Liquid Batch Culture Research Articles

Genetically encoded multimode reporter of adaptor complex 3 traffic in budding yeast.

AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway's importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

Directing Clostridium ljungdahlii fermentation products via hydrogen to carbon monoxide ratio in syngas

Clostridium ljungdahlii was grown on syngas of varying composition to investigate how syngas blends used in fermentation can be tailored toward desired product formation. Experiments were conducted with liquid batch cultures exposed to the same volume of gas with varying ratios of hydrogen to carbon monoxide (H2/CO). Formation of acetate was favored by higher concentrations of hydrogen in the headspace. The maximum acetate concentration of 35.21 ± 0.84 mM was obtained using an H2/CO ratio of 2.0. Generation of more reduced metabolites was favored by greater carbon monoxide concentrations in the headspace. Maximum ethanol (7.53 ± 0.58 mM) and 2,3-Butanediol (5.20 ± 0.79 mM) concentrations were achieved under an H2/CO ratio of 0.5. Linear relationships were obtained in that describe the effect of the syngas H2/CO ratios on acetate, ethanol, and 2,3-BD formation, respectively.

The Inoculation Method Could Impact the Outcome of Microbiological Experiments.

For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.IMPORTANCE Pure liquid cultures are fundamental to the field of microbiological research. These cultures are normally thought of as homogeneous mixtures of single-cell bacteria; the present study shows that this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcomes of experiments by altering the phenotype of the cultures. The study found a mechanism whereby preformed aggregates are able to recruit surrounding single cells in a form of snowball effect, creating more and larger aggregates in the cultures. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.

Digital Image Analysis of Yeast Single Cells Growing in Two Different Oxygen Concentrations to Analyze the Population Growth and to Assist Individual-Based Modeling.

Nowadays control of the growth of Saccharomyces to obtain biomass or cellular wall components is crucial for specific industrial applications. The general aim of this contribution is to deal with experimental data obtained from yeast cells and from yeast cultures to attempt the integration of the two levels of information, individual and population, to progress in the control of yeast biotechnological processes by means of the overall analysis of this set of experimental data, and to assist in the improvement of an individual-based model, namely, INDISIM-Saccha. Populations of S. cerevisiae growing in liquid batch culture, in aerobic and microaerophilic conditions, were studied. A set of digital images was taken during the population growth, and a protocol for the treatment and analyses of the images obtained was established. The piecewise linear model of Buchanan was adjusted to the temporal evolutions of the yeast populations to determine the kinetic parameters and changes of growth phases. In parallel, for all the yeast cells analyzed, values of direct morphological parameters, such as area, perimeter, major diameter, minor diameter, and derived ones, such as circularity and elongation, were obtained. Graphical and numerical methods from descriptive statistics were applied to these data to characterize the growth phases and the budding state of the yeast cells in both experimental conditions, and inferential statistical methods were used to compare the diverse groups of data achieved. Oxidative metabolism of yeast in a medium with oxygen available and low initial sugar concentration can be taken into account in order to obtain a greater number of cells or larger cells. Morphological parameters were analyzed statistically to identify which were the most useful for the discrimination of the different states, according to budding and/or growth phase, in aerobic and microaerophilic conditions. The use of the experimental data for subsequent modeling work was then discussed and compared to simulation results generated with INDISIM-Saccha, which allowed us to advance in the development of this yeast model, and illustrated the utility of data at different levels of observation and the needs and logic behind the development of a microbial individual-based model.

Plant Materials are Sustainable Substrates Supporting New Technologies of Plant-Only-Based Culture Media for in vitro Culturing of the Plant Microbiota.

