Biofilm Detachment Research Articles

Farnesol Emulsion as an Effective Broad-Spectrum Agent against ESKAPE Biofilms.

The family of ESKAPE pathogens is comprised of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter. Together they are the main contributors of nosocomial infections and are well established for their ability to "escape" antibiotics. Farnesol is an FDA-approved cosmetic and flavoring agent with significant anti-biofilm properties. In a proprietary emulsion, farnesol has been shown to be capable of disrupting S. aureus, P. aeruginosa, and A. baumannii biofilms. The current work demonstrates that this farnesol emulsion reduces the number of viable bacteria, while also leading to reductions in biomass, of the other three ESKAPE pathogens: Enterococcus faecium, Klebsiella pneumoniae, and Enterobacter, both in vitro and in an ex vivo human skin model. A concentration of 0.5 mg/mL was effective for impeding biofilm development of all three bacteria, while 1 mg/mL for E. faecium and K. pneumoniae, or 0.2 mg/mL for E. cloacae, was able to kill bacteria in established biofilms. Contrary to antibiotics, no resistance to farnesol was observed for E. faecium or K. pneumoniae. The results indicate that farnesol is effective for direct cell killing and also has the ability to induce biofilm detachment from surfaces, as confirmed using Live/Dead image analysis. Our findings confirm that farnesol emulsion is an effective broad-spectrum agent to impede ESKAPE biofilms.

Biofilm growth characteristic and footprint identification in gravity-driven ceramic membrane bioreactor with electro-coagulation under extreme conditions for roofing rainwater purification

The identification of biofilm growth footprints influencing on the biofilm detachment and breakup can advance research into how biofilms form. Thus, a gravity-driven ceramic membrane bioreactor (GDCMBR) was used to investigate the growth, detachment and breakup of biofilm using rainwater pretreated by electrocoagulation under 70-days continuous operation. The in-situ ultrasonic time-domain reflectometry (UTDR) technique was applied to non-invasively determine the biofilm thickness. Initially, the biofilm was slowly thickening, but it would collapse and became thinner after accumulating to a certain level, and then it thickened again in a later period, following a cyclic pattern of ‘thickening - collapsing – thickening’. This is because the biofilm growth is related with the accumulation of flocs, however, excessive floc formation results in the biofilm being overweight till reaching the thickness limit and thus collapsing. Subsequently, the biofilm gradually thickens again due to the floc production and continuous deposition. Although the biofilm was dynamically changing, the water quality of treatment of the biofilm always remained stable. Ammonia nitrogen and total phosphorus have been almost completely removed, while CODMn removal efficiency was around 25%. And total bacteria amount in the membrane concentrate was obviously higher than that in the influent with the greater microbial activity, demonstrating the remarkable enrichment effect on bacteria. The understanding of biofilm growth characteristic and footprint identification enables us to develop rational approaches to control biofilm structure for efficient GDCMBR performance and operation lifespan.

Isobutyric and n-butyric acid production from H2 and CO2 by Clostridium luticellarii in a hollow fiber membrane biofilm reactor: Reactor operation and process modelling

Clostridium luticellarii is an acetogen uniquely capable of producing acetic, n-butyric and isobutyric acid from H2 and CO2. Since n-butyric and isobutyric acid are currently produced from petrochemically derived propylene, establishing a direct production route from CO2 and H2 could greatly improve their sustainability as platform chemicals while simultaneously capturing carbon. Here, we demonstrate for the first time the production of these acids from H2 and CO2 by C. luticellarii in a continuously operated hollow fiber membrane biofilm reactor (HFMBR). The HFMBR was operated for 80 days while gradually increasing the H2:CO2 ratio from 2.5 to 5.0. Concentrations of acetic, n-butyric and isobutyric acid of respectively 8.93 ± 0.22 g L−1, 1.56 ± 0.04 g L−1 and 1.51 ± 0.06 g L−1 were achieved at a dilution rate of 0.25 d−1. Subsequently, a holistic process model was developed to gain deeper insights into the performance of the system and how to optimize its operation. The model described the autotrophic production of the acids, as well as mass transfer of H2 and CO2 through the membrane, biofilm diffusion and convective movement of particulates, and biofilm growth and detachment. Calibration against experimental data confirmed the model accuracy in reflecting the production dynamics (NRMSE of products < 0.1). Scenario analyses highlighted the importance of biofilm thickness control to minimize pH gradients and revealed that increased membrane surface area and optimized dilution rates are critical in enhancing volumetric production rates and steering product selectivity. Overall, these combined experimental results and model-generated insights can inform future optimization of HFMBRs for the production of added-value products from H2 and CO2.

