Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

STUDY ON BIOLOGICAL CHARACTERISTICS OF HETEROTROPHIC MARINE MICROALGA—SCHIZOCHYTRIUM MANGROVEI PQ6 ISOLATED FROM PHU QUOC ISLAND,KIEN GIANG PROVINCE,VIETNAM1

Schizochytrium sp. PQ6, a heterotrophic microalga isolated from Phu Quoc (PQ) Island in the Kien Giang province of Vietnam, contains a high amount of docosahexaenoic acid (DHA, C22:6n‐3). In this study, the culture conditions are developed to maximize biomass and DHA production. Nucleotide sequence analysis of partial 18S rRNA gene from genomic DNA showed that PQ6 has a phylogenetic relationship close to Schizochytrium mangrovei Raghu‐Kumar. The highest growth rate and DHA accumulation of this strain were obtained in 6.0% glucose, 1.0% yeast extract, 50% artificial seawater (ASW), and pH 7 at 28°C. In addition, carbon and nitrogen sources could be replaced by glycerol, ammonium acetate, sodium nitrate, or fertilizer N–P–K. Total lipid content reached 38.67% of dry cell weight (DCW), in which DHA and eicosapentaenoic acid (EPA, C20:5n‐3) contents accounted for 43.58% and 0.75% of the total fatty acid (TFA), respectively. In 5 and 10 L fermenters, the cell density, DCW, total lipid content, and maximum DHA yield were 46.50 × 10⁶ cells · mL^?1, 23.7 g · L^?1, 38.56% of DCW, and 8.71 g · L^?1 (in 5 L fermenter), respectively, and 49.71 × 10⁶ cells · mL^?1, 25.34 g · L^?1, 46.23% of DCW, and 11.55 g · L^?1 (in 10 L fermenter), respectively. Biomass of PQ6 strain possessed high contents of Na, I, and Fe (167.185, 278.3, and 43.69 mg · kg^?1 DCW, respectively). These results serve as a foundation for the efficient production of PQ6 biomass that can be used as a food supplement for humans and aquaculture in the future.     

Effects of varying levels of dietary vitamin E (α‐tocopherol) on growth performance,proximate and fatty acid composition of juvenile silver pomfret (Pampus argenteus Euphrasen, 1788)

This study investigated the effects of elevated dietary levels of vitamin E (α‐tocopherol) on growth performance, proximate composition and fatty acid profiles of juvenile silver pomfret, Pampus argenteus. Three semi‐purified experimental diets were formulated to contain 49% protein and 16% lipid. High docosahexaenoic acid (DHA) tuna oil was added to the diets to supplement DHA. A graded level of vitamin E (0‐, 50‐, and 100 mg kg^?1) was added to experimental diets 1 to 3, respectively. Analyzed vit. E levels were 155.2, 195.3 and 236.4 mg kg^?1 in diets 1, 2 and 3, respectively. The experiment was conducted for 12 weeks with juvenile silver pomfret (29.6 ± 7.6 g) using a flow‐through system consisting of nine 1‐m³ tanks. Each treatment had three replicates and fish were stocked at the rate of 20 m^?3. Growth performance and feed utilization parameters of fish fed diets 2 and 3 were significantly (P < 0.05) higher than in fish fed diet 1, but the parameters in diets 2 and 3 did not differ significantly (P > 0.05). Although whole body protein levels were not influenced by the dietary vit. E levels, whole body lipid in fish fed diet 2 was significantly higher than in fish fed the other diets. The whole body vit. E levels in fish fed diet 2 (22.6 mg kg^?1) and diet 3 (24.1 mg kg^?1) were significantly (P < 0.05) higher than in those fed diet 1 (18.2 mg kg^?1). Whole body total saturated fatty acids were significantly lower, and DHA levels higher in fish fed diets 2 and 3 than those fed diet 1. The results of the present study suggest that increasing dietary supplementation of vit. E in high lipid diets enhances the growth performance of fish and that a dietary level of 196 mg kg^?1 vit. E is suitable for the growth of silver pomfret.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Microalgae for phosphorus removal and biomass production: a six species screen for dual‐purpose organisms

