Showing papers in "Engineering in Life Sciences in 2021"

Recent applications of plant cell culture technology in cosmetics and foods.

Abstract: Plants have been used as the main source of phytochemicals with nutritional, medicinal, cultural and cosmetic applications since times immemorial. Nowadays, achieving sustainable development, global climate change, restricted access to fresh water, limited food supply and growing energy demands are among the critical global challenges faced by humanity. Plant cell culture technology has the potential to address some of these challenges by providing effective tools for sustainable supply of phyto-ingredients with reduced energy, carbon and water footprints. The main aim of this review is to discuss the recent trends in the development of plant cell culture technologies for production of plant-derived substances with application in food products and cosmetic formulations. The specific technological steps and requirements for the final products are discussed in the light of the advances in cultivation technologies used for growing differentiated and undifferentiated plant in vitro systems. Future prospects and existing challenges of the commercialization of plant cell culture-derived products have been outlined through the prism of the authors' point of view. We expect this review will encourage scientists, policymakers and business enterprises to join efforts for speeding-up the mass commercialization and popularization of plant cell culture technology as an eco-friendly alternative method for sustainable production of plant-derived additives with application in food and cosmetic products.

Purification and characterization of the receptor-binding domain of SARS-CoV-2 spike protein from Escherichia coli

Abstract: SARS-CoV-2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID-19. Spike protein of SARS-CoV-2 mediates viral entry into host cells by binding ACE2 through the receptor-binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD-1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD-1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD-1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD-2). The secondary structure and tertiary structure of RBD-1 were largely maintained without glycosylation. In particular, the major beta-sheet content of RBD-1 was almost unaltered. RBD-1 could strongly bind ACE2 with a dissociation constant (K-D) of 2.98 x 10(-8) M. Thus, RBD-1 was expected to apply in the vaccine development, screening drugs and virus test kit.

Ultra pH-sensitive detection of total and free prostate-specific antigen using electrochemical aptasensor based on reduced graphene oxide/gold nanoparticles emphasis on TiO2/carbon quantum dots as a redox probe.

Abstract: The development of a rapid, sensitive, and straightforward detection method of prostate-specific antigen (PSA) is indispensable for the early diagnosis of prostate cancer (PCa). This work relates an electrochemical method using functionalized single-stranded DNA aptamer to diagnose PCa and benign prostate hyperplasia. The sensing platform relies on PSA recognition by aptamer/Au/GO-nanohybrid-modified glassy carbon electrode. Besides ferrocyanide TiO2/carbon quantum dots (CQDs) probe is used to investigate the effect of nanoparticle-containing electrolyte. Optimization of incubation time of aptamer/Au/GO-nanohybrid and volume fraction of nafion were done using Design Expert 10 software reporting 42.4 h and 0.095% V/V, respectively. In ferrocyanide medium, PSA detection as low as 3, 2.96, and 0.85 ng mL-1 was achieved with a dynamic range from 0.5 to 7 ng ml-1, in accord with clinical values, using cyclic voltammetry, square wave voltammetry, and electrochemical impedance spectroscopy, respectively. Moreover, this sensor exhibited conspicuous performance in TiO2/CQDs-containing medium with different pH values of 5.4 and 8 to distinguish total PSA and free PSA, resulting in very low limit of detections, 0.028, and 0.007 ng ml-1, respectively. The results manifested the proposed system as a forthcoming sensor in a clinical and point of care analysis of PSA.

Challenges of influencing cellular morphology by morphology engineering techniques and mechanical induced stress on filamentous pellet systems-A critical review.

