Improved Bioethanol Production Using Fusants of Saccharomyces cerevisiae and Xylose-Fermenting Yeasts

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose?Cxylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8?±?0.31?g L^?1), ethanol productivity (1.06?g L^?1 h^?1) and ethanol yield (0.458?g g^?1) by fermentation of glucose?Cxylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08?±?0.142?g L^?1, ethanol yield of 0.44?g g^?1, productivity of 0.45?g L^?1?h^?1 and biomass yield of 0.40?g g^?1.     

Characterization of Bioimprinted Tannase and Its Kinetic and Thermodynamics Properties in Synthesis of Propyl Gallate by Transesterification in Anhydrous Medium

Tannase has been extensively applied to synthesize gallic acid esters. Bioimprinting technique can evidently enhance transesterification-catalyzing performance of tannase. In order to promote the practical utilization of the modified tannase, a few enzymatic characteristics of the enzyme and its kinetic and thermodynamics properties in synthesis of propyl gallate by transesterification in anhydrous medium have been studied. The investigations of pH and temperature found that the imprinted tannase holds an optimum activity at pH?5.0 and 40?°C. On the other hand, the bioimprinting technique has a profound enhancing effect on the adapted tannase in substrate affinity and thermostability. The kinetic and thermodynamic analyses showed that the modified tannase has a longer half-time of 1,710?h at 40?°C; the kinetic constants, the activation energy of reversible thermal inactivation, and the activation energy of irreversible thermal inactivation, respectively, are 0.054?mM, 17.35?kJ?mol^?1, and 85.54?kJ?mol^?1 with tannic acid as a substrate at 40?°C; the free energy of Gibbs (??G) and enthalpy (??H) were found to be 97.1 and 82.9?kJ?mol^-1 separately under the same conditions.     

Ethanol Production from Starch Hydrolyzates using <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and Glucoamylase Entrapped in Polyvinylalcohol Hydrogel

The glucoamylase from Aspergillus niger, immobilized into poly(vinylalcohol) hydrogel lens-shaped capsules LentiKats®, was used for simultaneous saccharification and fermentation (SSF) with Zymomonas mobilis in free form. This system was stable in both the repeated batch and continuous mode of SSF. The microorganism was found to adsorb on the capsules with immobilized enzyme. This increased the ethanol productivity of the repeated batch system with 5% w/v of immobilized glucoamylase almost 2.1 times (7.2 g l^?1 h^?1) compared to free enzyme–free microorganism system (3.5 g l^?1 h^?1). The continuous SSF with the immobilized glucoamylase (11.5% w/v) tested for 15 days had productivity 10 g l^?1 h^?1, which is comparable to continuous experiments on semi-defined glucose medium (10 g l^?1 h^?1). These two systems were stable in both glucoamylase activity and microorganism productivity.     

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.     

Rapid,sensitive fluorimetric assay for proteases using fluorescein-labelled albumin coupled to Fractogel activated with 2-fluoro-1-methylpyridinium salt in flow-injection analysis

A rapid and sensitive flow-injection method is described for the fluorimetric determination of the proteolytic activity of a number of enzymes. The substrate, fluorescein-labelled bovine serum albumin coupled to a 2-fluoro 1-methylpyridinium salt-activated Fractogel support, is packed in a small reactor (35 × 2 mm i.d.) and inserted into a flow manifold. Under the reaction conditions the amount of fluorescein released from enzymatic cleavage of the substrate is found to be directly proportional to the activity of the proteolytic enzyme. Sample throughput is 30–35 h^?1. The calibration range for trypsin is linear up to at least 0.35 μg ml^?1. Linear responses were also obtained for otherproteolytic enzymes such as papain, chymotrypsin, ficin and bromelain.     

An Elemental Phosphorus Photocatalyst with a Record High Hydrogen Evolution Efficiency

Semiconductive property of elementary substance is an interesting and attractive phenomenon. We obtain a breakthrough that fibrous phase red phosphorus, a recent discovered modification of red phosphorus by Ruck et al., can work as a semiconductor photocatalyst for visible‐light‐driven hydrogen (H₂) evolution. Small sized fibrous phosphorus is obtained by 1) loading it on photoinactive SiO₂ fibers or by 2) smashing it ultrasonically. They display the steady hydrogen evolution rates of 633 μmol h^?1 g^?1 and 684 μmol h^?1 g^?1, respectively. These values are much higher than previous amorphous P (0.6 μmol h^?1 g^?1) and Hittorf P (1.6 μmol h^?1 g^?1). Moreover, they are the highest records in the family of elemental photocatalysts to date. This discovery is helpful for further understanding the semiconductive property of elementary substance. It is also favorable for the development of elemental photocatalysts.     

