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Preview: Acta Biotechnologica

Engineering in Life Sciences

Wiley Online Library : Engineering in Life Sciences

Published: 2017-09-01T00:00:00-05:00


Optimization of cell culture-derived influenza A virus particles purification using sulfated cellulose membrane adsorbers


Downstream processing remains one of the biggest challenges in manufacturing of biologicals and vaccines. This work focuses on a Design of Experiments approach to understand factors influencing the performance of sulfated cellulose membrane adsorbers for the chromatographic purification of a cell culture-derived H1N1 influenza virus strain (A/Puerto Rico/8/34). Membranes with a medium ligand density together with low conductivity and a high virus titer in the feed stream resulted in optimum virus yields and low protein and DNA content in the product fraction. Flow rate and salt concentration in the buffer used for elution were of secondary importance while membrane permeability had no significant impact on separation performance. A virus loss of 2.1% in the flow through, a yield of 57.4% together with a contamination level of 5.1 pgDNA HAU−1 and 1.2 ngprot HAU−1 were experimentally confirmed for the optimal operating point predicted. The critical process parameters identified and their optimal settings should support the optimization of sulfated cellulose membrane adsorbers based purification trains for other influenza virus strains, streamlining cell culture-derived vaccine manufacturing. This article is protected by copyright. All rights reserved

Conversion of Glucose-Xylose Mixtures to Pyruvate using a Consortium of Metabolically Engineered Escherichia coli


Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose-xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed-batch process, both sugars in a glucose-xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g. This article is protected by copyright. All rights reserved

Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator


PHBs have attracted much attention due to their biodegradability and biocompatibility properties. The main drawback to the commercial production of them is their high cost. The recovery of PHB from bacterial cytoplasm significantly increases total processing costs. Efficient, economical and environment-friendly extraction of PHB from cells is required for its industrial production. In the present study, several non-halogenated organic solvents (ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, and acetic acid) were examined for their efficacy regarding recovery at different temperatures from culture broth containing Cupriavidus necator cells. The highest recovery percentage (98.6 %) and product purity (up to 98%) were seen to be those of ethylene carbonate-assisted extraction at 150°C within 60 min of incubation time. Average molecular weight of the recovered PHB (1.3 × 106) was not significantly affected by the extraction solvent and conditions. The melting point of PHB extracted using ethylene carbonate was measured to be 176.2°C with an enthalpy of fusion of 16.8 % and the corresponding degree of crystallinity of 59.2 %. NMR and GC analyses confirmed that the extracted biopolymer was PHB. The presented strategy can help researchers to reduce the cost to obtain the final product. This article is protected by copyright. All rights reserved

Advanced monitoring and control of pharmaceutical production processes with Pichia pastoris by using Raman spectroscopy and multivariate calibration methods


This contribution includes an investigation of the applicability of Raman spectroscopy as a PAT analyzer in cyclic production processes of a potential Malaria vaccine with Pichia pastoris. In a feasibility study, Partial Least Squares Regression (PLSR) models were created off-line for cell density and concentrations of glycerol, methanol, ammonia and total secreted protein. Relative cross validation errors RMSEcvrel range from 2.87 % (glycerol) to 11.0 % (ammonia). In the following, on-line bioprocess monitoring was tested for cell density and glycerol concentration. By using the nonlinear Support Vector Regression (SVR) method instead of PLSR, the error RMSEPrel for cell density was reduced from 5.01 % to 2.94 %. The high potential of Raman spectroscopy in combination with multivariate calibration methods was demonstrated by the implementation of a closed loop control for glycerol concentration using PLSR. The strong nonlinear behavior of exponentially increasing control disturbances was met with a feed-forward control and adaptive correction of control parameters. In general the control procedure works very well for low cell densities. Unfortunately, PLSR models for glycerol concentration are strongly influenced by a correlation with the cell density. This leads to a failure in substrate prediction, which in turn prevents substrate control at cell densities above 16 gl−1. This article is protected by copyright. All rights reserved

Parametric studies on droplet generation reproducibility for applications with biological relevant fluids


Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants-free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media. This article is protected by copyright. All rights reserved

Ultrasensitive SPR detection of miRNA-93 using antibody-enhanced and enzymatic signal amplification


MiRNAs are endogenous non-coding RNA molecules. They play important gene-regulatory roles by binding to the mRNA of target genes thereby leading to either transcript degradation or translational repression. In virtually all diseases, distinct alterations of miRNA expression profiles have been found thus suggesting miRNAs as interesting biomarkers. Here, we present a SPR biosensor that utilizes disposable, injection-molded sensor chip/microfluidic hybrids combined with a lateral imaging optical system for parallel analysis of three one-dimensional spot arrays to detect miRNA-93. To increase the sensitivity of the biosensor we used two different amplification strategies. By adding an RNA-DNA-hybrid antibody for primary signal amplification, a limit of detection of 10 pmol/l was achieved. Based on that method we demonstrate the detection of miRNA-93 in total RNA lysate from HEK-293 cells. Utilizing an enzymatic signal amplification with Poly(A) polymerase, the sensitivity could be increased even further leading to a limit of detection of 1 fmol/l. This article is protected by copyright. All rights reserved

