RESUMEN
A microaerophilic Gram-stain-negative bacilliform bacterial strain, FB-5 T, was isolated from activated sludge in Yokohama, Japan, that exhibited filamentous growth and formed a microtube (sheath). Cells were motile using a single polar flagellum. The optimum growth temperature and pH were 30 °C and 7.5, respectively. Strain FB-5 T was catalase-negative. Peptides and amino acids were utilized as energy and carbon sources. Sugars and organic acids were not utilized. Vitamin B12 enhanced the growth of strain FB-5 T. Sulfur-dependent lithotrophic growth was possible. Major respiratory quinone was UQ-8. Major fatty acids were C16:1ω7 and C16:0. The genomic DNA G + C content was 69.16%. Phylogenetic analysis of the 16S rRNA gene suggested that strain FB-5 T belongs to the genus Sphaerotilus. The close relatives were S. natans subsup. sulfidivorans and S. natans subsup. natans with 98.0% and 97.8% similarity based on the 16S rRNA gene analysis, respectively. The genome size (6.06 Mbp) was larger than that (4.39-5.07 Mbp) of the Sphaerotilus strains. The AAI values against the related strains ranged from 71.0 to 72.5%. The range of ANI values was 81.7 - 82.5%. In addition to these distinguishable features of the genome, the core genome and dDDH analyses suggested that this strain is a novel member of the genus Sphaerotilus. Based on its physiological properties and genomic features, strain FB-5 T is considered as a novel species of the genus Sphaerotilus, for which the name S. microaerophilus sp. nov. is proposed. The type strain is FB-5 T (= JCM 35424 T = KACC 23146 T).
Asunto(s)
Composición de Base , ADN Bacteriano , Ácidos Grasos , Filogenia , ARN Ribosómico 16S , Aguas del Alcantarillado , Aguas del Alcantarillado/microbiología , ARN Ribosómico 16S/genética , Ácidos Grasos/análisis , ADN Bacteriano/genética , Técnicas de Tipificación Bacteriana , Análisis de Secuencia de ADN , Japón , Genoma BacterianoRESUMEN
This review discusses critically how seasonal changes might affect the community composition and dynamics of activated sludge wastewater treatment plants (WWTP), and examines the factors thought more generally to control microbial community assembly, including the role of taxa-time relationships and stochastic and deterministic influences. The review also questions the differences in protocols used in these studies, which make any subsequent attempts at data comparisons problematic. These include bacterial DNA extraction and PCR methodologies, 16S rRNA sequencing and especially its depth, and subsequent statistical analyses of the data, which together often fail to reveal seasonal dynamic community shifts. Suggestions are given as to how experimental protocols need to be improved and standardized, and especially the requirement to examine bacterial populations at the species level. This review looks critically at what is known currently about seasonal influences on key members of this community, including viruses, the bacteria responsible for nitrogen and phosphorus removal and those causing bulking and foaming. The data show many of these species exhibit replicative seasonal abundances over several years, but not under all conditions, illustrating how complex these community dynamics are. Fungal and protozoal/metazoal seasonal community dynamics, less studied, are also discussed. The current data suggest that seasonal temperature fluctuations are responsible for most of seasonal community dynamics by selectively favouring or otherwise individual populations. However, more longer term studies carried out under much stricter controlled conditions are required.
RESUMEN
Amplicon sequence fingerprinting of communities in activated sludge systems have provided data revealing the true level of their microbial biodiversity and led to suggestions of which intrinsic and extrinsic parameters might affect the dynamics of community assemblage. Most studies have been performed in China and Denmark, and comparatively little information is available for plants in other countries. This study looked at how the communities of three plants in Victoria, Australia, treating domestic sewage changed with season. All were designed to remove nitrogen microbiologically. They were all located close together to minimise any influence that climate and demographics might have on their operation, and samples were taken at weekly intervals for 12 months. 16S rRNA amplicon sequencing revealed that each plant community was distinctively different to the others and changed over the 12-month sampling period. Many of the factors suggested in other similar studies to be important in determining community composition in activated sludge systems could not explain the changes noted here. The most likely influential factors were considered to be temperature and influent composition reflecting changes in dietary intake by the populations served by each plant, since in all three, the most noticeable changes corresponded to seasonal shifts. KEY POINTS: ⢠Monitoring microbial communities in 3 wastewater treatment plants removing nitrogen ⢠Temperature is the most influential factor in dynamic changes in community composition.