Abstract: Filamentous microorganisms are main producers of organic acids, enzymes, and pharmaceutical agents such as antibiotics and other active pharmaceutical ingredients. With their complex cell morphology, ranging from dispersed mycelia to dense pellets, the cultivation is challenging. In recent years, various techniques for tailor-made cell morphologies of filamentous microorganisms have been developed to increase product formation and have been summarised under the term morphology engineering. These techniques, namely microparticle-enhanced cultivation, macroparticle-enhanced cultivation, and alteration of the osmolality of the culture medium by addition of inorganic salts, the salt-enhanced cultivation, are presented and discussed in this review. These techniques have already proven to be useful and now await further proof-of-concept. Furthermore, the mechanical behaviour of individual pellets is of special interest for a general understanding of pellet mechanics and the productivity of biotechnological processes with filamentous microorganisms. Correlating them with substrate uptake and finally with productivity would be a breakthrough not to be underestimated for the comprehensive characterisation of filamentous systems. So far, this research field is under-represented. First results on filamentous pellet mechanics are discussed and important future aspects, which the filamentous expert community should deal with, will be presented and critically discussed.

Production of β-carotene with Dunaliella salina CCAP19/18 at physically simulated outdoor conditions

Abstract: Batch growth and β-carotene production of Dunaliella salina CCAP19/18 was investigated in flat-plate gas-lift photobioreactors with a light path of 2 cm, operated in physically simulated outdoor conditions. Dunaliella salina CCAP19/18 showed robust growth with respect to pH 8.0-9.0 and 15-35°C at increasing salinity, simulating the evaporation of open photobioreactors. The highest β-carotene concentration of 25 mg L-1 (3 mg gCDW -1) was observed in batch processes at pH 8.5, 15-30°C and increasing salinity up to 110 g L-1, simulating a typical Mediterranean summer climate. Intracellular β-carotene accumulation of D. salina CCAP19/18 was shown to be independent of light availability, although nutrient limitation (K2HPO4, MgSO4, and/or ammonium ferric citrate) seems to enable stable β-carotene content in the algal cells despite increasing cell densities in the photobioreactor. Fully controlled, lab-scale photobioreactors simulating typical climate conditions of any region of interest are valuable tools for enabling a realistic characterization of microalgae on a laboratory scale, for production processes projected in open photobioreactor systems (e.g. thin-layer cascade photobioreactors).

Nanofiltration membrane for bio‐separation: Process‐oriented materials innovation

Abstract: Nanofiltration (NF) with advantages of high efficiency and low-cost has attracted increasing attentions in bio-separation. However, the large-scale application is limited by the inferior molecular selectivity, low chemical stability and serious membrane fouling. Many efforts, thus, have been devoted in NF materials design for specific applications to enhance the separation efficiency of bio-products and increase membrane life-time, as well as reduce the operating cost. This review summarized the recent progress of NF applications in bio-separation, discussed various demands for NF membrane in the bio-products purification and corresponding material innovations, finally proposed several practical suggestions for future research, which provided directions and guidance toward further product development and process industrialization.

The pro-inflammatory response of macrophages regulated by acid degradation products of poly(lactide-co-glycolide) nanoparticles

Abstract: Poly(lactide-co-glycolide) (PLGA) shows great potentials in biomedical applications, in particular with the field of biodegradable implants and control release technologies. However, there are few systematic and detailed studies on the influence of PLGA degradation behavior on the immunogenicity. In this study, in order to develop a method for dynamically assessing the immunological response of PLGA throughout the implantation process, PLGA particles are fabricated using an o/w single-emulsion method. The physicochemical characterizations of the prepared PLGA particles during in vitro hydrolytic degradation are investigated. Then, a series of immunological effects triggered by PLGA by-products formed with degradation process are evaluated, including cell viability, apoptosis, polarization and inflammatory reaction. THP-1 human cell line is set as in vitro cell model. Our results show that PLGA degradation-induced acid environment decreases cell viability and increases cell apoptosis, which is a potential factor affecting cell function. In particular, the macrophages exhibit up-regulations in both M1 subtype related surface markers and pro-inflammatory cytokines with the degradation process of PLGA, which indicates the degradation products of PLGA can convert macrophages to the pro-inflammatory (M1) polarization state. All these findings provide the mechanism of PLGA-induced inflammation and lay the foundation for the design of next-generation PLGA-based biomaterials endowed with immunomodulatory functions.