Protein micropatterns printed on glass: Novel tools for protein-ligand binding assays in live cells


Micrometer-sized patterns of proteins on glass or silica surfaces are in widespread use as protein arrays for probing with ligands or recombinant proteins. More recently, they have been used to capture the surface proteins of mammalian cells seeded onto them, and to arrange these surface proteins into pattern structures. Binding of small molecule ligands or of other proteins, transmembrane or intracellular, to these captured surface proteins can then be quantified. However, reproducible production of protein micropatterns on surfaces can be technically difficult. In this review, we outline the wide potential and the current practical uses of printed protein micropatterns in a historical overview, and we detail some potential pitfalls and difficulties from our own experience, as well as ways to circumvent them.

Anionic lipopeptides from Bacillus mojavensis I4 as effective antihypertensive agents: Production, characterization, and identification


A new isolated Bacillus mojavensis strain I4 was found as producer of biosurfactants by different screening methods, such as parafilm M test, hemolytic activity, oil displacement test, emulsification index, surface tension, and lipase production assay. Enhanced biosurfactants production was obtained using glucose and glutamic acid as carbon and nitrogen sources, respectively. The optimal production of the biosurfactants was obtained by using a C/N ratio of 17, pH of 7.0, and temperature of 37°C. The surface tension was reduced to 29 mN/m and the emulsification index E24 of 62% was achieved after 72 h of culture. The purified biosurfactants showed stability with regard to surface tension reduction and emulsification in a wide range of temperatures (4–120°C), pH (4–10), and salinity (2–12% of NaCl). The thin-layer chromatography showed that the produced biosurfactants were lipopeptides. The biosurfactants were characterized as a group of anionic lipopeptides with zeta potential measurement. Chromatographic characterization using HPLC revealed that I4 lipopeptides contained numerous isoforms and surfactin was the major component. Moreover, the I4 lipopeptides showed interesting angiotensin-converting enzyme-inhibitory activity.

A soft computing tool for species classification and prediction of glucomannan content in Amorphophallus genus


The proposed work aims at designing a classification system for automatic identification of A. muelleri species, grown as a potential cash crop in many Asian countries, from the DNA fingerprints of Amorphophallus genus. Four sets of 48 DNA fingerprints belonging to 37 species of the Amorphophallus genus, developed with the help of four different primers are considered for the experiment, with an objective to identify only the fingerprints of the species of interest. A second experimental setup deals with the automatic classification of species containing high amounts of glucomannan from the same set of DNA fingerprints of the Amorphophallus genus. For each set of 48 DNA fingerprints generated with a specific primer, the DNA fingerprints are preprocessed to extract a 42 dimensional feature vector which is used to generate a k-Nearest Neighbor based classifier based on the Leave One Out Cross Validation protocol. Final classification based on outputs from individual classifiers constructed with respect to the four different primers is performed according to a n-star consensus strategy. The n-star consensus predicts species A. muelleri with cent per cent accuracy while it predicts species containing glucomannan with a more modest accuracy of 81.25%.

Detection of growth rate-dependent product formation in miniaturized parallel fed-batch cultivations


Saccharomyces cerevisiae is a popular expression system for recombinant proteins. In most cases, production processes are performed as carbon-limited fed-batch cultures to avoid aerobic ethanol formation. Especially for constitutive expression systems, the specific product formation rate depends on the specific growth rate. The development of optimal feeding strategies strongly depends on laboratory-scale cultivations, which are time and resource consuming, especially when continuous experiments are carried out. It is therefore beneficial for accelerated process development to look at alternatives. In this study, S. cerevisiae AH22 secreting a heterologous endo-polygalacturonase (EPG) was characterized in microwell plates with an enzyme-based fed-batch medium. Through variation of the glucose release rate, different growth profiles were established and the impact on EPG secretion was analyzed. Product formation rates of 200–400 U (gx h)−1 were determined. As a reference, bioreactor experiments using the change-stat cultivation technique were performed. The growth-dependent product formation was analyzed over dilution rates of D = 0.01–0.35 with smooth change of D at a rate of 0.003 h−2. EPG production was found to be comparable with a qp of 400 U (gx h)−1 at D = 0.27 h−1. The presented results indicate that parallel miniaturized fed-batch cultures can be applied to determine product formation profiles of putative production strains. With further automation and parallelization of the concept, strain characterization can be performed in shorter time.

