Development of a simple intensified fermentation strategy for growth of Magnetospirillum gryphiswaldense MSR-1:Physiological responses to changing environmental conditions

The development of a simple pH-stat fed-batch fermentation strategy for the production of Magnetospirillum gryphiswaldense MSR-1 and magnetosomes (nanoscale magnetic organelles with biotechnological applications) is described. Flow cytometry was exploited as a powerful analytical tool for process development, enabling rapid monitoring of cell morphology, physiology and polyhydroxyalkanoate production. The pH-stat fed-batch growth strategy was developed by varying the concentrations of the carbon source (lactic acid) and the alternative electron acceptor (sodium nitrate) in the feed. Growth conditions were optimized on the basis of biomass concentration, cellular magnetism (indicative of magnetosome production), and intracellular iron concentration. The highest biomass concentration and cellular iron content achieved were an optical density at 565 nm of 15.5 (equivalent to 4.2 g DCW·L−1) and 33.1 mg iron·g−1 DCW, respectively. This study demonstrates the importance of analyzing bacterial physiology during fermentation development and will potentially aid the industrial production of magnetosomes, which can be used in a wide range of biotechnology and healthcare applications

Reduction of aerobic and lactic acid bacteria in dairy desludge using an integrated compressed CO2 and ultrasonic process

International audienceAbstractCurrent treatment routes are not suitable to reduce and stabilise bacterial content in some dairy process streams such as separator and bactofuge desludges which currently present a major emission problem faced by dairy producers. In this study, a novel method for the processing of desludge was developed. The new method, elevated pressure sonication (EPS), uses a combination of low frequency ultrasound (20 kHz) and elevated CO2 pressure (50 to 100 bar). Process conditions (pressure, sonicator power, processing time) were optimised for batch and continuous EPS processes to reduce viable numbers of aerobic and lactic acid bacteria in bactofuge desludge by ≥3-log fold. Coagulation of proteins present in the desludge also occurred, causing separation of solid (curd) and liquid (whey) fractions. The proposed process offers a 10-fold reduction in energy compared to high temperature short time (HTST) treatment of milk

Exposure of Salmonella enterica Serovar Typhimurium to High Level Biocide Challenge Can Select Multidrug Resistant Mutants in a Single Step

Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux.Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide.These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations

Non-pathogenic Escherichia coli biofilms: effects of growth conditions and surface properties on structure and curli gene expression

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions

A Protein-Centric Mass Spectrometry Approach for Species Identification within Harmful Algal Blooms

Harmful algal blooms present severe environmental threats, impacting water quality, aquatic ecosystems, and human health. The frequency and intensity of these blooms are rising, largely driven by global warming and changing climatic conditions. There is an urgent need for innovative methods to monitor blue-green algae, also known as cyanobacteria, to enable the implementation of preventative measures. Here, we show that native mass spectrometry is an effective tool for detecting cyanobacteria directly from lake samples, both prior and during bloom formation. Our approach allows for the rapid characterization of cyanobacterial populations within lakes, offering valuable insights into the dynamics of cyanobacterial species associated with harmful algae blooms. Overall, we highlight the exceptional capability of native mass spectrometry in directly detecting and monitoring cyanobacterial blooms, which will support the development of more effective strategies to mitigate this growing environmental challenge.</p

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli

Nanovibrational Stimulation of <i>Escherichia coli </i>Mitigates Surface Adhesion by Altering Cell Membrane Potential

Mechanical forces shape living matter from the macro- to the microscale as both eukaryotic and prokaryotic cells are force wielders and sensors. However, whereas such forces have been used to control mechanically dependent behaviors in mammalian cells, we lack the same level of understanding in bacteria. Surface adhesion, the initial stages of biofilm formation and surface biofouling, is a mechanically dependent process, which makes it an ideal target for mechano-control. In this study, we employed nanometer surface vibrations to mechanically stimulate bacteria and investigate their effect on adhesion. We discovered that vibrational stimulation at the nanoscale consistently reduces surface adhesion by altering cell membrane potential. Our findings identify a link between bacteria electrophysiology and surface adhesion and provide evidence that the nanometric mechanical "tickling" of bacteria can inhibit surface adhesion.</p

Nanovibrational Stimulation of <i>Escherichia coli </i>Mitigates Surface Adhesion by Altering Cell Membrane Potential

Mechanical forces shape living matter from the macro- to the microscale as both eukaryotic and prokaryotic cells are force wielders and sensors. However, whereas such forces have been used to control mechanically dependent behaviors in mammalian cells, we lack the same level of understanding in bacteria. Surface adhesion, the initial stages of biofilm formation and surface biofouling, is a mechanically dependent process, which makes it an ideal target for mechano-control. In this study, we employed nanometer surface vibrations to mechanically stimulate bacteria and investigate their effect on adhesion. We discovered that vibrational stimulation at the nanoscale consistently reduces surface adhesion by altering cell membrane potential. Our findings identify a link between bacteria electrophysiology and surface adhesion and provide evidence that the nanometric mechanical "tickling" of bacteria can inhibit surface adhesion.</p

Engineering biofilms for biocatalysis

Biofilm, friend not foe: Single species biofilms can be engineered to form robust biocatalysts with greater catalytic activity and significantly improved catalytic longevity than purified and immobilised enzymes. We report the engineering, structural analysis and biocatalytic capability of a biofilm that can mediate the conversion of serine and haloindoles to halotryptophans