Population dynamics of a dual Pseudomonas putida-Pseudomonas fluorescens biofilm in a capillary bioreactor.

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72 h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48 h. Even when the inoculation by P. putida was delayed for 24 h, P. fluorescens was eliminated from the capillary within 48 h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.

Antifouling activity of enzyme-functionalized silica nanobeads.

The amelioration of biofouling in industrial processing equipment is critical for performance and reliability. While conventional biocides are effective in biofouling control, they are potentially hazardous to the environment and in some cases corrosive to materials. Enzymatic approaches have been shown to be effective and can overcome the disadvantages of traditional biocides, however they are typically uneconomic for routine biofouling control. The aim of this study was to design a robust and reusable enzyme-functionalized nano-bead system having biofilm dispersion properties. This work describes the biochemical covalent functionalization of silica-based nanobeads (hereafter referred to as Si-NanoB) with Proteinase K (PK). Results showed that PK-functionalized Si-NanoB are effective in dispersing both protein-based model biofilms and structurally altering Pseudomonas fluorescens biofilms, with significant decreases in surface coverage and thickness of 30.1% and 38.85%, respectively, while increasing surface roughness by 19 % following 24 h treatments on bacterial biofilms. This study shows that enzyme-functionalized nanobeads may potentially be an environmentally friendly and cost effective alternative to pure enzyme and chemical treatments.

Comparison of biomass detachment from biofilms of two different Pseudomonas spp. under constant shear conditions.

In the context of biofilm development, detachment is of practical importance when placed in a biofilm management perspective. The objective of the present study was to examine biofilm structure and biofilm detachment under controlled conditions for two distinct microorganisms grown under constant shear conditions. Detached biofilm biomass was regularly collected and analysed over the course of 72 h biofilm growth by Pseudomonas putida and Pseudomonas fluorescens cells, and biofilm structural development assessed using confocal microscopy. The two Pseudomonas spp., which had very similar specific growth rates in planktonic culture, presented notably different characteristics in terms of biofilm morphology but their detachment behaviours over time were very similar. These findings underline the intrinsic complexity of the detachment phenomenon.

Upon impact: the fate of adhering Pseudomonas fluorescens cells during nanofiltration.

Nanofiltration (NF) is a high-pressure membrane filtration process increasingly applied in drinking water treatment and water reuse processes. NF typically rejects divalent salts, organic matter, and micropollutants. However, the efficiency of NF is adversely affected by membrane biofouling, during which microorganisms adhere to the membrane and proliferate to create a biofilm. Here we show that adhered Pseudomonas fluorescens cells under high permeate flux conditions are met with high fluid shear and convective fluxes at the membrane-liquid interface, resulting in their structural damage and collapse. These results were confirmed by fluorescent staining, flow cytometry, and scanning electron microscopy. This present study offers a "first-glimpse" of cell damage and death during the initial phases of bacterial adhesion to NF membranes and raises a key question about the role of this observed phenomena during early-stage biofilm formation under permeate flux and cross-flow conditions.

The significance of calcium ions on Pseudomonas fluorescens biofilms - a structural and mechanical study.

The purpose of this study was to investigate the effects of calcium ions on the structural and mechanical properties of Pseudomonas fluorescens biofilms grown for 48 h. Advanced investigative techniques such as confocal laser scanning microscopy and atomic force spectroscopy were employed to characterize biofilm structure as well as biofilm mechanical properties following growth at different calcium concentrations. The presence of calcium during biofilm development led to higher surface coverage with distinct structural phenotypes in the form of a granular and heterogeneous surface, compared with the smoother and homogeneous biofilm surface in the absence of calcium. The presence of calcium also increased the adhesive nature of the biofilm, while reducing its elastic properties. These results suggest that calcium ions could have a functional role in biofilm development and have practical implications, for example, in analysis of biofouling in membrane-based water-treatment processes such as nanofiltration or reverse osmosis where elevated calcium concentrations may occur at the solid-liquid interface.

Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolates.

Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms on food-processing surfaces, a pathway leading to cross contamination of foods. The purpose of this study was to investigate the influence of environmental stress factors found during seafood production on the adhesion and biofilm-forming properties of S. aureus. Adhesion and biofilm assays were performed on 26 S. aureus isolated from seafood and two S. aureus reference strains (ATCC 6538 and ATCC 43300). Cell surface properties were evaluated by affinity measurements to solvents in a partitioning test, while adhesion and biofilm assays were performed in polystyrene microplates under different stress conditions of temperature, osmolarity, and nutrient content. The expression of genes implicated in the regulation of biofilm formation (icaA, rbf and σ( B )) was analyzed by reverse transcription and quantitative real time PCR. In general, S. aureus isolates showed moderate hydrophobic properties and a marked Lewis-base character. Initial adhesion to polystyrene was positively correlated with the ionic strength of the growth medium. Most of the strains had a higher biofilm production at 37 °C than at 25 °C, promoted by the addition of glucose, whereas NaCl and MgCl(2) had a lower impact markedly affected by incubation temperatures. Principal Component Analysis revealed a considerable variability in adhesion and biofilm-forming properties between S. aureus isolates. Transcriptional analysis also indicated variations in gene expression between three characteristic isolates under different environmental conditions. These results suggested that the prevalence of S. aureus strains on food-processing surfaces is above all conditioned by the ability to adapt to the environmental stress conditions present during food production. These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging, and storage of seafood products.

Death at the interface: Nanotechnology's challenging frontier against microbial surface colonization.

The emergence of antimicrobial-resistant bacterial strains has led to novel approaches for combating bacterial infections and surface contamination. More specifically, efforts in combining nanotechnology and biomimetics have led to the development of next-generation antimicrobial/antifouling nanomaterials. While nature-inspired nanoscale topographies are known for minimizing bacterial attachment through surface energy and physicochemical features, few studies have investigated the combined inhibitory effects of such features in combination with chemical alterations of these surfaces. Studies describing surface alterations, such as quaternary ammonium compounds (QACs), have also gained attention due to their broad spectrum of inhibitory activity against bacterial cells. Similarly, antimicrobial peptides (AMPs) have exhibited their capacity to reduce bacterial viability. To maximize the functionality of modified surfaces, the integration of patterned surfaces and functionalized exteriors, achieved through physical and chemical surface alterations, have recently been explored as viable alternatives. Nonetheless, these modifications are prone to challenges that can reduce their efficacy considerably in the long term. Their effectiveness against a wider array of microbial cells is still a subject of investigation. This review article will explore and discuss the emerging trends in biomimetics and other antimicrobials while raising possible concerns about their limitations and discussing future implications regarding their potential combined applications.

Cross-regional scale pollution of freshwater biofilms unveiled by antibiotic resistance genes.

A comprehensive global profile of the distribution of ARGs in freshwater biofilms is lacking. We utilized metagenomic approaches to reveal the diversity, abundance, transferability and hosts of ARGs in 96 freshwater biofilm samples from 38 sampling sites across four countries. The abundant ARGs were associated with bacitracin, multidrug, polymyxin macrolide-lincosamide-streptogramin (MLS) aminoglycoside, ß-lactam, chloramphenicol, sulfonamide and tetracycline resistance, consistent with the spectrum of antibiotics commonly used in human or veterinary medicine. As expected, the resistome in freshwater biofilm habitats was significantly influenced by geographical location and human footprint. Based on the co-occurrence pattern revealed by network analysis, mdtC, kdpE, and emrB were proposed as ARG indicators in freshwater biofilms that can be used to evaluate the abundance of 46 other co-occurring ARG subtypes quantitatively. Metagenomic assembly analysis revealed that the identified ARGs were hosted by more than 46 bacterial phyla, including various pathogens, which greatly expands the knowledge of resistome diversity in freshwater biofilms. Our study points to the central roles of biofilms in harbouring ARGs. The results could enhance understanding the distribution of ARGs in freshwater habitats, thereby strengthening the global environmental risk assessment and management of ARGs.

