Biofilm Growth Research Articles

Molecular characterization of virulence genes and influence of Xanthium strumarium extract against two Enterobacter species isolated from some soil invertebrates.

The development of bacterial antibiotic resistance poses adanger to healthcare systems worldwide. To reduce the spread of disease, researchers are looking for novel measures to control bacterial infections to reduce the spread of disease. The antibacterial properties of Xanthium strumarium methanolic and ethanolic extracts were evaluated against Enterobacter cloacae and E. hormaeche isolated from some invertebrates (Porcellio laevis, Armadillidium sp. (isopods), and Archispirostreptus syriacus). All Enterobacter strains tested positive for the presence of the virulence genes csgA, csgD, AcrAB, fimH, and Hsp60. Extracts of X. strumarium had significant anti-biofilm activity against E. cloacae and E. hormaechei. The disruption of established biofilm growth by the plant samples proved to be effective against E. cloacae and E. hormaechei. Both E. cloacae and E. hormaechei showed inhibition of biofilm formation and promotion of biofilm eradication in response to X. strumarium extract. Phenolic compounds such as ferulic acid, chlorogenic acid, and trans-cinnamic acid, and flavonoids such as kaempferol were the most abundant components in the extract and might play crucial roles in the extract's antibacterial and antibiofilm action. Results suggest that ethanolic leaf extracts from X. strumarium show potential as a novel approach to prevent infections caused by E. cloacae and E. hormaechei.

Enhanced bioenergy recovery by innovative application of chia seeds nanopowder for anode modification in microbial fuel cell treating hospital wastewater

ABSTRACT Microbial fuel cells (MFCs) are new bioelectrochemical techniques for conversion of organic waste materials into energy depending on the metabolic activity of the anodic biofilm, which acts as the biocatalyst in the anode compartment. Hence, the anode material is a priority for better growth of bacterial species and electrical conductivity as well. In this study, an innovative application of Chia seeds nanopowder (CSNP) was carried out for the first time with acid-activated multiwall carbon nanotubes (functionalised MWCNTs) for anode nanomodification by overlaying the surfaces of the graphite anodes (GA) in MFCs fuelled with real hospital wastewater (HWW). Two tubular-enclosed dual-chamber MFCs were constructed, setup and operated in a continuous mode for 3 months. MFC1 was assembled with CSNP/MWCNTs-GA, whereby MFC2 was assembled with MWCNTs-GA. The results revealed higher power output up to 2202.73 mW/m3 observed in MFC1 compared to 1271.57 mW/m3 in MFC2. The efficiencies of organic content (COD) removal were 86.1% and 82.9% obtained in MFC1 and MFC2, respectively. Although both efficiencies of COD removal were relatively comparable, however, a remarkable increase in COD removal efficiency was achieved in MFC1. These observations indicated the potential role of CSNP for enhancing the biofilm growth and increasing the electrode conductivity.

Engineering and Clinical Study of Surface Geometry of Clear Aligners at the Nanoscale

This paper investigates the evolution of the outer surface geometry of Invisalign®—clear orthodontic aligners—caused by degradation triggered by wearing. The obtained results served to confirm whether or not the aligners could continue to be used once their wear time in the therapeutic procedure had ended, taking both their geometric and mechanical features into account. The measurements were performed using atomic force microscopy which allowed the mapping of nanomechanical properties. The obtained images were then processed to determine statistical and functional surface geometry parameters in accordance with relevant ISO standards. The results revealed that the unrepeatability of the manufacturing process causes the surface shape parameters of new aligners to be irregular; however, these features become gradually consistent for worn samples. On the other hand, properly used aligners may change in two ways: the outer layer flattens and its thickness decreases, and at the same time the Young’s modulus of the material decreases. It follows that the degradation processes may be caused by tribological phenomena (abrasion of contact surfaces) and/or biochemical phenomena (biofilm growth, decomposition of the material under the influence of enzymes in the oral cavity).

