Development of xylose-fermenting yeasts for ethanol production at high acetic acid concentrations

Mutants resistant to comparatively high levels of acetic acid were isolated from the xylose-fermenting yeastsCandida shehatae andPichia stipitis by adapting these cultures to increasing concentrations of acetic acid grown in shake-flask cultures. These mutants were tested for their ability to ferment xylose in presence of high acetic acid concentrations, in acid hydrolysates of wood, and in hardwood spent sulfite liquor, and compared with their wild-type counterparts and between themselves. TheP. stipitis mutant exhibited faster fermentation times, better tolerance to acid hydrolysates, and tolerance to lower pH.     

Fractionation of casein hydrolysates using polysulfone ultrafiltration hollow fiber membranes

The objective of this study was to characterize the fractionation profile of casein hydrolysates obtained with polysulfone hollow fiber ultrafiltration membranes. The two-step ultrafiltration process developed by Turgeon and Gauthier [J. Food Sci., 55 (1990) 106] was used: a caseinate solution was submitted to proteolysis with chymotrypsin or trypsin, and the reaction mixture (RM) was subsequently ultrafiltered using a 30 kDa (MWCO) hollow-fiber polysulfone membrane. The total hydrolysate permeating from this first step was further fractionated using a 1 kDa (MWCO) membrane, producing the mixture of polypeptides (retentate) and the amino acid fraction (permeate). The effect of enzyme specificity and of membrane retentivitiy on the total composition (total nitrogen, fat, lactose, minerals) and amino acid profile of the fractions was studied. The overall composition of the fractions was not significantly affected by the nature of the enzyme but the degree of hydrolysis and the molecular weight distribution profile analyses showed a marked effect of the enzyme specificity, with trypsin giving a larger proportion of small peptides (< 200 Da) in the mixture of polypeptides. Amino acid profile analyses provided useful information on the phenomena governing the fractionation of amino acids with a polysulfone membrane: (1) the target amino acids of the enzyme are concentrated in the permeate as a result of their presence in all peptides produced by hydrolysis, (2) polar amino acids are retained by the membrane, (3) non-polar amino acids are not selectively rejected by the membrane. Our results suggest that the charge/hydrophobicity balance of the peptides produced is the predominant factor determining the fractionation of casein hydrolysates.     

Cross-linking epoxide resins with hydrolysates of chrome-tanned leather waste

Differential scanning calorimetry was employed to investigate the reaction of diglycidyl ethers of bisphenol A (DGEBA) of mean molecular mass 348–480 Da, with collagen hydrolysate of chrome-tanned leather waste in a solvent-free environment. The reaction leads to biodegradable polymers that might facilitate recycling of plastic parts in products of the automotive and/or aeronautics industry provided with protective films on this basis. The reaction proceeds in a temperature interval of 205–220°C, at temperatures approx. 30–40°C below temperature of thermal degradation of collagen hydrolysate. The found value of reaction enthalpy, 519.19 J g⁻¹ (= 101.24 kJ mol⁻¹ of epoxide groups) corresponds with currently found enthalpy values of the reaction of oxirane ring with amino groups. Reaction heat depends on the composition of reaction mixture (or on mass fraction of diglycidyl ethers in the reaction mixture); proving the dependence of kinetic parameters of the reaction (Arrhenius pre-exponential factor A (min⁻¹) and activation energy E _a (kJ mol⁻¹)) did not succeed. Obtained values of kinetic parameters are on a level corresponding to the assumption that reaction kinetics is determined by diffusion.     

A study of a polysulfone membrane for use in an in-situ tunable microdialysis probe during monitoring of starch enzymatic hydrolysates

We report a study made on a 5 kDa molecular weight cut-off (MWCO) polysulfone membrane (SPS 4005) for use in microdialysis sampling of starch enzymatic hydrolysates on-line coupled to column liquid chromatography with integrated pulsed electrochemical detection. Membrane characteristics were evaluated by examining both the membrane and membrane support layer using a scanning electron microscope (SEM). This study was made so as to elucidate the mechanism or mode by which a membrane exposed to different bioprocess conditions is either fouled or destroyed. The correlation of extraction fraction data with SEM was a confirmatory test for the observed change in the membrane characteristics. Examinations were made after keeping the membrane in pure water for 5, 10 and 30 min at room temperature or after perfusing the membrane with pure water continuously at room temperature, 60 and 90°C for 24 h. Extraction fractions were evaluated at these temperatures to see applicability of the SPS 4005 membrane to high temperature bioprocesses. Scanning electron microscopy studies were also made on membranes used for sampling and sample clean-up during on-line monitoring of the hydrolysis of soluble starch (according to Zulkowsky) for 32 h, and the hydrolysis of wheat starch at room temperature, 60 and 90°C for 6 h. Non-specific/directed protein-membrane interactions were evaluated by sampling maltoheptaose with a microdialysis probe fitted with a 10 mm SPS 4005 membrane, before and after treatment with an enzyme solution of Termamyl (endo-1,4-- -glucan glucanohydrolase EC 3.2.1.78).     

