Broth Rheology Research Articles

Investigating a Stirred Bioreactor: Impact of Evolving Fermentation Broth Pseudoplastic Rheology on Mixing Mechanisms

The culture medium in many fermentations is a non-Newtonian fluid. In bacterial alginate batch production, the broth becomes more pseudoplastic as the alginate concentration increases, which impairs the mixing process. This work characterizes the effect of the interaction between changing broth rheology and impeller mixing on a bioreactor fluid dynamics. Experimentally, a fermentation with evolving broth pseudoplastic rheology is reproduced. Three fermentation stages are mimicked using appropriate solutions of water and xanthan gum. Impeller torque measurements are reported. The weakening of the impellers’ interaction over the fermentation process is identified. To overcome the experimental limitations, CFD is applied to study the evolution of the fermentation fluid flow patterns, velocity field, dead zones, and vortical structures. Precessional vortex macro-instabilities are identified as being responsible for the unstable flow patterns identified at the earlier stages of the fermentation. A stable parallel flow pattern accounts for the weakest impellers’ interaction at the final stage. Overall, this work contributes with a complete workflow to adapt CFD models for characterization and aided design of stirred tanks with changing broth pseudoplastic rheology as well as an evolving flow regime.

Role of microbubbles coupling fibrous-bed bioreactor in butyric acid production by Clostridium tyrobutyricum using Brewer’s spent grain as feedstock

Microbubbles coupling fermentation is an efficient technology for improving oxygen transfer to aerobic microorganisms in a bioreactor containing an air sparger. However, the effect of this technology on anaerobic Clostridium tyrobutyricum, whose morphology develops viscous broth rheology and lumps impeding the nutrient mass transfer, has not been reported yet. Therefore, we evaluated the role of microbubbles coupling fibrous-bed bioreactor (MBFBB) on butyrate production by immobilized Clostridium tyrobutyricum cells obtained from renewable feedstock brewer’s spent grain (BSG). Compared with the conventional fibrous-bed bioreactor (FBB) fermentation, two-fold shorter fermentation time and two-fold higher butyrate productivity were achieved by MBFBB-immobilized C. tyrobutyricum cells. Furthermore, long-term stability and reliability for butyrate production were obtained in ten cycle process using BSG hydrolysate. Finally, fed-batch fermentation using BSG hydrolysate produced a high butyrate titer of 43.68 g/L, with a significantly higher selectivity of 11.67, reducing the production cost of downstream processing. The results also indicated that butyrate productivity of 5.40 g/(L h were successfully obtained in BSG medium, thereby representing the highest productivity reported to date in C. tyrobutyricum from low-cost feedstocks. To our knowledge, this is also the first report related to microbial production of butyrate from BSG. Based on techno-economic analysis, it was demonstrated that the MBFBB-based fermentation process with BSG, was robust, economic and should underpin the future development of commercial biobutyrate production.

Morphology and rheological behaviour of Yarrowia lipolytica: Impact of dissolved oxygen level on cell growth and lipid composition

Abstract Yarrowia lipolytica was investigated as potential ascomycetes yeast for higher lipid productivity. Moreover, lipogenesis seems to be very sensitive to oxygen variation and to achieve efficient microbial conversion of crude glycerol into energetic molecules (single cell oils, SCO) is a real challenge in the bioprocess. Dissolved oxygen (DO) was selected as a key parameter for translating results from shake flask to large scale for a successful bioprocess. Lipid production under different oxygenation rates was carried out to identify the ideal scenario required for higher accumulation rate. A combination of metabolomic, morphological and rheological profiling was investigated in the presence of crude glycerol as a carbon substrate. Results showed that cells require a dissolved oxygen concentration of 30% to achieve the highest lipid production of 44.8 ± 1.2% (w/w). The increase or decrease of oxygen level might affect the lipid content. However, cells exhibited the same rheological behaviour and presented unimodal distribution despite the oxygen variation. Furthermore, broth rheology was ultimately dependent on nutrient limitation and aeration. The variation of oxygen level (i.e. volumetric oxygen transfer coefficient (KLa) and oxygen uptake rate (OUR)) affects the biomass concentration, lipid accumulation and composition. These parameters played a crucial role in determining bioprocess performance.

