Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors.

Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55-2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2 /L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.

Simultaneous Clostridial fermentation, lipase-catalyzed esterification, and ester extraction to enrich diesel with butyl butyrate.

The recovery of 1-butanol from fermentation broth is energy-intensive since typical concentrations in fermentation broth are below 20 g L(-1). To prevent butanol inhibition and high downstream processing costs, we aimed at producing butyl esters instead of 1-butanol. It is shown that it is possible to perform simultaneously clostridial fermentation, esterification of the formed butanol to butyl butyrate, and extraction of this ester by hexadecane. The very high partition coefficient of butyl butyrate pulls the esterification towards the product side even at fermentation pH and relatively low butanol concentrations. The hexadecane extractant is a model diesel compound and is nontoxic to the cells. If butyl butyrate enriched diesel can directly be used as car fuel, no product recovery is required. A proof-of-principle experiment for the one-pot bio-ester production from glucose led to 5 g L(-1) butyl butyrate in the hexadecane phase. The principle may be extended to a wide range of esters, especially to longer chain ones.

Multi-dimensional fractionation and characterization of crude protein mixtures: toward establishment of a database of protein purification process development parameters.

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.

Bioprocess intensification: Cases that (don't) work.

Development of affordable and low carbon biobased manufacturing depends critically on strategies that reduce cost and emission profiles. This paper indicates that efforts around the reduction of capital costs by intensification of process equipment need to be carefully weighed against the inherently fast increasing financial and climate costs of driving forces used for the intensification. The fundamental relation between capital expenditures (CAPEX) and operational expenditures (OPEX) of intensified and non-intensified biobased processes and their financial and climatic impacts are emphasized and provisionally explored for a few industrial processes. General learnings flag the importance in particular of OPEX minimisation for sustainable bio-economic development.

A possible influence of extracellular polysaccharides on the analysis of intracellular metabolites from Trichoderma harzianum grown under carbon-limited conditions.

Intracellular metabolites were evaluated during the continuous growth of Trichoderma harzianum P49P11 under carbon-limited conditions. Four different conditions in duplicate were investigated (10 and 20 g/L of glucose, 5.26/5.26 g/L of fructose/glucose and 10 g/L of sucrose in the feed). Differences in the values of some specific concentrations of intracellular metabolites were observed at steady-state for the duplicates. The presence of extracellular polysaccharide was confirmed in the supernatant of all conditions based on FT-IR and proton NMR. Fragments of polysaccharides from the cell wall could be released due to the shear stress and since the cells can consume them under carbon-limited conditions, this could create an unpredictable carbon flow rate into the cells. According to the values of the metabolite concentrations, it was considered that the consumption of those fragments was interfering with the analysis.

Continuous production of enzymes under carbon-limited conditions by Trichoderma harzianum P49P11.

Carbon-limited chemostat cultures were performed using different carbon sources (glucose, 10 and 20 g/L; sucrose, 10 g/L; fructose/glucose, 5.26/5.26 g/L; carboxymethyl cellulose, 10 g/L; and carboxymethyl cellulose/glucose, 5/5 g/L) to verify the capability of the wild type strain Trichoderma harzianum to produce extracellular enzymes. All chemostat cultures were carried out at a fixed dilution rate of 0.05 h-1. Experiments using glucose, fructose/glucose and sucrose were performed in duplicate. Glucose condition was found to induce the production of enzymes that can catalyse the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside (PNPGase). A concentration of 20 g/L of glucose in the feed provided the highest productivity (1048 ± 16 U/mol h). Extracellular polysaccharides were considered the source of inducers. Based on the obtained results, a new PNPGase production process was developed using mainly glucose. This process raises interesting possibilities of synthesizing the inducer substrate and the induced enzymes in a single step using an easily assimilated carbon source under carbon-limited conditions.

Enzymatic Hydrolysis of Sugarcane Bagasse in Aqueous Two-Phase Systems (ATPS): Exploration and Conceptual Process Design.

