RESUMO
Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpoda sp.), a flagellate (Cercomonas sp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixing Sinorhizobium meliloti to infectible root tips. Co-inoculation of protists plus S. meliloti resulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated with S. meliloti alone. Co-inoculation of the ciliate, Colpoda sp., with S. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix. Colpoda sp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum of S. meliloti resulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling.IMPORTANCEProtists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria, Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S. meliloti alone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.
Assuntos
Bactérias , Plantas , Rizosfera , Solo , Nitrogênio , Raízes de Plantas/microbiologia , Microbiologia do SoloRESUMO
The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.
Assuntos
Bactérias , Cilióforos , Medicago truncatula , Raízes de Plantas , Rizosfera , Bactérias/metabolismo , Medicago truncatula/microbiologia , Medicago truncatula/parasitologia , Raízes de Plantas/microbiologia , Raízes de Plantas/parasitologia , Sinorhizobium meliloti/fisiologia , Solo/parasitologia , Simbiose , Cilióforos/metabolismoRESUMO
Recent research has demonstrated that chemotactic bacteria can disperse inside microsized pores while traveling toward favorable conditions. Microbe-microbe cotransport might enable nonmotile bacteria to be carried with motile partners to enhance their dispersion and reduce their deposition in porous systems. The aim of this study was to demonstrate the enhancement in the dispersion of nonmotile bacteria (Mycobacterium gilvum VM552, a polycyclic aromatic hydrocarbon-degrader, and Sphingobium sp. D4, a hexachlorocyclohexane-degrader, through micrometer-sized pores near the exclusion-cell-size limit, in the presence of motile Pseudomonas putida G7 cells. For this purpose, we used bioreactors equipped with two chambers that were separated with membrane filters with 3, 5, and 12 µm pore sizes and capillary polydimethylsiloxane (PDMS) microarrays (20 µm × 35 µm × 2.2 mm). The cotransport of nonmotile bacteria occurred exclusively in the presence of a chemoattractant concentration gradient, and therefore, a directed flow of motile cells. This cotransport was more intense in the presence of larger pores (12 µm) and strong chemoeffectors (γ-aminobutyric acid). The mechanism that governed cotransport at the cell scale involved mechanical pushing and hydrodynamic interactions. Chemotaxis-mediated cotransport of bacterial degraders and its implications in pore accessibility opens new avenues for the enhancement of bacterial dispersion in porous media and the biodegradation of heterogeneously contaminated scenarios.
Assuntos
Hidrocarbonetos Policíclicos Aromáticos , Pseudomonas putida , Fatores Quimiotáticos/metabolismo , Quimiotaxia , Dimetilpolisiloxanos/metabolismo , Hexaclorocicloexano/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Porosidade , Pseudomonas putida/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Rapid cell identification is achieved in a compact and field-portable system employing single random phase encoding to record opto-biological signatures of living biological cells of interest. The lensless, 3D-printed system uses a diffuser to encode the complex amplitude of the sample, then the encoded signal is recorded by a CMOS image sensor for classification. Removal of lenses in this 3D sensing system removes restrictions on the field of view, numerical aperture, and depth of field normally imposed by objective lenses in comparable microscopy systems to enable robust 3D capture of biological volumes. Opto-biological signatures for two classes of animal red blood cells, situated in a microfluidic device, are captured then input into a convolutional neural network for classification, wherein the AlexNet architecture, pretrained on the ImageNet database is used as the deep learning model. Video data was recorded of the opto-biological signatures for multiple samples, then each frame was treated as an input image to the network. The pre-trained network was fine-tuned and evaluated using a dataset of over 36,000 images. The results show improved performance in comparison to a previously studied Random Forest classification model using extracted statistical features from the opto-biological signatures. The system is further compared to and outperforms a similar shearing-based 3D digital holographic microscopy system for cell classification. In addition to improvements in classification performance, the use of convolutional neural networks in this work is further demonstrated to provide improved performance in the presence of noise. Red blood cell identification as presented here, may serve as a key step toward lensless pseudorandom phase encoding applications in rapid disease screening. To the best of our knowledge this is the first report of lensless cell identification in single random phase encoding using convolutional neural networks.
