Late Exponential Phase Research Articles

ZnO nanoparticle engineered Pseudomonas aeruginosa SOA1 towards enhanced Rhamnolipid production

Rhamnolipids, the potential biosurfactants, have widespread applications in pharmaceutics, food, and agriculture fields. However, its low biosynthesis efficiency limits surfactant industrialization. In this study, different nanoparticles (NPs) were immobilized over Pseudomonas aeruginosa (SOA1) and targeted towards increased rhamnolipids production by increasing the secretion (key step of rhamnolipids biosynthesis). It was observed that, upon adding ZnO NPs (4.0 g/L), rhamnolipids titer increased 4.87 times (from 4.1 to 19.98 g/L) in the 10 L bioreactor. This is due to an increment in the expression of the gene (present in the rhamnolipids biosynthesis process) with the introduction of ZnO NPs to SOA1. Further, with ZnO NPs, few rhamnolipid biosynthesis genes (rhlA and rhlB) were significantly upregulated. In contrast, the rhamnose biosynthetic genes (rmlA, rmlC, and algC) were down regulated during the early and late exponential growth phases as studied in Quantitative real-time PCR. The biosurfactant generation is regulated by quorum-sensing genes, which exhibit a hierarchical expression profile. Specifically, in the early log phase (ZnO NPs cultivation), the last gene was elevated, but both pqsE and rhlI were stimulated in the late log phase. This experiment paves a new path toward improving the biosynthesis of microbial metabolites via. NPs engineering.

Effect of temperature on the production and degradation of bromoform and other brominated methanes by marine microorganisms

Brominated methanes such as bromoform (CHBr3) are known to be important carriers of bromine from the ocean to the atmosphere. Bromine released from brominated methanes by photolysis has been shown to catalyze ozone depletion. Marine phytoplankton has been reported as a source of CHBr3 and marine bacteria as a sink for CHBr3. The effects of temperature on both CHBr3 production by phytoplankton and CHBr3 degradation by bacteria have yet to be investigated. We investigated the effects of temperature on CHBr3 production and CHBr3 degradation by marine microorganisms. The marine diatom Ditylum brightwellii (CCMP358) was cultured at 15 °C, 20 °C, 24 °C, and 30 °C. The maximum CHBr3 production rate at 24 °C was 1.57–2.39 pmol (μg chlorophyll a)−1 d−1, several times higher than that at 15 °C (0.25–0.41 pmol (μg chlorophyll a)−1 d−1). Higher rates of CHBr3, CHBr2Cl, and CHBrCl2 production were observed in the late exponential phase (and stationary phase) than in the early exponential phase at each temperature. These results suggest that temperature affects the rate of CHBr3 production during plankton growth. We then cultured the marine α-proteobacterium Phaeobacter gallaeciensis (JCM 21319) and the γ-proteobacterium Pseudomonas sp. HKF-4 at 10 °C, 15 °C, 20 °C, and 25 °C for up to 15 days to analyze temperature effects on spiked 13CHBr3 degradation. The degradation rate of 13CHBr3 by P. gallaeciensis increased with increasing temperature from 10 °C to 25 °C. The half-life of 13CHBr3 at 25 °C was about 1.1 d, which is about 6 times shorter than the half-life at 10 °C (about 6.9 d). On the other hand, the change in the half-life of the degradation of 13CHBr3 by HKF-1 was relatively small as the temperature increased from 10 °C (half-life: about 5.5 d) to 25 °C (half-life: about 1.8 d). Considering the rate of CHBr3 production and degradation at each temperature, we estimated how much of the CHBr3 produced by D. brightwellii for 7 days was degraded by the coexisting bacteria and how much remained after 7 days at each temperature. When coexisting with P. gallaeciensis, the residual CHBr3 concentration in the culture was relatively higher at 20–25 °C. Similarly, when coexisting with HKF-4, it was relatively higher at 20–25 °C. To estimate the impact of future warming on CHBr3 concentrations in the oceans, we assume a 5 °C increase in sea surface temperature, with two sea surface temperatures, 15 °C and 20 °C, changing to 20 °C and 25 °C, respectively. Under this assumption, the residual concentration of CHBr3 produced by D. brightwellii in seawater would be “increased” (or, less likely, “no change”) when P. gallaeciensis coexisted in these areas. Similarly, the total amount of CHBr3 residuals produced by D. brightwellii in the seawater areas where HKF-4 coexisted increased as the temperature increased by 5 °C. If, as the present results suggest, there is a temperature effect on microbial CHBr3 production and degradation in the oceans, then future increases in surface seawater temperature could result in an upward trend in CHBr3 concentrations in the open oceans, although such effects would be greatly influenced by both the phytoplankton species and coexisting bacterial species.

