Cost-effective Production Process Research Articles

Discovery of novel highly active and stable aspartate dehydrogenases

Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1–4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.

Fluid Imbibition-Coulped Laser Interferometry As a Tool for Optical Biosensing with Anodic Aluminum Oxide

Optofluidics is a technology based on the fusion between microfluidics and optics. It is related to a variety of phenomena in which an interaction between light and fluids is found. It can be applied to the control of fluid properties by light, to the design of variable geometry optical elements by means of the adaptability of the fluid or by using optics to interrogate fluid properties. On the other hand, anodic aluminum oxide (AAO) is a porous material obtained by the electrochemical anodization of aluminum with a growing interest in nanotechnology because of its cost-effective production process and its promising variety of applications[1]. Recently, we have introduced an optofluidic technique called fluid imbibition-coupled laser interferometry (FICLI), based on the study by optical means of the liquid infiltration dynamics into AAO. We demonstrated its high accuracy for the characterization of the material[2,3]. In this work, we explore the possibilities of using such a sensitive technique as a sensor of biological species. Fluid imbibition-coupled laser interferometry consists of the measurement of the capillary filling dynamics of a fluid into the AAO nanopores. Such measurement is performed by monitoring light intensity interferometric fluctuations of a laser beam in real-time as the liquid infiltrates the pores. An illustration of the measurement system can be seen in the figure. AAO membranes with 50 µm-long pores are used (see SEM pictures in the figure) and the laser beam is reflected at its top and bottom interfaces creating a time-resolved intereference. Light intensity fluctuations are measured by a photodetector and a high-speed data acquisition system, and are analyzed on the basis of mathematical models of the filling kinetics. This technique has shown enough accuracy to demonstrate that AAO nanopores are actually conical with an angle as small as 0.007º. The processing of the time-resolved interferograms, as the one shown in the figure, can be done in several ways. The simplest method consists of measuring the filling time of the pores from each side of the membrane and combine these measurements with the information about the fluid and the surface properties (viscosity, surface tension, contact angle) to obtain the values of the radius at each pore end. A more elaborate method permits the determination of the pore profile by the inversion of the filling dynamics integral equations provided accurate measurement of the time-resolved interferograms for the filling from each end of the pores are obtained. In this work we introduce an alternative method based on the discretization of the filling dynamics equations, what permits to obtain a direct and accurate determination of the average radius of the nanopores. In order to evaluate the accuracy of this alternative method we applied different modifications to the pore geometry and we measured the pore radius modification by the different methods. Pore radius increase was achieved by applying controlled wet etching to the as-produced AOO membranes with increasing time. On the other hand, pore radius decrease was achieved by depositing successive bilayers of polyelectrolytes. Results show that the proposed technique is accurate enough to sense pore radius variations as small as 4 nm, in the range of the size of many biological macromoleclues such as proteins, antibodies or aptamers. With this ability in mind, we have studied the application of the technique to sense such biological species. In this communication we will report our results on the measurement of the changes in the filling dynamics of AAO membranes after the functionalization of their surface and the attachment of the different molecules: i) proteins like bovine serum albumin (BSA), ii) the successive attachment of the IgG-aIgG antibody-antigen pair with a view to the application of the technique as an immunosensor and iii) the attachment of an aptamer and the detection of changes in their conformation.

A simple, rapid and cost-effective process for production of latex clearing protein to produce oligopolyisoprene molecules

