Effect of various parameters on viability and growth of bacteria immobilized in sol–gel-derived silica matrices

Immobilized bacteria are being extensively used for metabolite production, biocatalysts, and biosensor construction. However, long-term viability and metabolic activity of entrapped bacteria is affected by several conditions such as their physiological state, the presence of high-osmolarity environments, porous structure and shrinkage of the matrix. The aim of this work was to evaluate the effect of various parameters on bacteria immobilized in sol–gel-derived silica matrices. With this purpose, we evaluated the stress of immobilization over bacteria cultures obtained from different growing states, the effect of cell density and bacteria capability to proliferate inside matrices. Best results to attain longer preservation times were obtained when we immobilized suspensions with an optimized bacterial number of 1 × 10⁷ cfu/gel in the presence of LB medium using aqueous silica precursors. Furthermore, the impact of osmotic stress with the subsequent intracellular trehalose accumulation and the addition of osmolites were investigated. Shorter preservation times were found for bacteria immobilized in the presence of osmolites while trehalose accumulation in stressed cells did not produce changes on entrapped bacteria viability. Finally, nutrient addition in silica matrices was studied indicating that the presence of a carbon source without the simultaneous addition of nitrogen was detrimental for immobilized E. coli. However, when both carbon and nitrogen sources were present, bacteria were able to survive longer periods of time.     

Physiologically Stressed Cells of Fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> EKi as Better Option for Bioformulation Development for Management of Charcoal Rot Caused by <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> in Field Conditions

Bioformulation that supports the inoculant under storage condition and on application to field is of prime importance for agroindustry. Pseudomonas strain EKi having biocontrol activity against Macrophomina phaseolina was used in the study. EKi cells were pretreated by carbon starvation, osmotic stress (NaCl), and freeze drying conditions, and talc-based bioformulation was developed. Combined pretreatment with carbon starvation and osmotic stress was given to Pseudomonas cells. Bioformulation of untreated, freeze dried (FD), carbon starved, osmotic stressed, and combined pre-treated cells showed 50.36, 44.76, 45.95, 34.82, and 27.27% reduction in CFU counts after 6 months of storage. The osmotic stressed cells showed one over-expressed protein (11.5 kDa) in common with carbon starved cells responsible for its better shelf life. The plant growth promotory activity of bioformulations was determined taking Cicer arietinum as a test crop in M. phaseolina infested field. Carbon starved + osmotic stressed cells showed maximum enhancement of dry weight (272.56%) followed by osmotic stressed (230.74%), untreated (155.70%), FD (88.93%), and carbon starved (59.34%) cells over uninoculated control. Carbon starved + osmotic stressed, osmotic stressed, untreated, FD, and carbon starved cells showed 156.60, 100, 75, 40, and 16.67% reduction of charcoal rot disease over uninoculated control. The results clearly showed that combined pretreatment by carbon starvation and osmotic stress provides the bacteria potential of rapid adaptation to different environment conditions.     

Death of<Emphasis Type="Italic"> Escherichia coli</Emphasis> during rapid and s<Emphasis>e</Emphasis>vere dehydration is related to lipid phase transition

This study reports the effects of exposure to increasing osmotic pressure on the viability and membrane structure of Escherichia coli. Changes in membrane structure after osmotic stress were investigated by electron transmission microscopy, measurement of the anisotropy of the membrane fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene) inserted in E. coli, and Fourier infrared spectroscopy (FTIR). The results show that, above a critical osmotic pressure of 35 MPa, the viability of the bacterium is drastically reduced (2 log decrease in survivors). Electron micrographs revealed a severe contraction of the cytoplasm and the formation of membrane vesicles at 40 MPa. Changes in DPH anisotropy showed that osmotic dehydration to 40 MPa promoted a decrease in the membrane fluidity of integral cells of E. coli. FTIR measurements showed that at 10–40 MPa a transition from lamellar liquid crystal to lamellar gel among the phospholipids extracted from E. coli occurred. Bacterial death resulting from dehydration can be attributed to the conjunction between membrane deformation, caused by the volumetric contraction, and structural changes of the membrane lipids. The influence of the latter on the formation of membrane vesicles and on membrane permeabilization at lethal osmotic pressure is discussed, since vesiculation is hypothetically responsible for cell death.     

Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli

Aims: To investigate the effect of oxygen limitation, glucose-starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results: The effect of BAC on planktonic and sessile cells were investigated using the gfp-tagged E. coli K-12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l⁻¹ BAC). Decreasing cell densities increased the decay rate for BAC-exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane-compromised cells than biofilms grown in aerobic medium. Image analyses of BAC-exposed biofilms showed that membrane-compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions: Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study: The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.     