Regulation of potassium uptake in Caulobacter crescentus.

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Here, we describe the regulation of potassium uptake systems in the oligotrophic α-proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by mainly using two K+ transporters to maintain potassium homeostasis, the low-affinity Kup and the high-affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by increasing the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABCDE expression, suggest that the inner membrane sensor regulatory component KdpD mainly works as a phosphatase to limit the growth when cells reach late exponential phase. Our data therefore suggest that KdpE is phosphorylated by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability.IMPORTANCEPotassium (K+) is a key metal ion involved in many essential cellular processes. Here, we show that the oligotroph Caulobacter crescentus can support growth at micromolar concentrations of K+ by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Using genome-wide approaches, we also determined the entire set of genes required for C. crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike Escherichia coli, the inner membrane sensor regulatory component KdpD seems to work rather as a phosphatase on the phosphorylated response regulator KdpE~P.

Growth phase matters: Boosting immunity via Lacticasebacillus-derived membrane vesicles and their interactions with TLR2 pathways.

Lipid bi-layered particles known as membrane vesicles (MVs), produced by Gram-positive bacteria are a communication tool throughout the entire bacterial growth. However, the MVs characteristics may vary across all stages of maternal culture growth, leading to inconsistencies in MVs research. This, in turn, hinders their employment as nanocarriers, vaccines and other medical applications. In this study, we aimed to comprehensively characterize MVs derived from Lacticaseibacillus rhamnosus CCM7091 isolated at different growth stages: early exponential (6h, MV6), late exponential (12h, MV12) and late stationary phase (48h, MV48). We observed significant differences in protein content between MV6 and MV48 (data are available via ProteomeXchange with identifier PXD041580), likely contributing to their different immunomodulatory capacities. In vitro analysis demonstrated that MV48 uptake rate by epithelial Caco-2 cells is significantly higher and they stimulate an immune response in murine macrophages RAW 264.7 (elevated production of TNFα, IL-6, IL-10, NO). This correlated with increased expression of lipoteichoic acid (LTA) and enhanced TLR2 signalling in MV48, suggesting that LTA contributes to the immunomodulation. In conclusion, we showed that Lacticaseibacillus rhamnosus CCM7091-derived MVs from the late stationary phase boost the immune response the most effectively, which pre-destines them for therapeutical application as nanocarriers.

Gas Chromatography-Ion Mobility Spectrometry Reveals Acetoin as a Biomarker for Carbapenemase-Producing Klebsiella pneumoniae.

BACKGROUND This study aimed to detect the volatile organic compound (VOC), 3-hydroxy-2-butanone (acetoin) using gas chromatography-ion mobility spectrometry (GC-IMS) in antimicrobial-resistant Klebsiella pneumoniae (K. pneumoniae) carbapenemase (KPC)-producing bacteria. MATERIAL AND METHODS Using stromal fluid of blood culture bottles (BacT/ALERT® SA) as the medium, 3-hydroxy-2-butanone (acetoin) released by K. pneumoniae during growth was detected using GC-IMS. The impact of imipenem (IPM) and carbapenemase inhibitors [avibactam sodium or pyridine-2,6-dicarboxylic acid (DPA)] on the emission of 3-hydroxy-2-butanone (acetoin) from various carbapenemase-producing K. pneumoniae was further investigated. Subsequently, VOCal software was used to generate a pseudo-3D plot of 3-hydroxy-2-butanone (acetoin), and the relative peak volumes were exported for data analysis. Standard strains served as references, and the findings were validated with clinical isolates. RESULTS The pattern of temporal changes in the 3-hydroxy-2-butanone (acetoin) release from K. pneumoniae in the absence of IPM was consistent with the growth curve. After the IPM addition, carbapenemase-positive strains released significantly higher contents of 3-hydroxy-2-butanone (acetoin) than carbapenemase-negative strains at the late exponential growth phase (T2). Notably, adding avibactam sodium significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class A carbapenemase-producing strains as compared to the absence of the carbapenemase inhibitor. Conversely, adding DPA significantly decreased the 3-hydroxy-2-butanone (acetoin) content released from the class B carbapenemase-producing strains (both standard and clinical strains, all P<0.05). CONCLUSIONS This study demonstrated the potential of 3-hydroxy-2-butanone (acetoin) as a VOC biomarker for detecting carbapenemase-producing K. pneumoniae, as revealed by GC-IMS analysis.