Elucidating the Role of Biofilm-Forming Microbial Communities in Fermentative Biohydrogen Process: An Overview.

Amongst the biofuels described in the literature, biohydrogen has gained heightened attention over the past decade due to its remarkable properties. Biohydrogen is a renewable form of H2 that can be produced under ambient conditions and at a low cost from biomass residues. Innovative approaches are continuously being applied to overcome the low process yields and pave the way for its scalability. Since the process primarily depends on the biohydrogen-producing bacteria, there is a need to acquire in-depth knowledge about the ecology of the various assemblages participating in the process, establishing effective bioaugmentation methods. This work provides an overview of the biofilm-forming communities during H2 production by mixed cultures and the synergistic associations established by certain species during H2 production. The strategies that enhance the growth of biofilms within the H2 reactors are also discussed. A short section is also included, explaining techniques used for examining and studying these biofilm structures. The work concludes with some suggestions that could lead to breakthroughs in this area of research.

Diffusion of nanoparticles in biofilms is altered by bacterial cell wall hydrophobicity.

Diffusion of entities inside biofilm triggers most mechanisms involved in biofilm-specific phenotypes. Using genetically engineered hydrophilic and hydrophobic cells of Lactococcus lactis yielding similar biofilm architectures, we demonstrated by fluorescence correlation spectroscopy that bacterial surface properties affect diffusion of nanoparticles through the biofilm matrix.

Spatial competition with Lactococcus lactis in mixed-species continuous-flow biofilms inhibits Listeria monocytogenes growth.

Surfaces in industrial settings provide a home for resident biofilms that are likely to interact with the attachment, growth and survival of pathogens such as Listeria monocytogenes. Experimental results have indicated that L. monocytogenes cells were inhibited by the presence of a model resident flora (Lactococcus lactis) in dual-species continuous flow-biofilms, and are spatially restricted to the lower biofilm layers. Using a new, simplified individual-based model (IBM) that simulates bacterial cell growth in a three-dimensional space, the spatial arrangements of the two species were reconstructed and their cell counts successfully predicted. This model showed that the difference in generation times between L. monocytogenes and L. lactis cells during the initial stages of dual-species biofilm formation was probably responsible for the species spatialization observed and the subsequent inhibition of growth of the pathogen.

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods.

Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.

Ecotoxicological effects of erythromycin on a multispecies biofilm model, revealed by metagenomic and metabolomic approaches.

The presence of antibiotics such as erythromycin, even in trace amounts, has long been acknowledged for negatively impacting ecosystems in freshwater environments. Although many studies have focused on the impact of antibiotic pollution at a macroecological level, the impact of erythromycin on microecosystems, such as freshwater biofilms, is still not fully understood. This knowledge gap may be attributed to the lack of robust multispecies biofilm models for fundamental investigations. Here, we used a lab-cultured multispecies biofilm model to elucidate the holistic response of a microbial community to erythromycin exposure using metagenomic and metabolomic approaches. Metagenomic analyses revealed that biofilm microbial diversity did not alter following erythromycin exposure. Notably, certain predicted metabolic pathways such as cell-cell communication pathways, amino acid metabolism, and peptidoglycan biosynthesis, mainly by the phyla Actinobacteria, Alpha/Beta-proteobacteria, Bacteroidetes, and Verrucomicrobia, were found to be involved in the maintenance of homeostasis-like balance in the freshwater biofilm. Further untargeted metabolomics data highlighted changes in lipid metabolism and linoleic acid metabolism and their related molecules as a direct consequence of erythromycin exposure. Overall, the study presented a unique picture of how multispecies biofilms respond to single environmental stress exposures. Moreover, the study demonstrated the feasibility of using lab simulated multispecies biofilms for investigating their interaction and reactivity of specific bioactive compounds or pollutants at a fundamental level.

Questioning the source of identified non-foodborne pathogens from food-contact wooden surfaces used in Hong Kong's urban wet markets.

