Alteration of the phospho- or neutral lipid content and fatty acid composition in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> due to acid adaptation mechanisms for hydrochloric,acetic and lactic acids at pH 5.5 or benzoic acid at neutral pH

This study provides a first approach to observe the effects on Listeria monocytogenes of cellular exposure to acid stress at low or neutral pH, notably how phospho- or neutral lipids are involved in this mechanism, besides the fatty acid profile alteration. A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown at pH 5.5 in presence of hydrochloric, acetic and lactic acids, or at neutral pH 7.3 in presence of benzoic acid, is described relative to cells grown in acid-free medium. The results showed that only low pH values enhance the antimicrobial activity of an acid. We suggest that, irrespective of pH, the acid adaptation response will lead to a similar alteration in fatty acid composition [decreasing the ratio of branched chain/saturated straight fatty acids of total lipids], mainly originating from the neutral lipid class of adapted cultures. Acid adaptation in L. monocytogenes was correlated with a decrease in total lipid phosphorus and, with the exception of cells adapted to benzoic acid, this change in the amount of phosphorus reflected a higher content of the neutral lipid class. Upon acetic or benzoic acid stress the lipid phosphorus proportion was analysed in the main phospholipids present: cardiolipin, phosphatidylglycerol, phosphoaminolipid and phosphatidylinositol. Interestingly only benzoic acid had a dramatic effect on the relative quantities of these four phospholipids.     

Oil and eicosapentaenoic acid production by the diatom Phaeodactylum tricornutum cultivated outdoors in Green Wall Panel (GWP®) reactors

Phaeodactylum tricornutum is a widely studied diatom and has been proposed as a source of oil and polyunsaturated fatty acids (PUFA), particularly eicosapentaenoic acid (EPA). Recent studies indicate that lipid accumulation occurs under nutritional stress. Aim of this research was to determine how changes in nitrogen availability affect productivity, oil yield, and fatty acid (FA) composition of P. tricornutum UTEX 640. After preliminary laboratory trials, outdoor experiments were carried out in 40‐L GWP® reactors under different nitrogen regimes in batch. Nitrogen replete cultures achieved the highest productivity of biomass (about 18 g m^?2 d^?1) and EPA (about 0.35 g m^?2 d^?1), whereas nitrogen‐starved cultures achieved the highest FA productivity (about 2.6 g m^?2 d^?1). The annual potential yield of P. tricornutum grown outdoors in GWP® reactors is 730 kg of EPA per hectare under nutrient‐replete conditions and 5,800 kg of FA per hectare under nitrogen starvation. Biotechnol. Bioeng. 2017;114: 2204–2210. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

     

Increasing fatty acid production in <Emphasis Type="Italic">E. coli</Emphasis> by simulating the lipid accumulation of oleaginous microorganisms

Unlike many oleaginous microorganisms, E. coli only maintains a small amount of natural lipids in cells, impeding its utility to overproduce fatty acids. In this study, acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus was expressed in E. coli to redirect the carbon flux to the generation of malonyl-CoA, which resulted in a threefold increase in intracellular lipids. Moreover, providing a high level of NADPH by overexpressing malic enzyme and adding malate to the culture medium resulted in a fourfold increase in intracellular lipids (about 197.74 mg/g). Co-expression of ACC and malic enzyme resulted in 284.56 mg/g intracellular lipids, a 5.6-fold increase compared to the wild-type strain. This study provides some attractive strategies for increasing lipid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms, which could aid the development of a prokaryotic fatty acid producer.     