Growth phase-dependent changes in conformational and physicochemical properties of bacterial surface biopolymers lead to changes in bacterial adhesion strength at nanoscale and macroscale

This study presents a detailed investigation into conformational, physicochemical and adhesive characteristics of biopolymers on the surfaces of Escherichia coli and potential probiotic Bacillus subtilis harvested from middle-exponential phase (mid-EP), late-exponential phase (late-EP) and early-stationary phase (early-SP) of growth. The lengths to which bacterial surface biopolymers extend (biopolymer brush lengths), densities of grafted bacterial surface biopolymers indicating the amounts of molecules covering the bacterial surfaces (biopolymer grafting densities), adhesion forces of bacterial surface biopolymers to the model inert surfaces of silicon nitride (Si3N4), and the pull-off distances of biopolymers from Si3N4 were measured in water by atomic force microscopy (AFM). The Weibull analysis of AFM adhesion data showed that as the culture aged, the adhesive bonds between Si3N4 AFM tips and surface molecules of E. coli harvested from the culture were broken with a higher applied force. However, the highest applied force to break the bonds was required for B. subtilis in late-EP, followed by those required for cells in early-SP and mid-EP, respectively. The results of a steric model fitting to AFM approach force-distance (FD) curves and analysis of the pull-off distances in the AFM retraction FD curves showed higher biopolymer grafting density for E. coli in early-SP and longer biopolymer brush layer for B. subtilis in late-EP, which were associated with stronger adhesion to Si3N4 in water. The results of thermodynamic adhesion energy calculations based on the Wu model showed that polar interaction energy dominated the bacterial adhesion at the macroscale, the strength of which varied as a function of the growth phase for both E. coli and B. subtilis. The growth phase-dependent variation in polar components of thermodynamic adhesion energies between the bacteria and Si3N4 in water was consistent with the growth phase-dependent variation in the bond strengths between the bacteria and Si3N4 in water as revealed by the Weibull analysis of AFM adhesion data. Therefore, information obtained by Weibull analysis of nanoscale AFM bacterial adhesion data can be used to predict macroscale bacterial adhesion to the model inert Si3N4 surface in water.

A growth phase analysis on the influence of light intensity on microalgal stress and potential biofuel production

It is urgent to find alternatives to fossil fuels to mitigate the effects of climate change. High light intensity during microalgal growth can promote lipid and carbohydrate accumulation (feedstocks to biofuels). However, more research needs to be done concerning the effect of this factor on microalgal productivity and biochemical composition from a growth phase point of view. This study evaluated the impact of high light intensities (291, 527 and 1107 μmol m−2 s−1) on Chlorella vulgaris growth in synthetic wastewater, nutrient assimilation and biochemical composition in late-exponential and stationary growth phases. Under the highest light intensity, a higher growth rate (0.54 ± 0.02 d−1) and maximum productivity (166 ± 13 mg/L d−1) were achieved. Also, nutrient removal efficiencies at the late-exponential phase reached 97.0 ± 0.2 % and 98.2 ± 0.1 % for nitrogen and phosphorus, respectively. Under all light intensities tested at the stationary phase, microalgae also achieved high nutrient removal efficiencies (>95 %). Extreme light intensity (1107 μmol m−2 s−1) boosted the accumulation of carbohydrates (30 ± 3 % w/w) and lipids (13.0 ± 0.4 % w/w). Under lower light intensities, the content of these compounds did not change significantly between growth phases. The chlorophyll content decreased with the increasing light intensity and increasing exposure time. Exposing C. vulgaris to high light intensity seems promising to simultaneously treat wastewater and boost lipid and carbohydrate accumulation, which can be used for biofuel production.

Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ ChromatinImmunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messengerRNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

Exploring the Genomic Landscape of Bacillus paranthracis PUMB_17 as a Proficient Phosphatidylcholine-Specific Phospholipase C Producer.