CO2 utilization in the production of biomass and biocompounds by three different microalgae


The atmospheric CO2 increase is considered the main cause of global warming. Microalgae are photosynthetic microorganisms that can help in CO2 mitigation and at the same time produce value-added compounds. In this study, Scenedesmus obliquus, Chlorella vulgaris, and Chlorella protothecoides were cultivated under 0.035 (air), 5 and 10% (v/v) of CO2 concentrations in air to evaluate the performance of the microalgae in terms of kinetic growth parameters, theoretical CO2 biofixation rate, and biomass composition. Among the microalgae studied, S. obliquus presented the highest values of specific growth rate (μ = 1.28 d−1), maximum productivities (Pmax = 0.28 g L−1d−1), and theoretical CO2 biofixation rates (0.56 g L−1d−1) at 10% CO2. The highest oil content was found at 5% CO2, and the fatty acid profile was not influenced by the concentration of CO2 in the inflow gas mixture and was in compliance with EN 14214, being suitable for biodiesel purposes. The impact of the CO2 on S. obliquus cells’ viability/cell membrane integrity evaluated by the in-line flow cytometry is quite innovative and fast, and revealed that 86.4% of the cells were damaged/permeabilized in cultures without the addition of CO2.

Measurement of heat transfer coefficients in stirred single-use bioreactors by the decay of hydrogen peroxide


Single-use bioreactors are barely described by means of their heat transfer characteristics, although some of their properties might affect this process. Steady-state methods that use external heat sources enable precise investigations. One option, commonly present in stirred, stainless steel tanks, is to use adjustable electrical heaters. An alternative are exothermic chemical reactions that offer a higher flexibility and scalability. Here, the catalytic decay of hydrogen peroxide was considered a possible reaction, because of the high reaction enthalpy of –98.2 kJ/mole and its uncritical reaction products. To establish the reaction, a proper catalyst needed to be determined upfront. Three candidates were screened: catalase, iron(III)-nitrate and manganese(IV)-oxide. Whilst catalase showed strong inactivation kinetic and general instability and iron(III)-nitrate solution has a pH of 2, it was decided to use manganese(IV)-oxide for the bioreactor studies. First, a comparison between electrical and chemical power input in a benchtop glass bioreactor of 3.5 L showed good agreement. Afterwards the method was transferred to a 50 L stirred single-use bioreactor. The deviation in the final results was acceptable. The heat transfer coefficient for the electrical method was 242 W/m2/K, while the value achieved with the chemical differed by less than 5%. Finally, experiments were carried out in a 200 L single-use bioreactor proving the applicability of the chemical power input at technical relevant scales.

Bicistronic expression strategy for high-level expression of recombinant proteins in Corynebacterium glutamicum


Directly using the promoter associated with 5′-untranslated region of a high-protein-abundance gene from the genome may cause low expression activity of an expression system. A bicistronic expression part containing the short 5′ coding sequence of the source gene and an embedded Shine–Dalgarno sequence can cause higher expression levels of the recombinant gene in a bicistronic cassette. Here, we evaluated two methods to construct expression parts and exploited genomic sequence sources to provide specific functional sequences to complete the expression system. The architecture of the bicistronic part increased the expression levels of target genes and performed more reliably than conventional expression parts with the same promoter and 5′ untranslated region. For Corynebacterium glutamicum, the strongest bicistronic part, HP-BEP4, was obtained from a heterologous sequence source, leading to a 2.24-fold increase in the expression level of fluorescent protein over constitutively expressed pXMJ19 or the production of more than 100 mg/L single-chain variable fragment (scFv). It could meet the needs of overexpressing key genes in C. glutamicum.

Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor


The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40 μg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing.

Ultrasound-assisted swelling of bacterial cellulose


Bacterial cellulose (BC) was obtained by static cultivation using commercial BC gel from scoby. BC membranes (oven dried and freeze-dried) were swelled with 8% NaOH, in the absence and in the presence of ultrasound (US), for 30, 60, and 90 min. The influence of swelling conditions on both physico-chemical properties and molecules entrapment was evaluated. Considering the highest levels of entrapment, an optimum swelling procedure was established: 8% NaOH for 30 min at room temperature in the presence of US. Native and PEGylated laccase from Myceliophthora thermophila was immobilized on BC membranes and a different catalytic behaviour was observed after immobilization. Native laccase presented activity values similar to published reports (5–7 U/gBC) after immobilization whereas PEGylated enzymes showed much lower activity (1–2 U/gBC). BC swelled membranes are presented herein as a potential support for the preparation of immobilized enzymes for industrial applications, like phenolics polymerization.

