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.     

Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro‐bioreactor system

Monoclonal antibody production in commercial scale cell culture bioprocessing requires a thorough understanding of the engineering process and components used throughout manufacturing. It is important to identify high impact components early on during the lifecycle of a biotechnology‐derived product. While cell culture media selection is of obvious importance to the health and productivity of mammalian bioreactor operations, other components such as antifoam selection can also play an important role in bioreactor cell culture. Silicone polymer‐based antifoams were known to have negative impacts on cell health, production, and downstream filtration and purification operations. High throughput screening in micro‐scale bioreactors provides an efficient strategy to identify initial operating parameters. Here, we utilized a micro‐scale parallel bioreactor system to study an IgG1 producing CHO cell line, to screen Dynamis, ProCHO5, PowerCHO2, EX‐Cell Advanced, and OptiCHO media, and 204, C, EX‐Cell, SE‐15, and Y‐30 antifoams and their impacts on IgG1 production, cell growth, aggregation, and process control. This study found ProCHO5, EX‐Cell Advanced, and PowerCHO2 media supported strong cellular growth profiles, with an IVCD of 25‐35 × 10⁶ cells‐d/mL, while maintaining specific antibody production (Q_p > 2 pg/cell‐d) for our model cell line and a monomer percentage above 94%. Antifoams C, EX‐Cell, and SE‐15 were capable of providing adequate control of foaming while antifoam 204 and Y‐30 noticeably stunted cellular growth. This work highlights the utility of high throughput micro bioreactors and the importance of identifying both positive and negative impacts of media and antifoam selection on a model IgG1 producing CHO cell line. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:262–270, 2018     

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.     

Strategies to overcome foaming and wall-growth during the cultivation of Morinda elliptica cell suspension culture in a stirred-tank bioreactor

Strategies to overcome foaming and wall-growth during the cultivation of Morinda elliptica (Rubiaceae) cell suspension cultures in a stirred-tank bioreactor are described. Of all the strategies applied, only bubble-free aeration was successful in eliminating foaming by 100%. Despite the foaming effect of around 40% in G medium strategy with 0.012% (v/v) antifoam, the maximum dry cell weight attained (19.2 g l^-1) and anthraquinone (AQ) content (4.0 mg g^-1 DW) was nearly three times higher than that achieved in cultivation using 0.025% (v/v) antifoam. For continuous cell growth, the effect of inoculum age should also be considered when anti-foam is to be added. P medium strategy, without antifoam addition, not only promoted both growth (18 g l^-1) and AQ production (9.8 mg g^-1 DW), but also resulted in lower foaming and wall-growth (below 30% level), and higher foaming reduction (30–40%). This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Influence of n-hexadecane on penicillin fermentation with Penicillium chrysogenum: The morphological effect

Summary In addition to acting as an oxygen transfer enhancer, n-hexadecane plays an important role in dominating the mycelial morphology of Penicillium chrysogenum in a submerged penicillin fermentation. The cell morphology is related to the timing of adding n-hexadecane to the culture. n-Hexadecane also has a function of suppressing foaming and thus can be employed as an antifoam agent in the fermentation.     

Different feeding strategy for the production of biosurfactant from Pseudomonas aeruginosa USM AR2 in modified bioreactor

A locally-isolated Pseudomonas aeruginosa USM AR2 possessing the ability to produce glycolipid-type biosurfactant (rhamnolipid) was used in this research to explore fermentation technology for rhamnolipid production. Rhamnolipid concentration in 2.5 L fed-batch fermentation was improved from 0.173 to 8.06 g/L by manipulating the feeding strategy and cultivation protocol. The culture was fed with petroleum diesel and complex medium. The highest rhamnolipid concentration was achieved when the culture was initially fed with both petroleum diesel and complex medium, followed by feeding of petroleum diesel only at the end of the stationary phase. Severe foaming problem was resolved by modifying and integrating a foam recycler to the bioreactor. This successfully extended the cultivation period and increased the yield of final rhamnolipid. No antifoam agent was added as this modified bioreactor allowed cultivation to proceed even under foam generation. The viscosity measurement, surface tension activity test, and drop-collapse test were performed as an indirect measure of rhamnolipid presence.     