Assuntos
Eritrócitos/classificação , Holografia/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Redes Neurais de Computação , Imagem Óptica/instrumentação , Animais , Bovinos , Cavalos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/instrumentação , Aprendizado de MáquinaRESUMO
Margination refers to the migration of particles toward blood vessel walls during blood flow. Understanding the mechanisms that lead to margination will aid in tailoring the attributes of drug-carrying particles for effective drug delivery. Most previous studies evaluated the margination propensity of these particles via an adhesion mechanism, i.e., by measuring the number of particles that adhered to the channel wall. Although particle adhesion and margination are related, adhesion also depends on other factors. In this study, we quantified the margination propensity of particles of varying diameters (0.53, 0.84, and 2.11 µm) and apparent wall shear rates (30, 61, and 121 s-1) by directly tracking fluorescent particles flowing through a microfluidic channel. The margination parameter, M, is defined as the total number of particles found within the cell-free layers normalized by the total number of particles that passed through the channel. In this study, an M-value of 0.2 indicated no margination, which was observed for all particle sizes in water. In the case of blood, larger particles were found to have higher M-values and thus marginated more effectively than smaller particles. The corresponding M-values at the device outlet were 0.203, 0.223, and 0.285 for 0.53-, 0.84-, and 2.11-µm particles, respectively. At the inlet, the M-values for all particle sizes in blood were <0.2, suggesting that non-fully-developed flow and constriction may lead to demargination. For particle velocities transverse to the flow direction (vy), all particle sizes showed a larger standard deviation of vy as well as a higher effective diffusivity when the particles were suspended in blood relative to water. These higher values are attributed to collisions between the blood cells and particles, further supporting recent simulation results. In terms of flow rates, for a given particle size, the higher the flow rate, the higher the M-value.
Assuntos
Hemorreologia , Microfluídica , Animais , Sangue/metabolismo , Bovinos , Corantes Fluorescentes , Dispositivos Lab-On-A-Chip , Modelos Biológicos , Tamanho da Partícula , Água/química , Água/metabolismoRESUMO
Microbial processes in the subsurface can be visualized directly using micromodels to emulate pore-scale geometries. Here, emulated soil micromodels were used to measure transport of fluorescent beads in the presence and absence of the soil ciliate Colpoda sp. under quiescent conditions. Beads alone or beads with protists were delivered to the input wells of replicate micromodels that contained three 20 mm(2) channels emulating a sandy loam microstructure. Bead abundance in microstructured channels was measured by direct counts of tiled confocal micrographs. For channels with protists, average bead abundances were approximately 320, 560, 710, 830, and 790 mm(-2) after 1, 2, 3, 5, and 10 days, respectively, versus 0, 0, 0.3, 7.8, and 45 mm(-2) without protists. Spatial and temporal patterns of bead abundance indicate that protist-facilitated transport is not a diffusive-type process but rather a function of more complex protist behaviors, including particle uptake and egestion and motility in a microstructured habitat. Protist-facilitated transport may enhance particle mixing in the soil subsurface and could someday be used for targeted delivery of nanoparticles, encapsulated chemicals, or bacteria for remediation and agriculture applications.
Assuntos
Cilióforos , Dispositivos Lab-On-A-Chip , SoloRESUMO
Bacterial biofilms are a metabolically heterogeneous community of bacteria distributed in an extracellular matrix comprised primarily of hydrated polysaccharides. Effective inhibitory concentrations measured under planktonic conditions are not applicable to biofilms, and inhibition concentrations measured for biofilms vary widely. Here, we introduce a novel microfluidic approach for screening respiration inhibition of bacteria in a biofilm array morphology. The device geometry and operating conditions allow antimicrobial concentration and flux to vary systematically and predictably with space and time. One experiment can screen biofilm respiratory responses to many different antimicrobial concentrations and dosing rates in parallel. To validate the assay, onset of respiration inhibition following NaN3 exposure is determined optically using an O2-sensing thin film. Onset of respiration inhibition obeys a clear and reproducible pattern based on time for diffusive transport of the respiration inhibitor to each biofilm in the array. This approach can be used for high-throughput screening of antimicrobial effectiveness as a function of microbial characteristics, antimicrobial properties, or antimicrobial dosing rates. The approach may also be useful in better understanding acquired antimicrobial resistance or for screening antimicrobial combinations.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Técnicas Biossensoriais/métodos , Microquímica , Respiração/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Catalase/metabolismo , Simulação por Computador , Relação Dose-Resposta a Droga , Fluorescência , Peróxido de Hidrogênio/metabolismo , Testes de Sensibilidade Microbiana , Técnicas Analíticas Microfluídicas , Oxigênio/química , Oxigênio/metabolismo , Nitrito de Sódio/farmacologiaRESUMO
Environmental structure describes physical structure that can determine heterogenous spatial distribution of biotic and abiotic (nutrients, stressors etc.) components of a microorganism's microenvironment. This study investigated the impact of micrometre-scale structure on microbial stress sensing, using yeast cells exposed to copper in microfluidic devices comprising either complex soil-like architectures or simplified environmental structures. In the soil micromodels, the responses of individual cells to inflowing medium supplemented with high copper (using cells expressing a copper-responsive pCUP1-reporter fusion) could be described neither by spatial metrics developed to quantify proximity to environmental structures and surrounding space, nor by computational modelling of fluid flow in the systems. In contrast, the proximities of cells to structures did correlate with their responses to elevated copper in microfluidic chambers that contained simplified environmental structure. Here, cells within more open spaces showed the stronger responses to the copper-supplemented inflow. These insights highlight not only the importance of structure for microbial responses to their chemical environment, but also how predictive modelling of these interactions can depend on complexity of the system, even when deploying controlled laboratory conditions and microfluidics.
