High Acetate Concentrations Research Articles

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis.

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.

Contribution of Uncharacterized Target Genes of MxtR/ErdR to Carbon Source Utilization by Pseudomonas putida KT2440.

MxtR/ErdR is a two-component system that has been previously described as a regulator of the utilization of acetate in Vibrio cholerae and in some Pseudomonas species. Regulation is achieved by controlling the expression of the acs gene (acetyl-coenzyme A [CoA] synthetase). However, the physiological significance of other identified target genes is not fully understood. Here, we investigated the role of pp_0154 (scpC) and pp_0354/pp_0353 in the soil bacterium Pseudomonas putida KT2440. To this end, the genes were individually deleted and complemented in trans. Then, the growth of the resulting strains on different carbon sources was analyzed. To obtain information on protein function, a bioinformatic analysis was performed, and ScpC was purified and characterized in vitro. Our results indicated that scpC is important for P. putida KT2440 to cope with high concentrations of acetate. The encoded enzyme catalyzes the transfer of coenzyme A between acetate and succinate. On the contrary, pp_0353 and pp_0354 proved to be unimportant for the growth of the strain on acetate under our conditions. Extending the phenotypic analysis to other carbon sources led to the discovery that mxtR, erdR, and pp_0353 are important for the utilization of pyruvate as a carbon source. Taken together, the findings of this study expand the knowledge about the role of the MxtR/ErdR two-component system in carbon source utilization and about the specific functions of its target genes. IMPORTANCE MxtR/ErdR and homologous two-component systems play important roles in the regulatory networks that control cell metabolism and influence bacterial-host interactions. Using the MxtR/ErdR two-component system of the plant growth-promoting soil bacterium Pseudomonas putida KT2440 as a model, this work elucidates the function of previously uncharacterized target genes of MxtR/ErdR and extends the knowledge of the physiological significance of the two-component system. Our results suggest that the target gene scpC encodes an acetate:succinate CoA transferase that is involved in the detoxification of acetate when it is present in large amounts. Furthermore, it is shown that MxtR/ErdR controls the metabolism of not only acetate but also pyruvate. This control involves the target gene pp_0353 (putative exonuclease). These findings may facilitate the optimization of P. putida KT2440 as a chassis for biotechnological applications and may contribute to a better understanding of the regulatory network of pathogens like Pseudomonas aeruginosa.

Characterization of a new Saccharomyces cerevisiae isolated from banana stems and its mutant with l-leucine accumulation for awamori brewing.

We isolated a new strain of the yeast Saccharomyces cerevisiae, 35a14, from banana stems in Okinawa. This strain did not belong to any industrial yeast groups in a phylogenetic tree and produced high levels of alcohol. Furthermore, awamori, an Okinawa's traditional distilled alcoholic beverage, brewed with an l-leucine overproducing mutant derived from 35a14 showed a high concentration of isoamyl acetate.

Effect of Inoculum Microbial Diversity in Ex Situ Biomethanation of Hydrogen.

The effects of the inoculum origin, temperature or operational changes on ex situ biomethanation by complex microbial communities have been investigated; however, it remains unclear how the diversity of the inoculum influences the process and its stability. We explored the effect of microbial diversity of four inocula (coded as PF, WW, S37 and Nrich) on methane production, process stability and the formation of volatile fatty acids as by-products. The highest methane amounts produced were 3.38 ± 0.37 mmol, 3.20 ± 0.07 mmol, 3.07 ± 0.27 mmol and 3.14 ± 0.06 mmol for PF, WW, S37 and Nrich, respectively. The highest acetate concentration was found in less diverse cultures (1679 mg L-1 and 1397 mg L-1 for S37 and Nrich, respectively), whereas the acetate concentrations remained below 30 mg L-1 in the more diverse cultures. The maximum concentration of propionate was observed in less diverse cultures (240 mg L-1 and 37 mg L-1 for S37 and Nrich cultures, respectively). The highly diverse cultures outperformed the medium and low diversity cultures in the long-term operation. Methanogenic communities were mainly composed of hydrogenotrophic methanogens in all cultures. Aceticlastic methanogenesis was only active in the highly diverse sludge community throughout the experiment. The more diverse the inocula, the more methane was produced and the less volatile fatty acids accumulated, which could be attributed to the high number of microbial functions working together to keep a stable and balanced process. It is concluded that the inoculum origin and its diversity are very important factors to consider when the biomethanation process is performed with complex microbial communities.

