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.     

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.     

Foam and its mitigation in fermentation systems

Key aspects of foaming and its mitigation in fermentation systems are presented. Foam properties and behavior, conditions that affect foaming, and consequences of foaming are discussed, followed by methods to detect and prevent foam, both without and with the use of antifoam, and their implications. Antifoams were catalogued according to their class (e.g., polyalkylene glycols, silicone emulsions, etc.) to facilitate recognition of antifoams possessing similar base compositions. Relatively few published studies directly comparing antifoams experimentally are available, but those reports found only partially identify clear benefits/disadvantages of any one antifoam type. Consequently, desired characteristics, trends in antifoam application, and chemical types of antifoams are evaluated on the basis of a thorough review of available literature reports describing a specific antifoam's usage. Finally, examples of specific foaming situations taken from both the literature and from actual experience in an industrial fermentation pilot plant are examined for their agreement with expected behavior.     

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.     

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.     

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.     

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.     

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.     

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.     

Surfactant pretreatment of a polysulfone ultrafilter for reduction of antifoam fouling

Effectiveness of surfactant precoat treatment of the polysulfone ultrafilter was first investigated for reduction of membrane fouling in ultrafiltration of antifoam. Fifteen different surfactants, including alcohols and synthetic nonionic surfactants, were tested. In general, pretreatment with nonionic surfactant gave a larger flux than that with alcohol did. The flux increase by pretreatment with nonionic surfactant depended on a hydrophile lipophile balance (HLB) value and type of hydrophobic tail. The most effective surfactant for reducing antifoam fouling among the 15 surfactants was Brij-58 which has an HLB value of 16 and a straight alkyl hydrophobic chain. The ultrafiltration flux of the membrane treated with Brij-58 was almost three times larger than that of untreated membrane. The precoat treatment with Brij-58 was the most effective for reducing antifoam fouling in terms of rejection properties.Furthermore, flux was also improved by the surfactant pretreatment in ultrafiltration of model process streams, such as fermentation media, broth, and yeast suspension with or without antifoam. The surfactant Brij-58 was found to be more effective for reducing membrane fouling in ultrafiltration of model stream YG compared with ethanol or Brij-35. The mean flux increase by the pretreatment with Brij-58 was about 80% in ultrafiltration of the model stream without antifoam. When antifoam was added to the model stream, flux was almost doubled by the pretreatment with Brij-58. The effectiveness of surfactant precoat treatment for reducing membrane fouling was also confirmed in terms of rejection properties. (c) 1994 John Wiley & Sons, Inc.     

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     

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     

An investigation of the physico-chemical basis of foaming in fungal fermentations

A detailed physico-chemical analysis of two foaming fungal fermentations was carried out to identify that key groups of compounds responsible for foam formation. Fermentations were carried out on a 20-L scale in a stirred aerated tank, over 7 days, using a commercial, defined medium. The organisms investigated were Penicillium herqueii, a hyphomycete, and an unidentified Ingoldian fungus. Samples of broth and, where possible, foam were analyzed to determine which groups of compounds were concentrated into generated foams. Surface tension, bulk viscosity, and antifoam A concentration were additionally determined in broth samples. To date the cause of foaming in fermentations has been attributed to the surfactant properties of extracellular proteins. This assumption was tested and found to be incomplete as many additional groups of biochemicals were found to be enriched into the foam. The results of the investigation revealed the presence of proteins, carbohydrates, alpha-keto acids, and lipophilic biosurfactants, particularly extracellular pigments, enriched within stable foams. (c) 1994 John Wiley & Sons, Inc.     

Oxygenation of intensive cell-culture system

The abilities of various methods of oxygenation to meet the demands of high-cell-density culture were investigated using a spin filter perfusion system in a bench-top bioreactor. Oxygen demand at high cell density could not be met by sparging with air inside a spin filter (oxygen transfer values in this condition were comparable with those for surface aeration). Sparging with air outside a spin filter gave adequate oxygen transfer for the support of cell concentrations above 10⁷ ml^–1 in fully aerobic conditions but the addition of antifoam to control foaming caused blockage of the spinfilter mesh. Bubble-free aeration through immersed silicone tubing with pure oxygen gave similar oxygen transfer rates to that of sparging with air but without the problems of bubble damage and fouling of the spin filter. A supra-optimal level of dissolved oxygen (478% air saturation) inhibited cell growth. However, cells could recover from this stress and reach high density after reduction of the dissolved oxygen level to 50% air saturation.     

Processing and transport of environmental virus samples

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Processing and transport of environmental virus samples.

Poliovirus-seeded tap water, conditioned with MgCl2 and passed through virus-adsorbing filters, gave better poliovirus recovery than water identically treated but conditioned with AlCl3. Elution of several filter types with beef extract yielded higher recoveries than did elution with glycine. Seeded samples filtered through various filters and stored showed considerable virus loss in 2 days when stored at 4 degrees C, whereas those stored at -70 degrees C gave stable virus recovery up to 4 days. Additionally, the use of antifoam during the elution process reduced foaming and increased virus recovery by 28%.     

Apparatus for the Maintenance of Bacterial Cultures in the Steady State : II. Improved turbidity control and culture cell

(1) A more convenient method for maintaining constant turbidity in the culture cell is described. (2) An improved culture cell and cell wipers which do not cause foaming are described.     

The application of foaming for the recovery of Surfactin from B. subtilis ATCC 21332 cultures

Foaming, a proficient method for the recovery of surface active solutes from dilute solutions, was successfully applied for the concentration of the lipopeptide biosurfactant Surfactin from B. subtilis ATCC 21332 cell culture broths. Foaming was only partially successful in concentrating Surfactin when applied as a separate semi-batch unit downstream of the cell culture stage. Surfactin partitioned strongly into the foam during the latter stages of the semi-batch process, where enrichments of over 50 could be obtained. However, simultaneous high enrichments and recoveries of Surfactin could not be obtained as the majority of Surfactin (around 70% of the total recovered) was produced at a low concentration during the early stages of foaming. Foam fractionation was considered for both cell free and cell containing broths; the presence of cells increased the foamability of the solution and therefore yielded more dilute Surfactin preparations. More favourable recovery and enrichment of Surfactin occurred when foaming was integrated with the cell culture stage. The use of low stirrer speeds was essential in producing foam at a controlled rate. By collecting fractions of the foam produced between 10 and 30 hours, from systems stirred at 166 and 146 rpm, a highly concentrated Surfactin extract could be obtained. The Surfactin concentration in the foam was 1.22 and 1.67 g l(-1) respectively, which represented enrichments and percent recoveries of over 60. This study points to the utility of foaming as a method for the recovery of surface-active fermentation products, particularly when used in an integrated production/recovery system.     

Fish toxicity and surface tension of non-ionic surfactants: investigations of antifoam agents

Fish toxicity of etho-nonylphenol and of two non-ionic antifoam agents was tested using rainbow trout, Sulmo guirdneri , in 24 h tests. The surface tension of these three compounds was found to depend on concentration and ageing time of the test solutions. The toxicity of the surfactant was about twice as high as known values, with a LC⁵⁰ of 8.5 mg/1 at 15°C. The addition of antifoam agents to the surfactant solutions further lowered the surface tension but did not alter substantially the fish toxicity of the surfactant. Contrary to expectation, the fish survived in concentrated antifoam solutions at surface tensions as low as 31 dyn/cm for more than 24 h without any apparent damage. The relevance of the surface tension regarding the fish toxicity of detergents in general is critically discussed.