The Ethanol Tolerance of <Emphasis Type="Italic">Pachysolen tannophilus</Emphasis> in Fermentation on Xylose

The influence of ethanol on fermentation by Pachysolen tannophilus was studied. When xylose utilization rate was 80%, ethanol concentration began to decline. Fermentation of P. tannophilus was affected by ethanol addition in the beginning of fermentation; average xylose consumption rate was 0.065 g·l⁻¹·h⁻¹, and maximum specific growth rate was 0.07 h⁻¹ at 28 g·l⁻¹ ethanol, comparing with the average xylose consumption rate of 0.38 g·l⁻¹·h⁻¹ and maximum specific growth rate of 0.14 h⁻¹ in fermentation with no ethanol addition; P. tannophilus stopped growth at 40 g·l⁻¹ ethanol. When the initial ethanol concentration was 30 g·l⁻¹, the addition of glucose in xylose media made the growth of P. tannophilus better, and the most favorable glucose concentration was 15 g·l⁻¹ with the highest biomass of 1.51 g·l⁻¹ as compared with that of 0.95 g·l⁻¹ in pure xylose media.     

Assessment of Bermudagrass and Bunch Grasses as Feedstock for Conversion to Ethanol

Research is needed to allow more efficient processing of lignocellulose from abundant plant biomass resources for production to fuel ethanol at lower costs. Potential dedicated feedstock species vary in degrees of recalcitrance to ethanol processing. The standard dilute acid hydrolysis pretreatment followed by simultaneous sacharification and fermentation (SSF) was performed on leaf and stem material from three grasses: giant reed (Arundo donax L.), napiergrass (Pennisetum purpureum Schumach.), and bermudagrass (Cynodon spp). In a separate study, napiergrass, and bermudagrass whole samples were pretreated with esterase and cellulose before fermentation. Conversion via SSF was greatest with two bermudagrass cultivars (140 and 122 mg g⁻¹ of biomass) followed by leaves of two napiergrass genotypes (107 and 97 mg g⁻¹) and two giant reed clones (109 and 85 mg g⁻¹). Variability existed among bermudagrass cultivars for conversion to ethanol after esterase and cellulase treatments, with Tifton 85 (289 mg g) and Coastcross II (284 mg g⁻¹) being superior to Coastal (247 mg g⁻¹) and Tifton 44 (245 mg g⁻¹). Results suggest that ethanol yields vary significantly for feedstocks by species and within species and that genetic breeding for improved feedstocks should be possible.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

Continuous cultivation of dilute-acid hydrolysates to ethanol by immobilized Saccharomyces cerevisiae

The continuous cultivation of immobilized Saccharomyces cerevisiae CBS 8066 on dilute-acid hydrolysates of forest residuals was investigated. The yeast cells were immobilized in 2–4% Ca-alginate beads. The 2% beads were not stable. However, the 3 and 4% beads were stable for at least 3 wk when an extra resource of calcium ions was available in the medium. The continuous cultivation of a dilute-acid hydrolysate by the immobilized cells at dilution rates of 0.3, 0.5, and 0.6 h⁻¹ resulted in 86, 83, and 79% sugar consumption, respectively, and an ethanol yield between 0.45 and 0.48 g/g. The hydrolysate was fermentable at a dilution rate of 0.1 h⁻¹ in a free-cell system but washed out at a dilution rate of 0.2 h⁻¹. The continuous cultivation of a more inhibiting hydrolysate was not successful by either free- or immobilized-cell systems even at a low dilution rate of 0.07 h⁻¹. However, when the hydrolysate was overlimed, it was fermentable by the immobilized cells at a dilution rate of 0.2 h⁻¹.     