Anti-EGFR antibody-drug conjugate for triple-negative breast cancer therapy.

Abstract: Triple-negative breast cancers (TNBCs) are highly aggressive, metastatic and recurrent. Cytotoxic chemotherapies with limited clinical benefits and severe side effects are the standard therapeutic strategies, but, to date, there is no efficacious targeted therapy. Literature and our data showed that epidermal growth factor receptor (EGFR) is overexpressed on TNBC cell surface and is a promising oncological target. The objective of this study was to develop an antibody-drug conjugate (ADC) to target EGFR+ TNBC and deliver high-potency drug. First, we constructed an ADC by conjugating anti-EGFR monoclonal antibody with mertansine which inhibits microtubule assembly via linker Sulfo-SMCC. Second, we confirmed the TNBC-targeting specificity of anti-EGFR ADC by evaluating its surface binding and internalization in MDA-MB-468 cells and targeting to TNBC xenograft in subcutaneous mouse mode. The live-cell and live-animal imaging with confocal laser scanning microscopy and In Vivo Imaging System (IVIS) confirmed the TNBC-targeting. Finally, both in vitro toxicity assay and in vivo anti-cancer efficacy study in TNBC xenograft models showed that the constructed ADC significantly inhibited TNBC growth, and the pharmacokinetics study indicated its high circulation stability. This study indicated that the anti-EGFR ADC has a great potential to against TNBC.

Development of a 3D-printed single-use separation chamber for use in mRNA-based vaccine production with magnetic microparticles.

Abstract: Laboratory protocols using magnetic beads have gained importance in the purification of mRNA for vaccines. Here, the produced mRNA hybridizes specifically to oligo(dT)-functionalized magnetic beads after cell lysis. The mRNA-loaded magnetic beads can be selectively separated using a magnet. Subsequently, impurities are removed by washing steps and the mRNA is eluted. Magnetic separation is utilized in each step, using different buffers such as the lysis/binding buffer. To reduce the time required for purification of larger amounts of mRNA vaccine for clinical trials, high-gradient magnetic separation (HGMS) is suitable. Thereby, magnetic beads are selectively retained in a flow-through separation chamber. To meet the requirements of biopharmaceutical production, a disposable HGMS separation chamber with a certified material (United States Pharmacopeia Class VI) was developed which can be manufactured using 3D printing. Due to the special design, the filter matrix itself is not in contact with the product. The separation chamber was tested with suspensions of oligo(dT)-functionalized Dynabeads MyOne loaded with synthetic mRNA. At a concentration of cB = 1.6-2.1 g·L-1 in lysis/binding buffer, these 1 μm magnetic particles are retained to more than 99.39% at volumetric flows of up to 150 mL·min-1 with the developed SU-HGMS separation chamber. When using the separation chamber with volumetric flow rates below 50 mL·min-1, the retained particle mass is even more than 99.99%.

Food ingredients and food made with plant cell and tissue cultures: State‐of‐the art and future trends

Abstract: Climate change and an increasing world population means traditional farming methods may not be able to meet the anticipated growth in food demands. Therefore, alternative agricultural strategies should be considered. Here, plant cell and tissue cultures (PCTCs) may present a possible solution, as they allow for controlled, closed and sustainable manufacturing of extracts which have been or are still being used as colorants or health food ingredients today. In this review we would like to highlight developments and the latest trends concerning commercial PCTC extracts and their use as food ingredients or even as food. The commercialization of PCTC-derived products, however, requires not only regulatory approval, but also outstanding product properties or/and a high product titer. If these challenges can be met, PCTCs will become increasingly important for the food sector in coming years.

Metabolomic and kinetic investigations on the electricity-aided production of butanol by Clostridium pasteurianum strains.