RESUMO
Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (â¼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.
Assuntos
Bacillus subtilis/química , Espectrometria de Massas/métodos , Imagem Molecular/métodos , Bacillus subtilis/metabolismo , Biofilmes , Lasers , Metabolismo dos Lipídeos , Lipídeos/química , Espectrometria de Massas/instrumentação , Imagem Molecular/instrumentaçãoRESUMO
Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.
Assuntos
Biofilmes/crescimento & desenvolvimento , Optogenética/métodos , Polissacarídeos Bacterianos/biossíntese , Transdução de Sinais/efeitos da radiação , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Expressão Gênica/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz , Raízes de Plantas/microbiologia , Ribossomos/metabolismo , Microbiologia do Solo , Sphingomonadaceae/metabolismo , Simbiose/genética , Fatores de Transcrição/metabolismoRESUMO
We describe a simple and reliable fabrication method for producing multiple, manually activated microfluidic control valves in polydimethylsiloxane (PDMS) devices. These screwdriver-actuated valves reside directly on the microfluidic chip and can provide both simple on/off operation as well as graded control of fluid flow. The fabrication procedure can be easily implemented in any soft lithography lab and requires only two specialized tools-a hot-glue gun and a machined brass mold. To facilitate use in multi-valve fluidic systems, the mold is designed to produce a linear tape that contains a series of plastic rotary nodes with small stainless steel machine screws that form individual valves which can be easily separated for applications when only single valves are required. The tape and its valves are placed on the surface of a partially cured thin PDMS microchannel device while the PDMS is still on the soft-lithographic master, with the master providing alignment marks for the tape. The tape is permanently affixed to the microchannel device by pouring an over-layer of PDMS, to form a full-thickness device with the tape as an enclosed underlayment. The advantages of these Tape Underlayment Rotary-Node (TURN) valves include parallel fabrication of multiple valves, low risk of damaging a microfluidic device during valve installation, high torque, elimination of stripped threads, the capabilities of TURN hydraulic actuators, and facile customization of TURN molds. We have utilized these valves to control microfluidic flow, to control the onset of molecular diffusion, and to manipulate channel connectivity. Practical applications of TURN valves include control of loading and chemokine release in chemotaxis assay devices, flow in microfluidic bioreactors, and channel connectivity in microfluidic devices intended to study competition and predator/prey relationships among microbes.
Assuntos
Análise de Injeção de Fluxo/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , RotaçãoRESUMO
Mass balances on 10 polycyclic aromatic hydrocarbons (PAHs) in the New York-New Jersey Harbor (hereafter "the Harbor") were constructed using monitoring data from the water column, sediment, and atmosphere. Inputs considered included tributaries, atmospheric deposition, wastewater treatment plant discharges, combined sewer overflows (CSOs), and stormwater runoff. Removal processes examined included tidal exchange between the Harbor and the coastal Bight and Long Island Sound, volatilization, and accumulation or burial of sediment-bound PAHs in the Harbor. The PAHs investigated were fluorene, phenanthrene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, perylene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene, and dibenz[a,h]anthracene. The results show inputs and outputs are fairly well balanced for most compounds, a finding that suggests aerobic biodegradation may not be a key loss process in this Harbor, as has been assumed in other systems. The main pathway for inputs of all PAHs is stormwater runoff. Atmospheric deposition is an important conveyor of PAHs with molecular weights < or =202 g mol(-1). A principal objective of this report is to expose key data gaps, which include the need for comprehensive monitoring of both flow and PAH concentrations in stormwater and CSOs. An improved understanding of the key transmission routes of nonpoint source pollutants is essential for sustainable management of urban water resources.
