Deciphering biodegradable chelant-enhanced phytoremediation through microbes and nitrogen transformation in contaminated soils

Biodegradable chelant-enhanced phytoremediation offers an alternative treatment technique for metal contaminated soils, but most studies to date have addressed on phytoextraction efficiency rather than comprehensive understanding of the interactions among plant, soil microbes, and biodegradable chelants. In the present study, we investigated the impacts of biodegradable chelants, including nitrilotriacetate, S,S-ethylenediaminedisuccinic acid (EDDS), and citric acid on soil microbes, nitrogen transformation, and metal removal from contaminated soils. The EDDS addition to soil showed the strongest ability to promote the nitrogen cycling in soil, ryegrass tissue, and microbial metabolism in comparison with other chelants. Both bacterial community-level physiological profiles and soil mass specific heat rates demonstrated that soil microbial activity was inhibited after the EDDS application (between day 2 and 10), but this effect completely vanished on day 30, indicating the revitalization of microbial activity and community structure in the soil system. The results of quantitative real-time PCR revealed that the EDDS application stimulated denitrification in soil by increasing nitrite reductase genes, especially nirS. These new findings demonstrated that the nitrogen release capacity of biodegradable chelants plays an important role in accelerating nitrogen transformation, enhancing soil microbial structure and activity, and improving phytoextraction efficiency in contaminated soil.     

Plant responses to a phytomanaged urban technosol contaminated by trace elements and polycyclic aromatic hydrocarbons

Medicago sativa was cultivated at a former harbor facility near Bordeaux (France) to phytomanage a soil contaminated by trace elements (TE) and polycyclic aromatic hydrocarbons (PAH). In parallel, a biotest with Phaseolus vulgaris was carried out on potted soils from 18 sub-sites to assess their phytotoxicity. Total soil TE and PAH concentrations, TE concentrations in the soil pore water, the foliar ionome of M. sativa (at the end of the first growth season) and of Populus nigra growing in situ, the root and shoot biomass and the foliar ionome of P. vulgaris were determined. Despite high total soil TE, soluble TE concentrations were generally low, mainly due to alkaline soil pH (7.8–8.6). Shoot dry weight (DW) yield and foliar ionome of P. vulgaris did not reflect the soil contamination, but its root DW yield decreased at highest soil TE and/or PAH concentrations. Foliar ionomes of M. sativa and P. nigra growing in situ were generally similar to the ones at uncontaminated sites. M. sativa contributed to bioavailable TE stripping by shoot removal (in g ha^?1 harvest^?1): As 0.9, Cd 0.3, Cr 0.4, Cu 16.1, Ni 2.6, Pb 4, and Zn 134. After 1 year, 72 plant species were identified in the plant community across three subsets: (I) plant community developed on bare soil sowed with M. sativa; (II) plant community developed in unharvested plots dominated by grasses; and (III) plant community developed on unsowed bare soil. The shoot DW yield (in mg ha^?1 harvest^?1) varied from 1.1 (subset I) to 6.9 (subset II). For subset III, the specific richness was the lowest in plots with the highest phytotoxicity for P. vulgaris.     

Fate of antimicrobial resistance genes in response to application of poultry and swine manure in simulated manure-soil microcosms and manure-pond microcosms

This study aimed to determine the occurrence, abundance, and fate of nine important antimicrobial resistance genes (ARGs) (sul1, sul2, tetB, tetM, ermB, ermF, fexA, cfr, and Intl1) in the simulated soil and pond microcosms following poultry and swine manure application. Absolute quantitative PCR method was used to determine the gene copies. The results were modeled as a logarithmic regression (N?=?mlnt?+?b) to explore the fate of target genes. Genes sul1, Intl1, sul2, and tetM had the highest abundance following the application of the two manure types. The logarithmic regression model fitted the results well (R ² values up to 0.99). The reduction rate of all genes (except for the genes fexA and cfr) in manure-pond microcosms was faster than those in manure-soil microcosms. Importantly, sul1, intl1, sul2, and tetM had the lowest reduction rates in all the samples and the low reduction rates of tetM was the first time to be reported. These results indicated that ARG management should focus on using technologies for the ARG elimination before the manure applications rather than waiting for subsequent attenuation in soil or water, particularly the ARGs (such as sul1, intl1, sul2, and tetM investigated in this study) that had high abundance and low reduction rate in the soil and water after application of manure.     

