Journal of Bioprocessing & Biotechniques

There is a huge demand for developing new technologies for alternative energy sources due to the elevated costs of petroleum and its by-products, depletion of nonrenewable fuel sources, and to eliminate the disadvantages of geopolitical location and environmental pollution caused by high levels of carbon dioxide release. Science is striving to meet this demand and as molecular biology techniques have progressed, genetic engineering tools have been presented as promising future solutions in the form of optimizing the fermentation process to increase the ethanol yield from different carbon sources such as starch. As Saccharomyces cerevisiae is not naturally able to ferment starch, it can be genetically manipulated and modulated to improve the fuel production from starchy materials and the amount of cost that is required to produce ethanol would be decreased with these manipulations. General modifications in S. cerevisiae include specific gene expressions to gain new properties or improve existing pathways. This review aims to elicit the current status of ethanol production thorough alternative techniques from starch using current genetic engineering applications and to give further directions for high-throughput fermentations using genetically modified S. cerevisiae strains.

The ability to hydrolyze keratin, a rigid and strongly cross-linked protein in the waste of poultry feather and sheep wool, has made keratinase production by microorganisms highly important to the biotechnological industry. A proteindegrading bacterium (C4) was isolated from compost. Based on morphology and biochemical tests, along with 16S rRNA sequencing, the isolated C4 was tentatively identified as Bacillus sp. C4 (2008). The proteolytic activity of the Bacillus sp. C4 strain was broadly specific; it degraded keratinous and non-keratinous proteins to different degrees. Pea pods as substrate generated the highest protease production, followed by soybean meal and sheep wool. Notwithstanding, using wool keratin as a sole source of carbon and nitrogen yielded the highest level of soluble proteins. Furthermore, the C4 bacterium grew well, and produced a significant level of keratinase when using wool and feather as substrates. Supplementing the medium with yeast extract and peptone shortened the time required for feather degradation, but delayed the onset of the wool keratin hydrolysis with two days. The predominant amino acids released in feather hydrolysate were tyrosine, phenylalanine, and histidine. In contrast, the wool lysate was rich in aspartic acid, methionine, tyrosine, phenylalanine, histidine, and lysine. Results established that utilizing the C4 strain for keratin degradation in waste management holds considerable potential.

In this study, a QuEChERS method and gas chromatography coupled with time-of-flight mass spectrometry (GC×GCTOFMS) was developed for rapid extraction and simultaneous determination of 12 pesticide residues in honey. The GC×GC-TOFMS method was validated according to the SANCO guidance in terms of linearity, selectivity, reproducibility and recovery. Regarding the results, recovery rates ranged between 70-120% with relative standard deviations <20% in most cases. The method Limits of Quantification (LOQ) ranged between 6-26 ng/g. According to the estimated LOQ values the analytical procedure can be applied to analysis of real honey samples.

The structure and antioxidant activity of a polysaccharide from mycelia fermentation of Hirsutella sinensis were analyzed. The natural active component water-soluble polysaccharides was isolated from mycelia, and three polysaccharide fractions HSP-1, HSP-2, and HSP-3 were purified with chromatography and the structures were identified. The structural characteristics determination with a combination of chemical and instrumental analysis methods showed that the mainly component HSP-1 was about 1.7×104 Da, and composed of glucose, mannose and galactose at a molar ratio of 4.5:1.0:1.4. Further researches revealed that HSP-1 was a branched polysaccharide possessing a backbone of (1→4)-α-D-glucose residues (~70%), (1→4)-α-D-mannose residues (~15%) and (1→4)-α-D-galactose residues (~15%). The branches were at the (1,2,4,6→)-α-D-glucose residues (~8%) of the backbone, mainly composed of (1→4)-α-D-glucose residues, (1→4)-α-D-galcatose residues, (1→4)-α-D-mannose residues, and terminated with α-D-galactose residues. The in vitro antioxidant assay proved HSP-1 possessed the hydroxyl radical-scavenging activity with an IC50 value of 0.834 mg/mL.

