Continuous-flow Biofilm Research Articles

Microalgae biofilm photobioreactor and its combined process for long-term stable treatment of high-saline wastewater achieved high pollutant removal efficiency

High-saline wastewater treatment processes have a high energy demand, while microalgae-driven wastewater biotechnology is both economical and environmentally friendly, and the use of microalgae to treat high-saline wastewater can effectively reduce energy consumption. However, it is unclear whether the performance of microalgae reactor can be stable for a long time. In this study, an open continuous-flow biofilm photobioreactor using Dunaliella salina was innovatively constructed, and operated in combination with a membrane bioreactor (MBR) for high-saline wastewater treatment. The purpose of this study was to investigate the long-term performance of MBR, MBPBR alone and their combined operation for the removal of organic matter, phosphorus and nitrogen, explore the pathway and mechanism of nitrogen and phosphorus removal by MBPBR, and evaluate the feasibility of MBPBR-MBR integrated system for the treatment of saline wastewater. The MBR effectively removed most of the organic matter and converted ammonia nitrogen into nitrate nitrogen, which is a preferred nitrogen source for the growth of Dunaliella salina, thereby providing favorable water quality for Dunaliella salina in the subsequent MBPBR. The MBPBR, with a hydraulic retention time (HRT) of 72 h, achieved a NO3--N removal load of 74.9 g N/(m3∙d) and a removal efficiency of approximately 60 %, and the PO43--P removal load of 8.33 g P/(m3∙d) and removal efficiency of over 80 %. The MBR-MBPBR system demonstrated long-term stability, achieving removal efficiencies of 84.3 %, 81.6 %, and 91.0 % for COD, TN and PO43--P, respectively. Within the MBPBR, the growth of algae and their assimilation contributed 44.9 % and 38.7 % of the nitrogen and phosphorus removal, respectively. The abundance of Dunaliella salina in the MBPBR remained at approximately 90 % for over 40 days of operation, providing an opportunity for Dunaliella salina recovery. These findings provide valuable insights into the utilization of microalgae for the treatment of high-salinity wastewater, while also supporting the practical implementation of microalgae-based treatment methods for such wastewater.

Design and fabrication of TiO2/Nd polyurethane nanofibers based photoreactor: A continuous flow kinetics study for Estriol degradation and mechanism

Novel polyurethane (PU) nanofibers incorporated with the neodymium (Nd) doped TiO2 nanoparticles were fabricated by electrospinning process and employed for the photocatalytic degradation of the estriol (E3) hormone. Nanofibers possessed high porosity (0.47 μm) and uniform mesh-like structure while suitable incorporation of TiO2/Nd nanoparticles with increasing concentrations (5, 10, and 20 %) was obtained. The TiO2/Nd incorporated PU nanofiber photocatalytic activity under UV and visible light was evaluated using a continuous drip flow biofilm reactor. To establish sufficient adherence to the PU matrix necessary to maintain durability during crossflow reactor, the as-prepared nanofibers were thermally pressed on polyethylene terephthalate (PET) sheets to avoid any potential folding. The nanofibers performed better in UV than visible, with maximum degradation rates of ∼90.2 % and 30.4 %, respectively, at pH 7 in 120 min. The degradation performance of nanofibers increased with an increase in the number of layers and decreased with the E3 solution flow rate. The experimental data best fitted for the pseudo-first-order kinetic model with regression coefficient (R2) > 0.9. The highest degradation removal capacities were 2.436 and 0.847 mg/g under UV and visible light irradiation, respectively. The results showed that TiO2/Nd incorporated PU nanofibers exhibit a potential to degrade hormones from environmental water.

Novel hyperthermoacidic archaeal enzymes for removal of thermophilic biofilms from stainless steel.

