Biofilm architecture

Staphylococcus aureus strains of the 8325 lineage, especially 8325-4 and derivatives lacking prophage, have been used extensively for decades of research. We report herein the results of our deep sequence analysis of strain 8325-4. Assignment of sequence variants compared with the reference strain 8325 (NRS77/PS47) required correction of errors in the 8325 reference genome, and reassessment of variation previously attributed to chemical mutagenesis of the restriction-defective RN4220. Using an extensive strain pedigree analysis, we discovered that 8325-4 contains 16 single nucleotide polymorphisms (SNP) arising prior to the construction of RN4220. We identified 5 indels in 8325-4 compared with 8325. Three indels correspond to expected Φ11, 12, 13 excisions, one indel is explained by a sequence assembly artifact, and the final indel (Δ63bp) in the spa-sarS intergenic region is common to only a sub-lineage of 8325-4 strains including SH1000. This deletion was found to significantly decrease (75%) steady state sarS but not spa transcript levels in post-exponential phase. The sub-lineage 8325-4 was also found to harbor 4 additional SNPs. We also found large sequence variation between 8325, 8325-4 and RN4220 in a cluster of repetitive hypothetical proteins (SA0282 homologs) near the Ess secretion cluster. The overall 8325-4 SNP set results in 17 alterations within coding sequences. Remarkably, we discovered that all tested strains of the 8325-4 lineage lack phenol soluble modulin α3 (PSMα3), a virulence determinant implicated in neutrophil chemotaxis, biofilm architecture and surface spreading. Collectively, our results clarify and define the 8325-4 pedigree and reveal clear evidence that mutations existing throughout all branches of this lineage, including the widely used RN6390 and SH1000 strains, could conceivably impact virulence regulation.

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp -tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 × 10 5 μm 2 ) or microcolony size (1 × 10 6 μm 2 ) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to qu...

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP-dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeastto-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha : hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 /tpk2 strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 /tpk2 strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence.

h i g h l i g h t s Anaerobic systems provide for higher Cr(VI) removal rates compared to aerobic systems. Complete microbial Cr(VI) reduction is achieved under anaerobic conditions. Easily degradable substrates support higher Cr(VI) removal rates than complex ones. Cr(VI) removal rates follow the Arrhenius equation for temperatures between 12 Ce33 C. Sand filtration as a polishing step provides for complete Cr removal from groundwater.

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP-dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeastto-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha : hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 /tpk2 strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 /tpk2 strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence.

Candida albicans undergoes a reversible morphological transition from single yeast cells to pseudohyphal and hyphal filaments. In this organism, cAMP-dependent protein kinase (PKA), coded by two catalytic subunits (TPK1 and TPK2) and one regulatory subunit (BCY1), mediates basic cellular processes, such as the yeastto-hypha transition and cell cycle regulation. It is known that both Tpk isoforms play positive roles in vegetative growth and filamentation, although distinct roles have been found in virulence, stress response and glycogen storage. However, little is known regarding the participation of Tpk1p and/or Tpk2p in pseudohyphal development. This point was addressed using several C. albicans PKA mutants having heterozygous or homozygous deletions of TPK1 and/or TPK2 in different BCY1 genetic backgrounds. We observed that under hypha-only inducing conditions, all BCY1 heterozygous strains shifted growth toward pseudohyphal morphology; however, the pseudohypha : hypha ratio was higher in strains devoid of TPK2. Under pseudohypha-only inducing conditions, strains lacking TPK2 were prone to develop short and branched pseudohyphae. In tpk2 /tpk2 strains, biofilm architecture was markedly less dense, composed of short pseudohyphae and blastospores with reduced adhesion ability to abiotic material, suggesting a significant defect in cell adherence. Immunolabelling assays showed a decreased expression of adhesins Als1p and Als3p only in the tpk2 /tpk2 strain. Complementation of this mutant with a wild-type copy of TPK2 restored all the altered functions: pseudohyphae elongation, biofilm composition, cell aggregation and adhesins expression. Our study suggests that the Tpk2p isoform may be part of a mechanism underlying not only polarized pseudohyphal morphogenesis but also cell adherence.