Heavy metal and trace elements in riparian vegetation and macrophytes associated with lacustrine systems in Northern Patagonia Andean Range

Vegetation associated with lacustrine systems in Northern Patagonia was studied for heavy metal and trace element contents, regarding their elemental contribution to these aquatic ecosystems. The research focused on native species and exotic vascular plant Salix spp. potential for absorbing heavy metals and trace elements. The native species studied were riparian Amomyrtus luma, Austrocedrus chilensis, Chusquea culeou, Desfontainia fulgens, Escallonia rubra, Gaultheria mucronata, Lomatia hirsuta, Luma apiculata, Maytenus boaria, Myrceugenia exsucca, Nothofagus antarctica, Nothofagus dombeyi, Schinus patagonicus, and Weinmannia trichosperma, and macrophytes Hydrocotyle chamaemorus, Isöetes chubutiana, Galium sp., Myriophyllum quitense, Nitella sp. (algae), Potamogeton linguatus, Ranunculus sp., and Schoenoplectus californicus. Fresh leaves were analyzed as well as leaves decomposing within the aquatic bodies, collected from lakes Futalaufquen and Rivadavia (Los Alerces National Park), and lakes Moreno and Nahuel Huapi (Nahuel Huapi National Park). The elements studied were heavy metals Ag, As, Cd, Hg, and U, major elements Ca, K, and Fe, and trace elements Ba, Br, Co, Cr, Cs, Hf, Na, Rb, Se, Sr, and Zn. Geochemical tracers La and Sm were also determined to evaluate contamination of the biological tissues by geological particulate (sediment, soil, dust) and to implement concentration corrections.     

Microbial communities in pesticide-contaminated soils in Kyrgyzstan and bioremediation possibilities

In Kyrgyzstan, many former storehouses and dump sites for obsolete pesticides exist. In 2009/2010, an inventory and assessment of these sites including risks of environmental hazard has been conducted by FAO and the World Bank. Monitoring revealed high concentration of pesticides listed as persistent organic pollutants (POPs). The purpose of this research was to study the microbial structural complexes of the pesticide-contaminated soils in these dumping zones, and to search for and select microorganism’s destructors with cytochrome P450 genes for pesticide degradation. Culture-dependent and culture-independent approaches were used to determine the taxonomic composition of these bacterial communities. The universal primer set for the 16S ribosomal RNA (rRNA) gene and the specific primer set P450R were used to amplify the cytochrome P450 hydroxylase gene. In soils from Suzak A and B and soils from Balykchy dumping sites, the bacteria from the Actinobacteria phylum (Micrococcus genus) were dominant. These bacteria made up 32–47% of the indigenous local microflora; bacteria species from the Pseudomonas genus (Gammaproteobacteria phylum) made up 23% in Suzak, 12% in Balykchy soils. Bacillus species from the Firmicutes phylum were found only in Suzak soils. The 16S rRNA analyses and the specific primer set P450R have revealed bacteria with cytochrome genes which are directly involved in the degradation process of organic carbon compounds. Experiments were carried out to help select active degraders from the bacterial populations isolated and used to degrade Aldrin in laboratory. Active bacterial strains from the Pseudomonas fluorescens and Bacillus polymyxa population were selected which demonstrated high rates of degradation activity on Aldrin.     

Diversity of active microbial communities subjected to long-term exposure to chemical contaminants along a 40-year-old sediment core

In estuarine ecosystems, metallic and organic contaminants are mainly associated with fine grain sediments which settle on mudflats. Over time, the layers of sediment accumulate and are then transformed by diagenetic processes mainly controlled by microbial activity, recording the history of the estuary’s chemical contamination. In an environment of this specific type, we investigated the evolution of the chemical contamination and the structure of both total and active microbial communities, based on PhyloChip analysis of a 4.6-m core corresponding to a 40-year sedimentary record. While the archaeal abundance remained constant along the core, a decrease by one order of magnitude in the bacterial abundance was observed with depth. Both total and active microbial communities were dominated by Proteobacteria, Actinobacteria, and Firmicutes in all sediment samples. Among Proteobacteria, alpha-Proteobacteria dominated both total (from 37 to 60 %) and metabolically active (from 19.7 to 34.6 %) communities, including the Rhizobiales, Rhodobacter, Caulobacterales, and Sphingomonadales orders. Co-inertia analysis revealed a relationship between polycyclic aromatic hydrocarbons, zinc and some polychlorobiphenyls concentrations, and the structure of total and active microbial communities in the oldest and most contaminated sediments (from 1970 to 1975), suggesting that long-term exposure to chemicals shaped the structure of the microbial community.     