Abstract: In this contribution, we studied the effect of electro-fermentation on the butanol production of Clostridium pasteurianum strains by a targeted metabolomics approach. Two strains were examined: an electrocompetent wild type strain (R525) and a mutant strain (dhaB mutant) lacking formation of 1,3-propanediol (PDO). The dhaB-negative strain was able to grow on glycerol without formation of PDO, but displayed a high initial intracellular NADH/NAD ratio which was lowered subsequently by upregulation of the butanol production pathway. Both strains showed a 3-5 fold increase of the intracellular NADH/NAD ratio when exposed to cathodic current in a bioelectrochemical system (BES). This drove an activation of the butanol pathway and resulted in a higher molar butanol to PDO ratio for the R525 strain. Nonetheless, macroscopic electron balances suggest that no significant amount of electrons derived from the BES was harvested by the cells. Overall, this work points out that electro-fermentation can be used to trigger metabolic pathways and improve product formation, even when the used microbe cannot be considered electroactive. Accordingly, further studies are required to unveil the underlying (regulatory) mechanisms.

One-step selective affinity purification and immobilization of His-tagged enzyme by recyclable magnetic nanoparticles.

Abstract: The NiFe2O4 magnetic nanoparticles (NF-MNPs) were prepared for one-step selective affinity purification and immobilization of His-tagged recombinant glucose dehydrogenase (GluDH). The prepared nanoparticles were characterized by a Fourier-transform infrared spectrophotometer and microscopy. The immobilization and purification of His-tagged GluDH on NF-MNPs were investigated. The optimal immobilization conditions were obtained that mixed cell lysis and carriers in a ratio of 0.13 in pH 8.0 Tris-HCl buffer at 30℃ and incubated for 2 h. The highest activity recovery and protein bindings were 71.39% and 38.50 μg mg-1 support, respectively. The immobilized GluDH exhibited high thermostability, pH-stability and it can retain more than 65% of the initial enzyme after 10 cycles for the conversion of glucose to gluconolactone. Comparing with a commercial Ni-NTA resin, the NF-MNPs displayed a higher specific affinity with His-tagged recombinant GluDH.

Efficient phenol degradation by laccase immobilized on functional magnetic nanoparticles in fixed bed reactor under high‐gradient magnetic field

Abstract: Enzymatic degradation of emerging contaminants has gained great interest for the past few years. However, free enzyme often incurs high costs in practice. The immobilized laccase on the polyethylenimine (PEI)-functionalized magnetic nanoparticles (Fe3O4-NH2-PEI-laccase) was fabricated to efficiently degrade phenolic compounds continuously in a newly fixed bed reactor under a high-gradient magnetic field. The degradation rate of continuous treatment in the bed after 18 h was 2.38 times as high as that of batch treatment after six successive operations with the same treatment duration. Under the optimal conditions of volume fraction of nickel wires mesh, flow rate of phenol solution, phenol concentration, and Fe3O4-NH2-PEI-laccase amount, the degradation rate of phenol kept over 70.30% in 48 h continuous treatment. The fixed bed reactor filled with Fe3O4-NH2-PEI-laccase provided a promising avenue for the continuous biodegradation of phenolic compounds for industrial wastewater in practice.

pyFOOMB: Python framework for object oriented modeling of bioprocesses

Abstract: Quantitative characterization of biotechnological production processes requires the determination of different key performance indicators (KPIs) such as titer, rate and yield. Classically, these KPIs can be derived by combining black-box bioprocess modeling with non-linear regression for model parameter estimation. The presented pyFOOMB package enables a guided and flexible implementation of bioprocess models in the form of ordinary differential equation systems (ODEs). By building on Python as powerful and multi-purpose programing language, ODEs can be formulated in an object-oriented manner, which facilitates their modular design, reusability, and extensibility. Once the model is implemented, seamless integration and analysis of the experimental data is supported by various Python packages that are already available. In particular, for the iterative workflow of experimental data generation and subsequent model parameter estimation we employed the concept of replicate model instances, which are linked by common sets of parameters with global or local properties. For the description of multi-stage processes, discontinuities in the right-hand sides of the differential equations are supported via event handling using the freely available assimulo package. Optimization problems can be solved by making use of a parallelized version of the generalized island approach provided by the pygmo package. Furthermore, pyFOOMB in combination with Jupyter notebooks also supports education in bioprocess engineering and the applied learning of Python as scientific programing language. Finally, the applicability and strengths of pyFOOMB will be demonstrated by a comprehensive collection of notebook examples.