Effect of Corrosion Media on Biofilm Detachment and the Corrosion Mechanism of Serratia marcescens on Carbon Steel in River Water

Effect of Corrosion Media on Biofilm Detachment and the Corrosion Mechanism of Serratia marcescens on Carbon Steel in River Water

Enhancement of serrawettin W1-producing Serratia marcescens cell migration by resonant oscillation under alternating current electric field

Swarming migration is observed in flagellated bacteria on wet surfaces. As the secreted biosurfactant hydrates, friction between the cell and the wet surface is reduced, and weak flagellar movement appears to be a significant force for cell translocation. Developing an artificial swarming control method could greatly aid in establishing techniques for biofilm inhibition and detachment. In this study, the effect of forced cell vibration by an alternating current electric field (ACEF) on swarming motility was investigated using the serrawettin W1-producing Serratia marcescens strain. At frequencies close to the natural frequencies of microbial cells (12 MHz), swarming motion was effectively enhanced in biosurfactant-producing S. marcescens, but not in non-surfactant-producing Escherichia coli or non-flagellated Staphylococcus aureus. Electric field-assisted cell migration was significantly induced under swarming conditions with low friction resistance between the cell and gel surface. This finding suggests a direction for developing strategies to regulate biofilm formation and detachment using ACEF-assisted oscillation of cells attached to surfaces.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.

The mechanism of biofilm detachment in porous medium under flow field.

Biofilms are communities formed by bacteria adhering to surfaces, widely present in porous medium, and their growth can lead to clogging. Our experiment finds that under certain flow conditions, biofilms detach in pores and form a dynamically changing flow path. We define detachment that occurs far from the boundary of the flow path (with a distance greater than 200 μm) as internal detachment and detachment that occurs at the boundary of the flow path as external detachment. To understand the mechanism of biofilm detachment, we study the detachment behaviors of the Bacillus subtilis biofilm in a porous medium in a microfluidic device, where Bacillus subtilis strain is triple fluorescent labeled, which can represent three main phenotypes during the biofilm formation: motile cells, matrix-producing cells, and spores. We find that slow small-scale internal detachment occurs in regions with very few motile cells and matrix-producing cells, and bacterial movement in these areas is disordered. The increase in the number of matrix-producing cells induces clogging, and after clogging, the rapid detachment of the bulk internal biofilm occurs due to the increased pressure difference at the inlet and outlet. When both internal and external detachments occur simultaneously, the number of matrix-producing cells in the internal detachment area is 2.5 times that in the external detachment area. The results indicate that biofilm detachment occurs in areas with fewer matrix-producing cells, as matrix-producing cells can help resist detachment by secreting extracellular polymeric substances (EPSs).

Impact of operational conditions on drinking water biofilm dynamics and coliform invasion potential.

The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola. These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality.

Farnesol repurposing for prevention and treatment of Acinetobacter baumannii biofilms

Acinetobacter baumannii has emerged as a multidrug-resistant (MDR) superbug by causing severe infections, with high mortality rates. The ability of A. baumannii to form biofilms significantly contributes to its persistence in diverse environmental and hospital settings. Here we report that farnesol, an FDA-approved commercial cosmetic and flavoring agent, demonstrates efficacy for both inhibition of biofilm formation, and disruption of established A. baumannii biofilms. Moreover, no resistance to farnesol was observed even after prolonged culture in the presence of sub-inhibitory farnesol doses. Farnesol combats A. baumannii biofilms by direct killing, while also facilitating biofilm detachment. Furthermore, farnesol was safe, and effective, for both prevention and treatment of A. baumannii biofilms in an ex vivo burned human skin model. Since current treatment options for A. baumannii biofilm infections were mainly counted on the combination therapy of last-resort antibiotics, and clearly non-sustainable due to robust MDR phenotype of A. baumannii, we propose that farnesol alone can be repurposed as a highly effective agent for both preventing and treating life-threating biofilm-associated infections of A. baumannii due to its proven safety, convenient topical delivery, and excellent efficiency, plus its superiority of evading resistance development.

