Stationary Growth Phase Research Articles

Tuning metabolic efficiency for increased product yield in high titer fed-batch Chinese hamster ovary cell culture.

High demand in manufactured biologics drives the continued need for increased productivity. In this study elevated lactate metabolization resulted in improved metabolic efficiency and cellular productivity for a readily intensified high titer fed-batch process. Scheduled base or lactate feeds during the stationary growth phase led to increased titers (+9% and +8% respectively) without impacting the overall growth performance. The higher lactate consumption induced by either feed strategy substituted for glutamate catabolism and consequently reduced ammonia build-up. Direct correlation between increased titers and reduced ammonia levels was shown. Product quality attributes were impacted by both feeding strategies but could be matched with the control process by shortening the cell culture duration while maintaining titer constant.

Characterization of a New Pseudomonas Putida Strain Ch2, a Degrader of Toxic Anthropogenic Compounds Epsilon-Caprolactam and Glyphosate

In this work, a new Ch2 strain was isolated from soils polluted by agrochemical production wastes. This strain has a unique ability to utilize toxic synthetic compounds such as epsilon-caprolactam (CAP) as a sole carbon and energy source and the herbicide glyphosate (GP) as a sole source of phosphorus. Analysis of the nucleotide sequence of the 16S rRNA gene of Ch2 revealed that the strain belongs to the species Pseudomonas putida. This strain grew in the mineral medium containing CAP in a concentration range of 0.5 to 5.0 g/L and utilized 6-aminohexanoic acid and adipic acid, which are the intermediate products of CAP catabolism. The ability of strain Ch2 to degrade CAP is determined by a conjugative megaplasmid that is 550 kb in size. When strain Ch2 is cultured in a mineral medium containing GP (500 mg/L), more intensive utilization of the herbicide occurs in the phase of active growth. In the phase of declining growth, there is an accumulation of aminomethylphosphonic acid, which indicates that the C-N bond is the first site cleaved during GP degradation (glyphosate oxidoreductase pathway). Culture growth in the presence of GP during the early step of its degradation is accompanied by unique substrate-dependent changes in the cytoplasm, including the formation of vesicles of cytoplasmic membrane consisting of specific electron-dense content. There is a debate about whether these membrane formations are analogous to metabolosomes, where the primary degradation of the herbicide can take place. The studied strain is notable for its ability to produce polyhydroxyalkanoates (PHAs) when grown in mineral medium containing GP. At the beginning of the stationary growth phase, it was shown that, the amount and size of PHA inclusions in the cells drastically increased; they filled almost the entire volume of cell cytoplasm. The obtained results show that the strain P. putida Ch2 can be successfully used for the PHAs' production. Moreover, the ability of P. putida Ch2 to degrade CAP and GP determines the prospects of its application for the biological cleanup of CAP production wastes and in situ bioremediation of soil polluted with GP.

Changes in the metabolome of probiotics during the stationary phase increase resistance to lyophilization

In the probiotics manufacturing process, lyophilization damages the cell membrane of microorganisms, reducing their viability and delaying reactivation of their cellular metabolism. The growth phase of microorganisms at the time of lyophilization is thought to significantly affect their viability during storage and reactivation in the intestine. However, no study has systematically investigated the differences in overcoming mechanisms in microorganisms harvested at different growth phases against lyophilization. Therefore, in this study, we comparatively assessed the resistance mechanism of Bifidobacterium animalis ssp. lactis and Lacticaseibacillus rhamnosus harvested at exponential and stationary growth phases. We found that B. animalis ssp. lactis and L. rhamnosus harvested at the stationary phase had shorter lag phase in growth and higher survival rates during storage than those harvested at the exponential phase. As a result of metabolome analysis, the cells harvested at the stationary phase accumulated trehalose and arabinose, which function as cryoprotectants and protect cells from lyophilization-induced damage. These findings could provide a basis for establishing a manufacturing protocol for lyophilized probiotics that can be rapidly reactivated in the intestine to achieve optimal metabolic activity and induce health benefits.