To test the efficacy of novel hot/acid hyperthermoacidic enzyme treatments on the removal of thermophilic spore-forming biofilms from stainless steel surfaces. The present study measured the efficacy of hyperthermoacidic enzymes (protease, amylase, and endoglucanase) that are optimally active at low pH (≈3.0) and high temperatures (≈80°C) at removing thermophilic bacilli biofilms from stainless steel (SS) surfaces. Plate counts, spore counts, impedance microbiology, as well as epifluorescence microscopy and Scanning Electron Microscopy (SEM) were used to evaluate cleaning and sanitation of biofilms grown in a continuous flow biofilm reactor. Previously unavailable hyperthermoacidic amylase, protease, and the combination of amylase and protease were tested on Anoxybacillus flavithermus and Bacillus licheniformis; and endoglucanase was tested on Geobacillus stearothermophilus. In all cases, the heated acidic enzymatic treatments significantly reduced biofilm cells and their sheltering extracellular polymeric substances (EPS). Hyperthermoacidic enzymes and the associated heated acid conditions are effective at removing biofilms of thermophilic bacteria from SS surfaces that contaminate dairy plants.

Quorum sensing responses of r-/K-strategists Nitrospira in continuous flow and sequencing batch nitrifying biofilm reactors.

A better understanding of r-/K-strategists nitrifiers will help to balance the design and operation of bioprocesses for efficient pollution removal from wastewater. The objectives of study were to investigate the nitrite oxidation biokinetics, biofilm property, microbial community and quorum sensing (QS) of nitrifying biofilm in a continuously flow reactor (CFR) and a sequencing batch reactor (SBR). Results showed that nitrite-oxidizing bacteria were estimated to have a nitrite half saturation constant of 76.23 and 224.73 μM in CFR and SBR, respectively. High-throughput and metagenomic sequencing results showed that Nitrospira and Candidatus Nitrospira defluvii were the dominated nitrite-oxidizing taxa performing nitrite oxidation in both reactors. Nitrifying biofilm developed in CFR and SBR showed obviously different properties. Biofilm in SBR had an obviously higher ratio of polysaccharide and protein in extracellular polymeric substances, and higher thickness than in CFR. Metagenomics and chemical analysis revealed various types of acyl-homoserine lactone (AHL) circuit genes (e.g., luxI, lasI, hdtS) and four types of AHL signaling substances (e.g., C6-HSL, C8-HSL, C10-HSL and 3-oxo-C10-HSL) in nitrifying biofilm. The concentrations of these AHLs in biomass and water phases were obviously higher in SBR than that in CFR. Together, AHLs-based QS might affect the formation of nitrifying biofilm and thus contribute to the different biokinetics of Nitrospira in CFR and SBR. Our insights may reveal the molecular mechanism of Nitrospira for different biokinetics, and indicate the AHL association with Nitrospira adaptation to various conditions.

Influence of Fe2O3 and bacterial biofilms on Cu(II) distribution in a simulated aqueous solution: A feasibility study to sediments in the Pearl River Estuary (PR China)

This study investigated physico-chemical interactions among Cu(II), biogenic materials, and Fe2O3 in a continuous-flow biofilm reactor system under a well-controlled environment. The effects of Fe2O3 and bacterial biofilms on the distribution of Cu(II) in a simulated aquatic environment were studied. To control biological and abiotic elements in the marine environment, a biofilm reactor was designed to understand the metal speciation of Cu(II) and its distribution. The reactor consisted of a biofilm chamber equipped with glass slides for biofilms attachment. Due to its ability to grow as biofilm in the medium, Pseudomonas atlantica was cultivated to adsorb trace Cu(II) to attached and suspended cells. It was found that biofilms with 170–285 mequiv chemical oxygen demand (COD) concentration/m2 of total oxidizable materials accelerated the Cu(II) adsorption to the surface of the reactor significantly by a factor of five. A significant inhibition to the bacterial growth took place (p ≤ 0.05; t-test) when Cu(II) concentration was higher than 0.5 mg/L. In the absence of Cu(II), bacterial cells grew normally to 0.075 of optical density (OD). However, at the Cu(II) concentration of 0.2 mg/L, the cells grew to a lower OD of 0.58. The presence of glycine and EDTA substantially reduced the toxicity of Cu(II) on bacterial growth (p ≤ 0.05; paired t-test). Their complexation with Cu(II) rendered the metal ions less available to bacterial cells. This implies that the Fe2O3 and bacterial biofilm affected Cu(II) distribution and speciation in the aquatic environment.

