Outer Layers Of Biofilm Research Articles

High-resolution 3D spatial distribution of complex microbial colonies revealed by mass spectrometry imaging

IntroductionBacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples. ObjectivesThe study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies. MethodsThis article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization. ResultsCombined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains. ConclusionThese results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.

Model-based identification and testing of appropriate strategies to minimize N2O emissions from biofilm deammonification.

Based on a one-year pilot plant operation of a two-step biofilm nitritation-anammox pilot plant, N2O mitigation strategies were identified by applying a newly developed biofilm modeling approach. Due to adapted plant operation, the N2O emission could be diminished by 75% (8.8% → 2.3% of NH4-Noxidized_AOB). The results (measurement and simulation) confirm the huge importance of denitrification as an N2O source or N2O sink, depending on the boundary conditions. A significant reduction of N2O emissions could only be achieved with a one-step deammonification system, which is related to low nitrite and HNO2 concentrations. Increased oxygen concentrations in the bulk phase are not related to decreased emissions. N2O formation by ammonium-oxidizing bacteria (AOB) just shifts deeper into the biofilm; zones with low oxygen concentrations are not avoidable in biofilm systems. Low oxygen concentrations in the bulk phase, however, result in a reduction of the total net N2O formation due to increased activity of heterotrophic bacteria directly at the source of N2O formation (outer biofilm layer). For the model-based identification of mitigation strategies, the standard modeling approaches for biofilms were expanded by including the factor-based N2O formation and emission approach. The new model 'Biofilm/N2OISAH' was successfully validated using data from pilot-scale measurement campaigns. Altogether, the investigation confirms that the employed digital model can strongly support the development of N2O mitigation strategies without the need for specialized measurement inside the biofilm.

Higher performances of open vs. closed circuit microbial fuel cell sensor for nitrate monitoring in water

Microbial fuel cell (MFC) sensor exhibits attractive prospects for real-time monitoring of nitrate (NO3-) in water, with unique advantages of self-powering and simple structure; but its stability and sensitivity are unsatisfactory. In an attempt to address the limitations, here we employed an open-circuit MFC (O-MFC) sensor for real-time monitoring of NO3- in water, compared to a conventional closed-circuit MFC (C-MFC) sensor. The stability of the O-MFC sensor was confirmed when subject to the continuous fluctuation of interfering substances including organic matter (acetate, 2–20 mM), SO42- (250–400 mg/L) and Fe3+ (0.3–0.6 mg/L). For one-time and continuous monitoring of NO3- at a concentration of 1–40 mg/L, the O-MFC sensor both achieved higher sensitivity than that of the C-MFC sensor; especially at a low NO3- concentration of 1 mg/L, the changes of electrical signal (∆E) reached 10.0 ± 1.00 mV and 7.0 ± 0.85 mV respectively. In contrast, the C-MFC sensor failed to monitor at low NO3- concentrations of 1 and 5 mg/L. Moreover, good linear correlation between ∆E and NO3- concentration (R2 = 0.907 and 0.981) was obtained from the O-MFC sensor. However, owing to the highly activity of EAB that mainly grows in the outer layer of biofilm, the response time of the O-MFC sensor is too long. Microbial community analysis further revealed that the open-circuit condition is more suitable for NO3- monitoring in water, providing a new way to improve the performance of MFC sensors.

Dynamic Dispersal of Surface Layer Biofilm Induced by Nanosized TiO2 Based on Surface Plasmon Resonance and Waveguide.

Pollutant degradation is present mainly in the surface layer of biofilms, and the surface layer is the most vulnerable to impairment by toxic pollutants. In this work, the effects of nanosized TiO2 (n-TiO2) on the average thicknesses of Bacillus subtilis biofilm and on bacterial attachment on different surfaces were investigated. The binding mechanism of n-TiO2 to the cell surface was also probed. The results revealed that n-TiO2 caused biofilm dispersal and the thicknesses decreased by 2.0 to 2.6 μm after several hours of exposure. The attachment abilities of bacteria with extracellular polymeric substances (EPS) on hydrophilic surfaces were significantly reduced by 31% and 81% under 10 and 100 mg/liter of n-TiO2, respectively, whereas those of bacteria without EPS were significantly reduced by 43% and 87%, respectively. The attachment abilities of bacteria with and without EPS on hydrophobic surfaces were significantly reduced by 50% and 56%, respectively, under 100 mg/liter of n-TiO2 The results demonstrated that biofilm dispersal can be attributed to the changes in the cell surface structure and the reduction of microbial attachment ability.IMPORTANCE Nanoparticles can penetrate into the outer layer of biofilm in a relatively short period and can bind onto EPS and bacterial surfaces. The current work probed the effects of nanosized TiO2 (n-TiO2) on biofilm thickness, bacterial migration, and surface properties of the cell in the early stage using the surface plasmon resonance waveguide mode. The results demonstrated that n-TiO2 decreased the adhesive ability of both cell and EPS and induced bacterial migration and biofilm detachment in several hours. The decreased adhesive ability of microbes and EPS worked against microbial aggregation, reducing the effluent quality in the biological wastewater treatment process.

