Ufuk Bakir

Survival kinetics of lactic acid starter cultures during and after freeze drying

Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storage period after freeze drying was enhanced by the presence of dead microorganisms which reduce the interfacial area between the live cells and the external medium. Streptococcus thermophilus was found to be more resistant to freeze-drying conditions than Lactobacillus bulgaricus. Storage under vacuum or nitrogen was superior to storage under air. Poor survival rates under air was attributed to oxygen diffusion into the dry cells through the interfacial area.

Use of xylan, an agricultural by-product, in wheat gluten based biodegradable films: mechanical, solubility and water vapor transfer rate properties

The possibility of using xylan, as an agricultural by-product, for production of composite films in combinations with wheat gluten was investigated. Different levels of xylan (0-40% w/w) were incorporated into wheat gluten to form biodegradable composite films. Films were prepared at pH 4 and 11, and dried at either uncontrolled or controlled conditions. The mechanical properties, solubilities and water vapour transfer rate (WVTR) of the composite films were studied. Films were obtained with added xylan without decreasing film-forming quality. Xylan can be used as an additive, as much as 40% (w/w), in wheat gluten films. Changing pH, wheat gluten/xylan ratio, xylan type and drying conditions affected mechanical and solubility properties, however, WVTR was not affected by xylan additions. Wheat gluten/xylan composite films having different characteristics can be produced depending on xylan type, composition and process conditions.

An endo-β-1,4-xylanase from Rhizopus oryzae: production, partial purification and biochemical characterization.

Abstract An endoxylanase (1,4-β- d -xylan xylanohydrolase, EC 3.2.1.8) was produced by Rhizopus oryzae fermentation. Different xylan-containing agricultural byproducts such as wheat straw, wheat stems, cotton bagasse, hazelnut shells, corn cobs and oat sawdust were used as the carbon source, while soybean bagasse was used as both the nitrogen and carbon source in the enzyme production medium. Partial steam hydrolysis of the agricultural byproducts increased the enzyme yield of the microorganism. The highest xylanase activity, 260 IU/ml fermentation medium, was obtained by using a medium containing 3% hydrolyzed corn cobs, 1% hydrolyzed soybean bagasse, 1% ammonium sulfate and 0.5% sodium chloride at 35°C, pH 5, 350 rpm and under aerobic conditions in a 2-1 fermenter. A maximal cellulose activity of 0.06 IU/ml was observed. The enzyme was partially purified from the culture medium by ammonium sulfate precipitation and cation exchange filtration. A 55-fold purification was achieved, with the purified xylanase having a specific activity of about 50 IU/mg protein. The molecular weight of the enzyme is about 22 kDa by SDS-PAGE. The optimal pH and temperature values of the enzyme were about 4.5 and 55°C, respectively. The enzyme obeys Michaelis-Menten kinetics with K m and V max values being 18.5 mg xylan/ml and 90 IU/mg protein, respectively.

Immobilization of invertase in functionalized copolymer matrices

Abstract In this study, immobilization of invertase on functionalized polymer electrodes constructed with pyrrole-capped polyazotetrahydrofuran and polytetrahydrofuran-block-polystyrene copolymer matrices was performed. Immobilization in these enzyme electrodes was carried out by the entrapment of the enzyme in conducting polymer matrices during electrochemical polymerization of pyrrole. Sodium dodecyl sulphate was used as the supporting electrolyte in the preparation of enzyme electrodes. The effects of temperature and pH on the activity of the enzyme electrodes were examined, and re-use number studies were performed. The changes in the maximum reaction rate and the variations of the Michaelis–Menten constant were investigated.

Enhancing the value of nitrogen from rapeseed meal for microbial oil production

Rapeseed meal, a major byproduct of biodiesel production, has been used as a low-cost raw material for the production of a generic microbial feedstock through a consolidated bioconversion process. Various strategies were tested for the production of a novel fermentation medium, rich in free amino nitrogen (FAN): commercial enzymes (CEs) (2.7 mg g⁻¹ dry meal), liquid state fungal pre-treatment (LSF) using Aspergillus oryzae (4.6 mg g⁻¹), liquid state fungal pre-treatment followed by fungal autolysis (LSFA) (9.13 mg g⁻¹), liquid state pre-treatment using fungal enzymatic broth (EB) (2.1 mg g⁻¹), but the best strategy was a solid state fungal pre-treatment followed by fungal autolysis (34.5 mg g⁻¹). The bioavailability of the nitrogen sources in the novel medium was confirmed in fed-batch bioreactor studies, in which 82.3g dry cell L⁻¹ of the oleaginous yeast Rhodosporidium toruloides Y4 was obtained with a lipid content of 48%. The dry cell weight obtained was higher than that obtained using conventional yeast extract, due to a higher total nitrogen content in the novel biomedium. The fatty acids obtained from the microbial oil were similar to those derived from rapeseed oil.