Evaluating CO2 emissions from continuous flow and batch growth systems under autotrophic mode: Implications for GHG accounting of biological nutrient removal

The oxidation of ammonia by autotrophic bacteria is a central part of the nitrogen cycle and a fundamental aspect of biological nutrient removal (BNR) during wastewater treatment. Autotrophic ammonia oxidation produces protons and results in net-CO2 production due to the neutralizing effect of bicarbonate alkalinity. Attention must be paid to the propensity for this produced CO2 to be transferred to the atmosphere where it can act as a greenhouse gas (GHG). In the context of BNR systems, bicarbonate-derived CO2 emissions should be considered distinct from the biogenic CO2 that arises from cellular respiration, though this distinction is not made in current GHG accounting practices. The aim of this study was to evaluate the performance of two experimental systems operated under autotrophic mode and buffered with bicarbonate, to investigate the relationship between ammonia removal and gaseous CO2 emissions. The first system consisted of continuously aerated lab-scale batch reactors, which were effective in demonstrating the important link between ammonia oxidizer activity, pH, and gaseous CO2 production. Depletion of the buffer system always led to a rapid decline in system pH and cessation of CO2 emissions when the pH fell below 7.0. The second system was a tubular continuous-flow biofilm reactor which permitted comparison of ammonia removal and CO2 emission rates. A linear relationship between ammonia removal and CO2 emissions was demonstrated and the quantified CO2 production was relatively close to that which was predicted based on the stoichiometry of nitrification, with this CO2 being detected in the gas phase. It was apparent that this system offered minimal resistance to the mass transfer of CO2 from the liquid to gas, which is an important factor that determines how much of the bicarbonate-derived CO2 may contribute to greenhouse gas emissions in engineered systems such as those used for BNR.

Mannose Conjugated Polymer Targeting P.aeruginosa Biofilms.

Biofilms are one of the most challenging obstacles in bacterial infections. By providing protection against immune responses and antibiotic therapies, biofilms enable chronic colonization and the development of antibiotic resistance. As previous clinical observations and studies have shown, traditional antibiotic therapy alone cannot effectively treat and eliminate biofilm forming infections due to the protection conferred by the biofilm. A new strategy specifically targeting biofilms must be developed. Here, we specifically target and bind to the PAO1 biofilm and elucidate the molecular mechanism behind the interaction between a glycan targeted polymer and biofilm using a continuous flow biofilm model. The incubation of biofilms with fluorescent glycan targeted polymers demonstrated strong and persistent interactions with the mannose-containing polymer even after 24 h of continuous flow. To evaluate the role of major biofilm proteins LecB and CdrA, loss of function experiments with knockout variants established the dual involvement of both proteins in mannose targeted polymer retention. These results identify a persistent and specific targeting strategy to the biofilm, emphasizing its potential value as a delivery strategy and encouraging further exploration of biofilm targeted delivery.

