Pili and other surface proteins influence the structure and the nanomechanical properties of Lactococcus lactis biofilms.

Lactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globally the strains devoid of pili displayed smoother and stiffer biofilms (Young Modulus of 4-100 kPa) than those of piliated strains (Young Modulus around 0.04-0.1 kPa). Additional display of a mucus-binding protein did not affect the biofilm stiffness but made the biofilm smoother and more compact. Finally, we demonstrated the role of pili in the biofilm cohesiveness by monitoring the homotypic adhesion of bacteria to the biofilm surface. These results will help to understand the role of pili and mucus-binding proteins withstanding external forces.

Analysis of Homotypic Interactions of Lactococcus lactis Pili Using Single-Cell Force Spectroscopy.

Cell surface proteins of Gram-positive bacteria play crucial roles in their adhesion to abiotic and biotic surfaces. Pili are long and flexible proteinaceous filaments known to enhance bacterial initial adhesion. They promote surface colonization and are thus considered as essential factors in biofilm cohesion. Our hypothesis is that pili mediate interactions between cells and may thereby directly affect biofilm formation. In this study, we use single-cell force spectroscopy (SCFS) to quantify the force of the homotypic pili interactions between individual bacterial cells, using different Lactococcus lactis strains producing pili or not as model bacteria. Moreover the force-distance curves were analyzed to determine the physical and nanomechanical properties of L. lactis pili. The results for pili-devoided strains showed a weak adhesion between cells (adhesion forces and work in the range of 100 pN and 7 × 10-18 J, respectively). On the contrary, the piliated strains showed high adhesion levels with adhesion forces and adhesion work over 200 pN and 50 × 10-18 J, respectively. The force-extension curves showed multiple adhesion events, typical of the unfolding of macromolecules. These unfolding force peaks were fitted using the physical worm-like chain model to get fundamental knowledge on the pili nanomechanical properties. In addition, SCFS applied to a L. lactis isolate expressing both pili and mucus-binding protein at its surface and two derivative mutants revealed the capacity of pili to interact with other surface proteins including mucus-binding proteins. This study demonstrates that pili are involved in L. lactis homotypic interactions and thus can influence biofilm structuring.

Process-Oriented Review of Bacterial Quorum Quenching for Membrane Biofouling Mitigation in Membrane Bioreactors (MBRs).

Quorum Quenching (QQ) has been developed over the last few years to overcome practical issues related to membrane biofouling, which is currently the major difficulty thwarting the extensive development of membrane bioreactors (MBRs). QQ is the disruption of Quorum Sensing (QS), cell-to-cell communication enabling the bacteria to harmonize their behavior. The production of biofilm, which is recognized as a major part of the biocake formed on a membrane surface, and which leads to biofouling, has been found to be one of the bacterial behaviors controlled by QS. Since the enzymatic disruption of QS was reported to be efficient as a membrane biofouling mitigation technique in MBRs, the application of QQ to lab-scale MBRs has been the subject of much research using different approaches under different operating conditions. This paper gives an overview of the effectiveness of QQ in mitigating membrane biofouling in MBRs. It is based on the results of previous studies, using two microbial strains, Rhodococcus sp. BH4 and Pseudomonas sp. 1A1. The effect of bacterial QQ on the physical phenomena of the MBR process is analyzed, adopting an original multi-scale approach. Finally, the potential influence of the MBR operating conditions on QQ effectiveness is discussed.

Clarification of purple carrot juice: analysis of the fouling mechanisms and evaluation of the juice quality.

Purple carrot juice was clarified by microfiltration. Two modes of filtration, batch concentration and total recycle were tested and the effect of microfiltration process on permeate flux and membrane fouling was studied. Intrinsic membrane resistance was negligible compared with the fouling resistances, which was less than 5 % of total resistance. Determination of membrane hydraulic permeability showed that water cleaning could permit a recovery of about 7 % of initial hydraulic flux. The analysis of color parameters of feed, permeate and concentrate juice during filtration shows that the a* and b* values decrease for the permeate corresponding respectively to changes from green to red and from blue to yellow. The total sugar and reducing sugars increase in permeate and decrease in concentrate. This work showed that it was possible to clarify the purple carrot juice by microfiltration with a real amelioration of the juice appearance.

Mécano-Stimulation™ of the skin improves sagging score and induces beneficial functional modification of the fibroblasts: clinical, biological, and histological evaluations.

