High-throughput FTIR-based bioprocess analysis of recombinant cyprosin production

To increase the knowledge of the recombinant cyprosin production process in Saccharomyces cerevisiae cultures, it is relevant to implement efficient bioprocess monitoring techniques. The present work focuses on the implementation of a mid-infrared (MIR) spectroscopy-based tool for monitoring the recombinant culture in a rapid, economic, and high-throughput (using a microplate system) mode. Multivariate data analysis on the MIR spectra of culture samples was conducted. Principal component analysis (PCA) enabled capturing the general metabolic status of the yeast cells, as replicated samples appear grouped together in the score plot and groups of culture samples according to the main growth phase can be clearly distinguished. The PCA-loading vectors also revealed spectral regions, and the corresponding chemical functional groups and biomolecules that mostly contributed for the cell biomolecular fingerprint associated with the culture growth phase. These data were corroborated by the analysis of the samples’ second derivative spectra. Partial least square (PLS) regression models built based on the MIR spectra showed high predictive ability for estimating the bioprocess critical variables: biomass (R ² = 0.99, RMSEP 2.8%); cyprosin activity (R ² = 0.98, RMSEP 3.9%); glucose (R ² = 0.93, RMSECV 7.2%); galactose (R ² = 0.97, RMSEP 4.6%); ethanol (R ² = 0.97, RMSEP 5.3%); and acetate (R ² = 0.95, RMSEP 7.0%). In conclusion, high-throughput MIR spectroscopy and multivariate data analysis were effective in identifying the main growth phases and specific cyprosin production phases along the yeast culture as well as in quantifying the critical variables of the process. This knowledge will promote future process optimization and control the recombinant cyprosin bioprocess according to Quality by Design framework.     

Process optimization and production of polyhydroxybutyrate using palm jaggery as economical carbon source by marine sponge-associated Bacillus licheniformis MSBN12

The Polyhydroxybutyrate (PHB) producer, Bacillus licheniformis MSBN12 was isolated from the marine sponge Callyspongia diffusa. The PHB production of B. licheniformis MSBN12 was optimized using a four-factor Box-Behnken design to find the interactive effects of variables such as palm jaggery, wheat bran, seawater, and incubation temperature. The maximum yield of PHB (6.38 g/L) was achieved through response surface methodology-based optimization and the optimized conditions were further used for the batch and fed-batch fermentation. Maximum biomass was reached at 48 and 36 h of incubation with PHB accumulation of 62.91 and 67.16 % (w/w of dry cells) for batch and fed-batch process. The production of PHB under fed-batch process with B. licheniformis MSBN12 was increased threefold over shake flask culture when palm jaggery as sole carbon source. The ¹H NMR data was extrapolated with peaks of the PHB reference standard and confirmed as PHB analog.     

Ethanol effect on batch and fed-batch Arthrospira platensis growth

The ability of Arthrospira platensis to use ethanol as a carbon and energy source was investigated by batch process and fed-batch process. A. platensis was cultivated under the effect of a single addition (batch process) and a daily pulse feeding (fed-batch process) of pure ethanol, at different concentrations, to evaluate cell concentration (X) and specific growth rate (μ). A marked increase was observed in the cell concentration of A. platensis in runs with ethanol addition when compared to control cultures without ethanol addition. The fed-batch process using an ethanol concentration of 38 mg L^?1 days^?1 reached the maximum cell concentration of 2,393 ± 241 mg L^?1, about 1.5-fold that obtained in the control culture. In all experiments, the maximum specific growth rate was observed in the early exponential phase of cell growth. In the fed-batch process, μ decreased more slowly than in the batch process and control culture, resulting in the highest final cell concentration. Ethanol can be used as a feasible carbon and energy source for A. platensis growth via a fed-batch process.     

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L^?1 and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL^?1 was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL^?1) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L^?1 glucose led to a 6.2-fold increase (465.07 U mL^?1) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.     

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.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Enhancing isomaltulose production by recombinant Escherichia coli producing sucrose isomerase: culture medium optimization containing agricultural wastes and cell immobilization

Isomaltulose is a structural isomer of sucrose commercially used in food industries. In this work, recombinant Escherichia coli producing sucrose isomerase (SIase) was used to convert sucrose into isomaltulose. To develop an economical industrial medium, untreated cane molasses (10.63 g l^?1), yeast extract (25.93 g l^?1), and corn steep liquor (10.45 g l^?1) were used as main culture compositions for SIase production. The relatively high SIase activity (14.50 ± 0.11 U mg DCW^?1) was obtained by the recombinant cells. To the best of our knowledge, this is the first investigation on SIase production by engineered E. coli using untreated cane molasses. The recombinant E. coli cells expressing the SIase gene were immobilized in calcium alginate gel in order to improve the efficiency of recycling. The immobilization was most effective with 2 % (w/v) sodium alginate and 3 % (w/v) calcium chloride. The optimal initial biomass for immobilization was 20 % (w/v, wet wt.), with a hardening time of 8 h for cell immobilization. The immobilized E. coli cells exhibited good stability for 30 batches with the productivity of 0.45 g isomaltulose g pellet^?1 h^?1. A continuous isomaltulose formation process using a column reactor remained stable for 40 days with 83 ± 2 % isomaltulose yield, which would be beneficial for economical production of isomaltulose.     