Bacterial Community Shifts and Nitrogen Removal Characteristics for a SNAD Process Treating Anaerobic Digester Liquor of Swine Wastewater (ADLSW) in a Continuous-Flow Biofilm Reactor (CFBR)

To realize a simultaneous partial nitrification, ANAMMOX (anaerobic ammonium oxidation), and denitrification (SNAD) process treating anaerobic digester liquor of swine wastewater (ADLSW) in a continuous-flow biofilm reactor (CFBR), we first gradually increased the influent ammonium (NH4+-N) concentration, and then enhanced the ADLSW ratio in the influent during operation; dissolved oxygen (DO) was controlled at (0.4±0.1) mg·L-1 by adjusting the air flow rate, and the temperature was kept at (30±1)℃. Meanwhile, high-throughput sequencing and quantitative PCR (polymerase chain reaction) techniques were used to analyze the bacterial community shifts and the amount of dominant nitrogen removal bacteria. The results demonstrated that a successful start-up of the SNAD process was accomplished in 150 d, and replacement of the actual biogas slurry was completed in 298 d. The effluent (NO3--N+NO2--N)/ΔNH4+-N value was less than 0.11, and the average removal rates of NH4+-N and TN (total nitrogen) increased to 63.26% and 55.71%, respectively. Moreover, high-throughput sequencing results demonstrated that the dominant microbial populations at phylum level were Chloroflexi (with a relative abundance of 50.78%), Proteobacteria (13.34%), and Planctomycetes (9.26%). The relative abundance of Nitrosomonas increased from 1.55% to 1.98%. In addition, the relative abundance of Candidatus_Brocadia and Candidatus_Kuenenia increased from 0.01% and (<0.01%) to 4.66% and 4.18%, respectively, and the relative abundance of Denitratisoma increased from (<0.01%) to 2.06%. Meanwhile, qPCR analysis showed that the amounts of ammonia-oxidizing bacteria, ANAMMOX, and denitrifying bacteria increased significantly compared with the inoculated sludge. An efficient and stable nitrogen removal rate can be achieved, and the follow-up processing cost can be reduced, by application of the SNAD treatment process for ADLSW.

Effect of exposure time ratio on Intermittent Aerated Moving Bed Biofilm Reactor

This study combined the IA craft, and the goal is to achieve aerobic, anoxic and even anaerobic alternating cycles in the same reactor to enhance biological nitrogen and phosphorus removal. At present, there are many studies on the IA process, but less research on the IAMBBR craft. The IAMBBR craft combines intermittent aeration with a biofilm process, which can further improve the processing capacity of a continuous-flow biofilm reactor. Therefore, it is necessary to conduct a detailed study of the IAMBBR craft. In addition, the exposure time ratio is an important parameter for the operation of the IA craft. By adjusting the exposure time ratio, the DO concentration in the reactor can be controlled, which affects the operation effect of the reactor. Therefore, this chapter mainly explores the effect of exposure time ratio on the operating effect of IAMBBR. Six operating stages were connected: the ratio of CA and exposure time are 3h/3h, 1h/1h, 30min/30min, 15min/15min, and 5min/5min, respectively, to study the removal effect of pollutants.

Antimicrobial effect of herbal extract of Acacia arabica with triphala on the biofilm forming cariogenic microorganisms

BackgroundDental caries is a biofilm-related infectious disease with a multifactorial etiology, over five billion inhabitants have affected worldwide due to this disease. ObjectiveAntimicrobial efficacy of a mixed herbal powder extract (MHPE) against cariogenic microorganisms was investigated. Materials and methodsMIC, MBC, kinetics of killing, biofilm disruption and anticaries effect of MHPE were determined. For biofilm disruption, biofilms of Streptococcus mutans, Lactobacillus casei, Actinomyces viscosus and Candida albicans were treated with MHPE for 30 min and attached cells were quantified after staining. For live/dead staining biofilm assay, S. mutans biofilm treated with MHPE for 1min, 5min and 1 h was examined with confocal laser scanning system after live/dead staining. Efficacy was experimented by structural quality using Scanning Electron Microscope (SEM). Anticaries effect was determined by formation of caries-like lesion in continuous flow biofilm model. ResultsMHPE exhibited inhibition zones ranging from 12.5 to 24.0 mm. The highest inhibition zone was recorded at concentration of 50 μg/ml. MIC for S. mutans was between 12.23 and 36.7 μg/ml, while the MBC values ranged from 36.7 to 110.65 μg/ml. Inhibitory concentration of MHPE was three fold higher than CHLX. Significant reduction of cell count (49–95%) was observed with increasing time and higher concentration. Percentage biofilm reduction compare with negative control was 96.9% (A. viscosus), 94% (C. albicans), 99.8% (L. casei) and 91.7% (S. mutans). For MHPE-treated biofilm, live/dead staining demonstrated significant (p < 0.05) higher in deceased red fluorescence areas in all kinetics points from 53.6% (1min) to 85% (1h). SEM confirmed the damage in the outer layers of S. mutans. MHPE has components with effective antibacterial activity against caries-inducing microorganisms. ConclusionThe anti-adherence and anti-biofilm effect as well as the faster killing activity suggests that MHPE formula has effective antibacterial activity and could be a useful source of anti-cariogenic agents in near future.