Abiotic and biotic factors controlling sexual reproduction in populations of Pseudo-nitzschia pungens (Bacillariophyceae)

Pseudo-nitzschia pungens is a widely distributed marine pennate diatom. Hybrid zones, regions in which two different genotypes may interbreed, are important areas for speciation and ecology, and have been reported across the globe for this species. However, sexual reproduction between differing clades in the natural environment is yet to be observed and is difficult to predict. Here we carried out experiments using two mono-clonal cultures of P. pungens from different genotypes to measure the frequency and timing of sexual reproduction across varying biotic (growth phases and cell activity potential) and abiotic conditions (nutrients, light, turbulence). We found the mating rates and number of zygotes gradually decreased from exponential to late stationary growth phases. The maximum zygote abundance observed was 1,390 cells mL−1 and the maximum mating rate was 7.1%, both which occurred during the exponential growth phase. Conversely, only 9 cells mL−1 and a maximum mating rate of 0.1% was observed during the late stationary phase. We also found the higher the relative potential cell activity (rPCA) in parent cells, as determined by the concentration of chlorophyll a per cell and the ratio of colony formation during parent cultivations, revealed higher mating rates. Furthermore, sexual events were reduced under nutrient enrichment conditions, and mating pairs and zygotes were not formed under aphotic (dark) or shaking culture conditions (150 rpm). In order to understand the sexual reproduction of Pseudo-nitzschia in the natural environment, our results highlight that it is most likely the combination of both biotic (growth phase, Chl. a content) and abiotic factors (nutrients, light, turbulence) that will determine the successful union of intraspecific populations of P. pungens in any given region.

BIOCHEMICAL RESPONSES OF CHLORELLA VULGARIS AND SCENEDESMUS SP. UNDER NUTRIENT LIMITATION

Increasing microalgae carbohydrate content through nutrient limitation has been regarded as a potential strategy to optimize microalgae bioethanol production. This study assessed three different media in which nitrogen, phosphorus and sulfur concentration was decreased by 10-fold with regard to the control media. The performance of two robust microalgae, namely Chlorella vulgaris and Scenedesmus sp., were evaluated in terms of growth and biochemical composition. At the stationary growth phase, sulfur and phosphorus limited media supported the highest carbohydrate accumulation on Chlorella vulgaris and Scenedesmus sp., respectively (approximately 4-fold). Under these scenarios, Scenedesmus biomass did not exhibit arrested growth while Chlorella decreased its concentration by 10%. This decrease was attributed to the decreased photosynthetic performance that concomitantly affected nutrients uptake. After 15 days cultivation, the biochemical analysis revealed that out of the total carbohydrates obtained under those limited media (30% DW for Chlorella and 22% DW for Scenedesmus), only 5% was starch and therefore the main fraction of carbohydrates was contained in the cell wall. Likewise, the carbohydrate profile presented fast changes in short periods of time and therefore the importance of an appropriate harvesting schedule to obtain carbohydrate rich biomass was highlighted.

Isolation and characterisation of exosomes from Chinese hamster ovary (CHO) cells

Exosomes have previously been isolated from Chinese hamster ovary (CHO) cells and their anti-apoptotic properties reported. However, to further facilitate the study of CHO cell derived exosomes and allow their comparison across studies, it is necessary to characterise and define such exosomes using at least three criteria that can act as a reference for the generation of CHO cell produced exosomes. Here we report on the isolation of exosomes from CHO cells, an industrially relevant and widely used cell host for biopharmaceutical protein production, during the exponential and stationary phase of growth during batch culture using a Total Exosome Isolation (TEI) method. The resulting vesicles were characterized and visualized using a diverse range of techniques including Dynamic Light Scattering (DLS), Zeta potential, Electron Microscopy and immunoblotting, and their protein and RNA content determined. We also generated the lipid fingerprint of isolated exosomes using MALDI-ToF mass spectroscopy. We confirmed the presence of nano sized extracellular vesicles from CHO cells and their subsequent characterization revealed details of their size, homogeneity, surface charge, protein and RNA content. The lipid content of exosomes was also found to differ between exosomes isolated on different days of batch culture. This analysis provides a profile and characterisation of CHO cell exosomes to aid future studies on exosomes from CHO cells and improving the manufacturing of exosomes for biotherapeutic application.

