21025634

Biotechnol. Prog.
2007, 23, 767784
767
REVIEW
Foam and Its Mitigation in Fermentation Systems
Beth Junker*
Fermentation Development and Operations, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
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 antifoams 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.
Contents
Introduction Foam and Impact of Antifoams Properties and Behavior of Foam Conditions That Affect Foaming Consequences of Foam Detection of Foam Prevention of Foam without Adding Antifoam Impact of Antifoams Chemical Antifoams (Defoamers) Desired Characteristics General Trends in Application Types of Antifoams Specific Foaming Situations Literature Reports Qualitative Pilot-Scale Foam Observations Summary 767 767 767 768 770 771 771 772 773 773 775 776 780 780 780 781
a little about foams but few bother to know very much (Gaden and Kevorkian, 1956). Today, a similar lack of collective and systematic knowledge exists. Consequently, the purpose of this review article is to summarize past and current information concerning foam formation and its mitigation in fermentation applications.
Foam and Impact of Antifoams

Properties and Behavior of Foam. Foam is the dispersion of a gas in a continuous liquid phase, and thus foam dispersions possess bulk densities closer to that of a gas rather than a liquid (Vardar-Sukan, 1992). Foam is distinctly different from traditional gas holdup for which the gas-to-liquid volume ratio is smaller and bubbles are more spherical (Prins and vant Riet, 1987). Foam bubbles are located on top of rather than within the broth, as is the case for gas holdup (vant Riet and Tramper, 1991). A general definition of foam, applicable to bioreactors, determines foam to occur when gas holdup in a gas-liquid dispersion is greater than 90% (Schubert et al., 1993). Other authors have quantified the gas content of foam to be in the range of 60-90% (vant Riet and Tramper, 1991), with a gasto-liquid volume ratio >1and usually >3 (Prins and vant Riet, 1987). Foam generation is autocatalytic in some cases, exhibiting a balance between the forces creating and destroying it (VardarSukan, 1992). Foam layers are dynamic in nature, remaining the same size when the number of bubbles bursting equals the number of bubbles arriving in the foam layer (Lee et al., 1993). Two main types of foam are common in fermentations: (1) Froths, sea, or bubble bath foams are short-lived, transitory, and unstable, containing a wide range of bubble sizes. Bubbles are polyhedral and form a honeycomb structure (Prins and vant Riet, 1987). These foams contain significant amounts of entrained liquid and thus possess low gas-to-liquid volume
Introduction
Foaming is labeled a general nuisance in fermentation (Weng et al., 1997) because it is necessary to adequately aerate broth while keeping foam formation under control (VardarSukan, 1992). During industrial fermentations, foam control is perceived as an empirical art (Vardar-Sukan, 1992). Very few submerged fermentation processes are performed without the use of either natural or synthetic antifoam agents (Viesturs et al., 1982). Over 50 years ago, it was stated that everyone knows
* To whom correspondence should be addressed. Tel: (732) 594-7010. Fax: (732) 594-7698. Email: beth_junker@merck.com.
10.1021/bp070032r CCC: $37.00
2007 American Chemical Society and American Institute of Chemical Engineers Published on Web 06/13/2007
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Biotechnol. Prog., 2007, Vol. 23, No. 4
ratios. (2) Stable foams, resembling beer head, are uniform, long-lasting, and rigid. Bubbles are spherical or slightly ellipsoidal with diameters <2-3 mm (Lee et al., 1993). These foams are low in liquid content and thus have high gas-to-liquid volume ratios (Gaden and Kevorkian, 1956). Foam is stable if gas bubbles remain separated by thin liquid walls and do not coalesce (Gaden and Kevorkian, 1956). Drainage, the runoff of liquid between bubbles in foam, is dependent on the liquid viscosity and density (Gaden and Kevorkian, 1956). Bioprocess foams tend to be non-coalescing when the presence of surface-active agents stabilizes foams as they form (Vardar-Sukan, 1992); in other cases foams are unstable or metastable (Berovic, 1992). Foam stability is determined by the number of lamellae and the angles between them; foam is stable if three lamellae are present at an angle of 120 degrees (Ghildyal et al., 1988). Foam stability also is favored if the surface tension of the gas-liquid system is less that of the pure solvent of solution (i.e., the liquid phase) (Ghildyal et al., 1988). The surface tension of pure water is approximately 72 dyn/cm (Hall, 1971), whereas the surface tensions of most fermentation media range between 60 and 65 dyn/cm (Viesturs et al., 1982). The surface tension of water (or media) is further lowered by natural surfactants (such as proteins, lipoproteins, polypeptides, and fatty acids) to about 45-50 dyn/ cm; addition of surfactants can lower surface tensions substantially to values as low as 28 dyn/cm (Evans and Hall, 1971; Hall, 1971). Foams also can be stabilized by these surfactants (Jenkins et al., 1993). Conditions That Affect Foaming. Some amount of foaming during fermentation is acceptable, but excessive foaming requires some type of control action (van der Pol et al., 1983; Prins and Vant Riet, 1987). Conditions that affect the degree of foaming during fermentation include gas introduction (i.e., aeration), medium composition, cell growth, metabolite formation, surface-active substance formation, and indirectly, vessel geometry (Taticek et al., 1991; Vardar-Sukan, 1992). Knowledge of these conditions permits optimization of fermentation processes to minimize factors that may cause foam such as oxygen starvation and nitrogen limitation (Nobel et al., 1994b). Neural networks have been used to predict foam behavior and recommend specific action (i.e., add/not add antifoam, adjust process conditions) (Brown et al., 2001). Quantification. Foaminess is a characteristic of the solution having units of time, related to the mean residence time of gas in foam (Lee et al., 1993). Foaminess is defined as the equilibrium volume of foam/volumetric flowrate of gas or the height of foam/superficial velocity for cylindrical tanks (Bumbullis et al., 1979; Lee et al., 1993). Quantification of foam volume and sometimes even foam height is difficult at the large scale (Bumbullis et al., 1979). Foaminess is a measure of foaming capacity, independent of equipment geometry and measurement techniques but dependent on media ingredients, their relative concentrations, and various physical factors (Ghildyal et al., 1988). Raising gas flowrates to increase foaming for solutions with low foaminess is hampered by difficulty detecting the foam/liquid interface when high air flowrates are present (Edwards et al., 1982). Other relevant measures to characterize foaming include the liquid volume held in the foam (foam volume/liquid volume before foaming), volumetric foam overflow rate, and foam volume decrease over time (Wongsamuth and Doran, 1994; Abdullah et al., 2000). Fermentation Phase. The formation of foam differs depending on the fermentation phase. The two different foam phases are (1) early in the fermentation, for example, owing to proteins
present in complex media or when protein precipitated during sterilization is utilized, and specifically caused by some property of the fermentation medium, or (2) later in the fermentation, for example, owing to protein release from cell autolysis or some other property of the culture itself (Hastings, 1954; Schugerl, 1985; Stanbury and Whitaker, 1984; Konig et al., 1979; Su, 1995). This behavior parallels that found with activated sludge foam, namely, a white, frothy foam during start-up (first 3-4 days), followed by a sticky viscous foam later on when the culture becomes nutrient-limited (Jenkins et al., 1993). It also parallels the difference between frothy foam in wine fermentations owing to high rates of CO2 evolution and stable, strong foam in beer (Edwards et al., 1982). Foaming later in the fermentation process has been found easier to control using antifoams (Hastings, 1954). There are five distinctive patterns of foam behavior during fermentation (Stanbury and Whitaker, 1984 from Hall et al., 1973): the foam (1) remains at a constant level throughout the fermentation, (2) undergoes a steady fall during the initial fermentation phase and then remains constant, (3) falls slightly during the initial phase and then rises, (4) demonstrates low initial foaming and then rises, or (5) exhibits a combination of two or more of the above behaviors. The surface-active agents (e.g., proteins) that cause foam emerge from media components, extracellular enzymes, or cell lysis (vant Riet and Tramper, 1991). The presence of proteins causes foaminess at concentrations from 1 mg/L up to 1-10 g/L, after which further concentration increases decrease foaminess since proteins coagulate (vant Riet and Tramper, 1991). For example, for Penicillium cultivations in the absence of antifoam, foaminess has been found to increase, pass through a maximum, and then decrease (Konig et al., 1979). Medium Composition and the Presence of Cells, Particles, and Surfactants. Many aspects of the initial medium composition affect foam formation, including concentrations of salts, proteins, and sugars, as well as the presence of alcohols (VardarSukan, 1992). For example, for soybean-based media formulations, the acid-hydrolyzed product generated more foam, owing to its higher oil content, than the microbe-hydrolyzed product (Hall, 1971). Increases in glucose up to 24 wt % in medium containing 1-3% soybean meal increased foam stability, possibly by raising medium viscosity (Hall, 1971). Thus, the selection of medium components, including their method of manufacture and their concentration, can impact foaming. In fact, the protein content of growth medium has been cited as the most vulnerable factor for foaming (Vidyarthi et al., 2000). Salt-forming substances generally increase foaming and surface viscosity as well as decrease surface tension (Ghildyal et al., 1988). Although solution foaminess increases with increasing salt concentration, foam stability diminishes, most likely owing to changes in aqueous protein stability due to salt concentration (Vardar-Sukan, 1992; Kotsaridu et al., 1983a; Bumbullis et al., 1979). Specifically, protein solubility increases at lower salt concentrations (salting in effect), which in turn increases foam stability. Protein solubility decreases at higher salt concentrations, raising apparent protein concentrations in the foam layer, which in turn increases foaminess (Prins and vant Riet, 1987). Interestingly, low molecular weight (300010,000 Da) proteins and glycoproteins promoted foam more than higher molecular weight ones (>50,000 Da) (Hall, 1971). Short chain alcohols (such as methanol, ethanol, or propanol) increase foaminess of protein/water solutions with a maximum effect at 1-2% v/v (vant Riet and Tramper, 1991; VardarSukan, 1992). Alcohol additives also increase effective protein