Biodegradation of resin–dentin interfaces is dependent on the restorative material, mode of adhesion, esterase or MMP inhibition

ObjectiveTo measure the effect of simulated human salivary esterases (SHSE) and metalloproteinases (MMP) inhibition on the integrity of restoration–tooth interfaces made from traditional or polyacid-modified resin composites bonded to human dentin by either total-etch or self-etch adhesives. MethodsResin–dentin specimens, made from traditional (Z250) or polyacid-modified (Dyract-eXtra) composites were bonded to human dentin using total-etch (TE-Scotchbond) or self-etch (SE-EasyBond) adhesives. TE was applied with or without the MMP inhibitor galardin. Specimens were incubated in phosphate-buffer or SHSE (37°C/pH=7.0) for up to 180 days, then suspended in a continuous flow biofilm fermenter cultivating biofilms of Streptococcus mutans UA159. Interfacial bacterial penetration, biofilm biomass and viability were measured by confocal laser scanning microscopy and biomarker dyes and used as interfacial biodegradation markers. ResultsAll specimens showed increased biofilm penetration and biomass with time regardless of incubation condition. SHSE increased bacterial penetration in all experimental samples after 180days (p<0.05). Galardin reduced interfacial bacterial ingress and bacterial biomass vs. non-MMP-inhibited TE-bonded specimens (p<0.05). TE interfaces showed lower interfacial bacterial biomass vs. SE after 90-day and 180-day (p<0.05). Dyract-eXtra specimens showed lower bacterial cell viability within the interface vs. Z250 (p<0.05). SignificanceThe biodegradation of resin–tooth interfaces is accelerated by esterases, modulated by MMP inhibition and is dependent on the material’s chemistry and mode of adhesion. The in vitro bacterial growth model used in this study facilitates the elucidation of differences in interfacial integrity and biostability between different materials and techniques and is suitable for assessment of their performance prior to clinical evaluation.

Biodegradation and detoxification of naphthenic acids in oil sands process affected waters

After oil sands process affected water (OSPW) was treated in a continuous flow biofilm reactor, about 40% of the organic compounds in the acid extractable fraction (AEF) including naphthenic acids (NAs) were degraded resulting in a reduction of 73% in the Microtox acute toxicity and of 22% in the yeast estrogenic assay. Using effect directed analysis, treated and untreated OSPW were fractionated by solid phase extraction and the fractions with the largest decrease in toxicity and estrogenicity were selected for analysis by electrospray ionization combined with linear ion trap and a high-resolution Orbitrap mass spectrometer (negative ion mode). The aim of this study was to determine whether compositional changes between the untreated and treated fractions provide insight related to biodegradation and detoxification of NAs. The O2S, O3S and O4S compounds were either not major contributors of toxicity or estrogenicity or the more toxic or estrogenic ones were biodegraded. The O3- and O4-NAs seem to be more readily metabolized than O2NAs and their degradation would contribute to detoxification. The decrease in acute toxicity may be associated with the degradation of C12 and C13 bicyclic and C12–C14 tricyclic NAs while the decrease in estrogenicity may be linked to the degradation of C16 O2-NAs with double bond equivalents (DBE)=5 and 6, C16 and 17 O2-NAs with DBE=7, and C19-O2-NAs with DBE=8. The residual acute toxicity may be caused by recalcitrant components and/or degradation products such as the O2 bicyclic and tricyclic NAs, particularly the C14 and C15 bicyclic and C14–C16 tricyclic NAs as well as the polycyclic aromatic NAs (DBE≥5 compounds). The decrease in estrogenicity may be linked to the degradation of the O3 and O4 oxidized NAs while much of the residual estrogenicity may be due to the recalcitrant polycyclic aromatic O2-NAs. Hence, treatment to further detoxify OSPW should target these compounds.