Sonobactericide - An Adjunct Therapy for Infective Endocarditis: An In Vitro Proof-of-Concept

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent in combination with an antibiotic and a thrombolytic. We developed a model of IE vegetations using human whole-blood clots infected with S. aureus. Histology and live-cell imaging revealed an outer biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity®). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% clot width loss, which was greater than the other treatment arms. Size measurements of the effluent suggested low emboli risk. These results demonstrate that sonobactericide could be an effective therapeutic strategy for treating IE.

Study on the Structure of Candida Albicans-Staphylococcus Epidermidis Mixed Species Biofilm on Polyvinyl Chloride Biomaterial.

The objective of the study was to establish an in vitro model of Candida albicans-Staphylococcus epidermidis mixed species biofilm (BF) on polyvinyl chloride (PVC) material, and to investigate the formation and the structure of mixed species BF formation using a combined approach of confocal laser scanning microscope (CLSM), scanning electron microscope (SEM), and 3D image reconstruction technique. Mixed species BF is achieved by co-incubating Staphylococcus epidermidis bacteria (ATCC35984) and Candida albicans fungal (ATCC10231) with PVC pieces in Tris-buffered saline. BF formation was examined at 2, 6, 12, 24, 48, and 72h of co-culture. Thickness of these BFs and the number, and percentage of viable cells in BFs were measured. CT scan images of BFs were obtained using CLSM and SEM and reconstructed 3D images of mixed species BF were acquired, in an effort to examine structure of the BF. Staphylococcus epidermidis attached to various forms of candida albicans (spores, pseudohyphae, and hyphae), formed complex and dense mesh arrays. The BF is constituted of a large number of viable and dead pathogens, the surface of mixed species BF is uneven, with living pathogens predominating protrusive portions and dead pathogens aggregating in concaves. Mixed species BF formation on the surface of PVC material was found to be a dynamic process, with rapid growth being at 24h of co-culture, maximal thickness peaked at 48h. These mixed species BF matured at 48-72h. Significant differences were observed in the proportion of viable cells between interior, middle, and outer layers of BFs (p<0.05). Mixed species BF Candida albicans-Staphylococcus epidermidis is sophisticated in structure. The combined approach involving CLSM, SEM, and 3D image reconstruction technique is ideal for the investigation of mixed species BF on PVC material.

Microanatomy at Cellular Resolution and Spatial Order of Physiological Differentiation in a Bacterial Biofilm

ABSTRACTBacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σS (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as “anatomical” hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in “curli baskets.”

Effect of ZnCl2 on plaque growth and biofilm vitality

ObjectiveThe aim of this study was to evaluate the effects of ZnCl2 on plaque-growth and vitality pattern of dental biofilm and to determine the optimum zinc concentration for the inhibition of plaque formation. DesignData were collected from nine volunteers for whom a special-designed acrylic appliance was prescribed after a careful dental check up. The volunteers rinsed twice daily for 2min with ZnCl2 of 2.5, 5, 10, 20mM as treatment and double distilled water (DDW) as control in respective assigned test weeks. The plaque index (PI) was assessed after 48h of appliance wearing. The glass discs with the adhered biofilm were removed from the splints and stained with two fluorescent dyes. The biofilm thickness (BT) and bacterial vitality of the whole biofilm as well as the mean bacterial vitality (BV) of the inner, middle and outer layers of biofilm were evaluated under confocal laser scanning microscope (CLSM). ResultsPI, BT and BV of biofilms treated by various concentrations of ZnCl2 were reduced significantly when compared with the DDW group (p<0.05). PI, BT and BV of the 2.5mM ZnCl2 group was significantly higher than groups of 5, 10, 20mM ZnCl2 (p<0.05). The mean BV of the 3 layers (inner, middle and outer layers) showed that 2.5mM ZnCl2 was the lowest concentration to inhibit BV in the outer layer, 5mM was the lowest concentration to extend this inhibition of BV to the middle layer, and none of the concentrations investigated in this study has shown any effect on bacteria inhibition in the inner layer. ConclusionZinc ions exhibited possible inhibitory effects on plaque formation, and have a promising potential to be used as an antibacterial agent in future dentifrices and mouthrinses.