Impact of osmotic stress on protein diffusion in Lactococcus lactis

We measured translational diffusion of proteins in the cytoplasm and plasma membrane of the Gram‐positive bacterium Lactococcus lactis and probed the effect of osmotic upshift. For cells in standard growth medium the diffusion coefficients for cytosolic proteins (27 and 582 kDa) and 12‐transmembrane helix membrane proteins are similar to those in Escherichia coli. The translational diffusion of GFP in L. lactis drops by two orders of magnitude when the medium osmolality is increased by ～ 1.9 Osm, and the decrease in mobility is partly reversed in the presence of osmoprotectants. We find a large spread in diffusion coefficients over the full population of cells but a smaller spread if only sister cells are compared. While in general the diffusion coefficients we measure under normal osmotic conditions in L. lactis are similar to those reported in E. coli, the decrease in translational diffusion upon osmotic challenge in L. lactis is smaller than in E. coli. An even more striking difference is that in L. lactis the GFP diffusion coefficient drops much more rapidly with volume than in E. coli. We discuss these findings in the light of differences in turgor, cell volume, crowding and cytoplasmic structure of Gram‐positive and Gram‐negative bacteria.     

Control of Cell Elongation in Nitella by Endogenous Cell Wall pH Gradients: MULTIAXIAL EXTENSIBILITY AND GROWTH STUDIES

The multiaxial stress of turgor pressure was stimulated in vitro by inflating isolated Nitella cell walls with mercury. The initial in vitro extension at pH 6.5, 5 atmospheres pressure, returned the wall approximately to the in vivo stressed length, and did not induce any additional extension during a 15-minute period. Upon release of pressure, a plastic deformation was observed which did not correlate with cell growth rates until the final stages of cell maturation. Since wall plasticity does not correlate with growth rate, a metabolic factor(s) is implicated. Walls at all stages of development exhibited a primary yield stress between 0 and 2 atmospheres, while rapidly growing cells (1-3% per hour) exhibited a secondary yield stress of 4 to 5 atmospheres. The creep rate and plastic deformation of young walls were markedly enhanced by acid buffers (10 millimolar, pH ≤ 5.3).     

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review

Dps, the DNA‐binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far‐reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps‐like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.     

Zeta Potential of Selected Bacteria in Drinking Water When Dead, Starved, or Exposed to Minimal and Rich Culture Media

The zeta potentials of E. coli, GFP (green fluorescence protein)-labeled E. coli, Salmonella Newport, and Pseudomonas sp. in different states (nutrient-starved and dead) and grown in rich and minimal media were measured. Capillary electrophoresis experiments were conducted to measure the zeta potential of the different cells suspended in a drinking water sample. Salmonella Newport strain showed a lower zeta potential compared to E. coli, GFP-labeled E. coli, and Pseudomonas sp. Starved E. coli cells had a lower zeta potential compared to E. coli cells grown under rich media conditions. Salmonella Newport cells grown in minimal media also had a lower zeta potential compared to rich, starved, and dead cells. The different bacterial cell types exhibited differences in size as well. These results suggest that when bacterial cells are present in drinking water they can exhibit significant heterogeneity in the size and zeta potential, depending on their physiological state.     

Growth Inhibition, Turgor Maintenance, and Changes in Yield Threshold after Cessation of Solute Import in Pea Epicotyls

The dependence of stem elongation on solute import was investigated in etiolated pea seedlings (Pisum sativum L. var Alaska) by excising the cotyledons. Stem elongation was inhibited by 60% within 5 hours of excision. Dry weight accumulation into the growing region stopped and osmotic pressure of the cell sap declined by 0.14 megapascal over 5 hours. Attempts to assay phloem transport via ethylenediaminetetraacetate-enhanced exudation from cut stems revealed no effect of cotyledon excision, indicating that the technique measured artifactual leakage from cells. Despite the drop in cell osmotic pressure, turgor pressure (measured directly via a pressure probe) did not decline. Turgor maintenance is postulated to occur via uptake of solutes from the free space, thereby maintaining the osmotic pressure difference across the cell membrane. Cell wall properties were measured by the pressure-block stress relaxation technique. Results indicate that growth inhibition after cotyledon excision was mediated primarily via an increase in the wall yield threshold.     