Microalgae biofuel production can be feasible when a second function is added, such as wastewater treatment. Microalgae differ in uptake of phosphorus (P) and growth, making top performer identification fundamental. The objective of this screen was to identify dual‐purpose candidates capable of high rates of P removal and growth. Three freshwater – Chlorella sp., Monoraphidium minutum sp., and Scenedesmus sp. – and three marine – Nannochloropsis sp., N. limnetica sp., and Tetraselmis suecica sp. – species were batch cultured in 250 mL flasks over 16 days to quantitate total phosphorus (TP) removal and growth as a function of P loads (control, and 5, 10, and 15 mg L^?1 enrichment of control). Experimental design used 100 μmol m^?2 s^?1 of light, a light/dark cycle of 14/10 h, and no CO₂ enrichment. Phosphorus uptake was dependent on species, duration of exposure, and treatment, with significant interaction effects. Growth was dependant on species and treatment. Not all species showed increased P removal with increasing P addition, and no species demonstrated higher growth. Nannochloropsis sp and N. limnetica sp. performed poorly across all treatments. Two dual‐purpose candidates were identified. At the 10 mg L^?1 treatment Monoraphidium minutum sp. removed 67.1% (6.66 mg L^?1 ± 0.60 SE) of TP at day 8, 79.3% (7.86 mg L^?1 ± 0.28 SE) at day 16, and biomass accumulation of 0.63 g L^?1 ± 0.06 SE at day 16. At the same treatment Tetraselmis suecica sp. removed 79.4% (6.98 mg L^?1 ± 0.24 SE) TP at day 8, 83.0% (7.30 mg L^?1 ± 0.60 SE) at day 16, and biomass of 0.55 g L^?1 ± 0.02 SE at day 16. These species merit further study using high‐density wastewater cultures and lipid profiling to assess suitability for a nutrient removal and biomass/biofuel production scheme.     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 g_CDW L^?1) resulting in a recombinant protein titer of about 6.5 g L^?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used ¹³C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h^?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g^?1 h^?1 compared to 0.7 mmol g^?1 h^?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc.     

Metabolic engineering of Escherichia coli for agmatine production

Agmatine is a kind of important biogenic amine. The chemical synthesis route is not a desirable choice for industrial production of agmatine. To date, there are no reports on the fermentative production of agmatine by microorganism. In this study, the base Escherichia coli strain AUX4 (JM109 ?speC ?speF ?speB ?argR) capable of excreting agmatine into the culture medium was first constructed by sequential deletions of the speC and speF genes encoding the ornithine decarboxylase isoenzymes, the speB gene encoding agmatine ureohydrolase and the regulation gene argR responsible for the negative control of the arg regulon. The speA gene encoding arginine decarboxylase harboured by the pKK223‐3 plasmid was overexpressed in AUX4, resulting in the engineered strain AUX5. The batch and fed‐batch fermentations of the AUX5 strain were conducted in a 3‐L bioreactor, and the results showed that the AUX5 strain was able to produce 1.13 g agmatine L^?1 with the yield of 0.11 g agmatine g^?1 glucose in the batch fermentation and the fed‐batch fermentation of AUX5 allowed the production of 15.32 g agmatine L^?1 with the productivity of 0.48 g agmatine L^?1 h^?1, demonstrating the potential of E. coli as an industrial producer of agmatine.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.     

Continuous multistep versus fed‐batch production of ethanol and xylitol in a simulated medium of sugarcane bagasse hydrolyzates

