Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Improving glucose and xylose assimilation in Azotobacter vinelandii by adaptive laboratory evolution.

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.

Analysis of respiratory activity and carbon usage of a mutant of Azotobacter vinelandii impaired in poly-ß-hydroxybutyrate synthesis.

In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-ß-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).

Response of Bacillus velezensis 83 to interaction with Colletotrichum gloeosporioides resembles a Greek phalanx-style formation: A stress resistant phenotype with antibiosis capacity.

Plant growth-promoting rhizobacteria, such as Bacillus spp., establish beneficial associations with plants and may inhibit the growth of phytopathogenic fungi. However, these bacteria are subject to multiple biotic stimuli from their competitors, causing stress and modifying their development. This work is a study of an in vitro interaction between two model microorganisms of socioeconomic relevance, using population dynamics and transcriptomic approaches. Co-cultures of Bacillus velezensis 83 with the phytopathogenic fungus Colletotrichum gloeosporioides 09 were performed to evaluate the metabolic response of the bacteria under conditions of non-nutritional limitation. The bacterial response was associated with the induction of a stress-resistant phenotype, characterized by a lower specific growth rate, but with antimicrobial production capacity. About 12% of co-cultured B. velezensis 83 coding sequences were differentially expressed, including the up-regulation of the general stress response (sigB regulon), and the down-regulation of alternative carbon sources catabolism (glucose preference). Defense strategies in B. velezensis are a determining factor in order to preserve the long-term viability of its population. Mostly, the presence of the fungus does not affect the expression of antibiosis genes, except for those corresponding to surfactin/bacillomycin D production. Indeed, the up-regulation of antibiosis genes expression is associated with bacterial growth, regardless of the presence of the fungus. This behavior in B. velezensis 83 resembles the strategy used by the classical Greek phalanx formation: by sacrificing growth rate and metabolic versatility, resources can be redistributed to defense (stress resistant phenotype) while maintaining the attack (antibiosis capacity). The presented results are the first characterization of the molecular phenotype at the transcriptome level of a biological control agent under biotic stress caused by a phytopathogen without nutrient limitation.

The acetylation degree of alginates in Azotobacter vinelandii ATCC9046 is determined by dissolved oxygen and specific growth rate: studies in glucose-limited chemostat cultivations.

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (µ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02-0.15 h⁻¹). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of µ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low µ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.

Azotobacter vinelandii lacking the Na(+)-NQR activity: a potential source for producing alginates with improved properties and at high yield.

The mutant ATCN4 strain of Azotobacter vinelandii, which lacks the Na(+)-NQR activity and results in an alginate overproduction (highly mucoid phenotype), was cultured in shake flasks in minimal and rich medium, and the chemical composition and rheological properties of the alginate were determined. Mutant ATCN4 exhibited a high efficiency for sucrose conversion to alginate and PHB accumulation, reaching yields that were 3.6- and 1.6-fold higher than those obtained from the wildtype cultures in minimal medium (Burk's sucrose, BS). The alginate produced by ATCN4 in the minimal medium had a high degree of acetylation (≥4 %) and a low G/M ratio (=2) with respect to the polymer synthesised in the rich medium (BS with yeast extract) (degree of acetylation = 0 % and G/M ratio of 4.5). The alginate produced in the minimal medium exhibited a pronounced pseudoplastic behaviour and a higher G* module in comparison to that observed in the alginate obtained in the cultures using a rich medium. The ATCN4 mutant culture in the minimal medium promoted the synthesis of a polymer of improved rheological quality in terms of its mechanical properties. These characteristics make this mutant a valuable source for producing alginates with improved or special properties.

Oxygen transfer rate during the production of alginate by Azotobacter vinelandii under oxygen-limited and non oxygen-limited conditions.