Asunto(s)
Reactores Biológicos , Nitrógeno , Purificación del Agua , Bacterias/genética , China , Desnitrificación , ARN Ribosómico 16S/genética , Aguas del Alcantarillado , Victoria , Eliminación de Residuos Líquidos , Aguas ResidualesRESUMEN
The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g-1 of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.
Asunto(s)
Carga Bacteriana , Biota , Chloroflexi/aislamiento & purificación , Aguas del Alcantarillado/microbiología , Chloroflexi/genética , Análisis por Conglomerados , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Hibridación Fluorescente in Situ , Japón , Metagenoma , Filogenia , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , Aguas Residuales/microbiologíaRESUMEN
Previous studies have shown that enhanced biological phosphorus removal (EBPR) performance under continuous aerobic conditions always eventually deteriorates; however, the speed at which this happens depends on the carbon source supplied. The published data suggest that propionate is a better carbon source than acetate is for maintaining operational stability, although it is not clear why. A lab-scale sequencing batch reactor was run initially under conventional anaerobic/aerobic conditions with either acetate or propionate as the carbon source. Chemical and microbiological analyses revealed that both sources performed as expected for such systems. When continuous aerobic conditions were imposed on both these established communities, marked shifts of the "Candidatus Accumulibacter" clades were recorded for both carbon sources. Here, we discuss whether this shift could explain the prolonged EBPR stability observed with propionate.
Asunto(s)
Betaproteobacteria/clasificación , Betaproteobacteria/metabolismo , Biota , Carbono/metabolismo , Fósforo/metabolismo , Acetatos/metabolismo , Aerobiosis , Reactores Biológicos/microbiología , Propionatos/metabolismoRESUMEN
Fe3O4 magnetic nanoparticles (MgNPs-Fe3O4) are widely used in medical applications, including magnetic resonance imaging, drug delivery, and in hyperthermia. However, the same properties that aid their utility in the clinic may potentially induce toxicity. Therefore, the purpose of this study was to investigate the cytotoxicity and genotoxicity of MgNPs-Fe3O4 in A549 human lung epithelial cells. MgNPs-Fe3O4 caused cell membrane damage, as assessed by the release of lactate dehydrogenase (LDH), only at a high concentration (100 µg/mL); a lower concentration (10 µg/mL) increased the production of reactive oxygen species, increased oxidative damage to DNA, and decreased the level of reduced glutathione. MgNPs-Fe3O4 caused a dose-dependent increase in the CD44+ fraction of A549 cells. MgNPs-Fe3O4 induced the expression of heme oxygenase-1 at a concentration of 1 µg/mL, and in a dose-dependent manner. Despite these effects, MgNPs-Fe3O4 had minimal effect on cell viability and elicited only a small increase in the number of cells undergoing apoptosis. Together, these data suggest that MgNPs-Fe3O4 exert little or no cytotoxicity until a high exposure level (100 µg/mL) is reached. This dissociation between elevated indices of cell damage and a small effect on cell viability warrants further study.
Asunto(s)
Membrana Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Compuestos Férricos/toxicidad , Nanopartículas de Magnetita/toxicidad , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Células Epiteliales/metabolismo , Glutatión/metabolismo , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/efectos de los fármacos , Humanos , Receptores de Hialuranos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Pruebas de Mutagenicidad , Oxidación-Reducción/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages.
