Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

Palladium(II) Pyridinecarboxaldimine Complexes Derived from Unsaturated Amines

Pyridinecarboxaldimines (N–N′) derived from pyridin-2-ylcarboxaldehydes and unsaturated amines add to [PdCl₂(coe)]₂ (coe = cis-cyclooctene) to give complexes of the type PdCl₂(N–N′) in moderate yields. The palladium complexes have been investigated as substrates for hydroboration reactions and as antifungal agents against Aspergillus niger, A. flavus, Candida albicans, and Saccharomyces cerevisiae.     

Synthesis,characterization, and antimicrobial properties of two Cu(II) complexes derived from a benzimidazole ligand

Two copper(II) complexes, [Cu(L)₂](ClO₄)₂] and [Cu(L)(bipy)](ClO₄)₂, were prepared and characterized by the spectroscopic and analytic methods, where L is N-butylbenzimidazole and bipy is 2,2′-bipyridine. Single crystals of [Cu(L)(bipy)](ClO₄)₂ suitable for X-ray diffraction study were obtained by slow diffusion of diethyl ether into a DMF solution of the complex and the complex was found to crystallize as [Cu(L)(bipy)](ClO₄)₂·DMF. The asymmetric unit contains one [Cu(L)(bipy)]²⁺, two uncoordinated perchlorates, and one DMF solvate. Coordination geometry around Cu(II) is distorted square pyramidal with τ value of 0.31. Thermal properties of the complexes were examined by thermogravimetric analysis, indicating that the complexes are thermally stable to 310?°C. The metal complexes were screened for their in vitro antibacterial and antifungal activities Bacillus subtilis and Bacillus cereus (as Gram(+) bacteria), Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae (as Gram(–) bacteria), and Saccharomyces cerevisiae, Candida utilis, and Candida albicans (as yeasts). The complexes show antibacterial and antifungal activities against bacteria and yeasts.     

Dilute acid pretreatment of softwoods

Selective thinning of forests in the western United States will generate a large, sustainable quantity of softwood residues that can be an attractive feedstock for fuel ethanol production. The major species available from thinning of forests in northern California and the eastern Rocky Mountains include white fir (Abies concolor), Douglas fir (Pseudotsuga menziesii), and Ponderosa pine (Pinus ponderosa). Douglas fir chips were soaked in 0.4% sulfuric acid solution, then pretreated with steam at 200 – 230°C for 1 – 5 min. After pretreatment, 90 – 95% of the hemicellulose and as much as 20% of the cellulose was solubilized in water, and 90% of the remaining cellulose can be hydrolyzed to glucose by cellulase enzyme. The prehydrolysates, at as high as 10% total solid concentration, can be readily fermented by the unadapted yeastSaccharomyces cerevisiae D₅A.     

Uranium removal from acidic aqueous solutions by <Emphasis Type="Italic">Saccharomyces cerevisiae,Debaryomyces hansenii,Kluyveromyces marxianus</Emphasis> and <Emphasis Type="Italic">Candida colliculosa</Emphasis>

The sorption of uranium from acidic aqueous solutions (pH 4.5, C _init = 10 to 1000 mg U/L) by Saccharomyces cerevisiae, Debaryomyces hansenii, Kluyveromyces marxianus and Candida colliculosa was investigated using a batch technique. The U-sorption onto Saccharomyces cerevisiae and Debaryomyces hansenii followed a Langmuir, while that onto Kluyveromyces marxianus and Candida colliculosa a Freundlich isotherm. The results demonstrated that all investigated biomasses could effectively remove uranium from acidic aqueous solutions. From all sorbents, Saccharomyces cerevisiae appeared to be the most effective with a maximum sorption capacity of 127.7 mg U/g dry biomass.     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

Design,synthesis, and antimicrobial activities of new tanshinone IIA esters

Sixteen new ester derivatives with various partition coefficient (ClogP) values of tanshinone IIA (TSIIA), a major lipophilic component of Salvia miltiorrhiza, were designed and synthesised, including six aliphatic esters (3a–e, 5a), one phosphate ester (4c) and nine aromatic esters (5b–j). Their antimicrobial activities against three Gram-positive bacteria strains, Staphylococcus aureus, Bacillus subtilis, and Bacillus amyloliquefaciens, and two Gram-negative bacteria strains, Pseudomonas aeruginosa and Escherichia coli, as well as two fungi species, Candida albicans and Saccharomyces cerevisiae, were evaluated in vitro by broth microdilution susceptibility tests. The results showed that keeping ClogP values in a certain range is necessary for their antimicrobial activities. For those compounds with ClogP values between 5 and 10, their MIC values showed positive correlations with ClogP values. In particular, compound 3e exhibited fourfold and twofold higher potency than the standard drug amphotericin B against fungi C. albicans and S. cerevisiae with MIC values of 1.95 and 7.81 μg/mL, respectively.     