Aiming at finding feasible alternatives for rubber waste disposal, the partial enzymatic degradation of poly(cis-1,4-isoprene)-containing materials represents a potential solution. The use of rubber-degrading enzymes and the biotransformation of rubber into new materials is limited by the high costs associated with the production and purification of the enzyme and the complexity of the process. This study presents a simple and low-cost procedure to obtain purified latex clearing protein (Lcp), an enzyme capable of cleaving the double bonds of poly(cis-1,4-isoprene) in presence of oxygen to produce different size of oligomers with terminal aldehyde and ketone groups, respectively. The gene coding for Lcp1VH2 from Gordonia polyisoprenivorans strain VH2 was overexpressed in Escherichia coli C41 (DE3), and by using an auto-induction medium high protein yields were obtained. The cultivation process was described and compared with an IPTG-inducible medium previously used. Purification of the enzyme was performed using salting out precipitation with ammonium sulfate. Different salt concentrations and pH were tested in order to find the optimal for purification, obtaining a concentration of 60mg Lcp per l. The enzymatic activity of the purified enzyme was measured by an oxygen consumption assay in the presence of polyisoprene latex. Volumetric activities of 0.16Uml−1 were obtained at optimal conditions of temperature and pH. The results showed an active and partial purified fraction of Lcp1VH2, able to cleave the backbone of poly(cis-1,4-isoprene) and to produce degradation products that were identified with staining methodologies (Schiff reagent for aldehyde groups and 2,4-DNPH for carbonyl groups) and characterized using nuclear magnetic resonance (NMR). Thirteen different storage conditions were tested for the purified enzyme analyzing the enzymatic activity after 1 and 3 months. Lcp1VH2, as an ammonium sulfate precipitate, was stable, easy to handle and sufficiently active for further analysis. The described methodology offers the possibility to upscale the process and to produce large amounts of this protein.

Cold experiment of slag centrifugal granulation by rotary atomizer: Effect of atomizer configuration

Centrifugal granulation has recently been employed to produce small blast furnace slag particles, so as to recover the waste heat from the high-temperature molten blast furnace slag. An appropriate atomizer enables centrifugal granulation to become a better cost-effective process for particle production. Thus, increasing emphasis has been placed on influence of atomizer configuration on granulation. In present study, three groups of atomizers were specially designed and the granulation performance of each atomizer was experimentally tested during cold experiments. The influences of atomizer configuration on granulation modes and droplet characteristics were investigated visually. Two modified correlations were proposed to predict the granulating droplet size by means of data fitting. The results indicated that the rotary cup atomizers can inhibit the film formation in contrast to rotary disc atomizer. Moreover, atomizers with outer angle of 90° was capable of producing smaller droplets. The revised correlation as well as the newly-developed correlation including the influence of atomizer configurations, presented in good agreement with the experiment data. In addition, an analysis on atomizer design was conducted to provide a good insight for industrialization. It was recommended to adopt cup-like atomizer in granulation for its ability to produce fine particles with smaller atomizer size.

Development of a cost-effective microbial levan production process by Halomonas cultures

Development of a cost-effective microbial levan production process by Halomonas cultures

Field Assisted Sintering Technology Update, Part II

Abstract Field assisted sintering technology (FAST) enables hybrid components for aerospace to be designed with reduced weight and without sacrificing performance. FAST can be used to produce metal, ceramic, and composite components with tailored properties via a powder metallurgy approach. In many cases, it is a one-step, cost-effective manufacturing process for production of net-shape components.

Optimization of growth requirements of marine diatomChaetoceros muelleriusing Response Surface Methodology

The marine centric diatom Chaetoceros muelleri has been widely used as live feed in fish and shellfish aquaculture due to its excellent nutritional properties. The growth of microalgae is affected by various nutritional and environmental parameters, and species specific optimization of these parameters is essential for the development of cost-effective biomass production process. In this study, the growth of C. muelleri, was optimized using response surface methodology (RSM). The variables nitrate, phosphate, silicate, temperature, pH, salinity and agitation speed were initially screened for biomass production in C. muelleri using Plackett–Burman experimental design, and it was found that nitrate, phosphate, silicate, temperature and pH significantly influenced the biomass production. These variables were further optimized by central composite design of RSM for biomass production and nutrient composition, and the medium was re-constituted accordingly to have 180 mg L−1 nitrate, 7.5 mg L−1 phosphate, 30 mg L−1 silicate, with optimum growth conditions of temperature at 31°C and pH 6.46. At the end of 10 days culture period under the above conditions, biomass, protein, lipid and carbohydrate significantly increased from 0.360 ± 0.01 mg L−1, 9.41 ± 0.02%, 18.11 ± 0.01%, 0.6 ± 0.02% to 1.16 ± 0.01 mg L−1, 11.02 ± 0.01%, 19.58 ± 0.01% and 0.77 ± 0.01% respectively. The biomass production of C. muelleri could be increased 3.2-fold with an improved nutrient profile by modifying the growth factors, the study thus offering an optimized process for biomass production of C. muelleri.