Table 1. Summary of the Influence of Fermentation Operating Conditions on Foam Formation. element gas flow rate superficial velocity sparger orifice size agitation rate general influence increases with higher flow rates increases with higher velocities increases with smaller orifices mechanism greater amount of bubbles erupting from surface greater speed of bubbles erupting from surface smaller bubbles form smaller foam cell structures which collapse more slowly gas entrainment; cell lysis owing to high shear environment comments often foam maximum exists and then decreases if vvm held constant with scale up, superficial velocity increases sintered spargers may produce high amounts of foam depending on relative broth/impeller geometry, possibly can decrease foam by providing mechanical disturbance system-dependent behavior reference
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Vardar-Sukan, 1992; Vant Riet and Tramper, 1991 Pandit, 1989
Chisti, 1993
increases with higher rates
Hoeks et al., 1997,2003; Pandit, 1989
viscosity
increases as viscosity rises above initial water-like values decreases with higher temperature
decreases film drainage which increases foam decreases viscosity which increases liquid film drainage and reduces foam proteins least soluble near pI Maillard reaction products formed from sterilizing nitrogen sources and reducing sugars together
Prins and Vant Riet, 1987
temperature
foaming can increase when broth cooled awaiting harvest foaming can increase protein denaturation foam may decrease after aeration applied for a duration since Maillard reactions partially reversible
Prins and Vant Riet, 1987; Gaden and Kevorkian, 1956
broth pH sterilization
increases when near protein isoelectric point (pI) increases with longer sterilization hold times, higher temperature, higher pre-sterilization pH
Vardar-Sukan, 1992; vant Riet and Tramper, 1991 Kotsaridu et al., 1983b; Vardar-Sukan, 1992; Schugerl, 1985
concentrations in the foam layer, causing higher foaminess but reducing stability in some instances (Bumbullis and Schugerl, 1979; Wilde et al., 2003). In addition, carbohydrates and R-keto acids enhance protein-containing foam stability, presumably by affecting gas-liquid interfacial activities (Noble et al., 1994b). Components of distillery fermentation broths, which include sugars, electrolytes, proteins, and acids, concentrate at the gasliquid interface causing elastic foam that entraps fermentation gases (Romualdo et al., 2002). Lipids, consisting of longer chain fatty acids (g12 carbons), reduce foam in beer, competing with proteins to weaken liquid films and destabilize bubbles (Wilde et al., 2003). The presence of cells, as well as secreted compounds, can influence foam formation and stability. The presence of finely divided, insoluble particles can assist in foam stabilization (Schugerl, 1985) by concentrating their presence at gas-liquid interfaces (Gaden and Kevorkian, 1956). Specifically, aqueous solutions of colloidal materials adsorb at surfaces (i.e., interfaces) and add mechanical strength to the films formed (Gaden and Kevorkian, 1956). In fermentation applications, foams can be stabilized by hydrophobic particles, such as Nocardia cell solids, since this culture has long-chain, hydrophobic mycolic acids on its cell surface (Jenkins et al., 1993). Generally, thick cell suspensions exhibit less foaming than dilute broths perhaps as a result of higher viscosity, but at very high viscosities foam can reappear (vant Riet and Tramper, 1991). In some thick mycelial fermentations, there is only a gradual increase in the gas proportion in liquid from the bottom up to the top of the liquid height, making it harder to quantify foam levels. However, the effect of cells is hard to distinguish since cells are always present together with some amount of protein (vant Riet and Tramper, 1991). Foams in fermentations are likely derived from a variety of excreted products or cell lysis products and not solely from extracellular proteins (Vardar-Sukan, 1992; Noble et al., 1994ab).
For example, stable foams are formed when the yeast Moniliella became nitrogen-limited and are stabilized by secreted polysaccharides. This foam could not be suppressed using antifoam or mechanically destroyed using foam breakers (Burschapers et al., 2002). Removal of cells from medium due to foam formation can cause autolysis, which releases microbial proteins that enhance foam stability (Stanbury and Whitaker, 1984). Microorganisms trapped in foam experience oxygen and nutrient limitations (Pandit, 1989; Vogel, 1983), causing changes in microbial metabolism, protein denaturation, and/or microbial lysis (Vardar-Sukan, 1992). Proteins and microorganisms also are concentrated by froth flotation (Vardar-Sukan, 1992). Cells caught in stable foams form crusts or meringues, often adhering to walls (i.e., forming a ring) and cause sampling nonuniformity (Abdullah et al., 2000). This problem is especially prevalent in plant (Wongsamuth and Doran, 1994) and certain fungal cultures. Many plant cells secrete polysaccharides and/ or proteins, causing cultures to become sticky, entrapping bubbles in foam that then entraps cells forming a crust (Taticek et al., 1991). The onset of substantial cell growth reduces foaming, suggesting metabolism modifies the composition of surfaceactive agents (Bungay et al., 1960); thus some metabolites directly and others indirectly act as antifoams (Soifer et al., 1974). Foaming decreases in extent and duration with greater inoculum age during production fermentations of Actinomyces streptomycini owing to increases in antifoaming metabolite concentrations (Soifer et al., 1974). In fact, the bacteria Xenorhabdus has been demonstrated to produce antifoam during its normal metabolism (Jewell and Dunphy, 1996). The creation (via recombination by crossing strains) and/or selection (via mutation) of a non-foaming strain of a commercial organism helped control foaming late in fermentation (Stanbury and Whitaker, 1984). The use of such foam-negative mutants
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(Ishizuka et al., 1989) avoids changes in fermenter operating conditions and reduces antifoam additions. Fermentation Operating Conditions. Fermentation operating conditions strongly impact on the initiation and severity of foam formation. High air flowrates, coupled with foam-stabilizing proteins and carbohydrates present in the broth, make fermentation processes prone to foaming and particularly challenging applications for antifoams (Pelton, 2002). A summary of these trends is shown in Table 1. Foam levels generally increase in height with increasing gas flow rate since more bubbles erupt from the liquid surface and then are converted into foam (vant Riet and Tramper, 1991; Vardar-Sukan, 1992). In many cases, a maximum occurs after which foam layers decrease in size as gas flowrates become higher (Konig et al., 1979). This behavior is possibly due to a decrease in bubble monodispersity since higher flowrates raise coalescence rates (vant Riet and Tramper, 1991) or an increase in mechanical disturbances when higher flowrates disengage from the broth (Prins and vant Riet, 1987). The stable foam height generally increases directly with higher gas velocities and greater liquid depth above the sparger (Pandit, 1989). Gas superficial velocities themselves increase upon scale-up if specific aeration rates (i.e., volume air per volume broth per unit time) remain constant. Consequently, superficial velocities relevant to the large scale (e.g., 600 L) should be examined during smaller scale (e.g., 20 L) bioreactor process development work (Hoeks et al., 1997). Sparger orifice designs impact foaming by affecting bubble size. For hybridoma cells cultivated in serum-containing media, porous metal spargers (180-200 m, 0.00018-0.0002 m) produce foams with bubble sizes of about 0.002-0.003 m. These foams are challenging to control because they are densely packed. In contrast, foams produced by ring spargers, with holes <0.001 m that emit bubbles of about 0.01-0.02 m, are easier to control because the larger bubbles formed large foam cell structures and most bubbles collapsed quickly (Chisti, 1993). Larger holes in sparger rings also can reduce foaming in plant cultures (Taticek et al., 1991). Agitation often increases foam by increasing air entrapment and cell lysis. As impeller speed increases, foam cell size decreases and becomes more stable, which in turn increases the rate of foam buildup (Pandit, 1989). Once foam has formed, however, increased agitation sometimes reduces foam height owing to mechanical disturbance. However, the effectiveness of this measure depends highly on impeller position relative to broth level. There is additional anecdotal evidence for animal cell cultures that downward pumping hydrofoil impellers reduce foam formation relative to other impeller designs such as Rushton impellers. Foam level and persistence decreases as temperature increases potentially as a result of decreased broth viscosity (VardarSukan, 1992; Prins and vant Riet, 1987), increased drainage of liquid films (vant Riet and Tramper, 1991; Gaden and Kevorkian, 1956), and lower gas pressure within bubbles (Gaden and Kevorkian, 1956). These observations possibly explain foaming increases when broth is cooled while awaiting harvest. Other authors found that temperature increases decrease foam stability but enhance foaminess (Ghildyal et al., 1988) since protein denaturation increases (Vardar-Sukan, 1992). Foam formation is affected by the liquid (broth) properties, specifically viscosity, surface tension, and ionic strength (Vardar-Sukan, 1992). Foam increases when viscosity rises from 1 mPa-S up to 10-100 mPa-S; beyond 10-100 mPa-S foaming decreases, but at very high viscosities foaming starts again (Prins
and vant Riet, 1987). Liquid-phase surface tension directly affects stable foam height (Pandit, 1989). The liquid film thickness forming the foam bubble boundary is proportional to 0.57 (Pandit, 1989). Ionic strength primarily affects foaming by altering protein solubility as discussed previously. Broth pH affects the action of antifoam agents (Vardar-Sukan, 1992; vant Riet and Tramper, 1991) because it affects foams produced by colloidal agents such as proteins (Gaden and Kevorkian, 1956). If the pH is near the protein pI (isoelectric point) where proteins are least soluble (Vardar-Sukan, 1992), foam generally reaches its maximum extent (vant Riet and Tramper, 1991). In one example, broth pH decreases from 6 to 4 increased foaming and foam stability about 10-fold for a medium containing 4-5% soybean meal (Hall, 1971). In a second example, foam volume increased 4-fold and foam stability increased 6-fold for shake flask plant cell cultures of Atropa when broth pH was lowered from 7 to 6 (Wongsamuth and Doran, 1994). Sterilization increases the already high foaming capacity of complex nutrient media considerably (Vardar-Sukan, 1992; Schugerl, 1985). Heat causes nitrogen sources to become hydrolyzed or partially degraded, leading to Maillard reactions between reducing sugars and amino acids/proteins/peptides; these Maillard reaction products enhance foam formation (Vardar-Sukan, 1992). Maillard reaction products increase with higher sterilization temperatures, longer sterilization times, and higher presterilization pH values (Kotsaridu et al., 1983b). Overall Maillard reaction products can increase foaminess by a factor of 2100 during sterilization of potato-protein liquor and glucose medium (Ghildyal et al., 1988). During sterilization foam-stabilizing components form in soybean flour medium based on Maillard reaction product formation (Vardar-Sukan, 1992). Since the