Asunto(s)
Bacteriófagos , Podoviridae , Bacteriófagos/genética , Japón , Filogenia , Bases de Datos FactualesRESUMEN
Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [â4)-ß-d-GlcA-(1â4)-ß-d-Glc-(1â3)-ß-d-GalNAc-(1â4)-α-d-GalNAc-(1â4)-α-d-GalN-(1â]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.
Asunto(s)
Polisacárido Liasas/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Sphaerotilus/química , Paenibacillus/enzimologíaRESUMEN
Temperature is known to influence the operational efficiency of enhanced biological phosphorus removal (EBPR) systems. This study investigated the impact of thermal stress above 30°C on the properties of an EBPR community established with tropical inoculum. The results confirmed the stability of the 30°C EBPR system with high P-removal efficiency over 210 days. Accumulibacter was abundant in the community. When the EBPR sludge was subjected to a sudden temperature increase to 35°C under multiple cycles of anaerobic-aerobic phases, each lasting 4 h, high P-removal was maintained over 2 days, before gradually failing when the Competibacter appeared to outcompete Accumulibacter. These data suggested that the EBPR capacity is robust when subjected to occasional thermal stress. However, it could not be maintained even for a short time under temperature stress at 40°C. Thus, the threshold temperature for tropical EBPR failure is between 35°C and 40°C. PRACTITIONER POINTS: EBPR was stably maintained at 30°C with Accumulibacter being dominant. Good EBPR activities persisted for a short period at 35°C. EBPR was deteriorated at 40°C. The threshold temperature for tropical EBPR failure is between 35°C and 40°C.
Asunto(s)
Microbiota , Fósforo , Reactores Biológicos , Polifosfatos , Aguas del Alcantarillado , TemperaturaRESUMEN
Hair regenerative medicine is a promising approach for the treatment of hair loss and involves the transplantation of follicular stem cells into bald spots to regenerate hair. Various approaches have been investigated to engineer tissue grafts for use in hair regenerative medicine. Tissue-like three-dimensional aggregates, such as bioengineered hair follicle germs (HFGs), have shown great promise for hair regeneration, with normal tissue morphology and hair cycles. However, these approaches have not yet been applied in clinical settings, and further studies are needed to improve hair generation efficiency. The biological molecules in in vivo microenvironments around HFGs may provide cues for the in vitro preparation of HFGs with higher trichogenic functionalities. Activated platelet-rich plasma releasate (PRPr) is an autologous source of signaling molecules including growth factors and cytokines. In this study, we investigated the effects of PRPr on the preparation of HFGs in vitro. The presence of PRPr did not hinder the spontaneous formation of dumbbell-like HFGs from a suspension of embryonic skin-derived epithelial and mesenchymal cells in a custom-designed HFG culture plate. HFGs prepared with PRPr displayed greater levels of follicular gene expression compared to those prepared in the absence of PRPr. Moreover, the hair regeneration ability upon intracutaneous transplantation was significantly improved in the presence of PRPr. These results suggest that PRPr is beneficial for engineering HFGs for autologous hair regenerative medicine.
Asunto(s)
Folículo Piloso/citología , Folículo Piloso/fisiología , Plasma Rico en Plaquetas/metabolismo , Medicina Regenerativa/métodos , Humanos , Células Madre Mesenquimatosas/citología , Células Madre/citología , Ingeniería de TejidosRESUMEN
In this study, we assessed and optimized a low-dissolved-oxygen oxic-anoxic (low-DO OA) process to achieve a low-cost and sustainable solution for wastewater treatment systems in the developing tropical countries treating low chemical oxygen demand-to-nitrogen ratio (COD/N) wastewater. The low-DO OA process attained complete ammonia removal and the effluent nitrate nitrogen (NO3-N) was below 0.3 mg/L. The recommended hydraulic retention time and sludge retention time (SRT) were 16 h and 20 days, respectively. The 16S rRNA sequencing data revealed that long SRT (20 days) encouraged the growth of nitrite-oxidizing bacteria (NOB) affiliated with "Candidatus Nitrospira defluvii". Comammox made up 10-20% of the Nitrospira community. NOB and comammox related to Nitrospira were enriched at long SRT (20 days) to achieve good low-DO nitrification performance. The low-DO OA process was efficient and has simpler design than conventional processes, which are keys for sustainable wastewater treatment systems in the developing countries treating low COD/N wastewater.