Improved production of Daptomycin in an airlift bioreactor by morphologically modified and immobilized cells of Streptomyces roseosporus

The increased threat of drug resistance has challenged the existence of several conventional and non-conventional antibiotics in the recent times. Daptomycin is a novel cyclic-lipopeptide antibiotic produced by Streptomyces roseosporus that has progressed as a significant anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotic. But, the economic practicality of this highly valued secondary metabolite is deterred by its poor production and tedious processing methodology. The present study aims at strategic improvement of Daptomycin production through morphological variations of S. roseosporus cells. Free cells, pelletized cells and immobilized cells on ultra porous refractory brick and silk sachets were investigated for the production of Daptomycin in a lab-scale 2.0 l air-lift bioreactor. The effect(s) of nitrogen source, inoculum size and oxygen stress were analyzed for pellet formation of S. roseosporus. Interestingly, free cells produced 750 mg/l of Daptomycin in a single batch. But, the three phase broth viscosity increased due to vigorous growth of free cells which hampered the oxygen transfer rate. The cell degeneration over the time deterred pellet reusability. 1430 mg/l Daptomycin was produced using pellets for 2 batches. On the contrary, mechanical stability, buoyancy and reusability of refractory bricks and silk sachets were beneficial. Daptomycin production was recorded for 6–8 batches. Immobilized cells on refractory bricks and silk sachets led to 4895 mg/l and 3623 mg/l Daptomycin production respectively. Cell immobilization improved the three phase broth rheology and hence, the hydrodynamics within the reactor. Therefore, whole-cell immobilization could contribute to the ameliorated production of this life-saving drug.

Improvement of glucoamylase production using axial impellers with low power consumption and homogeneous mass transfer

Significantly influenced by complex cell morphology, glucoamylase fermentation using Aspergillus niger is characterized by high apparent viscosity and shear-thinning rheology. In this study, the influence of liquid flow field patterns on morphology, broth rheology, mass transfer and glucoamylase production was investigated by applying two different configurations with radial (Ruston Turbine, RT) and axial (Wide-blade hydrofoil upward-pumping, WHu) flow impellers. It was found that empirical correlations for averaged quantities, such as the mass transfer coefficient and viscosity, cannot reasonably explain the observations. Therefore, numerical simulation was carried out to study the detailed characteristics of local field in lab-scale bioreactors. The results showed, under similar glucose and oxygen uptake rates, that the WHu configuration formed relatively homogeneous viscosity and mass transfer fields, while the RT configuration was accompanied with significant heterogeneities. Under these conditions, the fraction of active mycelia in pellets could be highly correlated with enzyme production, and a novel parameter (Active Part Percentage, APP) was defined to introduce the effects of flow field on pelletized morphology. The WHu impellers facilitated the formation of pellets and hairy structures, with a higher APP of the pellets. As a result, the culture with the axial flow impeller configuration exhibited a larger glucoamylase production rate (+25%) and product yield on sugar (+23%) and yield on energy (+60%) in comparison to the radial flow impeller. Computational fluid models were proposed to in-depth understand such results based on local mass transfer and viscosity values, since the average values are similar over the entire fermentation processes.

Genetically shaping morphology of the filamentous fungus Aspergillus glaucus for production of antitumor polyketide aspergiolide A.

BackgroundFor filamentous fungi, the basic growth unit of hyphae usually makes it sensitive to shear stress which is generated from mechanical force and dynamic fluid in bioreactor, and it severely decreases microbial productions. The conventional strategies against shear-sensitive conundrum in fungal fermentation usually focus on adapting agitation, impeller type and bioreactor configuration, which brings high cost and tough work in industry. This study aims to genetically shape shear resistant morphology of shear-sensitive filamentous fungus Aspergillus glaucus to make it adapt to bioreactor so as to establish an efficient fermentation process.ResultsHyphal morphology shaping by modifying polarized growth genes of A. glaucus was applied to reduce its shear-sensitivity and enhance aspergiolide A production. Degenerate PCR and genome walking were used to obtain polarized growth genes AgkipA and AgteaR, followed by construction of gene-deficient mutants by homologous integration of double crossover. Deletion of both genes caused meandering hyphae, for which, ΔAgkipA led to small but intense curves comparing with ΔAgteaR by morphology analysis. The germination of a second germ tube from conidiospore of the mutants became random while colony growth and development almost maintained the same. Morphology of ΔAgkipA and ΔAgteaR mutants turned to be compact pellet and loose clump in liquid culture, respectively. The curved hyphae of both mutants showed no remarkably resistant to glass bead grinding comparing with the wild type strain. However, they generated greatly different broth rheology which further caused growth and metabolism variations in bioreactor fermentations. By forming pellets, the ΔAgkipA mutant created a tank environment with low-viscosity, low shear stress and high dissolved oxygen tension, leading to high production of aspergiolide A (121.7 ± 2.3 mg/L), which was 82.2% higher than the wild type.ConclusionsA new strategy for shaping fungal morphology by modifying polarized growth genes was applied in submerged fermentation in bioreactor. This work provides useful information of shaping fungal morphology for submerged fermentation by genetically modification, which could be valuable for morphology improvement of industrial filamentous fungi.