In order to improve the culturability and biomass production of rhizobacteria, we previously introduced plant-only-based culture media. We herein attempted to widen the scope of plant materials suitable for the preparation of plant-only-based culture media. We chemically analyzed the refuse of turfgrass, cactus, and clover. They were sufficiently rich to support good in vitro growth by rhizobacteria isolates representing Proteobacteria and Firmicutes. They were also adequate and efficient to produce a cell biomass in liquid batch cultures. These culture media were as sufficient as artificial culture media for the cultivation and recovery of the in situ rhizobacteria of barley (Hordeum murinum L.). Based on culture-dependent (CFU plate counting) and culture-independent analyses (qPCR), mowed turfgrass, in particular, supported the highest culturable population of barley endophytes, representing >16% of the total bacterial number quantified with qPCR. This accurately reflected the endophytic community composition, in terms of diversity indices (S′, H′, and D′) based on PCR-DGGE, and clustered the plant culture media together with the qPCR root populations away from the artificial culture media. Despite the promiscuous nature of the plant materials tested to culture the plant microbiome, our results indicated that plant materials of a homologous nature to the tested host plant, at least at the family level, and/or of the same environment were more likely to be selected. Plant-only-based culture media require further refinements in order to provide selectivity for the in vitro growth of members of the plant microbiome, particularly difficult-to-culture bacteria. This will provide insights into their hidden roles in the environment and support future culturomic studies.

Persistence of antibiotic resistance plasmids in bacterial biofilms.

The emergence and spread of antibiotic resistance is a crisis in health care today. Antibiotic resistance is often horizontally transferred to susceptible bacteria by means of multidrug resistance plasmids that may or may not persist in the absence of antibiotics. Because bacterial pathogens often grow as biofilms, there is a need to better understand the evolution of plasmid persistence in these environments. Here we compared the evolution of plasmid persistence in the pathogen Acinetobacter baumannii when grown under antibiotic selection in biofilms versus well‐mixed liquid cultures. After 4 weeks, clones in which the plasmid was more stably maintained in the absence of antibiotic selection were present in both populations. On average plasmid persistence increased more in liquid batch cultures, but variation in the degree of persistence was greater among biofilm‐derived clones. The results of this study show for the first time that the persistence of MDR plasmids improves in biofilms.

Quorum sensing systems differentially regulate the production of phenazine-1-carboxylic acid in the rhizobacterium Pseudomonas aeruginosa PA1201

Pseudomonas aeruginosa strain PA1201 is a newly identified rhizobacterium that produces high levels of the secondary metabolite phenazine-1-carboxylic acid (PCA), the newly registered biopesticide Shenqinmycin. PCA production in liquid batch cultures utilizing a specialized PCA-promoting medium (PPM) typically occurs after the period of most rapid growth, and production is regulated in a quorum sensing (QS)-dependent manner. PA1201 contains two PCA biosynthetic gene clusters phz1 and phz2; both clusters contribute to PCA production, with phz2 making a greater contribution. PA1201 also contains a complete set of genes for four QS systems (LasI/LasR, RhlI/RhlR, PQS/MvfR, and IQS). By using several methods including gene deletion, the construction of promoter-lacZ fusion reporter strains, and RNA-Seq analysis, this study investigated the effects of the four QS systems on bacterial growth, QS signal production, the expression of phz1 and phz2, and PCA production. The possible mechanisms for the strain- and condition-dependent expression of phz1 and phz2 were discussed, and a schematic model was proposed. These findings provide a basis for further genetic engineering of the QS systems to improve PCA production.

Enhanced Xylitol and Ethanol Yields by Fermentation Inhibitors in Steam-Pretreated Lignocellulosic Biomass

A systematic study was conducted on the effects of specific concentrations of fermentation inhibitors acetic acid, 5-hydroxymethylfurfural (5-HMF), furfurals, and phenolics on the fermentation of xylose to xylitol and hexoses to ethanol by Candida guilliermondii in water-soluble fractions (hydrolysates) from steam-pretreated sugarcane bagasse, hybrid poplar, switchgrass, mixed wood, and giant reed. The influence of different inhibitor concentrations on the growth and fermentation abilities of C. guilliermondii cultivated in lignocellulosic hydrolysates was measured during liquid batch cultures. In the presence of up to8 g/Lof acetic acid along with furfural, 5-HMF, and phenolics (found in hybrid poplar hydrolysate and sugarcane bagasse), the ethanol and xylitol yields increased up to 140% and 88%, respectively, compared to a control containing no inhibitors(100% and 68, respectively). The highest ethanol but lowest xylitol yields compared to controls were measured in hybrid poplar hydrolysate,which was then selected forthesystematic study for production of specific concentrationsof the fermentation inhibitors to enhance ethanol and xylitol yields. For all six steam pretreatment severities tested for hybrid poplar, the presence of inhibitors caused the ethanol yield to be enhanced by as much as 22% compared to the control. However, the xylitol yield was negatively affected by the higher concentrations of inhibitors in the six hybrid poplar hydrolysates. Overall this study showed that fermentation inhibitors are not necessarily harmful compounds. At specific concentrations, acetic acid, furfural, 5-HMF, and phenolics might be called fermentation enhancers rather than inhibitors.