Phospholipases find versatile applications across industries, including detergent production, food modification, pharmaceuticals (especially in drug delivery systems), and cell signaling research. In this study, we present a strain of Bacillus paranthracis for the first time, demonstrating significant potential in the production of phosphatidylcholine-specific phospholipase C (PC-PLC). The investigation thoroughly examines the B. paranthracis PUMB_17 strain, focusing on the activity of PC-PLC and its purification process. Notably, the PUMB_17 strain displays extracellular PC-PLC production with high specific activity during the late exponential growth phase. To unravel the genetic makeup of PUMB_17, we employed nanopore-based whole-genome sequencing and subsequently conducted a detailed genome annotation. The genome comprises a solitary circular chromosome spanning 5,250,970 bp, featuring a guanine-cytosine ratio of 35.49. Additionally, two plasmids of sizes 64,250 bp and 5845 bp were identified. The annotation analysis reveals the presence of 5328 genes, encompassing 5186 protein-coding sequences, and 142 RNA genes, including 39 rRNAs, 103 tRNAs, and 5 ncRNAs. The aim of this study was to make a comprehensive genomic exploration that promises to enhance our understanding of the previously understudied and recently documented capabilities of Bacillus paranthracis and to shed light on a potential use of the strain in the industrial production of PC-PLC.

B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Maximizing key biochemical products of Spirulina platensis: optimal light quantities and best harvesting time

Light management can optimize algal biomass and biochemical composition, but responses vary based on growth stage and harvesting time. A 22 factorial design along with 3 center point runs was used to investigate the effects of light intensity (50, 100, and 150 μmol photon m−2 s−1) and exposure time (10.5, 16, and 21.5 h) on lipid, protein, carbohydrate, and pigments content, density, and productivity of Spirulina platensis. By analyzing trends in cellular content, the time each key metabolite reached its maximum was chosen as its best harvesting time. Low factor levels during the mid-exponential phase resulted in the highest protein content, while high levels at the very early exponential phase showed the most carbohydrates. Low light intensity favored the accumulation of lipids, chlorophyll, and carotenoids at the end of exponential phase. The highest phycobiliproteins were obtained at intermediate factor levels during the late exponential phase. Harvesting at high levels of factors at the end of exponential phase led to the highest component density. The maximum productivities were observed at high factor levels. These findings emphasize the importance of carefully selecting the harvesting time in different light conditions to achieve the desired biochemical content, density, and productivity in Spirulina applications.

Karenia mikimotoi induced adverse impacts on abalone Haliotis discus hannai in Fujian coastal areas, China

Abstract Large-scale outbreaks of the dinoflagellate Karenia mikimotoi caused substantial mortality of abalone, Haliotis discus hannai in Fujian, China in 2012, resulting in 20 billion in economic losses to abalone industries. However, the mechanism behind the mortality, especially the reaction of abalone to this microalgal toxicity, which possibly differed significantly from the former ‘fish killer’ strain in the South China Sea (SCS). Our study revealed that K. mikimotoi FJ-strain exhibited a four-fold higher haemolytic toxicity than the SCS-strain during the late exponential phase. At the microalgal cell density of 3 × 107 cell L−1, the FJ-strain caused abalone mortality of 67% in 48 h, with decreased granulocyte–hyalinocyts ratio and phagocytic activity by 58.96% and 75.64%, respectively, increased haemocyte viability by 4.8-fold and severe gill damage. The toxic effect only worked for the haemolytic toxicity from active algal cells, which were probably produced under the contact of algal cells and abalone gills. However, under exposure to the SCS-strain, more than 80% of individuals survived under aeration. The results indicated that FJ-strain was a new K. mikimotoi ecotype with stronger toxicity. It evoked severe effects, with complete abalone mortality within 24 h under the cascading effect of non-aeration (dissolved oxygen declined to 2.0 mg L−1), when exposed to K. mikimotoi FJ-strain at the above density. Thus, apart from the microalgal toxicity, DO depletion exacerbated the mortality of abalone in the experiment. The massive abalone mortalities in Fujian were probably caused by the combination of microalgal toxic effects and oxygen depletion, leading to immunological depression and histopathological disruption.

The RNA chaperone Hfq has a multifaceted role in Edwardsiella ictaluri.

