Glycerol-containing Medium Research Articles

Влияние условий культивирования Acinetobacter calcoaceticus ІМВ B-7241 на антиадгезивные свойства поверхностно-активных веществ

To study the effect of growth factors and microelements in composition of ethanol-, n-hexadecane- and glycerol-containing media on antiadhesive properties of A cinetobacter calcoaceticus IMV B-7241 surfactants. Surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). The number (%) of attached cells (adhesion) was determined as a ratio of the optical density of the suspension obtained from the materials treated with surfactants to the optical density of the control samples (100 %). Dependence of surfactants antiadhesive properties on presence in the medium of A. calcoaceticus IMB B-7241 cultivation of growth factors and certain microelements, as well as the nature of the carbon source was established. Adhesion of bacteria (Escherichia coli IEM- 1, Bacillus subtilis BT-2) and yeast (Candida albicans D-6) on plastic, dutch tile, linoleum, and steel was a minimal (25-35 %) after surface treatment with surfactant (0.005 mg/ml) synthesized on ethanol in the presence of yeast autolysate and microelements. Replacement the yeast autolysate and microelement mixture in the composition of ethanol- and n-hexadecane-containing media by copper sulfate and iron sulfate and in the medium with glycerol by KCl, zinc sulfate and copper sulfate accompanied by decreasing antiadhesive properties of synthesized surfactants. The obtained data indicate that the increasing surfactant synthesis does not always the accompanied by the formation of product with the required biological properties and indicate the need for studies depending on biological properties of surfactants of the cultivation conditions of producer.

Control of Saccharomyces cerevisiae pre-tRNA processing by environmental conditions.

tRNA is essential for translation and decoding of the proteome. The yeast proteome responds to stress and tRNA biosynthesis contributes in this response by repression of tRNA transcription and alterations of tRNA modification. Here we report that the stress response also involves processing of pre-tRNA 3′ termini. By a combination of Northern analyses and RNA sequencing, we show that upon shift to elevated temperatures and/or to glycerol-containing medium, aberrant pre-tRNAs accumulate in yeast cells. For pre-tRNAUAUIle and pre-tRNAUUULys these aberrant forms are unprocessed at the 5′ ends, but they possess extended 3′ termini. Sequencing analyses showed that partial 3′ processing precedes 5′ processing for pre-tRNAUAUIle. An aberrant pre-tRNATyr that accumulates also possesses extended 3′ termini, but it is processed at the 5′ terminus. Similar forms of these aberrant pre-tRNAs are detected in the rex1Δ strain that is defective in 3′ exonucleolytic trimming of pre-tRNAs but are absent in the lhp1Δ mutant lacking 3′ end protection. We further show direct correlation between the inhibition of 3′ end processing rate and the stringency of growth conditions. Moreover, under stress conditions Rex1 nuclease seems to be limiting for 3′ end processing, by decreased availability linked to increased protection by Lhp1. Thus, our data document complex 3′ processing that is inhibited by stress in a tRNA-type and condition-specific manner. This stress-responsive tRNA 3′ end maturation process presumably contributes to fine-tune the levels of functional tRNA in budding yeast in response to environmental conditions.

Yeast Interspecies Comparative Proteomics Reveals Divergence in Expression Profiles and Provides Insights into Proteome Resource Allocation and Evolutionary Roles of Gene Duplication

Omics analysis is a versatile approach for understanding the conservation and diversity of molecular systems across multiple taxa. In this study, we compared the proteome expression profiles of four yeast species (Saccharomyces cerevisiae, Saccharomyces mikatae, Kluyveromyces waltii, and Kluyveromyces lactis) grown on glucose- or glycerol-containing media. Conserved expression changes across all species were observed only for a small proportion of all proteins differentially expressed between the two growth conditions. Two Kluyveromyces species, both of which exhibited a high growth rate on glycerol, a nonfermentative carbon source, showed distinct species-specific expression profiles. In K. waltii grown on glycerol, proteins involved in the glyoxylate cycle and gluconeogenesis were expressed in high abundance. In K. lactis grown on glycerol, the expression of glycolytic and ethanol metabolic enzymes was unexpectedly low, whereas proteins involved in cytoplasmic translation, including ribosomal proteins and elongation factors, were highly expressed. These marked differences in the types of predominantly expressed proteins suggest that K. lactis optimizes the balance of proteome resource allocation between metabolism and protein synthesis giving priority to cellular growth. In S. cerevisiae, about 450 duplicate gene pairs were retained after whole-genome duplication. Intriguingly, we found that in the case of duplicates with conserved sequences, the total abundance of proteins encoded by a duplicate pair in S. cerevisiae was similar to that of protein encoded by nonduplicated ortholog in Kluyveromyces yeast. Given the frequency of haploinsufficiency, this observation suggests that conserved duplicate genes, even though minor cases of retained duplicates, do not exhibit a dosage effect in yeast, except for ribosomal proteins. Thus, comparative proteomic analyses across multiple species may reveal not only species-specific characteristics of metabolic processes under nonoptimal culture conditions but also provide valuable insights into intriguing biological principles, including the balance of proteome resource allocation and the role of gene duplication in evolutionary history.