Fouling of microfiltration membranes by broth-free antifoam agents

The fouling tendencies of seven commercial antifoam agents used with microfiltration membranes were investigated in a stirred cell. Parameters such as viscosity, oil droplet size distribution, contact angle, work of adhesion (W(a)), membrane type, operating pressure, and feed concentration were examined. The results show that a silicone-based antifoam, G832, gave a significantly lower flux (相似文献   

Effect of antifoam addition on gas-liquid mass transfer in stirred fermenters

The influence of three well-known antifoaming agents (polypropylene glycol, silicone and soybean oil) on gas-liquid mass transfer in stirred tanks is studied, both in model and in fermentation media. The effect of antifoam concentration, ionic strength, viscosity, agitation speed and gas flow rate are investigated. It is found that antifoam addition at low concentrations markedly decreases the gas-liquid volumetric mass transfer coefficient, k_La, for the three antifoam agents tested. Although the major effect is on the film coefficient k_L, some effect is also detected on the specific area, a. It is found that the influence of viscosity and antifoam addition are not cumulative: each tends to attenuate the other's effect on mass transfer. Both for silicone and for soybean oil, but not for PPG in the concentration range studied, there is an antifoam concentration above which further antifoam addition starts to improve k_La.     

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.     

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.     

油脂在抗生素发酵工业中的应用

发酵工业中使用油已有较长的历史,由于(1)油的绝对价格比淀粉质原料的高;(2)油脂代谢需要消耗大量的氧,使供氧能耗上升;(3)不同的油或同一种油,生产批次不同可能对发酵造成影响等原因。油在发酵过程中主要作为消泡剂和碳源。事实上,在发酵工业中油具有多种用途 。     

Antifoam addition to shake flask cultures of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> increases yield

Background  

Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.     

Stirred tank bioreactor with a recirculation loop for improved L-lysine production

Summary A bioreactor equipped with a recirculation loop was used to investigate the fermentation for L-lysine production. The broth was pumped from the bottom of the fermenter and fed back to the top. With the recirculation stream, antifoam was not required as the fermentation proceeded and the proposed system significantly improved the productivity for L-lysine fermentation.     

Behavior of lipids in biological wastewater treatment processes

Lipids (characterized as oils, greases, fats and long-chain fatty acids) are important organic components of wastewater. Their amount, for example, in municipal wastewater is approximately 30–40% of the total chemical oxygen demand. The concern over the behavior of lipids in biological treatment systems has led to many studies, which have evaluated their removal, but still the exact behavior of lipids in these processes is not well understood. In this review, we discuss the current knowledge of how lipids/fatty acids affect both aerobic and anaerobic processes and specific methods that have been used in an attempt to enhance their removal from wastewater. Overall, the literature shows that lipids/fatty acids are readily removed by biological treatment methods, inhibitory to microbial growth as well as the cause of foaming, growth of filamentous bacteria and floc flotation.     

Wirkung von Ölen und Fettsäuren auf Wachstum und Enzymbildung bei Thermoactinomyees vulgaris. V. Untersuchungen zur Metabolisierung des Öles

The splitting of vegetable and animal oils used as antifoam agents and the metabolization of fatty acids released were analysed during fermentation of T. vulgaris in a 30 l jar fermenter. During cultivation time of 22 hours the oil amount decreased by 60 to 70%. The metabolization of fatty acids is 50 to 90% of the starting level depending on the kind of acid. The eventual effect of the fatty acids released from antifoam oils on the protease production is discussed.     

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     

A TWO LITRE SCALE CONTINUOUS CULTURE APPARATUS FOR MICRO-ORGANISMS

SUMMARY: The design and construction of a continuous culture apparatus with a 2 1. culture vessel are described. Aeration is achieved by means of a mechanical stirrer and injected air, automatic temperature and pH control are features of the apparatus, and foaming is controlled by adding antifoam through a manually operated valve. A high degree of agitation ensures that good mixing takes place. Accurate and easily variable control of the medium flow rate is obtained by means of the Mariotte bottle principle. The apparatus can be operated continuously with freedom from contamination for periods in excess of 1000 hr.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture

The growth and metabolism of Saccharomyces cerevisiae was studied in steady-state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady-state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady-state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro-aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.     

Inhibition of fungal exopolysaccharide production by chemical antifoams

Attempts to control foam production in exopolysaccharide synthesising cultures of Acremonium persicinum in aerobic fermentation with polypropylene glycol (PPG) 2025 led to a marked reduction in yield. Similar effects were seen with polysaccharide producing cultures of Epicoccum purpurascens , but not with Aureobasidium pullulans. The effects of other chemical antifoams on polysaccharide production in A. persicinum were examined, and no inhibition was noticed with any silicone-based compound.