Effects of sugars and growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth of <Emphasis Type="Italic">Dactylorhiza</Emphasis> species

The influence of sugars and growth regulators on shoot and root growth of Dactylorhiza species was studied under in vitro conditions. The seedling development was stimulated with the application of glucose and sucrose at concentration of 10 g dm⁻³ each. The improvement of shoot growth rate and shoot length was enhanced by cytokinins N ⁶-(2-isopentenyl)adenine or N ⁶-benzyladenine and their combination with auxin indolebutyric acid (IBA). The root growth rate and root length of seedlings increased in the presence of IBA and α-naphthaleneacetic acid. Individual Dactylorhiza species showed statistically significant differences in shoot and root development depending on sugar and growth regulator combinations.     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Relationship between glucose catabolism and xanthan production in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Two genes involved in central carbon metabolism were inactivated to modulate intracellular glucose 6-phosphate and to evaluate its effects on xanthan production in wild-type Xanthomonas oryzae pv. oryzae. Upon the inactivation of the phosphogluconate dehydratase gene (edd), intracellular glucose 6-phosphate increased from 0.05 to 1.17 mmol/g (dry cell wt). This was accompanied by increased xanthan production of up to 2.55 g/l (culture medium). In contrast, inactivation of 6-phosphogluconate dehydrogenase gene (gndA) did not influence intracellular glucose 6-phosphate nor xanthan production. The intracellular availability of glucose 6-phosphate is proposed as a rate-limiting factor in xanthan production, and it may be possible to increases production of xanthan by modulating the activities of enzymes in central carbon metabolism.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-alanine by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli W was genetically engineered to produce l-alanine as the primary fermentation product from sugars by replacing the native d-lactate dehydrogenase of E. coli SZ194 with alanine dehydrogenase from Geobacillus stearothermophilus. As a result, the heterologous alanine dehydrogenase gene was integrated under the regulation of the native d-lactate dehydrogenase (ldhA) promoter. This homologous promoter is growth-regulated and provides high levels of expression during anaerobic fermentation. Strain XZ111 accumulated alanine as the primary product during glucose fermentation. The methylglyoxal synthase gene (mgsA) was deleted to eliminate low levels of lactate and improve growth, and the catabolic alanine racemase gene (dadX) was deleted to minimize conversion of l-alanine to d-alanine. In these strains, reduced nicotinamide adenine dinucleotide oxidation during alanine biosynthesis is obligately linked to adenosine triphosphate production and cell growth. This linkage provided a basis for metabolic evolution where selection for improvements in growth coselected for increased glycolytic flux and alanine production. The resulting strain, XZ132, produced 1,279 mmol alanine from 120 g l⁻¹ glucose within 48 h during batch fermentation in the mineral salts medium. The alanine yield was 95% on a weight basis (g g⁻¹ glucose) with a chiral purity greater than 99.5% l-alanine. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning,expression, and characterization of xylose reductase with higher activity from <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Xylose reductase (XR) is a key enzyme in xylose metabolism because it catalyzes the reduction of xylose to xylitol. In order to study the characteristics of XR from Candida tropicalis SCTCC 300249, its XR gene (xyll) was cloned and expressed in Escherichia coli BL21 (DE3). The fusion protein was purified effectively by Ni²⁺-chelating chromatography, and the kinetics of the recombinant XR was investigated. The Km values of the C. tropicalis XR for NADPH and NADH were 45.5 μM and 161.9 μM, respectively, which demonstrated that this XR had dual coenzyme specificity. Moreover, this XR showed the highest catalytic efficiency (kcat=1.44×l0⁴ min⁻¹) for xylose among the characterized aldose reductases. Batch fermentation was performed with Saccharomyces serivisiae W303-lA:pYES2XR, and resulted in 7.63 g/L cell mass, 93.67 g/L xylitol, and 2.34 g/L · h xylitol productivity. This XR coupled with its dual coenzyme specificity, high activity, and catalytic efficiency proved its utility in in vitro xylitol production.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

The effect of CreA in glucose and xylose catabolism in<Emphasis Type="Italic"> Aspergillus nidulans</Emphasis>

The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivations on single carbon sources, it was demonstrated that xylose acted as a carbon catabolite repressor (xylose cultivations), while the enzymes in the xylose utilisation pathway were also subject to repression in the presence of glucose (glucose cultivations). In the wild type strain growing on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression had been relieved. Xylose was both a repressor and an inducer of xylanases at the same time. The creA mutation seemed to have pleiotropic effects on carbohydratases and carbon catabolism.     

Decolourisation and dephenolisation potential of selected <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Nigri</Emphasis> strains – <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> in olive mill wastewater

Aspergillus section Nigri strains Aspergillus aculeatus Ege-K 258, A. foeditus var. pallidus Ege-K156, A. niger Ege-K 4 and A. tubingensis Ege-K 265 were used to treat olive mill wastewater (OMW) in an investigation aimed at exploring their dephenolisation and decolourisation ability and, consequently, the economic feasibility of using any or all of these strains in a pre-treatment step in the processing of OMW. Of these strains A. tubingensis Ege-K 265 resulted in an 80% decolourisation of twofold-diluted OMW and a 30% decolourisation of undiluted OMW; in addition, it was able to remove approximately 30% of all phenolic compounds in both twofold-diluted and undiluted OMW. We conclude that A. tubingensis Ege-K 265 could be effectively used in the pre-treatment step of a combined aerobic-anaerobic process to solve the environmental problems caused by OMW in Mediterranean countries.     