Bacillus cihuensis sp. nov., isolated from rhizosphere soil of a plant in the Cihu area of Taiwan

A Gram-positive, moderately halotolerant, rod-shaped, spore forming bacterium, designated strain FJAT-14515^T was isolated from a soil sample in Cihu area, Taoyuan County, Taiwan. The strain grew at 10–35 °C (optimum at 30 °C), pH 5.7–9.0 (optimum at pH 7.0) and at salinities of 0–5 % (w/v) NaCl (optimum at 1 % w/v). The diagnostic diamino acid of the peptidoglycan of the isolated strain was meso-diaminopimelic acid and major respiratory isoprenoid quinone was MK-7. Major cellular fatty acids were anteiso-C_15:0 (40.6 %), iso-C_15:0 (20.7 %) and the DNA G+C content of strain FJAT-14515^T was 37.1 mol %. A phylogenetic analysis based on 16S rRNA gene sequences indicated that strain FJAT-14515^T belongs to the genus Bacillus, and was most closely related to the reference strains of Bacillus muralis DSM 16288^T (97.6 %) and Bacillus simplex DSM 1321^T (97.5 %). Levels of DNA–DNA relatedness between strain FJAT-14515^T and the reference strains of B. muralis DSM 16288^T and B. simplex DSM 1321^T were 27.9 % ± 3.32 and 44.1 % ± 0.57, respectively. Therefore, on the basis of phenotypic, chemotaxonomic and genotypic properties, strain FJAT-14515^T represents a novel species of the genus Bacillus, for which the name Bacillus cihuensis sp. nov. is proposed. The type strain is FJAT-14515^T (=DSM 25969^T = CGMCC 1.12697^T).     

Enhanced biotransformation of 1,3-dichloro-2-propanol to epichlorohydrin via resin-based in situ product removal process

Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH.     

Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures

Dissociated cells separated from a natural colony of Nostoc flagelliforme were cultivated heterotrophically in the darkness on glucose under fed-batch culture conditions. The effects of carbon sources (glucose, fructose, xylose, and sucrose) and concentrations on cell growth and extracellular polysaccharide (EPS) production were investigated. At harvest, the culture contained 1.325 g L^?1 of biomass and 117.2 mg L^?1 of EPS, respectively. The gravimetric EPS production rate was 16.7 mg g^?1 cell dry weight day^?1, which was 2.1 times higher than previously reported. Using sigmoid model, batch fermentation of N. flagelliforme was kinetically simulated to obtain equations including substrate consumption, biomass growth, and EPS accumulation. Results from a simulation correlated well with the experimental ones, indicating that this method could be useful in studying EPS production from batch and fed-batch cultures.     

Enhanced production of the nonribosomal peptide antibiotic valinomycin in Escherichia coli through small-scale high cell density fed-batch cultivation

Nonribosomal peptides (NRPs), a large family of natural products, possess numerous pharmaceutically significant bioactivities. However, many native microbial producers of NRPs are not cultivable or have low production yields making mass production infeasible. The recombinant production of natural products in a surrogate host has emerged as a strategy to overcome these limitations. De novo recombinant production of the NRP antibiotic valinomycin in an engineered Escherichia coli host strain was established with the necessary biosynthetic pathway constituents from Streptomyces tsusimaensis. In the present study, the initially modest valinomycin yields could be significantly increased from 0.3 up to 2.4 mg L^?1 by switching from a batch to an enzyme-based fed-batch mode in shake flasks. A subsequent design of experiment-driven optimization of parallel fed-batch cultivations in 24-well plates with online monitoring of dissolved oxygen and pH led to valinomycin yields up to 6.4 mg L^?1. Finally, repeated glucose polymer feeding to enzyme-based high cell density cultivations in shake flasks resulted in cell densities of OD₆₀₀ >50 and a valinomycin titer of appr. 10 mg L^?1. This represents a 33-fold improvement compared to the initial batch cultivations and is the highest concentration of a nonribosomal peptide which has been produced in E. coli without feeding of specific precursors so far to our knowledge. Also, such a small-scale optimization under fed-batch conditions may be generally applicable for the development and scale-up of natural product production processes in E. coli.     

Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content

A chemically defined platform basal medium and feed media were developed using a single Chinese hamster ovary (CHO) cell line that produces a monoclonal antibody (mAb). Cell line A, which showed a peak viable cell density of 5.9 × 10⁶ cells/mL and a final mAb titer of 0.5 g/L in batch culture, was selected for the platform media development. Stoichiometrically balanced feed media were developed using glucose as an indicator of cell metabolism to determine the feed rates of all other nutrients. A fed-batch culture of cell line A using the platform fed-batch medium yielded a 6.4 g/L mAb titer, which was 12-fold higher than that of the batch culture. To examine the applicability of the platform basal medium and feed media, three other cell lines (A16, B, and C) that produce mAbs were cultured using the platform fed-batch medium, and they yielded mAb titers of 8.4, 3.3, and 6.2 g/L, respectively. The peak viable cell densities of the three cell lines ranged from 1.3 × 10⁷ to 1.8 × 10⁷ cells/mL. These results show that the nutritionally balanced fed-batch medium and feeds worked well for other cell lines. During the medium development, we found that choline limitation caused a lower cell viability, a lower mAb titer, a higher mAb aggregate content, and a higher mannose-5 content. The optimal choline chloride to glucose ratio for the CHO cell fed-batch culture was determined. Our platform basal medium and feed media will shorten the medium-development time for mAb-producing cell lines.     

Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production

A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10^T, was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823^T (94.38 %). The temperature, pH, and NaCl range for growth was determined to be 20–45 °C (optimum 37 °C), 4.0–10.0 (optimum pH 7.3), and 0–3.0 % (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5 %) were identified as iso-C_15:0, C_16:0, C_16:0 dma, C_14:0, anteiso-C_15:0 and iso-C_13:0. No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10^T (=DSM 27788^T = JCM 19606^T = CICC 10730^T).     

Bizionia psychrotolerans sp. nov., a psychrophilic bacterium isolated from the intestine of a sea cucumber (Apostichopus japonicus)

A novel Gram-negative, non-flagellated, non-gliding, rod-shaped bacterial strain, designated PB-M7^T, was isolated from the intestine of a sea cucumber collected from Pohang, South Korea. Growth was observed at 4–30 °C (optimum, 25 °C), pH 6.0–9.0 (optimum, pH 7.0–8.0), and with 2.0–6.0 % (w/v) NaCl (optimum, 2.0 %). In a phylogenetic analysis based on 16S rRNA gene sequences, strain PB-M7^T was found to belong to the genus Bizionia and to be most closely related to Bizionia echini KMM 6177^T (99.0 % 16S rRNA gene sequence similarity), Bizionia hallyeonensis T-y7^T (97.9 %), Bizionia algoritergicola APA-1^T (97.5 %), Bizionia argentinensis JUB59^T (97.5 %) and Bizionia myxarmorum ADA-4^T (97.1 %). The predominant fatty acids of strain PB-M7^T were identified as iso-C_15:0 (22.2 %), iso-C_15:1 G (10.8 %), iso-C_17:0 3-OH (16.7 %) and summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c; 11.2 %). The major respiratory quinone was identified as menaquinone-6 (MK-6). The polar lipid profile of strain PB-M7^T was found to contain phosphatidylethanolamine, an unidentified phospholipid, three unidentified aminolipids and three unidentified lipids. The DNA G + C content of strain PB-M7^T was determined to be 33.4 mol% and the mean DNA–DNA relatedness values with the type strains of B. echini, B. hallyeonensis, B. algoritergicola, B. argentinensis, and B. myxarmorum were 52.9, 48.5, 46.5, 37.1 and 26.6 %, respectively. Based on the data presented, strain PB-M7^T represents a novel species of the genus Bizionia, for which the name Bizionia psychrotolerans sp. nov. is proposed. The type strain of B. psychrotolerans is PB-M7^T (= KCCM 43042 ^T = JCM 19924 ^T).     