Comparative metabolomic analysis of exudates of microcystin-producing and microcystin-free Microcystis aeruginosa strains.

Cyanobacterial harmful algal blooms (cHABs) dominated by Microcystis aeruginosa threaten the ecological integrity and beneficial uses of lakes globally. In addition to producing hepatotoxic microcystins (MC), M. aeruginosa exudates (MaE) contain various compounds with demonstrated toxicity to aquatic biota. Previously, we found that the ecotoxicity of MaE differed between MC-producing and MC-free strains at exponential (E-phase) and stationary (S-phase) growth phases. However, the components in these exudates and their specific harmful effects were unclear. In this study, we performed untargeted metabolomics based on liquid chromatography-mass spectrometry to reveal the constituents in MaE of a MC-producing and a MC-free strain at both E-phase and S-phase. A total of 409 metabolites were identified and quantified based on their relative abundance. These compounds included lipids, organoheterocyclic compounds, organic acid, benzenoids and organic oxygen compounds. Multivariate analysis revealed that strains and growth phases significantly influenced the metabolite profile. The MC-producing strain had greater total metabolites abundance than the MC-free strain at S-phase, whereas the MC-free strain released higher concentrations of benzenoids, lipids, organic oxygen, organic nitrogen and organoheterocyclic compounds than the MC-producing strain at E-phase. Total metabolites had higher abundance in S-phase than in E- phase in both strains. Analysis of differential metabolites (DMs) and pathways suggest that lipids metabolism and biosynthesis of secondary metabolites were more tightly coupled to growth phases than to strains. Abundance of some toxic lipids and benzenoids DMs were significantly higher in the MC-free strain than the MC-producing one. This study builds on the understanding of MaE chemicals and their biotoxicity, and adds to evidence that non-MC-producing strains of cyanobacteria may also pose a threat to ecosystem health.

Multiobjective optimization of frozen and freeze-dried Lactobacillus delbrueckii subsp. bulgaricus CFL1 production via the modification of fermentation conditions.

This study investigates the individual and combined effects of fermentation parameters for improving cell biomass productivity and the resistance to freezing, freeze-drying, and freeze-dried storage of Lactobacillus delbrueckii subsp. bulgaricus CFL1. Cells were cultivated at different temperatures (42°C and 37°C) and pH values (5.8 and 4.8) and harvested at various growth phases (mid-exponential, deceleration, and stationary growth phases). Specific acidifying activity was determined after fermentation, freezing, freeze-drying, and freeze-dried storage. Multiple regression analyses were performed to identify the effects of fermentation parameters on the specific acidifying activity losses and to generate the corresponding 3D response surfaces. A multiobjective decision approach was applied to optimize biomass productivity and specific acidifying activity. The temperature positively influenced biomass productivity, whereas low pH during growth reduced the loss of specific acidifying activity after freezing and freeze-drying. Furthermore, freeze-drying resistance was favored by increased harvest time. Productivity, and freezing and freeze-drying resistances of L. delbrueckii subsp. bulgaricus CFL1 were differentially affected by the fermentation parameters studied. There was no single fermentation condition that improved both productivity and resistance to freezing and freeze-drying. Thus, Pareto fronts were helpful to optimize productivity and resistance, when cells were grown at 42°C, pH 4.8, and harvested at the deceleration phase.