1. Burton, B., Indoor Air Pollution and Health (ed. Bardana, EJ), Marcel Dekker Inc., New York, 1997, pp. 127–135. 2. Gammage, RB and Berton, BA, Indoor Air Human Health, Lewis Publications, New York, 1996, 2nd edn. 3. Guao, H., Lee, SC, Li, WM and Cao, JJ, Source ...

ABSTRACTThe speciation of radionuclides and toxic metals in wastes subjected to microbial action is important in determining the extent of stabilization in a disposal environment. As part of an ongoing study, we investigated the reduction of uranium by a Clostridium sp. using X-ray absorption near edge spectroscopy (XANES) at the National Synchrotron Light Source (NSLS) and X-ray photoelectron spectroscopy (XPS). XPS analysis of uranyl acetate containing hexavalent uranium exhibited a binding energy of 382.0eV at the U 4f7/2 peak. The sample incubated in the presence of bacteria was shifted to lower binding energy (380.6eV), confirming the reduction of U6+ to U4+ at the bacterial surface. XANES analysis, using an electron yield detector, was performed at the Mv absorption edge (3d--> 5f). The absorption peak energy of the sample exhibited a shift from 3551.1eV to 3550.1eV which is higher than uranium metal (3549.6eV) but lower than U4+ (3550.4eV). This indicates the presence of U...

, cepa tolerante a cromato, presenta la capacidad de formar biopeliculas y transformar el 95 % de cromato presente en el medio a Cr(III), compuesto menos tóxico, después de 48h de incubación, a una velocidad de transformación de 1.67 mg L-1 h-1. S. marcescens puede ser utilizado en biorremediation de residuos contaminados con cromato.

The chromate-reducing ability of Pseudomonas aeruginosa A2Chr was compared in batch culture, with cells entrapped in a dialysis sac, and with cells immobilized in an agarose-alginate film in conjunction with a rotating biological contactor. In all three systems, the maximum Cr(VI) reduction occurred at 10 mg Cr(VI)/l. Whereas at 50 mg Cr(VI)/l concentration, only 16% of the total Cr(VI) was reduced, five spikings with 10 mg chromate/l at 2-h intervals led to 96% reduction of the total input of 50 mg Cr(VI) /l. Thus maximum Cr(VI) reduction was achieved by avoiding Cr(VI) toxicity to the cells by respiking with lower Cr(VI) concentrations. At 10 mg Cr(VI)/l, the pattern of chromate reduction in dialysis-entrapped cells was almost similar to that of batch culture and 86% of the bacterially reduced chromium was retained inside the dialysis sac. In electroplating effluent containing 100 mg Cr(VI)/l, however, the amount of Cr(VI) reduced by the cells immobilized in agarose-alginate biofilm was twice and thrice the amount reduced by batch culture and cells entrapped in a dialysis sac, respectively. Materials and methods Bacterial strain and culture conditions Pseudomonas aeruginosa A2Chr is a Cr(VI)-reducing strain isolated from the effluent of a leather-tanning unit located in Kanpur,

The chromate-reducing ability of Pseudomonas aeruginosa A2Chr was compared in batch culture, with cells entrapped in a dialysis sac, and with cells immobilized in an agarose-alginate film in conjunction with a rotating biological contactor. In all three systems, the maximum Cr(VI) reduction occurred at 10 mg Cr(VI)/l. Whereas at 50 mg Cr(VI)/l concentration, only 16% of the total Cr(VI) was reduced, five spikings with 10 mg chromate/l at 2-h intervals led to 96% reduction of the total input of 50 mg Cr(VI) /l. Thus maximum Cr(VI) reduction was achieved by avoiding Cr(VI) toxicity to the cells by respiking with lower Cr(VI) concentrations. At 10 mg Cr(VI)/l, the pattern of chromate reduction in dialysis-entrapped cells was almost similar to that of batch culture and 86% of the bacterially reduced chromium was retained inside the dialysis sac. In electroplating effluent containing 100 mg Cr(VI)/l, however, the amount of Cr(VI) reduced by the cells immobilized in agarose-alginate biofilm was twice and thrice the amount reduced by batch culture and cells entrapped in a dialysis sac, respectively. Materials and methods Bacterial strain and culture conditions Pseudomonas aeruginosa A2Chr is a Cr(VI)-reducing strain isolated from the effluent of a leather-tanning unit located in Kanpur,