Co‐cultures for simultaneous production of ethanol and xylitol were studied under different operation bioreactor modes using Candida tropicalis IEC5‐ITV and Saccharomyces cerevisiae ITV01‐RD in a simulated medium of sugarcane bagasse hydrolyzates. Xylitol and ethanol tolerance by S. cerevisiae and C. tropicalis, respectively, was evaluated. The results showed that C. tropicalis was sensitive to ethanol concentrations up to 30 g/L, while xylitol had no effect on S. cerevisiae viability and metabolism. The best condition found for simultaneous culture was S. cerevisiae co‐culture and C. tropicalis sequential cultivation at 24 h. Under these conditions, productivity and yield for ethanol were Q_EtOH = 0.72 g L^?1 h^?1 and Y_EtOH/s = 0.37 g/g, and for xylitol, Q_XylOH = 0.10 g L^?1 h^?1 and Y_XylOH/S = 0.31 g/g, respectively; using fed‐batch culture, the results were Q_EtOH = 0.87 g L^?1 h^?1 and Y_EtOH/s = 0.44 g L^?1 h^?1, and Q_EtOH = 0.27 g L^?1 h^?1 and Y_EtOH/s = 0.57 g/g, respectively. Maximum volumetric productivity in continuous multistep cultures of ethanol and xylitol was at dilution rates of 0.131 and 0.074 h^?1, respectively. Continuous multistep production, Q_EtOH increased up to 50% more than in fed‐batch culture, even though xylitol yield remained unchanged.     

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed‐batch synthesis of galacto‐oligosaccharides (GOS) from lactose with β‐galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed‐batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L^?1 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g^?1 in fed‐batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g^?1). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS‐5 and GOS‐6) was observed in the fed‐batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:59–67, 2014     

Improvement of docosahexaenoic acid fermentation from Schizochytrium sp. AB‐610 by staged pH control based on cell morphological changes

Schizochytrium sp. AB‐610 accumulates relatively higher amount of DHA‐rich lipid in the cells, and it was found that DHA yield was closely related to the cell morphology and pH value during fermentation period. DHA production from Schizochytrium sp. AB‐610 in fed‐batch fermentation was investigated and four growth stages were clarified as lag stage, balanced growth stage, lipid accumulation stage, and lipid turnover stage, based on the morphologic observation and key parameters changes. Then a simple strategy of two‐stage pH control was developed, in which pH 7.0 was kept until 12 h after the end of balanced growth stage, and then shifted to 5.0 for the rest period in fermentation. A maximal DHA production of 11.44g/L was achieved. This approach has advantage of easy scaling up for industrial DHA fermentation from Schizochytrium sp. cells.     

Effect of linear alkylbenzene sulfonate on Cu2+ removal by Spirulina platensis strain (FACHB‐834)

The removal efficiency of Cu²⁺ by Spirulina platensis (strain FACHB‐834), in viable and heat‐inactivated forms, was investigated in the presence and absence of linear alkylbenzene sulfonate (LAS). When the initial Cu²⁺ concentration was in the range of 0.5–1.5 mg · L^?1, a slight increase in growth rate of FACHB‐834 was observed. In contrast, when Cu²⁺ or LAS concentrations were at or higher than 2.0 or 6.0 mg · L^?1, respectively, the growth of FACHB‐834 was inhibited and displayed yellowing and fragmentation of filaments. The presence of LAS improved Cu²⁺ removal by ~20%, and accelerated attainment of Cu²⁺ retention equilibrium. For the 2‐ mg · L^?1 Cu²⁺ treatments, retention equilibrium occurred within 2 d and showed maximum Cu²⁺ removal of 1.83 mg · L^?1. In the presence of LAS, the ratio of extracellular bound Cu²⁺ to intracellular Cu²⁺ taken up by the cells was lower (1.05–2.26) than corresponding ratios (2.46–7.85) in the absence of LAS. The percentages of extracellular bound Cu²⁺ to total Cu²⁺ removal (both bound and taken up by cells) in the presence of LAS ranged from 51.2% to 69.3%, which was lower than their corresponding percentages (71.1%–88.7%) in the absence of LAS. LAS promoted biologically active transport of the extracellular bound form of Cu²⁺ into the cell. In contrast, the addition of LAS did not increase the maximum removal efficiency of Cu²⁺ (61.4% ± 5.6%) by heat‐inactivated cells compared to that of living cells (59.6% ± 6.0%). These results provide a theoretical foundation for designing bioremediation strategies using FACHB‐834 for use in surface waters contaminated by both heavy metals and LAS.     