Significant Changes in Cytoplasmic Amino Acid Composition Occur in the Transition between Mid-Exponential and Stationary Phases of Growth of Staphylococcus aureus: An Example of Adaptive Homeostasis in Response to Nutrient Limitations

The bacterial pathogen Staphylococcus aureus causes a wide range of infections that result in high morbidity and mortality rates worldwide. S. aureus is known for its capacity to survive harsh environments between hosts and certain strains are very efficient as opportunistic pathogens. It is important to understand their capacities for metabolic adaptation in response to changing environmental conditions. This investigation aimed to explore the alterations in the amino acid compositions of the cytoplasm as nutrients became limiting during the growth of S. aureus. Cells were grown under optimal growth conditions and harvested at the mid-exponential and stationary phases of growth and then extracted for the analyses of amino acids in the cytoplasm. The analyses revealed that the stationary phase cells had a significantly higher concentration of total cytoplasmic amino acids compared with cells at the mid-exponential phase and displayed substantial alterations in amino acid composition. Aspartic acid was the major amino acid in the stationary phase cells, whereas glutamic acid was the most abundant in the mid-exponential cells. The glutamic acid was reduced by 47% of its original value when the growth was extended to the stationary phase. Interestingly, certain amino acids were either absent or present depending on the phase of growth. These outcomes are in line with the premise that bacterial cells of S. aureus transition into a different form of metabolic homeostasis in the shift between the exponential and stationary phases of growth, as nutrients become depleted and waste products accumulate in the external medium. The ability of S. aureus to continually and promptly adapt to differences within growth phases may represent an essential strategy assisting its virulence as a successful opportunistic pathogen to establish infections. An understanding of the switch mechanisms controlling these obvious alterations in amino acids through the growth/life cycle of this virulent pathogen may provide novel clinical strategies to battle infection.

Возможная роль представителей кишечного микробиома в биотрансформации арил-, пиридилсодержащих фосфинов и их производных (по данным спектроскопии ЯМР 31Р)

Organophosphorus compounds (OPCs) are used in many areas of human activity. Unique physicochemical properties and high biological activity not only determine the applied value of OPCs but also give them the properties of xenobiotics that are harmful to human health, which largely depend on the state of the endogenous microbiota. The purpose of this work is to assess the effect of pyridyl- and aryl-containing phosphines, their oxides, and sulfides on the growth of Bifidobacterium bifidum and Escherichia coli and to identify possible patterns of biotransformation of OPCs using 31P NMR spectroscopy. Bacteria were cultivated in thioglycollate medium containing the tested phosphorus compounds. At the stationary growth phase was determined the cell concentration and were recorded spectra 31P NMR. It was shown that the most reliable (p<0,05) and opposite effect on the growth dynamics of both species of bacteria was exhibited by phosphine sulfides 1 and 3: PS1 (pyridyl) reduced the population reproduction rate by 35–40%, and PS3 (aryl) increased it by 45–60%. At the same time, OPCs themselves were most likely not metabolized. Phosphine oxides reduced the average titer of bacteria compared to the control, and PO3 caused complete cell elimination after 24 hours cultivation. In the medium with PO2 was identified PS2 appeared, which may be caused by the biologically mediated process of oxide-to-sulfide conversion. All phosphines are chemically labile and they were oxidized abiotically to oxides and sulfides. Formed sulfides could be the reason for a significant increase (to 35%) in the growth rate bacteria population both species. Five new unidentified organophosphorus compounds (UPC) were recorded on medias with B. bifidum, some of which may belong to the products of biotransformation of the original OPCs. Some organic phosphates, homologous to the phosphine oxides tested, have a δp value neighboring to the taken NMR spectra. This may indicate the way of their biological transformation: oxidation of the CP bond in the phosphine oxides to an ester bond of organic phosphates. This ability of bifidobacteria to metabolize OPCs will make it possible to develop a therapeutic strategy based on the use of B. bifidum as a microorganism that degrades phosphorus-containing xenobiotics.