<Emphasis Type="SmallCaps">l</Emphasis>(+)-Lactic acid production by co-fermentation of glucose and xylose with <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> obtained by low-energy ion beam irradiation

Low-energy ion beam irradiation (10–200 keV) has been proved to have a wide range of biological effects in recent years. When Rhizopus oryzae PW352 was irradiated with a 15-keV low-energy ion beam an l(+)-lactic acid high-yield mutant, RQ4015, was obtained. When 150 g/l glucose was used as the sole carbon source, l(+)-lactic acid of RQ4015 reached 121 g/l after 36 h shake-flask cultivation. However, the highest lactic acid concentration 74 g/l was obtained when 100 g/l xylose was present in the medium as the sole carbon source. When mixed xylose (25 g/l) and glucose (75 g/l) were present in a bubble column, l(+)-lactic acid production of RQ4015 reached 83 g. A high mutation rate and a wide mutation spectrum of low-energy ion implantation were observed in the experiment, suggesting that ion implantation can be a highly efficient mutagenic means for microorganism breeding in many commercial applications.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

The gene <Emphasis Type="Italic">bap</Emphasis>, involved in biofilm production,is present in <Emphasis Type="Italic">Staphylococcus</Emphasis> spp. strains from nosocomial infections

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Potential long-term storage of the predatory mite <Emphasis Type="Italic">Phytoseiulus</Emphasis><Emphasis Type="Italic">persimilis</Emphasis>

Increasing the ability to store mass-reared natural enemies during periods or seasons of low demand is a critical need of the biocontrol industry. We tested the hypothesis that chemicals can enhance long-term cold storage of a predatory mite Phytoseiulus persimilis Athias-Henriot. The research objective was to determine the effect of cryoprotectant and carbohydrate chemicals on in-storage survival of predators. In-storage survival at 8°C was greater for predators sprayed with glycerol (5%, v/v) or glucose (10% and 20%, v/v) than with water spray controls. After 74 days in the cryoprotectant experiment, predator survival declined to 11.5% in the 5% glycerol treatment and 7.8% in the water spray control. After 88 days in the carbohydrate experiment, predator survival declined to 22% in the 20% glucose treatment and 2% in the water spray control. Although many individuals expired within 50 days in both experiments, a few females survived more than 200 days. This research suggests that select cryoprotectants and carbohydrates have a limited capacity to facilitate long-term storage of P. persimilis.     

The incidence of mistletoe (<Emphasis Type="Italic">Viscum album</Emphasis> ssp. <Emphasis Type="Italic">abietis</Emphasis>) on silver fir (<Emphasis Type="Italic">Abies alba</Emphasis>) in Croatia

The research on incidence of mistletoe (Viscum album ssp. abietis) on silver fir (Abies alba) was carried out in natural fir stands in Croatia. In the area of Gorski Kotar 32.8 % of the examined dominant fir trees were infected with mistletoe. The mistletoe incidence was presented according to the damage degrees of silver fir from 0 (healthy trees) to 4 (dead trees), rated by the crown defoliation and needle discoloration. With the increase of incidence, mistletoe spreads more onto more vital, i.e. less damaged trees. In the Dinaric Alps 28.6% of the examined fir trees were infected, this percentage amounting to 27.1% for the mountainous regions between the Sava and Drava rivers. The site and stand parameters (exposure, elevation, site quality, forest community and crown closure) were analysed in order to establish whether there was a correlation between these parameters and mistletoe incidence. There was a negative correlation between the elevation and mistletoe incidence. Among the compartments with a closed stands there were considerably less compartments with higher mistletoe incidence than among compartments with a sparsely closed and understocked stands. The other analysed site and stand parameters individually had no significant influence on mistletoe incidence. The correlation of silver fir mortality in 2004 and mistletoe incidence in 2002/03 was analysed, and their strong correlation was established. Mistletoe could be considered as a bioindicator of silver fir decline, and probably a significant contributor to that decline. In the areas where mistletoe incidence is great it can be presumed that silver fir is significantly damaged.     

Xylitol production from xylose mother liquor: a novel strategy that combines the use of recombinant <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Candida maltosa</Emphasis>

Background  

Xylose mother liquor has high concentrations of xylose (35%-40%) as well as other sugars such as L-arabinose (10%-15%), galactose (8%-10%), glucose (8%-10%), and other minor sugars. Due to the complexity of this mother liquor, further isolation of xylose by simple method is not possible. In China, more than 50,000 metric tons of xylose mother liquor was produced in 2009, and the management of sugars like xylose that present in the low-cost liquor is a problem.     

A new name in <Emphasis Type="Italic">Pavetta</Emphasis> L. (<Emphasis Type="Italic">Rubiaceae</Emphasis>)

Summary  The name Pavetta modesta (Hiern) S. E. Dawson is a later homonym of P. modesta Bremek. Pavetta crystalensis is proposed as a new name.     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.