Microbial growth within porous gravity currents

The effect of microbial activity on buoyancy-driven flow within a porous layer is analysed. The input fluid provides an energy source for the growth of biofilms on the porous rock. At each location within the porous layer, the porosity and permeability begin to decrease once the input fluid has invaded. This leads to an evolving rock heterogeneity that depends on the passing time of the input fluid. Hence, the evolution of the flow is partly controlled by its own history. We present an axisymmetric gravity current model, accounting for this effect. In general, a reduction in permeability leads to the flow having a lesser extent in the radial direction and greater thickness (extent in the cross-flow direction), whilst a reduction in porosity has negligible effect on the thickness but leads to a much greater radial extent. The flow is fastest near the free surface where the permeability is greatest. In the case where the porosity and permeability reduce as power-law functions of fluid residence time, the evolution of the flow and the rock properties are self-similar. Consumption of the input fluid by the microbes is also incorporated in the model and it generally leads to flows with lesser radial extent but little change in the thickness. The three impacts of microbial growth (volume loss owing to consumption and the reduction in permeability and porosity) each influence the flow in substantially different ways and the interplay is analysed. A motivation of the study, the underground storage of hydrogen, is briefly discussed.

Bacterial single-cell RNA sequencing captures biofilm transcriptional heterogeneity and differential responses to immune pressure

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display metabolic and transcriptional diversity along with recalcitrance to antibiotics and host immune defenses. Here, we present an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. BaSSSh-seq captures extensive transcriptional heterogeneity during biofilm compared to planktonic growth. We quantify and visualize transcriptional regulatory networks across heterogeneous biofilm subpopulations and identify gene sets that are associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detects alterations in biofilm metabolism, stress response, and virulence induced by distinct immune cell populations. This work facilitates the exploration of biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.

Electrochemical Impedance Spectroscopy Sensors in the Sugar Bush to Detect Biofilms

The demand for maple syrup is increasing, and producers must improve efficiency to maintain high-quality products at a low cost. To meet transparency, density, color, and aroma standards, quality control systems are established throughout boiling processes. However, microbial growth in the harvesting infrastructure throughout the sugaring season has been a significant challenge, as biofilms reduce sap flow rates or cause clogging of sap lines. Microbial growth also degrades the product's quality by changing the syrup's color, taste, and viscosity. Therefore, smart farming is an essential solution that can bridge the gap between microbial growth monitoring and maple syrup quality. Wireless Sensor Networks in maple forests, also known as sugar bushes, are vital for monitoring processes such as sap harvesting using individual sensor nodes that send current biogeochemical information to a field station. A biofilm sensor is needed to indicate biofilm formation in real time.This presentation introduces a microfabricated electrochemical impedance spectroscopy sensor (EIS) tailored to measure biofilm formation in sap infrastructure during the harvesting season, defined by temperature fluctuations between -10 and 20 ˚C. Impedance spectroscopy sensors were fabricated using standard evaporation, lithography, and wet chemical etching techniques. To increase the sensitivity of biofilm formation, interdigitated electrodes with a 15 µm gap between two electrodes were utilized, and 50 gold electrode pairs were implemented on a borosilicate glass wafer. Diced EIS dies were integrated into an additively manufactured housing that allowed electrochemical measurements in maple sap in the laboratory or sugar bush.To test the efficacy of the EIS sensor, a Pseudomonas Sp. strain was extracted from maple sap harvested in 2023 at the Michigan State University Upper Peninsula Forestry Innovation Center and grown at room temperature. Sterile sap was filled into the flow cell and inoculated with microorganisms after 4 h. All experiments were performed in triplicates, and abiotic controls followed the same procedure minus the injection of cell culture enrichments.Impedance data were captured every 30 minutes for 7 days, and impedance changes to the abiotic signal were characterized at defined frequencies. After 1 hour, an impedance change of 15% was observed. The impedance magnitude decreased over 7 days, indicating biofilm maturation. Biofilm growth on the EIS surface was confirmed with confocal microscopy. An impedance increase was observed in some cases, indicating biofilm detachment. Additionally, a model has been developed that relates the impedance response of maple sap to the required outdoor temperature range of -10 and 20 ˚C. Ultimately, this paper shows EIS as a valuable tool to characterize biofilm formation caused by maple sap.