In this study, a phylogenic analysis was performed on pathogens previously identified in Hong Kong wet markets' cutting boards. Phylogenetic comparisons were made between phylotypes obtained in this study and environmental and clinical phylotypes for establishing the possible origin of selected bacterial species isolated from wet market cutting board ecosystems. The results reveal a strong relationship between wet market bacterial assemblages and environmental and clinically relevant phylotypes. However, our poor knowledge of potential cross-contamination sources within these wet markets is further exacerbated by failing to determine the exact or presumed origin of its identified pathogens. In this study, several clinically relevant bacterial pathogens such as Klebsiella pneumoniae, Streptococcus suis and Streptococcus porcinus were linked to cutting boards associated with pork; Campylobacter fetus, Staphylococcus aureus, Escherichia coli, and A. caviae in those associated with poultry; and Streptococcus varanii, A. caviae, Vibrio fluvialis, and Vibrio parahaemolyticus in those associated with seafood. Identifying non-foodborne clinically relevant pathogens in wet market cutting boards in this study confirms the need for safety approaches for wet market meat, including cold storage. The presented study justifies the need for future systematic epidemiological studies to determine identified microbial pathogens. Such studies should bring about significant improvements in the management of hygienic practices in Hong Kong's wet markets and work towards a One Health goal by recognizing the importance of wet markets as areas interconnecting food processing with animal and clinical environments.

Exploring the optimization of aerobic food waste digestion efficiency through the engineering of functional biofilm Bio-carriers.

The possibility of breaking down cellulose-rich food waste through biofilm engineering was investigated. Six previously isolated strains from naturally degrading fruits and vegetables, screened for biofilm-forming ability and cellulolytic activity, were selected to enrich a biocarrier seeding microbial consortium. The food waste model used in this study was cabbage which was aerobically digested under repeated water rinsing and regular effluent drainage. The engineered biocarrier biofilm's functionality was evaluated by tracing microbial succession following metagenomic sequencing, quantitative PCR, scanning electron microscopy, and cellulolytic activity before and after the digestion processes. The engineered microbial consortium demonstrated superior biofilm-forming ability on biocarriers than the original microbial consortium and generally displayed a higher cellulolytic activity. The presented study provides one of the few studies of food waste aerobic digestion using engineered biofilms. Insights presented in this study could help further optimize aerobic food waste digestion.

The Potential of Single-Cell Oils Derived From Filamentous Fungi as Alternative Feedstock Sources for Biodiesel Production.

Microbial lipids, also known as single-cell oils (SCOs), are highly attractive feedstocks for biodiesel production due to their fast production rates, minimal labor requirements, independence from seasonal and climatic changes, and ease of scale-up for industrial processing. Among the SCO producers, the less explored filamentous fungi (molds) exhibit desirable features such as a repertoire of hydrolyzing enzymes and a unique pellet morphology that facilitates downstream harvesting. Although several oleaginous filamentous fungi have been identified and explored for SCO production, high production costs and technical difficulties still make the process less attractive compared to conventional lipid sources for biodiesel production. This review aims to highlight the ability of filamentous fungi to hydrolyze various organic wastes for SCO production and explore current strategies to enhance the efficiency and cost-effectiveness of the SCO production and recovery process. The review also highlights the mechanisms and components governing lipogenic pathways, which can inform the rational designs of processing conditions and metabolic engineering efforts for increasing the quality and accumulation of lipids in filamentous fungi. Furthermore, we describe other process integration strategies such as the co-production with hydrogen using advanced fermentation processes as a step toward a biorefinery process. These innovative approaches allow for integrating upstream and downstream processing units, thus resulting in an efficient and cost-effective method of simultaneous SCO production and utilization for biodiesel production.

Biomimetic Polymer Surfaces by High Resolution Molding of the Wings of Different Cicadas.

