HPAEC‐PAD method for the analysis of alkaline hydrolyzates of Haemophilus influenzae type b capsular polysaccharide

A gradient method has been devised for the rapid analysis of alkaline hydrolyzates of Haemophilus influenzae type b (Hib) capsular polysaccharide‐based vaccines by high‐performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). As compared with published procedures, peak shape and sensitivity were significantly improved with this approach, analysis time was short and there was little interference from impurities. The limits of detection and quantification were established with a purified reference polysaccharide. We propose this method as a practical alternative for the analysis of minute amounts of Hib polysaccharide, which can be lower than with the conventional approaches. Copyright © 2013 John Wiley & Sons, Ltd.     

Influence of inhibitory compounds and minor sugars on xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.     

Fermentative Production of Fructo-Oligosaccharides Using Aureobasidium pullulans: Effect of Dissolved Oxygen Concentration and Fermentation Mode

Fructo-oligosaccharides (FOS) are prebiotics with numerous health benefits. So far, the dissolved oxygen (DO) concentration control strategy for fermentative production of FOS is still unknown. In order to improve FOS production, the effects of DO concentration and fermentation mode on FOS using Aureobasidium pullulans were investigated in this study. The greatest FOS production (123.2 ± 6.2 g/L), with a yield of 61.6% ± 3.0% (g FOS/g sucrose), was obtained in batch culture under high DO concentration. Furthermore, repeated-batch culture revealed that enzyme production and FOS production were not closely associated with cell growth. By keeping the DO concentration above 5% in the repeated-batch culture, a maximum FOS concentration of 548.3 ± 37.4 g/L and yield of 68.6% ± 2.6% (g FOS/g sucrose) were obtained, which were 3.45% and 11.4% times higher than those obtained in the batch culture without DO control, respectively. Additionally, the ratios of 1-fructofuranosyl nystose (GF4) and 1,1,1,1-kestohexose (GF5) were 33.8% and 23.2%, respectively, in the product of repeated-batch culture, but these compounds were not detected in batch culture. Thus, it can be concluded that the DO concentration affects not only the yield of FOS but also the composition of FOS with different degrees of polymerization, which is the key factor in the fermentative production of FOS with a high polymerization degree.     

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20°C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH₄ ⁺:NO₃ ⁻ ratio (total nitrogen concentration of 60 mM) and PO₄ ³⁻ did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH₄ ⁺:NO₃ ⁻ ratio, and PO₄ ³⁻) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.     

α-amylase production by free and immobilized Bacillus subtilis

The effect of glucose on the α-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific α-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the α-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific α-amylase production with the free-cell system was produced by the immobilized-cell batch culture.     

Effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans in an airlift reactor

The effect of the shear rate on pullulan production from beet molasses by Aureobasidium pullulans P56 in an airlift reactor was investigated. A maximum polysaccharide concentration (18.5 g/L), biomass dry weight (14.0 g/L), polysaccharide yield (38.5%), and sugar utilization (96%) was achieved at a shear rate of 42 s⁻¹. A. pullulans grown on beet molasses produced a mixture of pullulan and other polysaccharides. The highest value of pullulan proportion (30% of total polysaccharide) was obtained at a low shear rate (42 s⁻¹). The apparent viscosity of the fermentation broth increased as the shear rate increased up to 42 s⁻¹ and then decreased. On the other hand, the dissolved oxygen concentration and the volumetric mass transfer coefficient increased with the increase of the shear rate from 21 to 84 s⁻¹. The external addition of L-glutamic acid, olive oil, and Tween-80 improved significantly the production of crude polysaccharide (27.0 g/L), but the pullulan content of the polysaccharide was low (20%).     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA)=t(GA)_max/(W_GA +t), (SOR)=t (SOR)_max/(W_Sor+t), v_GA=[W_GA (GA)_max]/(W_GA+t)² and V_SOR=[W_SOR (SOR)_max]/(W_SOR+t)². Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)_max= 541 g/L, (SOR)_max=552 g/L, W_GA=4.8h, W_SOR=4.9h, υ_GA=112.7 g/L· and υ_SOR=112.7 g/L·.     