A 24-hour <i>Сandida sp.</i> biofilm formation dynamically assesed with modified macrometric method

The aim of the study was to study the dynamics of 24-hour Candida spp. biofilm formation by using a modified macrometric method. The proposed macrometric method solves the problem of accelerating and simplifying the quantitative assessment of the biofilm formation process, increases sensitivity due to allowing to avoid mistakes related to applied polystyrene material. The ease of implementing such a technique makes it accessible to any laboratory. Reference strains from the American Type Culture Collection (ATCC) and clinical isolates of Candida spp. isolated from the female reproductive tract with candida dysbiosis were used for the study. Biofilm formation of Candida spp. studied according to the OToole G.A. et al. method modified by us. The biological activity of Сandida sp. biofilm formation was monitored for 48 hours with 4-hour intervals, in winter season, the IV phase of the moon. A 48-hour fungal culture corresponding to relevant maximum adhesion on glass surface was used. The study chrono-design implied obtaining 6 diurnal measurements for the function evaluated with a 35-repetits of the experimental conditions. Amplitude-phase characteristics of the studied biorhythms were graphically represented using cosinor analysis serving as the basic method to identify and model cyclic processes in biological systems. The study proved that the ability of micromycete cells to adhere is significantly higher in the stationary vs. logarithmic growth phase (p 0.05). The chronobiological technique used here allowed to reveal the presence of diurnal fungal film-forming activity (p 0.05) and reveal the general patterns of manifestated properties in representatives of all candida species examined. It has been experimentally established that the sequence and consistency of the biological properties of clinical Сandida sp. isolates over time were not fundamentally dependent on the type of fungus. During the study, rhythmometric markers of the strain-related pathogenicity was established reflecting contribution of rhythm and the amplitude-phase characteristic. It has been proven that the activity of biofilm formation increases along the reference strains clinical isolates axis. For С. albicans the MannWhitney test data was 29, for C. tropicalis 26, and for C. krusei 30 (p 0.05). We believe that chronobiological method opens up new perspectives in the studying physiology of Candida spp. because it allows to dynamically predict state of microorganism and take into account features of urgent and long-term adaptation to various environmental factors. Identifying diurnal rhythms in biofilm-forming activity of various Сandida sp. strains opens up an opportunity to control viability of bacterial-fungal associations and predict related resistance to diverse antimicrobial agents.

Characterization of Neochloris oleoabundans under Different Cultivation Modes and First Results on Bioactivity of Its Extracts against HCoV-229E Virus

Microalgae are proposed in several biotechnological fields because of their ability to produce biomass enriched in high-value compounds according to cultivation conditions. Regarding the health sector, an emerging area focuses on natural products exploitable against viruses. This work deals with the characterization of the green microalga Neochloris oleoabundans cultivated under autotrophic and mixotrophic conditions as a source of whole aqueous extracts, tested as antivirals against HCoV-229E (Coronaviridae family). Glucose was employed for mixotrophic cultures. Growth and maximum quantum yield of photosystem II were monitored for both cultivations. Algae extracts for antiviral tests were prepared using cultures harvested at the early stationary phase of growth. Biochemical and morphological analyses of algae indicated a different content of the most important classes of bioactive compounds with antiviral properties (lipids, exo-polysaccharides, and total phenolics, proteins and pigments). To clarify which phase of HCoV-229E infection on MRC-5 fibroblast cells was affected by N. oleoabundans extracts, four conditions were tested. Extracts gave excellent results, mainly against the first steps of virus infection. Notwithstanding the biochemical profile of algae/extracts deserves further investigation, the antiviral effect may have been mainly promoted by the combination of proteins/pigments/phenolics for the extract derived from autotrophic cultures and of proteins/acidic exo-polysaccharides/lipids in the case of mixotrophic ones.