Electrodeposition of Molybdenum from Water-in-Salt Electrolytes

Molybdenum is attractive to the manufacturing industry due to its high melting point and extremely low thermal expansion. Generally, Mo can be produced by thermal spraying, sintering, and pressing at high temperatures. Electrodeposition has been used widely as low-temperature method to prepare metals and alloys as coatings or films. However, it is well-known that metallic molybdenum is difficult to deposit from aqueous solution due to its highly negative reversible potential. More recently, a few studies have successfully deposited metallic molybdenum from highly concentrated acetate solutions1,2 as a thick coating on metallic objects. In this work, we report a systematic study on Mo deposition using this electrolyte system under the umbrella of water-in-salt concept. Aqueous electrolytes with an extremely high concentration of salt, known as water-in-salt electrolytes, offer a special electrolyte environment for electrodeposition where free water molecules are reduced by the hydration of salt.This report focuses on the effects of substrate and electrolyte chemistry on the Mo deposition process as well as the film properties. In this study, Cu electrode is fabricated by depositing Cu layer on Pt electrode. The electrochemical behavior is studied using cyclic voltammetry on Pt and Cu disc electrodes, as shown in Figure 1. Mo deposition starts at about -2.0 V on both Pt and Cu. In the anodic scan, the deposited Mo film begins to dissolve at -0.7V. X-ray fluorescence is used to measure film thickness, which is consistent with the stripping charge according to Faraday’s laws. However, the amount of Mo deposited on Cu, represented by the anodic stripping charge excluding the charge for Cu stripping, is much higher than that on Pt electrode. On the other hand, the presence of CH3COOK is found extremely critical for Mo deposition despite of a high total concentration of acetate. It is believed to relate to a suppressed proton reduction in presence of alkaline metal cations in a super highly concentrated acetate electrolyte. This correlation between hydrogen evolution reaction and Mo deposition is also consistent with the promoted deposition rate on Cu than on Pt surface. DC deposition studies are carried out to further verify these observations and to allow characterization of deposited Mo. Details will be further discussed in the poster presentation.1 Morley, T. J. et al., "The deposition of smooth metallic molybdenum from aqueous electrolytes containing molybdate ions," Electrochemistry Communications 15, 78-80 (2012).2 Syed, R. et al., "Electrodeposition of thick metallic amorphous molybdenum coating from aqueous electrolyte," Surface & Coatings Technology 261, 15-20 (2015). Figure 1

Native Mass Spectrometry for Peptide-Metal Interaction in Picoliter Cell Lysate.

Native mass spectrometry, which takes a high concentration of ammonium acetate (NH4OAc) for ionization, coupled with tedious and solvent-consuming purification, which separates proteins from complicated environments, has shown great potential for proteins and their complexes. A high level of nonvolatile salts in the endogenous intracellular environment results in serious ion suppression and has been one of the bottlenecks for native mass spectrometry, especially for protein complexes. Herein, an integrated protocol utilizing the inner surface of a micropipette for rapid purification, desorption, and ionization of peptide-metal interaction at subfemtomole level in cell lysate was demonstrated for native mass spectrometry. The methods showed robust and reproducibility in protein measurement within 1 min from various buffers. The E. coli cells expressing with various proteins were lysed and used to test our method. The specific interaction between the peptide-metal complex in cell lysates could be reserved and distinguished by mass spectrometry.

Enhanced nitrogen and carbon removal in natural seawater by electrochemical enrichment in a bioelectrochemical reactor

Municipal and industrial wastewater discharges in coastal and marine environments are of major concern due to their high carbon and nitrogen loads and the resulted phenomenon of eutrophication. Bioelectrochemical reactors (BERs) for simultaneous nitrogen and carbon removal have gained attention owing to their cost efficiency and versatility, as well as the possibility of electrochemical enrich specific groups. This study presented a scalable two-chamber BERs using graphite granules as electrode material. BERs were inoculated and operated for 37 days using natural seawater with high concentrations of ammonium and acetate. The BERs demonstrated a maximum current density of 0.9 A m−3 and removal rates of 7.5 mg NH4+-N L−1 d−1 and 99.5 mg L−1 d−1 for total organic carbon (TOC). Removals observed for NH4+-N and TOC were 96.2% and 68.7%, respectively. The results of nutrient removal (i.e., ammonium, nitrate, nitrite and TOC) and microbial characterization (i.e., next-generation sequencing of the 16S rRNA gene and fluorescence in situ hybridization) showed that BERs operated with a poised cathode at −260 mV (vs. Ag/AgCl) significantly enriched nitrifying microorganisms in the anode and denitrifying microorganisms and planctomycetes in the cathode. Interestingly, the electrochemical enrichment did not increase the total number of microorganisms in the formed biofilms but controlled their composition. Thus, this work shows the first successful attempt to electrochemically enrich marine nitrifying and denitrifying microorganisms and presents a technique to accelerate the start-up process of BERs to remove dissolved inorganic nitrogen and total organic carbon from seawater.