Maillard reaction is partially reversible, a reduction of foaminess is observed when sterilized medium is aged by aeration with sterile air (Vardar-Sukan, 1992) or stored at low temperatures (Kotsaridu et al., 1983b). Other specific examples of these effects have been reported: (1) The foam stability of a medium containing 2% soybean meal, 4% glucose, and 0.5% CaCO3 increases 5-fold during a 90 min sterilization at 125 C (Hall, 1971). (2) The foaming coefficient of molasses increases 2-fold for a sterilization temperature increase from 110 to 130 C (Berovic, 1992). Consequences of Foam. Sterilization. Foam during sterilization can cause sterility problems during the subsequent cultivation (Vardar-Sukan, 1992). Foaming during sterilization splashes media (e.g., flour) particles on fermenter side walls that may be sterilized inadequately. Foam probes can be enabled during sterilization to detect foam formation. Working Volume. Foaming enhances gas holdup (VardarSukan, 1992), which expands broth volume (Berovic, 1992), often requiring reductions in the working (i.e., operating) volume of the fermenter below the maximum level to ensure sufficient headspace (Edwards et al., 1982; Brown et al., 2001). These effective liquid volume increases lead to broth (e.g., substrate, biomass) loss through air entrainment, creating several sterility as well as containment problems (Bryant, 1970; Vardar-Sukan, 1992; Berovic, 1992). Wetting of off-gas hydrophobic vent filters (Duitschaever et al., 1988) causes plugging and backpressure buildup and then potential release of pressure relief devices (Koller, 2004). Cells that become entrapped in foam via flotation (Bryant, 1970) or deposited on upper parts of the fermenter undergo autolysis, which releases surface-active agents that catalyze further foam formation (Ghildyal et al., 1988; Vardar-Sukan, 1992). Productivity also is reduced from
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foam-related depositions of substrate and/or product on nonwetted parts of the vessel and piping (Brown et al., 2001; Pandit, 1989). Mass Transfer. Mass transfer is enhanced when surface-active compounds improve bubble stability by reducing coalescence and increasing the surface area available for mass transfer (Noble et al., 1994b). However, two mechanisms exist for antifoams to reduce mass transfer: (1) creation of larger bubbles and decreased gas holdup due to antifoam-induced bubble coalescence, and (2) creation of a spread film by the defoamer on bubble surfaces, reducing oxygen diffusion rates (Pelton, 2002). In the latter mechanism, mass transfer is slowed due to antifoam accumulation at the gas-liquid interface causing an additional gas-liquid interfacial resistance and decreased gas diffusion (Vardar-Sukan, 1992). Mass transfer coefficients are relatively independent of DO (i.e., concentration driving force) without antifoam present; with antifoam present these mass transfer coefficients become linearly dependent on DO, possibly due to the oxygen diffusion coefficient through the antifoam layer becoming concentration-dependent, an effect noted for polymers (Bull and Kempe, 1971). Foam not only causes changes in air bubble size but also in composition, altering dissolved gas profiles due to heterogeneous gas dispersion (Vardar-Sukan, 1992). Foam increases bubble residence time in broth, resulting in bubbles becoming oxygendepleted (Stanbury et al., 1995) and accumulating carbon dioxide, thus delaying its removal (Berovic, 1992; Koch et al., 1995). Interestingly during early hybridoma animal cell cultivations at the 1 L scale, stable foams are observed to increase surface aeration by 90% (Ju and Armiger, 1990), but this effect is considered primarily an indirect result of reduced survival of cells present in foams. Power Input/Mixing. The presence of foam reduces apparent viscosity owing to higher gas holdup, thus decreasing power dissipation and circulation rates (Vardar-Sukan, 1992). Thus, permitting some foaming (and avoiding antifoam addition) can increase mass transfer substantially and reduce power consumption (Berovic, 1992). In one example, when medium is changed from a non-foaming to foaming system, a higher OTR is achieved at same power input (Adler and Fiechter, 1986). Instrumentation. Foam on the broth surface adversely affects level measurements, which makes continuous cultivation difficult (Berovic, 1992). The presence of small bubbles from foam also can interfere with electrode sensors (Vardar-Sukan, 1992; Pandit, 1989). Miscellaneous. In some difficult fermentations (no specific information given by authors), foam must be strictly avoided because certain broth components are rendered corrosive by high oxygen levels in foam (PharmaTec, 2004). Detection of Foam. Types of Foam Sensors. Several types of foam sensors exist to detect the presence and, in some cases, depth of foam. Conductive probes, based on conduction of electrical charge (i.e., DC voltage) between the probe and vessel by ions present in the liquid (Brown et al., 2001), are sensitive to biofilm (Hall, 1971), which causes a permanent short circuit due to the collection of material across the probes insulation (Ghildyal et al., 1988). Capacitance probes have less biofilm interference than conductive probes since the entire capacitance probe is covered with insulation (Hall, 1971). Capacitance is sensed as the foam becomes dielectric (Ghildyal et al., 1988). Both conductance and capacitance foam sensors are able to measure liquid (i.e., foam) level (Getchell, 1983; Brown et al., 2001), possessing a user-adjustable detection level rather than fixed point. Less common are admittance probes, which use
high-frequency signals with wide band measuring bridges and amplifiers able to measure foam even with a biofilm present (PharmaTec, 2004). Another alternative for foam detection is sonar level detection based on the presence of a foam layer in some cases interfering with sonar feedback. Control Strategies. Several early examples of automated foam detection and antifoam addition systems are noted in the literature (Pfeifer and Heger, 1957; Bartholomew and Koslow, 1957), forming the basis for how antifoam addition is controlled today. Since on-off foam detection only indicates that foam is present (Brown et al., 2001), it is being replaced by continuous level detection that gives the foam height and permits set point adjustment. For sensors linked to automated antifoam addition systems, small time lags are introduced to prevent over-dosing and control the stop/start operation of the pump (PharmaTec, 2004). The time lags also permit adjustment of (1) the amount of antifoam added, (2) the interval between antifoam additions to permit mixing and for the antifoam to take effect, and (3) the detection sensitivity threshold by requiring continuous detection for a designated time period to avoid additions caused by splashing (Reisman, 1988; Getchell, 1983). In one example strategy, antifoam addition begins if the foam signal persists for 5 s continuously, then alternately adds antifoam for 30 s and mixes without antifoam being added for 30 s, repeating this cycle until foam is not detected for 5 s before stopping. For early antifoam addition on/off cycles, incorporation of a time lag permitted mixing and reduced overall antifoam consumption when compared to manual addition methods (Bartholomew and Koslow, 1957; Dworschack et al., 1954). The antifoam addition tubing bore size and pump rate can be used to estimate the amount of antifoam added per shot (although this value is somewhat dependent on fermenter back pressure), or antifoam reservoirs can be weighed continuously. Prevention of Foam without Adding Antifoam. Foam prevention, without additional antifoam beyond what may be present in the initial medium composition, is preferred if it is reliably attainable with no deleterious effects on the process (Vardar-Sukan, 1992). A brief summary of available options is presented below. Physical and Mechanical Methods Including Foam Breakers. Physical methods to prevent foam such as ultrasound, thermal, or electrical treatment can adversely affect cells (Vardar-Sukan, 1992) so these are not discussed further. Mechanical methods reduce foam by subjecting it to shear stress, but these devices can have significant extra power requirements (Brown et al., 2001). In some instances, they can increase cell death despite similar biomass concentrations (Vrana and Seichert, 1988), and they may preferentially destroy larger over smaller foam bubbles. Common mechanical foam breakers use rotating elements such as discs, bladed wheels, or stirrers (Solomons, 1969; PharmaTec, 2004; Yasukawa et al., 1991; Takesono et al., 2001) or a simple bar attached to the agitator shaft above the liquid level. They often are complicated in design, have high running costs, and also are unreliable (Prins and vant Riet, 1987; Vardar-Sukan, 1992). Mechanical foam breakers are used when the process cannot tolerate chemical antifoams (Olivieri et al., 1993), as was the case for early animal cell cultivation processes (Chisti, 1992). Foam breakers, used in conjunction with antifoam addition, can reduce antifoam addition by 33-50% (system not given) (Yamashita, 1972). Agitation Rate. Although counterintuitive, stirring-as-foamdisruption (SAFD) reduces foam by causing a higher liquid velocity to be generated by an upper impeller strategically located near the broth-foam (dispersion) interface. Axial
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impellers are more effective at this task than Rushton impellers. Specifically, for the volume range where this technique is effective, the foam height is almost linearly dependent on broth mass for constant Vs and rpm (Boon et al., 2000; Hoeks et al., 1997,2003). The mechanism of SAFD is foam entrainment; the gas holdup of the broth rises sharply when the impeller knocks down foam. Thus, the aerated liquid volume relative to the upper impeller location becomes a key parameter for effectiveness (Boon et al., 2002). In this pilot plant facility, this technique is effective for a fungal Glarea fermentation, as well as more effective than P2000 antifoam addition for knocking down foam induced by addition of Tween 80 surfactant. Other Operating Conditions. Foam generally is reduced by increasing back-pressure, decreasing agitation, and/or decreasing aeration during cultivation. Reducing airflow or agitation rates to reduce foam usually adversely affects productivity (VardarSukan, 1992). Although productivity often is less affected by back-pressure, higher back-pressures may not always be attainable at production scales without limiting airflow rates. Increasing back-pressure from 10-15 to 30 psig decreases foam during production of itaconic acid by Aspergillus (Pfeifer et al., 1952). Increasing back-pressure from 1.1 to 1.5 kg/cm2 when applying air to end sterilization and until the batch is cooled to its cultivation temperature also served to reduce foam in this pilot plant facility. Impact of Antifoams. Several negative effects of antifoams are noted in the literature. In general, negative effects of antifoams decrease when they are added regularly to fermentation processes in low amounts rather than in fewer additions of higher amounts (Kovalev et al., 1982). Concentration. Foam formation can be enhanced by antifoams present in too high concentrations (Vardar-Sukan, 1992). Specifically, foam stability