Enzymatic saccharification of cellulose in aqueous-ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate-DMSO media

Ionic liquid (IL) 1-ethyl-3-methylimidazolium dimethylphosphate ([Emim]DMP) was chosen as an environment-friendly solvent to enzymatically hydrolyze cellulose in situ. Under optimal reaction condition, 80.2 % of cellulose (10 mg mL⁻¹) were converted to glucose in aqueous-IL-DMSO (φ _r = 74: 25: 1) media at 55°C in 18 h. Finally, fermentability of the recovered hydrolyzates was evaluated using Saccharomyces cerevisiae which is able to ferment hydrolyzates efficiently, the ethanol production was 0.44 g g⁻¹ of glucose within 24 h of the process. Such information is vital for the saccharification of more complex cellulose materials and for the fermentation of hydrolyzates into biofuel.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Study and Application of Electrochemical Behavior of Calcium-ARS on a Mercury Film Electrode

A sensitive complex absorptive wave of Ca-ARS was obtained by using differential pulse voltammetry when a mercury film glass carbon electrode was immersed in 0.1 mol L⁻¹ KOH and 4.5×10⁻⁴ mol L⁻¹ ARS solution. The peak potential obtained was −1.17 V (vs Ag-AgCl). The peak current was proportional to the concentration of calcium in the range of 5.0×10⁻⁸−4.2×10⁻⁵ mol L⁻¹. The detection limit was 2.0×10⁻⁸ mol L⁻¹. This method was applied successfully to determining traces of calcium in blood serum. The electrochemical behavior of the system was also studied by cyclic voltammetry, and the experiment results showed that the electrode process was an irreversible absorptive with two electrons participating. Translated from Journal of Beijing Normal University (Natural Science Edition), 2005, 41(2) (in Chinese)     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).     

Continuous Production of Thermostable α-Amylase by Immobilized Bacillus Cells in a Fluidized-Bed Reactor

The exponential cells ofBacillus cereus immobilized in calcium alginate and spun into microcylindrical particles were used in a fluidized-bed reactor for continuous synthesis of thermostable α-amylase. The reactor was operated over a period of 30 d with a dilution rate of 0.33 h^-1, producing 1000–1200 U/mL of enzyme. The productivity of the reactor was in the range of 330–396 kU/h. A 20-fold increase in the productivity with respect to batch fermentation with free cells was attained.     

Variable Volume Fed-Batch Fermentation for Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp<Emphasis Type="Italic">. lactis W28</Emphasis>

A feeding technology that was suitable for improving the nisin production by Lactococcus lactis subsp. lactis W28 was established. The effects of initial sucrose concentration (ISC) in the fermentation broth, feeding time, and feeding rate on the fermentation were studied. It was observed that a fed-batch culture (ISC = 10 g l⁻¹) with 100 ml sucrose solution (190 g l⁻¹) being evenly fed (9–10 ml h⁻¹) into the fermenter after 3-h fermentation gave the best performance in terms of biomass and nisin yield. Under these conditions, the total biomass and the total nisin yield were approximately 23% and 51% higher than those in batch fermentation, respectively. When the sucrose concentration was controlled at 5–10 g l⁻¹ in variable volume intermittent fed-batch fermentation (VVIF) with ISC = 10 g l⁻¹, the total biomass and the total nisin yield were 29% and 60% above those in batch fermentation, respectively. The VVIF proved to be effective to eliminate the substrate inhibition by maintaining sucrose at appropriate levels. It is also easy to be scaled up, since various parameters involved in industrial production were taken into account.     

Effect of Constant Glucose Feeding on the Production of Exopolysaccharides by <Emphasis Type="Italic">Tremella fuciformis</Emphasis> Spores

A fed-batch culture system with constant feeding (glucose 80 g L⁻¹, 0.25 ml min⁻¹) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L⁻¹) and exopolysaccharides production (3.12 g L⁻¹) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d⁻¹ and 0.14 g g⁻¹. Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L⁻¹; the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d⁻¹ and 0.093 g g⁻¹, respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.     

Hydrolysis behavior of a precursor for bridged polysilsesquioxane 1,4-bis(triethoxysilyl)benzene: a <Superscript>29</Superscript>Si NMR study

The hydrolysis behavior of 1,4-bis(triethoxysilyl)benzene (BTB), a precursor of bridged polysilsesquioxane, was investigated with high-resolution ²⁹Si nuclear magnetic resonance (²⁹Si NMR) spectroscopy at ambient temperature in a system with BTB:ethanol:water:HCl = 1:10:x:0.8 × 10⁻⁴ (x = 3, 6 or 9). Signals due to hydrolyzed triethoxysilyl groups as well as unhydrolyzed triethoxysilyl groups [−Si(OEt)₃, −Si(OEt)₂(OH), −Si(OEt)(OH)₂ and −Si(OH)₃ (OEt = OCH₂CH₃)] formed four sub-regions based on the number of hydroxyl groups bound to a silicon atom. In addition, one silicon environment influenced the other silicon environment by an intra-molecular interaction between two silicon atoms, and each sub-region for monomeric species thus contained four signals. Based on the development of signal intensity, it is revealed that one of the two triethoxysilyl groups in BTB is hydrolyzed preferentially. Thus, when a triethoxysilyl group is hydrolyzed, the −Si(OH) _x (OEt)_3−x (x = 1, 2) groups formed undergo further hydrolysis, which is opposite to the tendency expected from the hydrolysis behavior of organotrialkoxysilanes under acidic conditions.     