Challenges in industrial fermentation technology research.

Industrial fermentation processes are increasingly popular, and are considered an important technological asset for reducing our dependence on chemicals and products produced from fossil fuels. However, despite their increasing popularity, fermentation processes have not yet reached the same maturity as traditional chemical processes, particularly when it comes to using engineering tools such as mathematical models and optimization techniques. This perspective starts with a brief overview of these engineering tools. However, the main focus is on a description of some of the most important engineering challenges: scaling up and scaling down fermentation processes, the influence of morphology on broth rheology and mass transfer, and establishing novel sensors to measure and control insightful process parameters. The greatest emphasis is on the challenges posed by filamentous fungi, because of their wide applications as cell factories and therefore their relevance in a White Biotechnology context. Computational fluid dynamics (CFD) is introduced as a promising tool that can be used to support the scaling up and scaling down of bioreactors, and for studying mixing and the potential occurrence of gradients in a tank.

Evaluation of orange peel, an industrial waste, for the production of Aspergillus sojae polygalacturonase considering both morphology and rheology effects

Orange peel is an agroindustrial waste rich in pectin and known to be an inducer for pectinase production. The use of this low-cost substrate for the production of an industrially important enzyme, polygalacturonase (PG), can be an alternative way to turn this waste into a value-added product, contributing to the reduction of environmental waste disposal problems. Enzyme productions by fungal microorganisms are affected by environmental and nutritional factors, demanding the determination of optimum conditions for maximum enzyme production with the desired fungal morphology and broth rheology. Therefore, complex and additional carbon sources were optimized with respect to PG production by Aspergillus sojae using statistical approaches. Effect of pH, another significant parameter affecting the rheology and morphology of the strain, was investigated in the serial bioreactor system using the optimized medium composition. Highest PG enzyme yield and productivity together with the maximum PG enzyme production (93.48 U/mL) were obtained under uncontrolled pH conditions. Under these conditions, morphologically, pellet sizes exhibited a normal distribution ranging between 0.5-1.0 mm and 1.0-1.5 mm, and rheological measurements revealed that fermentation broths showed non-Newtonian flow. The low pH trend observed during the course of the fermentation was another important positive outcome for industrial fermentations, prone to contamination problems.

Relationship Between Morphology and Itaconic Acid Production by Aspergillus terreus

The morphology of filamentous fungi closely correlates with the productivity in submerged culture. Using itaconic acid (IA) production by Aspergillus terreus as a research model, the quantitative relationship between the growth form of A. terreus and IA production was investigated. IA fermentation was scaled up from shake flasks to a 7 L stirred tank bioreactor based on the quantitative relationship. Our results demonstrated the following: (1) Three morphologies of A. terreus were formed by changing the inoculum level and shape of the flask. (2) Investigation of the effects of the three morphologies on broth rheology and IA production revealed the higher yield of IA on dry cell weight (DCW, IA/DCW) and yield of glucose on DCW (consumed glucose/DCW) were achieved during clump growth of A. terreus. (3) By varying the KH2PO4 concentration and culture temperature, the relationships between clump diameter and IA production were established, demonstrating that the yield of IA on DCW (R(2) = 0.9809) and yield of glucose on DCW (R(2) = 0.9421) were closely correlated with clump diameter. The optimum clump diameter range for higher IA production was 0.40-0.50 mm. (4) When the clump diameter was controlled at 0.45 mm by manipulating the mechanical stress in a 7 L fermentor, the yield of IA on DCW and yield of glucose on DCW were increased by 25.1% and 16.3%, respectively. The results presented in this study provide a potential approach for further enhancement of metabolite production by filamentous fungi.