Nutritional Values and In Vitro Fermentation Characteristics of 29 Different Chinese Herbs

The present study evaluated the nutritional values and in vitro fermentation characteristics of 29 different Chinese herbs in order to develop effective feed additives for livestock nutrition. The Chinese herbs were analyzed for their chemical composition. The in vitro fermentation characteristics were evaluated using the gas production technique with swine cecal chyme inoculum. The results showed that the Allium tuberosum Rottl (Semen allii tuberosi, SAT) had the highest gross energy (GE), crude protein (CP), ether extract (EE), and total amino acid (TAA) contents, whereas Anemones raddeanae Rhizoma (Agstache rugosus, AR) had the highest crude fiber, neutral detergent fiber, and acid detergent fiber contents compared with the other Chinese herbs. The Rhus chinensis Mill (Chinese gall, CG), Scrophularia ningpoensis Hemsl (Radix scrophulariae, RS), Punica granatum L (Punica granatum L, PGL), Atractylodes macrocephala Koidz (White atractylodes rhizome, WAR), Sanguisorbae radix (Garden burnet, GB), Anemarrhena asphodeloides Bunge (Rhizoma anemarrhenaw, RA), and Rhei radix Et Rhizoma (Rheum officinale, RO) had a higher non-fiber carbohydrate content than the other Chinese herbs. The in vitro fermentation kinetic analysis showed that the RS, WAR, and RA had a higher gas production, a theoretical gas production, and a fractional rate of gas production than the other groups. Furthermore, the RA, WAR, RS, and Crataegi fructus (Crataegus pinnatifida Bun, CPB) had a higher total short-chain fatty acids (SCFAs) concentration, and CPB had the highest acetate concentration, whereas the WAR and RS had higher propionate and butyrate concentrations than the other groups. Moreover, the Isatis indigotica Fort (Folium isatidis, FI) and SAT had higher ammonia-N concentration than the other groups. In summary, among the 29 different Chinese herbs, the SAT had the highest nutritional value, which is reflected in the highest contents of GE, CP, EE, and TAA, whereas the RA, WAR, and RS showed better intestinal fermentation profiles, which is reflected in the higher fermentation degree and suitable SCFAs fermentation pattern. These findings provide a theoretical basis for using Chinese herbs as feed additives or microbial carbon sources in the intestines of animals.

Assessment of Spontaneous Fermentation and Non-Saccharomyces Sequential Fermentation in Verdicchio Wine at Winery Scale

The use of non-Saccharomyces yeasts in sequential fermentation is a suitable biotechnological process to provide specific oenological characteristics and to increase the complexity of wines. In this work, selected strains of Lachancea thermotolerans and Starmerella bombicola were used in sequential fermentations with Saccharomyces cerevisiae and compared with spontaneous and pure S. cerevisiae fermentation trials in Verdicchio grape juice. Torulaspora delbrueckii together with the other two non-Saccharomyces strains (L. thermotolerans, S. bombicola) in multi-sequential fermentations was also evaluated. Wines, obtained under winery vinification conditions, were evaluated for their analytical and sensorial profile. The results indicated that each fermentation gave peculiar analytical and aromatic features of the final wine. L. thermotolerans trials are characterized by an increase of total acidity, higher alcohols and monoterpenes as well as citric and herbal notes. S. bombicola trials showed a general significantly high concentration of phenylethyl acetate and hexyl acetate and a softness sensation while multi-sequential fermentations showed a balanced profile. Spontaneous fermentation was characterized by the production of acetate esters (ethyl acetate and isoamyl acetate), citrus and herbal notes, and tannicity. The overall results indicate that multi-starter fermentations could be a promising tool tailored to the desired features of different Verdicchio wine styles.

