Process Engineering for High-Cell-Density Cultivation of Lipid Rich Microalgal Biomass of <Emphasis Type="Italic">Chlorella</Emphasis> sp. FC2 IITG

In the present study, process engineering strategy was applied to achieve lipid-rich biomass with high density of Chlorella sp. FC2 IITG under photoautotrophic condition. The strategy involved medium optimization, intermittent feeding of limiting nutrients, dynamic change in light intensity, and decoupling growth and lipid induction phases. Medium optimization was performed using combinations of artificial neural network or response surface methodology with genetic algorithm (ANN-GA and RSM-GA). Further, a fed-batch operation was employed to achieve high cell density with intermittent feeding of nitrate and phosphate along with stepwise increase in light intensity. Finally, mutually exclusive biomass and lipid production phases were decoupled into two-stage cultivation process: biomass generation in first stage under nutrient sufficient condition followed by lipid enrichment through nitrogen starvation. The key findings were as follows: (i) ANN-GA resulted in an increase in biomass titer of 157 % (0.95 g L^?1) in shake flask and 42.8 % (1.0 g L^?1) in bioreactor against unoptimized medium at light intensity of 20 μE m^?2 s^?1; (ii) further optimization of light intensity in bioreactor gave significantly improved biomass titer of 5.6 g L^?1 at light intensity of 250 μE m^?2 s^?1; (iii) high cell density of 13.5 g L^?1 with biomass productivity of 675 mg L^?1 day^?1 was achieved with dynamic increase in light intensity and intermittent feeding of limiting nutrients; (iv) finally, two-phase cultivation resulted in biomass titer of 17.7 g L^?1 and total lipid productivity of 313 mg L^?1 day^?1 which was highest among Chlorella sp. under photoautotrophic condition.     

High-level extracellular production of d-Psicose-3-epimerase with recombinant Escherichia coli by a two-stage glycerol feeding approach

The aim of this study is to achieve high-level extracellular production of d-Psicose-3-epimerase (DPE) with recombinant Escherichia coli. High-level production of DPE is one of the key factors in d-Psicose production. In the present study, the gene AAL45544.1 from Agrobacterium tumefaciens str. C58 was modified by artificial synthesis for overexpression in E. coli. The total DPE activity reached 3.96 U mL^?1 after optimization of the media composition, induction temperature, and concentration of inducer. Furthermore, it was found that addition of glycine had a positive effect on the extracellular production of DPE, which reached 3.5 U mL^?1. Finally, a two-stage glycerol feeding strategy based on both the specific growth rate before induction and the amount of glycerol residues after induction was applied in a 3-L fermenter. After a series of optimal strategies in the 3-L fermenter, the total and extracellular DPE activity were 5.08- and 3.11-fold higher than that noted in the shake flask. The extracellular and intracellular DPE activity reached 10.9 and 13.2 U mL^?1, achieving 25.5 and 31.1 % conversion of d-fructose to d-psicose, respectively. The systemic strategies presented in this study provide valuable novel information for the industrial application of DPE.     

Development of an efficient process intensification strategy for enhancing <Emphasis Type="Italic">Pfu</Emphasis> DNA polymerase production in recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

An efficient induction strategy that consisted of multiple additions of small doses of isopropyl-β-D-thiogalactopyranoside (IPTG) in the early cell growth phase was developed for enhancing Pfu DNA polymerase production in Escherichia coli. In comparison to the most commonly used method of a single induction of 1 mM IPTG, the promising induction strategy resulted in an increase in the Pfu activity of 13.5 % in shake flasks, while simultaneously decreasing the dose of IPTG by nearly half. An analysis of the intracellular IPTG concentrations indicated that the cells need to maintain an optimum intracellular IPTG concentration after 6 h for efficient Pfu DNA polymerase production. A significant increase in the Pfu DNA polymerase activity of 31.5 % under the controlled dissolved oxygen concentration of 30 % in a 5 L fermentor was achieved using the multiple IPTG induction strategy in comparison with the single IPTG induction. The induction strategy using multiple inputs of IPTG also avoided over accumulation of IPTG and reduced the cost of Pfu DNA polymerase production.     

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.     

Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation

A codon-optimized Escherichia coli appA phytase gene was synthesized and expressed in Pichia pastoris. Two residue substitutions (Q258N, Q349N) were sequentially introduced to enhance its glycosylation activity. Secretion of appA-Q258N/Q349N was approx. 0.3 mg ml^?1 and enzyme activity reached 1,030 U ml^?1. Purified appA-Q258N/Q349N had a specific activity of 3,137 U mg^?1 with an MW of approx. 53 kDa. Compared with appA-WT, appA-Q258N/Q349N showed over 40 % enhancement in thermostability (85 °C for 10 min) and 4–5 °C increases in the melting temperatures (T_m). The K_m and K_cat of appA-Q258N/Q349N were 0.43 mM and 3,058 s^?1, respectively, which are similar with that of appA-WT. The mutant appA-Q258N/Q349N obtained in this study could be used for the large-scale commercial production of phytase.     

Iodine from bacterial iodide oxidization by Roseovarius spp. inhibits the growth of other bacteria

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 μg ml^?1 iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 μg ml^?1 iodide (I^?). When 10⁶ CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I₂), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 μg ml^?1, respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I^?), the molecular iodine concentration was estimated to be 0.76 μg ml^?1 after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 μg ml^?1.     

Efficient extracellular expression of <Emphasis Type="Italic">Bacillus deramificans</Emphasis> pullulanase in <Emphasis Type="Italic">Brevibacillus choshinensis</Emphasis>

In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 10³ U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.     