Asunto(s)
Eliminación de Residuos Líquidos/métodos , Amoníaco , Bacterias , Análisis de la Demanda Biológica de Oxígeno , Reactores Biológicos , Nitratos , Nitrificación , Nitrógeno , Oxígeno , ARN Ribosómico 16S , Aguas del Alcantarillado/microbiología , Aguas Residuales/químicaRESUMEN
This study reveals that the abundance of the filament Kouleothrix (Eikelboom type 1851) correlated positively with poor settleability of activated sludge biomass in a Japanese full-scale nutrient removal wastewater treatment plant sampled over a one-year period. 16S rRNA amplicon sequence data confirmed that Kouleothrix was the dominant filament in the plant, with a relative abundance of 3.06% positively correlated with sludge volume index (SVI) (R = 0.691). Moreover, Kouleothrix (type 1851) appeared to form interfloc bridges, typical of bulking sludge, regardless of season. Together with earlier studies that indicated the responsibility of Kouleothrix (type 1851) on bulking events, these data suggest that their high relative abundances alone may be responsible for sludge bulking. 16S rRNA qPCR data for this filament showed changes in its relative abundance correlated with changes in several operational parameters, including mixed liquor temperature, sludge retention time, and suspended solids concentration, and it may be that manipulating these may help control Kouleothrix bulking.
Asunto(s)
Chloroflexi/crecimiento & desarrollo , Purificación del Agua , Aerobiosis , Anaerobiosis , Chloroflexi/genética , Dosificación de Gen , ARN Ribosómico 16S/genética , Análisis de Regresión , Aguas del Alcantarillado/microbiología , Factores de TiempoRESUMEN
Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-beta-hydroxyalkanoates or polyphosphate.
Asunto(s)
Chloroflexi/clasificación , Chloroflexi/aislamiento & purificación , Aguas del Alcantarillado/microbiología , Australia , Chloroflexi/citología , Chloroflexi/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Filogenia , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
This study firstly introduced a silicone rubber membrane (SRM) into microbial fuel cell (MFC) for passive oxygen supply to simultaneously remove phenol and nitrogen from synthetic coke-oven wastewater diluted with seawater. Passive oxygen transport with biofilm on the membrane was improved by ~ 18-fold in comparison with the one without a biofilm. In addition, although the oxygen supply was passive, nitrification accounted for 34% of those aeration conditions. It was also found that silicone rubber membrane can control NO2--N and/or NO3--N production. A dual-chamber MFC treating the synthetic coke-oven wastewater achieved a maximum power density of 54 mW m-2 with a coulombic efficiency of 2.7%. We conclude that silicone rubber membrane is effective for sustainable coke-oven wastewater treatment in MFCs.