Thermotolerant yeast for simultaneous saccharification and fermentation of cellulose to ethanol

Ten promising microbial strains were screened for glucose fermentation over the temperature range of 37–47°C, and five temperature-tolerant yeasts (Saccharomyces cerevisiae SERI strain (D₅A),S. uvarum, andCandida generaacidothermophilium, brassicae, andlusitaniae), were chosen for SSF evaluation on Sigmacell-50 cellulose with Genencor 150 L cellulase enzyme.Brettanomyces clausenii (Y-1414) was included for comparison to previous studies both by itself and in mixed culture withS. cerevisiae (D₅A). Good conversion rates were achieved at temperatures as high as 43°C withC. brassicae andS. uvarum; mixed cultures of either of these yeasts with the thermotolerant cellobiose fermenting yeastC. lusitaniae achieved higher rates and yields than any of the three yeasts alone. However, the mixed culture ofB. clausenii andS. cerevisiae at 37°C achieved as high conversion rates and higher yields than any of the other yeasts tested.     

Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.     

Antifungal activity of novel catecholamine ruthenium(III) complexes

New catecholamine ruthenium(III) complexes with potentially interesting biological activity are described. The complexes were prepared by reacting catecholamine with [RuCl(NH₃)₅]Cl₂. The newly prepared species were formulated as [Ru^III(NH₃)₄(catecholamine-R)] ⁿ⁺ where catecholamine-R = noradrenaline, dopamine and isoproterenol, and were characterized by elemental analysis, e.p.r., electronic absorption and f.t.-i.r. They were moderately stable in water at room temperature for at least 1 week. In vitro tests of antifungal activity showed the ruthenium–catecholamine complexes had strong inhibitory action against the pathogenic yeast Candida albicans, Candida glabrata and Candida tropicalis. Free catecholamine does not show any antifungal activity.     

Xylitol production from hardwood hemicellulose hydrolysates by Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii

Three different yeasts, Pachysolen tannophilus, Debaryomyces hansenii, and Candida guilliermondii, were evaluated to ferment xylose solutions prepared from hardwood hemicellulose hydrolysates, among which P. tannophilus proved to be the most promising microorganism. However, the presence of both lignin-derived compounds (LDC) and acetic acid rendered a poor fermentation. To enhance the fermentation kinetics, different treatments to purify the hydrolysates were studied, including overliming, charcoal adsorption for LDC removal, and evaporation for acetic acid and furfural stripping. Under the best operating conditions assayed, 39.5g/L of xylitol were achieved after 96 h of fermentation, which corresponds to a volumetric productivity of 0.41 g/L·h and a yield of product on consumed substrate of 0.63 g _p /g_S.     

Simultaneous saccharification and fermentation of pretreated wheat straw to ethanol with selected yeast strains and β-glucosidase supplementation

Previous shake flask and stirred tank evaluations of temperature tolerant (37–43°C) yeasts in simultaneous saccharification and fermentation (SSF) on Sigmacell-50 cellulose substrates to ethanol have identified several good microorganisms for further SSF studies (27). Of these, the glucose fermenting yeastCandida acidothermophilum, C. brassicae, Saccharomyces cerevisiae, S. uvarum, and a mixed culture of the cellobiose fermenting yeastBrettanomyces clausenii withS. cerevisiae as a control were chosen for shake flask SSF screening experiments with pretreated wheat straw. This study indicates that theSaccharomyces strainscerevisiae anduvarum, give very good performance at high cellulase loadings or when supplemented with Novo-188 β-glucosidase. In fact, with the higher enzyme loadings these yeast will give complete conversion of cellulose to ethanol. Yet at the lower, more economical enzyme loadings, the mixed culture ofBrettanomyces clausenii andS. cerevisiae performs better than any single yeast.