Impact of metal stress on the production of secondary metabolites in <Emphasis Type="Italic">Pteris vittata</Emphasis> L. and associated rhizosphere bacterial communities

Plants adapt to metal stress by modifying their metabolism including the production of secondary metabolites in plant tissues. Such changes may impact the diversity and functions of plant associated microbial communities. Our study aimed to evaluate the influence of metals on the secondary metabolism of plants and the indirect impact on rhizosphere bacterial communities. We then compared the secondary metabolites of the hyperaccumulator Pteris vittata L. collected from a contaminated mining site to a non-contaminated site in Vietnam and identified the discriminant metabolites. Our data showed a significant increase in chlorogenic acid derivatives and A-type procyanidin in plant roots at the contaminated site. We hypothesized that the intensive production of these compounds could be part of the antioxidant defense mechanism in response to metals. In parallel, the structure and diversity of bulk soil and rhizosphere communities was studied using high-throughput sequencing. The results showed strong differences in bacterial composition, characterized by the dominance of Proteobacteria and Nitrospira in the contaminated bulk soil, and the enrichment of some potential human pathogens, i.e., Acinetobacter, Mycobacterium, and Cupriavidus in P. vittata’s rhizosphere at the mining site. Overall, metal pollution modified the production of P. vittata secondary metabolites and altered the diversity and structure of bacterial communities. Further investigations are needed to understand whether the plant recruits specific bacteria to adapt to metal stress.     

Phytoextraction of rare earth elements in herbaceous plant species growing close to roads

The aim of study was to determine the phytoextraction of rare earth elements (REEs) to roots, stems and leaves of five herbaceous plant species (Achillea millefolium L., Artemisia vulgaris L., Papaver rhoeas L., Taraxacum officinale and Tripleurospermum inodorum), growing in four areas located in close proximity to a road with varied traffic intensity. Additionally, the relationship between road traffic intensity, REE concentration in soil and the content of these elements in plant organs was estimated. A. vulgaris and P. rhoeas were able to effectively transport REEs in their leaves, independently of area collection. The highest content of REEs was observed in P. rhoeas leaves and T. inodorum roots. Generally, HREEs were accumulated in P. rhoeas roots and leaves and also in the stems of T. inodorum and T. officinale, whereas LREEs were accumulated in T. inodorum roots and T. officinale stems. It is worth underlining that there was a clear relationship between road traffic intensity and REE, HREE and LREE concentration in soil. No positive correlation was found between the concentration of these elements in soil and their content in plants, with the exception of T. officinale. An effective transport of REEs from the root system to leaves was observed, what points to the possible ability of some of the tested plant species to remove REEs from soils near roads.     

Effect of phosphorus addition on the reductive transformation of pentachlorophenol (PCP) and iron reduction with microorganism involvement

The transformation of phosphorus added to the soil environment has been proven to be influenced by the Fe biochemical process, which thereby may affect the transformation of organic chlorinated contaminants. However, the amount of related literatures regarding this topic is limited. This study aimed to determine the effects of phosphorus addition on pentachlorophenol (PCP) anaerobic transformation, iron reduction, and paddy soil microbial community structure. Results showed that the transformation of phosphorus, iron, and PCP were closely related to the microorganisms. Moreover, phosphorus addition significantly influenced PCP transformation and iron reduction, which promoted and inhibited these processes at low and high concentrations, respectively. Both the maximum reaction rate of PCP transformation and the maximum Fe(II) amount produced were obtained at 1 mmol/L phosphorus concentration. Among the various phosphorus species, dissolved P and NaOH-P considerably changed, whereas only slight changes were observed for the remaining phosphorus species. Microbial community structure analysis demonstrated that adding low concentration of phosphorus promoted the growth of Clostridium bowmanii, Clostridium hungatei, and Clostridium intestinale and Pseudomonas veronii. By contrast, high-concentration phosphorus inhibited growth of these microorganisms, similar to the curves of PCP transformation and iron reduction. These observations indicated that Clostridium and P. veronii, especially Clostridium, played a vital role in the transformation of related substances in the system. All these findings may serve as a reference for the complicated reactions among the multiple components of soils.     

