Enhanced lipid production by Rhodosporidium toruloides using different fed-batch feeding strategies with lignocellulosic hydrolysate as the sole carbon source

Additional file 3: Figure S3. Time course of dissolved oxygen (DO) value during the pulse fed-batch (FB) cultures

Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures

To increase the yield of heterologous production of the class II bacteriocin DvnRV41 with Escherichia coli Origami (DE3) (pLysS/pCR03), induction of bacteriocin gene expression was optimized by varying the inducer isopropyl β-d-thiogalactopyranoside (IPTG) concentration (0-2mM), and controlled batch and fed-batch cultures were tested on a 2-L scale. A concentration of 0.5mM IPTG was found to be optimal for cell growth and bacteriocin production. Shake flask cultivation of E. coli Origami (DE3) (pLysS/pCR03) gave biomass and bacteriocin yields of 1.54 ± 0.06g cdw/l and 18 ± 1mg DvnRV41/l, respectively. Biomass (2.70 ± 0.06 and 6.8 ± 0.6g cdw/l, respectively) and bacteriocin yields (30 and 74mg DvnRV41 per liter, respectively) were both increased with batch and fed-batch compared to shake flask cultures. Bacteriocin yields reported in this study are among the highest published for other heterologous expression systems in shake flask

Production of γ-decalactone by Yarrowia lipolytica : insights into experimental conditions and operating mode optimization

BACKGROUND γ-Decalactone production from ricinoleic acid biotransformation derived from the triglycerides in castor oil by Yarrowia lipolytica, has been widely described in literature in studies concerning lipidic metabolism that leads to lactones production, interactions of cells with the lipid substrate, toxicity of produced metabolites, selection of over-producing mutants and selection of environmental conditions. RESULTS In order to improve technological aspects of γ-decalactone production, oxygen transfer rate (OTR), cell density and oil concentration effects were investigated, in batch and step-wise fed-batch cultures of Yarrowia lipolytica W29. The best γ-decalactone concentration of 5.4 ± 0.5 g L-1 was obtained for batch cultures with 60 g L-1 of cells and substrate concentration. CONCLUSION The direct influence of aeration and agitation rates, thus of OTR, on production of γ-decalactone has been demonstrated. γ-Decalactone productivity of 215 ± 19 mg L-1 h-1 was obtained with 60 g L-1 of cells and castor oil concentration in batch and step-wise fed-batch cultures of Yarrowia lipolytica. The results obtained suggest that these two strategies are good alternatives for industrial production processes.The authors thank the Project 'BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028' co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER and Fundacao para a Ciencia e a Tecnologia (FCT) (SFRH/BD/63701/2009 PhD grant to Adelaide Braga) for the financial support provided and FCT Strategic Project PEst-OE/EQB/LA0023/2013

Comparison of batch and fed-batch lipase production from olive mill wastewater by Yarrowia lipolytica and Candida cilindracea

Olive mill wastewater (OMW) is a liquid waste that results in large amounts from the olive oil manufacturing industry. The quality and quantity of OMW constituents are dependent of many factors, such as olives type and maturity, climatic conditions and region of origin, cultivation methods, and technology used for oil extraction1. From the 3-phases centrifugation process around 1.6 cubic meters of OMW per ton of olives processed are generated. These liquid wastes present an environmental problem and many solutions have been proposed for it, such as its use as culture medium for different lipolytic yeast strains (Gonçalves et al, 2009; Lopes et al, 2009). The aim of this work is the comparison of batch and fed-batch mode of operation for the lipase production and the OMW degradation by two strains, Yarrowia lipolytica W29 and Candida cylindracea CBS 7869. OMW collected from 3-phase continuous olive mills were used (COD of 30 to 261 g/L). OMW used without dilution was supplemented with ammonium chloride and yeast extract proportionally to the COD values. Batch and fed-batch cultures were conducted in a fermenter of 2 L of capacity at pH 7.2, 500 rpm, with constant or variable aeration rate for batch or fed-batch operation, respectively. Batch operation was more adequate to lipase production than fed-batch for both strains but the difference was more significant for Candida cylindracea that revealed to be the most efficient strain for lipase production. However, the final media of the fed-batch cultures presented lower values of COD and sugars indicating a higher level of organic matter degradation

Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

Bioprocess Development for Lantibiotic Ruminococcin-A Production in Escherichia coli and Kinetic Insights Into LanM Enzymes Catalysis

Ruminococcin-A (RumA) is a peptide antibiotic with post-translational modifications including thioether cross-links formed from non-canonical amino acids, called lanthionines, synthesized by a dedicated lanthionine-generating enzyme RumM. RumA is naturally produced by Ruminococcus gnavus, which is part of the normal bacterial flora in the human gut. High activity of RumA against pathogenic Clostridia has been reported, thus allowing potential exploitation of RumA for clinical applications. However, purifying RumA from R. gnavus is challenging due to low production yields (120 mg L–1 for the chimeric construct and >150 mg L–1 for RumM. The correlation observed between microscale and lab-scale bioreactor cultivations suggests that the process is robust and realistically applicable to industrial-scale conditions.DFG, 53182490, EXC 314: Unifying Concepts in CatalysisDFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

