Final Cell Dry Weight Research Articles

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Lipid production by Rhodosporidium toruloides from biodiesel-derived glycerol in shake flasks and bioreactor: Impact of initial C/N molar ratio and added onion-peel extract

Rhodosporidium toruloides NRRL Y-27012 grown in glycerol, produced yeast biomass and secondary metabolites as lipids (L) and intra-cellular polysaccharides (IPS) in trials under nitrogen limitation (initial C/N molar ratios ≈ 50, ≈ 100, ≈ 160 and ≈ 240). The initial glycerol (Glol0) concentration for all experiments was ≈ 90 g/L. The yeast demonstrated significant dry cell weight (DCW) production irrespective of the C/N ratio employed, ranging between 19 and 29 g/L. The optimum C/N ratio medium was that of 100 moles/moles, where a DCWmax = 24.5 g/L containing c. 50% w/w of lipids was recorded. Endopolysaccharides presented indeed impressive values (≥ 50% w/w) even at the very early growth steps, while IPS/DCW values decreased as the fermentation proceeded, with simultaneous increase of L/DCW values. Experiments were also conducted in media with a hydroglycerolic extract of onion peels (at 40 mL/L), while a blank experiment (no extract) was also realized. The trial with the added extract was performed under both aseptic (previous sterilization) and non-aseptic (previous thermal treatment at T = 100 °C; 5 min) conditions. The aseptic experiment with the added extract resulted in a relative decrease in lipid production in relation to the control (L=8.6 g/L in the control vs L = 6.2 g/L with the extract). Under non-aseptic conditions, lipid production was slightly lower (L = 5.2 g/L). Finally, the optimum trial in shake flasks was scaled-up in a batch bioreactor, and higher glycerol assimilation rate, and biomass and lipid production occurred compared to the flask trial (final DCW and lipid values ≈27 g/L and 13.5 g/L respectively). Cellular lipids, rich in palmitic and oleic acid, were mainly composed of neutral fractions. Phospholipids were more saturated than the neutral lipids.

Bicarbonate for microalgae cultivation: a case study in a chlorophyte, Tetradesmus wisconsinensis isolated from a Norwegian lake

Bicarbonate was evaluated as an alternative carbon source for a green microalga, Tetradesmus wisconsinensis, isolated from Lake Norsjø in Norway. Photosynthesis, growth, and lipid production were studied using four inorganic carbon regimes: (1) aeration only, (2) 20 mM NaHCO3, (3) 5% (v/v) CO2 gas, and (4) combination of 20 mM NaHCO3 and 5% CO2. Variable chlorophyll a fluorescence analysis revealed that the bicarbonate treatment supported effective photosynthesis, while the CO2 treatment led to inefficient photosynthetic activity with a PSII maximum quantum yield as low as 0.31. Conversely, bicarbonate and CO2 treatments gave similar biomass and fatty acid production. The maximum growth rate, the final cell dry weight, and total fatty acids under the bicarbonate-only treatment were 0.33 (± 0.06) day−1, 673 (± 124) mg L−1 and 75 (± 5) mg g−1 dry biomass, respectively. The most abundant fatty acid components were α-linolenic acid and polyunsaturated fatty acids constituting 69% of the total fatty acids. The fatty acid profile eventuated in unsuitable biodiesel fuel properties such as high degree of unsaturation and low cetane number; however, it would be relevant for food and feed applications. We concluded that bicarbonate could give healthy growth and comparative product yields as CO2.

Microalgal production of zeaxanthin

Zeaxanthin is a carotenoid pigment used in the food industry as well as in supplements for age-related macular degeneration. There is potential for microalgae to be used for zeaxanthin production, however there is relatively little work examining this issue, particularly with respect to scale-up. Here two species of cyanobacteria (Synechococcus sp. PCC7002 and Synechocystis sp. PCC6803) were examined, along with a Rhodophyte (Rhodosorus sp.). At a light intensity of 80 μmol photons m−2 s−1 specific zeaxanthin contents were 2.30 ± 0.84, 1.61 ± 0.68 and 2.16 ± 0.63 mg g−1 for Synechococcus sp. PCC7002, Synechocystis sp. PCC6803 and Rhodosorus sp., respectively. Of the species examined the Synechococcus PCC7002 had the highest specific growth rate (0.74 ± 0.09 day−1). This species was used to further optimize the process, increasing the nutrient concentration in the medium and using an incremental light addition strategy led to an approximately 13-fold increase in the cell density, giving a final dry cell weight of 4.25 ± 1.42 g L−1. Results from this work are among the highest in the literature for zeaxanthin productivity (0.7 ± 0.5 mg L−1 day−1) and the approach used can be readily scaled-up. Additionally, the approach used in this work to achieve high cell densities of PCC7002 could be potentially applied to the production of other compounds (e.g. phycobilins or compounds produced using metabolic engineering).