Edwardsiella ictaluri is a Gram-negative, facultative intracellular bacterium that causes enteric septicemia in catfish (ESC). The RNA chaperone Hfq (host factor for phage Qβ replication) facilitates gene regulation via small RNAs (sRNAs) in various pathogenic bacteria. Despite its significance in other bacterial species, the role of hfq in E. ictaluri remains unexplored. This study aimed to elucidate the role of hfq in E.ictaluri by creating an hfq mutant (EiΔhfq) through in-frame gene deletion and characterization. Our findings revealed that the Hfq protein is highly conserved within the genus Edwardsiella. The deletion of hfq resulted in a significantly reduced growth rate during the late exponential phase. Additionally, EiΔhfq displayed a diminished capacity for biofilm formation and exhibited increased motility. Under acidic and oxidative stress conditions, EiΔhfq demonstrated impaired growth, and we observed elevated hfq expression when subjected to in vitro and in vivo stress conditions. EiΔhfq exhibited reduced survival within catfish peritoneal macrophages, although it had no discernible effect on the adherence and invasion of epithelial cells. The infection model revealed that hfq is needed for bacterial persistence in catfish, and its absence caused significant virulence attenuation in catfish. Finally, the EiΔhfq vaccination completely protected catfish against subsequent EiWT infection. In summary, these results underscore the pivotal role of hfq in E. ictaluri, affecting its growth, motility, biofilm formation, stress response, and virulence in macrophages and within catfish host.

DEVELOPMENT OF QUALITY INDICATORS OF TRICHODERMA ATROBRUNNEUM CULTURE FLUID EXTRACT VKPM F-1434

Relevance. It is relevant to solve the problems of quality control of medicinal substances, drugs, preparations by conducting pharmaceutical analy-sis. The purpose of the study – determination of physico-chemical and biological properties of Trichoderma atrobrunneum VKPM F-1434 culture fluid ex-tract. Development of a method for determining the mass concentration of culture fluid extract T. atrobrunneum VKPM F-1434 in bioproducts. Material and methods. The strain Trichoderma atrobrunneum VKPM F-1434 synthesizing low molecular weight biologically active compounds with bacteriostatic effect was used in the work. The extract was obtained by single-stage mixing and settling extraction with ethyl acetate from the culture liquid of T. atrobrunneum VKPM F-1434 after 5 days of deep cultivation at a temperature of 28 ° C, the seed material was the vegetative mycelium of the producer in the late exponential growth phase. The mass concentration of T. atrobrunneum VKPM F-1434 culture fluid extract was determined in a healing liniment for wound healing. Results. Based on the study of physico-chemical and biological properties of culture fluid extract T. atrobrunneum VKPM F-1434, its individual charac-teristics have been established. The method "Determination of the mass concentration of culture fluid extract T. atrobrunneum VKPM F-1434 in the fin-ished bioproduct" has been developed. Scope of the method: determination of the extract’s mass concentration at the stages of the technological pro-cess of preparation, as well as in ready-made dosage forms, therapeutic and preventive orientation of zootechnical and veterinary purposes. Character-istic of the method: the range of determined values – mass concentrtion (ω): 1 – 10%; repeatability: CV = 1.69%; reproducibility CV1 = 1.68%; CV2 = 1.78%; Fr = 1.09; Ft = 3.44; correctness: R = 100.1%. Conclusions. Physico-chemical and biological properties of culture fluid extract T. atrobrunneum VKPM F-1434 can be used to establish its authenticity and quality assessment. The method of determining the mass concentration of culture fluid extract T. atrobrunneum VKPM F-1434 can be considered suitable for a reliable assessment of the composition of the finished bioproduct.

Unravelling the impact of fat content on the microbial dynamics and spatial distribution of foodborne bacteria in tri-phasic viscoelastic 3D models