decreases up to a maximum surfactant concentration, and then subsequent surfactant additions actually increase foam stability (Vardar-Sukan, 1992). When too much antifoam is added, foam is destroyed, but the liquid becomes strongly coalescing (Prins and vant Riet, 1987). Mass Transfer. A tradeoff exists between minimizing foam height and maximizing mass transfer (vant Riet and van Sonsbeek, 1992). Antifoams present at low concentrations markedly decrease the volumetric mass transfer coefficient, KLa, with the major effect being on the overall gas-liquid mass transfer coefficient, KL, and some effect on the specific interfacial area, a (Morao et al., 1999; Kawase and Moo-Young, 1990). Conditions that cause bubble collapse in foam also promote coalescence of bubbles within the liquid phase, which results in larger bubbles with lower specific interfacial surface areas and thus lower mass transfer (vant Riet and van Sonsbeek, 1992). Different types and quantities of antifoams can affect dissolved gas dispersion to the culture and consequently overall respiration rate (Elander, 1989). When gas holdup and specific interfacial area are reduced suddenly by anti-foam addition, the broth DO drops quickly (Schugerl, 1988; Yagi and Yoshida, 1974; Lengyel and Nyiri, 1966) but also recovers quickly. In general, mass transfer rates can be lowered by antifoam up to 50% (Berovic and Cimerman, 1979; Solomons, 1966; Atkinson and Mavituna, 1983) or 60% (PharmaTec, 2004) or 75% (Phillips et al., 1960). For example, despite higher interfacial area owing to a 13% reduction in surface tension, 0.25% of 3% Alkaterge C in lard oil causes a 50% reduction in the gas (oxygen) absorption coefficient of filtered Penicillium broth using complex medium (Deindoerfer and Gaden, 1955). The amount of reduction varies depending on the fermentation class: for yeast fermentation, 25% decrease in mass transfer;
for penicillin fermentation, 50-70% decrease in mass transfer (Chain et al., 1966). The amount also varies depending on the antifoam class for fermentation of the yeast Torulopsis on complex medium containing sugar beet molasses: control 47; 0.1% Dow Corning Antifoam A (Dow-Corning, silicone-based) 18.8; 0.1% Hodag K-4 (Lambent, organic-based) 20.4; 0.1% lard oil (natural oil) 31.4, all in units of mmol/L-h (Phillips et al., 1960). Finally, the amount varies based on antifoam quantity present: specifically, the KLa of pure medium decreases sharply upon addition of a small amount of KM70 (silicone emulsion) antifoam (Shinetsu Kagaku); after about 200 ppm is added there is no further reduction (Takahashi and Yoshida, 1979). Similarly, mass transfer rates decrease from 0 to 0.01% antifoam with no effect between 0.01% and 0.1% for Desmophen 3600 (polyester polyol) added to protein solutions (Adler et al., 1980). A critical antifoam concentration does exist above which both mass transfer and gas holdup increase (Morao et al., 1999; Kawase and Moo-Young, 1990). Owing to greater gas holdup and lower circulation rates, foaming also can result in lower heat as well as mass transfer rates (Vardar-Sukan, 1988). When antifoam effects are measured in water or media without cells, there are steep decreases in KLa reported, most likely due to most of the antifoam accumulating around the gasliquid interface and interfering with mass transfer. In one example using 3000 L fermenters, the mass transfer decrease caused by antifoams is largest (70-85% decrease) in sulfite solutions, less (50-70% decrease) in P. chrysogenum, and even lower (25% decrease) in the yeast Torula utilis (Chain et al., 1966). These results suggest that to truly characterize the effects of antifoam on mass transfer, detrimental effects should be characterized comparing antifoam-containing and antifoam-free media containing cells (i.e., actual fermentation broth) since antifoam distribution is affected by the presence of hydrophobic cells. Power Input/Mixing. Depending on the antifoam concentration, sharp increases in power draw due to degassing or a drop in mixing power due to lowered gas entrainment is observed (Ghildyal et al., 1988). In one instance, an increased power draw of up to 20% for large fermenters is observed after addition of a natural oil antifoam (Bungay et al., 1960). Biological and Metabolic. The presence of antifoam can change the growth rate and even morphology of cells (Noble et al., 1994b; PharmaTec, 2004), also negatively affecting cell density and/or product concentration (PharmaTec, 2004). Certain antifoams are preferentially used as a carbon source by some cultures (Ghildyal et al., 1988). The broth pH profile can be altered by fatty acids released due to lipase action on oils which both changes culture metabolism and fermentation progression (Ghildyal et al., 1988; Elander, 1989). In one application, this behavior is used to cause crude pH adjustment by relying on the culture to produce fatty acids by metabolizing antifoam oils (Bungay et al., 1960). Instrumentation. In one report, antifoams are found to accumulate on the membrane surfaces of DO probes, increasing diffusion resistance (Chen and Wang, 1993). However, this study is conducted in the absence of cells; in the presence of cells, the cells themselves also are observed to cause significant resistance. There are other informal reports of antifoam accumulation when concentrations exceed 1 mL/L on the surface of filter probes, designed to permit cell-free sampling of fermentation broths. Downstream Isolation. Antifoams retain their surface-active nature during isolation steps, so their permissible levels during mid-cycle fermentation should be examined in conjunction with
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Table 2. Selected Silicone-Based Oil (S) and Emulsion (SE) Antifoams Noted in Fermentation Literaturea tradename past and/or current vendor
downstream processing requirements (Paul et al., 1981). Antifoams cause difficulty in extraction and subsequent purification owing to the formation of difficult-to-break emulsions in aqueous-solvent systems (Ghildyal et al., 1988), specifically impairing phase separation during whole broth extraction (Paul et al., 1981). Antifoams also can co-extract with the desired product (Pollard et al., 2006). Antifoams accumulate on microfiltration membranes causing membrane fouling and even destruction (PharmaTec, 2004; Liew et al., 1997). Often changing the filtration step temperature relative to the antifoams cloud point can enhance performance, however, and an inprocess assay to detect their removal is critical to efficiently developing these steps. Finally, antifoams interfere with the binding step in ion exchange separations. Because of these isolation interferences, extra separation steps, often specific in nature to the antifoam employed, are implemented to remove defoamers (Ghildyal et al., 1988).
Silicone (100%) [Poly(methylsiloxane) in Silicone Oil] Sag M-10 Dow Corning Sag 471 Union Carbide DC-A, A Dow Corning Antifoam A (concentrate) Sigma Mazu DF100, DF1005 Noveon, Mazur FD 101b Stepan Q7-2243 LVAb Dow Corning b C100F Basildon S184 Wacker Chemie Q10-335b Dow Corning TMA812 Toshiba Antaphron NM40 UK Biospumex FDA 165K Cognis Silicone Emulsions (Silicone Percentage) KM-70, M-7W-0 (37%) Shinetsu Kogaku Mazu DF 210, 210 S, 2105 (10%) Mazur/Basf/Noveon 1510 (10%) Dow Corning 1520 (30%) Dow Corning AF (30%) Dow Corning C (30%) Dow Corning DC-B (10%) Dow Corning FG-10 (10%) Dow Corning DSP (10%) Dow Corning RD (10%) Dow Corning b M30 (30%) Dow Corning Q7-2587 (30%) Dow Corning Hodag FD-62 (10%) Lambent Hodag FD-82 K (30%) Lambent A (A-5758) (30%) Sigma B (10%) Sigma, JT Baker C (30%) Sigma Chemax DF-10 (10%) Rutgers Chemax DF-30 (30%) Rutgers SE15 (10%) Sigma SE9 (10%) Wacker Chemie Sag 10 (10%) Union Carbide Sag 30 (30%) Union Carbide Silcolapse 5001 (10%) ACC/Rhodia Silcolapse 5000 (30%) ICI/Ambersil/Rhodia GE 60 (30%) GE Roth 0865 (UK%) Carlroth.de PD30 (30%)b Basildon RS-70426 (27%)b Rhodia Silicon-Based 1705-W Assaff III SO-25b Lion Rhone-Poulenc
Chemical Antifoams (Defoamers)

Antifoams and defoamers are fundamentally the same chemicals despite some differing implications noted in the literature (Pelton, 2002). Antifoams are defined as strongly surface-active substances which replace foam-forming components and lower surface tension of liquids (vant Riet and Tramper, 1991; vant Riet and van Sonsbeek, 1992). Antifoams are dispersed by stirring, and foam is destroyed by bubble coalescence, which decreases the available surface area for gas-liquid mass transfer (de Haut, 2001). Antifoams typically are added to medium or broth before foaming occurs (Ghildyal et al., 1988), and sometimes they are called foam inhibitors (Weng et al., 1997). In contrast, defoamers compete with other surface-active agents for the surface layer, but they do not support foam formation (Gaden and Kevorkian, 1956). Defoamers are self-dispersed, and foam is destroyed by surface action (de Haut, 2001). Typically, defoamers are used to knock down foam after it has formed (Ghildyal et al., 1988), and sometimes they are called foam breakers (Weng et al., 1997). For the purpose of this review, the term antifoam is used to cover both aspects. Antifoams act via four steps via antifoam droplets: (1) entering the liquid film around the foam bubble, (2) bridging the width of the liquid film, (3) dewetting by causing the liquid film to thin around where the antifoam is present, and (4) rupture of the liquid film (Garrett, 1993; Denkov et al., 1999; Jha et al., 2000; Christiano and Fey, 2003). Certain antifoams contain insoluble particles which reduce the viscosity of foam lamellae, causing bubbles to thin and drain in this fashion, and severely reducing foam stability (PharmaTec, 2004). Chemical antifoams are simple and economical (VardarSukan, 1992). Consequently, they have proliferated and an abundance of choices are commercially available, both currently and in the past. Tables 2, 3, and 4 compile examples of antifoams, many of which are cited in fermentation literature. Unfortunately, often trade names do not aid in understanding the nature of the compounds. Consequently, similar antifoams are grouped together by class within Tables 2, 3, and 4 so that available antifoams options for fermentation applications are readily accessible and assessable. Confidentiality concerns frequently preclude obtaining precise details about antifoam compositions. In a few cases, little or no information is obtainable. For those cases where an antifoam is composed of components from more than one antifoam class, it is listed with the class of its highest percentage composition. In instances where one antifoam class is dissolved in a carrier of a second antifoam class, the class of the solute is applied.
Complex Polyhydric Alcohol/Silicone Polymer Sigma
a Information obtained from company websites, including MSDSs, and handbooks (Ash and Ash, 2000a,b) in addition to publications. Information limited by proprietary disclosures. Silicone oil is a silicone polymer, also known as poly(dimethylsiloxane). Simethicone consists of 95% poly(dimethylsiloxane) (silicone oil) and 5% hydrophobic silica. Sigma Antifoam A/C emulsions are both 30% but use different emulsifiers. Sigma sells both Antifoam A concentrate (100% silicone) and emulsion (30%). Some authors do not distinguish which type was used. b Antifoams offered commercially but not cited in fermentation literature at this time.