Comparative study on light attenuation models of Galdieria sulphuraria for efficient production of phycocyanin

The thermo-acidophilic red microalga Galdieria sulphuraria is valuable especially for the intracellular thermostable phycocyanin with broad application prospects. Heterotrophy or photoinduction had been studied to enhance the production of phycocyanin at small-scale indoors. The aim of this study was to scale up the photoinduction process. The Cornet model could well describe and predict the light attenuation in G. sulphuraria suspension compared with the Lambert-Beer and hyperbolic models. Volume-averaged light intensity was the key parameter for photoinduction by testing respectively in 1-L column bioreactor, 3-L and 15-L flat plate photobioreactors indoors and outdoors, and supposed to be 600 μmol photons m−2 s−1. Accordingly, amplifying photoinduction in 1.35-m2 raceway pond outdoors was successfully carried out with the final cell dry weight of 2.37 g L−1 and phycocyanin up to 10.32%, which was comparable with that from Spirulina/Arthrospira and 3.82 times higher than that of heterotrophic cells of G. sulphuraria.

High-throughput microalgae cultivation with adjustable LED-module applying different colours for Nannochloropsis and Chlorella microcultures

Utilization of algae includes both macroalgae for human consumption dating back to thousands of years, as well as the application of microalgae in health promoting dietary supplements. The autotrophic growth of microalgae is slow, but can be accelerated by optimizing their cultivation conditions. Efficiency optimizations for time and economy should be performed in many parallel experiments. A new high-throughput microalgae cultivation method is presented here, applying 24-low-well microplate with varying illumination, in which the cell growth is followed via evaluation of scanned images. A strain of the genus Nannochloropsis and two Chlorella vulgaris species have been chosen as well described and frequently applied model organisms in order to test the recently developed cultivation system. In these scaled down experiments, the custom design lighting panel was tested by studying the effect of the colour of illumination on cell growth kinetics. RGB LEDs (i.e. light emitting diodes, red: 622 nm, green: 528 nm, and blue: 467 nm) were used individually or together providing red, green, blue, and white colours. While the effect of light’s colour on algae growth was evaluated, also the new system was proven to be suitable for comparing maximal growth rates for different microalgae strains. While the tested two Chlorella isolates reached 1.2–1.4 g l–1 concentrations, the Nannochloropsis strain reached 1.4 g l–1 final cell dry weight, and specific growth rates were observed between 0.58–0.62 day–1.

Mathematical modeling of fed-batch fermentation of Schizochytrium sp. FJU-512 growth and DHA production using a shift control strategy.

To obtain high-cell-density cultures of Schizochytrium sp. FJU-512 for DHA production, two stages of fermentation strategy were used and carbon/nitrogen ratio, DO and temperature were controlled at different levels. The final dry cell weight, total lipid production and DHA yield in 15l bioreactor reached 103.9, 37.2 and 16.0g/l, respectively. For the further study of microbial growth and DHA production dynamics, we established a set of kinetic models for the fed-batch production of DHA by Schizochytrium sp. FJU-512 in 15 and 100l fermenters and a compensatory parameter n was integrated into the model in order to find the optimal mathematical equations. A modified Logistic model was proposed to fit the cell growth data and the following kinetic parameters were obtained: µm=0.0525/h, Xm=100g/l and n=4.1717 for the 15l bioreactor, as well as µm=0.0382/h, Xm=107.4371g/l and n=10 for the 100l bioreactor. The Luedeking-Piret equations were utilized to model DHA production, yielding values of α=0.0648g/g and β=0.0014g/g/h for the 15l bioreactor, while the values of α and β obtained for the 100l fermentation were 0.0209g/g and 0.0030g/g/h. The predicted results compared with experimental data showed that the established models had a good fitting precision and were able to exactly depict the dynamic features of the DHA production process.