耐乙酸粘红酵母合成油脂

乙酸是木质纤维素在水解过程中的主要副产物,高浓度的乙酸严重影响产油微生物的生长和油脂合成。本文研究了粘红酵母对乙酸的耐受性及其利用乙酸合成微生物油脂的能力。结果表明,在初始葡萄糖、木糖浓度分别为6 g/L和44 g/L的混合糖培养基中,乙酸浓度低于10 g/L时,不会对菌体生长产生抑制作用,油脂合成还得到了促进。当乙酸添加量为10 g/L时,生物量、油脂产量、油脂含量较对照组分别提高了21.5%、171.2%和121.6%。进一步研究表明,粘红酵母具备利用乙酸合成油脂的能力,当以乙酸为唯一碳源,浓度为25 g/L时,油脂产量达到3.20 g/L,油脂质量得率为13%。微生物油脂成分分析表明,粘红酵母以乙酸为底物制得的油脂可以作为制备生物柴油的油脂原料,其主要成分为棕榈酸、硬脂酸、油酸、亚油酸和亚麻酸,其中饱和脂肪酸和不饱和脂肪酸含量分别为40.9%和59.1%。由于粘红酵母具有利用乙酸合成微生物油脂的能力,在以木质纤维素水解液为原料生产微生物油脂的脱毒过程中,一定浓度的乙酸可以不必脱除。     

FATTY ACID AND LIPID COMPOSITION OF THE ACIDOPHILIC GREEN ALGA CHLAMYDOMONAS SP.1

The lipid and fatty acid compositions of Chlamydomonas sp. isolated from a volcanic acidic lake and C. reinhardtii were compared, and the effects of pH of the medium on lipid and fatty acid components of Chlamydomonas sp. were studied. The fatty acids in polar lipids from Chlamydomonas sp. were more saturated than those of C. reinhardtii. The relative percentage of triacylglycerol to the total lipid content in Chlamydomonas sp. grown in medium at pH 1 was higher than that in other cells grown at higher pH. A probable explanation might be that Chlamydomonas sp. has two low pH adaptation mechanisms. One mechanism is the saturation of fatty acids in membrane lipids to decrease membrane lipid fluidity, and the other is the accumulation of triacylglycerol, as a storage lipid, to prevent the osmotic imbalance caused by high concentrations of H₂SO₄.     

The effect of acetic acid concentration on the growth and production of gamma-linolenic acid byMucor circinelloides CBS 203.28 in fed-batch culture

The effect of different initial acetic acid concentrations on the growth of and lipid and gamma-linolenic acid (GLA) production byMucor circinelloides CBS 203.28 was determined in a 14 litre stirred tank reactor operated in a fedbatch, pH-stat mode with acetic acid as carbon source and pH titrant. Increased acetic acid concentrations in the culture resulted in a significant increase in the crude oil content of the biomass. By contrast, all the other parameters such as the biomass concentration, GLA and oil yield on acetic acid, the GLA content of the biomass and oil, the growth rate and volumetric rate of GLA production decreased with an increase in acetic acid concentration. The best results were obtained with acetic acid at 2 g/1, which gave 39.8 mg GLA/g biomass and 15.6% GLA in the neutral lipid fraction, amounting to 340 mg GLA/1 culture. A decrease in the glyco- and phospho-lipid fractions during the cultivation coincided with an increase in the neutral lipid fraction. The GLA content of the biomass remained within rather narrow limits of 3.5% to 4% of the biomass, irrespective of the oil content of the biomass. The fatty acid profile was not greatly affected by the acetic acid concentration. The hyphae of the fungus were characterized by the accumulation of large intracellular oil droplets and some septa delimited the hyphae.     

Membrane lipid polyunsaturation mediated by FATTY ACID DESATURASE 2 (FAD2) is involved in endoplasmic reticulum stress tolerance in Arabidopsis thaliana

Unsaturation of membrane glycerolipid classes at their hydrophobic fatty acid tails critically affects the physical nature of the lipid molecule. In Arabidopsis thaliana, 7 fatty acid desaturases (FADs) differently desaturate each glycerolipid class in plastids and the endoplasmic reticulum (ER). Here, we showed that polyunsaturation of ER glycerolipids is required for the ER stress response. Through systematic screening of FAD mutants, we found that a mutant of FAD2 resulted in a hypersensitive response to tunicamycin, a chemical inducer of ER stress. FAD2 converts oleic acid to linoleic acid of the fatty acyl groups of ER‐synthesized phospholipids. Our functional in vivo reporter assay revealed the ER localization and distinct tissue‐specific expression patterns of FAD2. Moreover, glycerolipid profiling of both mutants and overexpressors of FAD2 under tunicamycin‐induced ER stress conditions, along with phenotypic screening of the mutants of the FAD family, suggested that the ratio of monounsaturated fatty acids to polyunsaturated fatty acids, particularly 18:1 to 18:2 species, may be an important factor in allowing the ER membrane to cope with ER stress. Therefore, our results suggest that membrane lipid polyunsaturation mediated by FAD2 is involved in ER stress tolerance in Arabidopsis.     