Desired Characteristics. No single antifoam can possess all of the ideal, desirable characteristics (Vardar-Sukan, 1992), thus a compromise often is needed. Antifoam efficiency is directly proportional to its foam suppression ability and inversely proportional to the amount consumed in the fermentation to suppress foam (Vardar-Sukan, 1988). This efficiency is determined by comparing the (1) minimum volume required and (2) maximum yield of product or absence of any microbial activity (Duitschaever, 1988). Overall antifoams must have properties that permit them to function as antifoams, be used with living
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Table 3. Selected Poly(alkylene glycol) (PAG)-Based Antifoams Noted in Fermentation Literature chemical poly(propylene glycol) (linear polyether of propylene oxide), PPG tradename P2000 PPG2000 PPG 2025 PPG1025 (mw 1000) PPG-24 butyl ethera Pluriol P2000a Pluracol P2000a Pluracol P2010 Emkaphyl Pluracol E PEG Typ 300 KFO F119, F161a KFO 673 UCON LB625 (mw 1500) Mazu DF60P Mazu DF7960 Mazu DF800 S, DF8005 Ucolub N115, N-7 or N-7/1 Struktol J647, SB2121 SP1 Tetronic 901 Pluronic F68 Pluronic L 61, PL-61, PE6100 Pluriol L81, PE8100 Pluronic L122 Pleuronic Adekanol LG-109, LG-107 Mazu DF204 Struktol J650a Breox FMT30 Tego KS911 Antifoam 289, 286 VP1133 Antifoam 204 XFO-371 Sag 5693, 5698 Disfoam GD Bioquest 1110/1120a Bioquest 1395a Hodag K-4 Troy 333 SAG 4130 Clerol FBA 622 DL-2000
vendor Dow Wacker-Chemie E. Merck BDH UK BASF BASF BASF Dow BASF Carl Roth Noveon KABO/Lubrizol/Emerald Dow Mazur Mazur Mazur Brenntag Schill and Seilacher Th. Goldschmidt BASF BASF Ugine Kuhlmann BASF BASF Fluka Fermenta AB, Asahi Denka Kogyo, Adeka PPG Ouvrie Schill and Seilacher Intl. Specialty Chemicals/Cognis Degussa, Goldschmidt Sigma Wacker-Chemie Sigma Ivanhoe Union Carbide, OSI Specialties Nihon Yushi/BASF Baker Hughes Baker Hughes UKb UK UK Cognis/Henkel Nopco UK
poly(ethylene glycol) (linear polyether of ethylene oxide), PEG poly(alkylene glycol) (e.g., linear polyether of ethylene and propylene oxides; poly(propylene glycol) ether of butyl alcohol)
difunctional ethylene/propylene oxide (EO/PO) block copolymers
polyalcohol based on EO/PO block copolymer poly(alkylene glycol) (PAG)-based polyether likely PAG-based: 99% organic defoamer, 1% silicon glycol to enhance spreadability PAG-based: 20% branched simethylsiloxane/3.5% dispersed silicone dioxide/76.5% PPG2000 PPG-based polyether dispersions PAG-based with small amount of silicone PAG-based: 95% polyalkylene/5% silicone PAG derivative alkyl poly(alkylene glycol) ether blend oxyalkylated alkylphenolic resins organic oil UK
Antifoams offered commercially but not cited in fermentation literature at this time. b UK ) unknown.
cells, and not to interfere with electrodes such as pH or DO sensors (Vardar-Sukan, 1992). Speed/LongeWity. Antifoam effectiveness is measured by the rate at which foam collapses and the duration of its action both initially upon addition and over the period it is present in the broth (Byrant, 1970). Since often antifoams are added on demand, initial instantaneous action is highly desirable (VardarSukan, 1992) to achieve fast foam-breaking (Stanbury and Whitaker, 1984, from Hall et al., 1973; Vardar-Sukan, 1992) or knock down (Berovic, 1992; Solomons, 1967). Considering its projected elaspsed time in the broth, a short-acting defoamer could be tolerated for fermentations that foam for only short periods (Corbett, 1985). In general though, antifoams should be long-lasting and non-metabolizable (Stanbury and Whitaker, 1984, from Hall et al., 1973; Vardar-Sukan, 1992; Berovic, 1992), as well as readily transferable to and dispersible in fermentation broth (Stanbury and Whitaker, 1984, from Hall et al., 1973). Concentration/Cost. Antifoams should be low cost and effective at low concentrations (Vardar-Sukan, 1992; Berovic, 1992; Stanbury and Whitaker, 1984, from Hall et al., 1973). In
some cases, a balance is explored between selection of a costlier but less utilized (and usually more effective) antifoam and selection of a cheaper but more highly utilized (and usually less effective) antifoam (Corbett, 1985). Solubility. Insolubility of antifoams is preferred to promote their activity at low concentrations (Hall, 1971). Together with low solubility, antifoams also should have a low critical micelle concentration to reduce foam stability through surface interactions (Lee and Tynan, 1988). They should be insoluble in the foaming medium, specifically possessing some hydrophobic character for low solubility but also having some hydrophilic character to ensure a low interfacial tension and thus a positive spreading coefficient (Vardar-Sukan, 1992; Sie and Schugerl, 1983). Determining where the antifoam resides in the fermenter during a post-batch examination generates insight as to how it works (Phillips et al., 1960) and its solubility. Specifically, for spreading coefficients >0, air-oil-water systems are more stable than air-water systems and oil likely exists as a thin film at the air-water interface; when the spreading coefficient is <0, oils exists as discrete droplets in the bulk liquid (Weng

Table 4. Selected Antifoams, Based on Fatty Acids/Esters (FE), Polyesters (PEP), and Oils (NE) Noted in Fermentation Literature chemical alkylpolyalcoxyester polyester polyol ester and polyalcohol fatty acid ester fatty acids, paraffin oil and non-ionic emulsifier alkoxylated fatty acid esters on vegetable base polyalkoxyester (EO/PO block polymer/ester) poly(ethylene glycol)-fatty acid ester and silicone oil glycerol alkyl oleate natural oils tradename Clerol FBA Desmophen 3600, 3900 Struktol SB2023 O-30 Contraspum 210 Struktol J673 Biospumex 153 K AF emulsion Atlas G 5600 various (lard, lard burning, soybean, corn, maize, cod liver, cottonseed (Proflo), olive, sunflower, safflower, peanut, ground nut, grape seed, linseed, poppyseed, caster, palm 8% Alkaterge in grapeseed oil 3% Alkaterge in lard oil 1% PPG in lard oil octadecanol (stearyl alcohol) 1% octadecanol in ethyl alcohol 3% octadecanol in soybean oil, lard oil, or peanut oil Alkaterge C 66 Alkaterge Lubrol Wa Glanapon Nopco (Foam Master) Prochem #51 Dehysan 2111 3107a vendor Cognis Bayer AG Schill and Seilacher Sigma Zschimmer and Schwarz Schill and Seilacher Cognis Nakarai Atlas Europol-ICI various
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additives in natural oils
various
substituted oxazolidine cetyl polyoxyethylene condensate likely PAG-based, some containing fatty acid esters non-silicone UKb
a
commercial solvents commercial solvents ICI Bussetti/Jastbolaget Cognis/Henkel Lambent Henkel
Antifoams offered commercially but not cited in fermentation literature at this time. b UK ) unknown.