Comparative assessment of the level of tolerance of <I>Bacillus subtilis</I> soil isolates in relation to chemical copper compounds

Relevance. The biological potential of microorganisms due to the high level of physiological and adaptive characteristics to the action of abiotic factors (the formation of resistant forms) and has a wider range of applications in contrast to substitution therapy. Our work presents experimental data on the assessment of the level of tolerance of soil isolates of Bacillus subtilis in relation to various chemical compounds of copper and the degree of influence of a massive cationic load on the growth of the most resistant strains.Methods. To implement the tasks set, we used the methods of isolation and identification of pure cultures of microorganisms, the diffusion method of agar wells (assessment of the inhibitory characteristics of chemical compounds), and the nephelometric method (the effect of heavy metals on the growth of microorganisms).Results. The most resistant strain to chemical stress is the B. subtilis P-8, isolate, which exhibits resistance to extremely high concentrations of copper: CuCl2 · 7H2O and CuSO4 — 0.125 M/l, Cu(CH3COO)2 — 0.250 M/l, while the strain B. subtilis P-6 is the most sensitive to two of the three chemicals. When conducting studies to assess the level of copper sulfate fusion on the growth of the population of strains selected by us, it was found that this compound has a pronounced prolonging effect on the growth of the cell population of B. subtilis P-6 and B. subtilis P-8 with an increase in the time of onset of the stationary phase of growth by 6 hours, relative to benchmarks.

The effectiveness of biofilm formation of daily cultures of clinically significant strains of opportunistic bacteria

Background. The formation of biofilm structures by microorganisms living in the hospital environment is a serious medical problem. To conduct correct experimental studies, it is necessary to know the speed and efficiency of biofilm formation by clinically significant species of opportunistic bacteria. Aim: to study the kinetics of plankton culture growth and the rate of biofilm formation by clinically significant pathogens of infections associated with medical care to substantiate the methodology of further research. Materials and methods. The strains from the working collection of the Laboratory for Microbiome and Microecology of the Scientific Сentre for Family Health and Human Reproduction Problems were used. Experiments were carried out with conditionally pathogenic microorganisms of the Enterobacteriaceae family and non-fermenting gram-negative bacteria. The optical density was measured, the total microbial number of the cell suspension was determined, and the morphological structure of the biofilm was evaluated using a light microscope on sterile cover glasses for thespecies Pseudomonas aeruginosa, Klebsiella pneumoniae and Serratia marcescens. Results. The duration of the lag phase of the kinetic curve of cell growth varied in isolates of S. marcescens, P. aeruginosa and K. pneumoniae from 1 to 4 and 6 hours of cultivation, respectively. Despite this, the exponential growth phase was the same for all tested isolates and amounted to 4 hours. Thus, isolates of clinically significant species entered the stationary growth phase after 5–10 hours of cultivation and were characterized as fast-growing. On abiotic surfaces, after 8 hours of incubation of the tested cultures, the initial stages of the formation of biofilm structures were observed, after 20 hours the formed multilayer biofilm was visualized, after 24 hours succession occurred, new single cells were attached to the place of the detached structures. Conclusion. The data obtained on the duration of the main stages of growth kinetics compared with the visualization of the formation of biofilm structures on abiotic surfaces should be taken into account when studying the effects of disinfectants, antiseptics and antibacterial drugs on planktonic cells and biofilm associations of clinically significant opportunistic microorganisms.

Production of Fluorescent Dissolved Organic Matter by Microalgae Strains from the Ob and Yenisei Gulfs (Siberia).

Dissolved organic matter (DOM) is an important component of aquatic environments; it plays a key role in the biogeochemical cycles of many chemical elements. Using excitation-emission matrix fluorescence spectroscopy, we examined the fluorescent fraction of DOM (FDOM) produced at the stationary phase of growth of five strains of microalgae sampled and isolated from the Ob and Yenisei gulfs. Based on the morphological and molecular descriptions, the strains were identified as diatoms (Asterionella formosa, Fragilaria cf. crotonensis, and Stephanodiscus hantzschii), green microalgae (Desmodesmus armatus), and yellow-green microalgae (Tribonema cf. minus). Three fluorescent components were validated in parallel factor analysis (PARAFAC): one of them was characterized by protein-like fluorescence (similar to peak T), two others, by humic-like fluorescence (peaks A and C). The portion of fluorescence intensity of humic compounds (peak A) to the total fluorescence intensity was the lowest (27 ± 5%) and showed little variation between species. Protein-like fluorescence was most intense (45 ± 16%), but along with humic-like fluorescence with emission maximum at 470 nm (28 ± 14%), varied considerably for different algae strains. The direct optical investigation of FDOM produced during the cultivation of the studied algae strains confirms the possibility of autochthonous production of humic-like FDOM in the Arctic shelf regions.