Process and Strain Development for Reduction of Broth Viscosity with Improved Yield in Coenzyme Q10 Fermentation by Agrobacterium tumefaciens ATCC 4452

Viscous nature of the fermentation broth has phenomenal influence on process conditions and parameters in a fermentor. Though broth rheology has attracted significant influence in process research, still there is a challenge to modify fluid dynamics of fermentation broth. During the production of coenzymeQ10 (CoQ10) by Agrobacterium tumefaciens ATCC 4452, the culture broth becomes highly viscous due to excessive synthesis of exopolysaccharides. This hinders the CoQ10 yield and complicates the downstream process. The present study describes how this problem was tackled by media modification and mutation. Induced mutants were generated using UV and EMS as mutagenic agents followed by rational selection based on antibiotic resistance. On screening of these mutants in sucrose based PM-2 medium, UV induced, vancomycin resistant mutant M-6, showed significant reduction (6.29 fold) in viscosity development in the broth. Mutant M-6(S), a natural variant of mutant M-6, resistant to high substrate concentration was further selected for the CoQ10 production. Cane molasses as carbon source was found to be best suitable for CoQ10 fermentation using mutant M-6(S). Replacing sucrose with cheaper cane molasses significantly reduced the broth viscosity with improved specific CoQ10 content, thereby generating cost effective fermentation process. The newly developed mutant strain produced 48.89 mg/L of CoQ10 with specific CoQ10 content of 1.87 mg/g of DCW at 25°C, 500 rpm agitation and 0.2 vvm aeration using continuous fed batch fermentation and newly formulated cane molasses medium.

Comprehension of viscous morphology—Evaluation of fractal and conventional parameters for rheological characterization of Aspergillus niger culture broth

The filamentous fungus Aspergillus niger is a widely used host in industrial processes from food, chemical to pharmaceutical industry. The most prominent feature of this filamentous microorganism in submerged cultivation is its complex morphology which comprises dense spherical pellets as well as viscous elongated filaments. Depending on culture conditions, the exhibited morphology has tremendous effect on the overall process, making a precise understanding of fungal growth and morphology indispensable. Morphology, however, is only industrially relevant as long as it can be linked to important cultivation characteristics of filamentous microorganisms such as culture broth flow behavior. In the present study, different conventional and fractal morphological parameters gained from automatic image analysis were tested for their eligibility to predict culture broth rheology from morphologic appearance. The introduced biomass independent rheological parameters KBDW and nBDW obtained by power law relationship were successfully estimated from morphology related fractal and conventional parameters. For improved characterization of morphologic appearance of filamentous fungi newly introduced fractal quotient and lacunarity were compared to conventional particle shape parameters in form of the earlier established Morphology number (MN).

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7Uml(-1), respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301mUml(-1), respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35UgDW(-1)h(-1). In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.

Influence of ultrasound amplitude and duty cycle on fungal morphology and broth rheology of Aspergillus terreus

Effects of ultrasound amplitude and duty cycle on cultures of Aspergillus terreus are reported in a 25 l slurry bubble column sonobioreactor. Fermentations were carried out batchwise. A 2 k -factorial design with added central points was used. Sonication at any cycle and amplitude level did not affect biomass growth rate and yield relative to nonsonicated control, but did affect growth morphology. Ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Production of lovastatin was reduced by medium- and high-cycle sonication. Sonication affected broth rheology. In view of these results, sonication can be used to modify growth morphology and broth rheology without affecting growth rate and yield of filamentous fungi.

Enhanced Hyaluronic Acid Production of Streptococcus zooepidemicus by Shifting Dissolved Oxygen Level Based on Broth Rheology and Oxygen Mass Transfer Characteristics

This research focused on a dissolved oxygen (DO)-shifting strategy to enhance the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. In this strategy, DO was controlled at above 10 ± 1% during 0–8 h and at 5 ± 0.5% during 8–18 h. The DO-shifting strategy increased the consistency index and oxygen mass transfer coefficient and decreased the flow behavior index n. With the proposed DO-shifting strategy, HA production increased from 5.0 ± 0.1 g/L to 6.3 ± 0.1 g/L. The enhanced HA production was attributed to the alleviation of carbon and energy competition between cell growth and HA synthesis.