Micro free-flow isoelectric focusing with integrated optical pH sensors


Recently, a new observation method for monitoring of pH gradients in microfluidic free-flow electrophoresis has emerged. It is based on the use of chip-integrated fluorescent or luminescent micro sensor layers. These are able to monitor pH gradients in miniaturized separations in real time and spatially resolved; this is particularly useful in isoelectric focusing. Here these multifunctional microdevices that feature continuous separation, monitoring, and in some instances other functionalities, are reviewed. The employed microfabrication procedures to produce these devices are discussed and the different pH sensor matrices that were integrated and their applications in the separation of different types of biomolecules. The procedures for obtaining spatially resolved information about the separated molecules and the pH at the same time and different detection modalities to achieve this such as deep UV fluorescence as well as time-resolved referenced pH sensing and the integration of a precolumn labeling step into these platforms are also highlighted.

Progress in enzyme inhibition based detection of pesticides


The previous few decades have seen the development of biosensors and their use in monitoring of pesticides in food and environmental samples. Although inhibition-based biosensors have been subject of several recent research works, their performance characteristics greatly depend on the type of immobilization and the presence of interfering compounds in the samples. Moreover, sensitivity, detection limits, and rapidity of the response are few of the other major features that need to be investigated further if they are to become operationally user-friendly. This review will highlight research carried out in the past on biosensors that are based on enzyme inhibition for determination of organophosphorus compounds and carbamate pesticides.

Stability of polymersomes with focus on their use as nanoreactors


The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) toward mechanical stress, typically prevailing in stirred-tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W/kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two-phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case.

Effect of propanil, linuron, and dicamba on the degradation kinetics of 2,4-dichlorophenoxyacetic acid by Burkholderia sp. A study by differential analysis of 2,4-dichlorophenoxyacetic acid degradation data


The successive application of distinct pesticides, or mixtures of them, is a frequent practice that could adversely affect the microbial species inhabiting soil and aquatic ecosystems. The ability of soil or aquatic microbiota to degrade a pesticide could be affected by the presence of another. If the degradation rate of the first compound is inhibited, its dissipation half-life in the environment could be hazardously enlarged. Few studies have been made to quantify the impact on the biodegradation rate of pesticides in soils or water by the presence of other pesticides. In this work, a method for assessing the effect of a pesticide on the biodegradation rate of another, measuring its effect on the biodegradation kinetics of a single bacterial strain is presented. The mathematical analysis is a powerful tool to study the stoichiometry and kinetics of microbial processes, which was used to evaluate independently, in detail, the effect of three pesticides (propanil, linuron, and dicamba) on the biodegradation kinetics of 2,4-dichlorophenoxyacetic acid by a strain of Burkholderia sp. It was evidenced that linuron and dicamba caused a decay of more than 40% in the top instantaneous degradation rate of 2,4-dichlorophenoxyacetic acid, while propanil showed a minimal effect.

Experimental characterization and simulation of amino acid and peptide interactions with inorganic materials


Inspired by nature, many applications and new materials benefit from the interplay of inorganic materials and biomolecules. A fundamental understanding of complex organic–inorganic interactions would improve the controlled production of nanomaterials and biosensors to the development of biocompatible implants for the human body. Although widely exploited in applications, the interaction of amino acids and peptides with most inorganic surfaces is not fully understood. To date, precisely characterizing complex surfaces of inorganic materials and analyzing surface–biomolecule interactions remain challenging both experimentally and computationally. This article reviews several approaches to characterizing biomolecule–surface interactions and illustrates the advantages and disadvantages of the methods presented. First, we explain how the adsorption mechanism of amino acids/peptides to inorganic surfaces can be determined and how thermodynamic and kinetic process constants can be obtained. Second, we demonstrate how this data can be used to develop models for peptide–surface interactions. The understanding and simulation of such interactions constitute a basis for developing molecules with high affinity binding domains in proteins for bioprocess engineering and future biomedical technologies.

Microtiter plate-based cultivation to investigate the growth of filamentous fungi


Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL-scale. In this paper, the application of a microtiter plate-based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose-like aroma compound 2-phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms.

Antibody fingerprints in lyme disease deciphered with high density peptide arrays


Lyme disease is the most common tick-borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface-exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE-positive patients by mapping the protein as overlapping peptides and subsequent in-depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE-positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein-Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.