Metabolome analysis reveals the effect of carbon catabolite control on the poly(γ-glutamic acid) biosynthesis of Bacillus licheniformis ATCC 9945

Poly(γ-glutamic acid) (PGA) is a polymer composed of L- and/or D-glutamic acids that is produced by Bacillus sp. Because the polymer has various features as water soluble, edible, non-toxic and so on, it has attracted attention as a candidate for many applications such as foods, cosmetics and so on. However, although it is well known that the intracellular metabolism of Bacillus sp. is mainly regulated by catabolite control, the effect of the catabolite control on the PGA producing Bacillus sp. is largely unknown. This study is the first report of metabolome analysis on the PGA producing Bacillus sp. that reveals the effect of carbon catabolite control on the metabolism of PGA producing Bacillus licheniformis ATCC 9945. Results showed that the cells cultivated in glycerol-containing medium showed higher PGA production than the cells in glucose-containing medium. Furthermore, metabolome analysis revealed that the activators of CcpA and CodY, global regulatory proteins of the intracellular metabolism, accumulated in the cells cultivated in glycerol-containing and glucose-containing medium, respectively, with CodY apparently inhibiting PGA production. Moreover, the cells seemed to produce glutamate from citrate and ammonium using glutamine synthetase/glutamate synthase. Pulsed addition of di-ammonium hydrogen citrate, as suggested by the metabolome result, was able to achieve the highest value so far for PGA production in B. licheniformis.

The effect of pH, aeration, and temperature on arachidonic acid synthesis by Mortierella alpina

The effects of pH, aeration, and temperature on the growth of fungal strain Mortierella alpina LPM-301 and the synthesis of lipids and arachidonic acid in glycerol-containing medium were studied. Arachidonic acid production in the stationary growth phase was found to depend considerably on the pH value; it reached the optimum at pH 6.0 and was irreversibly inhibited at a pH of 3.0. The PO2 values in a range from 10 to 50% showed no marked effect on mycelium growth or the synthesis of lipids and arachidonic acid. The temperature optimum for arachidonic acid production was 20-22 degrees C. Under continuous cultivation, the amount of arachidonic acid reached 29.8% of lipids and 7.4% of biomass. The arachidonic acid yield from the glycerol consumed was 4.1% by mass and 8.8% by energy. It is suggested that arachidonic acid synthesis at an unfavorable pH and elevated temperatures was limited by the activity of A-12-desaturase and by the conversion of linoleic to arachidonic acid, respectively.

The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner.

Production of Poly(3-hydroxyalkanoate)s by Pseudomonas putida Cultivated in a Glycerol/Nonanoic Acid-Containing Medium

The biosynthesis of poly(3-hydroxyalkanoate) (PHA) by Pseudomonas putida (JCM6160) cultivated in a medium containing glycerol, nonanoic acid, or a glycerol/nonanoic acid mixture as the sole carbon sources was investigated. The PHA content was ~20 % when glycerol was the carbon source. This relatively low content can be attributed to the glycerol end-cap effect and the absence of enzymes that can directly synthesize PHA from acetyl CoA, which is the major metabolite of glycerol. Fatty acids, containing even numbered carbons, are synthesized from acetyl CoA, and they can be used as substrates for PHA synthesis. However, this process also results in decreasing PHA content as fatty acids are siphoned off into other pathways. However, addition of 5 mM nonanoic acid into a 20 mM glycerol-containing medium dramatically increased the PHA content in P. putida, which was 1.3 times larger than the sum of the values found when glycerol and nonanoic acid were each used as the sole carbon source. The PHA, synthesized in the glycerol/nonanoic acid medium, contains 3-hydroxy alkanoate units that have 5, 6, 7, 8, 9, or 10 carbons. The units that contain the even numbered carbons are derived from fatty acids that were produced from glycerol; whereas, the PHA units with the odd numbered carbons are derived from nonanoic acid. Pentanoate units were also found in the polyester derived from glycerol and nonanoic acid, and must have been synthesized indirectly via β-oxidation of nonanoic acid with the assistance of glycerol because pentanoate units were not found in PHA when P. putida was cultivated in the presence of only nonanoic acid.

