Experimental and simulation analysis of community structure of nitrifying bacteria in a membrane-aerated biofilm

2007 ◽  
Vol 55 (8-9) ◽  
pp. 283-290 ◽  
Author(s):  
S. Matsumoto ◽  
A. Terada ◽  
Y. Aoi ◽  
S. Tsuneda ◽  
E. Alpkvist ◽  
...  
Keyword(s):  
Cluster Size ◽  
Laser Scanning ◽  
Simulation Analysis ◽  
Current Model ◽  
Membrane Surface ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Confocal Laser ◽  
Ammonia Oxidizing Bacteria ◽  
Microelectrode Measurements

Until now, only few attempts have been made to assess biofilm models simulating microenvironments in a biofilm. As a first step, we compare the microenvironment observed in a membrane aerated biofilm (MAB) to that derived from a two-dimensional computational model with individual ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) embedded in a continuum EPS matrix. Gradients of oxygen were determined by means of microelectrodes. The change in nitrifying bacterial populations with the biofilm depth was quantified using fluorescence in situ hybridization (FISH) in combination with a confocal laser scanning microscopy (CLSM). Microelectrode measurements revealed that oxic and anoxic or anaerobic regions exist within the MAB. The oxygen profile predicted by the model showed good agreement with that obtained by microelectrode measurements. The oxic part of the biofilm was dominated by NSO190 probe-hybridized AOB, which formed relatively large clusters of cells directly on the membrane surface, and by the NOB belonging to genus Nitrobacter sp. On the other hand, NOB belonging to genus Nitrospira sp. were abundant at the oxic-anoxic interface. The model prediction regarding AOB and Nitrobacter sp. distribution was consistent with the experimental counterpart. Measurements of AOB cluster size distribution showed that colonies are slightly larger adjacent to the membrane than at the inner part of the biofilm. The sizes predicted by the current model are larger than those obtained in the experiment, leading to the arguments that some factors not contained in the model would affect the cluster size.

scholarly journals Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

1998 ◽  
Vol 330 (2) ◽  
pp. 853-860 ◽  
Author(s):  
N. J. Silvia MORENO ◽  
Li ZHONG ◽  
Hong-Gang LU ◽  
Wanderley DE SOUZA ◽  
Marlene BENCHIMOL
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Dependent Manner ◽  
Cytoplasmic Ph ◽  
Bafilomycin A1

Cytoplasmic pH (pHi) regulation was studied in Toxoplasma gondii tachyzoites by using the fluorescent dye 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Their mean baseline pHi (7.07±0.06; n = 5) was not significantly affected in the absence of extracellular Na+, K+ or HCO3- but was significantly decreased in a dose-dependent manner by low concentrations of N,Nʹ-dicyclohexylcarbodi-imide (DCCD), N-ethylmaleimide (NEM) or bafilomycin A1. Bafilomycin A1 also inhibited the recovery of tachyzoite pHi after an acid load with sodium propionate. Similar concentrations of DCCD, NEM and bafilomycin A1 produced depolarization of the plasma membrane potential as measured with bis-(1,3-diethylthiobarbituric)trimethineoxonol (bisoxonol), and DCCD prevented the hyperpolarization that accompanies acid extrusion after the addition of propionate, in agreement with the electrogenic nature of this pump. Confocal laser scanning microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the vacuolar (V)-H+-ATPase of T. gondii tachyzoites is also located in the plasma membrane. Surface localization of the V-H+-ATPase was confirmed by experiments using biotinylation of cell surface proteins and immunoprecipitation with antibodies against V-H+-ATPases. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of these intracellular parasites and with an important role of this enzyme in the regulation of pHi homoeostasis in these cells.

scholarly journals Evaluating Protein Fouling on Membranes Patterned by Woven Mesh Fabrics

Membranes ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 730
Author(s):  
Anna Malakian ◽  
Scott M. Husson
Keyword(s):  
Polyvinylidene Fluoride ◽  
Laser Scanning ◽  
Hydrophobic Interactions ◽  
Membrane Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Transient Nature ◽  
Spatiotemporal Information ◽  
Protein Fouling ◽  
Flux Decline

