Batch Phase Research Articles

Aligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein

Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N6-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli. We used the orthogonal pyrrolysyl-tRNA synthetase/suppressor tRNACUAPyl pair from Methanosarcina mazei to incorporate Azk site-specifically. We characterized Azk uptake and Fab production at bench-scale under different fermentation conditions, varying timing and mode of Azk addition, Azk-to-cell ratio and induction time. Our results indicate that Azk uptake is comparatively efficient in the batch phase. We discovered that the time between Azk uptake and inducing its incorporation into the Fab must be kept short, which suggests that intracellular Azk is consumed and/or degraded. The results obtained in this study are an important step towards the development of efficient production methods for Azk-incorporated proteins in E. coli. The developed process is scalable and provides excellent yields of 2.95 mg functionalized Fab per g CDM, which corresponds to 80% of yield obtained with the wild type Fab. We also identified the cellular uptake of Azk being dependent on the physiological state of the cell as a potential bottleneck in production.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations

BackgroundFilamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology.ResultsThis study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{-1}$$\\end{document} in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h)-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{-1}$$\\end{document}, the lowest rate of 0.3 g (l h)-1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$^{-1}$$\\end{document} revealed a threefold increase in cellobiohydrolase and a fivefold increase in β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\upbeta$$\\end{document}-glucosidase activity compared to batch processes using the same type and amount of carbon sources.ConclusionWe successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

High-pressure batch reverse osmosis (RO) for zero liquid discharge (ZLD) in a Cr(III) electroplating process

A batch RO system was designed and built for high-pressure (120 bar) operation. The system was developed for a ZLD application involving treatment of metal plating wastewater from a Cr(III) electroplating process at a major industrial plant. Hybrid semi-batch/batch operation enabled a compact design to be achieved. To maximize water recovery without exceeding a set peak pressure, a method for controlling the switch point between semi-batch and batch phases was developed. The system was tested with feed representative of rinse water from the electroplating process. A range of feed concentrations (at 10–20× dilution of the plating bath), feed flows (0.21–0.46 m3/h), water fluxes (6–14 LMH) and water recoveries (87–95.7 %) were investigated. The system successfully recovered Cr(III) and restored its concentration to that of the electrolyte bath, thus meeting the requirements for reuse in the electroplating process. Rejection of most species was >99.8 %, sufficient for reuse of the permeate as rinse. However, rejection of boric acid was only 69–80 % such that a second RO pass may be needed to remove boric acid. Specific Energy Consumption was <2.25 kWh per m3 of treated rinse water, representing a 50-fold saving compared to the current method of treatment and disposal at the industrial plant.

Integrating microalgae-based wastewater treatment, biostimulant production, and hydroponic cultivation: a sustainable approach to water management and crop production.

A natural appearing microalgae-bacteria consortium was used to process urban wastewater. The process was done in an 80m2 raceway reactor and the results were compared to an identical reactor operated using freshwater supplemented with commercial fertilisers. The biomass harvesting was done using commercial ultrafiltration membranes to reduce the volume of culture centrifuged. The membrane allowed achieving a biomass concentration of ∼9-10gL-1. The process proposed avoids the use of centrifuges and the drying of the biomass, two of the most energy consuming steps of conventional processes. The specific growth rate in freshwater and the wastewater-based media was estimated as 0.30 ± 0.05 and 0.24 ± 0.02 days-1, respectively (p < 0.05). The maximum concentration reached at the end of the batch phase was 0.96 ± 0.03 and 0.83 ± 0.07gL-1 when the biomass was produced using freshwater and wastewater, respectively (p < 0.05). The total nitrogen removal capacity of the system was on average 1.35gm-2·day-1; nitrogen assimilation into biomass represented 60%-95% of this value. Furthermore, the P-PO4 3- removal capacity of the system varied from 0.15 to 0.68gm-2·day-1. The outlet effluent of the reactor was used as a nutrient source in the hydroponic production of zucchini seedlings, leading to an increase in the root dry weight and the stem diameter compared to the water alone. The produced biomass showed potential for use as feedstock to produce plant biostimulants with positive effects on root development and chlorophyll retention.