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye.

BackgroundSaccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.MethodsS. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. ResultsThe proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.ConclusionsyECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

Plant-based culture media: Efficiently support culturing rhizobacteria and correctly mirror their in-situ diversity

Our previous publications and the data presented here provide evidences on the ability of plant-based culture media to optimize the cultivability of rhizobacteria and to support their recovery from plant-soil environments. Compared to the tested chemically-synthetic culture media (e.g. nutrient agar and N-deficient combined-carbon sources media), slurry homogenates, crude saps, juices and powders of cactus (Opuntia ficus-indica) and succulent plants (Aloe vera and Aloe arborescens) were rich enough to support growth of rhizobacteria. Representative isolates of Enterobacter spp., Klebsiella spp., Bacillus spp. and Azospirillum spp. exhibited good growth on agar plates of such plant-based culture media. Cell growth and biomass production in liquid batch cultures were comparable to those reported with the synthetic culture media. In addition, the tested plant-based culture media efficiently recovered populations of rhizobacteria associated to plant roots. Culturable populations of >106–108cfug−1 were recovered from the ecto- and endo-rhizospheres of tested host plants. More than 100 endophytic culture-dependent isolates were secured and subjected to morphophysiological identification. Factor and cluster analyses indicated the unique community structure, on species, genera, class and phyla levels, of the culturable population recovered with plant-based culture media, being distinct from that obtained with the chemically-synthetic culture media. Proteobacteria were the dominant (78.8%) on plant-based agar culture medium compared to only 31% on nutrient agar, while Firmicutes prevailed on nutrient agar (69%) compared to the plant-based agar culture media (18.2%). Bacteroidetes, represented by Chryseobacterium indologenes, was only reported (3%) among the culturable rhizobacteria community of the plant-based agar culture medium.

English

Fructooligosaccharides are gaining importance by leaps and bounce every day due to their low caloric value and accompanying health benefits for the consumer. Hence, newer potential sources for the fructosyltransferase (Ftase) enzyme are thoroughly scrutinized. The present work was carried out for preliminary and secondary screening of the microbial isolates for determination of the Ftase producing potential. Three classes of microorganisms’ viz. molds, yeasts and bacterial isolates were qualitatively screened. The preliminary screening revealed molds as the most potent group exhibiting greater zone of hydrolysis in the range of 0.30 ± 0.10 to 2.58 ± 0.10 cm. The secondary screening of 20 selected mold isolates was performed in liquid batch culture. Four isolates identified as Aspergillus niger, Aspergillus flavus, Aspergillus stallus and Aspergillus versicolor exhibited higher fructosyltransferase : invertase ratio which is most critical for the synthesis of fructooligosaccharides due to higher transferase activity and low hydrolytic activity. A. niger exhibited highest Ftase activity, 36.88 ± 0.23 IU/mg. A. flavus produced 21.45 ± 0.33 IU/mg of Ftase. A. stallus produced 18.09 ± 0.14 IU/ mg and A. versicolor was found to produce 23.78 ± 0.12 IU/mg of Ftase without any cultural medium optimization. Key words: Prelimnary screening, secondary screening, fructosyltransferase, fructooligosaccharides.

Biodegradation of chloro- and bromobenzoic acids: Effect of milieu conditions and microbial community analysis

Monohalogenated benzoic acids often appear in industrial wastewaters where biodegradation can be hampered by complex mixtures of pollutants and prevailing extreme milieu conditions. In this study, the biodegradation of chlorinated and brominated benzoic acids was conducted at a pH range of 5.0–9.0, at elevated salt concentrations and with pollutant mixtures including fluorinated and iodinated compounds. In mixtures of the isomers, the degradation order was primarily 4-substituted followed by 3-substituted and then 2-substituted halogenated benzoic acids. If the pH and salt concentration were altered simultaneously, long adaptation periods were required. Community analyses were conducted in liquid batch cultures and after immobilization on sand columns. The Alphaproteobacteria represented an important fraction in all of the enrichment cultures. On the genus level, Afipia sp. was detected most frequently. In particular, Bacteroidetes were detected in high numbers with chlorinated benzoic acids.