In this study, we use extended X-ray absorption fine structure (EXAFS) spectroscopy measurements to examine the atomic environment of Cd bound onto two experimental bacterial consortia: one grown from river water, and one grown from a manufacturing gas plant site. The experiments were conducted as a function of pH and demonstrate that the complex mixtures of bacteria, containing both Gram-positive and Gram-negative species, yield relatively simple EXAFS spectra, a result which indicates that only a limited number of functional group types contribute to Cd binding for each bacterial consortium. The EXAFS spectra indicate that the average Cd binding environment in the river water consortium varies significantly with pH, but the manufacturing gas plant consortium exhibits a Cd binding environment that remains relatively constant over the pH range examined. The EXAFS data for the river water consortium were modeled using carboxyl, phosphoryl and sulfhydryl sites. However, only carboxyl and phosphoryl sites were required to model the manufacturing gas plant consortium data under similar experimental conditions. This is the first EXAFS study to identify and quantify the relative importance of metal binding sites in bacterial consortia. Although our results indicate differences in the binding environments of the two consortia, the data suggest that there are broad similarities in the binding environments present on a wide range of bacterial cell walls.

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day ؊1 g ؊1 , with reduction of 0.17 mM day ؊1 g ؊1 under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.

Due to the increasing concentrations of heavy metals in potable water and industrial wastewater governmental agencies have created stricter regulations for the treatment of metal contaminated waste. However, current technologies are both expensive and time consuming to use for water and waste treatment. As an alternative to the current technologies, phytofiltration has been proposed, but to make phytofiltration effective the process must be further studied to better understand the metal-biomass chemical interactions. This current study on the use of hops biomass to remove heavy metal ions from aqueous solutions uses X-ray absorption spectroscopy (XAS) to investigate the binding mechanism of heavy metals to hops biomass. The XAS studies showed that copper(II), zinc(II) and chromium(III) remained in the same oxidation state as when they were reacted with the hops biomass. However, the reaction of chromium(VI) with the hops biomass resulted in the reduction of chromium(VI) to chromium(III). Analysis of the XAS spectra showed that copper(II) bound to the hops biomass with a molecular geometry of copper(II) acetate; zinc(II) bound to the hops biomass with a molecular geometry of zinc(II) gluconate; and chromium(VI) bound to the biomass with the geometry of chromium(III) acetate. In addition, the XAS studies showed that the nearest neighboring atom of all the bound metal ions was an oxygen atom. ᮊ

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 ؋ 10 5 m 2 ) or microcolony size (1 ؋ 10 6 m 2 ) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 m above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM day ؊1 g ؊1 , with reduction of 0.17 mM day ؊1 g ؊1 under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.

We assessed the potential of aerobic granular sludge consisting of mixed species of bacteria to remove and immobilize uranium (VI) and chromium (VI). Microbial granules were cultivated in a laboratory sequencing batch reactor (SBR) by feeding with acetate-containing synthetic media. Microbial granules formed in the SBR exhibited excellent settling characteristics and predominantly consisted of rod/cocci shaped bacteria. The microbial granules immobilized 218 ± 2 mg of U(VI) g −1 dry granular biomass. X-ray photoelectron spectroscopy (XPS) showed the association of U(VI) with the microbial granules and transformation of U(VI) to U(IV). Microbial granules reduced Cr(VI) and immobilized to Cr(III) at 0.17 mmoles/d/g under anaerobic conditions. X-ray absorption near edge spectroscopy (XANES) of chromium associated with microbial granules revealed complete conversion of Cr(VI) to Cr(III). Extended X-ray absorption fi ne structure (EXAFS) analysis of the Cr-laden microbial granules showed similarity to Cr(III)-phosphate. This study demonstrates the biotransformation and immobilization of U(VI) and Cr(VI) by mixed species microbial granules.