Auxin and cytokinin relationships in 24 microalgal strains1

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection‐41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC‐712 the lowest biomass. The auxins, indole‐3‐acetic acid (IAA) and indole‐3‐acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49 nmol IAA · g^?1 DW and 0.18 to 99.83 nmol IAM · g^?1 DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29 nmol · g^?1 DW in Klebsormidium flaccidum MACC‐692 to 21.40 nmol · g^?1 DW in Stigeoclonium nanum MACC‐790. The general trend was that cis‐zeatin types were the predominant cytokinins; isopentenyladenine‐type cytokinins were present in moderate concentrations, while low levels of trans‐zeatin‐type and very low levels of dihydrozeatin‐type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O‐glucosides were low. Only one N‐glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2‐ to 4‐fold higher than the cytokinin content.     

Phototrophic cultivation of NaCl‐tolerant mutant of Spirulina platensis for enhanced C‐phycocyanin production under optimized culture conditions and its dynamic modeling

Commercial cultivation of Spirulina sp. is highly popular due to the presence of high amount of C‐phycocyanin (C‐PC ) and other valuable chemicals like carotenoids and γ‐linolenic acid. In this study, the pH and the concentrations of nitrogen and carbon source were manipulated to achieve improved cell growth and C‐PC production in NaCl‐tolerant mutant of Spirulina platensis . In this study, highest C‐PC (147 mg · L^?1) and biomass (2.83 g · L^?1) production was achieved when a NaCl‐tolerant mutant of S. platensis was cultivated in a nitrate and bicarbonate sufficient medium (40 and 60 mM, respectively) at pH 9.0 under phototrophic conditions. Kinetic study of wildtype S. platensis and its NaCl‐tolerant mutant was also done to determine optimum nitrate concentrations for maximum growth and C‐PC production. Kinetic parameter of inhibition (Haldane model) was fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that the maximum specific growth rate (μ_max) for NaCl‐tolerant mutant increased by 17.94% as compared to its wildtype counterpart, with a slight increase in half‐saturation constant (K_s), indicating that this strain could grow well at high concentration of NaNO₃. C‐PC production rate (C_max) in mutant cells increased by 12.2% at almost half the value of K_s as compared to its wildtype counterpart. Moreover, the inhibition constant (K_i) value was 207.85% higher in NaCl‐tolerant mutant as compared to its wildtype strain, suggesting its ability to produce C‐PC even at high concentrations of NaNO₃.     

Enhancement of total lipid yield by nitrogen,carbon, and iron supplementation in isolated microalgae

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG‐11 containing sodium carbonate concentration at 0.03 g · L^?1, and in normal BG‐11 containing iron concentration (IC) at 0.009 or 0.012 g · L^?1. Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L^?1 under the IC of 0.012 g · L^?1. Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.     

Influences of salinity on the physiology and distribution of the Arctic coralline algae,Lithothamnion glaciale (Corallinales,Rhodophyta)

In Greenland, free‐living red coralline algae contribute to and dominate marine habitats along the coastline. Lithothamnion glaciale dominates coralline algae beds in many regions of the Arctic, but never in Godthåbsfjord, Greenland, where Clathromorphum sp. is dominant. To investigate environmental impacts on coralline algae distribution, calcification and primary productivity were measured in situ during summers of 2015 and 2016, and annual patterns of productivity in L. glaciale were monitored in laboratory‐based mesocosm experiments where temperature and salinity were manipulated to mimic high glacial melt. The results of field and cold‐room measurements indicate that both L. glaciale and Clathromorphum sp. had low calcification and photosynthetic rates during the Greenland summer (2015 and 2016), with maximum of 1.225 ± 0.17 or 0.002 ± 0.023 μmol CaCO ₃ · g^?1 · h^?1 and ?0.007 ±0.003 or ?0.004 ± 0.001 mg O₂ · L^?1 · h^?1 in each species respectively. Mesocosm experiments indicate L. glaciale is a seasonal responder; photosynthetic and calcification rates increase with annual light cycles. Furthermore, metabolic processes in L. glaciale were negatively influenced by low salinity; positive growth rates only occurred in marine treatments where individuals accumulated an average of 1.85 ± 1.73 mg · d^?1 of biomass through summer. These results indicate high freshwater input to the Godthåbsfjord region may drive the low abundance of L . glaciale , and could decrease species distribution as climate change increases freshwater input to the Arctic marine system via enhanced ice sheet runoff and glacier calving.