Influence of the production system on the surface properties of influenza A virus particles


In this study, influenza A/Puerto Rico/8/34 H1N1 virus particles (VP) produced in adherent and suspension Madin Darby canine kidney cells were investigated with a broad analytical toolbox to obtain more information on the VP's surface properties potentially affecting their aggregation behavior. First, differences in aggregation behavior were revealed by VP size distributions obtained via differential centrifugal sedimentation and confirmed by dynamic light scattering. The VP produced in adherent cells showed increased levels of aggregation in a 20 mM NaCl 10 mM Tris-HCl pH 7.4 low-salt buffer. This included the formation of multimers (dimers up to pentamers), whereas VP produced in suspension cells displayed no tendency toward aggregate formation. To investigate the cause of these differences in aggregation behavior, the VP samples were compared based on their zeta potential, their surface hydrophobicity, their lipid composition, and the N-glycosylation of their major VP surface protein hemagglutinin. The zeta potential and the hydrophobicity of the VP produced in the adherent cells was significantly decreased compared to the VP produced in the suspension cells. The lipid composition of both VP systems was approximately identical. The hemagglutinin of the VP produced in adherent cells included more of the larger N-glycans, whereas the VP produced in suspension cells included more of the smaller N-glycans. These results indicate that differences in the glycosylation of viral surface proteins should be monitored to characterize VP hydrophobicity and aggregation behavior, and to avoid aggregate formation and product losses in virus purification processes for vaccines and gene therapy.

Rapid process synthesis supported by a unified modular software framework


Although known to be very powerful, the widespread application of model-based techniques is still significantly hampered in the area of bio-processes. Reasons for this situation can be found along the whole chain to set up and implement such approaches. In a time-consuming step, models are typically hand-crafted. Whether alternatives of better models exist to actually fulfill the final goals is undocumented, most often even unknown. In a next step, model-based process control methods are hand-coded in an error-prone procedure. For many of these methods given in the literature, only simulation studies are shown, leaving the interested reader with the unanswered question whether the implementation of a specific method in a real process is viable. As the potentially time-consuming implementation of such a method presents a risk for a rapid process development, promising candidates may be overlooked. To remediate this unsatisfactory situation, a combination of theoretical methods and information technology is proposed here. By an exemplarily realized software tool, it is shown how such an environment helps to promote model-based optimization, supervision, and control of bio-processes and allows for an inexpensive test of new ideas as well in real-life experiments. The contribution concentrates on an overview of a possible software architecture with respect to necessary methods and a meaningful information strategy, highlighting some of the more crucial building blocks. Experimental results exploiting parts of the proposed methods are given for a yeast strain synthesizing a product of industrial interest.

A microfluidic device for the delivery of enzymes into cells by liposome fusion


Liposomes are versatile carriers of drugs or biomolecules and are ideally suited to transport molecules into cells. However, mechanistic studies to understand and improve the fusion of liposomes with cell membranes and endosomes are difficult. Here, we report a method that allows for stable coimmobilization of liposomes and living cells, thereby bringing the membranes into close contact, which is essential for membrane fusion. The small unilamellar liposomes are tethered to the surface by a linker so that no modification of the liposome membrane for cell binding is required. The cells are positioned above the liposomes by posts that are integrated into the microfluidic device, and a pH drop induces the fusion of the cell-liposome membranes. Both membrane fusion and release of molecules into the cytosol are visualized by fluorescence dequenching assays. Furthermore, we proved the efficient delivery of the enzyme β-galactosidase into the cells when a fusogenic liposome composition was used. The device could be used for fusion studies but is also a versatile means for cell transfection.

Fast-track development of a lactase production process with Kluyveromyces lactis by a progressive parameter-control workflow


The time-to-market challenge is key to success for consumer goods affiliated industries. In recent years, the dairy industry faces a fast and constantly growing demand for enzymatically produced lactose-free milk products, mainly driven by emerging markets in South America and Asia. In order to take advantage of this opportunity, we developed a fermentation process for lactase (β-galactosidase) from Kluyveromyces lactis within short time. Here, we describe the process of stepwise increasing the level of control over relevant process parameters during scale-up that established a highly efficient and stable production system. Process development started with evolutionary engineering to generate catabolite-derepressed variants of the K. lactis wild-type strain. A high-throughput screening mimicking fed-batch cultivation identified a constitutive lactase overproducer with 260-fold improved activity of 4.4 U per milligram dry cell weight when cultivated in glucose minimal medium. During scale-up, process control was progressively increased up to the level of conventional, fully controlled fed-batch cultivations by simulating glucose feed, applying pH- and dissolved oxygen tension (DOT)-sensor technology to small scale, and by the use of a milliliter stirred tank bioreactor. Additionally, process development was assisted by design-of-experiments optimization of the growth medium employing the response surface methodology.

Online bioprocess data generation, analysis, and optimization for parallel fed-batch fermentations in milliliter scale


Bioprocess development, optimization, and control in mini-bioreactor systems require information about essential process parameters, high data densities, and the ability to dynamically change process conditions. We present an integration approach combining a parallel mini-bioreactor system integrated into a liquid handling station (LHS) with a second LHS for offline analytics. Non-invasive sensors measure pH and DO online. Offline samples are collected every 20 min and acetate, glucose, and OD620 subsequently analyzed offline. All data are automatically collected, analyzed, formalized, and used for process control and optimization. Fed-batch conditions are realized via a slow enzymatic glucose release system. The integration approach was successfully used to apply an online experimental re-design method to eight Escherichia coli fed-batch cultivations. The method utilizes generated data to select the following experimental actions online in order to reach the optimization goal of estimating E. coli fed-batch model parameters with as high accuracy as possible. Optimal experimental designs were re-calculated online based on the experimental data and implemented by introducing pulses via the LHS to the running fermentations. The LHS control allows for various implementations of advanced control and optimization strategies in milliliter scale.