Development of Hypertolerant Strain of Yarrowia lipolytica Accumulating Succinic Acid Using High Levels of Acetate.

Acetate is emerging as a promising feedstock for biorefineries as it can serve as an alternate carbon source for microbial cell factories. In this study, we expressed acetyl-CoA synthase in Yarrowia lipolytica PSA02004PP, and the recombinant strain grew on acetate as the sole carbon source and accumulated succinic acid or succinate (SA). Unlike traditional feedstocks, acetate is a toxic substrate for microorganisms; therefore, the recombinant strain was further subjected to adaptive laboratory evolution to alleviate toxicity and improve tolerance against acetate. At high acetate concentrations, the adapted strain Y. lipolytica ACS 5.0 grew rapidly and accumulated lipids and SA. Bioreactor cultivation of ACS 5.0 with 22.5 g/L acetate in a batch mode resulted in a maximum cell OD600 of 9.2, with lipid and SA accumulation being 0.84 and 5.1 g/L, respectively. However, its fed-batch cultivation yielded a cell OD600 of 23.5, SA titer of 6.5 g/L, and lipid production of 1.5 g/L with an acetate uptake rate of 0.2 g/L h, about 2.86 times higher than the parent strain. Cofermentation of acetate and glucose significantly enhanced the SA titer and lipid accumulation to 12.2 and 1.8 g/L, respectively, with marginal increment in cell growth (OD600: 26.7). Furthermore, metabolic flux analysis has drawn insights into utilizing acetate for the production of metabolites that are downstream to acetyl-CoA. To the best of our knowledge, this is the first report on SA production from acetate by Y. lipolytica and demonstrates a path for direct valorization of sugar-rich biomass hydrolysates with elevated acetate levels to SA.

Growth parameters and responses of green algae across a gradient of phototrophic, mixotrophic and heterotrophic conditions.

Many studies have shown that algal growth is enhanced by organic carbon and algal mixotrophy is relevant for physiology and commercial cultivation. Most studies have tested only a single organic carbon concentration and report different growth parameters which hampers comparisons and improvements to algal cultivation methodology. This study compared growth of green algae Chlorella vulgaris and Chlamydomonas reinhardtii across a gradient of photoautotrophic-mixotrophic-heterotrophic culture conditions, with five acetate concentrations. Culture growth rates and biomass achieved were compared using different methods of biomass estimation. Both species grew faster and produced the most biomass when supplied with moderate acetate concentrations (1–4 g L−1), but light was required to optimize growth rates, biomass yield, cell size and cell chlorophyll content. Higher acetate concentration (10 g L−1) inhibited algal production. The choice of growth parameter and method to estimate biomass (optical density (OD), chlorophyll a fluorescence, flow cytometry, cell counts) affected apparent responses to organic carbon, but use of OD at 600, 680 or 750 nm was consistent. There were apparent trade-offs among exponential growth rate, maximum biomass, and culture time spent in exponential phase. Different cell responses over 1–10 g L−1 acetate highlight profound physiological acclimation across a gradient of mixotrophy. In both species, cell size vs cell chlorophyll relationships were more constrained in photoautotrophic and heterotrophic cultures, but under mixotrophy, and outside exponential growth phase, these relationships were more variable. This study provides insights into algal physiological responses to mixotrophy but also has practical implications for choosing parameters for monitoring commercial algal cultivation.

Changes on the structure of the gill apparatus of Cyprynus carpio under the influence of lead and copper

One of the urgent problems of aquatic toxicology is the increasing impact of heavy metals on hydrobionts. The aim of the study was to analyze the effect of high concentrations of lead acetate and copper acetate on carp gills during short-term exposure European carp (Cyprinus carpio carpio) was selected for the study weighing 1250 ± 136 g. During the experiment, 3 groups of 10 individuals were formed - a control group contained in water without a toxic agent; 1 experimental group of fish was contained in a solution of lead acetate (Pb(CH3COO)2) with a concentration of 0.6 mg/l (exceeding the maximum permissible concentration of lead for fishery reservoirs by 100 times); 2 the experimental group of fish was contained in a solution of copper acetate (Cu(CH3COO)2) with a concentration of 0.1 mg/l (exceeding the MPC of copper for fishery reservoirs by 100 times). The structure of the carp gill apparatus was studied by making a temporary wet preparation of the gill petals and its subsequent microscopy. The observed deviations in the state of the gills for the concentrations of copper and lead used included swelling and swelling of the gills, thickening of the epithelium, hypersecretion of the mucous cells of the gills.

Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio

This study evaluated the role of C/N/P in the increase in the synthesis of carbohydrates, proteins, and lipids in two high-mountain strains of algae (Chlorella sp. UFPS019 and Desmodesmus sp. UFPS021). Three carbon sources (sodium acetate, sodium carbonate, and sodium bicarbonate), and the sources of nitrogen (NaNO3) and phosphate (KH2PO4 and K2HPO4) were analyzed using a surface response (3 factors, 2 levels). In Chlorella sp. UFPS019, the optimal conditions to enhance the synthesis of carbohydrates were high sodium carbonate content (3.53 g/L), high KH2PO4 and K2HPO4 content (0.06 and 0.14 g/L, respectively), and medium-high NaNO3 (0.1875 g/L). In the case of lipids, a high concentration of sodium acetate (1.19 g/L) coupled with high KH2PO4 and K2HPO4 content (0.056 and 0.131 g/L, respectively) and a low concentration of NaNO3 (0.075 g/L) drastically induced the synthesis of lipids. In the case of Desmodesmus sp. UFPS021, the protein content was increased using high sodium acetate (2 g/L), high KH2PO4 and K2HPO4 content (0.056 and 0.131 g/L, respectively), and high NaNO3 concentration (0.25 g/L). These results demonstrate that the correct adjustment of the C/N/P ratio can enhance the capacity of high-mountain strains of algae to produce high concentrations of carbohydrates, proteins, and lipids.

Batch screening of weak base ion exchange resins for optimized extraction of acetic acid under fermentation conditions

Acetic acid is a valuable chemical with high demand for applications in food, pharmaceutical, polymer and other industries. A total of 10% acetate in the world is produced through bio-based processes such as fermentation. Such processes require cost-effective separation technologies to make the bio-acetate product competitive on the markets. Ion exchange resins can efficiently extract acetate from the fermentation broths and due to their re-usability after regeneration, can be cost-effective with lower operation and maintenance costs. To facilitate better process design, kinetics of acetate adsorption by six different basic ion exchange resins: Amberlite FPA-53, Amberlite IRA-67, Dowex-66, Dowex Marathon WBA-2, Amberlite IRA-96 and Amberlite IRA-400; was studied using well-known linear and non-linear adsorption isotherm models. The study showed that Amberlite FPA-53 (213 mg/g wet resin) had the highest acetate adsorption capability followed by Amberlite IRA-67 (202 mg/g wet resin) and Dowex-66 (191 mg/g wet resin). Of the tested models, the acetate adsorption onto these resins was found to fit well with the Langmuir model. It was found that there was a significant effect of acetic acid concentration and pH on acetate adsorption using gel-type and macroporous resins, especially at high initial concentrations of acetate in the broth. The study further showed, that under fermentation conditions (pH of 5–7), the adsorption capacity of all the resins was lower than at the pKa for acetic acid.

Adaptive Laboratory Evolution of Halomonas bluephagenesis Enhances Acetate Tolerance and Utilization to Produce Poly(3-hydroxybutyrate).

Acetate is a promising economical and sustainable carbon source for bioproduction, but it is also a known cell-growth inhibitor. In this study, adaptive laboratory evolution (ALE) with acetate as selective pressure was applied to Halomonas bluephagenesis TD1.0, a fast-growing and contamination-resistant halophilic bacterium that naturally accumulates poly(3-hydroxybutyrate) (PHB). After 71 transfers, the evolved strain, B71, was isolated, which not only showed better fitness (in terms of tolerance and utilization rate) to high concentrations of acetate but also produced a higher PHB titer compared with the parental strain TD1.0. Subsequently, overexpression of acetyl-CoA synthetase (ACS) in B71 resulted in a further increase in acetate utilization but a decrease in PHB production. Through whole-genome resequencing, it was speculated that genetic mutations (single-nucleotide variation (SNV) in phaB, mdh, and the upstream of OmpA, and insertion of TolA) in B71 might contribute to its improved acetate adaptability and PHB production. Finally, in a 5 L bioreactor with intermittent feeding of acetic acid, B71 was able to produce 49.79 g/L PHB and 70.01 g/L dry cell mass, which were 147.2% and 82.32% higher than those of TD1.0, respectively. These results highlight that ALE provides a reliable method to harness H. bluephagenesis to metabolize acetate for the production of PHB or other high-value chemicals more efficiently.