The enzymatic conversion of lignocellulosic material to sugars can provide a carbon source for the production of energy (fuels) and a wide range of renewable products. However, the efficiency of this conversion is impaired due to product (sugar) inhibition. Even though several studies investigate how to overcome this challenge, concepts on the process to conduct the hydrolysis are still scarce in literature. Aqueous two-phase systems (ATPS) can be applied to design an extractive reaction due to their capacity to partition solutes to different phases in such a system. This work presents strategies on how to conduct extractive enzymatic hydrolysis in ATPS and how to explore the experimental results in order to design a feasible process. While only a limited number of ATPS was explored, the methods and strategies described could easily be applied to any further ATPS to be explored. We studied two promising ATPS as a subset of a previously high throughput screened large set of ATPS, providing two configurations of processes having the reaction in either the top phase or in the bottom phase. Enzymatic hydrolysis in these ATPS was performed to evaluate the partitioning of the substrate and the influence of solute partitioning on conversion. Because ATPS are able to partition inhibitors (sugar) between the phases, the conversion rate can be maintained. However, phase forming components should be selected to preserve the enzymatic activity. The experimental results presented here contribute to a feasible ATPS-based conceptual process design for the enzymatic conversion of lignocellulosic material.

Analysis of the proteins secreted by Trichoderma harzianum P49P11 under carbon-limited conditions.

The wild type strain Trichoderma harzianum was able to synthesize enzymes that can catalyse the hydrolysis of p-nitrophenyl-ß-D-glucopyranoside (PNPGase) in glucose-limited chemostat cultures. Fructose/glucose and sucrose conditions provided low levels of PNPGase activity. To investigate whether under these conditions other enzymes were produced, a shotgun proteomics analysis of their supernatants was performed. The analysis has indicated that the different carbon sources used influenced the amounts of proteins secreted including 1,3-beta-glucanosyltransferase, alpha-1,2-mannosidase, alpha-galactosidase and glucan 1,3-beta-glucosidase. The analysis has also suggested the presence of beta-glucosidase, which could also be represented by PNPGase activity. Intracellular metabolites were quantified during PNPGase production for the condition using 20 g/L of glucose in the feed and differences were observed, indicating that intracellular glucose could be inhibiting PNPGase production. SIGNIFICANCE: This work shows that sugars such as glucose, fructose/glucose and sucrose can be used as substrates for the continuous synthesis of different enzymes under carbon-limited conditions by Trichoderma harzianum. As far as we know, this is the first work about the continuous synthesis of enzymes under carbon-limited conditions suggesting that different easily assimilated carbon sources can be used to generate different enzymatic cocktails. Each enzyme or uncharacterized protein suggested by shotgun proteomics has the potential to become a promising product for biotechnological applications.

Protein self-interaction chromatography on a microchip.

This paper presents the development of a novel miniaturized experimental procedure for the measurement of protein-protein interactions through Self-Interaction Chromatography (SIC) on a microchip, without the use of chromatographic resins. SIC was recently demonstrated to be a relatively easy method to obtain quantitative thermodynamic information about protein-protein interactions, like the osmotic second virial coefficient B(22), which relates to protein phase behavior including protein crystallization. This successful miniaturization to microchip level of a measurement device for protein self-interaction data is a first key step to a complete microfluidic screening platform for the rational design of protein crystallizations, using substantially less expensive protein and experimentation time.

Precipitation of porcine insulin with carbon dioxide.

Recent works have pointed to the use of volatile electrolytes such as carbon dioxide (CO2) dissolved in aqueous solutions as a promising alternative to the precipitating agents conventionally used for protein recovery in the food and pharmaceutical industries. In this work we investigated experimental and theoretical aspects of the precipitation of porcine insulin, a biomolecule of pharmaceutical interest, using CO2 as an acid-precipitating agent. The solubility of porcine insulin in NaHCO3 solutions in pressurized CO2 was determined as a function of temperature and pressure, with a minimum being observed close to the protein isoelectric point. A thermodynamic model was developed and successfully utilized to correlate the experimental data. Insulin was considered a polyelectrolyte in the model and its self-association reactions were also taken into account. The biological activity of insulin was maintained after precipitation with CO2, although some activity can be lost if foam is formed in the depressurization step.