Pseudomonas veronii strain 7–41 degrading medium-chain n-alkanes and polycyclic aromatic hydrocarbons

Pollution of the environment by crude oil and oil products (represented by various types of compounds, mainly aliphatic, mono- and polyaromatic hydrocarbons) poses a global problem. The strain Pseudomonas veronii 7–41 can grow on medium-chain n-alkanes (C8–C12) and polycyclic aromatic hydrocarbons such as naphthalene. We performed a genetic analysis and physiological/biochemical characterization of strain 7–41 cultivated in a mineral medium with decane, naphthalene or a mixture of the hydrocarbons. The genes responsible for the degradation of alkanes and PAHs are on the IncP-7 conjugative plasmid and are organized into the alk and nah operons typical of pseudomonads. A natural plasmid carrying functional operons for the degradation of two different classes of hydrocarbons was first described. In monosubstrate systems, 28.4% and 68.8% of decane and naphthalene, respectively, were biodegraded by the late stationary growth phase. In a bisubstrate system, these parameters were 25.4% and 20.8% by the end of the exponential growth phase. Then the biodegradation stopped, and the bacterial culture started dying due to the accumulation of salicylate (naphthalene-degradation metabolite), which is toxic in high concentrations. The activity of the salicylate oxidation enzymes was below the detection limit. These results indicate that the presence of decane and a high concentration of salicylate lead to impairment of hydrocarbon degradation by the strain.

Optimized conversion of wheat straw into single cell oils by Yarrowia lipolytica and Lipomyces tetrasporus and synthesis of advanced biofuels

This paper deals with the optimized conversion of undetoxified wheat straw hydrolysates into microbial lipids by two oleaginous yeasts, Yarrowia lipolytica and Lipomyces tetrasporus. Wheat straw were pretreated by steam explosion at 203 °C for 300 s and hydrolysed at 20% solid-to-liquid ratio by using an enzymatic loading of 15 FPU/g substrate. The mixed wheat straw hydrolysates (WHS) contained 86 gL-1 glucose and 22 gL-1 xylose, 2.3 gL-1 acetic acid, 0.9 gL-1 furanic compounds. The fermentation process was optimized in terms of the inoculum age and density, medium composition, and bioreactor feeding strategy. In particular, the different capacity of the two yeasts to overcome the toxic effect of the biomass degradation by-products, in different inoculum ages, was deeply investigated. Two hydrolysates concentration were tested: WSH containing 86 gL-1 glucose and 22 gL-1 xylose and the diluted medium containing 40 gL-1 glucose and 22 gL-1 xylose. The results indicated that both yeasts were able to detoxify WSH and grow on undetoxified hydrolysates as effect of the intrinsic capacity to metabolize the furan derivatives. Y. lipolytica was able to detoxify the medium in all the investigated set-ups, while L. tetrasporus was able to detoxify the medium only if inoculated in the stationary phase of growth. After the process optimization in shaken flasks, the production of Single Cell Oils (SCOs) by L. tetrasporus was carried out in a medium-scale bioreactor of 10L obtaining lipid yield and cell content of 21% and 62%, respectively. The extracted SCOs, with high oleic and palmitic acid content, were converted into biodiesel displaying overall features in accordance with international biodiesel standards, namely ASTM and EN 14214.