<Emphasis Type="Italic">Ailanthus Altissima</Emphasis> and <Emphasis Type="Italic">Phragmites Australis</Emphasis> for chromium removal from a contaminated soil

The comparative effectiveness for hexavalent chromium removal from irrigation water, using two selected plant species (Phragmites australis and Ailanthus altissima) planted in soil contaminated with hexavalent chromium, has been studied in the present work. Total chromium removal from water was ranging from 55 % (Phragmites) to 61 % (Ailanthus). After 360 days, the contaminated soil dropped from 70 (initial) to 36 and 41 mg Cr/kg (dry soil), for Phragmites and Ailanthus, respectively. Phragmites accumulated the highest amount of chromium in the roots (1910 mg Cr/kg_{(dry tissue)}), compared with 358 mg Cr/kg_{(dry tissue)} for Ailanthus roots. Most of chromium was found in trivalent form in all plant tissues. Ailanthus had the lowest affinity for Cr^VI reduction in the root tissues. Phragmites indicated the highest chromium translocation potential, from roots to stems. Both plant species showed good potentialities to be used in phytoremediation installations for chromium removal.     

Accumulation of Cr,Cd, Pb,Cu, and Zn by plants in tanning sludge storage sites: opportunities for contamination bioindication and phytoremediation

Tanning sludge enriched with high concentrations of Cr and other metals has adverse effects on the environment. Plants growing in the metalliferous soils may have the ability to cope with high metal concentrations. This study focuses on potentials of using native plants for bioindication and/or phytoremediation of Cr-contaminated sites. In the study, we characterized plants and soils from six tanning sludge storage sites. Soil in these sites exhibited toxic levels of Cr (averaged 16,492 mg kg^?1) and other metals (e.g., 48.3 mg Cu kg^?1, 2370 mg Zn kg^?1, 44.9 mg Pb kg^?1, and 0.59 mg Cd kg^?1). Different metal tolerance and accumulation patterns were observed among the sampled plant species. Phragmites australis, Zephyranthes candida, Cynodon dactylon, and Alternanthera philoxeroides accumulated moderate-high concentrations of Cr and other metals, which could make them good bioindicators of heavy metal pollution. High Cr and other metal concentrations (e.g., Cd and Pb) were found in Chenopodium rubrum (372 mg Cr kg^?1), Aster subulatus (310 mg Cr kg^?1), and Brassica chinensis (300 mg Cr kg^?1), being considered as metal accumulators. In addition, Nerium indicum and Z. candida were able to tolerate high concentrations of Cr and other metals, and they may be used as preferable pioneer species to grow or use for restoration in Cr-contaminated sites. This study can be useful for establishing guidelines to select the most suitable plant species to revegetate and remediate metals in tanning sludge-contaminated fields.     

Endogenous <Emphasis Type="Italic">trans</Emphasis>-zeatin content in plants with different metal-accumulating ability: a field survey

A field survey was conducted to evaluate soil metal pollution and endogenous trans-zeatin content in the leaves of plants growing at six sites in a metal-polluted area located in Gejiu, Yunnan, China. Five plant species were collected, and the physicochemical properties and concentrations of five metals in the soil were analyzed. The trans-zeatin content in plant leaves was measured by high-performance liquid chromatography. Based on the Nemerow pollution index, the six sites were classified into four levels of pollution (i.e., low, medium, high, and severely high). The degree of soil metal pollution was cadmium (Cd) > arsenic (As) > lead (Pb) > zinc (Zn) > copper (Cu). The leaf trans-zeatin content in Pteris vittata (an arsenic hyperaccumulator) increased significantly by 98.6 % in soil with a severely high level of pollution compared with soil at a low level of pollution. However, in non-hyperaccumulators Bidens pilosa var. radiata and Ageratina adenophora, a significant decrease in leaf trans-zeatin content of 35.6 and 87.6 %, respectively, was observed. The leaf trans-zeatin content in Artemisia argyi also decreased significantly by 73.6 % in high metal-polluted soil compared with that in medium metal-polluted soil. Furthermore, significant correlations were observed between leaf trans-zeatin content in Pteris vittata and As, Pb, and Cd concentrations in the soil; however, either no correlation or a negative one was observed in the other plant species. Therefore, a high content of trans-zeatin in the leaves of Pteris vittata may play an important role in its normal growth and tolerance to metals.     

Enhanced UV-B and Elevated CO<Subscript>2</Subscript> Impacts Sub-Arctic Shrub Berry Abundance,Quality and Seed Germination

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO₂ on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO₂ stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO₂ increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B × CO₂ interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B × elevated CO₂ on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.     