Nitrite-oxidizing bacteria, Nitrospira, distribution in the outer layer of the biofilm from filter materials of a recirculating water system for the goldfish Carassius auratus

We used a clone library method to investigate changes in the biofilm microflora associated with the conditioning of filter materials in a recirculating water system for the culture of goldfish Carassius auratus. The microbial density was higher in the outer layer of biofilm from filter materials (1.7 × 10 10–3.0 × 10 10 cells/g) than in the inner layer (1.5 × 10 9–2.4 × 10 9 cells/g) throughout the experimental period. The clone library method using bacterial 16S rRNA genes collected from the outer layer of filter material yielded sequences from four (day 8), nine (day 15), twelve (day 22) and nine (day 64) taxonomic categories of bacteria including Acidobacteria, Actinobacteria, Bacilli, Bacteroidetes, Flavobacteria, Fusobacteria, Nitrospira, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Verrucomicrobia and unclassified bacteria. The inner layer yielded sequences from six (day 8), eight (day 15), five (day 22) and five (day 64) taxonomic categories of bacteria including Acidobacteria, Actinobacteria, Bacilli, Flavobacteria, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Sphingobacteria, Verrucomicrobia and unclassified bacteria. Bacteria in the outer layer of biofilm belonged predominantly to the genera Acinetobacter, Cetobacterium, Comamonas, Flectobacillus, Flavobacterium, Ideonella and Pseudomonas, whereas those in the inner layer were predominantly members of the genera Flavobacterium, Flexibacter, Ideonella, Janthinobacterium, Pedobacter and Pseudomonas. As the filter materials became conditioned, the population of nitrite-oxidizing bacteria related to Nitrospira was restricted to the outer layer of the biofilm. In addition, phylogenetic analysis indicated the presence of both an indigenous Nitrospira and a distinct type of Nitrospira that appeared after conditioning had begun.

Identification and quantification of nitrogen removal in a rotating biological contactor by 15N tracer techniques

High autotrophic nitrogen removal rates of 858 mg N L −1 day −1 or 1.55 g N m −2 day −1 were obtained in a lab-scale rotating biological contactor treating an ammonium rich influent. It was postulated that ammonium was removed as dinitrogen gas by a sequence of aerobic ammonium oxidation to nitrite taking place in the outer biofilm layer and anaerobic ammonium oxidation with nitrite as electron acceptor occuring in the deeper biofilm layer. Chemical evidence for anaerobic ammonium oxidation within intact biofilm sludge from a lab-scale rotating biological contactor could be provided, without direct identification of responsible organisms catalysing this reaction. 15N tracer techniques were used for identification and quantification of nitrogen transformations. In batch tests with biofilm sludge at dissolved oxygen concentrations lower than 0.1 mg L −1, ammonium and nitrite did react in a stoichiometric ratio of 1:1.43 thereby forming dinitrogen. 15N isotope dilution calculations revealed that anaerobic ammonium oxidation was the major nitrogen transformation leading to concomitant ammonium and nitrite removal. Isotopic analysis of the produced biogas showed that both ammonium-N and nitrite-N were incorporated in N 2.

Strategies for augmenting the pentachlorophenol degradation potential of UASB anaerobic granules

Anaerobic degradation of pentachlorophenol (PCP) is an example of a process that may benefit from enrichment or bioaugmentation. In one approach, enrichment acceleration was attempted by applying an on-line control-based selective stress strategy to a native anaerobic upflow sludge bed (UASB) system; this strategy linked PCP loading rate to methane production. As a result, the reactor biomass potential for PCP complete dechlorination reached a rate of 4 mg g(-1) volatile suspended solid (VSS) day(-1) within a period of 120 days. In another approach, a pure culture, Desulfitobacterium frappieri PCP-1, a strictly anaerobic Gram-positive bacterium, was used to augment the granular biomass of the UASB reactor. This also resulted in a specific degradation rate of 4 mg PCPg(-1) VSS day(-1); however, this potential was attained within 56 days. Fluorescent in situ hybridization (FISH) showed that the PCP-1 strain was able to rapidly attach to the granule and densely colonize the outer biofilm layer.