BACKGROUND: The oxygen transfer rate (OTR) and dissolved oxygen tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum oxygen transfer rate (OTRmax) in cultures where the dissolved oxygen tension (DOT) was kept constant. RESULTS: The results revealed that in the two dissolved oxygen conditions evaluated, strictly controlled by gas blending at 0.5 and 5% DOT, an increase in the agitation rate (from 300 to 700 rpm) caused a significant increase in the OTRmax (from 17 to 100 mmol L(-1) h(-1) for DOT of 5% and from 6 to 70 mmol L(-1) h(-1) for DOT of 0.5%). This increase in the OTRmax improved alginate production, as well as the specific alginate production rate (SAPR), reaching a maximal alginate concentration of 3.1 g L(-1) and a SAPR of 0.031 g(alg) g(biom)(-1) h(-1) in the cultures at OTRmax of 100 mmol L(-1) h(-1). In contrast, the mean molecular mass (MMM) of the alginate isolated from cultures developed under non-oxygen limited conditions increased by decreasing the OTRmax, reaching a maximal of 550 kDa at an OTRmax of 17 mmol L(-1) h(-1). However, in the cultures developed under oxygen limitation (0.5% DOT), the MMM of the polymer was practically the same (around 200 kDa) at 300 and 700 rpm, and this remained constant throughout the cultivation. CONCLUSIONS: Overall, our results showed that under oxygen-limited and non oxygen-limited conditions, alginate production and its molecular mass are linked to the OTRmax, independently of the DOT of the culture.

Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 26⁻² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.

Bacillus velezensis 83 increases productivity and quality of tomato (Solanum lycopersicum L.): Pre and postharvest assessment.

Bacillus spp. are well known plant growth promoting bacteria (PGPB) and biological control agents (BCA) due to their capacity to synthesize a wide variety of phytostimulant and antimicrobial compounds. B. velezensis 83 is a strain marketed in Mexico as a foliar biofungicide (Fungifree AB™) which has been used for biological control of five different genera of phytopathogenic fungi (Colletotrichum, Erysiphe, Botrytis, Sphaerotheca, Leveillula) in crops of agricultural importance such as mango, avocado, papaya, citrus, tomato, strawberry, blueberry, blackberry and cucurbits, among others. In this work, the potential of plant growth promotion of B. velezensis 83 was evaluated on different phenological stages of tomato plants as well as the biocontrol efficacy of B. velezensis 83 formulations (cells and/or metabolites) against B. cinerea infection on leaves and postharvest fruits. Greenhouse grown tomato plants inoculated with a high concentration (1 × 108 CFU/plant) of B. velezensis 83 yielded 254 tons/Ha•year of which the 64% was first quality tomato (≥100 g/fruit), while the control plants produced less than 184 tons/Ha•year with only 55% of first quality tomato. Additionally, in vitro assays carried out with leaves and fruits, shown that the B. velezensis 83 cells formulation had an efficacy of control of B. cinerea infection of ∼31% on leaves and ∼89% on fruits, while the metabolites formulation had an efficacy of control of less than 10%. Therefore, it was concluded that spores (not the metabolites) are the main antagonism factor of Fungifree AB™. The high effectivity of B. cinerea control on fruits by B. velezensis 83, opens the possibility for a postharvest use of this biofungicide.

Molecular weight and viscosifying power of alginates produced by mutant strains of Azotobacter vinelandii under microaerophilic conditions.

Alginates are polysaccharides that are of interest in various industrial applications. This is due to the viscosifying properties of alginates, which depends on the weight-average molecular weight. The aim of the present study was to evaluate the changes in alginate quality, in terms of the viscosifying power and weight-average molecular weight of the polymer produced by Azotobacter vinelandii mutant strains in shake flasks under microaerophilic conditions. In cultures developed at oxygen transfer rate (OTR) values close to 5 mmol L-1 h-1, the highest viscosifying power (1.75 L g-1) and weight-average molecular weight (3112 ±â€¯150 kDa) were achieved in cultures performed with the AT9 strain. These values were higher than those obtained for the alginates produced by the parental strain ATCC 9046 grown under similar OTR conditions. In contrast, the alginate produced by the GG9 and OPAlgU + exhibited a very low weight-average molecular weight and therefore a poor viscosifying power. Our results have shown that by the cultivation of AT9 strain under microaerophilic conditions it is possible to obtain a polymer having a high weight-average molecular weight and excellent viscosifying capacity. Therefore, it could be a viable strategy for producing alginates for industrial applications.

Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion.