Lipotoxic Effects of Gossypol Acetate Supplementation on Hepatopancreas Fat Accumulation and Fatty Acid Profile in Cyprinus carpio

Cottonseed meal is an excellent protein source with low price and abundant nutrients, but the existence of free gossypol restricts its wide application in animal feeds. To explore the toxic effects of free gossypol, the effects of gossypol acetate on the growth performance, hepatopancreatic fat accumulation, and muscle fatty acid composition of the common carp (Cyprinus carpio) were investigated in the present study. Carp were randomly divided into four groups, which were fed a diet supplemented with 0, 170, 340, and 680 mg/kg gossypol acetate, respectively. After 60 days of feeding, the gossypol residue in the tissue, growth performance, blood biochemical indices, hepatopancreas histology, fatty acid components, and expression of key genes involved in lipid metabolism pathways were measured. It was found that the concentrations of free gossypol were increased significantly in the serum, bile, and hepatopancreas of carp fed 340 mg/kg and 680 mg/kg gossypol acetate ( P < 0.05 ). Dietary gossypol acetate had a limited effect on growth performance, and high concentrations of gossypol acetate induced excessive hepatopancreatic fat deposition and hepatocyte injury in C. carpio. In addition, gossypol acetate decreased total polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid in muscle. In the hepatopancreas, decreased HSL, PPARα, and CD36 may result in fat accumulation. In the muscle, decreased FADS2 may be the key reason for the decrease in PUFAs. Taken together, these findings indicated that dietary gossypol acetate can cause lipid metabolism disorders, such as hepatopancreatic damage and significant changes in tissue fatty acid profile, which are regulated by the key lipid metabolic genes in common carp.

Enhancing Ethanol Tolerance via the Mutational Breeding of Pichia terricola H5 to Improve the Flavor Profiles of Wine

Although using non-Saccharomyces yeasts during alcoholic fermentation can improve the wine aroma, most of them are not ethanol tolerant; therefore, in 2017, this study screened 85 non-Saccharomyces yeasts isolated and identified from 24 vineyards in seven Chinese wine-producing regions, obtaining Pichia terricola strain H5, which displayed 8% ethanol tolerance. Strain H5 was subjected to ultraviolet (UV) irradiation and diethyl sulfate (DES) mutagenesis treatment to obtain mutant strains with different fermentation characteristics from the parental H5. Compared with strain H5, the UV-irradiated strains, UV5 and UV8, showed significantly higher ethanol tolerance and fermentation capacity. Modified aroma profiles were also evident in the fermentation samples exposed to the mutants. Increased ethyl caprate, ethyl caprylate, and ethyl dodecanoate content were apparent in the UV5 samples, providing the wine with a distinctly floral, fruity, and spicy profile. Fermentation with strain UV8 produced a high ethyl acetate concentration, causing the wine to present a highly unpleasant odor. To a certain extent, UV irradiation improved the ethanol tolerance and fermentation ability of strain H5, changing the wine aroma profile. This study provides a theoretical basis for the industrial application of non-Saccharomyces yeasts that can improve wine flavor.

Adaptive laboratory evolution and shuffling of Escherichia coli to enhance its tolerance and production of astaxanthin

BackgroundAstaxanthin is one of the strongest antioxidants in nature and has been widely used in aquaculture, food, cosmetic and pharmaceutical industries. Numerous stresses caused in the process of a large scale-culture, such as high acetate concentration, high osmolarity, high level of reactive oxygen species, high glucose concentration and acid environment, etc., limit cell growth to reach the real high cell density, thereby affecting astaxanthin production.ResultsWe developed an adaptive laboratory evolution (ALE) strategy to enhance the production of chemicals by improving strain tolerance against industrial fermentation conditions. This ALE strategy resulted in 18.5% and 53.7% increases in cell growth and astaxanthin production in fed-batch fermentation, respectively. Whole-genome resequencing showed that 65 mutations with amino acid substitution were identified in 61 genes of the shuffled strain Escherichia coli AST-4AS. CRISPR interference (CRISPRi) and activation (CRISPRa) revealed that the shuffled strain with higher astaxanthin production may be associated with the mutations of some stress response protein genes, some fatty acid biosynthetic genes and rppH. Repression of yadC, ygfI and rcsC, activation of rnb, envZ and recC further improved the production of astaxanthin in the shuffled strain E. coli AST-4AS. Simultaneous deletion of yadC and overexpression of rnb increased the production of astaxanthin by 32% in the shuffled strain E. coli AST-4AS.ConclusionThis ALE strategy will be powerful in engineering microorganisms for the high-level production of chemicals.