Adaptive Fed-batch Control of Escherichia coli Fermentation for Protein Production

A new adaptive linearizing control algorithm that stabilizes the carbon source concentration in a desired value is proposed. This algorithm is applied to recombinant protein production by Escherichia coli. A model for control of the investigated process is derived. The model identification is made based on experimental data of the batch phase of the process. The operating model includes two sub-models. Each of them describes one of the two physiological states through which the process passes. Switching from one model to another depends on the sign of a key parameter obtained from the acetate measurements. A cascade scheme of software sensors for the estimation of two biomass growth rates included in the structure of the proposed control algorithm is derived. Simulation studies of the developed closed system have been carried out. The results of the impact of an open-loop control system on the same object are compared.

Machine Learning Approaches for Phase Identification Using Process Variables in Batch Processes

AbstractSpecialty and fine chemicals are often manufactured in multipurpose batch production plants. Compared to continuous production, these plants offer increased flexibility at the cost of operational complexity. A recipe defines the sequence and process parameters of different batch phases that are needed to transform raw materials into the desired product. In some plants detailed information about the executed recipe is not always captured by data acquisition systems. Knowledge of these phases is essential for optimizing quality and throughput. State‐of‐the‐art data‐driven machine learning techniques can recognize recurrent patterns in noisy time series data, enabling automatic labeling of batch phases based on widely available sensor data. In this review paper, we provide an overview of several machine learning approaches that can be used in an industrial setting.

Generalizability of Soft Sensors for Bioprocesses through Similarity Analysis and Phase-Dependent Recalibration.

A soft sensor concept is typically developed and calibrated for individual bioprocesses in a time-consuming manual procedure. Following that, the prediction performance of these soft sensors degrades over time, due to changes in raw materials, biological variability, and modified process strategies. Through automatic adaptation and recalibration, adaptive soft sensor concepts have the potential to generalize soft sensor principles and make them applicable across bioprocesses. In this study, a new generalized adaptation algorithm for soft sensors is developed to provide phase-dependent recalibration of soft sensors based on multiway principal component analysis, a similarity analysis, and robust, generalist phase detection in multiphase bioprocesses. This generalist soft sensor concept was evaluated in two multiphase bioprocesses with various target values, media, and microorganisms. Consequently, the soft sensor concept was tested for biomass prediction in a Pichia pastoris process, and biomass and protein prediction in a Bacillus subtilis process, where the process characteristics (cultivation media and cultivation strategy) were varied. High prediction performance was demonstrated for P. pastoris processes (relative error = 6.9%) as well as B. subtilis processes in two different media during batch and fed-batch phases (relative errors in optimized high-performance medium: biomass prediction = 12.2%, protein prediction = 7.2%; relative errors in standard medium: biomass prediction = 12.8%, protein prediction = 8.8%).

Cloning, protein expression and biochemical characterization of Carica papaya esterase

BackgroundGDSL-like esterase/lipase proteins (GELPs) are enzymes that possess unique characteristics, they contain four invariable catalytic residues. Advances in the study of these proteins are interesting. The cloning and functional expression of a papaya esterase have not been reported. Therefore, in this work we evaluated the heterologous production of Carica papaya esterase CpEST in the yeast Komogataella phaffii (Pichia pastoris). ResultsThe cloning and expression of the protein was performed under the PAOX1 promoter, and productions of up to 43 AU/mL were achieved using residual glycerol from biodiesel in the batch phase and methanol for the induction phase. Enzyme activity assays determined that CpEST has a high preference for short-chain substrates (p-NP C4 and p-NP C8), and optimal activity conditions were observed at 30°C and pH 10. The enzyme showed the highest stability to acetone, ethanol and tert-butanol solvents, retaining approximately 55% of its initial enzymatic activity after 1 h of exposure. ConclusionsCloning and functional expression of papaya CpEST esterase was achieved. During fermentation, the yeasts used as a carbon source residual glycerol from biodiesel production. Based on the results obtained from the characterization of the esterase, it was found that it has a high potential for use in the bioenergy and detergent industry.How to cite: Reyes-Reyes AL, Valero F, Sandoval G. Cloning, protein expression and biochemical characterization of Carica papaya esterase. Electron J Biotechnol 2023;61. https://doi.org/10.1016/j.ejbt.2022.11.004.