Repurposing Farnesol for Combating Drug-Resistant and Persistent Single and Polymicrobial Biofilms.

Biofilm-associated infections caused by drug-resistant and persistent bacteria remain a significant clinical challenge. Here we report that farnesol, commercially available as a cosmetic and flavoring agent, shows significant anti-biofilm properties when dissolved in ethanol using a proprietary formulation emulsion technique. Farnesol in the new formulation inhibits biofilm formation and disrupts established biofilms for Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, including their polymicrobial biofilms, and, moreover, kills S. aureus persister cells that have developed tolerance to antibiotics. No resistance to farnesol was observed for S. aureus after twenty continuous passages. Farnesol combats biofilms by direct killing, while also facilitating biofilm detachment. Furthermore, farnesol was safe and effective for preventing and treating biofilm-associated infections of both types of bacteria in an ex vivo burned human skin model. These data suggest that farnesol in the new formulation is an effective broad-spectrum anti-biofilm agent with promising clinical potential. Due to its established safety, low-cost, versatility, and excellent efficacy-including ability to reduce persistent and resistant microbial populations-farnesol in the proprietary formulation represents a compelling transformative, translational, and commercial platform for addressing many unsolved clinical challenges.

Comparing DNA isolation and sequencing strategies for 16S rRNA gene amplicon analysis in biofilm containing environments

Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.

Bacterial biofilm growth and perturbation by serine protease from Bacillus sp.

In nature, bacteria are ubiquitous and can be categorized as beneficial or harmless to humans, but most bacteria have one thing in common which is their ability to produce biofilm. Biofilm is encased within an extracellular polymeric substance (EPS) which provides resistance against antimicrobial agents. Protease enzymes have the potential to degrade or promote the growth of bacterial biofilms. In this study, the effects of a recombinant intracellular serine protease from Bacillus sp. (SPB) on biofilms from Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa were analyzed. SPB was purified using HisTrap HP column and concentrated using Amicon 30 ultra-centrifugal filter. SPB was added with varying enzyme activity and assay incubation period after biofilms were formed in 96-well plates. SPB was observed to have contrasting effects on different bacterial biofilms, where biofilm degradations were observed for both 7-day-old A. baumannii (37.26%) and S. aureus (71.51%) biofilms. Meanwhile, SPB promoted growth of P. aeruginosa biofilm up to 176.32%. Compatibility between protein components in S. aureus biofilm with SPB as well as a simpler membrane structure morphology led to higher biofilm degradation for S. aureus compared to A. baumannii. However, SPB promoted growth of P. aeruginosa biofilm due likely to its degrading protein factors that are responsible for biofilm detachment and dispersion, thus resulting in more multi-layered biofilm formation. Commercial protease Savinase which was used as a comparison showed degradation for all three bacterial biofilms. The results obtained are unique and will expand our understanding on the effects that bacterial proteases have toward biofilms.

Dynamic immobilization of bacterial cells on biofilm in a polyester nonwoven chemostat

Cell immobilization plays an important role in biocatalysis for high-value products. It is necessary to maintain the viability of immobilized cells for bioconversion using viable cells as biocatalysts. In this study, a novel polyester nonwoven chemostat was designed for cell immobilization to investigate biofilm formation and the dynamic balance between adsorption and desorption of cells on polyester nonwoven. The polyester nonwoven was suitable for cell immobilization, and the cell numbers on the polyester nonwoven can reach 6.5 ± 0.38 log CFU/mL. After adding the polyester nonwoven to the chemostat, the fluctuation phenomenon of free bacterial cells occurred. The reason for this phenomenon was the balance between adsorption and desorption of bacterial cells on the polyester nonwoven. Bacterial cells could adhere to the surface of polyester nonwoven via secreting extracellular polymeric substances (EPS) to form biofilms. As the maturation of biofilms, some dead cells inside the biofilms can cause the detachment of biofilms. This process of continuous adsorption and desorption of cells can ensure that the polyester nonwoven chemostat has lasting biological activity.Graphical