Chromium has been widely used in various industries. Hexavalent chromium (Cr 6+ ) is a priority toxic, mutagenic and carcinogenic chemical, whereas its reduced trivalent form (Cr 3+ ) is much less toxic and insoluble. Hence, the basic process for chromium detoxification is the transformation of Cr 6+ to Cr 3+ . A number of aerobic and anaerobic microorganisms are capable of reducing Cr 6+ . In the presence of oxygen, microbial reduction of Cr 6+ is commonly catalyzed by soluble enzymes, except in Pseudomonas maltophilia O-2 and Bacillus megaterium TKW3, which utilize membrane-associated reductases. Recently, two soluble Cr 6+ reductases, ChrR and YieF, have been purified from Pseudomonas putida MK1 and Escherichia coli, respectively. ChrR catalyzes an initially one-electron shuttle followed by a two-electron transfer to Cr 6+ , with the formation of intermediate(s) Cr 5+ and/or Cr 4+ before further reduction to Cr 3+ . YieF displays a four-electron transfer that reduces Cr 6+ directly to Cr 3+ . The membrane-associated Cr 6+ reductase of B. megaterium TKW3 was isolated, but its reduction kinetics is as yet uncharacterized. Under anaerobic conditions, both soluble and membrane-associated enzymes of the electron transfer system were reported to mediate Cr 6+ reduction as a fortuitous process coupled to the oxidation of an electron donor substrate. In this process, Cr 6+ serves as the terminal electron acceptor of an electron transfer chain that frequently involves cytochromes (e.g., b and c). An expanding array of Cr 6+ reductases allows the selection of enzymes with higher reductive activity, which genetic and/or protein engineering may further enhance their efficiencies. With the advancement in technology for enzyme immobilization, it is speculated that the direct application of Cr 6+ reductases may be a promising approach for bioremediation of Cr 6+ in a wide range of environments. r

For the first time a method has been developed for the extended X-ray absorption fine structure (EXAFS) data analyses of biological samples containing multiple oxidation states of chromium. In this study, the first shell coordination and interatomic distances based on the data analysis of known standards of potassium chromate (Cr(VI)) and chromium nitrate hexahydrate (Cr(III)) were investigated. The standards examined were mixtures of the following molar ratios of Cr(VI):Cr(III), 0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, and 1:0. It was determined from the calibration data that the fitting error associated with linear combination X-ray absorption near edge structure (LC-XANES) fittings was approximately 610% of the total fitting. The peak height of the Cr(VI) pre-edge feature after normalization of the X-ray absorption (XAS) spectra was used to prepare a calibration curve. The EXAFS fittings of the standards were also investigated and fittings to lechuguilla biomass samples laden with different ratios of Cr(III) and Cr(VI) were performed as well. An excellent agreement between the XANES data and the data presented in the EXAFS spectra was observed. The EXFAS data also presented mean coordination numbers directly related to the ratios of the different chromium oxidation states in the sample. The chromium oxygen interactions had two different bond lengths at approximately 1.68 and 1.98 Å for the Cr(VI) and Cr(III) in the sample, respectively.

For the first time a method has been developed for the extended X-ray absorption fine structure (EXAFS) data analyses of biological samples containing multiple oxidation states of chromium. In this study, the first shell coordination and interatomic distances based on the data analysis of known standards of potassium chromate (Cr(VI)) and chromium nitrate hexahydrate (Cr(III)) were investigated. The standards examined were mixtures of the following molar ratios of Cr(VI):Cr(III), 0:1, 0.25:0.75, 0.5:0.5, 0.75:0.25, and 1:0. It was determined from the calibration data that the fitting error associated with linear combination X-ray absorption near edge structure (LC-XANES) fittings was approximately 610% of the total fitting. The peak height of the Cr(VI) pre-edge feature after normalization of the X-ray absorption (XAS) spectra was used to prepare a calibration curve. The EXAFS fittings of the standards were also investigated and fittings to lechuguilla biomass samples laden with different ratios of Cr(III) and Cr(VI) were performed as well. An excellent agreement between the XANES data and the data presented in the EXAFS spectra was observed. The EXFAS data also presented mean coordination numbers directly related to the ratios of the different chromium oxidation states in the sample. The chromium oxygen interactions had two different bond lengths at approximately 1.68 and 1.98 Å for the Cr(VI) and Cr(III) in the sample, respectively.