Intensified design of experiments for upstream bioreactors


Statistical Design of Experiments (DoE) is a widely adopted methodology in upstream bioprocess development (and generally across industries) to obtain experimental data from which the impact of independent variables (factors) on the process response can be inferred. In this work, a method is proposed that reduces the total number of experiments suggested by a traditional DoE. The method allows the evaluation of several DoE combinations to be compressed into a reduced number of experiments, which is referred to as intensified Design of Experiments (iDoE). In this paper, the iDoE is used to develop a dynamic hybrid model (consisting of differential equations and a feedforward artificial neural network) for data generated from a simulated Escherichia coli fermentation. For the case study presented, the results suggest that the total number of experiments could be reduced by about 40% when compared to traditional DoE. An additional benefit is the simultaneous development of an appropriate dynamic model which can be used in both, process optimization and control studies.

Design of experiments-based high-throughput strategy for development and optimization of efficient cell disruption protocols


Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high-throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments-based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram-negative bacteria, which is applicable in a high-throughput format in a short time. The concentrations of four lysis-inducing chemical agents EDTA, lysozyme, Triton X-100, and polymyxin B were optimized for maximal soluble protein concentration and ß-galactosidase activity in a 96-well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.

A scalable software framework for data integration in bioprocess development


Effectiveness in lab workflows—despite progresses made in automation and lab informatics—is often hindered by insufficient integration of devices and data. The iLAB software framework, a middleware connecting and integrating devices and data, provides a plugin architecture that can be adapted to individual lab environments. Integration of devices is preferably based on standardized data and communication protocols. In addition to device integration, process data and result data from different sources (e.g. readers) are converted to a standard format and administered by a powerful database for further processing. In this paper, the use of iLAB in a bioprocess development application is described. Process parameters and measured values of two high-throughput bioreactor systems (96-well plates and 10 mL reactor vessels) are collected in the iLAB database. Data from screening experiments and offline data are visualized, analyzed, and compared. A filter algorithm allows searching for matching parameters in experiments as well as the comparison of correlated datasets, independently from the used bioreactor system. The database model enables the consolidation of data, the transition from data to information, and a solid base for management decisions on enterprise level.

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches


The biopharmaceutical industry is at a turning point moving toward a more customized and patient-oriented medicine (precision medicine). Straightforward routines such as the antibody platform process are extended to production processes for a new portfolio of molecules. As a consequence, individual and tailored productions require generic approaches for a fast and dedicated purification process development. In this article, different effective strategies in biopharmaceutical purification process development are reviewed that can analogously be used for the new generation of antibodies. Conventional approaches based on heuristics and high-throughput process development are discussed and compared to modern technologies such as multivariate calibration and mechanistic modeling tools. Such approaches constitute a good foundation for fast and effective process development for new products and processes, but their full potential becomes obvious in a correlated combination. Thus, different combinatorial approaches are presented, which might become future directions in the biopharmaceutical industry.

Cover Picture: Engineering in Life Sciences 9'17


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Electrofiltration improves dead-end filtration of hyaluronic acid and presents an alternative downstream processing step that overcomes technological challenges of conventional methods


Hyaluronic acid (HA) dispersion obtained from the bacteria Streptococcus equi was concentrated by electrofiltration. In the conventional downstream processing of HA, extraction and precipitation lead to increase in environmental issues, structural changes, and time and energy related costs. Using electrofiltration as an alternative technology delivers solutions to these limitations. Experiments were conducted in order to test the applicability of electrofiltration to downstream processing of the negatively charged HA. The structural changes and molecular weight distributions, often a consequence of the employed separation method, were tested by analysis of the initial dispersions and final products. In comparison to the conventional filtration, concentration factors were increased up to almost four times without any detectable structural change in the final product.

