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.     

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.     

The effect of agitation and aeration on the synthesis and molecular weight of gellan in batch cultures of Sphingomonas paucimobilis

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated in batch fermenter cultures of the bacterium, Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth of S. paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did not always lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymer synthesis.Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). An increase in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration or dissolved oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan production and probably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxyalkanoates, (such as polyxydroxybutyrate) which were found in S. paucimobilis cells indicating the existence of a carbon storage system, which may contribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mass (nutrient) transfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good near real time estimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming gravimetric assay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecular weight (at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and is important for the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weight of the biopolymers is clearly an area needing further research.     

Inulinase biosynthesis using immobilized mycelium of Aspergillus niger

Conidia of Aspergillus niger 20 Osm producing extracellular inulinase were immobilized on pumice stones or polyurethane sponge and used in repeated-batch processes. Some factors affecting inulinase biosynthesis by the mycelium A. niger immobilized on pumice stones were investigated. Maximal inulinase production occurred in 50 ml of medium containing 0.5 g of carrier at 30 °C, pH 6.0 and at an agitation speed of 200 rpm. This procedure enabled repeated-batch enzyme production and as many as six subsequent 24 h batches could be fermented by using the same carrier. This is the first report on inulinase biosynthesis by mycelium of A. niger immobilized on polyurethane sponge using unconventional oxygenation of culture which ensures that the dissolved oxygen concentration remains constant.     

Fermentation strategies for the production of lipase by an indigenous isolate Burkholderia sp. C20

Burkholderia sp. C20 strain isolated from food wastes produces a lipase with hydrolytic activities towards olive oil. Fermentation strategies for efficient production of this Burkholderia lipase were developed using a 5-L bench top bioreactor. Critical factors affecting the fermentative lipase production were examined, including pH, aeration rate, agitation rate, and incubation time. Adjusting the aeration rate from 0.5 to 2 vvm gave an increase in the overall lipase productivity from 0.057 to 0.076 U/(ml h), which was further improved to 0.09 U/(ml h) by adjusting the agitation speed to 100 rpm. The production of Burkholderia lipase followed mixed growth-associated kinetics with a yield coefficient of 524 U/g-dry-cell-weight. The pH optimum for cell growth and lipase production was different at 7.0 and 6.0, respectively. Furthermore, stepwise addition of carbon substrate (i.e., olive oil) enhanced lipase production in both flask and bioreactor experiments.     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

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.     

Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production

We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 °C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h⁻¹. The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K = 0.2 h⁻¹. The results attained also showed that this process is promising for G6PD production.     

Scale-up of micro-aerobic 1,3-propanediol production with Klebsiella pneumonia CGMCC 1.6366

The conversion of glycerol to 1,3-propanediol (1,3-PD) using Klebsiella pneumoniae CGMCC 1.6366 under aerobic condition was scaled up from scale 5 to 50,000 l in series. Several parameters including power input P/V_l, agitation rate n, impeller tip speed nD, superficial gas velocity u_s, and Re_s were investigated as the criteria for scaling up. Impeller tip speed was chosen as the main criterion. It was also noticed less aeration was favored in that less electron will be shunted to electron transfer chain. The fermentation in 500 l bioreactor produced 66.8 g 1,3-PD with the yield of 0.55 mol mol^?1 at agitation rate and aeration of 130 rpm and 0.14 vvm air flow. Using these empirically obtained control concepts we successfully scaled up in 500–50,000 l pilot-scale reactors. The final 1,3-PD concentrations in 50,000 l bioreactor amounted to 63.3 g l^?1 with the yield of 0.5 mol mol^?1.     

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     

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.     

l-Lactic acid production from liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and optimization

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing l-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH₄)₂SO_4, KH₂PO₄, MgSO₄ and ZnSO₄·7H₂O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 °C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for l-lactic acid production from low-cost starchy substrates.     

Reprint of “Multiphase mixing characteristics in a microcarrier-based stirred tank bioreactor suitable for human mesenchymal stem cell expansion”

Large-scale human mesenchymal stem cell expansion calls for a bioreaction system, that provides a sufficient growth surface. An alternative to static cultivations systems like cell factories are disposable stirred tank reactors. Here, microcarriers provide the required growth surface, but these make it difficult to achieve a complete homogenization in the bioreactor, while avoiding shear stress. To gain insight into this process, we investigated the impact of different power inputs (0.02–2.6 W m⁻³) on the mixing time (t_m). Whereas t_m was inversely proportional to agitation in a one-phase-system, aeration resulted in a constant mixing time at 30–70 rpm. A high microcarrier concentration (30 g L⁻¹) and low stirrer speed (30 rpm) in the liquid-solid system caused a 50-fold increase in t_m and the formation of a discrete non-mixed upper zone. The effect of the microcarrier concentration on t_m became negligible at higher stirrer speeds. In the three-phase system, microcarrier settling was prevented by aeration and a minimal specific power input of 0.6 W m⁻³ was sufficient for complete homogenization. We confirmed that a low power input during stem cell expansion leads to inhomogeneity, which has not been investigated in the three-phase system up to date.     

Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus

Effects of pellet morphology on broth rheology are reported for pelleted submerged cultures of the lovastatin producing filamentous fungus Aspergillus terreus, growing in fluidized bed and stirred tank bioreactors. The pellet diameter and compactness were affected by the agitation intensity of the broth; however, the total biomass productivity was not affected. In fluidized beds and stirred tanks with agitation intensity of up to 300 rpm (impeller tip speed of 1.02 m s⁻¹), the fungal pellets were stable at diameters of up to about 2300 μm. In more intensely agitated stirred tanks (≥600 rpm; impeller tip speed of ≥2.03 m s⁻¹), the stable pellet size was only about ≤900 μm. The biomass concentration and the pellet diameter were the main factors that influenced the flow index and the consistency index of the power-law broths. Because the biomass productivity was the same in all experiments in a given type of reactor and the oxygen concentration was kept at ∼400% of air saturation, the pellet size and morphology were not influenced by oxygen mass transfer effects. Pellets were always dense in the core region and no necrosis of the biomass occurred.     

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett–Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 ± 65 U/ml.     

Xylitol production in a bubble column bioreactor: Influence of the aeration rate and immobilized system concentration

This work evaluated the xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a bubble column bioreactor using cells of the yeast Candida guilliermondii immobilized in calcium-alginate. The fermentation runs were performed according to a 2² full factorial design with three replicates at the center point in order to determine the effect of the variables: aeration rate (0.66–1.33 vvm) and immobilized system concentration (20–40% v/v), on the efficiency of xylose-to-xylitol conversion and on the xylitol volumetric productivity. The results indicated a significant influence of both variables on xylitol production. The highest conversion efficiency (41%) was attained using 1.33 vvm aeration rate and 40% immobilized system. Under these conditions, the volumetric productivity was 0.21 g l⁻¹ h⁻¹.     

Production of an oxidant and SDS-stable alkaline protease from an alkaophilic Bacillus clausii I-52 by submerged fermentation: Feasibility as a laundry detergent additive

An investigation was carried out on enzyme production of an oxidant and SDS-stable alkaline protease secreted by Bacillus clausii I-52 using the submerged fermentation and its application as a detergent additive. Maximum enzyme activity was produced when cells were grown under the submerged fermentation conditions at 37 °C for 48 h with an aeration rate of 1.5 vvm and agitation rate of 700 rpm in a medium (pH 10.65) containing (w/v): soybean meal, 20; wheat flour, 10; liquid maltose, 25; K₂HPO₄, 4; Na₂HPO₄, 1; MgSO₄·7H₂O, 0.1; NaCl, 4; FeSO₄·7H₂O, 0.5; Na₂CO₃, 6. The alkaline protease produced was found to be highly compatible and stable against not only the commercial detergent components such as α-orephin sulfonate and zeolite but also the commercial detergent preparations. Wash performance analysis using EMPA test fabrics revealed that BCAP exhibited high efficiency for the removal of protein stains in the presence of commercial detergents as well as surfactants. These results suggest that the alkaline protease produced from B. clausii I-52 which showed high stability against detergents has significance for an industrial perspective, especially, detergent additive.     

Production of butyric acid by a cellulolytic actinobacterium Thermobifida fusca on cellulose

Thermobifida fusca not only produces cellulases, hemicellulases and xylanases, but also excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.     

Lipase-mediated hydrolysis of corn DDGS oil: Kinetics of linoleic acid production

In this study, we investigated the kinetics of linoleic acid production via lipase-mediated hydrolysis of corn DDGS oil in a batch reactor with continuous mechanical agitation and developed a kinetic model that incorporated the product inhibition to study the complete hydrolysis. The model agreed very well with observed data; though situations with low enzyme dosage or low stirring rates were modeled successfully without product inhibition, actual product concentration in such situations was too low to exert any inhibitory effects. Increasing the enzyme concentration increased hydrolysis, and beyond certain enzyme concentrations, effects tended to fade away because of excessive enzyme desorption from the interface. An enzyme dosage within the range of 40–60 KLU/L of oil dispersion could be successfully applied for a substrate concentration of 25–50 g/L of DDGS oil. Increasing the agitation rates improved enzymatic hydrolysis, but a higher stirring rate of 1000 rpm moderately improved production of linoleic acid compared with a stirring rate of 750 rpm. Within the range of substrate concentrations studied, enzymatic inhibition was moderate but still evident. The high degree of hydrolysis (i.e., ∼96% of theoretical linoleic acid yield) from DDGS oil suggests this method has potential for commercial production of linoleic acid.     

Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments

Protease producing halotolerant bacterium was isolated from saltern pond sediment (Tuticorin) and identified as Bacillus licheniformis (TD4) by 16S rRNA gene sequencing. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like incubation time, pH, agitation, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that xylose and urea enhances the enzyme production. Thus, with selected C–N sources along with 1 M NaCl the maximum protease production (141.46 U/mg) was obtained in the period of 24 h incubation at pH 8 under 250 rpm compared to the initial enzyme production (89.87 U/mg).