et al., 1997). In addition, non-metabolizable synthetic antifoams are likely to coat vessel surfaces, thus requiring use of larger amounts. Chemical Properties and Toxicity. Antifoams should have low surface and interfacial tension (Vardar-Sukan, 1992) and should be non-volatile (Vardar-Sukan, 1992; Berovic, 1992). Antifoams should be non-toxic to humans (i.e., without handling hazards) and the environment (i.e., able to be sewered in reasonable quantities), and especially non-toxic to the organism being cultured (Stanbury and Whitaker, 1984, from Hall et al., 1973; Vardar-Sukan, 1992; Berovic, 1992). They should be non explosive and non-corrosive to vessels (either during sterilization or fermentation); they should have no or low flammability (Vardar-Sukan, 1992). Mass Transfer. There should be no appreciable adverse effect on oxygen transfer by antifoams (Vardar-Sukan, 1992; Stanbury and Whitaker, 1984, from Hall et al., 1973). However, carbon dioxide transfer as well as oxygen transfer is affected by the presence of antifoams with changes in the carbon dioxide removal rate causing changes in pH (Koch et al., 1995). Downstream Isolation. There should be little or no interference with subsequent product isolation or purification (Solomons, 1967; Stanbury and Whitaker, 1984, from Hall et al., 1973). They should be non-reactive with the product and not impart any odor or color to the isolated product (Vardar-Sukan, 1992). Assay. Selection of a specific antifoam also can be strongly influenced by whether a straightforward and reliable assay is available for its detection downstream or if a suitable assay can be developed readily. Two example methods are published: a reverse phase LC method with evaporative light scattering developed for simethicone (Moore et al., 2002) and a supercritical fluid chromatography method developed for UCONLB625 (Dehaven et al., 1997). Sterilization. Antifoams should be stable during sterilization and able to be reliably steam-sterilized (Vardar-Sukan, 1992; Stanbury and Whitaker, 1984, from Hall et al., 1973). Interest-
ingly, oil-based antifoams (e.g., polyalkyleneglycols, PAG) require longer sterilization times than silicon-in-water emulsion (SE) antifoams (Solomons, 1969). The early literature describes a variety of sterilization conditions for antifoams: (1) sterilization at 160 C for 2-3 h under dry heat conditions versus 127 C for 90 min versus direct steam for 2-3 h at 127 C, all successfully applied (Bungay et al., 1960), (2) autoclaving for 90 min at 125 or 127 C (both temperatures cited, 127 C corresponded to 1.5 atm pressure) sufficient for 2 L quantities but dry sterilization at 160 C for 3 h inadequate (Paladino et al., 1954), (3) sterilization of larger than laboratory amounts routinely conducted by live steam injection at 2 atm pressure (Paladino et al., 1954). Small amounts of condensate formed during autoclaving or provided by live steam are deemed sufficient to provide the moist heat environment necessary for microorganism destruction at lower steam sterilization temperatures (Paladino et al., 1954). Sterilization of antifoams to be added to fermentations midcycle can be difficult, especially when they are undiluted, owing to low vapor pressures and despite sometimes lower spore D-values in antifoam relative to water (Junker et al., 1999). [Specifically, the D-values of Antifoam C, UCON LB625, and P2000 relative to water are 0.8, 0.154, and 0.865, respectively (Junker, 2001).] Interestingly, some antifoams are designed to be added post-sterilization (i.e., they are not stable at sterilization temperatures) which means a second antifoam can be required during batching and sterilization. In other cases, antifoams (e.g., UCON HB) can be designed to become less water soluble as temperature increases (reverse solubility), thus becoming more effective antifoams in applications such as boiler feedwater (Currie, 1953). However, these novel antifoams are only transferable to fermentation applications if they are biocompatible. General Trends in Application. The selection of the appropriate antifoam agent for a specific fermentation application must be treated with caution and understanding (Duitschaever, 1988). No single antifoam has all the properties for all foam
776
control situations, and antifoam selection mostly is accomplished by trial and error (Solomons, 1969). Often the only available method of selection is experimentation (Berovic, 1992), making empirical tests of antifoams necessary for each individual system (Schugerl, 1985). Interestingly, antifoams capable of destroying foam in one application could stabilize foam in another application (Vardar-Sukan, 1991). Only a few authors indicate trends in antifoam usage depending on the fermentation process. Oily liquids such as polyglycols are most effective in fungal fermentations, and silicone-based antifoams are most effective in bacterial fermentations (Solomons, 1969). Specifically, PPG is effective at 0.2% in a fermentation of Penicillium sp., but silicone emulsion is not; PPG is not effective in a bacterial fermentation, but silicon emulsion is effective (Solomons, 1967). Silicone defoamers generally are not satisfactory in mold fermentations because they are inactivated by molds (Ghildyal et al., 1988; Solomons, 1967). poly(dimethylsiloxane)s are most suitable for bacterial fermentations at alkaline pH and for yeast fermentations regardless of pH (Ghildyal et al., 1988). Often only 10 ppm or less of a poly(dimethylsiloxane) is needed to be effective compared with 100 to 1000 ppm for lower cost organic antifoams (Ross, 1967). Soybean and lard oil antifoams disturb sugar utilization for sodium gluconate fermentation by A. niger, but octanol in ethyl alcohol does not (Blom et al., 1952). Finally, inert nonmetabolizable antifoams are preferred for enzyme production, but metabolizable antifoams are favored for secondary metabolite production (Schugerl, 1985). There are some but comparatively few reports of antifoam use in polysaccharide fermentations or plant cell cultivations, presumably owing to the high viscosity of these broths, which reduces foaming. Antifoams used in plant cell culture are sometimes used to control formation of crusts that prevent broth circulation (Bond et al., 1987). Silicon antifoam toxicity varies according to the type of plant culture (Bond et al., 1987). Similarly, only relatively few authors directly compare antifoams for their effect on production and other fermentation parameters (Table 5). These studies are summarized with the antifoam class listed according to Tables 2, 3, and 4 to facilitate study comparisons. In some cases, antifoams of the same class have substantially different effects, suggesting that the proprietary nature of antifoam composition can be an important influence. In other cases, the antifoam class uniquely impacts performance. When such studies are undertaken, some utilize fermenters, whereas several others utilize shake flasks. Generally small amounts of most antifoam agents increase yield over no antifoam addition in shake flask experiments since surface oxygen transfer is presumed higher when no foam was present (Stefaniak et al., 1946). It can be difficult to apply shake flask results to predict bioreactor performance, however, since no sparging is present in shake flasks. One interesting study attempts unsuccessfully to relate the fatty acid composition of natural oils to its foam suppression capabililty (Table 6). Consequently, there does not appear to be known methods to predict an antifoams performance on the basis of its chemical composition. A tabulation of the antifoam class and specific antifoam type is attempted for various fermentation process types to determine if the fermentation literature suggests any further trends in antifoam selection (tabulation details not included). The most recently published similar tabulation of which this author is aware is nearly 20 years old (Ghildyal et al., 1988). Several factors make this analysis challenging. Many antifoam product lines have changed vendors more than once, adversely impacting
traceability. Often a single trade name may encompass several types of antifoam of differing chemical composition so that the product number becomes the critical differentiating factor. The experimental sections of literature reports often have insufficient information regarding the antifoam employed (e.g., concentration, vendor, trade name and product number, chemical composition), and in some cases, antifoam is not used at small scale (shake flasks or stirred tank bioreactors less than 20 L), or if it is used, its description (e.g., type, amount, and addition conditions) is not specified. In other cases, antifoam is not explicitly reported as a fermentation ingredient, although it appeared likely used from the experiments context. Since in various organizations (e.g., academic, industrial, government), typically one main antifoam is used for processes of the same nature, only one literature report from each group for the same cultivation system was included in the analysis. Antifoams used with carriers (Berovic, 1992) or antifoams used synergistically for a greater combined effect (either mixed before addition or added alternately) than when used individually (Berovic, 1992) also are noted. A sufficient number of published reports are examined to determine if any trends in class of antifoam usage exist as summarized in Table 7. Regardless of culture type, for complex media, a majority of antifoams utilized are PAG, with silicone/ silicone emulsions forming a second significant grouping. For semi-defined media, both PAG and silicone/silicone emulsions are most commonly selected, although fewer studies are conducted using this media type. For defined media, primarily PAG antifoams are utilized with far fewer selections of silicone/ silicone emulsions. By far the most reports were collected for lab-scale bioreactors, although more studies are reported for pilot and production cultivations using complex media than other media types. In addition, a sufficient number of reports are examined where possible to acquire enough data to determine if any trends in the amount of initial antifoam usage exist as summarized in Table 8 using the same set of reports used for Table 7. Initial antifoam amounts are greater for fungal and filamentous cultures grown on complex media compared with semi-defined and defined media. For yeast, single cell bacterial and animal/other cells, no such difference based on medium type is apparent. Antifoam often is added initially to prevent foaming during media preparation, transfer, and sterilization, as well as during cultivation. Since foaming would be expected to decrease at laboratory scales, initial amounts likely might be skewed by the large fraction of laboratory reports in the set (Table 7). Types of Antifoams. Silicone-Based. Silicones are poly(dialkylsiloxane)s, most commonly poly(dimethylsiloxane)s (PDMS). Their typical viscosity is 60,000 cSt, and their typical average molecular weight is 270,000 (Moore et al., 2002; Hall, 1971). They are virtually insoluble in water, possessing low volatility (Hall, 1971). Their surface tension is about 21 dyn/ cm, and their interfacial tension is about 42 dyn/cm (Hall, 1971). These properties translate into low spreading coefficients and low dispersion in foaming systems, making them less effective when used in their pure form (Hall, 1971). Consequently, silicone-based antifoams contain finely divided solids, specifically silicas, creating high surface areas and an emulsifying agent to aid component distribution (Flannigan, 1984). Some authors feel that hydrophobic, highly dispersed, solid silica particles are the main active ingredient and must be present for silicone antifoams to break foam (Ghildyal et al., 1988). Simethicones are a complex mixture of high molecular