Bacillus velezensis 83 was isolated from mango tree phyllosphere of orchards located in El Rosario, Sinaloa, México. The assessment of this strain as BCA (biological control agent), as well as PGPB (plant growth-promoting bacteria), were demonstrated through in vivo and in vitro assays. In vivo assays showed that B. velezensis 83 was able to control anthracnose (Kent mangoes) as efficiently as chemical treatment with Captan 50 PH™ or Cupravit hidro™. The inoculation of B. velezensis 83 to the roots of maize seedlings yielded an increase of 12% in height and 45% of root biomass, as compared with uninoculated seedlings. In vitro co-culture assays showed that B. velezensis 83 promoted Arabidopsis thaliana growth (root and shoot biomass) while, under the same experimental conditions, B. velezensis FZB42 (reference strain) had a suppressive effect on plant growth. In order to characterize the isolated strain, the complete genome sequence of B. velezensis 83 is reported. Its circular genome consists of 3,997,902 bp coding to 3949 predicted genes. The assembly and annotation of this genome revealed gene clusters related with plant-bacteria interaction and sporulation, as well as ten secondary metabolites biosynthetic gene clusters implicated in the biological control of phytopathogens. Despite the high genomic identity (> 98%) between B. velezensis 83 and B. velezensis FZB42, they are phenotypically different. Indeed, in vitro production of compounds such as surfactin and bacillomycin D (biocontrol activity) and γ-PGA (biofilm component) is significantly different between both strains.

Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h-1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

The influence of circulation frequency on fungal morphology: a case study considering Kolmogorov microscale in constant specific energy dissipation rate cultures of Trichoderma harzianum.

The energy dissipation/circulation function (EDCF) is the product of the specific energy dissipation rate in the impeller swept volume (P/kD(3)) and the frequency of particle circulation (1/t(c)) through that volume. A direct relationship between mycelial fragmentation and EDCF has been reported. However, and although hyphal fragmentation is assumed to occur by hyphae-eddy interaction, Kolmogorov microscale (lambda) has not been shown to determine, at least directly, fungal morphology. In this work we studied the influence of lambda and EDCF evolution, as well as the individual effects of P/kD(3) and 1/t(c), on Trichoderma harzianum cultures in an attempt to elucidate the mechanistic interactions between parameters. T. harzianum cultures, conducted at equivalent yielding P/kD(3) conditions, were developed using two different Rushton turbines diameter sets. For the studied conditions, 1/t(c) had a greater effect over mycelial clump size and growth rate than P/kD(3). Consequently, broth viscosity, and hence Kolmogorov microscale, was a function of impeller diameter, even among cultures operated at equivalent specific energy dissipation rates. The latter could partially explain why Kolmogorv's theory has not been able to fully correlate morphological data, and highlights the importance of 1/t(c) on fungal bioprocesses. A theoretical approach to monitor lambda in large-scale bioreactors is proposed.

Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii.

Several aspects of alginate and PHB synthesis in Azotobacter vinelandii at a molecular level have been elucidated in articles published during the last ten years. It is now clear that alginate and PHB synthesis are under a very complex genetic control. Genetic modification of A. vinelandii has produced a number of very interesting mutants which have particular traits for alginate production. One of these mutants has been shown to produce the alginate with the highest mean molecular mass so far reported. Recent work has also shed light on the factors determining molecular mass distribution; the most important of these being identified as; dissolved oxygen tension and specific growth rate. The use of specific mutants has been very useful for the correct analysis and interpretation of the factors affecting polymerization. Recent scale-up/down work on alginate production has shown that oxygen limitation is crucial for producing alginate of high molecular mass, a condition which is optimized in shake flasks and which can now be reproduced in stirred fermenters. It is clear that the phenotypes of mutants grown on plates are not necessarily reproducible when the strains are tested in lab or bench scale fermenters. In the case of PHB, A. vinelandii has shown itself able to produce relatively large amounts of this polymer of high molecular weight on cheap substrates, even allowing for simple extraction processes. The development of fermentation strategies has also shown promising results in terms of improving productivity. The understanding of the regulatory mechanisms involved in the control of PHB synthesis, and of its metabolic relationships, has increased considerably, making way for new potential strategies for the further improvement of PHB production. Overall, the use of a multidisciplinary approach, integrating molecular and bioengineering aspects is a necessity for optimizing alginate and PHB production in A. vinelandii.

Living with heterogeneities in bioreactors: understanding the effects of environmental gradients on cells.