Characterization of cell growth and starch production in the marine green microalga Tetraselmis subcordiformis under extracellular phosphorus-deprived and sequentially phosphorus-replete conditions

Microalgal starch is a potential feedstock for biofuel production. Nutrient stress is widely used to stimulate starch accumulation in microalgae. Cell growth and starch accumulation in the marine green microalga Tetraselmis subcordiformis were evaluated under extracellular phosphorus deprivation with initial cell densities (ICD) of 1.5, 3.0, 6.0, and 9.0?×?10⁶ cells mL^?1. The intracellular stored phosphorus supported cell growth when extracellular phosphorus was absent. The maximum starch content of 44.1 % was achieved in the lowest ICD culture, while the maximum biomass productivity of 0.71 g L^?1 day^?1, starch concentration of 1.6 g L^?1, and starch productivity of 0.30 g L^?1 day^?1 were all obtained in the culture with the ICD of 3.0?×?10⁶ cells mL^?1. Appropriate ICD could be used to regulate the intracellular phosphorus concentration and maintain adequate photosynthetic activity to achieve the highest starch productivity, along with biomass and starch concentration. The recovery of phosphorus-deprived T. subcordiformis in medium containing 0.5, 1.0, or 6.0 mM KH₂PO₄ was also tested. Cell growth and starch accumulation ability could be recovered completely. A phosphorus pool in T. subcordiformis was shown to manipulate its metabolic activity under different environmental phosphorus availability. Though lower starch productivity and starch content were achieved under phosphorus deprivation compared with nitrogen- or sulfur-deprived conditions, the higher biomass and starch concentration make T. subcordiformis a good candidate for biomass and starch production under extracellular phosphorus deprivation.     

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.     

Expression and evaluation of enzymes required for the hydrolysis of galactomannan

The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-β-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger β-mannosidase (cMndA) and synthetic Cellvibrio mixtus β-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml^?1, pH 5.47), Agal (135 nkat ml^?1, pH 2.37), cMndA (12 nkat ml^?1, pH 3.40) and Man5A (8 nkat ml^?1, pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l^?1 mannose and 10 g l^?1 locust bean gum.     

Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria

Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 μg ml^?1 caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 μg ml^?1 and nisin A/polymyxin B at 4.64/0.12 μg ml^?1 decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log₁₀ CFU ml^?1, respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.     

High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg?l^?1 of enzyme with a biomass yield of 16.2 mg?g_cdw ^?1 and a productivity of 10.5 mg?l^?1?h^?1.     

Enhanced production of ATP-binding cassette protein exporter-dependent lipase by modifying the growth medium components of Pseudomonas fluorescens

The industrially-important thermostable lipase, TliA, was extracellularly produced in the recombinant Pseudomonas fluorescens by the homologous expression of TliA and its cognate ABC protein exporter, TliDEF. To increase the secretory production of TliA, we optimized the growth temperature and the culture medium of P. fluorescens. The total amount and the specific productivity of lipase was highest at 25 °C of cell growth temperature, although maximal cell growth was observed at 30 °C. Using the culture medium composed of 20 g dextrin l^?1, 40 g Tween 80 l^?1 and 30 g peptone l^?1, TliA was produced at a level of 2,200 U ml^?1 in a flask culture. The TliA production increased about 3.8-fold (8,450 U ml^?1) in batch fermentation using a 2.5 l fermentor, which was about 7.7-fold higher than that of previously reported TliA production.     

Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters

Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml^?1) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.     

Influence of the nutritional conditions on haloalcohol dehalogenase HheC production by recombinant Escherichia coli P84A/MC1061

In this research, we first determined the three most significant nutrient factors affecting haloalcohol dehalogenase HheC production by Escherichia coli P84A/MC1061. These were glycerol, yeast extract, and ammonium sulfate. The steepest ascent method was then applied to obtain the optimal design intervals of the three factors. An application of center composite design was used, and the ingredients of the optimized medium were 1.8 g l^?1 glycerol, 48 g l^?1 yeast extract, 2.2 g l^?1 ammonium sulfate, 5 g l^?1 compound phosphate, 1 g l^?1 magnesium sulfate, and 1.19?×?10^?5?g l^?1 ferric sulfate. The enzyme activity reached 109,365 U ml^?1 under the most favorable conditions, which is a 277.7 % increase compared with the control group. Our study of cellular respiration parameters (oxygen uptake rate and carbon dioxide emission rate) revealed that the metabolic activity of the strain was strongly promoted under these optimal nutrient conditions and that yeast extract had a positive effect on respiratory intensity and the expression levels of HheC.     

Expression and Characterization of Recombinant Sucrose Phosphorylase

SPase is widely used in the food, cosmetics, and pharmaceutical industries. Previously, a SPase gene was cloned from Bifidobacterium longum JCM1217 and constructed into Escherichia coli BL21. In this paper, its expression conditions were optimized. The results showed that several induction factors determined the expression efficiency of SPase. The initial cell density, IPTG concentration, and induction time and temperature significantly (p?<?0.01) affected the total protein content and activity of expressed SPase. The highest expression efficiency was obtained at an initial cell density of OD₆₀₀?=?0.5, with 0.05 mM IPTG, followed by shaking at 180 rpm and incubation at 30 °C for 15 h. The purified SPase had a specific activity of 122.1 U/mg, which was raised by 1.85 -fold more than that before optimization, and its recovery yield was 86%. Furthermore, SPase also showed higher thermostability. The results of this study provide essential information for the industrial production of SPase.     

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.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. N16-5 in <Emphasis Type="Italic">Escherichia coli</Emphasis> by the optimization of induction condition and fed-batch fermentation

To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K _m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k _cat/K _m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01–0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.