The present research was carried out with objective to study the effect of different types of Impellers and Baffles on mixing and to examine the correlation between mixing time and mass transfer of Aerobic Stirred Tank Fermenter. An Aerobic Stirred Tank fermenter was assembled with different Impellers and Baffles alternately. The four different Impellers used were Rushton Impeller, Marine Impeller, A320 Impeller and HE3 Impeller while walled and un walled baffles were used in combination with these Impellers. The Fermenter was assembled with Resistance Temperature Detector, pH Probe and Pressure Gauge. Tachometer was used to calculate the Mixing Time of Fermenter. Volumetric Mass Transfer Coefficient was experimentally determined and calculated by the respective formulae. MATLAB was used for Mathematical Modelling of CFD, while Autodesk Simulation CFD and ANSYS FLUENT were used to generate the simulations. Turbulence lengths and Trailing Vortices were used to understand flow patterns inside the fermenter created by Impellers and Baffles. The kLa values suggested that Rushton Impeller was the most efficient among all. Turbulent Kinetic Energy and Turbulent Dissipation Rate were to understand the mixing efficiency of every Impeller.

Biologics production using yeast or CHO with Yeastolate as cell culture additives often introduces β-glucan, which could potentially pose immunogenicity risk, if not adequately removed. Although a previous study has shown the effective clearance of Yeastolate-derived β-glucan by Protein A chromatography, the clearance pattern of yeast cell derived β-glucan remains unknown. In this study, we characterized the β-glucan clearance patterns during downstream processing of three monoclonal antibody (mAb) products, one mAb fragment from Pichia pastoris (mAb A) and two full mAbs from CHO expression system (mAb B and mAb C), by Glucatell assay. We demonstrated effective β-glucan clearance in both small (100 L) and large scale (5000 L) batches of mAb A as well as in one batch of mAb B. Protein A purification step removed an average of 97.74% (1.7 log10 reduction) of β-glucan detected in the two batches of mAb A microfiltration permeates (MFP) and 99.99% (3.9 log10 reduction) of β-glucan detected in mAb B clarified culture fluid harvest (HCCF). Residual β-glucans post Protein A purification in the two batches of mAb A were further removed by the two polishing chromatography steps (94.76% reduction on average). Residual β-glucan measured in the mAb A and mAb B drug substance ranges from 7.8 to 19 pg/mg, which is unlikely to alter physiological concentrations significantly in healthy adults when administered with typical intravenous doses. However, in mAb C, after almost complete removal (99.99%) by Protein A purification step, β-glucan level increased more than 20 fold in Viral Filtration (VF) product sample, indicating that it can be introduced from materials used in downstream process, such as cellulose-based filters and membranes. Our study results suggest that although β-glucan can be cleared by Protein A and other chromatographic steps such as AEX, monitoring β-glucan clearance during downstream process development remains very important to identify and avoid potential contaminations to the drug substance.

Neural Progenitor Cells (NPCs) have gathered more and more attention in the field of Neural Stem Cells (NSCs). However, the multilineage differentiating behavior of these cells and their contribution to tissue regeneration, almost in lower vertebrate taxa, remain unknown. Since the early 1970s, many comparative studies have been performed using immunocytochemical screening on the brains of several vertebrate taxa, including teleosts, in order to identify these cells, even if the data are sometimes contrasting. This study aims: (1) to investigate in vitro the potential proliferative role of NPCs and Radial Glia Progenitors (RGP) in seabream neurogenesis; (2) to reveal the strict ability of fish NSCs to undertake the multilineage development and differentiation in neurons, astrocytes and oligodendrocytes. By the use of double Immunofluorescence (IF) analysis and phase contrast microscopy, we identified the multilineage differentiation and the exact cell morphology. We demonstrated that NSC can self-renew and differentiate into different types of neurons or glial cells during extended culturing. Mature neurons expressed specific neuronal markers; they could differentiate during long term culturing, generating an extensive neurite growth. Glia was found highly mitotic and could developed mature astrocytes and oligodendrocytes. Glial cells were assessed by Glial Fibrillary Acidic Protein (GFAP) reactivity; neurons and myelinating oligodendrocytes were immunostained with cell-specific markers. This work provide that the multilineage differentiation potential of seabream neural cell progenitors might be a useful tool for neurodegenerative diseases, being a promising approach for repairing the CNS injuries, also in other animals, as a new coming strategy for function recovery of damaged nerves.

Diethylaminoethyl (DEAE) cellulose adsorbed Pointed Gourd Peroxidase (PGP) was employed in decolorization of synthetic dyes. The expressed activity of immobilized preparation on fifth repeated use was ~50% and decolorization achieved for synthetic dyes DR19 and dye mixture (DR19+DB9) was 64.9% and 61.5% respectively. Immobilized enzyme could effectively decolorize up to 88.2% and 77.4% of DR19 and dye mixture respectively in stirred batch process at 40°C whereas dye color removal monitored at 30°C and 50°C was comparatively low under similar conditions. Immobilized enzyme in the packed column used for the continuous removal of dye color could successfully decolorize DR19 and dye mixture to 69.4% and 51.4% after 50 d of operation. Thus, DEAE immobilized PGP is a simple, economical and effective preparation to remove color of synthetic dyes.