A dormancy state in nonspore-forming bacteria

While cultivation is a convenient way of proliferating and understanding bacteria, studies have shown the formation of nonculturable cells in nonspore-forming bacteria in response to environmental stress and thus in turn have generated immense interest. Whether these cells are in a state of dormancy or in a stage preceding cell death has been considered of paramount importance for the past couple of decades. In this study, osmotic-stress-induced dormant bacterial cells were separated by cell sorting and revived by osmotic down-shift in the absence of nutrients, source(s) that potentially could supply nutrients, and/or the external addition of resuscitation factor(s). Reversal of dormancy followed a definite pattern akin to population asynchrony of dormant cells, and the phenomenon was observed across three species, namely, Enterobacter sp. strain mcp11b, Klebsiella pneumonia strain mcp11d and Escherichia coli. In addition, our study precisely forecasted the presence of multiple subpopulations in dormant cells, which is explained by an emerging theory of survival mechanisms in stressful environments. These observations reveal that the state of dormancy induced by environmental stress in these nonspore-forming bacteria is “reversible” and also implies that it is an orderly and spontaneous adaptation to circumvent adverse conditions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of various environmental factors on the ethidium monazite and quantitative PCR method to detect viable bacteria

Aims: Ethidium monoazide in combination with quantitative PCR (EMA–qPCR) has been considered as a promising method to enumerate viable cells; however, its efficacy can be significantly affected by disinfection conditions and various environments. In this study, thermal disinfection, osmotic pressure and acids with different pH values were systematically investigated to achieve the optimum conditions. Methods and Results: EMA treatment of pure cultures at low concentration (10 μg ml^?1) for 20 min resulted in effective differentiation between viable and nonviable bacteria and had no effect on viable cells. Heating at 85°C for 35 min was the optimum condition that yields inactivated Escherichia coli (E. coli) cells that were not detected with EMA–qPCR. Performing EMA treatment in high‐salt ion environment (sodium chloride concentration ≥4%) could weaken EMA inhibition effect. Both strong and weak acid solutions could react with EMA, change its absorption spectra and influence EMA inhibition effect. Because of the sublethal acidification injury, underestimation of cell counts were found using EMA–qPCR method, and 40‐min incubation in Luria–Bertani medium could completely offset this error. Conclusion: Our results provided optimum EMA treatment, thermal disinfection and environment conditions for EMA–qPCR and demonstrated the feasibility of this method when enumerating viable cells under varied osmotic pressure and pH environment. Significance and Impact of the Study: Optimum EMA treatment, thermal disinfection and EMA‐treated environment will be successfully applied in EMA–qPCR. Osmotic pressure and acid‐induced injury can be detected by EMA–qPCR with optimization.     

Effects of entrapment on nucleic acid content,cell morphology,cell surface property,and stress of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.     

Stimulation of Plant Growth and Drought Tolerance by Native Microorganisms (AM Fungi and Bacteria) from Dry Environments: Mechanisms Related to Bacterial Effectiveness

In this study we tested whether rhizosphere microorganisms can increase drought tolerance to plants growing under water-limitation conditions. Three indigenous bacterial strains isolated from droughted soil and identified as Pseudomonas putida, Pseudomonas sp., and Bacillus megaterium were able to stimulate plant growth under dry conditions. When the bacteria were grown in axenic culture at increasing osmotic stress caused by polyethylene glycol (PEG) levels (from 0 to 60%) they showed osmotic tolerance and only Pseudomonas sp. decreased indol acetic acid (IAA) production concomitantly with an increase of osmotic stress (PEG) in the medium. P. putida and B. megaterium exhibited the highest osmotic tolerance and both strains also showed increased proline content, involved in osmotic cellular adaptation, as much as increased osmotic stress caused by NaCl supply. These bacteria seem to have developed mechanisms to cope with drought stress. The increase in IAA production by P. putida and B. megaterium at a PEG concentration of 60% is an indication of bacterial resistance to drought. Their inoculation increased shoot and root biomass and water content under drought conditions. Bacterial IAA production under stressed conditions may explain their effectiveness in promoting plant growth and shoot water content increasing plant drought tolerance. B. megaterium was the most efficient bacteria under drought (in successive harvests) either applied alone or associated with the autochthonous arbuscular mycorrhizal fungi Glomus coronatum, Glomus constrictum or Glomus claroideum. B. megaterium colonized the rhizosphere and endorhizosphere zone. We can say, therefore, that microbial activities of adapted strains represent a positive effect on plant development under drought conditions.     