Cloning, Expression and Characterization of Endo-β-1,4-Mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo‐β‐1,4‐mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA) and the alcohol oxidase (AOX1) promoters, respectively. The highest production of mannanase (352 U mL−1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL−1). The fold increase in mannanase production was estimated as ∼12‐fold and ∼2‐fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (∼350 U mg−1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C.

Evaluation of alkaline pretreatment temperature on a multi-product basis for the co-production of glucose and hemicellulose based films from lignocellulosic biomass.

Cotton stalks were subjected to alkaline pretreatment for the co-production of glucose and hemicellulose based films with a multi-product approach. Three pretreatment temperatures (25, 60 and 90 °C) were evaluated for their effects both on the glucose yield and on the properties of hemicellulose based films. Compared to untreated cotton stalks, the glucose yields were enhanced 3.9, 4.1 and 4.2 times for pretreatments conducted at 25, 60 and 90 °C, respectively. The pretreatment temperature of 90 °C was detrimental in terms of film formation. Tensile energy to break values of the films obtained after pretreatments conducted at 25, 60 and 90 °C were 1.1, 0.8, and 0.4 MJ/m3, respectively. The hemicellulosic part of the process, which considers the production of hemicellulose based films, should govern the pretreatment temperature since it was more responsive to the changes in the pretreatment temperature compared to the cellulosic part that accounts for glucose production.

Immobilization of glucose oxidase in polypyrrole/polytetrahydrofuran graft copolymers

Glucose oxidase (GOD) was immobilized in four different conducting polymer matrices, namely: polypyrrole, (PPy), poly(pyrrole-graft-polytetrahydrofuran), (1) and (3); and poly(pyrrole-graft-polystyrene/polytetrahydrofuran), (2). The kinetic parameters V(max) and K(m), and the optimum temperature were determined for both immobilized and native enzymes. The effect of electrolysis time and several supporting electrolytes, p-toluenesulfonic acid, p-toluene sulfonic acid (PTSA), sodium p-toluene sulfonate, sodium p-toluene sulfonate (NaPTS), and sodium dodecyl sulfate, sodium dodecyl sulfate (SDS), on enzyme immobilization were investigated. The high K(m) value (59.9 mM) of enzyme immobilized in PPy was decreased via immobilization in graft copolymer matrices of pyrrole. V(max), which was 2.25 mM/min for pure PPy, was found as 4.71 mM/min for compound (3).

Immobilization of urease in conducting thiophene-capped poly(methyl methacrylate)/pyrrole matrices

Urease was immobilized in conducting polypyrrole and block copolymers of thiophene-capped poly(methyl methacrylate) matrices by electropolymerization. Immobilization of the enzyme was achieved by application of 1.0 V constant potential in a solution of 0.02 M pyrrole, 2 mg/ml urease, 0.5 mg/ml supporting electrolyte. The optimum immobilization conditions, electrolysis time, electrolysis medium, enzyme concentration during electrolysis, type of supporting electrolyte were determined. The kinetic behaviors of free and immobilized urease were determined.

Selecting the right blood glucose monitor for the determination of glucose during the enzymatic hydrolysis of corncob pretreated with different methods.

In order to assess their accuracy for the determination of glucose during the enzymatic hydrolysis of pretreated lignocellulosic biomass, four different blood glucose monitors (BGMs), each utilizing a different enzymatic mechanism for the determination of glucose, were utilized in an experimental setup, which compares the efficiency of ionic liquid pretreatment with dilute acid and alkaline pretreatments applied on corncob. Among the tested devices, Optium Xceed was found to be the most accurate device for the determination of glucose where Accu-Chek Active was the least accurate BGM, yielding similar results to those obtained with DNS method. Based on the HPLC results, the % error values for Optium Xceed ranged between 3.9-10.5% for the determination of glucose concentration. Upon enzymatic hydrolysis, ionic liquid and alkaline pretreatments gave similar glucose yields, which were slightly higher than the dilute acid pretreatment, which were 31.9%, 31.0% and 27.8%, respectively, based on untreated corncob.