Enzymatic Hydrolysis of N-Acylated 1-Aminophosphonic Acids

Abstract

Penicillin acylase from E. coli (FC 3.5.1.11) was found to hydrolyse N-phenylacetylated 1-aminoalkylphosphonic acids and their esters. Enzyme preferentially converts the R-form of the substrates: the ratios of the bimolecular rate constants of penicillin acylase-catalysed hydrolysis of R-and S- forms of 1-(N-phenylacetaminol-ethylphosphonic acid and its dimethyl- and diisopropyl- esters are 58000, 2600, 1800; these derivatives were shown to have the greatest values of the catalytic constants for enzymatic hydrolysis of all known substrates of penicillin acylase: 237, 148, and 134 s; corresponding values of Michaelis constants are 3.7×10^?5, 6.8×10^?4, and 6.2×10^?4 M. The kinetics of the enzymatic hydrolysis of 1-(N-phenylacetaminol-ethylphosphonic acid was investigated up to high degrees of conversion. The inhibition of penicillin acylase by high concentrations of the R-form of the substrate (with substrate inhibition constant 0.07 Ml and competitive inhibition by the reaction product phenylacetic acid (K_i=3.5×10_?5 M) was observed. Penicillin acylase was shown to possess quite broad substrate specificity among N-acylated 1-aminoalkylphosphonic acids and was found to be capable of hydrolysing 1-(N-phenylacetaminol-substituted 2-phenylethyl-, 1-phenylmethyl- and 3-methylbutylphosphonic acids with high efficiency and enantioselectivity.     

Flow Injection Analysis of Tannic Acid with Inhibited Electrochemiluminescent Detection

ABSTRACT

A flow injection method has been developed for the determination of tannic acid based on its inhibition of the electrochemiluminescence of luminol. The method is simple, rapid and sensitive with a detection limit of 2×10^?8 mol/l. It is effective to determine tannic acid in the range 5×10^?8 - 1×10^?5 mol/l. The variation coefficient of eleven determinations is 1.4% for 5×10^?6 mol/l. The method has been successfully applied to the determination of tannic acid in real Chinese gall and hop pellet samples.     

Improvement of the Production Efficiency of l-(+)-Tartaric Acid by Heterogeneous Whole-Cell Bioconversion

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to l-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of l-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37?×?10^?3 g U^?1 h^?1 was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m³ stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the l-(+)-tartaric acid industrial production.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Culture conditions dictate protease and tannase production in submerged and solid-state cultures of Aspergillus niger Aa-20

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.     

Contribution of Tris Buffer on Xylitol Enzymatic Production

Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L^?1 h^?1 xylitol volumetric productivity with 99% xylose–xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.     

Aquaculture by-product: a source of proteolytic enzymes for detergent additives

Intestine proteases of Nile tilapia (Oreochromis niloticus) were partially purified by heat treatment (purification factor of 3.5, enzyme activity remained almost constant) to reach the maximum activity and stability within an alkaline pH range of 7.2–11.0. The optimum temperature and stability over a 120 min period were found to be at 55°C and at 35–45°C, respectively. The proteases’ activity was not affected by a 1 vol. % saponin surfactant, inactivated by 0.01 g mL^?1 sodium dodecylsulphate after 120 min, and it remained stable for 30 min in a 5 vol. % and 10 vol. % hydrogen peroxide solutions. The proteases were slightly activated by Ca²⁺, Mg²⁺, and K⁺ and the substrate most effectively hydrolysed was casein (40.0 U mg^?1). A 2⁴ full factorial design used to evaluated the influence of independent variables showed that the enzyme extract, detergent concentration and the incubation time had a significant influence on the enzymatic activity. The best conditions to be used concerning detergent additive were found with 0.3 mg mL^?1 of protein and 3.0 mg mL^?1 of detergent for 30 min in the presence of Astrus® detergent.     

Wetting films on a hydrophilic silica surface obtained from aqueous solutions of hydrophobically modified inulin polymeric surfactant

The thickness of wetting films on a hydrophilic silica surface was investigated using a microinterferometric technique. Aqueous solutions of hydrophobically modified inulin (INUTEC^®SP1) at various concentrations, in the presence or absence of NaCl or Na₂SO₄, were studied. The equilibrium film thickness (h _eq) showed a complex dependence on INUTEC^®SP1 concentration. At low electrolyte concentrations, h _eq decreased with an increase in INUTEC^®SP1 concentration, reaching a minimum at 10^?6 mol dm^?3. However, at high electrolyte concentrations, this dependence became less pronounced. At any given INUTEC^®SP1 concentration, the equilibrium film thickness decreased with an increase in electrolyte concentration as a result of the compression of the electrical double layer reaching a minimum value. After that, the film thickness showed a small decrease with further increase in electrolyte concentration. This indicates that the electrostatic component of disjoining pressure can be neglected, and the steric repulsion of the loops and tails of INUTEC^®SP1 determined the film thickness.     