Assuntos
Hidrocarbonetos Policíclicos Aromáticos/análise , Poluentes Químicos da Água/análise , Poluição Química da Água/análise , Atmosfera/análise , New Jersey , New York , Esgotos/análise , Volatilização , Movimentos da ÁguaRESUMO
Microfluidic devices permit direct observation of microbial behavior in defined microstructured settings. Here, the swimming speed and dispersal of individual marine ciliates in straight and bent microfluidic channels were quantified. The dispersal rate and swimming speed increased with channel width, decreased with protozoan size, and was significantly impacted by the channel turning angle.
Assuntos
Eucariotos/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Animais , Cilióforos/fisiologia , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
Additive manufacturing, or 3D-printing techniques have recently begun to enable simpler, faster, and cheaper production of millifluidic devices at resolutions approaching 100-200 µm. At this resolution, cell culture devices can be constructed that more accurately replicate natural environments compared with conventional culturing techniques. A number of microfluidics researchers have begun incorporating additive manufacturing into their work, using 3D-printed devices in a wide array of chemical, fluidic, and even some biological applications. Here, we describe a 3D-printed cell culture platform and demonstrate its use in culturing Pseudomonas putida KT2440 bacteria for 44 h under a differential substrate gradient. Polyethylene glycol diacrylate (PEGDA) hydrogel barriers are patterned in situ within a 3D-printed channel. Transport of the toluidine blue tracer dye through the hydrogel barriers is characterized. Nutrients and oxygen were delivered to cells in the culture region by diffusion through the PEGDA hydrogel barriers from adjacent media or saline perfusion channels. Expression of green fluorescent protein by P. putida KT2440 enabled real time visualization of cell density within the 3D-printed channel, and demonstrated cells were actively expressing protein over the course of the experiment. Cells were observed clustering near hydrogel barrier boundaries where fresh substrate and oxygen were being delivered via diffusive transport, but cells were unable to penetrate the barrier. The device described here provides a versatile and easy to implement platform for cell culture in readily controlled gradient microenvironments. By adjusting device geometry and hydrogel properties, this platform could be further customized for a wide variety of biological applications.
RESUMO
We present a comprehensive review of the applications of biosynthesized metallic nanoparticles (NPs). The biosynthesis of metallic NPs is the subject of a number of recent reviews, which focus on the various "bottom-up" biofabrication methods and characterization of the final products. Numerous applications exploit the advantages of biosynthesis over chemical or physical NP syntheses, including lower capital and operating expenses, reduced environmental impacts, and superior biocompatibility and stability of the NP products. The key applications reviewed here include biomedical applications, especially antimicrobial applications, but also imaging applications, catalytic applications such as reduction of environmental contaminants, and electrochemical applications including sensing. The discussion of each application is augmented with a critical review of the potential for continued development.
Assuntos
Anti-Infecciosos , Materiais Biocompatíveis , Técnicas Biossensoriais , Nanopartículas Metálicas , Animais , HumanosRESUMO
Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 microm. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.
Assuntos
Técnicas Microbiológicas/instrumentação , Técnicas Microbiológicas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Vibrio/fisiologia , Dimetilpolisiloxanos/química , Ecossistema , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Vibrio/crescimento & desenvolvimento , Vibrio/metabolismoRESUMO
Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 microm s(-1) (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 microm s(-1). Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.
Assuntos
Cilióforos/fisiologia , Sedimentos Geológicos/parasitologia , Movimento , Água do Mar/parasitologia , Animais , Cilióforos/crescimento & desenvolvimento , Meios de Cultura , Dimetilpolisiloxanos , Desenho de Equipamento , Filtros Microporos , Microscopia de Vídeo , Nylons , Parasitologia/instrumentação , Parasitologia/métodosRESUMO
A strong inverse correlation was observed between the polycyclic aromatic hydrocarbon (PAH) mass fraction desorbed, a surrogate measure of bioavailability, and relative carcinogenicity, as quantified by potency equivalency factors (PEFs), for two study sediments from the New York/New Jersey Harbor estuary. Because compounds with the highest toxicity, such as dibenz(a,h)anthracene and benzo(a)pyrene (BAP), also tended to be the least rapidly and least extensively desorbed, the U.S. Environmental Protection Agency (EPA) default guidance may dramatically overestimate risk from exposure to PAH-contaminated soils or sediments. A "relative risk index" (RRI) was developed to account for the combined effects of compound-specific bioavailability and toxic potency in estimating excess cancer risk. Using this approach, estimated excess cancer risk may be diminished by as much as a factor of 159 times versus default EPA guidance. Also, the hierarchy of estimated risk between study sediments and among treatment fractions of study sediments differed using the two approaches, implying that the default approach may inaccurately determine site clean-up priorities. The percentage contribution of each potentially carcinogenic priority PAH to total excess cancer risk was computed under various scenarios. In each case, the contribution of BAP to total excess cancer risk was remarkably invariable, for example, ranging from 48% to 52% in one sediment, and 44% to 54% in the other, over four different exposure durations. These results suggest that BAP may be an excellent indexing compound for gauging relative exposure risk across sediments. Other important contributors to total excess cancer risk were benz(a)anthracene and dibenz(a,h)anthracene. Together, these three compounds comprised nearly 90% of total excess cancer risk from all PAHs in every scenario. This integrated RRI approach may enable regulators to more accurately gauge relative risks and make more informed sediment management decisions.