Dynamics of luminescence characteristics of Haematococcus lacustris cultures in different cultivation conditions.

Luminescence of microalgae cultures is a valuable property for the fast diagnostics of their physiological state; however, it has been rarely used in algaculture practice. In this work, luminescence spectrum characteristics of two-stage batch cultures of the green carotenogenic microalga Haematococcus lacustris (Girod-Chantrans) Rostafinski 1875 (Chlorophyceae, Chlamydomonadales) under conditions of autotrophic and mixotrophic growth were investigated. The dynamics of the heterotrophy indices in cultures at different stages of their development in different growth media was determined. The transition of H. lacustris cultures from the initially autotrophic to mixotrophic growth regime was registered during the induction of the astaxanthin biosynthesis by complex physicochemical stressing, including nutritional deficiencies, exposure to high concentrations of sodium acetate and chloride and increased illuminance and temperature. The applicability of luminescence spectrometry in vivo for a rapid assessment of the state of H. lacustris cultures in various growth media and with different methods of secondary carotenogenesis induction was shown. The results obtained can be used in experimental studies on optimizing cultivation methods for this species, as well as for the express control of the physiological state of its industrial cultures.

P095 Anti-inflammatory effect of high acetate concentration on organoid-derived epithelial monolayer from patients with Ulcerative Colitis

Abstract Background Short-chain fatty acids (SCFA), such as acetate and butyrate, have gained increasing interest for their potential to suppress IBD inflammation1,4. Most data have been generated for butyrate, showing beneficial effects on the gut microbiome composition, intestinal barrier function and immune system1. The effects of acetate are less known, despite its lower toxicity on epithelial cells and its ability to support growth of butyrate-producing bacteria by metabolic cross-feeding2. Furthermore, the probiotic effect of the biotherapeutic yeast Saccharomyces cerevisiae var. boulardii might be linked to its high acetic acid production3. Here, we studied the effect of acetate on inflammatory markers and barrier integrity using an organoid-derived epithelial monolayer system obtained from Ulcerative Colitis (UC) patients. Methods Confluent Transwell® organoid-derived monolayers evaluated by Transepithelial electrical resistance (TEER) of 8 UC patients were basolaterally stimulated with control medium (CTRL) or with an inflammatory mix (INFL) [100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin] (Fig. 1). After 24h, the monolayer was stimulated apically with control medium or with high acetate (HA) [100mM] and TEER was measured at 0, 24, and 48h post-stimulation. After 48h, RNA extraction of the epithelial cell monolayers and qPCR for selected target genes (IL8, TNFα, CLDN1, CLDN2, HIF1A, ZO1, OCLDN, MUC2 and MKI67) was performed. Cytokine measurement was performed using Mesoscale Discovery (Proinflammatory panel 1) on apical and basolateral media. Data were analyzed using GraphPad Prism 9 (D’Agostino & Pearson test for normality followed by Wilcoxon tests). Results Transcriptomic analysis of the monolayer cells showed a decrease in expression of IL8 (p=0.03), TNFα (p=0.08) and increase of HIF1A (p=0.03) upon HA administration in inflamed conditions (Fig. 2) whereas the proliferation marker MKI-67 was upregulated (p=0,03). Moreover, these findings were supported by significant decreases in pro-inflammatory cytokines measured apically after HA stimulation (Fig. 3A) such as IFNγ, TNFα, IL6, IL8 and IL1β (all p=<0.01). Basolaterally, IL8 (p=0.02) and IL13 (p=<0.01) increased significantly upon HA stimulation (Fig.3B). Administration of HA was associated with a numerical though non-significant increase in TEER-values (p=0.15) (Fig. 4) suggesting a potential for improved barrier integrity. Conclusion In this UC patient-derived epithelial cell culture model, high acetate administration positively affected barrier integrity gene expression and TEER-values and suppressed expression and production of pro-inflammatory cytokines.