Illumina sequencing-based analysis of a microbial community enriched under anaerobic methane oxidation condition coupled to denitrification revealed coexistence of aerobic and anaerobic methanotrophs

Methane is produced in anaerobic environments, such as reactors used to treat wastewaters, and can be consumed by methanotrophs. The composition and structure of a microbial community enriched from anaerobic sewage sludge under methane-oxidation condition coupled to denitrification were investigated. Denaturing gradient gel electrophoresis (DGGE) analysis retrieved sequences of Methylocaldum and Chloroflexi. Deep sequencing analysis revealed a complex community that changed over time and was affected by methane concentration. Methylocaldum (8.2%), Methylosinus (2.3%), Methylomonas (0.02%), Methylacidiphilales (0.45%), Nitrospirales (0.18%), and Methanosarcinales (0.3%) were detected. Despite denitrifying conditions provided, Nitrospirales and Methanosarcinales, known to perform anaerobic methane oxidation coupled to denitrification (DAMO) process, were in very low abundance. Results demonstrated that aerobic and anaerobic methanotrophs coexisted in the reactor together with heterotrophic microorganisms, suggesting that a diverse microbial community was important to sustain methanotrophic activity. The methanogenic sludge was a good inoculum to enrich methanotrophs, and cultivation conditions play a selective role in determining community composition.     

Biostimulation of the autochthonous microbial community for the depletion of polychlorinated biphenyls (PCBs) in contaminated sediments

In this study, the effect of the biostimulation of the autochthonous microbial community on the depletion of polychlorinated biphenyls (PCBs) in historically contaminated sediments (6.260?±?9.3 10^?3?μg PCB/ g dry weight) has been observed. Biostimulation consisted of (1) the amendment of an electron donor to favor the dehalogenation of the high-chlorinated PCBs and (2) the vegetation of sediments with Sparganium sp. plants to promote the oxidation of the low-chlorinated PCBs by rhizodegradation. The effects of the treatments have been analyzed in terms of both PCB depletion and changes of the autochthonous bacterial community structure. The relative abundance of selected bacterial groups with reference to untreated sediments has been evaluated by quantitative real-time PCR. The amendment of acetate determined the enrichment of anaerobic dechlorinators like Dehalococcoides sp. Vegetation with Sparganium sp. plants determined the enrichment of either (3) the dechlorinators, Dehalococcoides and the Chloroflexi o-17/DF-1 strains or (4) the Acidobacteria, β-Proteobacteria, Actinobacteria, α-Proteobacteria, Bacteroidetes, and Firmicutes. The combination of the two biostimulation strategy determined the 91.5 % of abatement of the initial PCB content.     

Modeling the dynamics of DDT in a remote tropical floodplain: indications of post-ban use?

Significant knowledge gaps exist regarding the fate and transport of persistent organic pollutants like dichlorodiphenyltrichloroethane (DDT) in tropical environments. In Brazil, indoor residual spraying with DDT to combat malaria and leishmaniasis began in the 1950s and was banned in 1998. Nonetheless, high concentrations of DDT and its metabolites were recently detected in human breast milk in the community of Lake Puruzinho in the Brazilian Amazon. In this work, we couple analysis of soils and sediments from 2005 to 2014 at Puruzinho with a novel dynamic floodplain model to investigate the movement and distribution of DDT and its transformation products (dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD)) and implications for human exposure. The model results are in good agreement with the accumulation pattern observed in the measurements, in which DDT, DDE, and DDD (collectively, DDX) accumulate primarily in upland soils and sediments. However, a significant increase was observed in DDX concentrations in soil samples from 2005 to 2014, coupled with a decrease of DDT/DDE ratios, which do not agree with model results assuming a post-ban regime. These observations strongly suggest recent use. We used the model to investigate possible re-emissions after the ban through two scenarios: one assuming DDT use for IRS and the other assuming use against termites and leishmaniasis. Median DDX concentrations and p,p′-DDT/p,p′-DDE ratios from both of these scenarios agreed with measurements in soils, suggesting that the soil parameterization in our model was appropriate. Measured DDX concentrations in sediments were between the two re-emission scenarios. Therefore, both soil and sediment comparisons suggest re-emissions indeed occurred between 2005 and 2014, but additional measurements would be needed to better understand the actual re-emission patterns. Monte Carlo analysis revealed model predictions for sediments were very sensitive to highly uncertain parameters associated with DDT degradation and partitioning. With this model as a tool for understanding inter-media cycling, additional research to refine these parameters would improve our understanding of DDX fate and transport in tropical sediments.     