     

Application of fentonś reaction to steam explosion prehydrolysates from poplar biomass

The application of Fenton’s reaction to enhance the fermentability of prehydrolysates obtained from steam explosion pretreatment of poplar biomass was studied. Reaction conditions of temperature and H₂O₂ and Fe(II) concentrations were studied. The fermentability of prehydrolysate treated by Fenton’s reaction was tested by using different inoculum sizes of thermotolerant strain Kluyveromyces marxianus CECT 10875. The highest percentages of toxic compound degradation (ranging from 71 to 93% removal) were obtained at the highest H₂O₂ concentration tested (50 mM). However, a negative effect on fermentability was observed at this H₂O₂ concentration at the lower inoculum loading. An increase in inoculum size to 0.6 g/L resulted in an enhanced ethanol fermentation yield of 95% relative to control.     

Vibrational and microbiological study on alkaline metal picolinates and o-iodobenzoates

FT–IR and Raman experimental data were assigned to appropriate bond vibrations and used to compare the different electronic charge distributions in the aromatic rings and carboxylic anions of various lithium, sodium, potassium, rubidium and caesium o-iodobenzoates and picolinates. Then principal component analysis (PCA) was applied in order to attempt to distinguish the biological activities of these compounds according to selected band wavenumbers. The growth of the bacteria Escherichia coli and Bacillus subtilis and the yeasts Saccharomyces cerevisiae and Hansenula anomala under optimal growth conditions were measured after 24 hours of incubation by the classical plate method. The influence of the picolinates and o-iodobenzoates on the growth of these microorganisms, again after 24 hours of incubation, was also measured and compared to the effect of sodium benzoate, which was used as a reference material. In general, the o-iodobenzoates exhibited more activity against the microorganisms than the picolinates. A statistically significant linear correlation between the spectral data and the degree of influence of a given compound on microorganism growth was established. The correlation coefficients for the o-iodobenzoates were 0.696, –0.628, 0.693 and 0.755 for E. coli, B. subtilis, H. anomala and S. cerevisiae, respectively, and for the picolinates they were 0.818, 0.826, 0.821 and 0.877 for E. coli, B. subtilis, H. anomala and S. cerevisiae, respectively. Therefore, IR spectroscopy is shown to be a rapid and reliable analytical tool for preliminary estimation of the antimicrobial properties of newly synthesized compounds, that can be applied before microbial performance tests.     

Synthesis,spectral characterization and antimicrobial activity of macrocyclic Schiff-base copper(II) complexes containing polycrystalline nanosized grains

Schiff-base copper(II) complexes were prepared using macrocyclic ligands, synthesized by condensation of diethylmalonate with Schiff bases derived from o-phenylenediamine and Knoevenagel condensed β-ketoanilides (obtained by the condensation of acetoacetanilide and substituted benzaldehydes). The ligands and their copper complexes were characterized by microanalytical, mass, UV–Vis, IR, ¹H-NMR, ESR and CV studies, as well as conductivity data. Microanalytical, mass and magnetic moment analyses are consistent with formation of monomeric [CuL]Cl₂. Spectral studies indicate square-planar geometry around copper. The smaller grain sizes found from XRD data suggest that these complexes are polycrystalline with nanosized grains. The SEM images of [CuL¹]Cl₂ have leaf-like morphology. The in vitro biological screening of the investigated compounds against the bacteria Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus aureus and fungi Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola and Candida albicans were tested by the well diffusion method to assess growth inhibition. A comparative study of MIC values of the Schiff-base ligands and their complexes indicate that the complexes exhibit higher antimicrobial activity than the free ligands.     

Mesoporous structures in never-dried softwood cellulose fibers investigated by nitrogen adsorption

Nitrogen adsorption was used to characterize mesoporous structures in never-dried softwood cellulose fibers. Distinct inflections in desorption isotherms were observed over the relative vapor pressure (P/P₀) range of 0.5–0.42 for never-dried cellulose fibers and partially delignified softwood powders. The reduction in N₂ adsorption volume was attributed to cavitation of condensed N₂ present in mesopores formed via lignin removal from wood cell walls during delignification. The specific surface areas of significantly delignified softwood powders were ~150 m² g^?1, indicating that in wood cell walls 16 individual cellulose microfibrils, each 3–4 nm in width, form one cellulose fibril bundle surrounded with a thin layer of lignin and hemicelluloses. Analysis of N₂ adsorption isotherms indicates that mesopores in the softwood cellulose fibers and partially delignified softwood powders had peaks ranging from 4 to 20 nm in diameter.     