Flow-injection biamperometry of pyrogallol compounds

A biamperometric method for the direct determination of pyrogallol compounds has been designed for flow-injection analysis. The method is based on the electrocatalytic oxidation of pyrogallol compounds at one pretreated platinum electrode and the reduction of platinum oxide at the other pretreated platinum electrode to form a biamperometric detection system with the applied potential difference of 10 mV. Three important compounds, pyrogallol, gallic acid and tannic acid, have been detected by the method. The linear relationships between currents and the concentrations of pyrogallol, gallic acid and tannic acid are obtained over the range 1.0×10⁻⁶-1.0×10⁻⁴, 1.0×10⁻⁶-1.0×10⁻⁴ and 1.0×10⁻⁶-2.0×10⁻⁴ mol l⁻¹ with the detection limit of 6.0×10⁻⁷, 6.0×10⁻⁷ and 8.0×10⁻⁷ mol l⁻¹ (S/N=2), respectively. The R.S.D. observed for 30 successive determinations of 5.0×10⁻⁵ mol l⁻¹ pyrogallol, gallic acid and tannic acid are 1.9, 2.5 and 2.0%, respectively. Most ions and organic compounds tested are found not to cause significant interference in the determinations. The method is simple, selective and efficient (180 h⁻¹), performing well as a routine assay, and has been validated by the determination of pyrogallol compounds in tea and Chinese gall.     

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30?g?g^?1) and productivity (0.19?g?L^?1?h^?1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3?g?L^?1 resulted in high biomass production. The highest biomass concentration (21?g?L^?1), yield (0.45?g?g^?1) and productivity (0.31?g?L^?1?h^?1), as well as ribonucleotide production (13.13?mg?g^?1), were observed at 700?rpm and 0.5?vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.     

Continuous flow extraction of indium with bis(2-ethylhexyl)phosphoric acid in 4-methylpentane-2-one coupled on-line with flame atomic absorption spectrometry

Extraction in liquid-liquid segmented flow is used for preconcentration of indium from dilute nitric acid solutions into bis(2-ethylhexyl)phosphoric acid dissolved in 4-methylpentane-2-one. The extraction setup is coupled on-line with flame spectrometry to give a fully mechanized system. The detection limit of the method is 0.03 mg l^?1, the calibration plot is linear up to 1.75 mg l^?1. Repeatability is 1.5% RSD measured at 1 mg l^?1. Sample throughput is 60 h^?1.     

Flow injection spectrophotometric determination of ascorbic acid in soft drinks and beer

Two spectrophotometric methods, a photochemical and a non-photochemical, for the determination of ascorbic acid in soft drinks and beer using a flow-injection system are proposed. The non-photochemical method is based on the redox reaction that takes place between ascorbic acid and Fe(III), yielding dehydroascorbic acid and Fe(II). Fe(II) reacts with 1,10-phenantroline, originating the reddish orange Fe(phen)₃ ²⁺ complex (ferroin). This complex is spectrophotometrically monitored at 512 nm, and the signal is directly related to the concentration of ascorbic acid in the sample. The photochemical method has the same basis, nevertheless, uses the irradiation with visible light to enhance the redox reaction and so achieve higher sensitivities in the analysis. The non-photochemical method shows a linear range between 5 and 80 μg mL^?1, with a relative standard deviation of 1.6% (n = 11), a detection limit of 2.7 μg mL^?1 and a sample throughput of ¶60 samples h^?1. The photochemical method shows a linear range between 1 and 80 μg mL^?1, with a relative standard deviation of 1.0% (n = 11), a detection limit of 0.5 μg mL^?1 and a sample throughput of 40 samples h^?1.     

Improving the Performance of a Continuous Process for the Production of Ethanol from Starch

In a previous work, a continuous simultaneous saccharification and fermentation process to produce ethanol from cassava starch was studied, using a set of fixed-bed reactors. The biocatalyst consisted of glucoamylase immobilized in silica particles and co-immobilized with S. cerevisiae in pectin gel. Using 3.8 U mL^?1 _reactor and 0.05 g_{wet yeast} mL^?1 _reactor at start-up, starch hydrolysis was the rate-limiting step. Maximum ethanol productivity was 5.8 g_ethanol L^?1 h^?1, with 94.0% conversion of total reducing sugars (TRS) and 83.0% of the ethanol theoretical yield. In this work, the molar mass of the substrate and the biocatalyst particle size were reduced in an attempt to improve the bioreactor performance. The diameters of silica and pectin gel particles were reduced from 100 μm and 3–4 mm, respectively, to 60 μm and 1–1.5 mm, and the degree of substrate prehydrolysis by α-amylase was increased. The bioreactor performance was assessed for different loads of immobilized glucoamylase (2.1, 2.8, and 3.8 U mL^?1 _reactor), for the same initial cell concentration (0.05 g_{wet yeast.}mL^?1 _reactor). Feeding with 154.0 g L^?1 of TRS and using 3.8 U mL^?1 _reactor, fermentation became the rate-limiting step. Productivity reached 11.7 g L^?1 h^?1, with 97.0% of TRS conversion and 92.0% of the ethanol theoretical yield. The reactor was operated during 275 h without any indication of destabilization.     

Sensitive flow-injection determination of l-lactate in human blood with immobilized enzyme columns and fluorimetric detection

Enzyme columns prepared by packing l-lactate oxidase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series. Hydrogen peroxide formed in the enzymatic conversion of l-lactate in the first column is mixed with 3-(p-hydroxyphenyl)propionic acid before passage through the peroxidase column and fluorimetric measurement. Linear calibration was obtained for 0.5–500 pmol of L-lactate in 20 μl of 1000-fold diluted, deproteinated whole blood. A rapid sampling rate (60 h^?1) was possible.