The aim of the current study is to develop and characterise novel complex multi-phase in vitro 3D models, for advanced microbiological studies. More specifically, we enriched our previously developed bi-phasic polysaccharide (Xanthan Gum)/protein (Whey Protein) 3D model with a fat phase (Sunflower Oil) at various concentrations, i.e., 10%, 20%, 40% and 60% (v/v), for better mimicry of the structural and biochemical composition of real food products. Rheological, textural, and physicochemical analysis as well as advanced microscopy imaging (including spatial mapping of the fat droplet distribution) of the new tri-phasic 3D models revealed their similarity to industrial food products (especially cheese products). Furthermore, microbial growth experiments of foodborne bacteria, i.e., Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Lactococcus lactis on the surface of the 3D models revealed very interesting results, regarding the growth dynamics and distribution of cells at colony level. More specifically, the size of the colonies formed on the surface of the 3D models, increased substantially for increasing fat concentrations, especially in mid- and late-exponential growth phases. Furthermore, colonies formed in proximity to fat were substantially larger as compared to the ones that were located far from the fat phase of the models. In terms of growth location, the majority of colonies were located on the protein/polysaccharide phase of the 3D models. All those differences at microscopic level, that can directly affect the bacterial response to decontamination treatments, were not captured by the macroscopic kinetics (growth dynamics), which were unaffected from changes in fat concentration. Our findings demonstrate the importance of developing structurally and biochemically complex 3D in vitro models (for closer proximity to industrial products), as well as the necessity of conducting multi-level microbial analyses, to better understand and predict the bacterial behaviour in relation to their biochemical and structural environment. Such studies in advanced 3D environments can assist a better/more accurate design of industrial antimicrobial processes, ultimately, improving food safety.

Effect of carbon and nitrogen concentrations on lipid accumulation and regulation of acetyl-CoA carboxylase in Papiliotrema laurentii.

Biodiesel is an interesting alternative to petroleum diesel as it is renewable, biodegradable, and has a low pollutant content. Yeast oils can be used for biodiesel production instead of edible oils, mitigating the use of arable land and water for biodiesel production. Maximum lipid accumulation is reached at 48h of cultivation by the oleaginous yeast Papiliotrema laurentii UFV-1. Nevertheless, the effects of carbon and nitrogen concentrations on lipid accumulation, as well as the regulation of lipid metabolism in this yeast are still not well-characterised. Therefore, this work evaluated the effects of carbon and nitrogen concentrations on the lipid accumulation in P. laurentti, the expression of the ACC gene, and the activity of the enzyme acetyl-CoA carboxylase (ACCase) in different carbon:nitrogen ratios (C:N) and glucose concentrations. The variation of ammonium sulfate concentration did not affect the growth and lipid accumulation in P. laurentii UFV-1. On the other hand, glucose concentration remarkably influenced biomass and lipid production by this yeast. Therefore, the carbon concentration is more important than the nitrogen concentration for lipid production by P. laurentii UFV-1. Importantly, the levels of both ACC gene expression and ACCase activity were maximum during the late-exponential growth phase and decreased after reaching the highest lipid contents, which was easier evidenced during the accumulation and maximum lipid levels. As such, the reduction of ACCase enzyme activity seems to be related to the decrease in the expression level of the ACC gene.

Effect of NaCl stress on exoproteome profiles of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H.

Salt stress can affect survival, multiplication and ability of plant growth promoting microorganisms to enhance plant growth. Changes in a microbe's proteome profile is one of the mechanisms employed by PGPM to enhance tolerance of salt stress. This study was focused on understanding changes in the exoproteome profile of Bacillus amyloliquefaciens EB2003A and Lactobacillus helveticus EL2006H when exposed to salt stress. The strains were cultured in 100 mL M13 (B. amyloliquefaciens) and 100 mL De man, Rogosa and Sharpe (MRS) (L. helveticus) media, supplemented with 200 and 0 mM NaCl (control), at pH 7.0. The strains were then incubated for 48 h (late exponential growth phase), at 120 rpm and 30 (B. amyloliquefaciens) and 37 (L. helveticus) °C. The microbial cultures were then centrifuged and filtered sterilized, to obtain cell free supernatants whose proteome profiles were studied using LC-MS/MS analysis and quantified using scaffold. Results of the study revealed that treatment with 200 mM NaCl negatively affected the quantity of identified proteins in comparison to the control, for both strains. There was upregulation and downregulation of some proteins, even up to 100%, which resulted in identification of proteins significantly unique between the control or 200 mM NaCl (p ≤ 0.05), for both microbial species. Proteins unique to 200 mM NaCl were mostly those involved in cell wall metabolism, substrate transport, oxidative stress tolerance, gene expression and DNA replication and repair. Some of the identified unique proteins have also been reported to enhance plant growth. In conclusion, based on the results of the work described here, PGPM alter their exoproteome profile when exposed to salt stress, potentially upregulating proteins that enhance their tolerance to this stress.