Removal of sugars in wastewater from food production through heterotrophic growth of Galdieria sulphuraria.

Abstract: The unicellular extremophilic red alga Galdieria sulphuraria is capable of chemoheterotrophy and its growth has been investigated on some defined and undefined substrates. In this study, the removal of sugars in wastewater from fruit‐salad production with G. sulphuraria strain SAG 21.92 was analyzed. Growth and sugar consumption were determined under variation of temperature, pH‐value and concentration of a model substrate, containing sucrose, glucose and fructose. In shake flask cultivation maximum specific growth rate and specific substrate consumption rate of 1.53±0.09 day−1 and 2.41±0.14 gSub·gDW −1·day−1 were measured at pH 2 and 42°C. A scale‐up of this process was conducted in a 3 L stirred tank reactor (STR). Wastewater from fruit‐salad production was diluted to 15 g·L−1 total sugar concentration, supplemented with micronutrients and ammonia and pH was set to 3. Determined growth rate and substrate consumption were 1.21 day−1 and 1.88 gSub·gDW −1·day−1, respectively. It was demonstrated, that high sugar concentrations in wastewater streams from food production processes can be significantly reduced with G. sulphuraria SAG 21.92. This strain could achieve substrate consumption rates in wastewater, equal to the more common strain 074G, but at higher pH values. Generated biomass can be used for production of phycocyanin, a valuable nutraceutical.

Interrogation of the perturbed gut microbiota in gouty arthritis patients through in silico metabolic modeling.

Abstract: Recent studies have shown perturbed gut microbiota associated with gouty arthritis, a metabolic disease characterized by an imbalance between uric acid production and excretion. To mechanistically investigate altered microbiota metabolism associated with gout disease, 16S rRNA gene amplicon sequence data from stool samples of gout patients and healthy controls were computationally analyzed through bacterial community metabolic models. Patient-specific community models constructed with the metagenomics modeling pipeline, mgPipe, were used to perform k-means clustering of samples according to their metabolic capabilities. The clustering analysis generated statistically significant partitioning of samples into a Bacteroides-dominated, high gout cluster and a Faecalibacterium-elevated, low gout cluster. The high gout cluster was predicted to allow elevated synthesis of the amino acids D-alanine and L-alanine and byproducts of branched-chain amino acid catabolism, while the low gout cluster allowed higher production of butyrate, the sulfur-containing amino acids L-cysteine and L-methionine, and the L-cysteine catabolic product H2S. By expanding the capabilities of mgPipe to provide taxa-level resolution of metabolite exchange rates, acetate, D-lactate and succinate exchanged from Bacteroides to Faecalibacterium were predicted to enhance butyrate production in the low gout cluster. Model predictions suggested that sulfur-containing amino acid metabolism generally and H2S more specifically could be novel gout disease markers.

A novel downstream process for highly pure 1,3-propanediol from an efficient fed-batch fermentation of raw glycerol by Clostridium pasteurianum.

Abstract: An efficient downstream process without prior desalination was developed for recovering 1,3-propanediol (1,3-PDO) with high purity and yield from broth of a highly productive fed-batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3-PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3-PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3-PDO ( 99% reduction of 1,3-PDO esters was achieved. This step conveniently provided free 1,3-PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3-PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3-PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high-salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3-PDO esters are crucial for the efficient separation of highly pure 1,3-PDO with acceptable yield from fermentation broth of raw glycerol.

Parameter and state estimation of backers yeast cultivation with a gas sensor array and unscented Kalman filter.