Assuntos
Neoplasias/induzido quimicamente , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes do Solo/toxicidade , Benzo(a)pireno/toxicidade , Disponibilidade Biológica , Carcinógenos Ambientais/toxicidade , Sedimentos Geológicos/análise , Humanos , Modelos Estatísticos , Hidrocarbonetos Policíclicos Aromáticos/farmacocinética , Medição de Risco , Poluentes do Solo/farmacocinéticaRESUMO
Polycyclic aromatic hydrocarbon (PAH) contaminated sediments from Piles Creek (PC) and Newtown Creek (NC) in the NY/NJ Harbor estuary were separated into size fractions and further separated into low (<1.7 g cm(-3)) and high (>1.7 g cm(-3)) density fractions. The fractionated sediments were characterized for carbon content pore structure, surface area, and PAH concentration. Most PAHs (50-80%) in both sediments were associated with the low-density fraction, which represents only 3-15% of total sediment mass, at levels greater than expected based on equilibrium partitioning. PC low-density sediment had 10 times greater organic carbon-normalized equilibrium partitioning coefficients (Koc) than the other size fractions and whole sediment. Characterization of the sediment organic matter suggested that the preferential sequestration observed in PC sediment was not correlated with soot carbon but was likely due to the presence of detrital plant debris, an important food source for benthic animals. Fractional PAH desorption from whole PC sediment was significantly higher than from NC sediment after 3 months. For both sediments, a smaller percentage of the total PAHs was desorbed from the low-density fraction. However, because PAH concentrations were greatly elevated in these fractions, more PAH mass was desorbed than from the corresponding bulk and high-density fractions. These results demonstrate that PAHs are preferentially sequestered in a separable, low-density fraction at levels not predictable by equilibrium partitioning theory. Further, the low-density fraction apparently controls whole-sediment PAH release. Although plant debris appears to be an important sorbent for PAHs, this material may readily release PAHs into the aqueous phase.
Assuntos
Carbono/química , Poluentes Ambientais/análise , Sedimentos Geológicos/química , Hidrocarbonetos Policíclicos Aromáticos/análise , Hidrocarbonetos Policíclicos Aromáticos/química , Animais , Disponibilidade Biológica , Invertebrados , Compostos Orgânicos , Plantas Comestíveis/química , Solubilidade , Fatores de TempoRESUMO
Biodegradation kinetics for three- and four-ring PAHs by Mycobacterium sp. strain PC01 were measured in whole and density-fractionated estuarine sediments and in a system without intra-aggregate mass transport limitations. The biokinetic data in the systems with and without intra-aggregate mass transport limitations were compared with abiotic PAH desorption kinetics. The results indicate that intra-aggregate mass transport limitations, and not the intrinsic bacterial PAH utilization capacity, were most important in controlling the rate of biodegradation of sediment-sorbed PAHs. Achievable extent of biodegradation could be predicted by the independently measured traction of desorbable PAHs in the fast-diffusion regime of a two-domain intra-aggregate mass transport model. A closed-form mathematical model was developed to describe sediment-pore water partitioning and rapid aqueous-phase diffusion of PAHs through the macropore and mesopore network of sediment aggregates, followed by first-order biodegradation of desorbed PAHs in the bulk aqueous domain. The model effectively predicted independent biodegradation kinetics of PAHs field-aged in two estuarine sediments. Despite low aqueous solubility of PAHs, macropore and mesopore diffusion may be an important mechanism controlling intra-aggregate mass transport and bioavailability of the most readily and extensively desorbed PAHs in sediments.