Effect of flow fluctuation on water pollution in drinking water distribution systems

The detachment of biofilm caused by changes in hydraulic conditions is an essential reason for the pollution of water in the drinking water distribution system (DWDS). In this research, the effect of flow fluctuation on bulk water quality was studied. The turbidity, iron concentration, manganese concentration, the total number of bacteria, biodegradable dissolved organic carbon (BDOC), bacterial community structure, and pathogenic genes in bacteria of bulk water were analyzed. The results indicate that the detachment of biofilm caused by fluctuant flow and reverse flow (especially instant reverse flow) can lead to the pollution of water. Throughout the entire experimental period, the turbidity under fluctuant flow velocity is 4.92%∼49.44% higher than that under other flow velocities. BDOC concentration is 5.68%∼53.99% higher than that under low and high flow velocities. The flow fluctuation increases bacterial regrowth potential (BRP) and reduces the biological stability of the bulk water. Low flow velocity is more conducive to the expression of pathogenic functional genes. In the short term, the water quality under low flow velocity is the best. Nevertheless, in a long-term operation (about seven days later), the water quality under high flow velocity is better than that under other flow velocities. This research brings new knowledge about the fluctuant hydraulic conditions on the bulk water quality within the DWDS and provides data support for stable drinking water distribution.

Neem-hypericum-bacterial cellulose wound care paste characterized in vitro and in Galleria mellonella in vivo model

Surgical wound infections represent a global health emergency because of antibiotic-resistant bacteria. Commensal and pathogenic bacteria can grow in the form of biofilms on damaged skin, and often exhibit an antibiotic-resistance profile. For this reason, the development of new strategies is necessary for the management of skin wounds, which are based not only on antimicrobial activity but also on the inhibition of bacterial biofilm formation or biofilm removal. In this study, we show the development and the biofilm-detaching properties of a novel bacterial cellulose formulation for wound healing. This paste is made by mixing bacterial cellulose pulp with neem-hypericum oil/water emulsion. Once its biocompatibility on murine fibroblasts has been determined, its efficacy was evaluated in vitro against Pseudomonas aeruginosa and Staphylococcus aureus biofilms and in vivo by using Galleria mellonella larvae as a burn wound infection model. Unlike pure BC, the paste showed promising biofilm detachment properties in vitro and a good biocompatibility activity in vivo, setting the stage for developing new strategies for the treatment of chronic wound infections.

A versatile micromodel technology to explore biofilm development in porous media flows.

Bacterial biofilms that grow in porous media are critical to ecosystem processes and applications ranging from soil bioremediation to bioreactors for treating wastewater or producing value-added products. However, understanding and engineering the complex phenomena that drive the development of biofilms in such systems remains a challenge. Here we present a novel micromodel technology to explore bacterial biofilm development in porous media flows. The technology consists of a set of modules that can be combined as required for any given experiment and conveniently tuned for specific requirements. The core module is a 3D-printed micromodel where biofilm is grown into a perfusable porous substrate. High-precision additive manufacturing, in particular stereolithography, is used to fabricate porous scaffolds with precisely controlled architectures integrating flow channels with diameters down to several hundreds of micrometers. The system is instrumented with: ultraviolet-C light-emitting diodes; on-line measurements of oxygen consumption and pressure drop across the porous medium; camera and spectrophotometric cells for the detection of biofilm detachment events at the outlet. We demonstrate how this technology can be used to study the development of Pseudomonas aeruginosa biofilm for several days within a network of flow channels. We find complex dynamics whereby oxygen consumption reaches a steady-state but not the pressure drop, which instead features a permanent regime with large fluctuations. We further use X-ray computed microtomography to image the spatial distribution of biofilms and computational fluid dynamics to link biofilm development with local flow properties. By combining the advantages of additive manufacturing for the creation of reproducible 3D porous microarchitectures with the flow control and instrumentation accuracy of microfluidics, our system provides a platform to study the dynamics of biofilm development in 3D porous media and to rapidly test new concepts in process engineering.

