Lipopeptide production from Bacillus sp. GB16 using a novel oxygenation method

During lipopeptide (biosurfactant) production by Bacillus sp. GB16, conventional aeration method using added antifoam agent was unsuccessful due to the excessive formation of foam and the inhibitory effects of the antifoam chemical. A novel integrated method was developed to increase the dissolved oxygen concentration during the microbial production of biosurfactant lipopeptides. This novel method consisted of adding hydrogen peroxide to the medium, which decomposed to oxygen and water by cell's catalase and adding a vegetable oil and Ca-stearate to the medium as antifoam agents, as well as oxygen vectors. The dissolved oxygen concentration could be controlled by the automatic addition of hydrogen peroxide to the bioreactor. A significant improvement, i.e., suppression of foaming and, therefore, a three-fold extension of the cultivation time and, consequently, remarkable increase in the lipopeptide production could be achieved. This result showed that a novel aeration method was effective, especially when excessive foaming caused problems during microbial production of biosurfactant.     

Characterisation of HFBII biosurfactant production and foam fractionation with and without antifoaming agents

The effects of foaming on the production of the hydrophobin protein HFBII by fermentation have been investigated at two different scales. The foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed-batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without the antifoam Struktol J647. In the presence of antifoam, HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam, a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment, averaged over all overflowed material throughout the whole fermentation, of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L⁻¹ in the fermenter. It is also shown that uncontrolled foaming resulted in reduced concentration of biomass in the fermenter vessel, affecting total production. This study illustrates the potential of foam fractionation for efficient recovery of HFBII through simultaneous high enrichment and recovery which are greater than those reported for similar systems.     

Comparison of single- and three-stage tower loop reactors

Summary Hansenula polymorpha was cultured for long periods in 254 cm high single and three-stage countercurrent tower loop reactors 20 cm in diameter using ethanol as a substrate in the absence and presence of antifoam agents (Desmophen 3600 and/or soy oil). In the absence of antifoam agents in the three-stage column, much higher volumetric mass transfer coefficients were attained than in the corresponding single-stage column. The cell productivity in the former, however, was only slightly higher than in the single-stage column due to considerable enrichment of the cells in the foam and nonuniform cell concentration distribution in the three-stage column. In the presence of antifoam agents the three-stage column has a higher cell productivity, OTR, k_L a and a lower specific energy requirement with regard to the absorbed oxygen and/or produced cell mass than the single stage column. The reactor performance is especially high if the bubbling layer height is reduced to 20 cm. Soy oil has considerably less foam eliminating property than Desmophen. Since the soy oil is metabolized by the yeast, large amounts are needed to operate these reactors.     

Strategies for improved dCO2 removal in large-scale fed-batch cultures

Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process. A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO(2) was measured using a fiber optic based probe. dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.). Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value. A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg). Titer at day 10 of culture improved by 1.5-fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.     

Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams

Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc.     

Cultivation of Bacillus thuringiensis in an airlift reactor with wire mesh draft tubes

An aeration strategy was proposed for foam control in an airlift reactor with double wire mesh draft tubes. The airlift reactor was employed in the cultivation of Bacillus thuringiensis for thuringiensin production. The aeration strategy involved two situations. If the foam rose and touched the foam probe, the air flow rate was dropped to a low value for a certain period. However, if the DO value was already below 10% of the saturation when the air flow rate was dropped, the conventional foam control was employed. The production of thuringiensin based on the proposed strategy was up to 70% higher than that of using the conventional cultivation method with addition of antifoam agents for foam control.     

OBSERVATIONS ON FOAMING AND ITS INHIBITION IN A BACTERIAL CULTURE

SUMMARY: Inhibition of foaming in a continuous culture of bacteria has been studied. At first, foaming was inhibited by the addition of antifoam to the culture only when a foam layer was present. When the antifoam was added in this way foaming often became intense and antifoam additions had to be more and more frequent. In a preferred method the antifoam was added at regular intervals which were sufficiently short to inhibit foaming completely throughout the intervening periods. This method required less antifoam than the other. The effects of foaming in cultures, its causes, and allied problems are discussed.     