Linking lipid transfer with reduced arbuscule formation in tomato roots colonized by arbuscular mycorrhizal fungus under low pH stress

Arbuscules are the core structures of arbuscular mycorrhizae (AM), and arbuscule development is regulated by environmental stress, e.g., low pH. Recent studies indicate that lipid transfer from plants is essential for AM fungal colonization; however, the role of lipid transfer in arbuscule formation and the dynamics of lipid accumulation in arbuscules under low pH stress are far from well understood. In the symbiosis of tomato and Rhizophagus intraradices under contrasting pH conditions (pH 4.5 vs. pH 6.5), we investigated arbuscule formation, nutrient uptake, alkaline phosphatase activity and lipid accumulation; examined the gene expression involved in phosphate transport, lipid biosynthesis and transfer and sugar metabolism; and visualized the lipid dynamics in arbuscules. Low pH greatly inhibited arbuscule formation, in parallel with reduced phospholipid fatty acids accumulation in AM fungus and decreased P uptake. This reduction was supported by the decreased expression of plant genes encoding lipid biosynthesis and transfer. More degenerating arbuscules were observed under low pH conditions, and neutral lipid fatty acids accumulated only in degenerating arbuscules. These data reveal that, under low pH stress, reduced lipid transfer from hosts to AM fungi is responsible for the inhibited arbuscule formation.     

Multi-stage biofilm reactor for acetic acid production at high concentration

Summary Continuous production of acetic acid by liquid surface culture ofAcetobacter aceti M7 was investigated using a Multi-Stage Biofilm Reactor (MSBFR) composed of ten shallow flow horizontal reactors of laboratory scale. With varying dilution rate in the range from 0.049 to 0.2 h^–1, the maximum exit acetic acid concentration reached was as high as 98.0 g/l at the lowest dilution rate with step feed of ethanol-rich medium to stages 3, 5, and 7. The production rate (4.3 g/l/h) was rather high considering the inhibitory effect of high acetic acid concentration. This may be ascribed to non-homogeneous distribution of acetic acid concentration in the bioreactor and step feed of ethanol-rich medium.     

Lipid modulation by environmental stresses in two models of extremophiles isolated from Antarctica

Thermoacidophilic and halotolerant microorganisms from the Antarctic continent were studied for their lipid modulation under stress growth conditions. Temperature-induced changes in complex lipids and fatty acids of four strains belonging to the genus Alicyclobacillus involved the relative proportions of different polar lipids and the synthesis of ω-cyclohexyl-acyl chains, which were favoured by high temperatures. Studies were carried out on the lipid composition of four strains of extremely halotolerant bacteria belonging to the genus Micrococcus grown at different salt concentrations from 0 up to 4.5 M NaCl. The main lipids found were two unidentified glycolipids and two phospholipids: 1,2 diacylglycero-3-phosphoryl-glycerol (PG) and cardiolipin (DPG). Among the strains analysed, the lipids of the Micrococcus strain Erebus were shown to be strongly influenced by salt concentrations, in that DPG and one glycolipid were absent at a low salt molarity while, under these conditions, PG was the main lipid found. The predominant fatty acids in all halotolerant strains were of the anteiso type; growth under increasing salinity gave rise to an increase in long chain fatty acids and of straight chain fatty acids, while a decrease in iso fatty acids occurred. Accepted: 20 May 2000     