Disrupting Biofilms From Titanium with a CHG Antimicrobial Irrigation System

Category: Other Introduction/Purpose: Orthopedic device-related infections can be difficult to treat and decrease the success of surgical interventions. The implant provides a surface for bacterial attachment and biofilm formation, which decreases antimicrobial efficacy. This study was conducted to assess the ability of the Chlorhexidine Gluconate (CHG) Antimicrobial Irrigation System to disrupt biofilms from titanium coupons using an adapted ASTM E2871-19 method, 'Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single Tube Method.' Methods: CDC Biofilm Reactor was inoculated with Staphylococcus epidermidis (ATCC 14990TM), methicillin-resistant Staphylococcus aureus (ATCC 43300TM), Pseudomonas aeruginosa (ATCC 15442TM), or Escherichia coli (ATCC 25922TM). The reactors were run in a batch phase for 24 h, followed by continuous flow phase for an additional 24 h to produce single-species biofilms on titanium coupons. After coupons were washed with PBS to remove planktonic bacteria, growth control coupons were placed into standard sampling solution with 1% Tamol (SST) (neutralizer) or treated with CHG Antimicrobial Irrigation System according to Instructions for Use (IFU) followed by a 60 sec dwell or no dwell. Coupons were rinsed with normal saline following treatment and placed in SST. Then coupons were sonicated and vortexed to release bacteria and plated for enumeration. A coupon from each group was fixed for SEM imaging. Results: The CHG Antimicrobial Irrigation System disrupted biofilms of all organisms from titanium coupons with the greatest log reduction per coupon in the 60 sec dwell treatment group (log10 reduction 2.02 to 4.56 CFU/coupon from controls) (Table). Overall, S. epidermidis and E. coli biofilms had the greatest reductions in bacterial densities. SEM images confirmed biofilm disruption from the titanium surface. Conclusion: his study determined that the CHG Antimicrobial Irrigation System disrupted biofilms from titanium coupons when used according to the instructions for use.

CPV of the Future: AI-Powered Continued Process Verification for Bioreactor Processes.

According to the standard guidelines by the FDA, process validation in biopharma manufacturing encompasses a life cycle consisting of three stages: process design (PD), process qualification (PQ), and continued process verification (CPV). The validity and efficiency of the analytics methods employed during the CPV require extensive knowledge of the process. However, for new processes and new drugs, such knowledge is often not available from Process performance qualification and Validation (PPQV). In this work, the suitability of methods based on machine learning/artificial intelligence (ML/AI) for the CPV applied in bioprocess monitoring and cell physiological control of the yeast Pichia pastoris (Komagataella phaffii) was studied with limited historical data. In particular, the production of recombinant Candida rugosa lipase 1 (Crl1) under hypoxic conditions in fed-batch cultures was considered as a case study. Supervised and unsupervised machine learning models using data from fed-batch bioprocesses with different gene dosage clones under normoxic and hypoxic conditions were evaluated. Firstly, a multivariate anomaly detection (isolation forest) model was applied to the batch phase of the bioprocess. Secondly, a supervised random forest model for prediction of required operator's control actions during the semiautomated fed-batch phase under hypoxic conditions was assessed to maintain the respiratory quotient (RQ) within the desired range for maximizing the specific production rate (qP ). The performance of these models was tested on historical data using independent evaluation of the process by the process control engineer (subject matter expert-SME), and on real-time data in the case of manual action prediction, where the model was implemented to guide the control of the bioprocess. The work presented here constitutes a proof-of-concept that multivariate analytics methods, based on machine learning, can be a valuable tool for real-time monitoring and control of biopharma manufacturing bioprocesses to improve its efficiency and to assure product quality.

Mineral medium design based on macro and trace element requirements for high cell density cultures of Priestia megaterium DSM 509

To assess the suitability of a mineral medium selected from the literature for high cell density cultivations of Priestia megaterium DSM 509 (ultimately for producing PHA), fermentations were planned and tested in a (2 L) bioreactor. After few hours of a batch culture, growth was limited despite an efficient oxygenation and the relative abundance of macronutrients, suggesting trace elements (TE) deficiencies. They had to be increased to achieve higher growth rates and cell densities. Growth was improved by adding up to 10 times more TE as maximum growth rate increased from 0.20 to 2.36 g L−1 h−1, but eventually growth limitations occurred. Major and trace minerals were therefore determined by ICP-OES. These analyses showed that the selected medium was deficient mainly in Co, Mn and Zn. Requirements for 11 minerals could then be assessed and used to formulate a new mineral medium. Tested in a fed-batch culture assigned to a specific growth rate of 0.2 h−1, it allowed P. megaterium to grow in 8 h during the batch phase without being limited until the exhaustion of glucose. In the fed-batch phase, high cell densities of 53.6 g L−1 were reached after 10 h with a space-time yield of 2.96 g L−1 h−1.