Spatial distribution of respiratory activity in Pseudomonas putida 54G biofilms degrading volatile organic compounds (VOC).

All over the world, Microbial systems are used to clean soils, waters and air streams that have been contaminated with volatile organic compounds (VOC). Information about the structure and function of the microbes that metabolize these contaminants can be gained by studying these microbial systems. Here we describe the spatial patterns of respiratory activity in Pseudomonas putida 54G aerobic biofilms degrading two VOC, toluene and ethanol. Oxygen concentration profiles within the biofilm were measured using microsensors. These profiles are thought to be most accurate reflection of the structure and function of aerobic microbial biofilms. The degrading process certainly imposed a structural and functional patterns on the microbial biofilm community growing at the expense of the VOC substrate. Cryosectioning coupled with the staining of biofilm samples confirmed a high respiratory activity near the substratum, that decreased towards the biofilm/fluid interface. The accumulation of inactive cells in the outer biofilm layer protects the inner biofilm from high concentrations of toxic compounds and also limits the degradation rate. This stratification phenomenon appeared to be a general pattern for P. putida 54G biofilms degrading VOC.

Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of nitrifiers and heterotrophs

A bench-scale biological aerated filter (BAF) was operated under a range of substrate loadings and C/N influent ratios. Start-up with a substrate loading of 4.8 COD kg m−3 d−1 and an influent C/N ratio of 1.5 gave no nitrification, due to the high biofilm detachment rate caused by the daily backwashing. Subsequent runs with lower substrate loadings and backwashing rates allowed stable nitrification. A final run with the initial substrate loading and backwash rate gave stable nitrification. Oligonucleotide probes targeted to the 16S rRNA of the nitrifiers, as well as to other types of microorganisms, were used to characterize how the changes in loading and detachment affected the microbial structure in the biofilms. The proportion of nitrifiers decreased with increasing C/N ratio. The biofilm remaining after backwashing also contained a higher fraction of nitrifiers than did the backwash solids stripped from the outer layers of biofilm, which consisted mainly of active heterotrophs. Thus, stable nitrification was possible only when the nitrifiers became established deeper inside the biofilm.

Influence of substrate C/N ratio on the structure of multi-species biofilms consisting of nitrifiers and heterotrophs

A bench-scale biological aerated filter (BAF) was operated under a range of substrate loadings and C/N influent ratios. Start-up with a substrate loading of 4.8 COD kg m-3 d-1 and an influent C/N ratio of 1.5 gaveno nitrification, due to the high biofilm detachment rate caused by the daily backwashing. Subsequent runs with lower substrate loadings and backwashing rates allowed stable nitrification. A final run with the initial substrate loading and backwash rate gave stable nitrification. Oligonucleotide probes targeted to the 16S rRNA of the nitriflers, as well as to other types of microorganisms, were used to characterize how the changes in loading and detachment affected the microbial structure in the biofilms. The proportion of nitrifiers decreased with increasing C/N ratio. The biofilm remaining after backwashing also contained a higher fraction of nitrifiers than did the backwash solids stripped from the outer layers of biofilm, which consisted mainly of active heterotrophs. Thus, stable nitrification was possible only when the nitrifiers became established deeper inside the biofilm.

Evaluation of multiple-species biofilm and floc processes using a simplified aggregate model

A simplified model of microbial aggregates is developed to evaluate how the key features of aggregation affect the stability of nitrification in multiple-species biofilms and flocs. The foundation of the model is a layered system in which each layer contains a different type of biomass, includes mass-transport resistance for all substrates (i.e., COD, NH4+, and O2), and included formation and consumption of soluble microbial products. The model describes how the outer biofilm layer of heterotrophs protects the inner layers of nitrifiers and inert biomass from detachment, so that an inner layer can have a low specific detachment-loss rate. This protecting function is not important for suspended flocs, because the entire floc is wasted. Comparison of biofilm and suspended flocs shows that the protecting function of the biofilms provides greater nitrification stability. The model also demonstrates that extensive heterotrophic growth induced by high organic loading can cause a deterioration of nitrification when the dissolved oxygen is depleted in the inner layer of the aggregate. Although it is based on a simplified spatial distribution, the aggregate model identifies key differences among biofilm, floc, and dispersed-growth processes.