Effects of pulse feeding of beet molasses on recombinant benzaldehyde lyase production by Escherichia coli BL21(DE3)

The effect of fed-batch operation (FBO) strategy was investigated using pretreated-beet molasses, containing galactose that induces the lac promoter, on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21(DE3)pLySs. After batch cultivation with 30 g l^?1 pretreated-beet molasses consisting of 7.5 g l^?1 glucose and 7.5 g l^?1 fructose, three FBO strategies were applied at dissolved oxygen (=40%) cascade to air-flow rate. In FBO1 when air-flow rate decreased considerably, feed was given to the system in pulses in such a way that pretreated-beet molasses concentration increased by 10 kg m^?3 (containing 2.5 g l^?1 glucose+2.5 g l^?1 fructose); however, decrease in air-flow rate demonstrated only the absence of glucose but not fructose. Thus, in FBO2 when fructose and glucose were completely utilized, pretreated-beet molasses was pulse-fed and its concentration increased by 10 g l^?1. In FBO3 with the decreased amount of pretreated-beet molasses (6 g l^?1), shift response time from glucose to fructose consumption was avoided, and glucose and fructose consumptions were well correlated with air-flow rate, and the highest C _X (8.04 g l^?1) and BAL (2,315 U ml^?1) production were obtained (t?=?24 h) with the highest substrate yield on cell and product formation.     

Nitratireductor shengliensis sp. nov., Isolated from an Oil-Polluted Saline Soil

Two Gram-negative, non-motile, short-rod-shaped bacterial isolates, designated 110399^T and 110248, were isolated from an oil-polluted saline soil in Shengli Oilfield, Eastern China. The two strains shared 99.9 % 16S rRNA gene sequence similarity with the DNA–DNA relatedness value being 80.0 %. They were both capable to grow at 20–40 °C, pH 7–9, and 1–9 % (w/v) NaCl with the optimum growth happened at 30 °C, pH 8, and 2–6 % (w/v) NaCl. The phylogenetic analysis based on 16S rRNA gene sequences revealed that the two strains were members of Nitratireductor and most closely related to Nitratireductor pacificus pht-3B^T and N. basaltis J3^T with the 16S rRNA gene sequence similarities being 97.1 and 97.0 %. The DNA–DNA relatedness between the novel strains and two type strains were below 27 ± 7 %. The strains 110399^T and 110248 also differed from N. pacificus and N. basaltis in nitrate reduction, salt tolerance, enzyme activities, and utilization of carbon sources. The major cellular fatty acids of strain 110399^T were C_19:0ω8c cyclo (10.5 %) and Summed Feature 8 (C_18:1ω7c and/or C_18:1ω6c, 41.5 %) which are typical in the genus Nitratireductor. The predominant ubiquinone was Q-10. The genome DNA G+C content of strain 110399^T and 110248 was 61.1 and 61.7 mol%. On the basis of genetic, phenotypic, and chemotaxonomic analyses, strains 110399^T and 110248 represent a novel species within the genus Nitratireductor, for which the name Nitratireductor shengliensis sp. nov. is proposed. The type strain is 110399^T (=CGMCC 1.12519^T = LMG 27405^T).     

Improving lactate metabolism in an intensified CHO culture process: productivity and product quality considerations

In this study, we discussed the development and optimization of an intensified CHO culture process, highlighting medium and control strategies to improve lactate metabolism. A few strategies, including supplementing glucose with other sugars (fructose, maltose, and galactose), controlling glucose level at <0.2 mM, and supplementing medium with copper sulfate, were found to be effective in reducing lactate accumulation. Among them, copper sulfate supplementation was found to be critical for process optimization when glucose was in excess. When copper sulfate was supplemented in the new process, two-fold increase in cell density (66.5 ± 8.4 × 10⁶ cells/mL) and titer (11.9 ± 0.6 g/L) was achieved. Productivity and product quality attributes differences between batch, fed-batch, and concentrated fed-batch cultures were discussed. The importance of process and cell metabolism understanding when adapting the existing process to a new operational mode was demonstrated in the study.     

Comparison of biomass production and total lipid content of freshwater green microalgae cultivated under various culture conditions

The growth and total lipid content of four green microalgae (Chlorella sp., Chlorella vulgaris CCAP211/11B, Botryococcus braunii FC124 and Scenedesmus obliquus R8) were investigated under different culture conditions. Among the various carbon sources tested, glucose produced the largest biomass or microalgae grown heterotrophically. It was found that 1 % (w/v) glucose was actively utilized by Chlorella sp., C. vulgaris CCAP211/11B and B. braunii FC124, whereas S. obliquus R8 preferred 2 % (w/v) glucose. No significant difference in biomass production was noted between heterotrophic and mixotrophic (heterotrophic with light illumination/exposure) growth conditions, however, less production was observed for autotrophic cultivation. Total lipid content in cells increased by approximately two-fold under mixotrophic cultivation with respect to heterotrophic and autotrophic cultivation. In addition, light intensity had an impact on microalgal growth and total lipid content. The highest total lipid content was observed at 100 μmol m^?2s^?1 for Chlorella sp. (22.5 %) and S. obliquus R8 (23.7 %) and 80 μmol m^?2s^?1 for C. vulgaris CCAP211/11B (20.1 %) and B. braunii FC124 (34.9 %).     

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.