Bacteria attached to surfaces in biofilms are responsible for the contamination of industrial processes and many types of microbial infections and disease. Once established, biofilms are notoriously difficult to eradicate. A more complete understanding of how biofilms form and behave is crucial if we are to predict, and ultimately control, biofilm processes. A major breakthrough in biofilm research came in the early 1990's when confocal scanning laser microscopy (CSLM) showed that biofilms formed complex structures which could facilitate nutrient exchange. We have recently found that biofilms growing in turbulent flow can also be temporally complex. Structures such as cell clusters and ripples can migrate downstream along solid surfaces. Further, biofilm viscoelasticity allows the biofilm to structurally deform when exposed to varying shear stresses.

Mixed-species bio®lms, consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas uorescens and Stenotrophomonas maltophilia, were grown in glass¯ow cells under either laminar or turbulent¯ow. The bio®lms grown in laminar¯ow consisted of roughly circular-shaped microcolonies separated by water channels. In contrast, bio®lm microcolonies grown in turbulent¯ow were elongated in the downstream direction, forming ®lamentous streamers'. Moreover, bio®lms growing in turbulent ow developed extensive patches of ripple-like structures between 9 and 13 days of growth. Using timelapse microscopic imaging, we discovered that the bio®lm ripples migrated downstream. The morphology and the migration velocity of the ripples varied with short-term changes in the bulk liquid¯ow velocity. The ripples had a maximum migration velocity of 800 mm h À1 (2.2´10 À7 m s À1 ) when the liquid¯ow velocity was 0.5 m s À1 (Reynolds number 1800). This work challenges the commonly held assumption that bio®lm structures remain at the same location on a surface until they eventually detach.

Bacteria attached to surfaces in biofilms are responsible for the contamination of industrial processes and many types of microbial infections and disease. Once established, biofilms are notoriously difficult to eradicate. A more complete understanding of how biofilms form and behave is crucial if we are to predict, and ultimately control, biofilm processes. A major breakthrough in biofilm research came in the early 1990's when confocal scanning laser microscopy (CSLM) showed that biofilms formed complex structures which could facilitate nutrient exchange. We have recently found that biofilms growing in turbulent flow can also be temporally complex. Structures such as cell clusters and ripples can migrate downstream along solid surfaces. Further, biofilm viscoelasticity allows the biofilm to structurally deform when exposed to varying shear stresses.

Mixed-species bio®lms, consisting of Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas uorescens and Stenotrophomonas maltophilia, were grown in glass¯ow cells under either laminar or turbulent¯ow. The bio®lms grown in laminar¯ow consisted of roughly circular-shaped microcolonies separated by water channels. In contrast, bio®lm microcolonies grown in turbulent¯ow were elongated in the downstream direction, forming ®lamentous streamers'. Moreover, bio®lms growing in turbulent ow developed extensive patches of ripple-like structures between 9 and 13 days of growth. Using timelapse microscopic imaging, we discovered that the bio®lm ripples migrated downstream. The morphology and the migration velocity of the ripples varied with short-term changes in the bulk liquid¯ow velocity. The ripples had a maximum migration velocity of 800 mm h À1 (2.2´10 À7 m s À1 ) when the liquid¯ow velocity was 0.5 m s À1 (Reynolds number 1800). This work challenges the commonly held assumption that bio®lm structures remain at the same location on a surface until they eventually detach.

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 ؋ 10 5 m 2 ) or microcolony size (1 ؋ 10 6 m 2 ) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 m above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-à-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

The oxidation state of intracellular chromium has been determined directly in mammalian lung cells exposed to mutagenic and carcinogenic chromium compounds. Microprobe X-ray absorption spectroscopy (XAS) experiments on single V79 Chinese hamster lung cells showed that Cr(VI) and Cr(V) complexes were reduced completely (>90%) to Cr(III) within 4 h of exposure of the cells. This result provides direct evidence for the hypothesis that these genotoxic oxidants react rapidly with intracellular reductants.