Tolerance of Chemoorganotrophic Bioleaching Microorganisms to Heavy Metal and Alkaline Stresses

The bioleaching potential of the bacterium Bacillus mucilaginosus and the fungus Aspergillus niger towards industrial residues was investigated by assessing their response towards various heavy metals (including arsenic, cadmium, cobalt, chromium, nickel, lead, and zinc) and elevated pH. The plate diffusion method was performed for each metal to determine the toxicity effect. Liquid batch cultures were set up for more quantitative evaluation as well as for studying the influence of basicity. Growth curves were prepared using bacterial/fungal growth counting techniques such as plate counting, optical density measurement, and dry biomass determination. Cadmium, nickel, and arsenite had a negative influence on the growth of B. mucilaginosus, whereas A. niger was sensitive to cadmium and arsenate. However, it was shown that growth recovered when microorganisms cultured in the presence of these metals were inoculated onto metal-free medium. Based on the findings of the bacteriostatic/fungistatic effect of the metals and the adaptability of the microorganisms to fairly elevated pH values, it is concluded that both strains have potential applicability for further research concerning bioleaching of alkaline waste materials.

Osmoadaptation Strategy of the Most Halophilic Fungus, Wallemia ichthyophaga, Growing Optimally at Salinities above 15% NaCl

Wallemia ichthyophaga is a fungus from the ancient basidiomycetous genus Wallemia (Wallemiales, Wallemiomycetes) that grows only at salinities between 10% (wt/vol) NaCl and saturated NaCl solution. This obligate halophily is unique among fungi. The main goal of this study was to determine the optimal salinity range for growth of the halophilic W. ichthyophaga and to unravel its osmoadaptation strategy. Our results showed that growth on solid growth media was extremely slow and resulted in small colonies. On the other hand, in the liquid batch cultures, the specific growth rates of W. ichthyophaga were higher, and the biomass production increased with increasing salinities. The optimum salinity range for growth of W. ichthyophaga was between 15 and 20% (wt/vol) NaCl. At 10% NaCl, the biomass production and the growth rate were by far the lowest among all tested salinities. Furthermore, the cell wall content in the dry biomass was extremely high at salinities above 10%. Our results also showed that glycerol was the major osmotically regulated solute, since its accumulation increased with salinity and was diminished by hypo-osmotic shock. Besides glycerol, smaller amounts of arabitol and trace amounts of mannitol were also detected. In addition, W. ichthyophaga maintained relatively small intracellular amounts of potassium and sodium at constant salinities, but during hyperosmotic shock, the amounts of both cations increased significantly. Given our results and the recent availability of the genome sequence, W. ichthyophaga should become well established as a novel model organism for studies of halophily in eukaryotes.

Effect of Different Carbon Sources on Decolourisation of an Industrial Textile Dye Under Alkaline–Saline Conditions

White-rot fungal strains of Trametes versicolor and Phanerochaete chrysosporium were selected to study the decolourisation of the textile dye, Reactive Black 5, under alkaline-saline conditions. Free and immobilised T. versicolor cells showed 100% decolourisation in the growth medium supplemented with 15gl(-1) NaCl, pH 9.5 at 30°C in liquid batch culture. Continuous culture experiments were performed in a fixed-bed reactor using free and immobilised T. versicolor cells and allowed 85-100% dye decolourisation. The immobilisation conditions for the biomass and the additional supply of carbon sources improved the decolourisation performance during a long-term trial of 40days. Lignin peroxidase, laccase and glyoxal oxidase activities were detected during the experiments. The laccase activity varied depending on carbon source utilized and glycerol-enhanced laccase activity compared to sucrose during extended growth.