Enhanced growth and total fatty acid production of microalgae under various lighting conditions induced by flashing light


Microalgae are gaining importance as a source of high-value bioproducts. However, data regarding optimization of algal productivity via variation of environmental factors are lacking. Here, we evaluated a novel lighting method for the enhancement of biomass and total fatty acid (TFA) productivities during algal cultivation. We cultivated six different algal strains (Chlorella vulgaris KCTC AG10002, Acutodesmus obliquus KGE18, Uronema sp. KGE03, Micractinium reisseri KGE19, Fragilaria sp., and Spirogyra sp.) under various lighting conditions—continuous light (CL), light-dark cycle (LD), and continuous dark (CD)—with or without additional flashing light. We monitored dry cell weight (DCW) and TFA concentrations during cultivation. For each algal strain, the growth rate showed markedly different responses to the various lighting modes. The growth rates of C. vulgaris KCTC AG10002 (1.34-fold DCW increase, LD with flash), A. obliquus KGE18 (5.16-fold DCW increase, LD with flash), Uronema sp. KGE03 (2.77-fold DCW increase, CL with flash), and M. reisseri KGE19 (1.52-fold DCW increase, CL with flash) markedly increased in response to flashing light. Additionally, in some algal strains cultivated under the LD mode, the flashing light treatment induced increased TFA concentrations (C. vulgaris, 1.19-fold increase; A. obliquus, 2.59-fold increase; and M. reisseri, 3.31-fold increase). Phytohormone analysis of M. reisseri revealed increases in growth rate and TFA concentrations, associated with phytohormone induction via flashing light (e.g. 2.93-fold increase in gibberellic acid); hence, flashing light can promote substantial alterations in algal metabolism.

Improvement of docosahexaenoic acid fermentation from Schizochytrium sp. AB-610 by staged pH control based on cell morphological changes


Schizochytrium sp. AB-610 accumulates relatively higher amount of DHA-rich lipid in the cells, and it was found that DHA yield was closely related to the cell morphology and pH value during fermentation period. DHA production from Schizochytrium sp. AB-610 in fed-batch fermentation was investigated and four growth stages were clarified as lag stage, balanced growth stage, lipid accumulation stage, and lipid turnover stage, based on the morphologic observation and key parameters changes. Then a simple strategy of two-stage pH control was developed, in which pH 7.0 was kept until 12 h after the end of balanced growth stage, and then shifted to 5.0 for the rest period in fermentation. A maximal DHA production of 11.44g/L was achieved. This approach has advantage of easy scaling up for industrial DHA fermentation from Schizochytrium sp. cells.

Engineering bi-functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration


This study reports the application of peptide linker in the construction of bi-functional formate dehydrogenase (FDH) and leucine dehydrogenase (LeuDH) enzymatic complex for efficient cofactor regeneration and L-tert leucine (L-tle) biotransformation. Seven FDH-LeuDH fusion enzymes with different peptide linker were successfully developed and displayed both parental enzyme activities. The incorporation order of FDH and LeuDH was investigated by predicting three-dimensional structures of LeuDH-FDH and FDH-LeuDH models using the I-TASSER server. The enzymatic characterization showed that insertion of rigid peptide linker obtained better activity and thermal stability in comparison with flexible peptide linker. The production rate of fusion enzymatic complex with suitable flexible peptide linker was increased by 1.2 times compared with free enzyme mixture. Moreover, structural analysis of FDH and LeuDH suggested the secondary structure of the N-, C-terminal domain and their relative positions to functional domains was also greatly relevant to the catalytic properties of the fusion enzymatic complex. The results show that rigid peptide linker could ensure the independent folding of moieties and stabilized enzyme structure, while the flexible peptide linker was likely to bring enzyme moieties in close proximity for superior cofactor channeling.

Production of biopesticide azadirachtin using plant cell and hairy root cultures


The extensive use of nondegradable chemical pesticides for pest management has developed serious environmental hazards. This has necessitated the urgent need to switch over to an alternative mode of biopesticide development for mass agriculture and field crop protection. Azadirachta indica A. Juss (commonly known as neem) houses a plethora of bioactive secondary metabolites with azadirachtin being the most active constituent explored in the sector of ecofriendly and biodegradable biopesticides characterized by low toxicity toward nontarget organisms. It has been reported that the highest content of azadirachtin and related limonoids is present in the seeds, available once in a year. Moreover, the inconsistent content and purity of the metabolites in whole plant makes it imperative to tap the potential of in vitro plant tissue culture applications, which would allow for several controlled manipulations for better yield and productivities. This review gives a summarized literature of the applied research and achievements in plant cell/hairy cultures of A. indica A. Juss mainly in context with the biopesticide azadirachtin and applications thereof.

Inulinolytic activity of broths of Aspergillus niger ATCC 204447 cultivated in shake flasks and stirred tank bioreactor


It is the first detailed study of an inulinolytic fungus Aspergillus niger ATCC 204447 since its discovery, covering submerged cultivations both in shake flasks and a stirred tank bioreactor. Various carbon sources were applied to induce the inulinolytic activity in shake flask cultures. The highest volumetric and specific (per gram of biomass) activities (respectively 0.68 U/mL and 184 U g/X) were observed for the initial inulin and sucrose concentrations equal to 20 g/L. The fungus grew as large (>3 mm) spherical pellets. The influence of inoculum density and application of microparticle-enhanced cultivation (MPEC) were studied in the batch bioreactor cultivations. Inoculum density moderately affected the inulinolytic activities, whose highest values were 0.7 U/mL and 165 U g/X at the lowest studied spore density of 3.33·108 L−1. Dispersed hyphae evolved in the bioreactor made the broth difficult to aerate due to high apparent viscosity (exceeding 200 Pa sn at shear rate about 0.05 s−1) and shear thinned properties (flow behavior index below 0.2). In MPEC (10 μm talc microparticles) the pellets of diameter between 1 and 2 mm were formed, which facilitated the aeration of the broth and increased the specific inulinolytic activity 3.5-fold.