Table 5. Effect of Antifoam Selection and Concentration on Production and Other Fermentation Parametersa process bioconversion of avermectin to 27-OH avermectin by Nocardia antifoam class PAG SE PAG PAG polyoxamine NO NO NO N/A SE S UK PAG PAG SE SE SE NO specific antifoam P2000 FD62 Pluronic L 122 Pluracol P2010 Tetronic 901 lard burning oil Proflo oil FD62 lard burning oil control FD 62 SAG 471 SAG 4130 P2000 SAG 5693 Mazu 210 S Chemax DF-10 Chemax DF-30 cod liver oil corn oil olive oil soybean oil lard oil control silicone oil groundnut oil octadecanol soybean oil lard oil 1% octanol in ethyl alcohol control P2000 none lard oil soybean oil 3% octadecanol in soybean oil none 3% octadecanol in lard oil Nopco (type N/A) corn oil P2000 FD-62 SAG-471 UCON-LB625 MAZU-DF201 A/Sigma B/Sigma C/Sigma Antifoam 204 UCON-LB625 parameter (units) 0 (titer, % control) 91 23 18 21 61 21 330 (titer, U/L) 410 100 (titer, % control) 100 127 94 102 100 94 92 102 89 22 89 80 58 56.8 (titer, g/L) 57.5 73.8 55.9 0.12 (% glucose utiliization/h) 0.04 0.86 17 (titer, mg/L) 53 90 (peak titer, mg/L) 112 98 95 97 101 121 142 -64 (titer, ( % max) +<10 -<10 +40 -15 -<10 +30 +13 +<10 47.6 (titer, mg/L) 42.7 69.1 93.8 0 (titer, ( % max) -49 -87 -95 100 (cell mass, g/L) 119 121 108 0.004 (titer, U/L) 0.0019 0.00224 0.00048 10,374 (titer, IU/L) 11,690 12,348 13,020 11,946 11,886 amount 1 g/L SF scale reference Chartrain et al., 1990
777
5 mL/L 15 g/L 5 mL/L 0 mL/L 5 mL/L
SF 14 L BR SF Marcin et al., 1993
lipase production by Pseudomonas aeruginosa
1 mL/L
citric acid production by Aspergillus sodium gluconate by Aspergillus anti-parasitic production byStreptomyces anti-infective production by Penicillium
N/A S NO ALC NO NO ALC N/A PAG N/A NO NO ALC N/A ALC UK NO PAG SE S PAG SE SE SE SE PAG-based dispersion PAG
no concn given 0.4 mL/L 0.15 mL/L 15 mL/L 0 mL/L 2.5 mL/L 0 1 mL/L
1 L BR
Kamal et al., 1999
1100 L BR
Blom et al., 1952
SF SF
Burg et al., 1979 Stefaniak et al., 1946
0 1 mL/L
antifungal production by Micromonospora
1 mL/L
SF
Sigmund and Hirsch, 1998
PAG
P2000
bakers yeast (cell mass) recombinant protein production by E. coli biopesticide production by Bacillus
SE PAG PAG NO PAG S PAG/S SE PAG SE NO
not given
0 mL/L 1 mL/L 3 mL/L 4 mL/L 0 mL/L 0.5 mL/L 1.5 mL/L 2.0 mL/L 0.1 g/L
SF
SF
not given
Yasukochi, 1993
PPG 2000 S184 VP1133 SE9 PPG A canola oil peanut oil olive oil soybean oil
1 g/L 2 g/L 1 g/L 5 g/L AAN
2 L BR
Koch et al., 1995
10 L BR
Vidyarthi et al., 2000
778
Table 5. (Continued) process antibody production by hybridoma in serum- containing medium human cell line (E1A complementing) antifoam class SE w/PAG specific antifoam C plus 0.2% w/v Pluronic F-68 parameter (units) 37 (titer, mg/L) 40 45 48 amount 0.01 g/L 0.05 g/L 0.1 g/L 0.2 g/L
scale 2 L BR
reference Zhang et al., 1992
N/A control 1.0 (normalized viable cell) 0 ppm SF Meis et al., 2004 SE A 1.6 100 SE B 2.15 100 SE SE15 1.6 200 PAG P2000 1.6 1000 PAG LB-625 0.3 1000 FE O-30 0 400 plant cell culture N/A control 12 (titer, g/L) 0 SF Wongsamuth and Doran, 1994 of Atropa SE C 12 0.3 g/L PAG PPG 1025 12 PAG Pluronic PE6100 7.5 plant cell culture N/A control 4.2 (g/L dcw) 0 ppm 8 L BR Bond et al., 1987 of Catharanthus SE 1510 8.7 0.1 g/L N/A N/A 2.8 0 ppm PAG P2000 10.6 (g/L dcw) 0.1 g/L plant cell S DC-A 99.4 (relative growth) 0.1 g/L SF Wang and Staba, 1963 culture of Mentha SE DC-B 99.3 0.1 g/L SE FD-62 99.5 0.1 g/L SE GE-60 98.2 0.3 g/L UK Troy-333 99.1 0.1 g/L NO safflower 109.7 0.1 g/L a 15,000 ppm of FD62 required upon scale up to 14 L to control foam (appearing upon bioconversion substrate addition) which lowered titer. Lard oil (5 mL/L in medium charge and 6 mL/L added mid-cycle) used for 500 L fermentations with aeration rate of 50 Lpm (Chartrain et al., 1990). Abbreviations: PAG, poly(alkalene glycol); S, silicone (100%); SE, silicone emulsion; NO, natural oil; ALC, alcohol; PEP, polyester polyol; FE, fatty acid or fatty acid esters including synthetic oils; OX, oxazolidine; UK, unknown (not enough information available either from vendors or from literature); SF, shake flask; BR, bioreactor. Table 6. Major Fatty Acid Composition of Natural Oilsa saturated fatty acids (%) natural oil palmitic stearic arachidic palmitoleic unsaturated fatty acids (%) oleic linoleic 52.0 50.0 15.0 7.0 54.0 42.0 linolenic 8.0 0.1 51.0 1.0 0 0.3
soybean 8.0 6.0 0.6 0.2 22.0 cottonseed 24.0 2.0 0.3 0.9 21.0 linseed 6.5 5.5 0 0.2 21.0 olive 9.5 2.0 0.3 1.6 81.0 sunflower 6.0 4.0 0.6 0.2 36.0 sesame 8.0 6.0 0.5 0.4 40.0 a Listed in decreasing order of effectiveness in suppressing foam in unsterilized medium (Vardar-Sukan, 1988).
Table 7. Summary of Antifoam Selection and Fermentation Scale for Various Culture/Media Combinations Based on Published Fermentation Studiesa culture fungal media total reports (class/scale) S SE antifoam class (% reports) PAG PEP NO FE ALC OX UK flask scale (% reports) lab pilot prod UK
complex 53/59 11 9 52 6 6 4 6 6 0 7 56 29 5 3 semi-defined 7/10 0 4 4 2 0 0 0 0 0 0 70 30 0 0 defined 12/15 8 8 60 8 8 8 0 0 0 0 80 20 0 0 filamentous bacterial complex 38/42 18 13 45 5 10 0 0 0 9 7 36 48 9 0 semi-defined 5/5 20 0 40 0 40 0 0 0 0 20 40 20 20 0 defined 9/9 0 11 89 0 0 0 0 0 0 11 78 11 0 0 yeast complex 12/14 17 17 58 0 0 8 0 0 0 7 57 36 0 0 semi-defined 10/10 20 40 30 0 0 10 0 0 0 0 70 30 0 0 defined 24/28 8 8 67 0 0 13 0 0 4 0 89 11 0 0 single cell bacterial complex 40/47 13 25 55 5 0 0 0 0 2 2 79 15 2 2 semi-defined 19/19 16 21 42 5 0 0 0 0 16 0 100 0 0 0 defined 34/40 6 3 85 0 0 0 0 0 6 2 73 18 5 2 animal and other cells complex 3/3 0 67 33 0 0 0 0 0 0 0 100 0 0 0 semi-defined 1/1 0 100 0 0 0 0 0 0 0 0 0 0 0 100 defined 3/3 0 67 33 0 0 0 0 0 0 0 33 67 0 0 a Higher number of scale reports due to some reports containing studies at multiple scales. Reports that compare multiple antifoams in Table 5 are omitted from this analysis.
weight PDMS oligomers such as dimethicon with particulate silicon dioxide added (Moore et al., 2002). Mixtures of mineral or vegetable oils directly with silicone fluids and finely divided solids prove difficult to maintain homogeneous conditions prior to use (Flannigan, 1984). Consequently, finely divided silica particles are dispersed in PDMS
oil-in-water emulsions of 10-30 vol % (Solomons, 1967), formulated to overcome problems with diluting antifoams suitably for addition to fermentations (Keill et al., 1976). The end result of this emulsification with surfactants and stabilizers is to raise hydrophilicity and improve dispersibility (Hall, 1971). Water-based silicone emulsions can be less effective in control-

Table 8. Summary of Antifoam Amount for Various Culture/Media Combinations Based on Published Fermentation Studiesa antifoam amount (% reports) culture fungal filamentous bacterial yeast single cell bacterial animal and other cells media complex semi-defined defined complex semi-defined defined complex semi-defined defined complex semi-defined defined complex semi-defined defined
a
779
total reports 54 9 13 36 5 9 11 10 26 40 19 35 3 1 2
UK 2 11 7 0 0 0 0 0 0 5 10 6 0 0 0
AANb 13 11 46 22 20 22 27 20 30 30 37 34 0 0 0
e0.01% e0.1 mL/L 9 33 8 14 0 11 28 30 15 25 10 17 33 0 100
0.01<Xe0.1% 0.1<Xe1 mL/L 31 33 39 42 60 45 45 50 35 30 38 40 67 100 0
0.1<Xe0.2% 1<Xe2 mL/L 24 12 0 8 0 11 0 0 8 5 0 0 0 0 0
0.2<Xe1.0% 2<Xe10 mL/L 29 0 0 14 20 11 0 0 8 3 5 3 0 0 0
1.0<Xe2.0% 10<Xe20 mL/L 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0
>2% >20 mL/L 0 0 0 0 0 0 0 0 4 2 0 0 0 0 0
Reports that compare multiple antifoams in Table 5 are omitted from this analysis. b Added as needed.
ling foam than pure silicone, although the emulsions can be readily added to fermentations (Keill et al., 1976). These emulsions require some form of mixing to avoid settling during prolonged stagnant periods with settling occurring faster at higher dilutions. In addition, silicone emulsions tend to demulsify and break up after prolonged sterilization (Ghildyal et al., 1988; Solomons, 1967). PDMS-organic copolymers have been created containing PDMS (MW g 2000) and organic PAG (polyoxypropylene of MW g 800 and polyoxypropylene/polyoxyethylene of MW g 1500) components (Keill et al., 1976). Polyethers. Polyethers typically are composed exclusively of ethylene or propylene oxides or random and block copolymers of ethylene and propylene oxides to achieve a greater range of surface activities (Hall, 1971). Polyalkylene glycols are polyethers of molecular weights typically in the range of 400-2500 (although the molecular weight of Pluronic F68 is 8400, which may account for its more modest effectiveness as an antifoam), with sufficient molecular weight to decrease solubility and thus control foam effectively (Dow; Lee et al., 1993). These antifoams have low solubility and reach gas-liquid interfaces by diffusion (Lee et al., 1993). Specifically at 20 C, poly(propylene glycol) solubility in water is 15.5 g/L and polypropylene/polyethylene (copolymer) solubility in water is 3.4 g/L (Pollard et al., 2006). Lower molecular weight polymers are too water-soluble for foam control (Hall, 1971). Viscosities of polyethers with molecular weights of 2000 are about 340 mPas (Bayer AG). One interesting report describes use of a mixture of PPGs of various molecular weights. Specifically, PPG1025 (BDH), PPG 2025 (BDH), and FoamMaster PPG (Henkel) are used in a ratio of 2:2:1, diluted in water about 25% v/v, and then added to a fungal Fusarium fermentation in defined medium to final concentrations of 0.4-0.7% v/v (Wiebe et al., 2001). Another report describes dispersion of larger amounts of PPG using a non-ionic surfactant, an all-in-one formulation, which solubilizes the PPG (e.g., EEA-142 Henkel-Nopco) (Lee et al., 1993). Natural Oils. Natural oils are the earliest antifoams (Hall, 1971), serving as one type of water-insoluble organic liquid (Vardar-Sukan, 1992). These oils generally are esters of glycerol with long chain saturated or unsaturated monobasic acids (triglycerides) along with some free fatty acids (Hall, 1971; Gutcho, 1973). Typical compositions of natural oils are shown