Control of bacterial contamination in microalgae cultures integrated with wastewater treatment by applying feast and famine conditions

The integration of microalgae production with wastewater treatment can significantly increase economic and environmental sustainability of the treatment process and of the microalgal biomass production. However, a major bottleneck of this strategy is the control of contamination by heterotrophic bacteria, which compete with microalgae for the organic substrate and can negatively affect the quality of the produced biomass. Here, a strategy to control bacterial contamination is proposed, whereby a first batch phase with the medium replete in the organic substrate (feast) is followed by a second batch phase without the organic substrate (famine). Permeates from microfiltration and ultrafiltration of cheese whey were used as sources of organic substrates, with different C/N. Biomass production and pollutant removal were analyzed, and the growth kinetics of microalgae and bacteria were characterized and modeled to determine the specific growth rate (µmax) during the feast phase and the decay rate (kD) during the famine phase. Bacteria had µmax (0.16 h−1) about two folds higher than microalgae (0.07 h−1), however, bacteria lysed in larger fraction during the famine phase, allowing to reduce contamination. A remarkable finding was that, for both microalgae and bacteria, only a cell subpopulation experienced lysis during the famine phase. The fraction of resistant cells was higher for microalgae and decreased with increasing the C/N ratio in the initial medium, indicating that cell-to-cell heterogeneity for carbon storage is crucial in determining cell resistance. Guidelines are derived to maximize microalgae biomass productivity and pollutant removal while maintaining a prescribed bacterial contamination.

Production, purification, and characterization of recombinant rabies virus glycoprotein expressed in PichiaPink™ yeast

The commonly used host for industrial production of recombinant proteins Pichia pastoris, has been used in this work to produce the rabies virus glycoprotein (RABV-G). To allow a constitutive expression and the secretion of the expressed recombinant RABV-G, the PichiaPink™ commercialized expression vectors were modified to contain the constitutive GAP promoter and the α secretion signal sequences. Recombinant PichiaPink™ strains co-expressing the RABV-G and the protein chaperone PDI, have been then generated and screened for the best producer clone. The influence of seven carbon sources on the expression of the RABV-G, has been studied under different culture conditions in shake flask culture. An incubation temperature of 30°C under an agitation rate of 250 rpm in a filling volume of 10:1 flask/culture volume ratio were the optimal conditions for the RABV-G production in shake flask for all screened carbon sources. A bioreactor Fed batch culture has been then carried using glycerol and glucose as they were good carbon sources for cell growth and RABV-G production in shake flask scale. Cells were grown on glycerol during the batch phase then fed with glycerol or glucose defined solutions, a final RABV-G concentration of 2.7 µg/l was obtained with a specific product yield (YP/X) of 0.032 and 0.06 µg/g(DCW) respectively. The use of semi-defined feeding solution enhanced the production and the YP/X to 12.9 µg/l and 0.135 µg/g(DCW) respectively. However, the high cell density favored by these carbon sources resulted in oxygen limitation which influenced the glycosylation pattern of the secreted RABV-G. Alternatively, the use of sucrose as substrate for RABV-G production in large scale culture, resulted in less biomass production and a YP/X of 0.310 µg/g(DCW) was obtained. A cation exchange chromatography was then used for RABV-G purification as one step method. The purified protein was correctly folded and glycosylated and able to adopt trimeric conformation. The knowledges gained through this work offer a valuable insight into the bioprocess design of RABV-G production in Pichia pastoris to obtain a correctly folded protein which can be used during an immunization proposal for subunit Rabies vaccine development.

Stress-induced increase of monoclonal antibody production in CHO cells.