Electrooptical Determination of Polarizability for On-Line Viability and Vitality Quantification of Lactobacillus plantarum Cultures

The rapid assessment of cell viability is crucial for process optimization, e.g., during media selection, determination of optimal environmental growth conditions and for quality control. In the present study, the cells' electric anisotropy of polarizability (AP) as well as the mean cell length in Lactobacillus plantarum batch and fed-batch fermentations were monitored with electrooptical measurements coupled to fully automated sample preparation. It was examined, whether this measurement can be related to the cells' metabolic activity, and thus represents a suitable process analytical technology. It is demonstrated that the AP is an early indicator to distinguish between suitable and unsuitable growth conditions in case of a poor energy regeneration or cell membrane defects in L. plantarum batch and fed-batch cultivations. It was shown that the applied method allowed the monitoring of physiological and morphological changes of cells in various growth phases in response to a low pH-value, substrate concentration changes, temperature alterations, exposure to air and nutrient limitation. An optimal range for growth in batch mode was achieved, if the AP remained above 25·10−28 F·m2 and the mean cell length at ~2.5 μm. It was further investigated, in which way the AP develops after freeze-drying of samples, which were taken in different cultivation phases. It was found that the AP increased most rapidly in resuspended samples from the retardation and late stationary phases, while samples from the early stationary phase recovered slowly. Electrooptical measurements provide valuable information about the physiologic and morphologic state of L. plantarum cells, e.g., when applied as starter cultures or as probiotic compounds.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität BerlinEC/H2020/643056/EU/Rapid Bioprocess Development/Biorapi

Model Predictive Feeding Rate Control in Conventional and Single-use Lab-scale Bioreactors: A Study on Practical Application

A developed solution for fed-batch process modeling and model predictive control (MPC), facilitating good manufacturing practice (GMP) based on process elaboration, control, and validation, is presented in the paper. The step-by-step evolution of the so-called “golden batch” optimal biomass growth profile and its control during the process is demonstrated. The case study of an advanced fed-batch control was performed on the recombinant E. coli BL21 lab-scale (5.4 L) biomass production process using the conventional stirred tank glass reactor. Additionally, a test experiment for control reproducibility and applicability assessment of the proposed approach was carried out in a single-use stirred tank reactor (5.7 L). Four sequentially performed experiments are demonstrated as an example for desirable feeding profile evolution for E. coli BL21 biomass production in a glucose-limited fed-batch process. Under different initial biomass and glucose conditions, as well as for different reference feeding profiles selected in the explorative experiments, good tracking quality of preset reference trajectories by the MPC system has been demonstrated. Estimated and experimentally measured biomass mean deviations from the preset reference value at the end of the processes were 4.6 and 3.8 %, respectively. Biomass concentration of 93.6 g L–1 (at 24 h) was reached in the most productive run. Better process controllability and safer process run, in terms of avoiding culture overfeeding but still maintaining a sufficiently high growth rate, was suggested for the process with biomass yield of 79.8 g L–1 (at 24 h). Practical recommendations on the approach application and adaptation for fed-batch cultures of interest are provided

High-level fed-batch fermentative expression of an engineered Staphylococcal protein A based ligand in E. coli: purification and characterization

The major platform for high level recombinant protein production is based on genetically modified microorganisms like Escherichia coli (E. coli) due to its short dividing time, ability to use inexpensive substrates and additionally, its genetics is comparatively simple, well characterized and can be manipulated easily. Here, we investigated the possibilities of finding the best media for high cell density fermentation, by analyzing different media samples, focusing on improving fermentation techniques and recombinant protein production. Initial fermentation of E. coli BL21 DE3:pAV01 in baffled flasks showed that high cell density was achieved when using complex media, Luria–Bertani (LB) and Terrific medium broth (TB) (10 and 14 g/L wet weight, respectively), as compared to mineral media M9, modified minimal medium (MMM) and Riesenberg mineral medium (RM) (7, 8 and 7 g/L, respectively). However, in fed-batch fermentation processes when using MMM after 25 h cultivation, it was possible to yield an optical density (OD600) of 139 corresponding to 172 g/L of wet biomass was produced in a 30 L TV Techfors-S Infors HT fermenter, with a computer controlled nutrient supply (glucose as a carbon source) delivery system, indicating nearly 1.5 times that obtained from TB. Upon purification, a total of 1.65 mg/g of protein per gram cell biomass was obtained and the purified AviPure showed affinity for immunoglobulin. High cell density fed batch fermentation was achieved by selecting the best media and growth conditions, by utilizing a number of fermentation parameters like media, fermentation conditions, chemical concentrations, pO2 level, stirrer speed, pH level and feed media addition. It is possible to reach cell densities higher than shake flasks and stirred tank reactors with the improved oxygen transfer rate and feed.Fil: Kangwa, Martin. Jacobs University; AlemaniaFil: Yelemane, Vikas. Jacobs University; AlemaniaFil: Polat, Ayse Nur. Jacobs University; AlemaniaFil: Gorrepati, Kanaka Durga Devi. Jacobs University; AlemaniaFil: Grasselli, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Fernández Lahore, Marcelo. Jacobs University; Alemani