Investigation of autotrophic, heterotrophic, and mixotrophic modes of cultivation on lipid and biomass production in Chlorella vulgaris

Background: The cultivation mode plays a great role in biomass production and composition in the green microalga Chlorella vulgaris. Aims and Objectives: The effects of three different cultivation modes including autotrophic, heterotrophic, and mixotrophic growth conditions on the biomass and lipid production and also on the biomass composition in the unicellular photosynthetic microalga C. vulgaris were investigated. Materials and Methods: BG-11 culture medium was used for preservation and analysis of the studied microalgal strain. Effects of three different cultivation strategies including autotrophic, heterotrophic, and mixotrophic modes of cultivation were investigated on biomass and lipid production in C. vulgaris. Results: The maximum amount of cell dry weight was obtained in mixotrophic mode (3.91 g L−1) while autotrophic and heterotrophic modes achieved a final cell dry weight of 2.47 g L−1 and 1.52 g L−1, respectively. Besides, the biomass production in mixotrophic was increased 158% and 257% in 21 days, compared with autotrophic and heterotrophic conditions. The heterotrophic cultivation mode achieved the highest lipid contents (48.68%) which was 110.01% and 143.13% times higher than the mixotrophic (44.25%) and autotrophic (34.01%) conditions. The protein accumulation in autotrophic mode with 41.7% had the highest value. Conclusion: Considering the biomass produced in mixotrophic condition in the studied microalgal strain, this cultivation mode is recommended for lipid production which can be used for green energy production.

Metabolic engineering of Escherichia coli for the synthesis of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose

Escherichia coli was metabolically engineered to effectively produce a series of biopolymers consisted of four types of monomers including glycolate, lactate, 3-hydroxybutyrate and 4-hydroxybutyrate from glucose as the carbon source. The biosynthetic route of novel quadripolymers was achieved by the overexpression of a range of homologous and heterologous enzymes including isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, glyoxylate/hydroxypyruvate reductase, propionyl-CoA transferase, β-ketothiolase, acetoacetyl-CoA reductase, succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, CoA transferase and PHA synthase. In shake flask cultures using Luria-Bertani medium supplemented with glucose, the recombinant E. coli reached 7.10g/l cell dry weight with 52.60wt% biopolymer content. In bioreactor study, the final cell dry weight was 19.61g/l, containing 14.29g/l biopolymer. The structure of the produced polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the quadripolymer possessed decreased crystallinity and improved toughness, in comparison to poly-3-hydroxybutyrate homopolymer. This is the first study reporting efficient microbial production of the quadripolymer poly(glycolate-co-lactate-co-3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose.

Simultaneous treatment of food-waste recycling wastewater and cultivation of Tetraselmis suecica for biodiesel production.

There is an increasing interest in the use of cultivated microalgae to simultaneously produce biodiesel and remove nutrients from various wastewaters. For this purpose, Tetraselmis suecica was cultivated in flasks and fermenters using diluted food-waste recycling wastewater (FRW). The effect of FRW dilution on T. suecica growth and nutrient removal was initially tested in flasks. The maximal microalgal concentration after 14 days was in medium with a twofold dilution (28.3 × 10(6) cells/mL) and a fivefold dilution (25.5 × 10(6) cells/mL). When fivefold diluted medium was used in fermenters, the final dry cell weight of T. suecica was 2.0 g/L. The removal efficiencies of ammonium and phosphate in the fermenters were 99.0 and 52.3%, respectively. In comparison with the results of previous studies, the growth data of T. suecica in the FRW medium indicate that microalgal cultivation system incorporating removal of nutrients in FRW is feasible at the field level.

High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply.

High Cell Density (HCD) cultivation of bacteria is essential for the majority of industrial processes to achieve high volumetric productivity (g L(-1) h(-1) ) of a bioproduct of interest. This study developed a fed batch bioprocess using glucose as sole carbon and energy source for the HCD of the well described biocatalyst Pseudomonas putida KT2440 without the supply of oxygen enriched air. Growth kinetics data from batch fermentations were used for building a bioprocess model and designing feeding strategies. An exponential followed by linearly increasing feeding strategy of glucose was found to be effective in maintaining biomass productivity while also delaying the onset of dissolved oxygen (supplied via compressed air) limitation. A final cell dry weight (CDW) of 102 g L(-1) was achieved in 33 h with a biomass productivity of 3.1 g L(-1) h(-1) which are the highest ever reported values for P. putida strains using glucose without the supply of pure oxygen or oxygen enriched air. The usefulness of the biomass as a biocatalyst was demonstrated through the production of the biodegradable polymer polyhydroxyalkanoate (PHA). When nonanoic acid (NA) was supplied to the glucose grown cells of P. putida KT2440, it accumulated 32% of CDW as PHA in 11 h (2.85 g L(-1) h(-1) ) resulting in a total of 0.56 kg of PHA in 18 L with a yield of 0.56 g PHA g NA(-1) .