The presence of spatial gradients in fundamental culture parameters, such as dissolved gases, pH, concentration of substrates, and shear rate, among others, is an important problem that frequently occurs in large-scale bioreactors. This problem is caused by a deficient mixing that results from limitations inherent to traditional scale-up methods and practical constraints during large-scale bioreactor design and operation. When cultured in a heterogeneous environment, cells are continuously exposed to fluctuating conditions as they travel through the various zones of a bioreactor. Such fluctuations can affect cell metabolism, yields, and quality of the products of interest. In this review, the theoretical analyses that predict the existence of environmental gradients in bioreactors and their experimental confirmation are reviewed. The origins of gradients in common culture parameters and their effects on various organisms of biotechnological importance are discussed. In particular, studies based on the scale-down methodology, a convenient tool for assessing the effect of environmental heterogeneities, are surveyed.

Diffusional and transcriptional mechanisms involved in laccases production by Pleurotus ostreatus CP50.

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

Development of advanced image analysis techniques for the in situ characterization of multiphase dispersions occurring in bioreactors.

Fermentation bioprocesses typically involve two liquid phases (i.e. water and organic compounds) and one gas phase (air), together with suspended solids (i.e. biomass), which are the components to be dispersed. Characterization of multiphase dispersions is required as it determines mass transfer efficiency and bioreactor homogeneity. It is also needed for the appropriate design of contacting equipment, helping in establishing optimum operational conditions. This work describes the development of image analysis based techniques with advantages (in terms of data acquisition and processing), for the characterization of oil drops and bubble diameters in complex simulated fermentation broths. The system consists of fully digital acquisition of in situ images obtained from the inside of a mixing tank using a CCD camera synchronized with a stroboscopic light source, which are processed with a versatile commercial software. To improve the automation of particle recognition and counting, the Hough transform (HT) was used, so bubbles and oil drops were automatically detected and the processing time was reduced by 55% without losing accuracy with respect to a fully manual analysis. The system has been used for the detailed characterization of a number of operational conditions, including oil content, biomass morphology, presence of surfactants (such as proteins) and viscosity of the aqueous phase.

Characterization of Azotobacter vinelandii aggregation in submerged culture by digital image analysis.

A simple and accurate method for determining the distribution of sizes of single cells and aggregates of Azotobacter vinelandii by image analysis has been developed. A staining procedure using methylene blue helps to enhance the contrast between aggregates and background without altering aggregate size distribution. Sample dilution affected the distribution of the population and therefore should be avoided. Mixing and aeration conditions during culture play an important role in the aggregation of A. vinelandii. Cells grown under mild mixing conditions (unbaffled flasks) presented a thick slime layer and formed aggregates of up to 35 microm of average equivalent diameter. In contrast, under strong agitation conditions (baffled flasks) practically no aggregates were formed throughout cultivation. The method described can be used for the characterization of aggregation of other microbial cultures.

Reproducing shake flasks performance in stirred fermentors: production of alginates by Azotobacter vinelandii.

Keeping equal the initial power drawn (0.27 W l(-1)) in shake flasks and in a stirred fermentor did not reproduce the behaviour of alginate production by Azotobacter vinelandii. A lower mean molecular weight (1.1x10(6) Da) of the polymer was obtained in the bioreactor as compared to that obtained in shake flasks (1.9x10(6) Da). The reasons for this can reside in the fact that the evolution of the power drawn in the shake flasks could be considerably different to that observed in the stirred bioreactor. A drastic drop in the specific power drawn is expected in the shake flasks as a consequence of the increased viscosity, which caused the liquid not following the movement of the shaker. This was supported by the fact that cultures developed in the fermentor at lower initial power drawn (as low as 0.027-0.056 W l(-1)) or in a culture in which the power drawn was deliberately reduced along cultivation, produced alginates with similar molecular characteristics as that obtained in shake flasks.

Phases dispersion and oxygen transfer in a simulated fermentation broth containing castor oil and proteins.

The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the concentration of soluble protein (bovine serum albumin [BSA] and lipase, as model proteins) in a three-phase system using a simulated fermentation medium (aqueous salt solution, castor oil, and air). Small amounts of proteins (<0.02 g/L) caused an important decrease in oil drops and bubbles sizes, together with a pronounced decrease in surface tension. The extent and profiles of this decrease seem to be determined by the conformation of the protein at the interface. The kLa value increased considerably for increasing concentration (up to 0.02 g/L) of the two proteins but was very different (2-fold higher for the lipase) at the highest concentrations tested (0.5 g/L), a phenomenon that can be caused by the extent to which bubbles are trapped within oil drops.