Recent studies have shown that insect wings have evolved to have micro- and nanoscale structures on the wing surface, and biomimetic research aims to transfer such structures to application-specific materials. Herein, we describe a simple and cost-effective method of replica molding the wing topographies of four cicada species using UV-curable polymers. Different polymer blends of polyethylene glycol diacrylate and polypropylene glycol diacrylate were used as molding materials and a molding chamber was designed to precisely control the x, y, and z dimensions. Analysis by scanning electron microscopy showed that structures ranged from 148 to 854 nm in diameter, with a height range of 191-2368 nm, and wing patterns were transferred with high fidelity to the crosslinked polymer. Finally, bacterial cell studies show that the wing replicas possess the same antibacterial effect as the cicada wing from which they were molded. Overall, this work shows a quick and simple method for patterning UV-curable polymers without the use of expensive equipment, making it a highly accessible means of producing microstructured materials with biological properties.

Enhanced surface colonization by Escherichia coli O157:H7 in biofilms formed by an Acinetobacter calcoaceticus isolate from meat-processing environments.

A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 x 10(6) microm(3)) in a dynamic cocultured biofilm than in a monoculture (3.0 x 10(3) microm(3)), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.

Micro ecosystems from feed industry surfaces: a survival and biofilm study of Salmonella versus host resident flora strains.

BACKGROUND: The presence of Salmonella enterica serovars in feed ingredients, products and processing facilities is a well recognized problem worldwide. In Norwegian feed factories, strict control measures are implemented to avoid establishment and spreading of Salmonella throughout the processing chain. There is limited knowledge on the presence and survival of the resident microflora in feed production plants. Information on interactions between Salmonella and other bacteria in feed production plants and how they affect survival and biofilm formation of Salmonella is also limited. The aim of this study was to identify resident microbiota found in feed production environments, and to compare the survival of resident flora strains and Salmonella to stress factors typically found in feed processing environments. Moreover, the role of dominant resident flora strains in the biofilm development of Salmonella was determined. RESULTS: Surface microflora characterization from two feed productions plants, by means of 16 S rDNA sequencing, revealed a wide diversity of bacteria. Survival, disinfection and biofilm formation experiments were conducted on selected dominant resident flora strains and Salmonella. Results showed higher survival properties by resident flora isolates for desiccation, and disinfection compared to Salmonella isolates. Dual-species biofilms favored Salmonella growth compared to Salmonella in mono-species biofilms, with biovolume increases of 2.8-fold and 3.2-fold in the presence of Staphylococcus and Pseudomonas, respectively. CONCLUSIONS: These results offer an overview of the microflora composition found in feed industry processing environments, their survival under relevant stresses and their potential effect on biofilm formation in the presence of Salmonella. Eliminating the establishment of resident flora isolates in feed industry surfaces is therefore of interest for impeding conditions for Salmonella colonization and growth on feed industry surfaces. In-depth investigations are still needed to determine whether resident flora has a definite role in the persistence of Salmonella in feed processing environments.

Effects of sodium citrate on the structure and microbial community composition of an early-stage multispecies biofilm model.

In recent years, most biofilm studies have focused on fundamental investigations using multispecies biofilm models developed preferentially in simulated naturally occurring low-nutrient medium than in artificial nutrient-rich medium. Because biofilm development under low-nutrient growth media is slow, natural media are often supplemented with an additional carbon source to increase the rate of biofilm formation. However, there are knowledge gaps in interpreting the effects of such supplementation on the resulting biofilm in terms of structure and microbial community composition. We investigated the effects of supplementation of a simulated freshwater medium with sodium citrate on the resulting structure, bacterial community composition, and microbial network interactions of an early-stage multispecies biofilm model. Qualitative and quantitative analyses of acquired confocal laser scanning microscopy data confirmed that sodium citrate supplementation distinctly increased biofilm biomass. Sequencing data revealed that the microbial community structure of biofilms grown in sodium citrate-supplemented conditions was characterized with increased relative abundance and dominance of Proteobacteria compared with that of biofilms grown in sodium citrate-free conditions. Our findings suggest that the supplementation of a low-nutrient medium with a carbon source in experiments involving multispecies biofilms may lead to structural and compositional biases of the microbial community, causing changes in biofilm phenotype.