Asunto(s)
Fuentes de Energía Bioeléctrica , Carbono/administración & dosificación , Membranas Artificiales , Nitrógeno/administración & dosificación , Oxígeno/administración & dosificación , Elastómeros de Silicona/químicaRESUMEN
Many wastewater treatment plants (WWTPs) operating in biological nitrogen removal activated sludge process in the tropics are facing the pressure of increasingly stringent effluent standards while seeking solutions to reduce the plants' energy consumption and operating cost. This study investigated the feasibility of applying low-dissolved oxygen (low-DO) nitrification and utilizing slowly-biodegradable chemical oxygen demand (sbCOD) for denitrification, which helps to reduce energy usage and operating cost in treating low soluble COD-to-nitrogen tropical wastewater. The tropical wastewater was first characterized using wastewater fractionation and respirometry batch tests. Then, a lab-scale sequencing batch reactor (SBR) was operated to evaluate the long-term stability of low-DO nitrification and utilizing sbCOD for denitrification in an anoxic-oxic (AO) process treating tropical wastewater. The wastewater fractionation experiment revealed that particulate settleable solids (PSS) in the wastewater provided slowly-biodegradable COD (sbCOD), which made up the major part (51⯱â¯10%) of the total COD. The PSS hydrolysis rate constant at tropical temperature (30⯰C) was 2.5 times higher than that at 20⯰C, suggesting that sbCOD may be utilized for denitrification. During the SBR operation, high nitrification efficiency (93⯱â¯6%) was attained at low-DO condition (0.9⯱â¯0.1â¯mg O2/L). Utilizing sbCOD for post-anoxic denitrification in the SBR reduced the effluent nitrate concentration. Quantitative polymerase chain reaction, 16S rRNA amplicon sequencing and fluorescence in-situ hybridization revealed that the genus Nitrospira was a dominant nitrifier. 16S rRNA amplicon sequencing result suggested that 50% of the Nitrospira-related operational taxonomic units were affiliated with comammox, which may imply that the low-DO condition and the warm wastewater promoted their growth. The nitrogen removal in a tropical AO process was enhanced by incorporating low-DO nitrification and utilizing sbCOD for post-anoxic denitrification, which contributes to an improved energy sustainability of WWTPs.
Asunto(s)
Nitrógeno/metabolismo , Eliminación de Residuos Líquidos/métodos , Análisis de la Demanda Biológica de Oxígeno , Desnitrificación , Nitrificación , Oxígeno , Aguas Residuales/químicaRESUMEN
Bacteriophages (phages) are biological entities that have attracted a great deal of attention in recent years. They have been reported as the most abundant biological entities on the planet and their ability to impact the composition of bacterial communities is of great interest. In this review, we aim to explore where phages exist in natural and artificial environments and how they impact communities. The natural environment in this review will focus on the human body, soils, and the marine environment. In these naturally occurring environments there is an abundance of phages suggesting a role in the maintenance of bacterial community homeostasis. The artificial environment focuses on wastewater treatment plants, industrial processes, followed by pharmaceutical formulations. As in natural environments, the existence of bacteria in manmade wastewater treatment plants and industrial processes inevitably attracts phages. The presence of phages in these environments can inhibit the bacteria required for efficient water treatment or food production. Alternatively, they can have a positive impact by eliminating recalcitrant organisms. Finally, we conclude by describing how phages can be manipulated or formulated into pharmaceutical products in the laboratory for use in natural or artificial environments.
RESUMEN
Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.
