<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis>: effect of oxygen on growth and acetic acid production

The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1). In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.     

Identification of a second <Emphasis Type="Italic">PAD1</Emphasis> in <Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> LAMAP2480

Volatile phenols are aromatic compounds produced by some yeasts of the genus Brettanomyces as defense against the toxicity of hydroxycinnamic acids (p-coumaric acid, ferulic acid and caffeic acid). The origin of these compounds in winemaking involves the sequential action of two enzymes: coumarate decarboxylase and vinylphenol reductase. The first one converts hydroxycinnamic acids into hydroxystyrenes, which are then reduced to ethyl derivatives by vinylphenol reductase. Volatile phenols derived from p-coumaric acid (4-vinylphenol and 4-ethylphenol) have been described as the major contributors to self-defeating aromas associated with stable, gouache, wet mouse, etc., which generates large economic losses in the wine industry. The gene responsible for the production of 4-vinylphenol from p-coumaric acid has been identified as PAD1, which encodes a phenylacrylic acid decarboxylase. PAD1 has been described for many species, among them Candida albicans, Candida dubliniensis, Debaryomyces hansenii and Pichia anomala. In Brettanomyces bruxellensis LAMAP2480, a 666 bp reading frame (DbPAD) encodes a coumarate decarboxylase. Recent studies have reported the existence of a new reading frame belonging to DbPAD called DbPAD2 of 531 bp, which could encode a protein with similar enzymatic activity to PAD1. The present study confirmed that the transformation of Saccharomyces cerevisiae strain BY4722 with reading frame DbPAD2 under the control of the B. bruxellensis ACT1 promoter, encodes an enzyme with coumarate decarboxylase activity. This work has provided deeper insight into the origin of aroma defects in wine due to contamination by Brettanomyces spp.     

<Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> yeasts: impact on wine and winemaking

Yeasts belonging to the Brettanomyces/Dekkera genus are non-conventional yeasts, which affect winemaking by causing wine spoilage all over the world. This mini-review focuses on recent results concerning the presence of Brettanomyces bruxellensis throughout the wine processing chain. Here, culture-dependent and independent methods to detect this yeast on grapes and at the very early stage of wine production are encompassed. Chemical, physical and biological tools, devised for the prevention and control of such a detrimental species during winemaking are also presented. Finally, the mini-review identifies future research areas relevant to the improvement of wine safety and sensory profiles.     

Interaction effects of lactic acid and acetic acid at different temperatures on ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in corn mash

The combined effects of lactic acid and acetic acid on ethanol production by S. cerevisiae in corn mash, as influenced by temperature, were examined. Duplicate full factorial experiments (three lactic acid concentrations × three acetic acid concentrations) were performed to evaluate the interaction between lactic and acetic acids on the ethanol production of yeast at each of the three temperatures, 30, 34, and 37°C. Corn mash at 30% dry solids adjusted to pH 4 after lactic and acetic acid addition was used as the substrate. Ethanol production rates and final ethanol concentrations decreased (P<0.001) progressively as the concentration of combined lactic and acetic acids in the corn mash increased and the temperature was raised from 30 to 37°C. At 30°C, essentially no ethanol was produced after 96 h when 0.5% w/v acetic acid was present in the mash (with 0.5, 2, and 4% w/v lactic acid). At 34 and 37°C, the final concentrations of ethanol produced by the yeast were noticeably reduced by the presence of 0.3% w/v acetic acid and ≥2% w/v lactic acid. It can be concluded that, as in previous studies with defined media, lactic acid and acetic acid act synergistically to reduce ethanol production by yeast in corn mash. In addition, the inhibitory effects of combined lactic and acetic acid in corn mash were more apparent at elevated temperatures.     

Screening of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine strains for the production of acetic acid

In this study 80 wine strains of Saccharomyces cerevisiae were characterized for the production of acetic acid. A significant variability in the production levels was determined among the strains, which produced from a few mg/l to more than 1 g/l. Fifteen strains, differing in acetic acid production, were tested in fermentation of grape musts of different varieties (Aglianico Basilicata, Aglianico Apulia, Cannonau, Bombino nero, Nero d'Avola, Vermentino, Fiano). The results emphasized a great strain variability, but the best strain behaviour was strictly related to the must composition, which is a determinant factor on the expression of the best strain potentiality. Therefore, this study, confirming the high/low production of acetic acid as a strain characteristic, demonstrated also that the inoculated fermentation becomes more advantageous when the starter culture is chosen in relation to the interaction of yeast strain/vine variety.     

The fate of linoleic acid on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> metabolism under aerobic and anaerobic conditions

Introduction

Saccharomyces cerevisiae has been widely used for fermenting food and beverages for over thousands years. Its metabolism together with the substrate composition play an important role in determining the characteristics of the final fermented products. We previously showed that the polyunsaturated fatty acid, linoleic acid, which is present in the grape juice at trace levels, significantly affected the development of aroma compounds of the wines. However, the effect of linoleic acid on the overall cell metabolism of S. cerevisiae is still not clear. Therefore, we aimed to unlock the metabolic response of S. cerevisiae to linoleic acid using metabolomics and isotope labelling experiments.

Methods

We cultured the cells on a minimal mineral medium supplementing them with linoleic acid isomers and 13C-linoleic acid. Both intracellular and extracellular metabolite profiles were determined using gas chromatography coupled to mass spectrometry (GC–MS) to investigate which S. cerevisiae pathways were affected by linoleic acid supplementation.

Results

The utilisation of linoleic acid by S. cerevisiae had a significant impact on the primary carbon metabolism increasing the glucose consumption and the ethanol production under anaerobic condition. The energetic state of the cell was, therefore, affected and the glycolytic pathway, the TCA cycle and the amino acid production were up-regulated. We also observed that linoleic acid was transported into the cell and converted into other fatty acids affecting their profile even under anaerobic condition.