Changes in membrane functions during short-term starvation of Vibrio fluvialis strain NCTC 11328

The marine bacterium Vibrio fluvialis strain NCTC 11328 responded to starvation conditions by forming ultramicrocells of dwarf bacteria. The viability of starved cells began to decrease after 2–3 days. During this time the respiratory potential of the bacteria decreased by four- or fivefold, most probably as a result of a decrease in the specific activity of NADH and succinate dehydrogenases. Although respiratory potential in starving cells was lower than in growing cells, bacteria starved for 1 or 2 days maintained a proton motive force that was slightly larger than that of growing bacteria. Starved bacteria contained substantial concentrations of ATP although the UTP and GTP concentrations were much lower in starved than in growing cells. Two or three proteins that were not present in membranes of growing cells, were evident in the membranes of starved bacteria.Abbreviations MMS modified Morita's salts - MMSGC modified Morita's salts plus 20 mM glucose and 0.1% (w/v) casamino acids - MMST modified Morita's salts buffered with 50 mM tricine, (pH 8.5) - NM broth nutrient modified Morita's salts - CFU colony-forming unit - TPP tetraphenylphosphonium - STM 0.1 M tricine, (pH 8.0) plus 0.25 M sucrose and 0.02 M magnesium acetate - DCPIP dichlorophenolindophenol - CCCP carbonyl cyanidem-chlorophenylhydrazone - PMF proton motive force     

Water stress induced by PEG decreases the maximum growth pressure of the roots of pea seedlings

Roots of plants growing in dry soil often experience large mechanical impedance because the decreased soil water content is associated with increased in soil strength. The combined effect of mechanical impedance and water stress hinders the establishment of seedlings in many soils, but little is known about the interaction between these two stresses. A method has been designed that, for the first time, measured the maximum axial force exerted by a root growing under controlled water stress. Using this technique the axial force exerted by a pea radicle was measured using a shear beam, while the seedling was suspended in an aerate solution of polyethylene glycol 20 000 at osmotic potentials between 0 and -0.45 MPa. The maximum growth force was then divided by the cross-sectional area of the root to give the maximum axial growth pressure. The value of maximum axial growth pressure decreased linearly from 0.66 and 0.35 MPa as the osmotic potentials of the solution of PEG decreased from 0 to -0.45 MPa. In dry soil, therefore, the maximum strength of soil that a root can penetrate is decreased because of the decrease in maximum growth pressure. The elongation rates of unimpeded roots were similar whether the roots were subject to either a matric potential in soil or to an osmotic potential in a solution of PEG.Key words: Pisum sativum L, pea, mechanical impedance, axial growth pressure, water stress, PEG 20 000.      

Nutrient Starvation Induces Cross Protection against Heat,Osmotic, or H2O2 Challenge in Vibrio parahaemolyticus

Glucose-starved cells of Vibrio parahaemolyticus were compared with non-starved counterparts with respects to heat, osmotic, and oxidative challenges. The starved cells demonstrated greater thermal and oxidative resistance than did the non-starved cells. The starved cells also showed greater resistance against low osmotic challenge than did the non-starved cells although both cells showed a comparable resistance against high osmotic challenge.     

Osmotic Shock Induction of the Expression of Vibrio fischeri lux Genes in Escherichia coli Cells

The effect of osmotic shock on the expression of genes in the lux regulon of marine bacteria Vibrio fischeri was studied in cells ofEscherichia coli. Bioluminescence of cells was shown to drastically increase, when cells were exposed to osmotic shock at the early logarithmic growth phase, at far lower optic densities as compared to the critical optic density characteristic. The expression of lux genes induced by osmotic shock is determined by the two-component regulatory system RcsC–RcsB. A nucleotide sequence in the regulatory region of the luxR gene homologous to the RcsB-box consensus of E. coli is assumed to be a primary site for this system.     

Lactobacillus,Bifidobacterium and Lactococcus response to environmental stress: Mechanisms and application of cross-protection to improve resistance against freeze-drying

The review deals with lactic acid bacteria in characterizing the stress adaptation with cross-protection effects, mainly associated with Lactobacillus, Bifidobacterium and Lactococcus. It focuses on adaptation and cross-protection in Lactobacillus, Bifidobacterium and Lactococcus, including heat shocking, cold stress, acid stress, osmotic stress, starvation effect, etc. Web of Science, Google Scholar, Science Direct, and PubMed databases were used for the systematic search of literature up to the year 2020. The literature suggests that a lower survival rate during freeze-drying is linked to environmental stress. Protective pretreatment under various mild stresses can be applied to lactic acid bacteria which may enhance resistance in a strain-dependent manner. We investigate the mechanism of damage and adaptation under various stresses including heat, cold, acidic, osmotic, starvation, oxidative and bile stress. Adaptive mechanisms include synthesis of stress-induced proteins, adjusting the composition of cell membrane fatty acids, accumulating compatible substances, etc. Next, we reveal the cross-protective effect of specific stress on the other environmental stresses. Freeze-drying is discussed from three perspectives including the regulation of membrane, accumulation of compatible solutes and the production of chaperones and stress-responsive proteases. The resistance of lactic acid bacteria against technological stress can be enhanced via cross-protection, which improves industrial efficiency concerning the survival of probiotics. However, the adaptive responses and cross-protection are strain-dependent and should be optimized case by case.