Membrane surface patterning is one approach used to mitigate fouling. This study used a combination of flux decline measurements and visualization experiments to evaluate the effectiveness of a microscale herringbone pattern for reducing protein fouling on polyvinylidene fluoride (PVDF) ultrafiltration membranes. Thermal embossing with woven mesh stamps was used for the first time to pattern membranes. Embossing process parameters were studied to identify conditions replicating the mesh patterns with high fidelity and to determine their effect on membrane permeability. Permeability increased or remained constant when patterning at low pressure (≤4.4 MPa) as a result of increased effective surface area; whereas permeability decreased at higher pressures due to surface pore-sealing of the membrane active layer upon compression. Flux decline measurements with dilute protein solutions showed monotonic decreases over time, with lower rates for patterned membranes than as-received membranes. These data were analyzed by the Hermia model to follow the transient nature of fouling. Confocal laser scanning microscopy (CLSM) provided complementary, quantitative, spatiotemporal information about protein deposition on as-received and patterned membrane surfaces. CLSM provided a greater level of detail for the early (pre-monolayer) stage of fouling than could be deduced from flux decline measurements. Images show that the protein immediately started to accumulate rapidly on the membranes, likely due to favorable hydrophobic interactions between the PVDF and protein, followed by decreasing rates of fouling with time as protein accumulated on the membrane surface. The knowledge generated in this study can be used to design membranes that inhibit fouling or otherwise direct foulants to deposit selectively in regions that minimize loss of flux.

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

2007 ◽  
Vol 53 (5) ◽  
pp. 559-571 ◽  
Author(s):  
Amy J. Pogue ◽  
Kimberley A. Gilbride
Keyword(s):  
Activated Sludge ◽  
Laser Scanning ◽  
Ammonia Oxidation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Batch Reactors ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrate Accumulation ◽  
Protozoan Grazing ◽  
Nitrite Nitrate

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.

scholarly journals Statistical Analysis of Pseudomonas aeruginosa Biofilm Development: Impact of Mutations in Genes Involved in Twitching Motility, Cell-to-Cell Signaling, and Stationary-Phase Sigma Factor Expression

2002 ◽  
Vol 68 (4) ◽  
pp. 2008-2017 ◽  
Author(s):  
Arne Heydorn ◽  
Bjarne Ersbøll ◽  
Junichi Kato ◽  
Morten Hentzer ◽  
Matthew R. Parsek ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Current Model ◽  
Flow Chamber ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Time Points ◽  
Biofilm Development

ABSTRACT Four strains of Pseudomonas aeruginosa (wild type, ΔpilHIJK mutant, lasI mutant, and rpoS mutant) were genetically tagged with the green fluorescent protein, and the development of flow chamber-grown biofilms by each of them was investigated by confocal laser scanning microscopy. The structural developments of the biofilms were quantified by the computer program COMSTAT (A. Heydorn, A. T. Nielsen, M. Hentzer, C. Sternberg, M. Givskov, B. K. Ersbøll, and S. Molin, Microbiology 146:2395-2407, 2000). Two structural key variables, average thickness and roughness, formed the basis for an analysis of variance model comprising the four P. aeruginosa strains, five time points (55, 98, 146, 242, and 314 h), and three independent rounds of biofilm experiments. The results showed that the wild type, the ΔpilHIJK mutant, and the rpoS mutant display conspicuously different types of temporal biofilm development, whereas the lasI mutant was indistinguishable from the wild type at all time points. The wild type and the lasI mutant formed uniform, densely packed biofilms. The rpoS mutant formed densely packed biofilms that were significantly thicker than those of the wild type, whereas the ΔpilHIJK mutant formed distinct microcolonies that were regularly spaced and almost uniform in size. The results are discussed in relation to the current model of P. aeruginosa biofilm development.

Spatial Architecture of Nitrifying Bacteria Biofilm Immobilized on Polyurethane Foam in an Automatic Biodetector for Water Toxicity

2010 ◽  
Vol 16 (5) ◽  
pp. 550-560 ◽  
Author(s):  
Andrzej Woznica ◽  
Jagna Karcz ◽  
Agnieszka Nowak ◽  
Aleksander Gmur ◽  
Tytus Bernas
Keyword(s):  
Polyurethane Foam ◽  
Laser Scanning ◽  
Spatial Organization ◽  
3D Visualization ◽  
Three Dimensional ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Confocal Laser ◽  
X Ray

