Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

Bioprocess technology is a multidisciplinary industry that combines knowledge of biology and chemistry with process engineering. It is a growing industry because its applications have an important role in the food, pharmaceutical, diagnostics and chemical industries. In addition, the current pressure to decrease our dependence on fossil fuels motivates new, innovative research in the replacement of petrochemical products. Bioprocesses are processes that utilize cells and/or their components in the production of desired products. Bioprocesses are already used to produce fuels and chemicals, especially ethanol and building-block chemicals such as carboxylic acids. In order to enable more efficient, sustainable and economically feasible bioprocesses, the raw materials must be cheap and the bioprocesses must be operated at optimal conditions. It is essential to measure different parameters that provide information about the process conditions and the main critical process parameters including cell density, substrate concentrations and products. In addition to offline analysis methods, online monitoring tools are becoming increasingly important in the optimization of bioprocesses. Capillary electrophoresis (CE) is a versatile analysis technique with no limitations concerning polar solvents, analytes or samples. Its resolution and efficiency are high in optimized methods creating a great potential for rapid detection and quantification. This work demonstrates the potential and possibilities of CE as a versatile bioprocess monitoring tool. As a part of this study a commercial CE device was modified for use as an online analysis tool for automated monitoring. The work describes three offline CE analysis methods for the determination of carboxylic, phenolic and amino acids that are present in bioprocesses, and an online CE analysis method for the monitoring of carboxylic acid production during bioprocesses. The detection methods were indirect and direct UV, and laser-induced frescence. The results of this work can be used for the optimization of bioprocess conditions, for the development of more robust and tolerant microorganisms, and to study the dynamics of bioprocesses.

Beyond defoaming:the effects of antifoams on bioprocesses productivity

Antifoams are often added to bioprocesses with little knowledge of their impact on the cells or product. However, it is known that certain antifoams can affect the growth rates of both prokaryotic and eukaryotic organisms in addition to changing surface properties such as lipid content, resulting in changes to permeability. This in turn can be beneficial to a recombinant protein production system for soluble proteins, as has been demonstrated by increased secretion of a-amylase and GFP, or achievement of greater yields of protein due to increased biomass. However, in some cases, certain concentrations of antifoams appear to have a detrimental effect upon cells and protein production, and the effects vary depending upon the protein being expressed. These findings emphasise the importance of optimising and understanding antifoam addition to bioprocesses.

Comparison of oxygen mass transfer coefficient in simple and extractive fermentation systems

Aeration and agitation are important variables to ensure effective oxygen transfer rate during aerobic bioprocesses: therefore, the knowledge of the volumetric mass transfer coefficient (k(L)a) is required. In view of selecting the optimum oxygen requirements for extractive fermentation in aqueous two-phase system (ATPS), the k(L)a values in a typical ATPS medium were compared in this work with those in distilled water and in a simple fermentation medium. in the absence of biomass. Aeration and agitation were selected as the independent variables using a 2(2) full factorial design. Both variables showed statistically significant effects on k(L)a, and the highest values of this parameter in both media for simple fermentation (241 s(-1)) and extractive fermentation with ATPS (70.3 s(-1)) were observed at the highest levels of aeration (5 vvm) and agitation (1200 rpm). The k(L)a values were then used to establish mathematical correlations of this response as a function of the process variables. The exponents of the power number (N(3)D(2)) and superficial gas velocity (V(s)) determined in distilled water (alpha = 0.39 and beta = 0.47, respectively) were in reasonable agreement with the ones reported in the literature for several aqueous systems and close to those determined for a simple fermentation medium (alpha=0.38 and beta=0.41). On the other hand, as expected by the increased viscosity in the presence of polyethylene glycol, their values were remarkably higher in a typical medium for extractive fermentation (alpha=0.50 and beta=1.0). A reasonable agreement was found between the experimental data of k(L)a for the three selected systems and the values predicted by the theoretical models, under a wide range of operational conditions. (C) 2009 Elsevier B.V. All rights reserved.