Integrated Vacuum Stripping and Adsorption for the Efficient Recovery of (Biobased) 2-Butanol.

Biobased 2-butanol offers high potential as biofuel, but its toxicity toward microbial hosts calls for efficient techniques to alleviate product inhibition in fermentation processes. Aiming at the selective recovery of 2-butanol, the feasibility of a process combining in situ vacuum stripping followed by vapor adsorption has been assessed using mimicked fermentation media. The experimental vacuum stripping of model solutions and corn stover hydrolysate closely aligned with mass transfer model predictions. However, the presence of lignocellulosic impurities affected 2-butanol recovery yields resulting from vapor condensation, which decreased from 96 wt % in model solutions to 40 wt % using hydrolysate. For the selective recovery of 2-butanol from a vapor mixture enriched in water and carbon dioxide, silicalite materials were the most efficient, particularly at low alcohol partial pressures. Integrating in situ vacuum stripping with vapor adsorption using HiSiv3000 proved useful to effectively concentrate 2-butanol above its azeotropic composition (>68 wt %), facilitating further product purification.

A robotic platform to screen aqueous two-phase systems for overcoming inhibition in enzymatic reactions.

Aqueous two-phase systems (ATPS) can be applied to enzymatic reactions that are affected by product inhibition. In the biorefinery context, sugars inhibit the cellulolytic enzymes in charge of converting the biomass. Here, we present a strategy to select an ATPS (formed by polymer and salt) that can separate sugar and enzymes. This automated and miniaturized method is able to determine phase diagrams and partition coefficients of solutes in these. Tailored approaches to quantify the solutes are presented, taking into account the limitations of techniques that can be applied with ATPS due to the interference of phase forming components with the analytics. The developed high-throughput (HT) platform identifies suitable phase forming components and the tie line of operation. This fast methodology proposes to screen up to six different polymer-salt systems in eight days and supplies the results to understand the influence of sugar and protein concentrations on their partition coefficients.

Options for biochemical production of 4-hydroxybutyrate and its lactone as a substitute for petrochemical production.

Options are discussed for biochemical production of 4-hydroxybutyrate (4-HB) and its lactone, gamma-butyrolactone (GBL), from renewable sources. In the first part of the study, the thermodynamic feasibility of four potential metabolic pathways from glucose to 4-HB are analyzed. The calculations reveal that when the pathways are NAD(+) dependent the intermediate succinate semialdehyde (SSA) accumulates leading to low 4-HB yields at equilibrium. For NADP(+) dependent pathways the calculated yield of 4-HB improves, up to almost 100%. In the second part of this study, continuous removal of 4-HB from the solution is considered to shift SSA conversion into 4-HB so that SSA accumulation is minimized. One option is the enzymatic production of GBL from 4-HB. Candida antarctica Lipase B shows good lactonization rates at pH 4, but unfortunately this conversion cannot be performed in-vivo during 4-HB production because of the neutral intracellular pH.

Solvent-impregnated resins as an in situ product recovery tool for phenol recovery from Pseudomonas putida S12TPL fermentations.

The sustainable production of fine/bulk chemicals is often hampered by product toxicity and inhibition to the producing micro-organisms. Consequently, the product must be removed from the micro-organisms' environment. To achieve this, so-called solvent-impregnated resins (SIRs) as well as commercial resins have been added to a Pseudomonas putida S12TPL fermentation that produces phenol as a model compound from glucose. The SIRs contained an ionic liquid which extracts phenol effectively. It was observed that the addition of these particles resulted in an increased phenol production of more than a fourfold while the commercial resin (XAD-4) which is widely used in aromatic removal from aqueous phases, only gave a 2.5-fold increase in volumetric production.

Lab-scale fermentation tests of microchip with integrated electrochemical sensors for pH, temperature, dissolved oxygen and viable biomass concentration.