Keywords: Aerobic granular sludge (GS) 2-Fluorophenol (2-FP) Bioaugmentation Sequencing batch reactor (SBR) Wastewater treatment a b s t r a c t Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by

The oxidation state of intracellular chromium has been determined directly in mammalian lung cells exposed to mutagenic and carcinogenic chromium compounds. Microprobe X-ray absorption spectroscopy (XAS) experiments on single V79 Chinese hamster lung cells showed that Cr(VI) and Cr(V) complexes were reduced completely (>90%) to Cr(III) within 4 h of exposure of the cells. This result provides direct evidence for the hypothesis that these genotoxic oxidants react rapidly with intracellular reductants.

The oxidation state of intracellular chromium has been determined directly in mammalian lung cells exposed to mutagenic and carcinogenic chromium compounds. Microprobe X-ray absorption spectroscopy (XAS) experiments on single V79 Chinese hamster lung cells showed that Cr(VI) and Cr(V) complexes were reduced completely (>90%) to Cr(III) within 4 h of exposure of the cells. This result provides direct evidence for the hypothesis that these genotoxic oxidants react rapidly with intracellular reductants.

Keywords: Aerobic granular sludge (GS) 2-Fluorophenol (2-FP) Bioaugmentation Sequencing batch reactor (SBR) Wastewater treatment a b s t r a c t Aerobic granular sludge is extremely promising for the treatment of effluents containing toxic compounds, and it can economically compete with conventional activated sludge systems. A laboratory scale granular sequencing batch reactor (SBR) was established and operated during 444 days for the treatment of an aqueous stream containing a toxic compound, 2-fluorophenol (2-FP), in successive phases. Initially during ca. 3 months, the SBR was intermittently fed with 0.22 mM of 2-FP added to an acetate containing medium. No biodegradation of the target compound was observed. Bioaugmentation with a specialized bacterial strain able to degrade 2-FP was subsequently performed. The reactor was thereafter continuously fed with 0.22 and 0.44 mM of 2-FP and with 5.9 mM of acetate (used as co-substrate), for 15 months. Full degradation of the compound was reached with a stoichiometric fluoride release. The 2-FP degrading strain was successfully retained by

Remediation measures for hexavalent chromium [Cr(VI)] are required for a safe environment. As a recent development in microbiology, bacterial biofilms are being studied as effective bioremediation agents. When bacteria are in fungal surface-attached biofilm mode, they are called fungal–bacterial biofilms (FBBs). They have not been tested for bioremediation so far. Hence, this study was conducted to develop FBBs and glass-wool-attached bacterial biofilms (BBs), and to evaluate Cr(VI) tolerability and removal of bacterial monocultures, BBs and FBBs. FBBs showed a significantly high level of Cr(VI) tolerance and resistance compared with its BBs or monocultures. After 10 days, up to 90% of Cr(VI) had been removed, which was significantly higher than that of BBs or its monocultures. Thus, it is clear that FBBs can be used as a novel tool to decontaminate Cr(VI) both in situ and ex situ.

This paper examines the development of aerobic granular sludge in the presence of a synthetic chelating agent, nitrilotriacetic acid (NTA), in sequencing batch reactors (SBR). The growth of seed sludge at 0.26 mM, 0.52 mM and 1.05 mM of NTA was found to be significantly lower as compared to that in the absence of NTA. Aerobic granulation was significantly enhanced in the three SBRs (R2, R3 and R4), which were fed with 0.26 mM, 0.52 mM and 1.05 mM of NTA as a co-substrate, in comparison to the acetate-alone fed SBR (R1). After 2 months of operation, the mean diameter of the biomass stabilized at 0.35 mm in R1 (acetate alone), as compared to 2.18 mm in R4 (1.05 mM NTA þ acetate). NTA degradation was established in SBRs, with almost complete removal during the SBR cycle. Batch experiments also showed efficient degradation of NTA by the aerobic granules.

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CITATIONS 46 READS 44 6 authors, including: Some of the authors of this publication are also working on these related projects: Effect of nitrate and sulfate on selenate bioreduction and influence of pH on reactor operation View project Biohydrometallurgy of critical/scarce/precious metals from waste materials View project Development of mixed-culture microbial granules under aerobic conditions in a sequencing batch reactor (SBR), capable of completely degrading a recalcitrant metal chelating agent is reported. In laboratory-scale reactor studies, the microbial granules degraded 2 mM of free nitrilotriacetic acid (NTA) and Fe(III)-NTA completely in 14 and 40 h, respectively.