BACKGROUND: Loss of mechanical tension appears to be the major factor underlying decreased collagen synthesis in aged skin. Numerous in vitro studies have shown the impact of mechanical forces on fibroblasts through mechanotransduction, which consists of the conversion of mechanical signals to biochemical responses. Such responses are characterized by the modulation of gene expression coding not only for extracellular matrix components (collagens, elastin, etc.) but also for degradation enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). A new device providing a mechanical stimulation of the cutaneous and subcutaneous tissue has been used in a simple, blinded, controlled, and randomized study. MATERIALS AND METHODS: Thirty subjects (aged between 35 years and 50 years), with clinical signs of skin sagging, were randomly assigned to have a treatment on hemiface. After a total of 24 sessions with Mécano-Stimulation™, biopsies were performed on the treated side and control area for in vitro analysis (dosage of hyaluronic acid, elastin, type I collagen, MMP9; equivalent dermis retraction; GlaSbox(®); n=10) and electron microscopy (n=10). Furthermore, before and after the treatment, clinical evaluations and self-assessment questionnaire were done. RESULTS: In vitro analysis showed increases in hyaluronic acid, elastin, type I collagen, and MMP9 content along with an improvement of the migratory capacity of the fibroblasts on the treated side. Electron microscopy evaluations showed a clear dermal remodeling in relation with the activation of fibroblast activity. A significant improvement of different clinical signs associated with skin aging and the satisfaction of the subjects were observed, correlated with an improvement of the sagging cheek. CONCLUSION: Mécano-Stimulation is a noninvasive and safe technique delivered by flaps microbeats at various frequencies, which can significantly improve the skin trophicity. Results observed with objective measurements, ie, in vitro assessments and electron microscopy, confirm the firming and restructuring effect clinically observed.

Experimental Study of Membrane Fouling during Crossflow Microfiltration of Yeast and Bacteria Suspensions: Towards an Analysis at the Microscopic Level.

Microfiltration of model cell suspensions combining macroscopic and microscopic approaches was studied in order to better understand microbial membrane fouling mechanisms. The respective impact of Saccharomyces cerevisiae yeast and Escherichia coli bacteria on crossflow microfiltration performances was investigated using a multichannel ceramic 0.2 µm membrane. Pure yeast suspensions (5 µm ovoid cells) and mixtures of yeast and bacteria (1 to 2.5 µm rod shape cells) were considered in order to analyse the effect of interaction between these two microorganisms on fouling reversibility. The resistances varied significantly with the concentration and characteristics of the microorganisms. Membrane fouling with pure yeast suspension was mainly reversible. For yeast and bacteria mixed suspensions (6 g L-1 yeast concentration) the increase in bacteria from 0.15 to 0.30 g L-1 increased the percentage of normalized reversible resistance. At 10 g L-1 yeast concentration, the addition of bacteria tends to increase the percentage of normalized irreversible resistance. For the objective of performing local analysis of fouling, an original filtration chamber allowing direct in situ observation of the cake by confocal laser scanning microscopy (CLSM) was designed, developed and validated. This device will be used in future studies to characterize cake structure at the microscopic scale.

Fluorescent proteins as in-vivo and in-situ reporters to study the development of a Saccharomyces cerevisiae yeast biofilm and its invasion by the bacteria Escherichia coli.

This work deals with the bacterial contamination of yeast, both as biofilm and in the planktonic phase. A model continuous system using self-fluorescent microorganisms was proposed to perform in vivo and in situ studies of a mixed biofilm. The yeast strain was inoculated first while the bacteria were added few days later to mimic a contamination. Supports sampled during the experiment were observed by scanning confocal laser microscopy. The behaviour of the microorganisms in real process conditions was then analysed without any treatment that could modify their physiology and/or damage the community structure. Using image analysis, the characteristics of biofilm development (microorganism ratio, 3D-organisation, growth rates) were studied and compared to the behaviour of the suspended cells in the bioreactor. Based on the biovolumes (volume occupied by each microorganism), the growth rates in biofilm for the bacteria and the yeasts were determined at 0.10 and 0.03 h(-1) respectively, while the imposed dilution rate was 0.10 h(-1). Even though the ability of yeast to develop biofilm was demonstrated, its capacity remained very low compared to that of the bacteria which quickly invaded and covered the whole yeast biofilm. This approach makes an original and powerful tool to study the competition phenomena occurring in model biofilms.

Why and how membrane bioreactors with unsteady filtration conditions can improve the efficiency of biological processes.

A membrane bioreactor (MBR), an association of a bioreactor with a crossflow filtration unit, enables continuous processes with total cell retention within the reactor to be realized. Provided that high dilution rates can be applied and that inhibition processes are avoided, very high biomass concentrations can be reached, thereby improving the volumetric productivities. These membrane bioreactors have been successfully applied to various microbial bioconversion, such as alcoholic fermentation, solvents, organic acid production, starters, and wastewater treatment. On the basis of the biological reaction characteristics and bibliographic results, the potentialities and bottlenecks of this methodology are discussed. Depending on the application, it is shown how the performance of the membrane bioreactor can be enhanced by acting either on the biological reaction achievement, by controlling the balance between cell growth and death, or on the dilution rate, by increasing the permeate flux through the filtration unit. This discussion is based on results obtained in specific biological treatments applied to polluted liquid and gas.