Abstract: Real-time information about the concentrations of substrates and biomass is the key to accurate monitoring and control of bioprocess. However, on-line measurement of these variables is a challenging task and new measurement systems are still required. An alternative are software sensors, which can be used for state and parameter estimation in bioprocesses. The software sensors predict the state of the process by using mathematical models as well as data from measured variables. The Kalman filter is a type of such sensors. In this paper, we have used the Unscented Kalman Filter (UKF) which is a nonlinear extension of the Kalman filter for on-line estimation of biomass, glucose and ethanol concentration as well as for estimating the growth rate parameters in S. cerevisiae batch cultivation, based on infrequent ethanol measurements. The UKF algorithm was validated on three different cultivations with variability of the substrate concentrations and the estimated values were compared to the off-line values. The results obtained showed that the UKF algorithm provides satisfactory results with respect to estimation of concentrations of substrates and biomass as well as the growth rate parameters during the batch cultivation.

New developments in biological phosphorus accessibility and recovery approaches from soil and waste streams

Abstract: Phosphorus (P) is a non-renewable resource and is on the European Union's list of critical raw materials. It is predicted that the P consumption peak will occur in the next 10 to 20 years. Therefore, there is an urgent need to find accessible sources in the immediate environment, such as soil, and to use alternative resources of P such as waste streams. While enormous progress has been made in chemical P recovery technologies, most biological technologies for P recovery are still in the developmental stage and are not reaching industrial application. Nevertheless, biological P recovery could offer good solutions as these technologies can return P to the human P cycle in an environmentally friendly way. This mini-review provides an overview of the latest approaches to make P available in soil and to recover P from plant residues, animal and human waste streams by exploiting the universal trait of P accumulation and P turnover in microorganisms and plants.

Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells

Abstract: The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the "Warburg" effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation-dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody-producing CHO DP-12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl-CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate-based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.

Recent advances in microbial capture of hydrogen sulfide from sour gas via sulfur-oxidizing bacteria.

Abstract: Biological desulfurization offers several remarkably environmental advantages of operation at ambient temperature and atmospheric pressure, no demand of toxic chemicals as well as the formation of biologically re-usable sulfur (S0), which has attracted increasing attention compared to conventionally physicochemical approaches in removing hydrogen sulfide from sour gas. However, the low biomass of SOB, the acidification of process solution, the recovery of SOB, and the selectivity of bio-S0 limit its industrial application. Therefore, more efforts should be made in the improvement of the BDS process for its industrial application via different research perspectives. This review summarized the recent research advances in the microbial capture of hydrogen sulfide from sour gas based on strain modification, absorption enhancement, and bioreactor modification. Several efficient solutions to limitations for the BDS process were proposed, which paved the way for the future development of BDS industrialization.

Micro-aerobic production of isobutanol with engineered Pseudomonas putida.

Abstract: Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled-up the production of isobutanol with P. putida from shake flask to fed-batch cultivation in a 30 L bioreactor. The design of a two-stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L-1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2-ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro-aerobic conditions during production doubled the integral glucose-to-isobutanol conversion yield to 60 mgisobutanol gglucose -1 while preventing undesired carbon loss as 2-ketogluconic acid.

A novel twin-column continuous chromatography approach for separation and enrichment of monoclonal antibody charge variants

Abstract: Downstream processing of mAb charge variants is difficult owing to their similar molecular structures and surface charge properties. This study aimed to apply a novel twin-column continuous chromatography (called N-rich mode) to separate and enrich acidic variants of an IgG1 mAb. Besides, a comparison study with traditional scaled-up batch-mode cation exchange (CEX) chromatography was conducted. For the N-rich process, two 3.93 mL columns were used, and the buffer system, flow rate and elution gradient slope were optimized. The results showed that 1.33 mg acidic variants with nearly 100% purity could be attained after a 22-cycle accumulation. The yield was 86.21% with the productivity of 7.82 mg/L/h. On the other hand, for the batch CEX process, 4.15 mL column was first used to optimize the separation conditions, and then a scaled-up column of 88.20 mL was used to separate 1.19 mg acidic variants with the purity of nearly 100%. The yield was 59.18% with the productivity of 7.78 mg/L/h. By comparing between the N-rich and scaled-up CEX processes, the results indicated that the N-rich method displays a remarkable advantage on the product yield, i.e. 1.46-fold increment without the loss of productivity and purity. Generally, twin-column N-rich continuous chromatography displays a high potential to enrich minor compounds with a higher yield, more flexibility and lower resin cost.