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 II: production of xylitol and ethanol in the presence of inhibitors

A systematic study was conducted characterizing the effect of furfural, 5-hydroxymethylfurfural (5-HMF), and acetic acid concentration on the production of xylitol and ethanol by a novel endophytic yeast, Rhodotorula mucilaginosa strain PTD3. The influence of different inhibitor concentrations on the growth and fermentation abilities of PTD3 cultivated in synthetic nutrient media containing 30 g/l xylose or glucose were measured during liquid batch cultures. Concentrations of up to 5 g/l of furfural stimulated production of xylitol to 77 % of theoretical yield (10 % higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in the presence of 5-HMF at concentrations of up to 3 g/l. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99 % of theoretical yield (15 % higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5 g/l of furfural, 5-HMF, and acetic acid, respectively. This yeast would be a potent candidate for the bioconversion of lignocellulosic sugars to biochemicals given that in the presence of low concentrations of inhibitors, its xylitol and ethanol yields are stimulated, and it is capable of metabolizing pretreatment degradation products.

Production of the 14C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce (14)C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-(14)C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L(-1). Concentrations of 12.6 g (14)C-labeled glycerol L(-1) (1 % v/v) resulted in the production of 17.1 mg (14)C-Cry1Ab L(-1) cultivation medium. (14)C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total (14)C originated from Cry1Ab. In the presence of 2.04 MBq (14)C-labeled carbon sources, a specific activity of up to 268 Bq mg(-1) (14)C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that (14)C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple (14)C-labeled carbon source. It also provides a general strategy to produce (14)C-labeled proteins useful for soil metabolic studies.

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 I: production of xylitol and ethanol

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.

Developmental profile of storage reserve accumulation in soybean somatic embryos

Soybean somatic embryos were developed as a model for investigating the developmental relationships of lipid biosynthesis and accumulation in this important crop. Batch cultures of embryos grown for 8 wk in liquid culture medium exhibited typical sigmoidal growth kinetics as they passed through characteristic globular, heart, torpedo, and cotyledon stages. Exponential growth occurred for the first 4 wk in culture with net growth terminating when total embryo fresh weight per culture flask reached a maximum of 4–4.5 g at 6 wk. This was followed by a slight decrease in embryo fresh weight (FW) and the onset of apparent tissue senescence as judged by yellowing and browning of embryos. On a FW basis, embryos accumulated up to 4% protein, 2.5% soluble sugars, 1.9% starch, and 1.5% lipid relatively early in development. Levels decreased to 0.8% protein, 0.5% soluble sugars, 0.03% starch, and 0.09% lipid at the end of the culture period. On a mass percent basis, lipid extracts were comprised of approximately 80–90% polar lipid early in embryo development. This shifted to 56% storage lipid (triacylglycerol) and 44% polar lipid after 4–5 wk in culture and then reverted back to 91% and 9% polar vs. storage lipid, respectively, by the end of the 8-wk culture period. On the average, polar and storage lipid fractions were comprised of 24% palmitic acid, 7% stearic acid, 8% oleic acid, 36% linoleic acid, and 26% linolenic acid. However, the amounts of linoleic and linolenic acids declined sharply during embryo senescence at the end of the culture period, with corresponding increases in the combined amounts of palmitic and stearic acids. This is the first report that documents the progress of storage reserve accumulation in soybean somatic embryos in relation to their continuous growth in liquid batch cultures.

Fusarium oxysporum degradation and detoxification of a new textile-glycoconjugate azo dye (GAD)

Degradation and detoxification of textile dyes are of interest due to the huge environmental impact of such chemicals. An isolate of Fusarium oxysporum was used to degrade and to detoxify a new chemical class of textile dyes called Glycoconjugate Azo Dye (GAD). After 6d of growth in a liquid batch culture, the fungus degraded the dye and the culture medium at the end of incubation period showed a ˜100% detoxification compared to the initial dye solution. Increasing the initial fungal inoculum, the dye was totally decolourized after 24h of incubation. The degradation ability was found to be common among various isolates of F. oxysporum suggesting this as a specific trait of this species. Degrading rate was enhanced in concomitancy to the glucose depletion and the beginning of the stationary phase of growth, suggesting that the shift from the primary to the secondary metabolism may be the trigger of the degradation pathway. The Daphnia magna acute toxicity test demonstrated a strong detoxification of GAD-4 by F. oxysporum, resulting in non-toxic metabolite production. Fusarium oxysporum could, therefore, be taken into consideration to develop new remediation strategies of textile effluents.