AbstractWe describe the architecture of nitrifying bacteria biofilms immobilized on a three-dimensional (3D) polyurethane foam that permits efficient water flow through a bioreactor. The 3D spatial organization of immobilized bacterial colonies is characterized on three resolution levels with X-ray tomography, light confocal microscopy, and scanning electron microscopy (SEM). Using these techniques we demonstrate biofilm distribution in the foam and the existence of several modes of binding of bacteria to the foam. Computed X-ray tomography permits observation of the distribution of the biofilm in the whole open cellular polyurethane material volume and estimation of biofilm volume. SEM and confocal laser scanning microscopy techniques permit 3D visualization of biofilm structure. Three distinct immobilization patterns could be observed in the open cellular polyurethane material: (1) large irregular aggregates of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) spherical (pom-pom) aggregates of bacteria localized on the external surface of biofilm; and (3) biofilm threads adherent to the surface of polyurethane foam. Finally, we demonstrate that immobilized bacteria exhibit metabolic activity and growth.

Microbial population structure changes in a suspended carrier biofilm reactor

2006 ◽  
Vol 54 (9) ◽  
pp. 145-153
Author(s):  
R.C. Wang ◽  
X.H. Wen ◽  
Y. Qian
Keyword(s):  
Population Structure ◽  
Microbial Population ◽  
Biofilm Reactor ◽  
Laser Scanning Microscopy ◽  
Nitrifying Bacteria ◽  
Confocal Laser ◽  
Ammonia Oxidizing Bacteria ◽  
N Removal ◽  
Quinone Profile ◽  
Suspended Carrier

The changes of microbial population structure in biofilm caused by variations of COD/NHNH+4-N ratio in influent were investigated in a suspended carrier biofilm reactor (SCBR). When COD/NHNH+4-N was 10, the highest COD removal reached was about 73.8 per cent, whereas the highest NH+4-N removal, about 55.9 per cent, was reached when COD/NH+4-N was 5. By using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) observation, it was found that the thickness of the biofilm formed on the carrier was about 80 to 120 μm, and ammonia-oxidizing bacteria (AOB) and nitrifying bacteria existed mainly in the upper layer about 20 to 30 μm. The results of the quinone profile analysis showed that when the COD/NH+4-N increased, the microbial diversity (DQ) increased, while the proportions of AOB and nitrifying bacteria in the biofilm communities decreased.

Reduction of Listeria monocytogenes on Green Peppers (Capsicum annuum L.) by Gaseous and Aqueous Chlorine Dioxide and Water Washing and Its Growth at 7°C†

2001 ◽  
Vol 64 (11) ◽  
pp. 1730-1738 ◽  
Author(s):  
Y. HAN ◽  
R. H. LINTON ◽  
S. S. NIELSEN ◽  
P. E. NELSON
Keyword(s):  
Listeria Monocytogenes ◽  
Laser Scanning ◽  
Membrane Surface ◽  
Fluorescein Isothiocyanate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Green Pepper ◽  
Gas Treatment ◽  
Water Washing ◽  
Significant Difference

Reduction of Listeria monocytogenes Scott A on uninjured and injured surfaces of green peppers after 0.3- and 3-mg/liter gaseous and aqueous ClO2 treatment and water washing for 10 min at 20°C was studied. Growth of the L. monocytogenes untreated or treated with 0.6 mg/liter ClO2 gas for 30 min at 20°C on green peppers also was investigated. A membrane-surface-plating method was used for resuscitation and enumeration of L. monocytogenes treated with ClO2. The bacterial viability on pepper surfaces was visualized using confocal laser scanning microscopy (CLSM). Live and dead cells of L. monocytogenes were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively. More than 6 log CFU/5 g L. monocytogenes on uninjured surfaces and about 3.5 log CFU/5 g on injured surfaces were inactivated by both 3-mg/liter and 0.6-mg/liter ClO2 gas treatments. The 3-mg/liter aqueous ClO2 treatment achieved 3.7- and 0.4-log reductions on uninjured and injured surfaces, respectively; whereas, water washing alone showed 1.4- and 0.4-log reductions, respectively. ClO2 gas treatment was the most effective in reducing L. monocytogenes on both uninjured and injured green pepper surfaces, when compared with aqueous ClO2 treatment and water washing. The significant difference (P < 0.05) between log reductions on uninjured and injured surfaces and the results from CLSM analysis suggested that injured surfaces protected more bacteria from sanitation treatments than did uninjured surfaces. Not only could L. monocytogenes grow on green pepper surfaces at 7°C, bacteria that survived the 0.6-mg/liter ClO2 gas treatment also could grow.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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