Asunto(s)
Folículo Piloso/citología , Medicina Regenerativa/métodos , Animales , Folículo Piloso/trasplante , Ratones Endogámicos C57BL , Ratones Desnudos , MicrotecnologíaRESUMEN
Thiothrix strains are filamentous sulfur-oxidizing bacteria common in activated sludge. Some of the members, including Thiothrix nivea and T. fructosivorans, are known to form a microtubular sheath that covers a line of cells. The sheaths are assemblages of [â4)-ß-d-GlcN-(1â4)-ß-d-Glc-(1â]n modified with unusual deoxy sugars. In an attempt to elucidate the sheath-forming mechanism, the patterns of sheath formation and cell proliferation were determined in this study. Prior to analysis, both sheaths were confirmed to be highly de-N-acetylated. Sheaths in viable filaments were N-biotinylated followed by cultivation and then fluorescently immunostained. Epifluorescence microscopy of the filaments revealed ubiquitous elongation of the sheaths. For visualization of the cell proliferation pattern, the cell membrane was fluorescently stained. The epifluorescence images demonstrated that cell proliferation also proceeds ubiquitously, suggesting that sheath elongation proceeds surrounding an elongating cell. In addition, the fine structure of the Thiothrix filaments was analyzed by transmission electron microscopy employing a freeze-substitution technique. The micrographs of freeze-substituted filaments showed that the sheaths were thin and single layered. In contrast, the sheaths in chemically fixed filaments appeared thick and multilayered. Treatment with glutaraldehyde probably caused deformation of the sheaths. Supporting this possibility, the sheaths were found to be deformed or solubilized by N-acetylation.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Aguas del Alcantarillado/microbiología , Thiothrix/química , Acetilación , Secuencia de Carbohidratos , Reactivos de Enlaces Cruzados/química , Desoxiazúcares/química , Desoxiazúcares/metabolismo , Colorantes Fluorescentes/química , Glutaral/química , Humanos , Hidrólisis , Coloración y Etiquetado/métodos , Propiedades de Superficie , Thiothrix/crecimiento & desarrollo , Thiothrix/ultraestructura , Eliminación de Residuos LíquidosRESUMEN
We developed a gold-coated membrane substrate modified with an oligopeptide layer that can be used to grow and subsequently detach a thick cell sheet through an electrochemical reaction. The oligopeptide CCRRGDWLC was designed to contain a cell adhesive domain (RGD) in the center and cysteine residues at both terminals. Cysteine contains a thiol group that forms a gold-thiolate bond on a gold surface. Cells attached to gold-coated membrane substrates via the oligopeptide layer were readily and noninvasively detached by applying a negative electrical potential to cleave the gold-thiolate bond. Because of the effective oxygen supply, fibroblasts vigorously grew on the membrane substrate and the thickness of the cell sheets was â¼60 µm at 14 days of culture, which was 2.9-fold greater than that of cells grown on a conventional culture dish. The cell sheets were detached after 7 min of electrical potential application. Using this approach, five layers of cell sheets were stacked sequentially with thicknesses reaching >200 µm. This approach was also beneficial for rapidly and readily transplanting cell sheets. Grafted cell sheets secreted collagen and remained at the transplanted site for at least 2 months after transplantation. This simple electrochemical cell sheet engineering technology is a promising tool for tissue engineering and regenerative medicine applications.
RESUMEN
Hydrogels that can be rapidly cross-linked under physiological conditions are beneficial for the engineering of vascularized 3-dimensional (3D) tissues and organs, in particular when cells are embedded at a high cell density or tissues are fabricated using bottom-up processes, including bioprinting and micromolding. Here, we prepared a gelatin-carboxymethylcellulose (CMC) hydrogel that cross-linked rapidly (<30 s) by mixing hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified CMC (CMC-CHO). Vascular endothelial cells encapsulated in the gelatin-CMC hydrogels were viable and sprouted readily, indicating that the hydrogels and their cross-linking reactions were cytocompatible and provided a suitable microenvironment for angiogenesis. Sprouting length of the vascular endothelial cells was modulated by altering the stiffness of the hydrogels and varying the concentrations of the two hydrogel components. Furthermore, we used an electrochemical reaction to detach cells from a gold electrode surface. In this approach, cells that were seeded on a gold surface via the oligopeptide layer, detached rapidly along with the electrochemical desorption of the layer and transferred to the hydrogel. Owing to the rapid gelation of the hydrogels and rapid electrochemical detachment of cells, cell transfer was completed within 10 min (including 30 s of gelation and 5 min of potential application). Rapid cell transfer was observed not only on a flat surface but also on different shapes, such as cylindrical needles. Vascular endothelial cells were transferred from needles onto the hydrogel to fabricate endothelial cell-enveloped microchannels. In subsequent perfusion culture, the transferred endothelial cells migrated and formed luminal structures in the hydrogel. This in situ cross-linkable hydrogel may be useful for the rapid fabrication of perfusable vascular networks to engineer vascularized and cell-dense 3D tissues and organs.