Enhanced probiotic potential of Lactobacillus kefiranofaciens OSU-BDGOA1 through co-culture with Kluyveromyces marxianus bdgo-ym6.

Due to the increasing consumer demand for the development and improvement of functional foods containing probiotics, new probiotic candidates need to be explored as well as novel means to enhance their beneficial effects. Lactobacillus kefiranofaciens OSU-BDGOA1 is a strain isolated from kefir grains that has demonstrated probiotic traits. This species is the main inhabitant of kefir grains and is responsible for the production of an exopolysaccharide (EPS) whit vast technological applications and potential bioactivities. Research has shown that interkingdom interactions of yeast and lactic acid bacteria can enhance metabolic activities and promote resistance to environmental stressors. Comparative genomic analyses were performed to distinguish OSU-BDGOA1 from other strains of the same species, and the genome was mined to provide molecular evidence for relevant probiotic properties. We further assessed the cumulative effect on the probiotic properties of OSU-BDGOA1 and Kluyveromyces marxianus bdgo-ym6 yeast co-culture compared to monocultures. Survival during simulated digestion assessed by the INFOGEST digestion model showed higher survival of OSU-BDGOA1 and bdgo-ym6 in co-culture. The adhesion to intestinal cells assessed with the Caco-2 intestinal cell model revealed enhanced adhesion of OSU-BDGOA1 in co-culture. The observed increase in survival during digestion could be associated with the increased production of EPS during the late exponential and early stationary phases of co-culture that, by enhancing co-aggregation between the yeast and the bacterium, protects the microorganisms from severe gastrointestinal conditions as observed by SEM images. Immune modulation and barrier function for recovery and prevention of flagellin-mediated inflammation by Salmonella Typhimurium heat-killed cells (HKSC) in Caco-2 cells were also measured. OSU-BDGOA1 in mono- and co-culture regulated inflammation through downregulation of pro-inflammatory cytokine expression and increased membrane barrier integrity assessed by TEER, FD4 permeability, and expression of tight junctions. The results of the study warrant further research into the application of co-cultures of yeast and LAB in functional probiotic products and the potential to increase EPS production by co-culture strategies.

Significance of cell culture phases for utilizing 3D cultures of A549 cells for in vitro studies.

Three-dimensional (3D) culture systems of human cancer cell lines have become popular experimental models for a variety of applications including drug screening. It is understood that the 2D and 3D cultures of the same cell line behave differently in several aspects. One such difference is in the duration of cell culture phases (the lag, log, plateau and the decline stages). We obtained 3D cultures of A549 cells on agarose hydrogels. We observed and compared the morphological differences in the progression of 2D and 3D cultures of A549 cells in a time-dependent manner. The morphological features along with the cell counts and viabilities obtained for the 2D and 3D cultures at different time intervals clearly indicate that the cell culture phases occurred as more extended one for the 3D cultures compared to that of the 2D counterparts. The plateau stage for the 2D and 3D cultures occurred at 48 and 69 h, respectively. Such cell culture phase durations can be different for different cell lines as a function of their doubling times. We propose that the cell culture phase durations for any cell line should be first established before using them for drug testing or for studies involving toxicity to obtain useful results from 3D cell cultures. Also, we propose that the late-exponential (lag) phase of 3D cultures of cancer cell lines is the most ideal one for drug testing owing to the various optimal features of the aggregates in this cell culture phase.

Continuous production of an antimicrobial metabolite with the terrestrial cyanobacterium Chroococcidiopsis cubana

The terrestrial cyanobacterium Chroococcidiopsis cubana was identified as a producer of an antimicrobial substance inhibiting growth of the Gram-positive bacterium Micrococcus luteus and the yeast Candida auris. It was found that the production is initiated by nitrogen limitation, usually occurring during late exponential growth phase of the cyanobacterium. This effect was used for implementation of a continuous production set-up, which led to a significantly increased formation of the antimicrobial metabolite. The produced bioactive culture supernatant was purified by reverse phase chromatography; the most potent fraction caused a growth inhibition of over 90 % for both indicator organisms. The metabolite remained active until a temperature of approx. 45 °C; at increasing temperatures, the inhibiting effect decreased significantly. By identification of the trigger initiating the compound synthesis, a first scale-up could be implemented, leading to a reliable production of the antimicrobial metabolite. As Candida auris is an emerging pathogen causing serious infections, this work can be the first step to the development of a potent antifungal drug.