Monoclonal antibodies (mAbs) are of great interest to the biopharmaceutical industry due to their widely used application as human therapeutic and diagnostic agents. As such, mAb require to exhibit human‐like glycolization patterns. Therefore, recombinant Chinese hamster ovary (CHO) cells are the favored production organisms; many relevant biopharmaceuticals are already produced by this cell type. To optimize the mAb yield in CHO DG44 cells a corelation between stress‐induced cell size expansion and increased specific productivity was investigated. CO2 and macronutrient supply of the cells during a 12‐day fed‐batch cultivation process were tested as stress factors. Shake flasks (500 mL) and a small‐scale bioreactor system (15 mL) were used for the cultivation experiments and compared in terms of their effect on cell diameter, integral viable cell concentration (IVCC), and cell‐specific productivity. The achieved stress‐induced increase in cell‐specific productivity of up to 94.94.9%–134.4% correlates to a cell diameter shift of up to 7.34 μm. The highest final product titer of 4 g/L was reached by glucose oversupply during the batch phase of the process.

Linking process and metabolic modelling for the estimation of carbon flux distribution in Corynebacterium glutamicum growth in spent sulfite liquor

Process monitoring in microbial cultures became feasible thanks to the development of accurate measurement devices, including in-situ probes to monitor biomass growth, oxygen, carbon dioxide and sugar consumption. In comparison, estimating the metabolic fluxes of the cell factories still rely on analysis based on an under-determined set of equations, and requires an expensive and time consuming methods of verification. This problem intensifies in the presence of complex substrates, in which different sugars are utilized in parallel by the cell factories. In the present study, a growth experiment of Corynebacterium glutamicum in spent sulfite liquor was studied. The bioprocess was monitored during batch and fed-batch phases, and a parameter estimation routine was conducted to define a process model and the corresponding uptake rates. A tracking optimization algorithm minimized the error between the measured process fluxes and the equivalent fluxes of the elementary flux modes. The results indicate that the optimization technique obtained a set of elementary modes that are closer to reality than the computed from the metabolic analysis. Taken together, we show that an online estimation of metabolic flux distribution of C glutamicum based on a set of process measurement signals was possible with an optimization function that links the process and metabolic model. The procedure can be complementary to the sophisticated and expensive C NMR experimental analytical technique.

Fed-Batch mcl- Polyhydroxyalkanoates Production in Pseudomonas putida KT2440 and ΔphaZ Mutant on Biodiesel-Derived Crude Glycerol.

Crude glycerol has emerged as a suitable feedstock for the biotechnological production of various industrial chemicals given its high surplus catalyzed by the biodiesel industry. Pseudomonas bacteria metabolize the polyol into several biopolymers, including alginate and medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs). Although P. putida is a suited platform to derive these polyoxoesters from crude glycerol, the attained concentrations in batch and fed-batch cultures are still low. In this study, we employed P. putida KT2440 and the hyper-PHA producer ΔphaZ mutant in two different fed-batch modes to synthesize mcl-PHAs from raw glycerol. Initially, the cells grew in a batch phase (μmax 0.21 h–1) for 22 h followed by a carbon-limiting exponential feeding, where the specific growth rate was set at 0.1 (h–1), resulting in a cell dry weight (CDW) of nearly 50 (g L–1) at 40 h cultivation. During the PHA production stage, we supplied the substrate at a constant rate of 50 (g h–1), where the KT2440 and the ΔphaZ produced 9.7 and 12.7 gPHA L–1, respectively, after 60 h cultivation. We next evaluated the PHA production ability of the P. putida strains using a DO-stat approach under nitrogen depletion. Citric acid was the main by-product secreted by the cells, accumulating in the culture broth up to 48 (g L–1) under nitrogen limitation. The mutant ΔphaZ amassed 38.9% of the CDW as mcl-PHA and exhibited a specific PHA volumetric productivity of 0.34 (g L–1 h–1), 48% higher than the parental KT2440 under the same growth conditions. The biosynthesized mcl-PHAs had average molecular weights ranging from 460 to 505 KDa and a polydispersity index (PDI) of 2.4–2.6. Here, we demonstrated that the DO-stat feeding approach in high cell density cultures enables the high yield production of mcl-PHA in P. putida strains using the industrial crude glycerol, where the fed-batch process selection is essential to exploit the superior biopolymer production hallmarks of engineered bacterial strains.

Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli

• VHb reduced overflow metabolism. • VHb improved pDNA production under oxygen limitations. • VHb improved pDNA yields even at lower RNAII/RNAI ratios. • Higher lactate production may be caused by stronger NAD + demand. Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In this study, Vitreoscilla hemoglobin (VHb) was expressed in the chromosome of E. coli as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the wild type strain. The culture was shifted from aerobic to microaerobic conditions during the fed-batch phase. pDNA yields increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the wild type under microaerobic conditions. Unexpectedly, the ratio of positive/negative replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the wild type strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the fluxes in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD + in glycolysis under microaerobic conditions.

Optimization of carbon source efficiency for lipid production with the oleaginous yeast Saitozyma podzolica DSM 27192 applying automated continuous feeding

BackgroundBiotechnologically produced microbial lipids are of interest as potential alternatives for crude and plant oils. Their lipid profile is similar to plant oils and can therefore be a substitute for the production of biofuels, additives for food and cosmetics industry as well as building blocks for oleochemicals. Commercial microbial lipids production, however, is still not profitable and research on process optimization and cost reduction is required. This study reports on the process optimization using glucose or xylose with the unconventional oleaginous yeast Saitozyma podzolica DSM 27192 aiming to reduce the applied carbon source amount without sacrificing lipid productivity.ResultsBy optimizing the process parameters temperature and pH, lipid productivity was enhanced by 40%. Thereupon, by establishing a two-phase strategy with an initial batch phase and a subsequent fed-batch phase for lipid production in which a constant sugar concentration of about 10 g/L was maintained, resulted in saving of ~ 41% of total glucose and ~ 26% of total xylose. By performing the automated continuous sugar feed the total sugar uptake was improved to ~ 91% for glucose and ~ 92% for xylose and thus, prevented waste of unused carbon source in the cultivation medium. In addition, reduced glucose cultivation resulted in to 28% higher cell growth and 19% increase of lipid titer. By using xylose, the by-product xylonic acid was identified for the first time as by-product of S. podzolica.ConclusionsThese findings provide a broad view of different cultivation process strategies with subsequent comparison and evaluation for lipid production with S. podzolica. Additionally, new biotechnological characteristics of this yeast were highlighted regarding the ability to produce valuable organic acids from sustainable and renewable sugars.

Mixed glycerol and orange peel-based substrate for fed-batch microbial biodiesel production

The aqueous extraction of orange peel waste (OPW), the byproduct of the juice extraction process generated annually in massive amounts (21 Mton), yields a carbohydrate-rich liquid fraction, termed orange peel extract (OPE). Several studies highlight that the combination of glycerol, a biodiesel byproduct, with carbohydrate mixtures might boost microbial lipid production. This study performed first a shaken flask screening of 15 oleaginous yeast strains based on their growth and lipid-producing abilities on OPE- and glycerol-based media. This screening enabled the selection of R. toruloides NRRL 1091 for the assessment of the process transfer in a stirred tank reactor (STR). This assessment relied, in particular, on either single- and double-stage feeding fed-batch (SSF-FB and DSF-FB, respectively) processes where OPE served as the primary medium and nitrogen-containing glycerol-OPE mixtures as the feeding one. The continuous supply mode at low dilution rates (0.02 and 0.01 h−1 for SSF-FB and DSF-FB, respectively) starting from the end of the exponential growth of the initial batch phase enabled the temporal extension of biomass and lipid production. The SSF-FB and DSF-FB processes attained high biomass and lipid volumetric productions (LVP) and ensured significant lipid accumulation on a dry cell basis (YL/X). The SSF-FB process led to LVP of 20.6 g L−1 after 104 h with volumetric productivity (rL) of 0.20 g L−1 h−1 and YL/X of 0.80; the DSF-FB process yielded LVP, rL and YL/X values equal to 15.92 g L−1, 0.11 g L−1 h−1 and 0.65, respectively. The fatty acid profiles of lipids from both fed-batch processes were not significantly different and resembled that of Jatropha oil, a vastly used feedstock for biodiesel production. These results suggest that OPE constitutes an excellent basis for the fed-batch production of R. toruloides lipids, and this process might afford a further option in OPW-based biorefinery.

Optimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations

BackgroundSmall-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved.ResultsCustomization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24–0.90 mg/h per well.ConclusionsThe optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.