d-xylose transport by Candida succiphila and Kluyveromyces marxianus

The kinetics and regulation of d-xylose uptake were investigated in the efficient pentose fermentor Candida succiphila, and in Kluyveromyces marxianus, which assimilate but do not ferment pentose sugars. Active high-affinity (K _m ∼ 3.8 mM; V _max ∼ 15 nmol/[mg·min]) and putative facilitated diffusion low-affinity (K _m ∼ 140 mM; V _max ∼ 130 nmol/[mg·min]) transport activities were found in C. succiphila grown, respectively, on xylose or glucose. K. marxianus showed facilitated diffusion low-affinity (K _m ∼ 103 mM; V _max ∼ 190 nmol/[mg·min]) transport activity when grown on xylose under microaerobic conditions, and both a low-affinity and an active high-affinity (K _m ∼ 0.2 mM; V _max ∼ 10 nmol/[mg·min]) transport activity when grown on xylose under fully aerobic conditions.     

Lactic acid production through cell-recycle repeated-batch bioreactor

The effect of various nitrogen sources on cell growth and lactic acid production was investigated. The most effective nitrogen source was yeast extract; more yeast extract gave higher cell growth and lactic acid productivity. Yeast extract dosage and cell growth were proportional up to a yeast extract concentration of 30 g/L, and lactic acid productivity was linearly correlated up to a yeast extract dosage of 25 g/L. However, increasing the yeast extract content raises the total production cost of lactic acid. Therefore, we attempted to find the optimum yeast extract dosage for a repeated-batch operation with cell recycling. The results show that when using Enterococcus faecalis RKY1 only 26% of the yeast extract dosage for a conventional batch fermentation was sufficient to produce the same amount of lactic acid, whereas the lactic acid concentration in the product stream (92–94 g/L) and lactic acid productivity (6.03–6.20 g/[L·h]) were similar to those of a batch operation. Furthermore, long-term stability was established.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Comparative energetics of glucose and xylose metabolism in recombinant Zymomonas mobilis

Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote “coupled growth”), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell…h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell…h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar respectively. The corresponding value of Y_ATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. Y_ATP for the wild-type culture CP4 with glucose was 10.4g of DCM/mol of ATP. For single substratechem ostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max Y_a/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the “maintenance coefficient” (m) was determined as the y-axis intercept. For xylose, values of max Y _s/s and m were 0.0417g of DCM/g of xylose (Y_ATP=6.25) and 0.04g of, xylose/(g of cell…h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D>0.1/h, Y_ATP=8.71 and m=2.05g of glu/(g of cell…h) whereas at D<0.1/h, Y_ATP=4.9g of DCM/mol of ATP and m=0.04g of glu/(g of cell…h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt's maintenance coefficient is a constant that is in dependent of the growth rate. Collectively, these observations with individual sugars and the values assign ed to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes.     

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.     

Degradation processes in the cellulose/<Emphasis Type="Italic">N</Emphasis>-methylmorpholine-<Emphasis Type="Italic">N</Emphasis>-oxide system studied by HPLC and ESR. Radical formation/recombination kinetics under UV photolysis at 77 K

Degradation processes of N-methylmorpholine-N-oxide monohydrate (NMMO), cellulose and cellulose/NMMO solutions were studied by high performance liquid chromatography (HPLC) and electron spin resonance (ESR) spectroscopy. Kinetics of radical accumulation processes under UV (λ = 248 nm) excimer laser flash photolysis was investigated by ESR at 77 K. Beside radical products of cellulose generated and stabilized at low temperature, radicals in NMMO and cellulose/NMMO solutions were studied for the first time in those systems and attributed to nitroxide type radicals ∼CH₂–NO^•–CH₂∼ and/or ∼CH₂–NO^•–CH₃∼ at the first and methyl ^•CH₃ and formyl ^•CHO radicals at the second step of the photo-induced reaction. Kinetic study of radicals revealed that formation and recombination rates of radical reaction depend on cellulose concentration in cellulose/NMMO solutions and additional ingredients, e.g., Fe(II) and propyl gallate. HPLC measurements showed that the concentrations of ring degradation products, e.g., aminoethanol and acetaldehyde, are determined by the composition of the cellulose/NMMO solution. Results based on HPLC are mainly maintained by ESR that supports the assumption concerning a radical initiated ring-opening of NMMO.     