Production of recombinant Chikungunya virus envelope 2 protein in Escherichia coli

Chikungunya, a mosquito-borne viral disease caused by Chikungunya virus (CHIKV), has drawn substantial attention after its reemergence causing massive outbreaks in tropical regions of Asia and Africa. The recombinant envelope 2 (rE2) protein of CHIKV is a potential diagnostic as well as vaccine candidate. Development of cost-effective cultivation media and appropriate culture conditions are generally favorable for large-scale production of recombinant proteins in Escherichia coli. The effects of medium composition and cultivation conditions on the production of recombinant Chikungunya virus E2 (rCHIKV E2) protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed-batch process was also carried out for high cell density cultivation of E. coli expressing rE2 protein. Expression of rCHIKV E2 protein in E. coli was induced with 1 mM isopropyl-beta-thiogalactoside (IPTG) at ~23 g dry cell weight (DCW) per liter of culture and yielded an insoluble protein aggregating to form inclusion bodies. The final DCW after fed-batch cultivation was ~35 g/l. The inclusion bodies were isolated, solubilized in 8 M urea and purified through affinity chromatography to give a final product yield of ~190 mg/l. The reactivity of purified E2 protein was confirmed by Western blotting and enzyme-linked immunosorbent assay. These results show that rE2 protein of CHIKV may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rE2 protein in E. coli with high yield may also offer a promising method for production of other viral recombinant proteins.

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l(-1) and 138.8 mg l(-1) h(-1), respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.

Production of polyhydroxybutyrate (PHB) by Alcaligenes latus using sugarbeet juice

The practicality of using sugarbeet juice as medium to grow Alcaligenes latus (ATCC 29714) for production of polyhydroxybutyrate (PHB), a biodegradable plastic, was explored in this study. Dilute sugarbeet juice, sugarbeet juice with partial and complete addition of nutrients other than sugar were used as culture media. Media with partial nutrient addition was shown to be optimal for PHB production, with final dry cell weight (DCW) 10.30±1.01g/L, PHB concentration 4.01±0.95g/L, PHB content 38.66±7.28%, Yp/x (gPHB produced per g dry cell weight) 0.39±0.07 and a maximum PHB productivity of 0.22±0.01g/Lh. The melting temperature of PHB extracted from sugarbeet juice-grown cells supplemented with partial nutrients was measured to be 151.46°C with crystallinity of 43.12% and the corresponding crystallinity temperature of 45.42°C. Thermal degradation of extracted PHB occurred from 255.14 to 283.69°C with the degradation peak at 273.86°C.

Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

BackgroundPseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs). To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain.ResultsThe genes encoding xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW) of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions.ConclusionsSequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

γ-Heptalactone is an endogenously produced quorum-sensing molecule regulating growth and secondary metabolite production by Aspergillus nidulans

Microbes monitor their population density through a mechanism termed quorum sensing. It is believed that quorum-sensing molecules diffuse from the microbial cells and circulate in the surrounding environment as a function of cell density. When these molecules reach a threshold concentration, the gene expression of the entire population is altered in a coordinated manner. This work provides evidence that Aspergillus nidulans produces at least one small diffusible molecule during its growth cycle which accumulates at high cell density and alters the organism's behaviour. When added to low-density cell cultures, ethyl acetate extracts from stationary phase culture supernatants of A. nidulans resulted in the abolition of the lag phase, induced an earlier deceleration phase with 16.3% decrease in the final cell dry weight and resulted in a 37.8% increase in the expression of ipnA::lacZ reporter gene construct, which was used as a marker for penicillin production compared to non-treated controls. The bioactive molecule present in the stationary phase extract was purified to homogeneity and was identified by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy to be γ-heptalactone. This study provides the first evidence that A. nidulans produces γ-heptalactone at a high cell density and it can alter the organism's behaviour at a low cell density. γ-Heptalactone hence acts as a quorum-sensing molecule in the producing strain.