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, 9 to reduce chromate and remove it from synthetic minimal medium. In batch experiments, 10 acetate-fed granular biofilms incubated aerobically reduced 0.2 mM of Cr (VI) from a 11 minimal medium at 0.15 mM d -1 g -1 , and 0.17 mM d -1 g -1 under anaerobic conditions. 12

The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration.

We assessed the potential of mixed microbial consortia, in the form of granular biofilms, to reduce chromate and remove it from synthetic minimal medium. In batch experiments, acetate-fed granular biofilms incubated aerobically reduced 0.2 mM Cr(VI) from a minimal medium at 0.15 mM/day/g, with reduction of 0.17 mM/day/g under anaerobic conditions. There was negligible removal of Cr(VI) (i) without granular biofilms, (ii) with lyophilized granular biofilms, and (iii) with granules in the absence of an electron donor. Analyses by X-ray absorption near edge spectroscopy (XANES) of the granular biofilms revealed the conversion of soluble Cr(VI) to Cr(III). Extended X-ray absorption fine-structure (EXAFS) analysis of the Cr-laden granular biofilms demonstrated similarity to Cr(III) phosphate, indicating that Cr(III) was immobilized with phosphate
on the biomass subsequent to microbial reduction. The sustained reduction of Cr(VI) by granular biofilms was confirmed in fed-batch experiments. Our study demonstrates the promise of granular-biofilm-based systems in treating Cr(VI)-containing effluents and wastewater.

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation.

Development of mixed-culture microbial granules under aerobic conditions in a sequencing batch reactor (SBR), capable of completely degrading a recalcitrant metal chelating agent is reported. In laboratory-scale reactor studies, the microbial granules degraded 2mM of free nitrilotriacetic acid (NTA) and Fe(III)-NTA completely in 14 and 40 h, respectively. Free NTA was degraded at a specific rate of 0.7 mM (gMLSS)(-1)h(-1), while Fe(III)-NTA was degraded at a specific rate of 0.37 mM (gMLSS)(-1)h(-1). Achievement of significant degradation rates of NTA and ferric-NTA in double-distilled water suggests that the microbial metabolism is not constrained by lack of essential elements. Efficient degradation of recalcitrant synthetic chelating agents by aerobic microbial granules suggests their potential application in a variety of situations where heavy metals or radionuclides are to be co-disposed with metal chelating agents.

Aerobic microbial granules, self-immobilized microbial
consortia cultured in aerobically operated bioreactors,
primarily consist of mixed species of bacteria
ensconced in an extracellular polymeric matrix of
their own creation. Such aerobically grown microbial
granules have attracted considerable research interest
in environmental biotechnology. In recent times, it has
been demonstrated that the granules could be used for
efficient degradation of recalcitrant organic compounds
and for the treatment of a growing number of wastes.
The objective of this study was to investigate whether
aerobic granules could be used as novel biomass material
for biosorption of uranium from aqueous solutions.
The granular biomass for biosorption experiments was
cultivated in a laboratory-scale sequencing batch reactor
by feeding with synthetic wastewater. Biosorption
of uranium [U(VI)] was studied at different initial pH
values (1 to 8) and different initial uranium concentrations
(6 to 750 mg l–1). Biosorption was observed to
be rapid (<1 h) in acidic pH range (1 to 6) compared
to that at pH 7.0 or above. Almost complete removal of
uranium was observed in the range 6–100 mg l–1 in
less than 1 h. Redlich–Peterson model gave the best fit
when the experimental data were analysed using different
adsorption isotherm equations. The maximum
biosorption capacity of U(VI) was determined to be
218 ± 2 mg g–1 dry granular biomass. Further, it wasobserved that cations such as Na+, K+, Mg+2, and Ca+2
were simultaneously released into the bulk solution
during U(VI) biosorption, indicating the involvement
of ion exchange mechanism in radionuclide uptake.
Live and dead biomass did not show significant difference
in U removal, indicating the involvement of a passive
sorption process. The study suggests that aerobic
granular biomass has the potential to be employed as an
effective biosorbent material for recovering/removing
uranium (and probably other radionuclides) from dilute
nuclear wastes.