Butanol production from glycerol by recombinant Escherichia coli

Escherichia coli MG1655 (DE3) with the ability to synthesize butanol from glycerol was constructed by metabolic engineering. The genes thil, adhe2, bcs operon (crt, bcd, etfB, etfA, and hbd) were cloned into the plasmid vectors, pETDuet-1 and pACYCDuet-1, then the two resulting plasmids, pACYC-thl-bcs and pET-adhe2, were transferred to E. coli, and the recombinant strain was able to synthesize up to 18.5 mg/L butanol on a glycerol-containing medium. After the glycerol transport protein gene GlpF was expressed, the butanol production was improved to 22.7 mg/L. The competing pathway of byproducts, such as ethanol, succinate, and lactate, was subsequently deleted to improve the 1-butanol production to 97.9 mg/L. Moreover, a NADH regeneration system was introduced into the E. coli, and finally a 154.0 mg/L butanol titer was achieved in a laboratory-scale shake-flask experiment.

Single-Cell Pharmacodynamic Monitoring of Ribosomal Protein S6 Phosphorylation During AC220 Therapy Demonstrates in Vivo FLT3 Inhibition in Circulating Leukemic Blasts and Accurately Detects the Development of AC220 Resistance.

AbstractAbstract 2453 Background:Quizartinib (AC220) is a potent, highly selective inhibitor of FLT3, KIT, and FMS tyrosine kinases with promising clinical activity in AML patients, particularly those with FLT3 internal tandem duplication (FLT3-ITD) mutations. However, limited biochemical FLT3 inhibition in leukemic blasts in vivo by AC220 has been previously described (Perl, et al ASH 2012 #3502). As a biomarker for FLT3 inhibition, we assessed monitoring of phosphorylated ribosomal protein S6 (pS6) at serines 235/236 by flow cytometry. S6 is a downstream target of the PI3 kinase/mammalian target of rapamycin (mTOR) pathway, is constitutively phosphorylated in nearly all FLT3-ITD+ samples, and its phosphorylation shows dynamic changes in response to FLT3 ligand (FL) or FLT3 inhibitors in vitro. We hypothesized that S6 phosphorylation would be a biomarker for FLT3 inhibition and here provide final analysis of AC220 phamacodynamic monitoring at our center. Methods:Serial peripheral blood samples were collected during a phase 2 AC220 clinical trial (Cortes, et al. ASH 2011, #2576). Blood was aliquoted within four hours of collection and a subset was exposed to signaling inhibitors (ex vivo AC220, rapamycin × 30 min.) or activators (phorbol ester/PMA or FL × 10 min.) to establish dynamic controls. Following incubation, samples were formaldehyde-fixed and red cells lysed with the permeabilizing agent triton X-100. Samples were stored at −20C in glycerol-containing medium. After collecting all time points, samples were simultaneously thawed, denatured with ice-cold methanol, and stained for flow cytometry. Blasts were identified by using CD45 and side scatter and confirmed by expression of multiple surface markers (CD33, CD34, CD117, HLA-DR, etc.). Constitutive S6 phosphorylation was defined by comparing unstained (fluorescence minus one, FMO) and PMA and/or cytokine-stimulated cells. Biochemical sensitivity to AC220 was defined as a reduction in the percentage of positive phospho-S6 blasts by >50% as compared to baseline. Results:21 subjects provided whole blood samples (15 FLT3-ITD+, 6 FLT3-WT), 15 had evaluable peripheral blasts (>100 blasts/microliter) for p-S6 monitoring. Only one subject with FLT3-WT had sufficient circulating blasts for analysis. By contrast, 13/15 FLT3-ITD+ subjects’ blasts were evaluable. As previously described by our group and others using flow cytometry, pS6 is heterogeneous in primary AML samples and, at basal state, frequently only demonstrable in a subset of blasts. The mean percentage of blasts demonstrating S6 phosphorylation (pS6+) prior to AC220 therapy was 15% (median 4%, range <0.5–97%). To determine if AC220 inhibits FLT3 kinase in vivo, we serially monitored pS6 in subjects prior to and following their initial dose of AC220. 10/13 subjects in which AC220 reduced or eliminated tumor burden in blood and/or marrow were biochemically sensitive to AC220 by flow. The mean percentage of pS6+ blasts 2 hours following a single dose of AC220 was 3% (median 1%, range <0.5–11%). Samples evaluated at predicted drug peak and trough concentration gave similar reductions in pS6, suggesting the drug inhibits signaling throughout the entire dosing interval. Importantly, all subjects with biochemically-sensitive leukemia cleared their peripheral blasts by day 29. In contrast, in the only subject with de novo clinical resistance to AC220, no reduction of S6 phosphorylation was observed. Re-activation of S6 phosphorylation occurred in 5/5 subjects who relapsed during AC220 and for whom samples were available. 4/5 subjects had treatment-emergent FLT3 mutations (e.g. FLT3-D835) in addition to a demonstrable FLT3-ITD; the fifth showed clonal selection for a FLT3-WT clone. S6 phosphorylation in these relapse samples was not potently suppressed by additional ex vivo AC220 in 3/4, suggesting that the cells developed primary resistance to the drug and that drug failure was not secondary to pharmacokinetic changes over time. Conclusions:We demonstrate the feasibility and utility of flow cytometry for phospho-S6 to monitor the biochemical efficacy of FLT3 inhibitors. The potential of these methods to predict clinical response/resistance paired with the rapid turnaround time of flow cytometry suggests potential future application of this technology in the screening evaluation of patients being considered for therapy with novel signal transduction inhibitors. Disclosures:Perl:Astellas Pharmaceuticals: Consultancy. Carroll:GlaxoSmithKlein: Research Funding.