Sucrose Lipids of Arthrobacteria,Corynebacteria and Nocardia Grown on Sucrose

Arthrobacter paraffineus KY 4303, when grown on sucrose as the sole carbon source, produced novel glycolipids, either of which was different from trehalose lipid produced from n-alkane by the same microorganism. Two kinds of glycolipids were isolated by chromatography on silicic acid columns. Major components of these lipids were sucrose and α-branched β-hydroxy fatty acid. One of the lipid (SL–1, having high polarity) was identified as 6-O-monofattyacyl glucosly-β-fructoside. Another (SL–2, having low polarity) was partly characterized as sucrose ester of at least two moles of the fatty acid.

Formation of sucrose lipids was also demonstrated in sucrose-grown cells of several microorganisms of Corynebacteria, Nocardia and Brevibacteria, which were isolated as hydrocarbon-utilizing bacteria and could produce a considerable amount of trehalose lipid from n-alkane.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Oleic acid transformations by selected strains of Sphingobacterium thalpophilum and Bacillus cereus from composted manure

In a survey of 186 randomly selected microbial strains isolated from composted manure, 63 transformed oleic acid into three types of products: hydroxy fatty acid, fatty amide, and less polar oleyl lipid. Selection of oleic acid-transforming microorganisms was enhanced in nutrient agar supplemented with 0.1% (vol/vol) oleic acid at pH 7.2. Most of the 63 diverse isolates elicited inconsistent and poorly reproduced transformations. However, strains 142b (NRRL B-14797) transformed oleic acid to 10-hydroxystearic acid consistently, and strain 229b (NRRL B-14812) produced an octadecenamide. Taxonomic studies indicated that NRRL strain B-14797, possessing 1,3-dihydroxy-2-amino-15-methylhexadecane and sphinganine bases, was closely related to Sphingobacterium thalpophilum, and NRRL B-14812 was identified as Bacillus cereus.     

Production of acetic acid by Dekkera/Brettanomyces yeasts under conditions of constant pH

Sixty yeast strains were previously screened for their ability to produce acetic acid, in shaken flask batch culture, from either glucose or ethanol. Seven of the strains belonging to the Brettanomyces and Dekkera genera, from the ARS Culture Collection, Peoria, IL, were further evaluated for acetic acid production in bioreactor batch culture at 28 °C, constant aeration (0.75 v/v/m) and pH (6.5). The medium contained either 100 g glucose/l or 35 g ethanol/l as the carbon/energy source. Dekkera intermedia NRRL YB-4553 produced 42.8 and 14.9 g acetic acid/l from the two carbon sources, respectively, after 64.5 h. The optimal pH was determined to be 5.5. When the initial glucose concentration was 150 or 200 g/l, the yeast produced 57.5 and 65.1 g acetic acid/l, respectively.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

Growth,biochemical, fatty acid composition,and mRNA levels of hepatic enzymes in genetically improved farmed tilapia (GIFT,Oreochromis niloticus) (Linnaeus, 1758) at different stocking densities

Investigated were the effects of stocking density on juvenile growth performance, serum biochemistry, fatty acid composition, and mRNA levels of enzymes involved in lipid metabolism in genetically improved farmed tilapia (GIFT), Oreochromis niloticus. Juvenile fish (n = 900) were distributed into 15 tanks at five initial stocking densities (75, 150, 225, 300, and 375 g/m³ per 800‐L tank, represented as D1–D5, respectively). Temperature was maintained at 28 ± 0.3°C, and water flow rate into the tanks was about 8 L/min. The fish accepted floating pelleted feed (crude protein 28%, crude lipid 6%, and gross energy 149.2 KJ/g diet). The feed amount offered was 4%–8% of body weight. At the end of the 60‐day experiment, the final stocking densities were 1,454.18, 2,010.60, 2,913.13, 3,819.73, and 4,380.80 g/m³, respectively. Growth and body mass coefficient of variation were significantly affected (p < .05). No differences in feed conversion rate or survival were found (p > .05). Muscle lipid contents decreased with stocking density from D1 to D5. Serum total protein and cholesterol levels were not affected by the stocking density but the D5 group produced the highest serum cortisol and lactate levels and the lowest glucose levels compared to those of the other groups. Lower muscle saturated fatty acid and n?3 polyunsaturated fatty acid contents were observed in the D4 group than in the other groups, whereas the reverse was found in the n?6 polyunsaturated fatty acid content. Hepatic glucose 6‐phosphate dehydrogenase, fatty acid synthetase, hormone‐sensitive triglyceride lipase, and lipoprotein lipase mRNA levels were significantly upregulated in response to a high density. These data suggest that the GIFT adapted to high‐density conditions by altering the composition of muscle fatty acid and stimulating lipid metabolic enzymes. These mechanisms may help fish respond to high stocking density stress in intensive aquaculture systems.     