Corrosion Enhanced By Hydrogenotrophic Methanogens Differently Enriched on Copper Alloys

Copper and its alloys, such as brass and bronze, are widely used in heat exchangers valves, fitting, and other components of industrial facilities due to their corrosion resistance, high conductivity, and biocide properties. However, they can suffer of microbial corrosion like or more than other non-toxic materials, as several bacteria and archaea can settle and form thick biofilm on them. Nowadays, there is a growing interest in studying the metabolisms of methanogens and their effects for materials [1], due to the pivotal role that these microorganisms play in the biotechnological processes. The production of methane by CO2 and H2, for instance, is one of the most promising bioprocesses addressing the energy transition [2].In this context, the aim of this work is to evaluate, and underline, the corrosiveness of biofilm growth on different copper alloys, starting from a common microbial pool enriched of bacteria and hydrogenotrophic archaea, treated at different temperatures and autoclaved. The study was evaluated based on 16 days test on the surface of two different copper materials, a pure Cu and a CuZn brass (40 wt. % Zn). Electrochemical investigations were performed using a three-electrode cell consisting of a working electrode (test alloy), a reference electrode (saturated Ag/AgCl electrode) and a counter electrode (titanium mesh). Investigations included open circuit potential (OCP) monitoring and electrochemical impedance spectroscopy (EIS). Chemical characterizations of the corrosion products and post-experiment observations were performed by SEM and micro-Raman spectroscopy (μRS). Molecular analysis by next generation sequencing (NGS) of 16S RNA was performed by swabbing the surface of the material and identifying the microorganisms constituting the microbial communities for each case. Results evidenced a different corrosion behavior of materials, which corresponded to different enrichment of microorganisms and different corrosion products, depending on the material and the treatment of the inoculum. [1] Lahme S, Mand J, Longwell J, Smith R, Enning D. Severe Corrosion of Carbon Steel in Oil Field Produced Water Can Be Linked to Methanogenic Archaea Containing a Special Type of [NiFe] Hydrogenase. Appl Environ Microbiol. 2021 Jan 15;87(3) [2] Hirano SI, Ihara S, Wakai S, Dotsuta Y, Otani K, Kitagaki T, Ueno F, Okamoto A. Novel Methanobacterium Strain Induces Severe Corrosion by Retrieving Electrons from Fe0 under a Freshwater Environment. Microorganisms. 2022 Jan 25;10(2):270.

Influence of biofilm growth on permeability evolution in gravel layers of leachate collection system: A pore-scale analysis using Darcy-Brinkman-Stokes approach

Biofilm formation within the gravel layer of leachate collection systems (LCS) poses a significant challenge by reducing permeability, potentially leading to clogging. Understanding the impact of biofilm growth on the hydraulic performance of LCS is essential for optimizing gravel layer design. This study utilized the Navier-Stokes-Brinkman equations to develop a numerical model that describes fluid flow at the pore scale within both biofilm-covered and open pore regions of the LCS gravel layer. The model was validated using previous experimental and numerical results. A geometric model of a porous network composed of randomly packed spherical particles, reflecting actual particle sizes in LCS, was then established. This model was used to investigate the evolution of the flow field and changes in permeability under varying conditions of particle size, biofilm thickness, and permeability characteristics. The results showed that increasing biofilm thickness reduced channel cross-sectional area, elevated flow velocity, increased pressure drop, and caused an exponential decrease in permeability. The study also confirmed that the permeability and porosity of the biofilm significantly influenced the extent of permeability reduction in the LCS gravel layer. Additionally, smaller particle size combinations had a more pronounced effect on permeability reduction. These findings provide valuable insights at the pore scale, helping to predict the impact of biofilm growth on gravel layer permeability in LCS and offering essential guidance for future design and management strategies.

Flash chromatographic isolation of garcinol and isogarcinol from Garcinia indica Choisy (kokum) fruit and evaluation of their potential antibiofilm activity

The present study describes the isolation and separation of isogarcinol and garcinol from kokum fruit by flash chromatography using water (containing 0.1 % formic acid) and methanol mixed with acetonitrile (1:1) with UV detection at 254 nm and was characterised by HR-MS and NMR studies. These were further subjected to antimicrobial studies on Staphylococcus aureus FR1722 and Listeria monocytogenes Scott A by agar diffusion assay and broth microdilution method wherein the MIC (Minimum Inhibitory Concentration) of garcinol and isogarcinol were, 20 μg/mL and 50 μg/mL for S. aureus FR1722 and 50 μg/mL and 100 μg/mL for L. monocytogenes Scott A respectively. Further, studies on the control of biofilm growth were tested using MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay followed by Crystal Violet (CV) assay, which showed up to 80 % inhibition. In addition, the biofilm eradication as evaluated by bright field microscopy and the metabolic activity were also carried out against these bacterial strains and the experimental details are presented.