Screening of environmental samples for bacteria producing 1,3-propanediol from glycerol.

Twenty nine environmental samples were screened for the presence of anaerobic microorganisms fermenting glycerol with 1,3-propanediol as a final product. Seven samples were then selected for the next step of our research and eight bacteria strains were cultured anaerobically. Seven of them produced 1,3-propanediol with a yield of 0.47-0.58. Six of the the isolated microorganisms were then classified as Clostridium butyricum (four strains), C. lituseburense (one strain), and C. sartagoforme (one strain). We suggest that of all these strains C. butyricum 2CR371.5 is the best 1,3-propanediol producer as producing no lactate as a by-product and growing well on a glycerol-containing medium.

Promising directions for utilization of glycerol-containing waste from biodiesel fuel production

The provided examples of utilization of glycerol and glycerol-containing media are far from completely reflecting the diversity of research works dedicated to this problem. Nevertheless, the presented works provide sufficiently convincing evidence on broad prospects for utilization of excess glycerol for obtainment of practically important products in the course of chemical and biotechnological processes.

Screening of Novel Basidiomycetes for the Production of Lignocellulolytic Enzymes During Fermentation of Food Wastes

Nine species of white-rot basidiomycetes isolated from the forests of Georgia have been screened for the endoglucanase, xylanase, laccase, and manganese peroxidase production in both glycerol-containing synthetic medium and mandarin peels-based medium. Irpex lacteus and Trametes ochracea expressed the highest hydrolases activity in submerged and solid-state fermentation (SSF), respectively, of ethanol production residue (EPR). Lenzites betulina appeared to be very promising producer of laccase in both synthetic (52.5 U ml -1 ) and complex (76.7 U ml -1 ) media. The tested white-rot fungi exhibited quite different responses to growth substrates used. Mandarin peelings, ethanol production residue, and wheat bran appeared to be excellent growth substrates for the production of complex preparations of hydrolases and oxidases. The same substrates as well as walnut pericarp enhanced laccase and MnP secretion. In addition, the ratio of individual enzymes in final preparations signifi cantly depended on growth substrate. Thus, the laccase/MnP ratio changed from 3 to 548 with substitution of EPR by walnut pericarp in submerged fermentation by L. betulina. Usually, the submerged fermentation of lignocellulosic materials was preferable for the target enzyme secretion. However, the SSF of wheat bran was essential for the CMCase production by I. lacteus, L. betulina, and S. hirsutum. The SSF of EPR was preferable for the laccase production by G. resinaceum, L. betulina, and T. ochracea while the SSF of wheat bran favored MnP accumulation as compared to submerged cultivation.