Application of a pH Feedback-Controlled Substrate Feeding Method in Lactic Acid Production

Substrate concentration in lactic acid fermentation broth could not be controlled well by traditional feeding methods, including constant, intermittent, and exponential feeding methods, in fed-batch experiments. A simple feedback feeding method based on pH was proposed to control pH and substrate concentration synchronously to enhance lactic acid production in fed-batch culture. As the linear relationship between the consumption amounts of alkali and that of substrate was concluded during lactic acid fermentation, the alkali and substrate in the feeding broth were mixed together proportionally. Thus, the concentration of substrate could be controlled through the adjustment of pH automatically. In the fed-batch lactic acid fermentation with Lactobacillus lactis-11 by this method, the residual glucose concentration in fermentation broth was controlled between 4.1 and 4.9 g L⁻¹, and the highest concentration of lactic acid, maximum cell dry weight, volumetric productivity of lactic acid, and yield were 96.3 g L⁻¹, 4.7 g L⁻¹, 1.9 g L⁻¹ h⁻¹, and 0.99 g lactic acid per gram of glucose, respectively, compared to 82.7 g L⁻¹, 3.31 g L⁻¹, 1.7 g L⁻¹ h⁻¹, and 0.92 g lactic acid per gram of glucose in batch culture. This feeding method was simple and easily operated and could be feasible for industrial lactic acid production in the future.     

A Thermodynamic Study on the Binding of Calcium Ion with Myelin Basic Protein

The interaction of myelin basic protein (MBP) from the bovine central nervous system with divalent calcium ion was studied by isothermal titration calorimetry at 27 °C in aqueous solution. The extended solvation model was used to reproduce the enthalpies of Ca²⁺-MBP interaction over the whole range of Ca²⁺ concentrations. The solvation parameters recovered from the solvation model were attributed to the structural change of MBP due to the metal ion interaction. It was found that there is a set of two identical and non-interacting binding sites for Ca²⁺ ions. The association equilibrium constant is 0.021 μmol⋅dm⁻³. The molar enthalpy of binding is ΔH=−15.10 kJ⋅mol⁻¹.     

An automatic flow injection analysis procedure for photometric determination of ethanol in red wine without using a chromogenic reagent

An automatic reagentless photometric procedure for the determination of ethanol in red wine is described. The procedure was based on a falling drop system that was implemented by employing a flow injection analysis manifold. The detection system comprised an infrared LED and a phototransistor. The experimental arrangement was designed to ensure that the wine drop grew between these devices, thus causing a decrease in the intensity of the radiation beam coming from the LED. Since ethanol content affected the size of the wine drop this feature was exploited to develop an analytical procedure for the photometric determination of ethanol in red wine without using a chromogenic reagent. In an attempt to prove the usefulness of the proposed procedure, a set of red wines were analysed. No significant difference between our results and those obtained with a reference method was observed at the 95% confidence level. Other advantages of our method were a linear response ranging from 0.17 up to 5.14 mol L⁻¹ (1.0 up to 30.0%) ethanol (R=0.999); a limit of detection of 0.05 mol L⁻¹ (0.3%) ethanol; a relative standard deviation of 2.5% (n=10) using typical wine sample containing 2.14 mol L⁻¹ (12.5%) ethanol; and a sampling rate of 50 determinations per hour.     