Aqueous two-phasemicellar systems in an oscillatory flowmicro-reactor: study of perspectives and experimental performance

BACKGROUND: Aqueous two-phase micellar systems (ATPMS) are micellar surfactant solutions with physical properties that make them very efficient for the extraction/concentration of biological products. In this work the main proposal that has been discussed is the possible applicability and importance of a novel oscillatory flow micro-reactor (micro-OFR) envisaged for parallel screening and/or development of industrial bioprocesses in ATPMS. Based on the technology of oscillatory flow mixing (OFM), this batch or continuous micro-reactor has been presented as a new small-scale alternative for biological or physical-chemical applications. RESULTS: ATPMS experiments were carried out in different OFM conditions (times, temperatures, oscillation frequencies and amplitudes) for the extraction of glucose-6-phosphate dehydrogenase (G6PD) in Triton X-114/buffer with Cibacron Blue as affinity ligand. CONCLUSION: The results suggest the potential use of OFR, considering this process a promising and new alternative for the purification or pre-concentration of bioproducts. Despite the applied homogenization and extraction conditions have presented no improvements in the partitioning selectivity of the target enzyme, when at rest temperature they have influenced the partitioning behavior in Triton X-114 ATPMS. (C) 2011 Society of Chemical Industry

Wall material properties of yeast cells. Part II. Analysis

In the preceding paper (Part I) force-deformation data were measured with the compression experiment in conjunction with the initial radial stretch ratio and the initial wall-thickness to cell-radius ratio for baker's yeast (Saccharomyces cerevisiae). In this paper, these data have been analysed with the mechanical model of Smith et al. (Smith, Moxham & Middelberg (1998) Chemical Engineering Science, 53, 3913-3922) with the wall constitutive behaviour defined a priori as incompressible and linear-elastic. This analysis determined the mean Young's modulus ((E) over bar), mean maximum von Mises stress-at-failure (<(sigma)over bar>(VM,f)) and mean maximum von Mises strain-at failure (<(epsilon)over bar>(VM,f)) to be (E) over bar = 150 +/- 15 MPa, <(sigma)over bar>(VM,f) = 70 +/- 4 MPa and <(epsilon)over bar>(VM,f) = 0.75 +/- 0.08, respectively. The mean Young's modulus was not dependent (P greater than or equal to 0.05) on external osmotic pressure (0-0.8 MPa) nor compression rate (1.03-7.68 mu m/s) suggesting the incompressible linear-elastic relationship is representative of the actual cell-wall constitutive behaviour. Hydraulic conductivities were also determined and were comparable to other similar cell types (0-2.5 mu m/MPa s). The hydraulic conductivity distribution was not dependent on external osmotic pressure (0-0.8 MPa) nor compression rate (1.03-7.68 mu m/s) suggesting inclusion of cell-wall permeability in the mechanical model is justified. <(epsilon)over bar>(VM,f) was independent of cell diameter and to a first-approximation unaffected (P greater than or equal to 0.01) by external osmotic pressure and compression rate, thus providing a reasonable failure criterion. This criterion states that the cell-wall material will break when the strain exceeds <(epsilon)over bar>(VM,f) = 0.75 +/- 0.08. Variability in overall cell strength during compression was shown to be primarily due to biological variability in the maximum von Mises strain-at-failure. These data represent the first estimates of cell-wall material properties for yeast and the first fundamental analysis of cell-compression data. They are essential for describing cell-disruption at the fundamental level of fluid-cell interactions in general bioprocesses. They also provide valuable new measurements for yeast-cell physiologists. (C) 2000 Elsevier Science Ltd. All rights reserved.

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.