A fluorescence-based yeast sensor for monitoring acetic acid.

Abstract: Accumulation of acetic acid indicates an imbalance of the process due to a disturbed composition of the microorganisms. Hence, monitoring the acetic acid concentration is an important parameter to control the biogas process. Here, we describe the generation and validation of a fluorescence-based whole cell sensor for the detection of acetic acid based on the yeast Saccharomyces cerevisiae. Acetic acid induces the transcription of a subset of genes. The 5´-regulatory sequences (5´ URS) of these genes were cloned into a multicopy plasmid to drive the expression of a red fluorescent reporter gene. The 5´ URS of YGP1, encoding a cell wall-related glycoprotein, led to a 20-fold increase of fluorescence upon addition of 30 mM acetic acid to the media. We show that the system allows estimating the approximate concentration of acetic acid in condensation samples from a biogas plant. To avoid plasmid loss and increase the long-term stability of the sensor, we integrated the reporter construct into the yeast genome and tested the suitability of spores for long-term storage of sensor cells. Lowering the reporter gene's copy number resulted in a significant drop of the fluorescence, which can be compensated by applying a yeast pheromone-based signal amplification system.

Assessment of extraction options for a next-generation biofuel: Recovery of bio-isobutanol from aqueous solutions.

Abstract: Isobutanol is a widely used platform compound and a raw material for synthesizing many high value-added compounds. It also has excellent fuel properties and is an ideal gasoline additive or substitute with a very broad development space. Isobutanol production by biological fermentation has the advantages of a comprehensive source of raw materials, low cost, environmental protection, and sustainability. However, it also has disadvantages such as many impurities, low isobutanol concentration, and difficulty separating the water + isobutanol azeotrope. Thus, it is necessary to explore an appropriate downstream separation process for the water + isobutanol azeotrope. K2CO3 with a strong salting-out effect was used as the salting-out agent, and the salting-out of isobutanol from aqueous solutions was investigated at 298.15 K. The effect of the initial salt concentration in the aqueous solution, the recovery of isobutanol, and the effect of dehydration were investigated in detail. The e-NRTL-RK model was employed to generate the binary parameters for isobutanol and water, and electrolyte pair parameters for water/isobutanol and ions to reproduce the phase diagram with high accuracy. The processes of solvent extractive distillation, and salting-out + distillation were simulated by Aspen Plus. The energy consumptions for the solvent-based and salting-out-based processes were compared. The salting-out + distillation process turned out to be more energy-saving than the solvent extraction process.

Implementation of QbD strategies in the inoculum expansion of a mAb production process.

Abstract: The quality by design approach was introduced to the biopharmaceutical industry over 15 years ago. This principle is widely implemented in the characterization of monoclonal antibody production processes. Anyway, the early process phase, namely the inoculum expansion, was not yet investigated and characterized for most processes. In order to increase the understanding of early process parameter interactions and their influence on the later production process, a risk assessment followed by a design of experiments approach was conducted. The DoE included the critical parameters methotrexate (MTX) concentration, initial passage viable cell density and passage duration. Multivariate data analysis led to mathematical regression models and the establishment of a designated design space for the studied parameters. It was found that the passage duration as well as the initial viable cell density for each passage during the inoculum expansion have severe effects on the growth rate and viability of the early process phase. Furthermore, the variations during the inoculum expansion directly influenced the production process responses. This carry-over of factor effects highlights the crucial impact of early process failures and the importance of process analysis and control during the first part of mAb production processes.