For successful commercialization of biomass to ethanol process, the techno- economic hurdles have to be overcome. Complete substrate utilization is the key for making economics favorable. Existing enzyme preparations do not give high saccharification efficiency, thus, a broader suite of hydrolases is required. Xylanase supplementation in enzyme cocktail is an important strategy to increase sugar yields. In our study, hyperxylanolytic Aspergillus awamori F18 was used to produce xylanase. A. awamori F18 expressed high levels of xylanase during Solid state fermentation of corncob and also, Carboxy Methyl Cellulase (CMCase), filter paper activity (FP lyase) and β-glucosidase (524.43, 43.95, 8.64 and 29.81 IU mg-1 proteins respectively). A 10.6-12.5 fold concentration of these activities was achieved by single-step acetone precipitation. Supplementing concentrated xylanase to Accellerase®1500 resulted in 69.5% enhancement in sugars released after 72 h of saccharification of steam-pretreated rice straw. HPLC analysis of hydrolysates showed higher glucose levels along with the presence of xylose and arabinose. There was a 12.4% increase in the amounts of glucose as well as total sugars released when xylanase was supplemented to commercial cellulase and β-glucosidase.

Aim of this study is to investigate the effect of sonication as a pretreatment on the efficiency of anaerobic digestion of Laying Hen Manure (LHM) under mesophilic conditions. In this study,the pretreatment studies were carried out using two different sonotrode (BS2d22 and BS2d40) and two different types of booster (B2-1,4 and B2-1,8) at four different amplitudes (9, 31, 40, 81 μm) at two time settings (5 and 15 min duration). Biochemical Methane production Potential (BMP) test protocol was also employed in order to evaluate the effect of sonication under varying amplitudes on the biogas production for 50 days. The BMP results obtained in this work suggested that sonication significantly enhanced the biogas productivity of chicken manure which was much lower if it was digested alone. Between 12-70% increase in methane production depending on the sonolysis matrix used was obtained in comparison to control group which had no sonication as pretreatment. The best result was obtained at an amplitude of 81 μm and 5 min sonication duration which is a combination of BS2d22/B2-1,4. The results of experiment demonstrated that use of this pretreatment technology could significantly enhance the biogas production from chicken manure.

Design and optimization of Immobilized Metal Affinity Chromatography (IMAC) processes require deep knowledge of driving factors responsible for interaction between immobilized metal and biomolecules. Based on this requirement, interactions between lactoferrin from cheese whey and IDA-Cu2+-cryogel system was investigated. Data from adsorption of lactoferrin in the system at pH 6, 7 and 8, as well as NaCl concentration from 200 to 1000 mmol L−1 were adjusted Langmuir, Freundlich, Temkin and Langmuir-Freundlich isotherm models. Although all models were able to explain the interaction lactoferrin-cryogel system, the Langmuir-Freundlich model was the most accurate one. In addition, it could explain quantitatively the cooperativity and heterogeneity of the bounds between protein and matrix. The methods used in this project are useful for both better understanding of the protein-immobilized metal interactions and developing preparative scale IMAC.

The main objective of this research was to develop a mathematical model of heat and mass balance of the bread baking process to predict the temperature and water content of bread at different heating temperatures and times. The model was able to predict the bread temperature and water content at different oven temperatures (180, 190, 200, 210 and 220°C). The results showed that the bread temperature and weight loss of bread increase with increasing oven temperature, when, the oven temperature increased from 180 to 220°C, the temperature of bread increased from 112.73 to 168.49°C and the weight loss of bread increased from 22.40 to 52.46%. The results also showed that the bread temperature and weight loss of bread increase with increasing time, this increment starts to decline after 18-20 min, until it reach equilibrium after 30 min. The weight loss of bread increases with increasing bread temperature. It indicates that when the bread temperature increased from 20.00 to 131.69°C, the weight loss of bread increased from 0.00 to 40.79% at 200°C oven temperature. The model was validated with an experimental data and showed a reasonable agreement with those measured, where; it ranged 20.00 to 131.69°C theoretically while it was from 25.00 to 119.00°C experimentally during the baking at 200°C oven temperature. The weight loss data was in a reasonable agreement with those measured, where; it ranged 0.00 to 40.79% theoretically while it was from 0.00 to 48.69% experimentally during the baking at 200°C oven temperature.