Production of recombinant nonstructural 1 protein in Escherichia coli for early detection of Japanese encephalitis virus infection

SummaryJapanese encephalitis is a major public health problem in South‐East Asia and Western Pacific countries. The recombinant nonstructural 1 (rNS1) protein of Japanese encephalitis virus is a potential diagnostic as well as vaccine candidate. Developments of cost‐effective and simple culture media as well as appropriate culture conditions are generally favourable for large‐scale production of recombinant proteins. The effects of medium composition and cultivation conditions on the production of rNS1 protein were investigated in shake flask culture as well as batch cultivation of Escherichia coli. Further, the fed‐batch process was also carried out for high cell density cultivation (HCDC) of E. coli expressing rNS1 protein. Isopropyl‐β‐d‐thiogalactopyranoside (IPTG) was used to induce the expression of rNS1 protein at ∼ 13 g dry cell weight per litre of culture. The final dry cell weight after fed‐batch cultivation was ∼ 17 g l−1. The Inclusion bodies were isolated and purified through affinity chromatography to give a final product yield of ∼ 142 mg l−1. The reactivity of purified protein was confirmed by Western blotting and Enzyme linked immunosorbent assay. These results show that rNS1 protein may be used as a diagnostic reagent or for further prophylactic studies. This approach of producing rNS1 protein in E. coli with high yield may also offer promising method for production of other viral recombinant proteins.

Polyhydroxyalkanoate Production by &amp;lt;i&amp;gt;Pseudomonas putida&amp;lt;/i&amp;gt; KT217 on a Condensed Corn Solubles Based Medium Fed with Glycerol Water or Sunflower Soapstock

Pseudomonas putida KT217 was grown on a complex medium comprised of co-products of the ethanol and biodiesel industries to assess the organism's capability to produce medium-chain-length polyhydroxyalkanoate (mcl-PHA). The growth phase was carried out in a medium containing 400 g/L condensed corn solubles (CCS), supplemented with ammonium hydroxide as a nitrogen source. Following the exponential phase, co-products of the biodiesel industry (soapstock and glycerin) were fed into the reactor to trigger PHA production. When glycerin was added to the bioreactor (75 g/L total addition), the final cell dry weight (CDW) and PHA content were 30 g/L and 31%, respectively. The monomeric composition in the PHA formed was relatively uniform throughout incubation with 3-hydroxydecanoate dominating. When a total of 153 g/L of sunflower soapstock was added to the bioreactor in a fed-batch manner, the final CDW and PHA content were 17 g/L and 17%, respectively. Following addition of soapstock the monomeric composition of the polymer changed dramatically, with the 3-hydroxyoctanoate monomer becoming dominant and greater unsaturation present in the PHA.

Heterologous production of active ribonuclease inhibitor in Escherichia coli by redox state control and chaperonin coexpression

BackgroundEukaryotic Ribonuclease inhibitor (RI), belonging to the RNH1 family, is distinguished by unique features - a high sensitivity to oxidation due to the large number of reduced cysteins and a high hydrophobicity, which made most production approaches so far unsuccessful or resulted in very low yields. In this work efficient in vivo folding of native RI in the Escherichia coli cytoplasm was obtained by external addition of a reducing agent in tandem with oxygen limitation and overproduction of a molecular chaperonin. After optimisation of the production conditions in the shake flask scale the process was scaled up to high cell densities by applying a glucose limited fed-batch procedure.ResultsRI production in a T7 RNA polymerase based system results in accumulation of aggregated inactive product in inclusion bodies. Combination of addition of the reductant DTT, low production temperature and coexpression of the chaperonin GroELS resulted in high level production of approximately 25 mg g-1 CDW active RI in E. coli ER2566 pET21b, corresponding to approximately 800 kU g-1 cell wet weight. Further conditional screening under fed-batch-like conditions with the EnBase® technology and scale up into the bioreactor scale resulted in an efficient high cell density glucose and oxygen limited fed-batch process with a final cell dry weight of 25 g L-1 and a total RI yield of app. 625 mg L-1 (volumetric activity of 80,000 kU L-1). The E. coli based production constructs showed a very high robustness. The recombinant culture maintained its productivity despite the combination of the toxic growth conditions, the substrate limited production mode in tandem with a high level expression of several recombinant proteins, the set of molecular chaperonins and the target protein (RI).ConclusionsHigh level production of active RI in E. coli in a T7 RNA polymerase expression system depends on the following factors: (i) addition of a reducing agent, (ii) low production temperature, (iii) oxygen limitation, and (iii) co-overexpression of the chaperonin GroELS. The study indicates the strength of applying fed-batch cultivation techniques for the efficient optimisation of production factors already at the screening stage for fast and straight forward bioprocess development even for target proteins which show a complex folding behaviour. In our case none of the approaches alone would have resulted in significant accumulation of active RI.

Growth and recombinant protein expression with Escherichia coli in different batch cultivation media

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.