Antibiofouling polyethersulfone ultrafiltration membranes functionalized with MoS2 nanosheets and self-cleaning with hydrogen peroxide

The development of biofouling mitigation and cleaning strategy is of paramount importance for ultrafiltration (UF) membrane biofouling. In this study, polyethersulfone (PES) UF membrane was functionalized, namely MoS2/PES composite membrane. The MoS2/PES composite membrane exhibited superior antifouling property as evidenced by antibacterial circle, static and dynamic biofouling experiments. The permeate fluxes of the pristine PES and MoS2/PES composite membrane respectively decreased by 76% and 31% at the end of 3-h filtration. After exposure of E. coli to MoS2, inhibited cell growth led to reduced membrane biofouling. The biofilm formation was retarded under the dynamic flow condition, which were further confirmed by Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) observations. As for the enhanced biofouling removal by cleaning with H2O2, 62% of the flux recovery rate for the MoS2/PES composite membrane was much higher than the pristine PES membrane (24%). The peroxidase-like properties of MoS2 were responsible for the enhancement of the superior cleaning performance. The use of H2O2 to trigger the in-situ catalytic function of the MoS2 nano-enzyme generated more ROS and O2 for inducing bacterial cells death and facilitating detachment of biofilm. This work will be helpful to provide important insights into developing more effective strategies for biofouling control and cleaning using advanced nanomaterials in membrane-based water treatment processes.

The porous structure induced heterogeneous and localized failure of the biofilm in microfluidic channels.

Understanding the mechanism of biofilm distribution and detachment is very important to effectively improve water treatment and prevent blockage in porous media. The existing research is more related to the local biofilm evolving around one or few microposts and the lack of the integral biofilm evolution in a micropost array for a longer growth period. This study combines microfluidic experiments and mathematical simulations to study the distribution and detachment of biofilm in porous media. Microfluidic chips with an array of microposts with different sizes are designed to simulate the physical pore structure of soil. The research shows that the initial formation and distribution of biofilm are influenced by bacterial transport velocity gradients within the pore space. Bacteria prefer to aggregate areas with smaller microposts, leading to the development of biofilm in those regions. Consequently, impermeable blockage structures form in this area. By analyzing experimental images of biofilm structures at the later stages, as well as coupling fluid flow and porous medium, and the finite element simulation, we find that the biofilm detachment is correlated with the morphology and permeability (kb) (from 10-15 to 10-9 m2) of the biofilm. The simulations show that there are two modes of biofilm detachment, such as internal detachment and external erosion.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans-C. albicans SC5314 and 3147 (ATCC 10231)-in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.

Enhanced mechanistic insights and performance optimization: Controlling methane and sulfide in sewers using nitrate dosing strategies

Nitrate has been used for nearly 80 years as a chemical to control problematic gases in the sewer system. However, few studies have explored simultaneous control in biofilm and sediment using different strategies. Here, we introduced a nitrate dosing method involving an initial high shock followed by low level dosing, tested at two distinct frequencies in a lab-scale sewer reactor <110 days. Long-term investigation revealed that the more frequent 20 min interval dosing slightly surpassed the 1 h interval method when applying the same hourly dose of 30 mg N/L (sulfide control: 98.3 ± 1.7 % vs 97.9 ± 1.5 %; methane control: 89.8 ± 4.5 % vs 83.4 ± 6.7 %). 16 s rRNA gene amplicon sequencing revealed biofilm detachment and sediment stratification, which can be attributed to the differing effects of nitrate dosing on biofilm and sedimentary microbial interactions. Dominant bacteria such as Thauera and Thiobacillus performed autotrophic denitrification and nitrate-reducing sulfide-oxidation in conjunction with methane oxidizers. These microbes collaboratively control sulfide and methane emissions from sediment. Our findings suggest that nitrate supports the diversity and versatility of their metabolism in the sewer system.