Pseudomonas aeruginosa Exhibits Deficient Biofilm Formation in the Absence of Class II and III Ribonucleotide Reductases Due to Hindered Anaerobic Growth.

Chronic lung infections by the ubiquitous and extremely adaptable opportunistic pathogen Pseudomonas aeruginosa correlate with the formation of a biofilm, where bacteria grow in association with an extracellular matrix and display a wide range of changes in gene expression and metabolism. This leads to increased resistance to physical stress and antibiotic therapies, while enhancing cell-to-cell communication. Oxygen diffusion through the complex biofilm structure generates an oxygen concentration gradient, leading to the appearance of anaerobic microenvironments. Ribonucleotide reductases (RNRs) are a family of highly sophisticated enzymes responsible for the synthesis of the deoxyribonucleotides, and they constitute the only de novo pathway for the formation of the building blocks needed for DNA synthesis and repair. P. aeruginosa is one of the few bacteria encoding all three known RNR classes (Ia, II, and III). Class Ia RNRs are oxygen dependent, class II are oxygen independent, and class III are oxygen sensitive. A tight control of RNR activity is essential for anaerobic growth and therefore for biofilm development. In this work we explored the role of the different RNR classes in biofilm formation under aerobic and anaerobic initial conditions and using static and continuous-flow biofilm models. We demonstrated the importance of class II and III RNR for proper cell division in biofilm development and maturation. We also determined that these classes are transcriptionally induced during biofilm formation and under anaerobic conditions. The molecular mechanism of their anaerobic regulation was also studied, finding that the Anr/Dnr system is responsible for class II RNR induction. These data can be integrated with previous knowledge about biofilms in a model where these structures are understood as a set of layers determined by oxygen concentration and contain cells with different RNR expression profiles, bringing us a step closer to the understanding of this complex growth pattern, essential for P. aeruginosa chronic infections.

USMB-induced synergistic enhancement of aminoglycoside antibiotics in biofilms

This study evaluated the effect of combining antibiotics with ultrasound and microbubbles (USMB) toward the eradication of biofilms. Pseudomonas aeruginosa PAO1 biofilms were treated with the antibiotics gentamicin sulfate or streptomycin sulfate, or a combination of USMB with the respective antibiotics. Biofilm structure was quantified using confocal laser scanning microscopy with COMSTAT analysis, while activity was measured as whole-biofilm CO2 production in a continuous-flow biofilm model. The combined antibiotic–USMB treatment significantly impacted biofilm biomass, thickness and surface roughness compared to antibiotics alone (p<0.05). USMB exposure caused the formation of craters (5–20μm in diameter) in the biofilms, and when combined with gentamicin, activity was significantly lower, compared to gentamicin, USMB or untreated controls, respectively. Interestingly, the CO2 production rate following combined streptomycin–USMB treatment was higher than after streptomycin alone, but significantly lower than USMB alone and untreated control. These results show strong evidence of a synergistic effect between antibiotics and USMB, although the varied response to different antibiotics emphasize the need to optimize the USMB exposure conditions to maximize this synergism and ultimately transfer this technology into clinical or industrial practice.

