Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O₂ transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O₂ transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.     

Scopolamine,anisodamine and hyoscyamine production by Brugmansia candida hairy root cultures in bioreactors

Hyoscyamine, anisodamine (6β-hydroxyhyoscyamine) and scopolamine are tropane alkaloids produced by plants belonging to the Solanaceae family such as Brugmansia candida. These alkaloids were traditionally used in medicine because of their anticholinergic activity. Further therapeutical properties for anisodamine were recently described renewing the interest in these alkaloids. The scaling-up of hairy root cultures is an interesting strategy for the pharmaceutical production of these compounds instead of the isolation from plants. In this work, B. candida hairy roots were cultured in a modified 1.5 L stirred tank in order to analyze an alternative production system of scopolamine and anisodamine. It was found that these cultures produced an increased biomass and alkaloids concentration compared to the processes carried out in Erlenmeyer flasks. Anisodamine was the predominant alkaloid reaching a maximum concentration of 10.05 ± 0.76 mg/g DW in modified bioreactor culture system. The results obtained in this work are potentially applicable for the rational scale-up of the process.     

Enhanced production of (+)-terrein by Aspergillus terreus strain PF26 with epigenetic modifier suberoylanilide hydroxamic acid

New strategies for improving the fermentation yield of (+)-terrein which is a fungal metabolite with multiple bioactivities are very urgent. In this study, the effect of suberoylanilide hydroxamic acid, one kind of epigenetic modifier, on the biosynthesis of (+)-terrein by Aspergillus terreus strain PF26 isolated from the marine sponge Phakellia fusca was investigated. It was found that suberoylanilide hydroxamic acid exhibited a positive impact on (+)-terrein production, resulting from promoting the biosynthesis of 6-hydroxymellein, the precursor of (+)-terrein. Through optimization of feeding concentration and time of suberoylanilide hydroxamic acid, 5.58 g/L (+)-terrein could be obtained in shake flask cultivation, 29.5% higher than the control. Correspondingly, the fermentation of A. terreus strain PF26 in 7.5-L stirred bioreactor with feeding suberoylanilide hydroxamic acid (900 μM, day 4) yielded 9.07 g/L (+)-terrein, 77.1% higher than the control. These results showed that the epigenetic modifier-suberoylanilide hydroxamic acid could be utilized to enhance the production of (+)-terrein, which laid the foundation of massive production of (+)-terrein by fermentation.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

An integrated bioreactor-expanded bed adsorption system for the removal of acetate to enhance the production of alpha-interferon-2b by Escherichia coli

A stirred tank bioreactor (STB) integrated with an expanded bed adsorption (EBA) system containing anion-exchange resin (Diaion WA30) was developed for in situ removal of acetate to increase the production of α-interferon-2b (α-PrIFN-2b) by Escherichia coli (E. coli). Although the total acetate (9.79 g/L) secreted by E. coli in the integrated STB/EBA system was higher than that in a bioreactor with dispersed resin or a conventional batch bioreactor, cell growth (14.97 g/L) and α-PrIFN-2b production (867.4 μg/L) were significantly improved owing to the high efficiency of acetate removal from the culture. The production of α-PrIFN-2b in the integrated STB/EBA system was improved by 3-fold and 1.4-fold over that obtained in a conventional batch bioreactor and a bioreactor containing dispersed resins, respectively.     

Lipolytic enzyme production by Thermus thermophilus HB27 in a stirred tank bioreactor

In this study, lipolytic enzyme production by Thermus thermophilus HB27 at bioreactor scale has been investigated. Cultivation was performed in a 5-L stirred tank bioreactor in discontinuous mode, at an agitation speed of 200 rpm. Different variables affecting intra- and extra-cellular lipolytic enzyme production such as culture temperature and aeration rate have been analysed. The bacterium was able to grow within the temperature range tested (from 60 to 70 °C) with an optimum value of 70 °C for intra- and extra-cellular lipolytic enzyme production.On the other hand, various aeration levels (from 0 to 2.5 L/min) were employed. A continuous supply of air was necessary, but no significant improvement in biomass or enzyme production was detected when air flow rates were increased above 1 L/min. Total lipolytic enzyme production reached a maximum of 167 U/L after 3 days, and a relatively high concentration of extra-cellular activity was detected (40% of the total amount). Enzyme yield was around 158 U/g cells. Moreover, it is noteworthy that the lipolytic activity obtained operating at optimal conditions (70 °C and air flow of 1 L/min) was about five-fold higher than that attained in shake flask cultures     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Production of gluconic acid from glucose by Aspergillus niger: growth and non-growth conditions