Saccharification of biopretreated paddy straw with indigenous holocellulase and fermentation with Saccharomyces cerevisiae LN1 under optimized conditions

The current study was aimed at optimizing the fermentation conditions for efficient ethanol production from biologically pretreated paddy straw. The yeast strain Saccharomyces cerevisiae LN1 showed highest fermentation efficiency at pH 5.0 and temperature 30 °C. Paddy straw pretreated with fungus Myrothecium roridum LG7 was saccharified with indigenous holocellulase from Aspergillus niger SH3 producing total sugar yield of 26.14 mg/ml with 19.23 mg/ml of glucose. Enzymatic hydrolysate was then fermented using S. cerevisiae LN1 to observe the effect of nutrient supplementation (yeast extract, MgSO4·7H2O and (NH4)2SO4) on ethanol production. Higher ethanol was produced from saccharified material fermented without supplementation of any nutrient source. With the scale-up of ethanol production under optimized conditions in 7L bioreactor, 4.46 g/l of ethanol was produced with fermentation efficiency of 47.2 %. TLC of enzymatic hydrolysate confirmed the presence of p-coumaric acid, ferulic acid, vanillic acid, gallic acid and many other aromatic compounds and inhibitors in the saccharified material which limit fermentation efficiency of yeast strain. Thus, optimization of fermentation conditions can lead to development of a cost-effective process for efficient ethanol production, exploitation of which also requires removal of aromatic compounds and inhibitors which may hinder the ethanol production efficiency.

ChemInform Abstract: Synthesis of γ‐Valerolactone from Carbohydrates and Its Applications

AbstractReview: 88 refs.

Diammonium phosphate stimulates transcription of L-lactate dehydrogenase leading to increased L-lactate production in the thermotolerant Bacillus coagulans strain

Exploration of cost-effective fermentation substrates for efficient lactate production is an important economic objective. Although some organic nitrogen sources are also cheaper, inorganic nitrogen salts for lactate fermentation have additional advantages in facilitating downstream procedures and significantly improving the commercial competitiveness of lactate production. In this study, we first established an application of diammonium phosphate to replace yeast extract with a reduced 90% nitrogen cost for a thermotolerant Bacillus coagulans strain. In vivo enzymatic and transcriptional analyses demonstrated that diammonium phosphate stimulates the gene expression of L-lactate dehydrogenase, thus providing higher specific enzyme activity in vivo and increasing L-lactic acid production. This new information provides a foundation for establishing a cost-effective process for polymer-grade L-lactic acid production in an industrial setting.

In Situ Characterization Techniques Based on Synchrotron Radiation and Neutrons Applied for the Development of an Engineering Intermetallic Titanium Aluminide Alloy

Challenging issues concerning energy efficiency and environmental politics require novel approaches to materials design. A recent example with regard to structural materials is the emergence of lightweight intermetallic TiAl alloys. Their excellent high-temperature mechanical properties, low density and high stiffness constitute a profile perfectly suitable for their application as advanced aero-engine turbine blades or as turbocharger turbine wheels in next-generation automotive engines. As the properties of TiAl alloys during processing as well as during service are dependent on the phases occurring, detailed knowledge of their volume fractions and distribution within the microstructure is of paramount importance. Furthermore, the behavior of the individual phases during hot deformation and subsequent heat treatments is of interest to define reliable and cost-effective industrial production processes. In situ high-energy X-ray diffraction methods allow tracing the evolution of phase fractions over a large temperature range. Neutron diffraction unveils information on order-disorder transformations in TiAl alloys. Small-angle scattering experiments offer insights into the materials’ precipitation behavior. This review attempts to shine a light on selected in situ diffraction and scattering techniques and the ways in which they promoted the development of an advanced engineering TiAl alloy.