Foam control in biopesticide production from sewage sludge

Several antifoam agents were evaluated for the ability to control foam in the production of Bacillus thuringiensis-based biopesticides using sewage sludge as a raw material. Experiments were conducted in shake flasks as well as in 15 l fermentors with controlled parameters. Polypropylene glycol (PPG), the most commonly used antifoam agent in B. thuringiensis fermentation, inhibited cell growth, sporulation and decreased the entomotoxicity yield even at a concentration of 0.1% (v/v) in sewage sludge medium. About 40% reduction in entomotoxicity was observed when PPG was used at 0.3% (v/v). The impact of PPG on sporulation and toxin synthesis in tryptic soy yeast broth (TSYB) medium was also studied. The inhibitory effects were less severe in TSYB than in sludge medium. Another silicone-based antifoam agent, “Antifoam A”, showed less severe effect on growth and stendotoxin production. The problem of the inhibitory effect of chemical antifoam agents on growth and endotoxin production was minimised substantially with the use of vegetable oils such as canola, olive, and peanut oils. Canola and peanut oil stimulated both sporulation and δ-endotoxin synthesis. The stimulus effect varies with the monounsaturated fat contents of oils. Journal of Industrial Microbiology & Biotechnology (2000) 25, 86–92. Received 09 February 2000/ Accepted in revised form 06 June 2000     

Modification of physiological properties in Saccharomyces uvarum and Kluyveromyces bulgaricus grown in presence of polyoxyalkylene glycoĺ-oleic acid condensates

Summary Condensates of polyoxyalkylene glycol of diverse molecular weight esterified by oleic acid, used as antifoam agents in fermentors, were tested on Saccharomyces uvarum and Kluyveromyces bulgaricus. These compounds, used at a concentration of 0.1% (V/V) in the culture medium, stimulated the aerobic growth of the yeasts, and adding oleic acid (up to a concentration of 0.005% V/V in the medium) to the antifoam compounds further increased the final biomass.the presence of the antifoam agents during the development of yeasts increased their viability at the end of the culture and reinforced this viability for a further conservation by freezing. Antifoam agents also stimulated respiration in K. bulgaricus and to a lesser degree in S. uvarum. Flocculation of both yeasts was decreased.Over and above their physico-chemical foam — inhibiting action, polyoxyalkylene glycol compounds had a beneficial effect on the metabolism of yeasts. These compounds have a more positive action on yeasts than colza oil, another industrial antifoam agents.     

Gas holdup, power consumption, and oxygen absorption coefficient in a stirred-tank fermentor under foam control

For a laboratory stirred-tank fermentor (STF) with foaming system of 0.5M sulfite solution containing an anionic soft detergent, the performing of a foam-breaking apparatus with a rotating disk (FARD)fitted to the STF was evaluated. The gas holdup in a mechanical foam-control system (MFS), i.e., the stirred-tank fermentor with the rotating disk foambreaker, was confirmed to be larger than that in a nonfoaming system (NS), i.e., the STF with an antifoam agent added. The agitation power in the mechanical foam control system was found to be smaller compared with the agitation power in the nonfoaming system, due to the increased gas holdup. Comparison of the oxygen absorption coefficient between the mechanical foam control system and the nonfoaming system in terms of the specific power input also demonstrated the superiority of the mechanical foam control system, not only in oxygen transfer performance but also in power input economy.     

Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris

A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.     

The cyclone column culture vessel for batch and continuous,synchronous or asynchronous,culture of microorganisms

A simple inexpensive apparatus with a working volume of 10 liters of culture is described. Details of construction and procedures for operation of the cyclone column vessel are given. The vessel is self-contained, so that experimental parameters of temperature and aeration are individually controlled; homogeneous mixing and representative sampling of the culture, besides control of foam without need for antifoam, are obtained. The vessel may be used in single or multistage systems for aerobic or anaerobic cultivation of organisms in batch, chemostat, or phased cultures.     

Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms

Photosynthesis for the generation of fuels and chemicals from cyanobacteria and microalgae offers the promise of a single host organism acting both as photocatalyst and processor, performing sunlight absorption and utilization, as well as CO(2) assimilation and conversion into product. However, there is a need to develop methods for generating, sequestering, and trapping such bio-products in an efficient and cost-effective manner that is suitable for industrial scale-up and exploitation. A sealed gaseous/aqueous two-phase photobioreactor was designed and applied for the photosynthetic generation of volatile isoprene (C(5)H(8)) hydrocarbons, which operates on the principle of spontaneous diffusion of CO(2) from the gaseous headspace into the microalgal or cyanobacterial-containing aqueous phase, followed by photosynthetic CO(2) assimilation and isoprene production by the transgenic microorganisms. Volatile isoprene hydrocarbons were emitted from the aqueous phase and were sequestered into the gaseous headspace. Periodic replacement (flushing) of the isoprene (C(5)H(8)) and oxygen (O(2)) content of the gaseous headspace with CO(2) allowed for the simultaneous harvesting of the photoproducts and replenishment of the CO(2) supply in the gaseous headspace. Reduction in practice of the gaseous/aqueous two-phase photobioreactor is offered in this work with a fed-batch and a semi-continuous culturing system using Synechocystis sp. PCC 6803 heterologously expressing the Pueraria montana (kudzu) isoprene synthase (IspS) gene. Constitutive isoprene production was observed over 192 h of experimentation, coupled with cyanobacterial biomass accumulation. The diffusion-based process in gaseous/aqueous two-phase photobioreactors has the potential to be applied to other high-value photosynthetically derived volatile molecules, emanating from a variety of photosynthetic microorganisms.     

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized “cell-free” protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These “hydrofoam” reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are “cell-free” and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.     

Equilibrium oxygen concentration in fermentation fluids

Summary The equilibrium oxygen concentration (C₀) was determined in a complex fermentation media containing sucrose, lysine, molasses, corn steep liquor, antifoam agents and biomass. In simple systems, with all the components being dissolved, C₀ represents the oxygen solubility and linearly decreases with increasing solute concentrations. In complex solutions with multi-phase structure an increase in C₀ can be detected. It suggests that C₀ consists of two components — one being oxygen solubility, the other being determined by the amount of oxygen adsorbed on the interphase and bound by macromolecules. The presence of biomass leads to a decrease in C₀.     

Silicone antifoam performance enhancement by nonionic surfactants in potato medium

The ability of a silicone antifoam to retard foaming in a liquor prepared from potatoes is enhanced by the addition of ethoxylated nonionic surfactants. The enhancement is non-linear for surfactant concentration, with all 12 surfactants tested possessing a concentration at which foam heights strongly diminish, referred to as the surfactant critical antifoaming concentration (SCAFC). SCAFCs vary between surfactants, with lower values indicating better mass efficiency of antifoaming enhancement. SCAFCs decrease with degree of ethoxylation and decrease with the hydrophilic–lipophilic balance for ethoxylated nonionic surfactants. Surfactant addition produces a mixed water-surface layer containing surfactant and surface-active components in the potato medium. Surface tension reduction does not correlate well with antifoam performance enhancement. A model is proposed where surfactant adsorption promotes desorption of surface-active potato medium components from the water surface. At the SCAFC, desorption is not complete, yet the rate of bubble rupture is sufficiently enhanced to provide excellent foam control. Electronic Publication     

Foaming and media surfactant effects on the cultivation of animal cells in stirred and sparged bioreactors.

Foam formation and the subsequent cell damage/losses in the foam layer were found to be the major problems affecting cell growth and monoclonal antibody (MAb) production in stirred and sparged bioreactors for both serum-supplemented and serum-free media. Surfactants in the culture media had a profound effect on cell growth by changing both the properties of bubbles and the qualities of foam formed. Comparable cell growth and MAb production in sparged bioreactors and in stirred and surface-aerated control cultures were observed only in Pluronic F-68 containing culture media. In media devoid of Pluronic F-68, cells became more sensitive to direct bubble aeration in the presence of antifoam agent which was used to suppress foam formation. Compared with serum-supplemented medium, more severe cell damage effects were observed in serum-free medium. In addition, serum-free medium devoid of cells was partially degraded under continuous air sparging. The mechanism of this damage effect was not clear. Pluronic F-68 provided protective effect to cells but not to the medium. A theoretical model based on the surface active properties of Pluronic F-68 was proposed to account for its protective effect on cell growth. Optimum media surfactant composition in terms of maximum cell growth and minimum foam formation was proposed for stirred and sparged animal cell bioreactor.