Spectroscopic and Analytic Properties of New Copper(II) Complex of Antiviral Drug Valacyclovir

The complexation processes between Cu^II and the antiviral drug, valacyclovir hydrochloride (H₂L), were studied under different reaction conditions, using UV-Vis, IR and mass spectra, magnetochemical, thermogravimetric, atomic absorption, conductivity, and elemental analysis data. Using the Job method, the compositions of these complexes were determined. Under the ligand (H₂L) excess (M:L = 1:1–1:10). In aqueous solutions, a binuclear violet complex Cu₂(HL)₂L(H₂O)₄ is formed, with an octahedral structure. Under comparable amounts of the reagents, in MeOH solutions, a green mononuclear complex Cu(H₂L)Cl₂ is formed. Protonation constants of the H₂L, and stability constants of its Cu²⁺ complexes, were determined by potentiometric titration in H₂O media at 25 ± 0.02 °C under a N₂ atmosphere and ionic strength of 0.1 m NaCl. It has been observed that H₂L has three protonation constants. The divalent metal ion Cu²⁺ forms stable 2:1 and 2:2 complexes with H₂L. The antimicrobial activity studies of the valacyclovir and it’s complexes have been studied against some gram positive species: (Corynebacterium xerosis, Bacillus brevis, Bacillus megaterium, Bacillus cereus, Mycobacterium smegmatis, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis) and gram negative (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Yersinia enterocolitica, Kluyveromyces fragilis, Saccharomyces cerevisiae, Candida albicans) bacteriaz.     

Electrofusion of an industrial Baker’s yeast strain with a sour dough yeast

A method for hybridization of yeast protoplasts ofSaccharomyces cerevisiae andCandida holmii by electrofusion was optimized. The hybrids were screened on maltose-acetate agar plates. The average fusion frequency was 1.1xl0-³. Two hybrids of 132 collected from selection plates were found to be stable over 15 sequential shake flask cultivations. However, the strains reverted during production of baker’s yeast in a laboratory scale process imitating the industrial process.     

Xylulokinase activity in various yeasts includingSaccharomyces cerevisiae containing the cloned xylulokinase gene

d-Xylose is a major constituent of hemicellulose, which makes up 20–30% of renewable biomass in nature.d-Xylose can be fermented by most yeasts, includingSaccharomyces cerevisiae, by a two-stage process. In this process, xylose is first converted to xylulose in vitro by the enzyme xylose (glucose) isomerase, and the latter sugar is then fermented by yeast to ethanol. With the availability of an inexpensive source of xylose isomerase produced by recombinantE. coli, this process of fermenting xylose to ethanol can become quite effective. In this paper, we report that yeast xylose and xylulose fermentation can be further improved by cloning and overexpression of the xylulokinase gene. For instance, the level of xylulokinase activity in S.cerevisiae can be increased 230fold by cloning its xylulokinase gene on a high copy-number plasmid, coupled with fusion of the gene with an effective promoter. The resulting genetically-engineered yeasts can ferment xylose and xylulose more than twice as fast as the parent yeast.     

Enzymatic Resolution of Ethyl α-Hydroxyphosphinates in a Modified Reaction Environment

The enzymatic resolutions of two racemic ethyl hydroxyalkane(P-phenyl)phosphinates were performed by both esterification and hydrolysis approaches. The first reaction was performed in anhydrous diisopropyl ether with triethylamine or pyridine as additives by using lipases from three different sources (Candida cylindracea, Aspergillus niger, and Mucor javanicus). The increase in enantioselectivity was observed when NEt₃ was applied. The second reaction—lipase-catalysed hydrolysis of ethyl butyryloxyalkane(P-phenyl)phosphinates—was carried out by Candida cylindracea lipase in diisopropyl ether saturated with water or in aqueous solutions containing MgCl₂, LiCl, or Triton X-100. The usefulness of biphasic systems consisting of diisopropyl ether and water or aqueous solution of MgCl₂, LiCl, or Triton X-100 also were tested. The use of biphasic system in the presence of Triton X-100 resulted in the higher conversion of the substrates.