Effects of Doping Elements in β-FeSi<sub>2</sub> Prepared Utilizing Cast Iron Scrap Chips

In this work, the thermoelectric properties of p- and n-type β-FeSi2, prepared utilizing cast iron scrap chips, have been characterized by measuring the Seebeck coefficient, electrical conductivity and thermal conductivity at temperatures ranging from room temperatures to 800°C. In a previous study, the upgrade recycling of cast iron scrap chips into β-FeSi2 thermoelectric materials was proposed as an eco-friendly and costeffective production process. By doping with different substitution concentrations of Co, Mn and Al, the conduction type and properties of β-FeSi2 can be modified and improved using cast iron scrap chips as a starting material. The effects of the doping elements are discussed for preparing β-FeSi2 utilizing cast iron scrap chips. Cast iron scrap chips could be preferable as a starting material to replace pure Fe for n- and ptype β-FeSi2 thermoelectric materials. An optimum composition for n-type β-FeSi2 0.94C.I.-0.06Co-1.86Si shows that the largest ZT value of 0.22 occurs at 700°C, whereas for p-type β-FeSi2 0.92C.I.-0.08Mn-1.86Si, the largest ZT value of 0.17 occurs at 800°C.

Synthesis of γ‐Valerolactone from Carbohydrates and its Applications

γ-Valerolactone (GVL) is a valuable chemical intermediate that can be obtained by catalytic reduction of levulinic acid (LA) or alkyl levulinates (AL). There are many reports on the synthesis of GVL from LA or AL. However, the demand for the large-scale synthesis of GVL requires more environmentally friendly and cost-effective production processes. This article focuses on the recent advance in the synthesis of GVL from carbohydrates or lignocellulosic biomass. In addition, application of GVL as the reaction solvents, fuel additives, and as precursor for the synthesis of jet fuel and polymer monomers is also discussed.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-based tuberculosis vaccine

Background Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. About 1.4 million people die of TB each year. Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A adenovirus, a new promising tuberculosis vaccine candidate, has been studied with mouse, guinea pig, goat and cow animal models, and was shown to be effective against Mycobacterium tuberculosis (Mtb) infection [1]. A phase I trial on Ad5Ag85A adenovirus was also conducted, which demonstrated that AdAg85A adenovirus was safe and highly immunogenic [2]. To further evaluate the efficacy of this vaccine and reduce the cost of this promising vaccine candidate, a feasible and cost-effective large-scale cell culture production process had to be developed for manufacturing large quantities of AdAg85A adenovirus required for further clinical trials. Furthermore, the process had to be designed to meet all requirements for industrialization and commercialization of this vaccine candidate. Here we report our study on optimization of cell culture conditions, scale up of AdAg85A adenovirus production in 60L bioreactor and purification of the AdAg85A adenovirus at different scales. The optimized conditions for AdAg85A adenovirus production and purification were transferred to a GMP facility for manufacturing of AdAg85A adenovirus for further clinical trials. Materials and methods Four commercial serum-free cell culture media (SFM4HEK-293 and SFM4Transfx-293 from HyClone; Adenovirus Expression Medium (AEM) and CD 293 from Life Technologies), were evaluated for supporting the growth of HEK293SF-3F6 cell in suspension and also for the production of AdAg85a adenovirus in 125 mL shake flask cultures under various experimental conditions. The production of the AdAg85A adenovirus was then scaled up to 3L controlled bioreactor under the optimized conditions obtained from the shake flask experiment, further validated in a 60L bioreactor. Purification of the AdAg85A adenovirus was accomplished through many different steps. Some of the critical steps include cell lysis, benzonase treatment, Q-Sepharose HP anion exchange chromatography for capture of adenovirus/purification, Capto Core 700 multimodal chromatography for polishing, concentration and diafiltration into formulation buffer. The purification processes were also scaled up from 3L to 60 L production scale.

Novel and cost-effective refolding of a recombinant receptor binding domain of Plasmodium falciparum EBA-175

Plasmodium falciparum erythrocyte binding antigen (EBA-175), which is a 175kDa protein, binds with sialic acid residues on glycophorin A during invasion of human erythrocytes. Receptor-binding residues of EBA-175 lie in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins. Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by rapid dilution in presence of proline and purified to homogeneity by ion-exchange chromatography. Recombinant PfF2 was refolded at higher concentrations with improved refolding efficiency and yield. The refolded protein was characterized using biochemical methods and shown to be pure, homogenous and functional as it binds human erythrocytes with specificity. Immunization of mice and rabbits with recombinant PfF2 formulated with Freund’s and Montanide ISA720 adjuvants, elicited high and potent antibody responses. These observations support the development of a novel and cost-effective process for production of recombinant PfF2, a blood stage vaccine candidate for P. falciparum malaria.