Promotion of glycerol utilization using ethanol and 1-propanol in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe does not grow in media containing glycerol as a sole carbon source but uses glycerol in the presence of ethanol. Ethanol, but not glycerol, triggered upregulation of gld1+ and fbp1+ during glucose starvation even though gld1+ and fbp1+ are essential for growth on glycerol. This upregulation occurred at a very low concentration of ethanol. The transcriptional regulation of gld1+ was tested in the presence of various alcohols, and both ethanol and 1-propanol were found to induce gld1+ and to support growth in glycerol-containing media. We suggest that S. pombe has a novel ethanol and/or 1-propanol recognition mechanism that upregulates glycerol utilization during glucose starvation.

Caloramator boliviensis sp. nov., a thermophilic, ethanol-producing bacterium isolated from a hot spring

A novel moderately thermophilic, anaerobic, ethanol-producing bacterial strain, 45B(T), was isolated from a mixed sediment water sample collected from a hot spring at Potosi, Bolivia. The cells were straight to slightly curved rods approximately 2.5 µm long and 0.5 µm wide. The strain was Gram-stain-variable, spore-forming and monotrichously flagellated. Growth of the strain was observed at 45-65 °C and pH 5.5-8.0, with optima of 60 °C and pH 6.5. The substrates utilized by strain 45B(T) were xylose, cellobiose, glucose, arabinose, sucrose, lactose, maltose, fructose, galactose, mannose, glycerol, xylan, carboxymethylcellulose and yeast extract. The main fermentation product from xylose and cellobiose was ethanol (0.70 and 0.45 g ethanol per gram of consumed sugar, respectively). Acetate, lactate, propionate, carbon dioxide and hydrogen were also produced in minor quantities. 1,3-Propanediol was produced when glycerol-containing medium was supplemented with yeast extract. The major cellular fatty acids were anteiso-C(15:0), C(16:0), iso-C(16:0), C(15:1), iso-C(14:0), C(13:0) and C(14:0). The polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an aminoglycolipid and 15 other unidentified lipids were predominant. The DNA G+C content of strain 45B(T) was 32.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence similarity revealed that strain 45B(T) is located within the Gram-type positive Bacillus-Clostridium branch of the phylogenetic tree. On the basis of morphological and physiological properties and phylogenetic analysis, strain 45B(T) represents a novel species, for which the name Caloramator boliviensis sp. nov. is proposed; the type strain is 45B(T) (=DSM 22065(T)=CCUG 57396(T)).

Production of 1,2-Propanediol from Glycerol in Saccharomyces cerevisiae

Glycerol has become an attractive carbon source in the biotechnology industry owing to its low price and reduced state. However, glycerol is rarely used as a carbon source in Saccharomyces cerevisiae because of its low utilization rate. In this study, we used glycerol as a main carbon source in S. cerevisiae to produce 1,2-propanediol. Metabolically engineered S. cerevisiae strains with overexpression of glycerol dissimilation pathway genes, including glycerol kinase (GUT1), glycerol 3-phosphate dehydrogenase (GUT2), glycerol dehydrogenase (gdh), and a glycerol transporter gene (GUP1), showed increased glycerol utilization and growth rate. More significant improvement of glycerol utilization and growth rate was accomplished by introducing 1,2-propanediol pathway genes, mgs (methylglyoxal synthase) and gldA (glycerol dehydrogenase) from Escherichia coli. By engineering both glycerol dissimilation and 1,2-propanediol pathways, the glycerol utilization and growth rate were improved 141% and 77%, respectively, and a 2.19 g 1,2- propanediol/l titer was achieved in 1% (v/v) glycerolcontaining YEPD medium in engineered S. cerevisiae.

Hyper-production of beta-glucosidase and beta-xylosidase by Aspergillus niger NCIM 1207 in xylan-containing media