Fractionation of follicle stimulating hormone charge isoforms in their native form by preparative electrophoresis technology

Complex glycoprotein biopharmaceuticals, such as follicle stimulating hormone (FSH), erythropoietin and tissue plasminogen activator consist of a range of charge isoforms due to the extent of sialic acid capping of the glycoprotein glycans. Sialic acid occupies the terminal position on the oligosaccharide chain, masking the penultimate sugar residue, galactose from recognition and uptake by the hepatocyte asialoglycoprotein receptor. It is therefore well established that the more acidic charge isoforms of glycoprotein biopharmaceuticals have higher in vivo potencies than those of less acidic isoforms due to their longer serum half-life. Current strategies for manipulating glycoprotein charge isoform profile involve cell engineering or altering bioprocesss parameters to optimise expression of more acidic or basic isoforms, rather than downstream separation of isoforms. A method for the purification of a discrete range of bioactive recombinant human FSH (rhFSH) charge isoforms based on Gradiflow(TM) preparative electrophoresis technology is described. Gradiflow(TM) electrophoresis is scaleable, and incorporation into glycoprotein biopharmaceutical production bioprocesses as a potential final step facilitates the production of biopharmaceutical preparations of improved in vivo potency. (C) 2005 Elsevier B.V. All rights reserved.

Design analysis for refolding monomeric protein

Renaturation of protein expressed as inclusion bodies within Escherichia coli is a key step in many bioprocesses. Operating conditions for the refolding step dramatically affect the amount of protein product recovered, and hence profoundly influence the process economics. The first systematic comparison of refolding conducted in batch, fed-batch and continuous stirred-tank reactors is provided Refolding is modeled as kinetic competition between first-order refolding (equilibrium reaction) and irreversible aggregation (second-order). Simulations presented allow direct comparison between different flowsheets and refolding schemes using a dimensionless economic objective. As expected from examination of the reaction kinetics, batch operation is the most inefficient merle. For the base process considered, the overall cost of fed-batch and continuous refolding is virtually identical (less than half that of the batch process). Reactor selection and optimization of refolding using overall economics are demonstrated to be vitally important.

In situ NIR spectroscopy monitoring of plasmid production processes effect of producing strain, medium composition and the cultivation strategy

BACKGROUNDWhile the pharmaceutical industry keeps an eye on plasmid DNA production for new generation gene therapies, real-time monitoring techniques for plasmid bioproduction are as yet unavailable. This work shows the possibility of in situ monitoring of plasmid production in Escherichia coli cultures using a near infrared (NIR) fiber optic probe. RESULTSPartial least squares (PLS) regression models based on the NIR spectra were developed for predicting bioprocess critical variables such as the concentrations of biomass, plasmid, carbon sources (glucose and glycerol) and acetate. In order to achieve robust models able to predict the performance of plasmid production processes, independently of the composition of the cultivation medium, cultivation strategy (batch versus fed-batch) and E. coli strain used, three strategies were adopted, using: (i) E. coliDH5 cultures conducted under different media compositions and culture strategies (batch and fed-batch); (ii) engineered E. coli strains, MG1655endArecApgi and MG1655endArecA, grown on the same medium and culture strategy; (iii) diverse E. coli strains, over batch and fed-batch cultivations and using different media compositions. PLS models showed high accuracy for predicting all variables in the three groups of cultures. CONCLUSIONNIR spectroscopy combined with PLS modeling provides a fast, inexpensive and contamination-free technique to accurately monitoring plasmid bioprocesses in real time, independently of the medium composition, cultivation strategy and the E. coli strain used.

Modeling and optimization of extracellular polysaccharides production by Enterobacter A47