Batch fermentation of glucose to gluconic acid was conducted using Aspergillus niger under growth and non-growth conditions using pure oxygen and air as a source of oxygen for the fermentation in 2 and 5 l stirred tank reactors (batch reactor). Production of gluconic acid under growth conditions was conducted in a 5 l batch reactor. Production and growth rates were higher during the period of supplying pure oxygen than that during supplying air, and the substrate consumption rate was almost constant. For the production of gluconic acid under non-growth conditions, conducted in the 2 l batch reactor, the effect of the pure oxygen flow rate and the biomass concentration on the gluconic acid production was investigated and an empirical equation suggested to show the dependence of the production rate r_p on the biomass concentration C_x and oxygen flow rate Q, at constant operating conditions (30 °C, 300 rpm and pH 5.5). Biomass concentration had a positive effect on the production rate r_p, and the effect of Q on r_p was positive at high biomass concentrations.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.     

Astaxanthin from Jerusalem artichoke: Production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics

Jerusalem artichoke extract or powder was used for astaxanthin production using Phaffia rhodozyma without acidic or enzymatic inulin hydrolysis. The culture medium containing Jerusalem artichoke as carbon source was optimized, and feeding strategies, including constant, exponential, pH-stat, and substrate feedback fed-batch fermentations, were also compared for enhancing the cell biomass and astaxanthin synthesis by P. rhodozyma. Substrate-feedback fed-batch fermentation resulted in the highest dry cell weight of 83.60 g/L, with a carotenoid concentration and yield of 982.50 mg/L and 13.30 mg/g, respectively, under optimized medium components using Jerusalem artichoke extract as carbon source in a 3-L stirred-tank bioreactor. Moreover, 482.50 mg/L of carotenoids and 253.10 mg/L of astaxanthin were obtained by continuous feeding of Jerusalem artichoke powder, which was used as carbon source. Astaxanthin essence with high DPPH-scavenging activity was obtained from the extracted astaxanthin, and the DPPH free radical scavenging rate of 40 ppm astaxanthin essence reached 76.29%. When stored at 4 °C, astaxanthin essence showed the highest stability, with a minimum k value of 0.0099 week⁻¹ and maximum half-life (t_1/2) value of 70 weeks.     

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor

Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Submerged culture production of chitinase by Trichoderma harzianum in stirred tank bioreactors – the influence of agitator speed

The chitinase fermentation process utilizing chitin as the sole carbon source was investigated in a stirred tank bioreactor. Agitator speed of 224 rpm was found to be most suitable for cell growth as well as for chitinase production. Chitinase yield decreased rapidly at higher agitator speed, while decrease in cell yield at higher agitator speed was not rapid. Probably, mass transfer limitation was predominant in the fermentation process at lower agitator speed. Higher agitator speed appears to reduce chitinase production.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.     

Effect of process parameters on lipase production by Candida cylindracea in stirred tank bioreactor using renewable palm oil mill effluent based medium

A two-level full factorial design (FFD) was employed to determine the effects of process parameters on lipase production by Candida cylindracea ATCC 14830 in palm oil mill effluent (POME)-based medium. Ten experimental runs based on three parameters (temperature, agitation and aeration) as indicated by the FFD were carried out in a stirred-tank bioreactor. On statistical analysis of the results, the optimum temperature, aeration and agitation rates were found to be 30 °C, 1.0 vvm and 400 rpm respectively, with a maximum activity of 41.46 U/ml after 36 h of fermentation. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.999, indicating a satisfactory fit of the model with the experimental data. All the three parameters were statistically significant at p < 0.05. The validation experiment also confirmed that apart from lipase production, there was an increase in chemical oxygen demand (COD) removal throughout the fermentation period.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation

This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34 cm diameter and 50 cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586 ± 63 U gds⁻¹) was obtained in the fed-batch mode using saturated air after 24 h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57 °C and 4.8–5.2, respectively. For a temperature range from 40 to 70 °C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47 h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622 h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.