Plant biomass as a source of low-temperature yeasts

More than 40 yeast strains were isolated from various types of plant biomass and then evaluated for potential applications in biotechnological processes conducted at low temperature. Adaptation to low temperature was tested by passaging the isolates at decreasing temperatures, from 30 to 15 °C. Only the strains that were able to adapt to the final temperature and reached the stationary growth phase relatively quickly were submitted to further experimentation. These included eight environmental yeast isolates from four types of materials of plant origin: wheat, rye, and cucumber, containing glucose, fructose, sucrose, and starch; yeast-fermentable sugars; red beetroot, containing large amounts of glucose and fructose; and fruits (grapes and apples) containing glucose, fructose, and sucrose. The strains were identified and then subjected to a series of experiments to assess their suitability for use in low-temperature biotechnological industrial processes incorporating microbial biomass. The growth dynamics and assimilation profiles of the yeast strains were investigated, as well as their ability to produce volatile compounds.

Heterologous Lignan Production in Stirred-Tank Reactors—Metabolomics-Assisted Bioprocess Development for an In Vivo Enzyme Cascade

Towards establishing a prospective industrial microbial lignan production process, we set up and investigated the biotransformation of coniferyl alcohol to secoisolariciresinol with recombinant Escherichia coli in a stirred-tank reactor (STR). Initially, we tested different cofactor concentrations and antifoam additions in shake flasks. Next, we designed an STR batch bioprocess and tested aeration rates, pH regulation, and substrate-feeding strategies. Targeted metabolomics of phenylpropanoids and lignans assisted the bioprocess development by monitoring the lignan pathway activity. We found that the copper concentration and the substrate-feeding strategy had considerable impact on lignan production. Furthermore, time-resolved monitoring of pathway metabolites revealed two maximal intracellular lignan concentrations, the first shortly after induction of gene expression and the second after the cells entered the stationary growth phase. During STR cultivation, a maximal intracellular titer of 130.4 mg L−1 secoisolariciresinol was achieved, corresponding to a yield coefficient of 26.4 mg g−1 and a space–time yield of 2.6 mg L−1 h−1. We report for the first time the in-depth evaluation of microbially produced lignans in a well-controlled STR bioprocess. Monitoring of the lignan pathway activity showed that lignan accumulation is highly dynamic during the cultivation and points towards the need for a more efficient coniferyl alcohol dimerization system for optimal microbial production conditions.

Green Extracellular Synthesis of Silver Nanoparticles by Pseudomonas alloputida, Their Growth and Biofilm-Formation Inhibitory Activities and Synergic Behavior with Three Classical Antibiotics.

Bacterial resistance to antibiotics is on the rise and hinders the fight against bacterial infections, which are expected to cause millions of deaths by 2050. New antibiotics are difficult to find, so alternatives are needed. One could be metal-based drugs, such as silver nanoparticles (AgNPs). In general, chemical methods for AgNPs' production are potentially toxic, and the physical ones expensive, while green approaches are not. In this paper, we present the green synthesis of AgNPs using two Pseudomonas alloputida B003 UAM culture broths, sampled from their exponential and stationary growth phases. AgNPs were physicochemically characterized by transmission electron microscopy (TEM), total reflection X-ray fluorescence (TXRF), infrared spectroscopy (FTIR), dynamic light scattering (DLS), and X-ray diffraction (XRD), showing differential characteristics depending on the synthesis method used. Antibacterial activity was tested in three assays, and we compared the growth and biofilm-formation inhibition of six test bacteria: Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We also monitored nanoparticles' synergic behavior through the growth inhibition of E. coli and S. aureus by three classical antibiotics: ampicillin, nalidixic acid, and streptomycin. The results indicate that very good AgNP activity was obtained with particularly low MICs for the three tested strains of P. aeruginosa. A good synergistic effect on streptomycin activity was observed for all the nanoparticles. For ampicillin, a synergic effect was detected only against S. aureus. ROS production was found to be related to the AgNPs' antibacterial activity.