Ultrasound affects fungal morphology and broth rheology of Aspergillus terreus

Ultrasound affects fungal morphology and broth rheology of Aspergillus terreus

Changes in morphology of Rhizopus chinensis in submerged fermentation and their effect on production of mycelium-bound lipase

In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.

Effect of Aeration and Agitation Regimes on Lipase Production by Newly Isolated Rhodotorula mucilaginosa–MTCC 8737 in Stirred Tank Reactor Using Molasses as Sole Production Medium

The influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation has been studied in 1.5-l stirred tank reactor for lipase production using Rhodotorula mucilaginosa MTCC 8737. Molasses, as sole production medium, is used for lipase production by varying aeration (1, 2, and 3 vvm) and agitation speeds (100, 200, and 300 rpm). Maximum lipase activity of 72 U/ml was obtained during 96 h of fermentation at 2 vvm, 200 rpm, pH 7, and 25 +/- 2 degrees C temperature. Lipase production kinetics with respect to dry cell weight of biomass showed Y (P/S) of 25.71 U/mg, specific product formation of 10.9 U/mg DC, and Y (X/S) 2.35 mg/mg. Maximum lipase activity (MC 2) of 56 U/ml was observed at 1% molasses, and a further increase in the molasses concentration of (%) 1.5 and 2 inhibited the product formation of lipase with 15 and 8.5 U/ml, respectively. The production kinetics of molasses media showed Y (P/X) was 14 U/mg DC, Y (P/S) 16 U/mg, and Y (X/S) 1.14 mg/mg during 96 h of bioreactor operation. The k(L)a values for all batches (MC 1-MC 4) at 96 h of fermentation were 32, 28, 21, and 19/h, and the |oxygen transfer rate were 54.4, 56, 35.7, and 17.29 mg/l h, respectively. Increase in molasses concentration resulted in decreased lipase activity by increase in viscosity of the fermentation broth.

Effects of ultrasound on culture of Aspergillus terreus

AbstractBACKGROUND: Fermentations of Aspergillus terreus are commercially used to produce lovastatin. How ultrasound might influence this fermentation is unknown. While high‐intensity ultrasound is effective in disrupting microbial cells, ultrasound of low intensity is known to improve productivity of some fermentation processes without damaging cells. Mechanisms behind productivity improvements have not been clearly identified in earlier studies. This work reports on the effects of ultrasound on A. terreus fermentation for low (957 W m−3), medium (2870 W m−3) and high (4783 W m−3) values of sonication power input in a slurry bubble column sonobioreactor.RESULTS: Sonication at any power level did not affect biomass growth profiles in comparison with negative controls. In contrast, medium‐ and high‐intensity sonication greatly reduced production of lovastatin and substantially altered the growth morphology. At medium and high intensity, ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Sonication affected broth rheology because rheology depends on the morphology of the suspended biomass.CONCLUSION: Sonication can be used to modify growth morphology and broth rheology without affecting growth of filamentous fungi. Sonication appears to influence the primary growth metabolism and secondary metabolism differently in different situations. Copyright © 2008 Society of Chemical Industry

Effect of Various Process Parameters on Morphology, Rheology, and Polygalacturonase Production by Aspergillus sojae in a Batch Bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.

Relationship between morphology, rheology and polygalacturonase production by Aspergillus sojae ATCC 20235 in submerged cultures

A full factorial statistical design, with the factors of, two taxonomically different strains, seven types of seed culture formulations (slants) and two types of fermentation media were used to investigate the effect of these parameters on the morphology and polygalacturonase production. The rheology of the final fermentation medium was analyzed and appropriate mathematical model was applied to calculate suspension viscosity. It was found that most fermentation broths showed non-Newtonian flow behavior. According to statistical analysis, factors of strain types and fermentation media and the interaction between them were found significant on the enzyme activity. The effect of seed culture formulations (slants) were found insignificant at the significance level of 1%. Interaction of slants with strain types and fermentation media were also found insignificant. Considering the morphology of the final culture, Aspergillus sojae with the desired pellet morphology in a complex media, inoculated with a seed culture prepared from molasses resulted in maximum polygalacturonase enzyme activity (0.2 U/ml) and lowest suspension viscosity with a broth rheology close to Newtonian flow behavior.