The biofilm produced by Cryptococcus neoformans protects the fungus from the antifungal and anti-melanin effects of cyclosporine

Understanding Cryptococcus neoformans pathogenesis requires a detailed analysis of the various virulence factors that contribute to its ability to cause disease. Cyclosporine, calcineurin inhibitor, impairs C. neoformans production of a polysaccharide capsule and secretion of urease, which are critical for cryptococcal pathogenesis. Two particularly important virulence factors are the production of cell wall melanin and formation of biofilm. In this study, we investigated cyclosporine's effects on melanin production and biofilm formation in C. neoformans. Initially, we examined melanin production in planktonic cells treated with cyclosporine using an L-DOPA containing melanin-inducing medium. Visual inspection and optical microscopy revealed a notable reduction in the characteristic dark coloration of cultures treated with cyclosporine, which indicate decreased melanin production in daughter cells compared to mother cells. Spectrophotometric analysis also demonstrated a significantly altered ultraviolet–visible (UV/vis) absorption spectra in cyclosporine-treated yeast cells, indicative of structural changes in melanin. Additionally, cyclosporine-treated cells exhibited reduced conductance (P-value < 0.0001), suggesting altered cellular ionic properties. The impact of cyclosporine on biofilm formation and mature biofilm disruption was also assessed. Despite cyclosporine's efficacy in modifying virulence factors during planktonic growth, cyclosporine did not inhibit biofilm formation or melanization under biofilm growth conditions, nor did it disrupt mature biofilms in terms of biomass or metabolic activity. However, there was a significant reduction in extracellular matrix production in cyclosporine-treated non-melanized biofilms. Our findings underscore the complex interplay between cyclosporine and C. neoformans, highlighting its differential effects on melanization and biofilm dynamics, which provides new insights into the shortcomings of cyclosporin for combatting cryptococcosis and informs pathways for future therapeutic strategies against cryptococcosis.

Acoustic Fingerprinting and Nanoslip Dynamics of Biofilms

AbstractIt is reported that bacteria can generate nanomotion, but understanding the complex dynamics of bacterial colony gliding on solid interfaces has remained unresolved. Here, this work captures the real‐time development and gliding of bacterial biofilms on vibrating solids made of piezoelectric quartz. The gliding, characterized by liquid slips, is measured in form of frequency and dissipation changes of the vibrating solid. These vibrations enable the generation of distinct acoustic fingerprints (sound/ music) of the three phases of biofilm development: viscoelastic strengthening, biofilm growth and biofilm stability. In adition, the effect of extracellular matrix secretion on the rigidity of the film and its nanoslip in each of the distinct biofilm developmental phases is quantified. This work provides a real‐time, label‐free method of quantifying bacteria biofilm dynamics and paves the way for developing libraries of acoustic signatures of bacteria and their metabolic products.

Surface remodeling and inversion of cell-matrix interactions underlies community recognition and dispersal in Vibrio cholerae biofilms.

Biofilms are ubiquitous surface-associated bacterial communities embedded in an extracellular matrix. While it is commonly assumed that biofilm-dwelling cells are glued together by the matrix, how the cell-matrix interaction depends on the specific biochemistry of the matrix components and how this interaction varies during biofilm growth remains unclear. Here, we investigated cell-matrix interactions in Vibrio cholerae ( Vc ), the causative agent of cholera. We combine genetics, microscopy, simulation, and biochemical tools to show that Vc cells are not attractive to V ibrio p oly s accharide (VPS), the main matrix component, but they can be bridged with each other and to the VPS network through crosslinking by Bap1. Downregulation of VPS and surface trimming by the polysaccharide lyase RbmB cause surface remodeling as biofilms age, shifting the nature of cell-matrix interactions from attractive to repulsive and facilitating cell dispersal as aggregated groups. Our results suggest a new conceptual model in understanding the intricate cell-matrix interaction as the major driver for biofilm development, which is potentially generalizable to certain other biofilm-forming species and exopolysaccharides.