Polysaccharides are gaining increasing attention as potential environmental friendly and sustainable building blocks in many fields of the (bio)chemical industry. The microbial production of polysaccharides is envisioned as a promising path, since higher biomass growth rates are possible and therefore higher productivities may be achieved compared to vegetable or animal polysaccharides sources. This Ph.D. thesis focuses on the modeling and optimization of a particular microbial polysaccharide, namely the production of extracellular polysaccharides (EPS) by the bacterial strain Enterobacter A47. Enterobacter A47 was found to be a metabolically versatile organism in terms of its adaptability to complex media, notably capable of achieving high growth rates in media containing glycerol byproduct from the biodiesel industry. However, the industrial implementation of this production process is still hampered due to a largely unoptimized process. Kinetic rates from the bioreactor operation are heavily dependent on operational parameters such as temperature, pH, stirring and aeration rate. The increase of culture broth viscosity is a common feature of this culture and has a major impact on the overall performance. This fact complicates the mathematical modeling of the process, limiting the possibility to understand, control and optimize productivity. In order to tackle this difficulty, data-driven mathematical methodologies such as Artificial Neural Networks can be employed to incorporate additional process data to complement the known mathematical description of the fermentation kinetics. In this Ph.D. thesis, we have adopted such an hybrid modeling framework that enabled the incorporation of temperature, pH and viscosity effects on the fermentation kinetics in order to improve the dynamical modeling and optimization of the process. A model-based optimization method was implemented that enabled to design bioreactor optimal control strategies in the sense of EPS productivity maximization. It is also critical to understand EPS synthesis at the level of the bacterial metabolism, since the production of EPS is a tightly regulated process. Methods of pathway analysis provide a means to unravel the fundamental pathways and their controls in bioprocesses. In the present Ph.D. thesis, a novel methodology called Principal Elementary Mode Analysis (PEMA) was developed and implemented that enabled to identify which cellular fluxes are activated under different conditions of temperature and pH. It is shown that differences in these two parameters affect the chemical composition of EPS, hence they are critical for the regulation of the product synthesis. In future studies, the knowledge provided by PEMA could foster the development of metabolically meaningful control strategies that target the EPS sugar content and oder product quality parameters.

Modification of microfiltration membranes by hydrogel impregnation for pDNA purification

The huge efforts for the achievement of highly purified biomolecules are growing every day. A great number of efficient techniques, such as chromatography, are already available in laboratory for separation processes. However, membrane-based technologies are the best match to assure simplicity, efficiency and easy scale-up procedures. Herein we report the modification of a commercial microfiltration membrane for plasmid DNA purification by agarose gel impregnation. The membrane was characterized by SEM, ATR-FTIR, EDS, contact angle, and porosity measurements. Additionally, the membrane pore radius was estimated from observed rejections of different proteins and with that information the rejection of a 6050 bp plasmid DNA (pDNA) molecule was estimated for different values of flux using a theoretical model of large flexible molecules in membranes with parallel cylindrical pores, which is applicable to pDNA ultrafiltration in conventional membranes, as recently shown in the literature. The experimental results show that the modified membrane has higher pDNA rejections than the predicted by the model, suggesting that the different type of porous structure that a hydrogel has, may have a positive effect on pDNA rejections as compared to other biomolecules with more rigid structures, making this type of modified membranes potential better candidates to be used for the selective recovery of pDNA in this type of bioprocesses.

Carbon materials as new generation of new electron shuttles for the anaerobic degradation of environmental xenobiotics

Tese de Doutoramento em Engenharia Química e Biológica

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite (R) 545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI. 3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I. 3, requiring a refolding step, was poorly immobilized on all supports tested ( best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.

The chemistry involved in the steam treatment of lignocellulosic materials

Pretreatment of lignocellulosic materials is essential for bioconversion because of the various physical and chemical barriers that greatly inhibit their susceptibility to bioprocesses such as hydrolysis and fermentation. The aim of this article is to review some of the most important pretreatment methods developed to date to enhance the conversion of lignocellulosics. Steam explosion, which precludes the treatment of biomass with high-pressure steam under optimal conditions, is presented as the pretreatment method of choice and its mode of action on lignocellulosics is discussed. The optimal pretreatment conditions for a given plant biomass are defined as those in which the best substrate for hydrolysis is obtained with the least amount of soluble sugars lost to side reactions such as dehydration. Therefore, pretreatment optimization results from a compromise between two opposite trends because hemicellulose recovery in acid hydrolysates can only be maximized at lower pretreatment severities, whereas the development of substrate accessibility requires more drastic pretreatment conditions in which sugar losses are inevitable. To account for this heterogeneity, the importance of several process-oriented parameters is discussed in detail, such as the pretreatment temperature, residence time into the steam reactor, use of an acid catalyst, susceptibility of the pretreated biomass to bioconversion, and process design.