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.     

Thermophilic fermentation of hydrolysates

Lignocellulosic biomass has great potential as a cheap feedstock in biological processes to produce biofuels or chemicals; however, dilute acid pretreatment at high temperatures produces undesirable compounds. Toxicity tests were done with inhibitors in standard media, to predict the growth-limiting effects on thermophilic strains. The 22 inhibitors included furfural, levulinic acid, acetic acid, and cinnamaldehyde. Neutralizing reagents and additional treatment steps have been tested.     

Calcium-binding ability of soy protein hydrolysates

This present study investigated the ability of various soy protein hydrolysates(SPHs)in binding calcium.It was demonstrated that the amount of Ca-bound depended greatly on the SPHs obtained using different proteases,which included:neutrase, flavourzyme,protease M and pepsin.The maximum level of Ca-bound(66.9 mg/g)occurred when protease M was used to hydrolyze soy protein.Peptide fragments exhibiting high Ca-binding capacity had molecular weights of either 14.4 or 8-9 kDa.The level of Ca-bound increased linearly with the increment of carboxyl content in SPHs,and further deamidation on SPHs from protease M improved Ca-binding of the hydrolysate.     

Curing of urea-formaldehyde adhesives with collagen type hydrolysates under acid condition

Condensation of dimethylol urea and its mixtures with urea or hydrolysate of chrome-tanned leather waste (mass fraction in mixture 0.05) in the presence of a variable quantity of phthalic acid, as acid curing agent (within mass fraction limits 0.01-0.1), was studied through TG technique. During condensation of sole dimethylol urea or of its mixture with urea, oxy-methylene as well as methylene cross-links are produced at a ratio of approx. 1:1, presence of leather waste hydrolysate reduces this ratio to approx. 1:2 in favour of more stable methylene bridges. Higher concentrations of phthalic acid (above mass fraction 0.05) lead to formation of a new TG mass decrement wave in the 150-180°C temperature interval, which probably relates to transition of more unstable oxy-methylene bridges to more stable methylene bridges, with splitting-off of formaldehyde. Oxy-methylene bridges may be regarded as a potential source of formaldehyde emissions by cured urea-formaldehyde adhesive films. Hydrolysate of chrome-tanned leather waste and a suitable concentration of acid curing agent distinctly reduce formaldehyde emission from cured urea-formaldehyde adhesive films. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.     

Bioactive Hydrolysates from Chlorella vulgaris: Optimal Process and Bioactive Properties

Microalgae have been described as a source of bioactive compounds, such as peptides. Microalgae are easy to produce, making them a sustainable resource for extracting active ingredients for industrial applications. Several microalgae species have interesting protein content, such as Chlorella vulgaris with around 52.2% of protein, making it promising for peptide hydrolysate production. Therefore, this work focused on the production of water-soluble hydrolysates rich in proteins/peptides from the microalgae C. vulgaris and studied bioactive properties. For that, a design of experiments (DOE) was performed to establish the optimal conditions to produce hydrolysates with higher levels of protein, as well as antioxidant and antihypertensive properties. Four experimental factors were considered (cellulase percentage, protease percentage, hydrolysis temperature, and hydrolysis duration) for three responses (protein content, antioxidant activity, and antihypertensive activity). The optimal conditions determined by the DOE allowed producing a scaled-up hydrolysate with 45% protein, with antioxidant activity, measured by oxygen radical absorbance capacity assay, of 1035 µmol TE/g protein, IC₅₀ for angiotensin-converting enzyme inhibition activity of 286 µg protein/mL, and α-glucosidase inhibition of 31% (30 mg hydrolysate/mL). The obtained hydrolysates can be used as functional ingredients for food and nutraceuticals due to their antioxidant, antihypertensive, and antidiabetic potential. Moreover, the antioxidant potential of the extracts may be relevant for the cosmetic industry, especially in antiaging formulations.