Rapid and efficient bioconversion of chicory inulin to fructose by immobilized thermostable inulinase from Aspergillus tubingensis CR16

Abstract Background Fructose, a monosaccharide, has gained wide applications in food, pharmaceutical and medical industries because of its favourable properties and health benefits. Biocatalytic production of fructose from inulin employing inulinase is the most promising alternative for fructose production. For commercial production, use of immobilized inulinase is advantageous as it offers reutilization of enzyme and increase in stability. In order to meet the demand of concentrated fructose syrup, inulin hydrolysis at high substrate loading is essential. Results Inulinase was immobilized on chitosan particles and employed for fructose production by inulin hydrolysis. Fourier transform infrared spectroscopy (FTIR) analysis confirmed linkage of inulinase with chitosan particles. Immobilized biocatalyst displayed significant increase in thermostability at 60 and 65 °C. Statistical model was proposed with an objective of optimizing enzymatic inulin hydrolytic process. At high substrate loading (17.5 % inulin), using 9.9 U/g immobilized inulinase at 60 °C in 12 h, maximum sugar yield was 171.1 ± 0.3 mg/ml and productivity was 14.25 g/l/h. Immobilized enzyme was reused for ten cycles. Raw inulin from chicory and asparagus was extracted and supplied in 17.5 % for enzymatic hydrolysis as a replacement of pure inulin. More than 70 % chicory inulin and 85 % asparagus inulin were hydrolyzed under optimized parameters at 60 °C. Results of high performance liquid chromatography confirmed the release of fructose after inulin hydrolysis. Conclusions The present findings prove potentiality of immobilized thermostable inulinase from Aspergillus tubingensis CR16 for efficient production of fructose syrup. Successful immobilization of inulinase on chitosan increased its stability and provided the benefit of enzyme reutilization. Box-Behnken design gave a significant model for inulin hydrolysis. Extraction of raw inulin from chicory and asparagus and their enzymatic hydrolysis using immobilized inulinase suggested that it can be a remarkable cost-effective process for large-scale fructose production.

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin.

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

Studies on biodiesel production from Jatropha Curcas oil using chemical and biochemical methods – A mathematical approach

As fossil fuel declining worldwide, attention is grown up to develop a cost effective process for biodiesel production from renewable sources such as Jatropha Curcas Plant. Biodiesel can be produced by transesterification of Jatropha Curcas oil using chemical catalyst or biocatalyst. In this research article, we formulate and analyze two mathematical models of transesterification reaction with respect to chemical catalyst and enzyme catalyst respectively. The model systems are solved numerically by Runge–Kutta method. A comparative mathematical study has been made between these two processes with respect to product efficiency, reaction conditions and mass transfer. We also apply control theoretic approach in mixing intensity through biocatalytic process for cost effective production of biodiesel from Jatropha Curcas oil. The validity of both the models is established by experimental results available in literatures.

Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin–superoxide dismutase–catalase–carbonic anhydrase

We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb–SOD–CAT–CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol–chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb–SOD–CAT–CA to 2, 4, or 6 times that of RBC.

Novel binder-less hierarchical ZSM-5 catalyst for olefins catalytic cracking to produce propylene

Developing new technology for high efficiency conversion of petroleum is great significance to maintain sustainable development of petrochemical industry. Due to the growing demand for propylene and abundant supply of C4/C5 hydrocarbon, new cost effective process for production of propylene by conversion of streams containing C4/C5 olefins is required. A novel binderless hierarchical ZSM-5 catalyst and OCC process, which produces propylene by catalytic cracking of C4/C5 olefins, were developed by Sinopec through ten-years research and development. A commercial OCC unit with C4/C5 hydrocarbon capacity 60 kt/a started up in Puyang, China in 2009. Novel binder-less hierarchical ZSM-5 catalyst, as a new catalytic material, commercialized for the first time on the world. As a new route for on-purpose propylene production, OCC boosts the development of propylene production technology.