Optically accessible, 3D-printed flow chamber with integrated sensors for the monitoring of oral multispecies biofilm growth in vitro.

The formation of pathogenic multispecies biofilms in the human oral cavity can lead to implant-associated infections, which may ultimately result in implant failure. These infections are neither easily detected nor readily treated. Due to high complexity of oral biofilms, detailed mechanisms of the bacterial dysbiotic shift are not yet even fully understood. In order to study oral biofilms in more detail and develop prevention strategies to fight implant-associated infections, in vitro biofilm models are sorely needed. In this study, we adapted an in vitro biofilm flow chamber model to include miniaturized transparent 3D-printed flow chambers with integrated optical pH sensors - thereby enabling the microscopic evaluation of biofilm growth as well as the monitoring of acidification in close proximity. Two different 3D printing materials were initially characterized with respect to their biocompatibility and surface topography. The functionality of the optically accessible miniaturized flow chambers was then tested using five-species biofilms (featuring the species Streptococcus oralis, Veillonella dispar, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis) and compared to biofilm growth on titanium specimens in the established flow chamber model. As confirmed by live/dead staining and fluorescence in situ hybridization via confocal laser scanning microscopy, the flow chamber setup proved to be suitable for growing reproducible oral biofilms under flow conditions while continuously monitoring biofilm pH. Therefore, the system is suitable for future research use with respect to biofilm dysbiosis and also has great potential for further parallelization and adaptation to achieve higher throughput as well as include additional optical sensors or sample materials.

Application of a novel phage vB_CjeM_WX1 to control Campylobacter jejuni in foods

Campylobacter jejuni is one of the leading causes of human gastroenteritis. Phage biocontrol is recognized as a natural, environmentally friendly technique that effectively targets pathogens in various foods. In this study, a novel C. jejuni phage named vB_CjeM_WX1 (WX1) was isolated from chicken feces. According to the morphology and genomic analysis, the phage belongs to the Eucampyvirinae genus within the subfamily of Caudoviricetes WX1 exhibited favorable physiological characteristics, as it could maintain its activity even under extreme conditions such as high temperatures (70 °C), acidity (pH = 4), alkalinity (pH = 12), NaCl concentration (1000 mM) and was UV-resistant for 50 min. WX1 could lyse 35 strains of C. jejuni, all of which are highly virulent and multi-drug resistant. Among them, 10 strains of C. jejuni exhibit strong biofilm formation, a critical factor in bacterial persistence and resistance to environmental stressors. The lysis rate of WX1 reached up to 47.3 % in 76 strains of C. jejuni. Phage WX1 inhibited the growth of multi-drug resistant, high virulence and strong biofilm C. jejuni 178-2B in NZCYM broth, as well as greatly reduced biofilm formation on stainless-steel, polyethylene surfaces, and glass. Moreover, phage WX1 decreased the number of C. jejuni in chicken skin to below the detection limit within 48 h. Therefore, phage WX1 can be used in food processing environments and poultry farming, both primary production and during slaughter with a great prospect.

Integrating Biofilm Growth and Degradation into a Model of Microplastic Transport in the Arctic Ocean

Integrating Biofilm Growth and Degradation into a Model of Microplastic Transport in the Arctic Ocean

“On-demand” nanosystem-integrated microneedles for amplified triple therapy against recalcitrant bacteria and biofilm growth