A Novel Membrane Process for Autotrophic Denitrification

Biological denitrification by heterotrophic bacteria is common in the wastewater industry in the U.S. and in drinking water processing in Europe.To facilitate heterotrophic denitrification, organic compounds such as methanol, ethanol and acetic acid are added to provide a carbon source for the bacteria. The resulting organic carbon residual may create problems with chlorination. The addition of these carbon compounds is expensive and results in added sludge production. This study focused on the use of autotrophic hydrogen oxidizing bacteria for denitrification. The method transfers hydrogen gas to solution via microporous hollow fiber membranes. Typically, gases are supplied to a system using conventional bubble diffusers. The conventional bubble aeration system has a low gas transfer efficiency, and, as a result, the cost of dissolving the required amount of gas is very high. In this study, microporous hollow fiber membranes were employed to supply hydrogen gas to hydrogen oxidizing autotrophic bacteria. Laboratory scale membrane modules were constructed and mass transfer studies were carried out to develop the design correlations for hydrogen gas transfer. A mixed culture was obtained and acclimated for batch denitrification studies. Both Sodium carbonate and carbon dioxide were used to deliver inorganic carbon. Bench scale continuous flow biofilm reactors containing plastic media were operated to remove nitrate from water. The required hydrogen gas was supplied at a constant rate via gas transfer modules, containing sealed end microporous hollow fiber membranes. The reactors were optimized for removal of nitrate and nitrite by varying the recycle ratios and hydraulic detention time. Experimental results indicated the presence of hydrogen oxidizing denitrifiers in wastewater sludge. Adequate pH control was possible and the pH averaged around 6.95. Gas transfer studies indicated that hydrogen transfer was primarily controlled by liquid film diffusion. Hydrogen gas was successfully delivered to the reactor via the hollow fiber membrane gas transfer module. Nitrate and hydrogen concentration measurements indicated that the system did not experience hydrogen limitations at detention times of 3.25 hours or greater. The use of hollow fiber membrane module appears to be a viable technology for transferring hydrogen gas to water. The research results in this report provide valuable information for pilot and full-scale studies for the water/wastewater community focusing on membrane processes for autotrophic denitrification. This title belongs to WERF Research Report Series ISBN: 9781843396437 (Print) ISBN: 9781780403229 (eBook)

Nitrite accumulation from simultaneous free-ammonia and free-nitrous-acid inhibition and oxygen limitation in a continuous-flow biofilm reactor.

To achieve nitrite accumulation for shortcut biological nitrogen removal (SBNR) in a biofilm process, we explored the simultaneous effects of oxygen limitation and free ammonia (FA) and free nitrous acid (FNA) inhibition in the nitrifying biofilm. We used the multi-species nitrifying biofilm model (MSNBM) to identify conditions that should or should not lead to nitrite accumulation, and evaluated the effectiveness of those conditions with experiments in continuous flow biofilm reactors (CFBRs). CFBR experiments were organized into four sets with these expected outcomes based on the MSNBM as follows: (i) Control, giving full nitrification; (ii) oxygen limitation, giving modest long-term nitrite build up; (iii) FA inhibition, giving no long-term nitrite accumulation; and (iv) FA inhibition plus oxygen limitation, giving major long-term nitrite accumulation. Consistent with MSNBM predictions, the experimental results showed that nitrite accumulated in sets 2-4 in the short term, but long-term nitrite accumulation was maintained only in sets 2 and 4, which involved oxygen limitation. Furthermore, nitrite accumulation was substantially greater in set 4, which also included FA inhibition. However, FA inhibition (and accompanying FNA inhibition) alone in set 3 did not maintained long-term nitrite accumulation. Nitrite-oxidizing bacteria (NOB) activity batch tests confirmed that little NOB or only a small fraction of NOB were present in the biofilms for sets 4 and 2, respectively. The experimental data supported the previous modeling results that nitrite accumulation could be achieved with a lower ammonium concentration than had been required for a suspended-growth process. Additional findings were that the biofilm exposed to low dissolved oxygen (DO) limitation and FA inhibition was substantially denser and probably had a lower detachment rate.