Development of a flow injection method for monitoring cell membrane damage of wine lactic acid bacteria

A flow injection analysis (FIA) system was developed for the determination of phosphate efflux from wine lactic acid bacteria (Oenococus oeni and Lactobacillus hilgardii) as an indication of cell membrane damage. The system allowed the direct injection of the cell suspension, avoiding the filtration step, with minimum sample treatment and minimized reagent consumption. The developed system is characterized by a linear concentration zone between 3.23 × 10⁻⁵ and 4.84 × 10⁻⁴ mol L⁻¹ PO₄ ³⁻ and repeatability better than 2.9%. Bacterial suspensions were exposed to a chemical stress with phenolic acids and injected in the FIA system at regular intervals. The extracellular concentration of phosphate was measured spectrophotometrically. The experimental results obtained indicate that hydroxycinnamic acids (p-coumaric, ferulic and caffeic) induced faster phosphate leakage rates than hydroxybenzoic acids (vanillic and p-hydroxybenzoic) in both strains tested, which could be related to their higher lipophilic character.     

Overproduction of Glucoamylase by a Deregulated Mutant of a Thermophilic Mould <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae, a glucoamylase-producing thermophilic mould, was mutagenised using nitrous acid and gamma (⁶⁰Co) irradiation in a sequential manner to isolate deregulated mutants for enhanced production of glucoamylase. The mutants were isolated on Emerson YpSs agar containing a non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) for selection. The preliminary screening for glucoamylase production using starch–iodine plate assay followed by quantitative confirmation in submerged fermentation permitted the isolation of several variants showing varying levels of derepression and glucoamylase secretion. The mutant strain T. indicae-seudaticae CR19 was able to grow in the presence of 0.5 g l⁻¹ 2-DG and produced 1.8-fold higher glucoamylase. As with the parent strain, glucoamylase production by T. indicae-seudaticae CR19 in 250-ml Erlenmeyer flasks attained a peak in 48 h of fermentation, showing higher glucoamylase productivity (0.67 U ml⁻¹ h⁻¹) than the former (0.375 U ml⁻¹ h⁻¹). A large-scale cultivation in 5-l laboratory bioreactor confirmed similar fermentation profiles, though the glucoamylase production peak was attained within 36 h attributable to the better control of process parameters. Although the mutant grew slightly slow in the presence of 2-DG and exhibited less sporulation, it showed faster growth on normal Emerson medium with a higher specific growth rate (0.138 h⁻¹) compared to the parent strain (0.123 h⁻¹). The glucoamylase produced by both strains was optimally active at 60 °C and pH 7.0 and displayed broad substrate specificity by cleaving α-1,4- and α-1,6-glycosidic linkages in starch, amylopectin, amylose and pullulan. Improved productivity and higher specific growth rate make T. indicae-seudaticae CR19 a useful strain for glucoamylase production.     

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture

The production of pullulan from beet molasses by a pigment-free strain of Aureobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by ¹³C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (M _w =2.1−4.1×10⁵), root mean square of the radius of gyration (R _g =30−38 nm), and polydispersity index (M _w /M _n =1.4−2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8−10.0 (w/v), the solution’s rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s⁻¹ at 20°C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.     

Removal and recovery of copper (II) ions by bacterial biosorption

Studies were conducted toinvestigate the removal and recovery of copper (II) ions from aqueous solutions by Micrococcus sp., which was isolated from a local activated sludge process. The equilibrium of copper biosorption followed the Langmuir isotherm model very well with a maximum biosorption capacity (q_max) of 36.5 mg of Cu²⁺/gofdry cell at pH 5.0 and 52.1 mg of Cu²⁺/g of dry cell at pH 6.0. Cells harvested at exponential growth phase and stationary phase showed similar biosorption characteristics for copper, Copper uptake by cells was negligible at pH 2.0 and then increased rapidly with increasing pH un til 6.0. In multim etal systems, Micrococcus sp. exhibited a preferential biosorption order: Cu−Pb>Ni−Zn. There is virtually no interference with copper uptake by Micrococcus sp. from solutions bearing high concentrations of Cl⁻, SO ₄ ²⁻ , and NO3/− (0–500 mg/L). Sulfuric acid (0.05 M) was the most efficient desorption medium, recovering >90% of the initial copper sorbed. The copper capacity of Micrococcus sp. remained unchanged after five successive sorption and desorption cycles. Immobilization of Micrococcus sp. in 2% calcium alginate and 10% polyacrylamide gel beads increased copper uptake by 61%. Biomass of Micrococcus sp. may be applicable to the development of potentially cost-effective biosorbent for removing and recovering copper from effluents.     

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.