Phototherapy has emerged to eradicate recalcitrant bacteria without causing drug resistance, but it is often accompanied by considerable limitations owing to a high tolerance of recalcitrant bacteria to heat and oxidative damage, leading to low efficiency of monotherapy and unwanted side effects. Assuming that employing antimicrobial peptides (AMPs) to disrupt bacterial membranes could reduce bacterial tolerance, a multifunctional “on-demand” nanosystem based on zeolitic imidazolate framework-8 (ZIF-8) with metal ions for intrinsic antibacterial activity was constructed to potently kill methicillin-resistant Staphylococcus aureus (MRSA). Then, microneedles (MNs) were used to transdermally deliver the ZIF-8-based nanosystem for localized skin infection. After MNs insertion, the nanoplatform could specifically deliver the loaded therapeutic components to bacterial infection sites through employing hyaluronic acid (HA) as a capping agent, thus realizing the “on-demand” payload release triggered by excess hyaluronidase secreted by MRSA. The prepared nanosystem and MNs were confirmed to exert an amplified triple therapy originating from membranolytic effect, phototherapy, and ion therapy, thus displaying a powerful bactericidal and MRSA biofilm destruction ability. This intelligent antimicrobial strategy may bring a dawn of hope for eradicating multidrug-resistant bacteria and biofilms.

A ‘Tuba Drain’ incorporated in sink drains reduces counts of antibiotic-resistant bacterial species at the plughole: a blinded, randomized trial in 36 sinks in a hospital outpatient department with a low prevalence of sink colonization by antibiotic-resistant species

Multi-resistant Gram-negative bacteria (GNB) survive in hospital drains in traps that contain water and may ascend into the sink because of splashes, or biofilm growth. To investigate whether the 'Tuba Drain' (TD) a long, bent, continually descending copper tube between the sink outlet and the trap prevents the ascent of bacteria. After initial laboratory tests confirmed that the TD prevented bacteria in the U-bend from splashing upwards into the sink outlet, TDs were assessed in a randomized, blinded trial in a hospital outpatient department built in 2019. Sinks were paired into those with a similar clinical exposure and each member of each pair was randomized to receive either new, standard plumbing up to and including the trap (18 sinks) or the same new standard plumbing but including the TD inserted between the sink outlet and trap. Bacterial counts in swabs from the sink outlets were determined blindly before and monthly after the plumbing change for a year. GNB that are associated with clinical infection and carriage of resistance genes, Pseudomonas aeruginosa, Acinetobacter baumanii, Stenotrophomonas maltophilia and all Enterobacterales were the organisms of primary interest and termed target bacteria. The TDs fitted into the required spaces and functioned without problems. The geometric means (over months) of the counts of target bacteria in TD-plumbed sinks was lower than those in their paired controls, P=0.012 (sign test, two-tailed). Prevalence of target bacteria in sinks was low. TDs were effective for reducing target bacteria in sinks.

Light intensity effects on bioproduct recovery from fuel synthesis wastewater using purple phototrophic bacteria in a hybrid biofilm-suspended growth system

This research looked at how three different light intensities (1600, 4300, and 7200 lx) affect the biomass development, treatment of fuel synthesis wastewater and the recovery of valuable bioproducts between biofilm and suspended growth in a purple-bacteria enriched photobioreactor. Each condition was run in duplicate using an agricultural shade cloth as the biofilm support media in a continuously mixed batch reactor. The results showed that the highest chemical oxygen demand (COD) removal rate (56.8 ± 0.9 %) was found under the highest light intensity (7200 lx), which also led to the most biofilm formation and highest biofilm biomass production (1225 ± 95.7 mg). The maximum carotenoids (Crts) and bacteriochlorophylls (BChls) content occurred in the suspended growth of the 7200 lx reactor. BChls decreased with light intensity in suspended growth, while in biofilm both Crts and BChls were relatively stable between light conditions, likely due to an averaging effect as biofilm thickened at higher light intensity. Light intensity did not affect protein content of the biomass, however, biofilm showed a lower average (41.2 % to 43.7 %) than suspended biomass (45.4 % to 47.7 %). For polyhydroxybutyrate (PHB) the highest cell concentration in biofilm occurred at 1600 lx (11.4 ± 2.4 %), while for suspended growth it occurred at 7200 lx (22.7 ± 0.3 %), though total PHB productivity remained similar between reactors. Shading effects from the externally located biofilm could explain most variations in bioproduct distribution. Overall, these findings suggest that controlling light intensity can effectively influence the treatment of fuel synthesis wastewater and the recovery of valuable bioproducts in a biofilm photobioreactor.