Denitrification in presence of benzene,toluene, and <Emphasis Type="Italic">m</Emphasis>-xylene

Denitrification of the electron donors toluene-C(15–100 mg/L), m-xylene-C (15–70 mg/L), benzene-C (5–25 mg/L), and acetate-C as experimental reference (50–140 mg/L) was carried out in batch culture. An initial concentration of 1.1±0.15 g of volatile suspended solids/L of denitrifying sludge without previous exposure to aromatic compounds was used as inoculum. The results showed toluene and nitrate consumption efficiency (E _T and E _N′ respectively) of 100%. Toluene was completely mineralized (oxidized) to CO₂. In all cases, the N₂ (γ_N2) and HCO ₃ ⁻ yields (γ_HCO3) were 0.97±0.01 and 0.8±0.05, respectively. The consumption efficiency (E _x ) of m-xylene (53±5.7%) was partial. The γ_N2 and γ_HCO3 were 0.96±0.01 and 0.86±0.02, respectively. Benzene was not consumed under denitrifying conditions. The specific consumption rates of toluene (q _T ) and m-xylene (q _X ) were lower than that of acetate (q _A ). The differences in specific consumption rates were probably owing to the negative effect of benzene, toluene, and isomers of xylene on the cell membrane.     

A sensitive nicotine sensor based on molecularly imprinted electropolymer of o-aminophenol

A sensitive nicotine sensor based on a molecularly imprinted electropolymer of o-aminophenol is proposed and its configuration and performance are studied in detail. On the condition of weak acidity, the sensitive layer was prepared by electropolymerization of o-aminophenol on a gold electrode in the presence of the template (nicotine). The sensor exhibits good selectivity and sensitivity to nicotine. The determination limit is 2.0×10⁻⁷ mol/L and a linear relationship between the current and concentration is found in the range of 4.0×10⁻⁷ ∼ 3.3 ×10⁻⁵ mol/L. The sensor has also been applied to the analysis of nicotine in tobacco samples with recovery rates ranging from 99.0% to 102%. __________ Translated from Journal of Hunan university (Nature Science), 2005, 32(3) (in Chinese)     

Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media

The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and 65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable of glucose and xylose utilization.     

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D _max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D _max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D _max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D _max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D _max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).     

Evaluation of carob pod as a substrate for pullulan production byAureobasidium pullulans

The production of extracellular polysaccharides from carob pod extract by Aureobasidium pullulans in batch fermentation was investigated. Optimum conditions for polysaccharide productivity, polysaccharide yield, and fermentation efficiency were: initial sugar concentration of 25 g/L, initial pH 6.5, and temperature 25–30°C. A maximum polysaccharide concentration (6.5 g/L), polysaccharide productivity (2.16 g/L/d), total biomass concentration (6.3 g/L), and polysaccharide yield (30%) were obtained with inoculum at 10% (v/v), initial sugars in carob pod extract of 25 g/L, pH 6.5, and 25°C. The highest values of pullulan proportion (70% of total polysaccharides) and fermentation efficiency (89%) were assumed at initial sugar concentration of 25 g/L, pH 6.5 and 30°C. Structural characterization of purified pullulan samples by monosaccharide and methylation analyses, 13C-NMR, and pullulanase digestion combined with size-exclusion chromatography revealed the presence of mainly α-(l → 4) (68%) and α-(l → 6) (31%) glucosidic linkages; however, small amounts (<1%) of triply linked (1, 3, 4-and 1, 4, 6-Glc) glucose residues were detected. The molecularsize distribution and intrinsic viscosity of pullulan derived from culture grown at pH 4.5 (30°C) showed a higher molecular weight than its counterpart obtained at pH 6.5 (30°C).     

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.