Changes of γ-linolenic acid content inRhizopus arrhizus duringl(+)-lactic acid fermentation

Effects of nitrogen source, temperature and pH onl(+)-lactic acid production and γ-linolenic acid (GLA) accumulation byRhizopus arrhizus were examined. The nitrogen source had a minor effect on lactate synthesis but influenced the total lipid content and the fatty acid composition in fungus. Higher temperature favorably influenced the rate of both lactic acid production and lipid formation in the biomass and caused a decrease in the yields of oligounsaturated fatty acids. At higher temperature and after glucose exhaustion, degradation of lactate increased. A low pH value negatively affected the formation of lipids and lactate synthesis. The highest value of GLA in the lipid (25.5%,W/W) was reached at the end of lactate synthesis, but maximum yields of total lipids were achieved when the cultivation continued in the presence of lactate until polyols were exhausted.     

Reaction engineering analysis of hydrogenotrophic production of acetic acid by Acetobacterium woodii

Great interest has emerged in biological CO₂‐fixing processes in the context of current climate change discussions. One example for such a process is the hydrogenotrophic production of acetic acid by anaerobic microorganisms. Acetogenic microorganisms make use of carbon dioxide in the presence of hydrogen to produce acetic acid and biomass. In order to establish a process for the hydrogenotrophic production of acetic acid, the formation of acetate by Acetobacterium woodii was studied in a batch‐operated stirred‐tank bioreactor at different hydrogen partial pressures (pH₂) in the gas phase. The volumetric productivity of the batch processes increased with increasing hydrogen partial pressure. A maximum of the volumetric productivity of 7.4 g_acetate L⁻¹ day⁻¹ was measured at a pH₂ of 1,700 mbar. At this pH₂ a final acetate concentration of 44 g L⁻¹ was measured after a process time of 11 days, if the pH was controlled at pH 7.0 (average cell density of 1.1 g L⁻¹ cell dry weight). The maximum cell specific actetate productivity was 6.9 g_acetate g day⁻¹ under hydrogenotrophic conditions. Biotechnol. Bioeng. 2011;108: 470–474. © 2010 Wiley Periodicals, Inc.     

Improvement of acetic acid tolerance and fermentation performance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by disruption of the <Emphasis Type="Italic">FPS1</Emphasis> aquaglyceroporin gene

The FPS1 gene coding for the Fps1p aquaglyceroporin protein of an industrial strain of Saccharomyces cerevisiae was disrupted by inserting CUP1 gene. Wild-type strain, CE25, could only grow on YPD medium containing less than 0.45% (v/v) acetic acid, while recombinant strain T12 with FPS1 disruption could grow on YPD medium with 0.6% (v/v) acetic acid. Under 0.4% (v/v) acetic acid stress (pH 4.26), ethanol production and cell growth rates of T12 were 1.7 ± 0.1 and 0.061 ± 0.003 g/l h, while those of CE25 were 1.2 ± 0.1 and 0.048 ± 0.003 g/l h, respectively. FPS1 gene disruption in an industrial ethanologenic yeast thus increases cell growth and ethanol yield under acetic acid stress, which suggests the potential utility of FPS1 gene disruption for bioethanol production from renewable resources such as lignocelluloses.