Saprotrophic soil fungi to improve phosphorus solubilisation and release: In vitro abilities of several species

Modern agriculture is dependent on phosphate rock (PR), which is a nonrenewable resource. Improvement of phosphorus (P) availability for crops in agricultural soils represents a key strategy to slow down the depletion of PR. The aim of this study was to identify potential P biofertilisers among saprotrophic fungal species. We tested 30 fungal strains belonging to 28 taxa (4 Zygomycota and 24 Ascomycota) and with different life strategies. The study showed that many saprotrophic fungi have the ability to mobilise P from insoluble forms according to a variety of mechanisms. Our results expand the pool of P solubilising fungal species, also suggesting a new solubilisation index and shedding light on parameters that could be basic in the selection of efficient soil P-biofertilisers fungi. Rhizopus stolonifer var. stolonifer, Aspergillus niger and Alternaria alternata were found to be the best performing strains in terms of amounts of TCP solubilisation.     

Evaluation of bioaugmentation and biostimulation on arsenic remediation in soil through biovolatilization

Arsenic (As) removal through microbially driven biovolatilization can be explored as a potential method for As bioremediation. However, its effectiveness needs to be improved. Biostimulation with organic matter amendment and bioaugmentation with the inoculation of genetic engineered bacteria could be potential strategies for As removal and site remediation. Here, the experiments were conducted to evaluate the impacts of rice straw and biochar amendment, inoculation of genetic engineered Pseudomonas putida KT2440 (GE P. putida) with high As volatilization activity, on microbial mediated As volatilization and removal from three different arseniferous soils. In general, the addition of rice straw (5%) significantly enhanced As methylation and volatilization in comparison with corresponding non-amended soils. Biochar amendments and inoculation of the GE P. putida increased As methylation and volatilization, respectively, but less than that of rice straw addition. The effectiveness of As volatilizations are quite different in the various paddy soils. The combined amendments of rice straw and GE P. putida exhibited the highest As removal efficiency (483.2 μg/kg/year) in Dayu soil, with 1.2% volatilization of the total As annually. The highest water-soluble As concentration (0.73 mg/kg) in this soil could be responsible for highest As volatilization besides the rice straw and bacteria in this soil.     

Molecular characterization of microbial communities and quantification of <Emphasis Type="Italic">Mycobacterium immunogenum</Emphasis> in metal removal fluids and their associated biofilms

A number of human health effects have been associated with exposure to metal removal fluids (MRFs). Multiple lines of research suggest that a newly identified organism, Mycobacterium immunogenum (MI), appears to have an etiologic role in hypersensitivity pneumonitis (HP) in case of MRFs exposed workers. However, our knowledge of this organism, other possible causative agents (e.g., Pseudomonads), and the microbial ecology of MRFs in general, is limited. In this study, culture-based methods and small subunit ribosomal RNA gene clone library approach were used to characterize microbial communities in MRF bulk fluid and associated biofilm samples collected from fluid systems in an automobile engine plant. PCR amplification data using universal primers indicate that all samples had bacterial and fungal contaminated. Five among 15 samples formed colonies on the Mycobacteria agar 7H9 suggesting the likely presence of Mycobacteria in these five samples. This observation was confirmed with PCR amplification of 16S rRNA gene fragment using Mycobacteria specific primers. Two additional samples, Biofilm-1 and Biofilm-3, were positive in PCR amplification for Mycobacteria, yet no colonies formed on the 7H9 cultivation agar plates. Real-time PCR was used to quantify the abundance of M. immunogenum in these samples, and the data showed that the copies of M. immunogenum 16S rRNA gene in the samples ranges from 4.33?×?10⁴ copy/ml to 4.61?×?10⁷ copy/ml. Clone library analysis revealed that Paecilomyces sp. and Acremonium sp. and Acremonium-like were dominant fungi in MRF samples. Various bacterial species from the major phylum of proteobacteria were found and Pseudomonas is the dominant bacterial genus in these samples. Mycobacteria (more specifically MI) were found in all biofilm samples, including biofilms collected from inside the MRF systems and from adjacent environmental surfaces, suggesting that biofilms may play an important role in microbial ecology in MRFs. Biofilms may provide a shield or sheltered microenvironment for the growth and/or colonization of Mycobacteria in MRFs.