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.

Bioremediation of polluted surface water by using biofilms on filamentous bamboo

Polluted surface water was remediated in a bioreactor using biofilms on filamentous bamboo in batch and continuous flow modes. The CODcr (Chemical Oxygen Demand, using K2Cr2O7 as oxidizer) removal rate of the enhanced systems increased more than 13% relative to a controlled system. In the batch reactor with 4-h cycles, the CODcr was mainly removed by the biofilm on the filamentous bamboo. The removal rate of the CODMn (permanganate index), NH4+–N (ammonia nitrogen), turbidity, and total bacteria were 11.2–74.3%, 2.2–56.1%, 20–100%, and more than 88.6% in a continuous flow biofilm reactor with a 3.5h retention time. The biofilms contained diverse organisms including protozoa and metazoa.

Generation of Variants in Listeria monocytogenes Continuous-Flow Biofilms Is Dependent on Radical-Induced DNA Damage and RecA-Mediated Repair

The food-borne pathogen Listeria monocytogenes is a Gram-positive microaerophilic facultative anaerobic rod and the causative agent of the devastating disease listeriosis. L. monocytogenes is able to form biofilms in the food processing environment. Since biofilms are generally hard to eradicate, they can function as a source for food contamination. In several occasions biofilms have been identified as a source for genetic variability, which potentially can result in adaptation of strains to food processing or clinical conditions. However, nothing is known about mutagenesis in L. monocytogenes biofilms and the possible mechanisms involved. In this study, we showed that the generation of genetic variants was specifically induced in continuous-flow biofilms of L. monocytogenes, but not in static biofilms. Using specific dyes and radical inhibitors, we showed that the formation of superoxide and hydroxyl radicals was induced in continuous-flow biofilms, which was accompanied with in an increase in DNA damage. Promoter reporter studies showed that recA, which is an important component in DNA repair and the activator of the SOS response, is activated in continuous-flow biofilms and that activation was dependent on radical-induced DNA damage. Furthermore, continuous-flow biofilm experiments using an in-frame recA deletion mutant verified that RecA is required for induced generation of genetic variants. Therefore, we can conclude that generation of genetic variants in L. monocytogenes continuous-flow biofilms results from radical-induced DNA damage and RecA-mediated mutagenic repair of the damaged DNA.

Chloroform aerobic cometabolism by butane-growing Rhodococcus aetherovorans BCP1 in continuous-flow biofilm reactors

This work focuses on chloroform (CF) cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow biofilm reactors. The goals were to obtain preliminary information on the feasibility of CF biodegradation by BCP1 in biofilm reactors and to evaluate the applicability of the pulsed injection of growth substrate and oxygen to biofilm reactors. The attached-cell tests were initially conducted in a 0.165-L bioreactor and, then, scaled-up to a 1.772-L bioreactor. Glass cylinders were utilized as biofilm carriers. The continuous supply of growth substrate (butane), which led to the attainment of the highest CF degradation rate (8.4 mg(CF) day(-1) m (biofilm surface)(-2)), was compared with four schedules of butane and oxygen pulsed feeding. The pulsed injection technique allowed the attainment of a ratio of CF mass degraded per unit mass of butane supplied equal to 0.16 mg(CF) mg (butane)(-1), a value 4.4 times higher than that obtained with the continuous substrate supply. A procedure based on the utilization of integral mass balances and of average concentrations along the bioreactors resulted in a satisfactory match between the predicted and the experimental CF degradation performances, and can therefore be utilized to provide a guideline for optimizing the substrate pulsed injection schedule.