This article shows the development and testing of a microchip with integrated electrochemical sensors for measurement of pH, temperature, dissolved oxygen and viable biomass concentration under yeast cultivation conditions. Measurements were done both under dynamic batch conditions as well as under prolonged continuous cultivation conditions. The response of the sensors compared well with conventional measurement techniques. The biomass sensor was based on impedance spectroscopy. The results of the biomass sensor matched very well with dry weight measurements and showed a limit of detection of approximately 1 g/L. The dissolved oxygen concentration was monitored amperometrically using an ultra-microelectrode array, which showed an accuracy of approximately 0.2 mg/L and negligible drift. pH was monitored using an ISFET with an accuracy well below 0.1 pH unit. The platinum thin-film temperature resistor followed temperature changes with approximately 0.1 degrees C accuracy. The dimensions of the multi sensor chip are chosen as such that it is compatible with the 96-well plate format.

Selection of pH-related parameters in ion-exchange chromatography using pH-gradient operations.

This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.5 or anion-exchange chromatography (AEX) at pH 7.2 without lowering the salt concentration in the CCS. The performance of both CEX and AEX was predicted to be equal for this particular MAb capture.

Perspectives for the microbial production of methyl propionate integrated with product recovery.

A new approach was studied for bio-based production of methyl propionate, a precursor of methyl methacrylate. Recombinant E. coli cells were used to perform a cascade reaction in which 2-butanol is reduced to butanone using alcohol dehydrogenase, and butanone is oxidized to methyl propionate and ethyl acetate using a Baeyer-Villiger monooxygenase (BVMO). Product was removed by in situ stripping. The conversion was in line with a model comprising product formation and stripping kinetics. The maximum conversion rates were 1.14 g-butanone/(L h), 0.11 g-ethyl acetate/(L h), and 0.09 g-methyl propionate/(L h). The enzyme regioselectivity towards methyl propionate was 43% of total ester. Starting from biomass-based production of 2-butanol, full-scale ester production with conventional product purification was calculated to be competitive with petrochemical production if the monooxygenase activity and regioselectivity are enhanced, and the costs of bio-based 2-butanol are minimized.

Integration of Gas Enhanced Oil Recovery in Multiphase Fermentations for the Microbial Production of Fuels and Chemicals.

In multiphase fermentations where the product forms a second liquid phase or where solvents are added for product extraction, turbulent conditions disperse the oil phase as droplets. Surface-active components (SACs) present in the fermentation broth can stabilize the product droplets thus forming an emulsion. Breaking this emulsion increases process complexity and consequently the production cost. In previous works, it has been proposed to promote demulsification of oil/supernatant emulsions in an off-line batch bubble column operating at low gas flow rate. The aim of this study is to test the performance of this recovery method integrated to a fermentation, allowing for continuous removal of the oil phase. A 500 mL bubble column is successfully integrated with a 2 L reactor during 24 h without affecting cell growth or cell viability. However, higher levels of surfactants and emulsion stability are measured in the integrated system compared to a base case, reducing its capacity for oil recovery. This is related to release of SACs due to cellular stress when circulating through the recovery column. Therefore, it is concluded that the gas bubble-induced oil recovery method allows for oil separation and cell recycling without compromising fermentation performance; however, tuning of the column parameters considering increased levels of SACs due to cellular stress is required for improving oil recovery.

pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations.

This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.

Optimization of biopharmaceutical downstream processes supported by mechanistic models and artificial neural networks.

Downstream process development is a major area of importance within the field of bioengineering. During the design of such a downstream process, important decisions have to be made regarding the type of unit operations as well as their sequence and their operating conditions. Current computational approaches addressing these issues either show a high level of simplification or struggle with computational speed. Therefore, this article presents a new approach that combines detailed mechanistic models and speed-enhancing artificial neural networks. This approach was able to simultaneously optimize a process with three different chromatographic columns toward yield with a minimum purity of 99.9%. The addition of artificial neural networks greatly accelerated this optimization. Due to high computational speed, the approach is easily extendable to include more unit operations. Therefore, it can be of great help in the acceleration of downstream process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:696-707, 2017.