Conclusion

Our data clearly shows that linoleic acid supplementation in growth medium increased glucose consumption and ethanol production by S. cerevisiae under anaerobic condition. We also suggest that S. cerevisiae might be able to perform an alternative anaerobic pathway to β-oxidation, which has not been reported yet.

     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Fermentation and growth kinetic study of <Emphasis Type="Italic">Aeromonas caviae</Emphasis> under anaerobic conditions

Although Aeromonas caviae is pathogenic to a broad range of invertebrates including human, frequent in aquatic environments, and potentially vital for acidogenesis in anaerobic digestion, virtually no biokinetic information on its anaerobic growth is at hand. Therefore, this study focused on evaluating its anaerobic growth kinetics on glucose. To provide a set of relevant biokinetic coefficients for modeling, a combination of curve fitting and numerical modeling was used. Microcultivations were performed at eight different initial glucose concentrations of 0.1 to 2.5 g l⁻¹ to establish a function of specific growth rate versus substrate concentration. A batch anaerobic bioreactor was then operated to collect a data set for the numerical analysis. Kinetic coefficients were estimated from three different biomass growth profiles monitored by optical density, volatile suspended solids (VSS), or DNA measurement, and applied for simulating continuous operations at various hydraulic retention times (HRTs). Assuming the influent glucose concentration is 5,000 mg l⁻¹, the substrate utilization efficiency predicted to be 77.2% to 92.0% at 17 to 36 h HRTs. For the VSS-model-based simulation, the washout HRT was estimated to be 16.6 h, and similar for the other models. Overall, the anaerobic biokinetic coefficients of A. caviae grown on glucose were successfully estimated and found to follow a substrate inhibition model.     

An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Modeling of growth kinetics for <Emphasis Type="Italic">Pseudomonas putida</Emphasis> during toluene degradation

Glucose has been often used as a secondary substrate to enhance the degradation of primary substrate as well as the increase of biomass, especially for the inhibitory range of substrate concentration. In this study, we investigated the effect of glucose concentration on growth kinetics of Pseudomonas putida during toluene degradation for a wide concentration range (60–250 mg/l). Batch microcosm studies were conducted in order to monitor bacterial growth for three different initial concentrations (2, 5, 10 mg/ml) of glucose for a given toluene concentration. Modeling of growth kinetics was also performed for each growth curve of glucose dose using both Monod and Haldane kinetics. Batch studies revealed that bacterial growth showed a distinct inhibitory phase above some limit (∼170 mg/l) for the lowest (2 mg/ml) glucose dose, but the degree of inhibition decreased as the glucose dose increased, leading to three different growth patterns. The bacterial growth followed each of the modified Wayman and Tseng, Wayman and Tseng, and Luong model as the glucose dose increased from 2 to 10 mg/ml. This indicates that glucose has a prominent influence on bacterial growth during toluene degradation and that different kinetics should be adopted for each broth condition.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Selective loss of <Emphasis Type="Italic">lin</Emphasis> genes from hexachlorocyclohexane-degrading <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ITRC-5 under different growth conditions

The chlorinated insecticide γ-hexachlorocyclohexane (γ-HCH) is sequentially metabolized by the products of linA, linB, linC, linD, linE, and linF genes to β-ketoadipate, which is subsequently mineralized. Two or more copies of these genes are present in the bacterium Pseudomonas aeruginosa ITRC-5 that was isolated earlier by selective enrichment on technical-HCH. At least one copy of linA, linB, linC, linD, and possibly linE is lost from ITRC-5 upon its growth on γ-HCH. All the lin genes, however, are lost when the bacterium was grown in Luria–Bertani (LB) medium. The loss of lin genes is accompanied with the loss/rearrangement of insertion sequence IS6100 genes. Concomitant to the loss of lin genes, the degradation of HCH-isomers by “γ-HCH grown cells” is slower, when compared with “technical-HCH grown cells”, and is completely lost by “LB-grown cells”. The selective loss of lin genes during different growth conditions has not been reported before and is expected to help in understanding the dynamism of degradative genes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Biotechnological production of <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid and its application

Glycerol is currently produced in large amounts as a by-product during fat splitting and biodiesel fuel production. Over the past decade, both chemical and biotechnological processes to convert glycerol to value-added chemicals have been increasingly explored. This mini-review provides recent information about the biotechnological production of a glycerol derivative, d-glyceric acid (d-GA), and its possible applications. Little is known about GA as a bioproduct, but it is naturally found in different kinds of plants as a phytochemical constituent and is reported to have some biological activity. A racemic mixture of dl-GA can be obtained from glycerol via chemical oxidation; however, d-GA is mainly biotechnologically produced with the aid of bacteria. Under aerobic conditions, some acetic acid bacteria transform glycerol into d-GA, and optimization of initial glycerol concentration and aeration rate provided a yield of more than 80 g/l d-GA, using a strain of Gluconobacter frateurii.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Mediated electrochemical measurement of the inhibitory effects of furfural and acetic acid on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Candida shehatae</Emphasis>

The toxic effects of furfural and acetic acid on two yeasts, Saccharomyces cerevisiae and Candida shehatae, were evaluated using an electrochemical method. Intracellular redox activities were lowered by 40% and 78% for S. cerevisiae and C. shehatae, respectively, by 8 g furfural l^–1, and by 46% and 67%, respectively, by 8 g acetic acid l^–1. The proposed method can accurately measure the effects of inhibitors on cell cultures.Revisions requested 27 September 2004/17 November 2004; Revisions received 15 November 2004/10 December 2004     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.