Production of naringenin from D-xylose with co-culture of E. coli and S. cerevisiae


Heterologous production of naringenin, a valuable flavonoid with various biotechnological applications, was well studied in the model organisms such as Escherichia coli or Saccharomyces cerevisiae. In this study, a synergistic co-culture system was developed for the production of naringenin from xylose by engineering microorganism. A long metabolic pathway was reconstructed in the co-culture system by metabolic engineering. In addition, the critical gene of 4-coumaroyl-CoA ligase (4CL) was simultaneously integrated into the yeast genome as well as a multi-copy free plasmid for increasing enzyme activity. On this basis, some factors related with fermentation process were considered in this study, including fermented medium, inoculation size and the inoculation ratio of two microbes. A yield of 21.16 ± 0.41 mg/L naringenin was produced in this optimized co-culture system, which was nearly eight fold to that of the mono-culture of yeast. This is the first time for the biosynthesis of naringenin in the co-culture system of S. cerevisiae and E. coli from xylose, which lays a foundation for future study on production of flavonoid.

The green alga Dictyosphaerium chlorelloides biomass and polysaccharides production determined using cultivation in crossed gradients of temperature and light


The green microalga Dictyosphaerium chlorelloides was identified as promising microorganism for biotechnological production of exopolysaccharides (EPS). In stationary phase the culture suspension solidifies to thick gel, with very high viscosity and high content of EPS which may be interesting for many biotechnological applications. To develop cultivation protocol for maximum biomass/polysaccharide production, the optimum conditions for growth and polysaccharides production were determined in this study using the crossed gradient cultivation method. Temperature and irradiance requirements of Dictyosphaerium chlorelloides were evaluated by statistical analyses for growth rate/biomass, extracellular (EPS) and intracellular (IPS) polysaccharides contents in crossed gradients of temperature (4–45°C) and irradiance (2–18 W/m2, 9.1 – 82.3 μmol/(m2 s)). The maximum relative growth rate was observed at temperatures around 19.2°C and relatively low irradiances in range 2.6–11 W/m2 (11.9–50.3 μmol/(m2 s)). The maximum IPS production was observed at temperatures around 19.2°C and irradiance around 11 W/m2 (50.3 μmol/(m2 s)). The maximum production of EPS was observed at temperatures around 25.7°C and similar irradiances as IPS production. Due to temperature separation of growth and EPS production, development of cultivation protocol based controlled temperature manipulation is possible.

Artificial consortium that produces riboflavin regulates distribution of acetoin and 2,3-butanediol by Paenibacillus polymyxa CJX518


The introduction of an NADH/NAD+ regeneration system can regulate the distribution between acetoin and 2,3-butanediol. NADH regeneration can also enhance butanol production in coculture fermentation. In this work, a novel artificial consortium of Paenibacillus polymyxa CJX518 and recombinant Escherichia coli LS02T that produces riboflavin (VB2) was used to regulate the NADH/NAD+ ratio and, consequently, the distribution of acetoin and 2,3-butanediol by P. polymyxa. Compared with a pure culture of P. polymyxa, the level of acetoin was increased 76.7% in the P. polymyxa and recombinant E. coli coculture. Meanwhile, the maximum production and yield of acetoin in an artificial consortium with fed-batch fermentation were 57.2 g/L and 0.4 g/g glucose, respectively. Additionally, the VB2 production of recombinant E. coli could maintain a relatively low NADH/NAD+ ratio by changing NADH dehydrogenase activity. It was also found that 2,3-butanediol dehydrogenase activity was enhanced and improved acetoin production by the addition of exogenous VB2 or by being in the artificial consortium that produces VB2. These results illustrate that the coculture of P. polymyxa and recombinant E. coli has enormous potential to improve acetoin production. It was also a novel strategy to regulate the NADH/NAD+ ratio to improve the acetoin production of P. polymyxa.

En route to economical eco-friendly solvent system in enhancing sustainable recovery of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer


Separation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco-balance as P(3HB-co-4HB) is widely used for biomedical applications. Therefore, an efficient and eco-based extraction of P(3HB-co-4HB) using a combination of NaOH and Lysol in digesting the non-polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB-co-4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.