Different management strategies for artificial substrates on nitrification, microbial composition, and growth of Penaeus vannamei in a super-intensive biofloc system

The aim of this study was to test the effect of different management strategies for artificial substrates on the nitrification process, microbial composition of water and biofilm, and growth of Penaeus vannamei in a super-intensive biofloc system. The research was conducted over 60 days using experimental units of 200 L and had the following treatments: T1 – artificial substrate + biofloc + shrimp + aeration (control), T2 - artificial substrate + water + aeration, T3 - artificial substrate + water, and T4 - only artificial substrate. The experiment was divided into two phases. Phase 1 involved maintaining the artificial substrates under the management strategy for 30 days. Phase 2 involved the use of substrates from phase 1 in a shrimp nursery for 30 days. Pre colonized artificial substrates (Needlona®) were employed at a ratio of 200 % of the tank's lateral area to promote biofilm growth. In the phase 1, T1 used a stocking density of 500 shrimp m−3 (9.72 ± 0.50 g). In phase 2, shrimp weighing 0.10 ± 0.05 g were stocked at a density of 1750 shrimp m−3 and all treatments included artificial substrates from phase 1, aeration, water, and shrimp. Molasses was the carbon source organic used. In phase 2, ammonia control was observed in treatments T2, T3, and T4 from the 10th day onwards. T4 had a nitrite spike, controlled from day 14 onwards, suggesting recovery of the nitrifying bacteria community. At the end of the phase 2, T2 and T4 showed higher abundance of coccoid bacteria in the biofilm compared to T1 and T3. T4 also had more bacillus. The shrimp final weight was higher in T2 compared to the other treatments. These findings suggest that maintaining the substrate submerged in water (T3) can be considered a practical management for artificial substrates and that it does not limit the nitrification process between culture cycles. Furthermore, exposure of artificial substrates to air (T4) also did not affect the nitrification process, leading to the recovery of the bacterial community, and the proliferation of various bacterial groups.

Environmental pH and compound structure affect the activity of short-chain carboxylic acids against planktonic growth, biofilm formation, and eradication of the food pathogen Salmonella enterica.

Short-chain carboxylic acids (SCCAs) that are naturally produced by microbial fermentation play an essential role in delaying microbial spoilage. SCCAs are structurally diverse, but only a few of them are routinely used in food biopreservation. This study investigated the effects of environmental pH and intrinsic properties of 21 structurally different SCCAs on the antimicrobial and antibiofilm activity against Salmonella enterica. Inhibition of SCCA toward planktonic and biofilm growth of S. enterica was higher in an acidic environment (pH 4.5) that is common in fermented products, and for SCCA that possessed both a high acid dissociation strength (pKa) (>4.0) and a positive hydrophobicity [octanol/water partition coefficient (log Kow)]. Crotonic and caproic acids were identified as SCCAs with potential as biopreservatives even at near-neutral pH. SCCA with hydrophilic groups such as lactic acid did not inhibit S. enterica at concentrations up to 50 mM, while SCCA with benzene or methyl groups or a double bond prevented S. enterica growth and biofilm formation. Stimulation of biofilm formation was observed for formic, acetic, and propionic acid close to the minimum inhibitory concentration to reduce 50% of cell density (MIC50) of planktonic cells, and for citric and isocitric acid with an MIC50 of ≥50 mM. The presence of low concentrations of formic and propionic acids during biofilm formation conferred protection during eradication possibly due to a pre-adaptation effect, yet two consecutive acid treatments were successful in eradicating biofilms if the first acid treatment was two- to threefold of the MIC50.IMPORTANCEThis study provides a systematic comparison on the antimicrobial and antibiofilm activity of more than 20 structurally different SCCAs against a common food pathogen. We tested the antimicrobial activity at controlled pH and identified the structure-dependent antimicrobial effects of SCCA without the confounding influence of acidification. The combined effect of pKa and log Kow was identified as an important feature that should be considered when deciding for a specific SCCA in the application as antimicrobial. Our results imply that additional phenomena such as the use of SCCA as substrate and cellular pre-adaption effects have to be taken into consideration. We finally present a two-step treatment as an efficient approach to eradicate biofilms, which can be applied for the disinfection of contact surfaces and manufacturing equipment. Results obtained here can serve as guidelines for application of SCCA to avoid the growth of food pathogens and/or to develop biopreserved food systems.