Aspergillus niger NCIM 1207 produced significantly high levels of β-glucosidase and β-xylosidase activities in submerged fermentation. Cellulose induced only β-glucosidase, while xylan induced both β-glucosidase and β-xylosidase activities. Both the enzymes of this strain were found to undergo catabolite repression in the presence of high concentrations of glucose and glycerol. The sudden drop in pH of the fermentation medium below 3.5 caused the inactivation of enzymes when the fungus was grown in glycerol-containing media at lower temperatures. The growth of the organism at 36 oC led to an increase in pH of the fermentation above 6.0 that affected β-xylosidase activity significantly. Highest levels of β-glucosidase ((19 IU mL-1 or 633 IU g-1 of substrate) and β-xylosidase (18.7 IU/mL-1 or 620 IU g-1 of substrate) activities were detected when A. niger was grown at 30 oC for first five days followed by further incubation at 36 oC. Such a process of growing the organism at lower temperatures (growth phase) followed by producing the enzymes at higher temperatures (production phase) in case of fungal systems has not been reported so far. The zymogram staining of the β-glucosidase demonstrated that A. niger produced only single species of β-glucosidase. We feel that A. niger NCIM 1207 is a potential candidate to produce both β-glucosidase and β-xylosidase in high amounts that can be used to supplement commercial cellulase preparation.

Life with and without AtTIP1;1, an Arabidopsis aquaporin preferentially localized in the apposing tonoplasts of adjacent vacuoles

The Arabidopsis thaliana Tonoplast Intrinsic Protein 1;1 (AtTIP1;1) is a member of the tonoplast aquaporin family. The tissue-specific expression pattern and intracellular localization of AtTIP1;1 were characterized using GUS and GFP fusion genes. Results indicate that AtTIP1;1 is expressed in almost all cell types with the notable exception of meristematic cells. The highest level of AtTIP1;1 expression was detected in vessel-flanking cells in vascular bundles. AtTIP1;1-GFP fusion protein labelled the tonoplast of the central vacuole and other smaller peripheral vacuoles. The fusion protein was not found evenly distributed along the tonoplast continuum but concentrated in contact zones of tonoplasts from adjacent vacuoles and in invaginations of the central vacuole. Such invaginations may result from partially engulfed small vacuoles. A knockout mutant was isolated and characterized to gain insight into AtTIP1;1 function. No phenotypic alteration was found under optimal growth conditions indicating that AtTIP1;1 function is not essential to the plant and that some members of the TIP family may act redundantly to facilitate water flow across the tonoplast. However, a conditional root phenotype was observed when mutant plants were grown on a glycerol-containing medium.

A novel finding thatStreptomyces clavuligeruscan produce the antibiotic clavulanic acid using olive oil as a sole carbon source

This study aims to establish whether commercially available food oils can be used by Streptomyces clavuligerus as sole carbon sources for growth and clavulanic acid production. Batch cultures in bioreactors showed that Strep. clavuligerus growth and clavulanic acid yields in a P-limited medium containing 0.6% (v/v) olive oil were respectively 2.5- and 2.6-fold higher than in a glycerol-containing medium used as control. Glycerol- and olive oil-grown cells present different macromolecular composition, particularly lipid and protein content. Streptomyces clavuligerus uses olive oil as the sole carbon and energy source for growth and clavulanic acid production. Yields and production rates in olive oil are comparable to those reported for oil-containing complex media. Differences in yields and in the macromolecular composition indicate that different metabolic pathways convert substrate into product. This is the first report of oils being used as the sole carbon source by Strep. clavuligerus. Apart from economic benefits, interesting questions are raised about Strep. clavuligerus physiology. Defined culture media allow physiological studies to be performed in the absence of interference by other compounds. Understanding how Strep. clavuligerus catabolises oils may have an economic impact in clavulanic acid production.

Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation

Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O2 limitation (below 1% of saturation). N starvation per se, or in combination with O2 limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O2-limited cells, depending on the time span of the limitation. O2-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O2 limitation hindered the O2-dependent desaturase(s) and favored the O2-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.

Changes in the intracellular Ca 2+ content in human red blood cells in the presence of glycerol

Changes of the intracellular Ca 2+ content in human red blood cells (RBCs) in glycerol-containing solutions and after freeze–thawing the cells with glycerol and subsequent deglycerolization were investigated with the Ca 2+-sensitive fluorescent dye fluo-4 using fluorescence microscopy. In the glycerol-containing solutions the Ca 2+ content increased when compared with a physiological medium (Hepes buffered saline solution (HBSS)). This effect was most likely a result of an inhibition of the Ca 2+ pump. After inhibiting the Ca 2+ pump using o-vanadate, the Ca 2+ uptake was not significantly different in the cells in glycerol-containing and physiological medium. Freeze–thawing and deglycerolization of RBCs resulted in a more pronounced increase in the Ca 2+ content. Also in this case, the Ca 2+ pump seemed to play a major role.