic Products

This review gives a brief overview about microbial contamination in pharmaceutical products. We discuss the distribution and potential sources of microorganisms in different areas, ranging from manufacturing sites, pharmacy stores, hospitals, to the post-market phase. We also discuss the factors that affect microbial contamination in popular dosage forms (e.g., tablets, sterile products, cosmetics). When these products are contaminated, the microorganisms can cause changes. The effects range from mild changes (e.g., discoloration, texture alteration) to severe effects (e.g., changes in activities, toxicity). The most common method for countering microbial contamination is the use of preservatives. We review some frequently used preservatives, and we describe the mechanisms by which microorganisms develop resistance to these preservatives. Finally, because preservatives are inherently toxic, we review the efforts of researchers to utilize water activity and other non-preservative approaches to combat microbial contamination.     

An exometabolomics approach to monitoring microbial contamination in microalgal fermentation processes by using metabolic footprint analysis

The early detection of microbial contamination is crucial to avoid process failure and costly delays in fermentation industries. However, traditional detection methods such as plate counting and microscopy are labor-intensive, insensitive, and time-consuming. Modern techniques that can detect microbial contamination rapidly and cost-effectively are therefore sought. In the present study, we propose gas chromatography-mass spectrometry (GC-MS)-based metabolic footprint analysis as a rapid and reliable method for the detection of microbial contamination in fermentation processes. Our metabolic footprint analysis detected statistically significant differences in metabolite profiles of axenic and contaminated batch cultures of microalgae as early as 3 h after contamination was introduced, while classical detection methods could detect contamination only after 24 h. The data were analyzed by discriminant function analysis and were validated by leave-one-out cross-validation. We obtained a 97% success rate in correctly classifying samples coming from contaminated or axenic cultures. Therefore, metabolic footprint analysis combined with discriminant function analysis presents a rapid and cost-effective approach to monitor microbial contamination in industrial fermentation processes.     

Metalworking fluids biodiversity characterization

Aims: Hypersensitivity pneumonitis of machinists associated with metalworking fluids (MWF) was recently linked to Mycobacterium immunogenum. In addition to Mycobacterium, impacts of continuous and massive contact to other micro‐organisms, such as Pseudomonas, were little studied. This report intended to quantify and characterize the microbial load of 44 in‐use MWF. Methods and Results: The main biodiversity of MWF was assessed using cultural methods, quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE). Total bacteria concentrations ranged from undetectable to 10⁹ 16S rRNA gene copies per millilitre. Concentrations obtained by qPCR were up to five orders of magnitude higher than by culture, suggesting that MWF contamination is generally underestimated. Two samples showed high concentrations of Myco. immunogenum (1·55 × 10⁷ and 3·49 × 10⁵ 16S rRNA gene copies per millilitre). The overall biodiversity was low, as observed by culture and DGGE, and was comparable to data found in the literature. Pseudomonas pseudoalcaligenes was by far the main bacteria found in MWF samples (33 out of 44), followed by Ochrobactrum anthropi (32 out of 44). There was no significant relationship between the biodiversity profiles and the kind of MWF or equipment used, making it difficult to predict which micro‐organisms will colonize each particular MWF. Conclusions: Very high concentrations of bacteria were found in most MWF studied and limited biodiversities were observed. Many species of micro‐organisms were retrieved from MWF samples, but they were mostly colonized by Pseudomonas pseudoalcaligenes and Ochrobactrum anthropi. Significance and Impact of the Study: The major micro‐organisms observed or recovered in this study from in‐use MWF were present in very high concentrations, and thus further studies are needed to confirm their role in workers’ respiratory disorders or health‐related problems.     

Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Use of PCR analysis for detecting low levels of bacteria and mold contamination in pharmaceutical samples

PCR assays were developed and compared to standard methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples were artificially contaminated with less than 10 CFU of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus niger. Bacterial DNA was extracted from each enrichment broth by mild lysis in Tris-EDTA-Tween 20 buffer containing proteinase K while mold DNA was extracted by boiling samples in Tris-EDTA-SDS buffer for 1 h. A 10-microl aliquot of extracted DNA was added to Ready-To-Go PCR beads and specific primers for E. coli, S. aureus, and P. aeruginosa. However, 50-microl aliquots of extracted mold DNA were used for amplification of specific A. niger DNA sequences. Standard methods required 6-8 days while PCR detection of all microorganisms was completed within 27 h. Low levels of microbial contamination were detected in all raw materials and products using PCR assays. Rapid quality evaluation of pharmaceutical samples resulted in optimization of product manufacturing, quality control, and release of finished products.     

Rhizosphere microflora of plants used for the phytoremediation of bitumen-contaminated soil

The microbial communities and their degradative potential in rhizospheres of alfalfa (Medicago sativa) and reed (Phragmites australis) and in unplanted soil in response to bitumen contamination of soil were studied in pot experiments. According to the results of fluorescence microscopy, over a period of 27 months, bitumen contamination of soil reduced the total number of microorganisms more significantly (by 75%) in unplanted than in rhizosphere soil (by 42% and 7% for reed and alfalfa, respectively) and had various effects on some important physiological groups of microorganisms such as actinomycetes as well as nitrogen-fixing, nitrifying, denitrifying, ammonifying, phosphate-solubilizing, sulphur-oxidizing, cellulolytic and hydrocarbon-degrading microorganisms. The changes in the physiological structure of the microbial community under bitumen contamination were found to hinge on not merely the presence of plants but also their type. It was noted that the rhizosphere microflora of alfalfa was less inhibited by hydrocarbon pollution and had a higher degradative potential than the rhizosphere microflora of reed.     

中药饮片有害微生物负载及鉴定技术研究进展

中药饮片在种植、采收、加工和储藏等过程中均有可能受到多种微生物的污染,特别是有害微生物如致病细菌、产毒真菌等的存在会影响中药饮片的安全性。本文综述了近年来中药饮片有害微生物的污染情况,比较不同国家药典对中药饮片微生物限度和真菌毒素限量的差异,归纳当前用于不同微生物鉴定方法的适用性,以期为中药饮片质量安全研究工作提供参考。     

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.     

Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers

Biodegradation of anthropogenic pollutants in shallow aquifers is an important microbial ecosystem service which is mainly brought about by indigenous anaerobic microorganisms. For the management of contaminated sites, risk assessment and control of natural attenuation, the assessment of in situ biodegradation and the underlying microbial processes is essential. The development of novel molecular methods, “omics” approaches, and high-throughput techniques has revealed new insight into complex microbial communities and their functions in anoxic environmental systems. This review summarizes recent advances in the application of molecular methods to study anaerobic microbial communities in contaminated terrestrial subsurface ecosystems. We focus on current approaches to analyze composition, dynamics, and functional diversity of subsurface communities, to link identity to activity and metabolic function, and to identify the ecophysiological role of not yet cultured microbes and syntrophic consortia. We discuss recent molecular surveys of contaminated sites from an ecological viewpoint regarding degrader ecotypes, abiotic factors shaping anaerobic communities, and biotic interactions underpinning the importance of microbial cooperation for microbial ecosystem services such as contaminant degradation.