Química Sem Fronteiras: o desafio da energia

Coal, natural gas and petroleum-based liquid fuels are still the most widely used energy sources in modern society. The current scenario contrasts with the foreseen shortage of petroleum that was spread out in the beginning of the XXI century, when the concept of "energy security" emerged as an urgent agenda to ensure a good balance between energy supply and demand. Much beyond protecting refineries and oil ducts from terrorist attacks, these issues soon developed to a portfolio of measures related to process sustainability, involving at least three fundamental dimensions: (a) the need for technological breakthroughs to improve energy production worldwide; (b) the improvement of energy efficiency in all sectors of modern society; and (c) the increase of the social perception that education is a key-word towards a better use of our energy resources. Together with these technological, economic or social issues, "energy security" is also strongly influenced by environmental issues involving greenhouse gas emissions, loss of biodiversity in environmentally sensitive areas, pollution and poor solid waste management. For these and other reasons, the implementation of more sustainable practices in our currently available industrial facilities and the search for alternative energy sources that could partly replace the fossil fuels became a major priority throughout the world. Regarding fossil fuels, the main technological bottlenecks are related to the exploitation of less accessible petroleum resources such as those in the pre-salt layer, ranging from the proper characterization of these deep-water oil reservoirs, the development of lighter and more efficient equipment for both exploration and exploitation, the optimization of the drilling techniques, the achievement of further improvements in production yields and the establishment of specialized training programs for the technical staff. The production of natural gas from shale is also emerging in several countries but its production in large scale has several problems ranging from the unavoidable environmental impact of shale mining as well as to the bad consequences of its large scale exploitation in the past. The large scale use of coal has similar environmental problems, which are aggravated by difficulties in its proper characterization. Also, the mitigation of harmful gases and particulate matter that are released as a result of combustion is still depending on the development of new gas cleaning technologies including more efficient catalysts to improve its emission profile. On the other hand, biofuels are still struggling to fulfill their role in reducing our high dependence on fossil fuels. Fatty acid alkyl esters (biodiesel) from vegetable oils and ethanol from cane sucrose and corn starch are mature technologies whose market share is partially limited by the availability of their raw materials. For this reason, there has been a great effort to develop "second-generation" technologies to produce methanol, ethanol, butanol, biodiesel, biogas (methane), bio-oils, syngas and synthetic fuels from lower grade renewable feedstocks such as lignocellulosic materials whose consumption would not interfere with the rather sensitive issues of food security. Advanced fermentation processes are envisaged as "third generation" technologies and these are primarily linked to the use of algae feedstocks as well as other organisms that could produce biofuels or simply provide microbial biomass for the processes listed above. Due to the complexity and cost of their production chain, "third generation" technologies usually aim at high value added biofuels such as biojet fuel, biohydrogen and hydrocarbons with a fuel performance similar to diesel or gasoline, situations in which the use of genetically modified organisms is usually required. In general, the main challenges in this field could be summarized as follows: (a) the need for prospecting alternative sources of biomass that are not linked to the food chain; (b) the intensive use of green chemistry principles in our current industrial activities; (c) the development of mature technologies for the production of second and third generation biofuels; (d) the development of safe bioprocesses that are based on environmentally benign microorganisms; (e) the scale-up of potential technologies to a suitable demonstration scale; and (f) the full understanding of the technological and environmental implications of the food vs. fuel debate. On the basis of these, the main objective of this article is to stimulate the discussion and help the decision making regarding "energy security" issues and their challenges for modern society, in such a way to encourage the participation of the Brazilian Chemistry community in the design of a road map for a safer, sustainable and prosper future for our nation.