in Table 6. Lard oil, composed mainly of glycerides of oleic and stearic acids, is one frequently used natural oil antifoam (Rolinson and Lumb, 1953). Recently, vegetable oils often are selected for products with food applications since they can be made kosher and are non-animal-sourced. Oils used as antifoams tend to oxidize forming aldehydes, carboxylic acids, peroxides, and other compounds with negative effects on fermentation performance, necessitating co-addition of non-toxic antioxidants (Rakyta et al., 1980) or short expiry dates. Oils increase the bubble size of foams, making them less stable (Rols and Goma, 1991). Their low hydrophilicity limits their spreading/dispersion abilities and their high viscosity limits their antifoaming capabilities (Hall, 1971; Vardar-Sukan, 1992). The best oil source for antifoam applications is defined as the source with the lowest concentration resulting in reasonably fast foam collapse (Vardar-Sukan, 1991). However, foam suppression (preventing/inhibiting foam) as well as foam collapse (eliminating/breaking foam) must be considered (Vardar-Sukan, 1991). All natural oils suppress foam formation (Jones and Porter, 1998) to some degree, depending partly on the difference between their surface tension and that of the broth (Ross, 1967). Natural oils are energy-containing, metabolizable nutrients (Jones and Porter, 1998). Their addition to cultivations can increase product yields (Vardar-Sukan, 1992) or adversely affect cultures (Ishida and Isono, 1952), particularly for genuses (such as Mucor, Aspergillus, and Penicillium) with high lipase activities (Ishida and Isono, 1952). For example, soybean oil is more efficient as a foam breaker (short-lived application) than a foam inhibitor (long-lived application) due to its consumption by the mold under cultivation (Weng et al., 1997). The effectiveness of natural oils in suppressing foam varies with medium type and state (Vardar-Sukan, 1988). For 0.51.0% v/v natural oil added to unsterilized soybean flourcontaining medium, foam height decreases in the following order: linseed > cotton seed > sesame > olive > soybean > sunflower (Vardar-Sukan, 1992). For various natural oils added to unsterilized soybean flour-containing medium, efficiency coefficients (based on oil cost and the amount required to reduce foam to a minimum height) decrease in the following order: soybean > cotton seed > linseed > olive > sunflower > > > > sesame (Vardar-Sukan, 1992). For various natural oils added to sterilized soybean flour-containing medium, efficiency coefficients decrease in the following order: cotton seed > > olive > > soybean > sunflower > linseed > sesame (Vardar-Sukan, >
780
1992). Finally, when 0.1 or 0.2% v/v of any one of several natural oils (e.g., caster, corn, linseed, poppy seed, soybean) are added to either unsterilized or sterilized complex media (either 5% sugar beet cosette or soybean flour), the foam collapse time decreases from minutes to seconds (Vardar-Sukan, 1991). Depending on fermenter agitation and aeration rates, natural oils increase, not decrease, volumetric mass transfer coefficients up to 90% when added at 0.25% v/v to S. cereVisiae in complex medium, recovering some of the rate reduction observed for amounts <0.25% v/v relative to when no oil was present (Liu et al., 1994). Below that threshold, volumetric mass transfer coefficients are found to decrease by up to 60%, with the biggest jump between 0 and 0.005% v/v (Liu et al., 1994). In this yeast fermentation, 1% v/v olive oil controls foam as well as 0.1% v/v PPG, with the olive oil yielding a similar cell density but a much higher KLa (Liu et al., 1994). In Hansenula cultivation using defined medium, soy (soybean) oil is less effective as an antifoam than Desmophen 3600; the foam does not disappear, but rather it exhibits increased bubble sizes and becomes less stable (Voigt and Schugerl, 1981). Polyalcohols. Aliphatic alcohols, such as n-octanol, act as antifoams and are attractive to use in fundamental studies since they are comprised of a single molecular species rather than a mixture; however, they are more volatile than higher molecular weight polymers (Lee et al. 1993). Higher molecular weight alcohols such as octadecyl alcohol (also known as 1-octadecanol or stearyl alcohol) also possess antifoam properties but require a carrier since they are waxy solids. Early on, soybean oil, lard oil, or lard oil containing 1% octadecyl alcohol were successfully used with complex medium containing corn steep solids for bacterial fermentations (Dworschack et al., 1954)
Specific Foaming Situations

Literature Reports. A few specific reports describing foaming during cultivations as a function of media composition are available. Selection of medium components and their concentrations notably affects foaming. In a very early study at the 30 L bioreactor scale with several fermentation systems, 4-10-fold more antifoam was required for complex than for synthetic media, but this comparison appears clouded since different cultures were used (i.e., no report of the same culture being employed for both synthetic and complex media) (Rivett et al., 1950). Foaming during cultivations of Actinomyces in complex medium is minimized by varying media composition, specifically using the same components at different relative concentrations (Soifer et al., 1974). Overall, the effect of individual medium components on foaming requires further study (Ghildyal et al., 1988). In a model aqueous solution, soybean meal both strongly foams and serves as foam stabilizer depending on its concentration with its maximum ability to stabilize foam at 5 wt % and its foaming being lower above and below 5% (Szarka and Magyar, 1969). Glycerol also acts as a foam stabilizer (Szarka and Magyar, 1969). Furthermore, glucose concentrations from 1% to 22% stabilize foam in 1-3% solutions of soybean meal (Szarka and Magyar, 1969). Foaminess of cell-free medium is believed caused mainly by the presence of corn steep liquor (CSL) and a linear relationship between CSL concentration and foaminess exists (Burschapers et al., 2002). Additional reports are available describing foaming of media during sterilization or immediately afterward. Certain media ingredients cause foam upon sterilization, particularly complex or natural ingredients. The elimination of foam-forming medium
constituents when possible (e.g., oat flour, tomato paste, animal extracts, meals) is the most effective way to reduce foaming without adding antifoam. Autoclaving, batch, or continuous sterilization potentially causes foaming of fermentation media (Bailey and Ollis, 1977). During sterilization, an increase in sterilization hold temperature from 110 to 130 C (30 min sterilization time) of molassescontaining media causes the foaming coefficient (foam height scaled by the ratio of foam disintegration to formation time) to double (Viesturs, 1982). Foaming of potato protein liquor at pH 5.2 (Emsland-starke, GmbH, FRG; protein content 30%, ash 17%, potassium content 7%, reducing sugar 8%) increases 2000fold during sterilization at 120 C for 30 min (Schugerl, 1985; Kotsaridu et al., 1983b). For a complex medium autoclaved without sugars (20 g/L Pharmamedia, 2.5 g/L corn starch, 10 g/L ammonium sulfate, 1.3 g/L potassium monophosphate, 15 g/L salts), foaminess is low during the subsequent fermentation; when the same medium is autoclaved with 120 g/L lactose and 2.5 g/L glucose, foaminess is considerable (Koch et al., 1995). Foaminess during sterilization can be a function of sterilization temperature, hold time duration, pre-sterilization pH, and antifoam concentration. Foaminess decreases from 2000 to 60 as hold temperature decreases from 120 C to 80, and hold duration decreases from 30 to 12.5 min (Kotsaridu et al., 1983b). Foaminess decreases from 737 to 66 to 45 as pre-sterilization pH decreased from 5.2 to 4.0 to 3.0, after sterilization at 120 C for 30 min (Kotsaridu et al., 1983b). Finally, foaminess decreases from 2000 to 67 as antifoam (Desmophen 3600) concentration increases from 0 to 5% after sterilization at 120 C for 30 min (Kotsaridu et al., 1983b). Qualitative Pilot-Scale Foam Observations. Several seed and production media containing complex and in some cases particulate media components were examined during and after batch sterilization in pilot plant fermenters to qualitatively assess foaming characteristics. Batch sterilization is conducted by heating up medium using jacket steam until a temperature of 95 C is reached. At 95 C, the jacket steam is discontinued and sparger steam is applied. At 105 C, steam is applied through the other internals (such as the inoculation and medium transfer lines) until the temperature is a few degrees shy of the target hold temperature, typically 122-123 C. During the 40 min (seed vessels) or 45 min (production vessels) sterilization hold period, the sparger steam remains flowing, but steam through the other internals is discontinued. Temperature is controlled through small, manual changes in fermenter back-pressure. At the conclusion of the sterilization, vessel back-pressure is increased to 1.5 kg/cm2 to control foaming, sparger air is turned on at a low flowrate, followed by shutting off the sparger steam and applying cooling water to the vessel jacket. Once the temperature falls to below 40 C, operating conditions are slowly adjusted to preinoculation set points. In general, at the 600 L pilot scale, complex media have a tendency to foam during heat up, prior to reaching the desired sterilization hold temperature. In cases where foam is somewhat controllable [Medium 1 ) 30 g/L Stadex 60 (dextrin, A.E. Stalely, Tate and Lyle, Decatur, IL), 20 g/L Pharmamedia (cotton seed flour, Traders Protein, Memphis, TN), 2.5 g/L Amberex pH (autolyzed yeast extract, Sensient Flavors, Indianopolis, IN), 7.5 g/L sucrose, 5.5 g/L cerelose (glucose monohydrate), 2.0 mL/L P2000 (Dow, Freeport, TX)], a few inches of foam typically appears during heat up and/or when sparger steam is applied. When internals are applied a few minutes later, foam decreases and often dissipates only to
781
Table 9. Qualitative Observations of Foaming during Various Stages of Batch Media Sterilization for Medium 3 in 600-L Pilot-Scale Vesselsa medium all components heat up observations when sparger steam applied, foam rose to top within a minute; foam decreased significantly shortly after (<1 min) internals applied foam to top when sparger steam applied; minimal when applyng internals minimal foam observed sterilization temp 122-124 C (more foam) normal hold time observations foam gradually increased to top, then subsided within next 3-5 min;lowering pressure, even in small increments, results in immediate foam foam to top during first 10 min, then subsided leaving 8 headspace minimal foam cool down observations despite pressure increase to 1.5 kg/cm2 prior to introducing sparger air, significant foaming observed foam leaving 14 in. headspace foam to top, then subsided leaving 12 in. headspace no foam
all components all components all components (including 2 mL/2000) plus 20 mL/L soybean oil
a
normal 60 min normal
All sterilization hold times were 45 min and temperatures were 124-126 C unless otherwise noted. Heat up and cool down procedures were normal.
reappear, sometimes rising considerably, when internals are shut off and to foam slightly during the sterilization hold phase. Foam then increases slightly when airflow is applied or in the best situations decreases or dissipates. In less optimal cases, foaming is less controllable, rising to the vessel sightglass (top) at various points during the sterilization process. For a complex medium [Medium 2 ) 20 g/L Stadex 60, 5 g/L Pharmamedia, 3 g/L NZ-Amine A (Quest International, Hoffman Estates, IL), 2 g/L yeast extract 106 (autolyzed, BioSpringer USA, Minneapolis, MN), 5.5 g/L cerelose, 2 mL/L P2000], increasing the pre-sterilization P2000 concentration to 6 mL/L or adding 10 mL/L soybean oil prevents foam rising to the top when internals are applied. Neither of these solutions is amenable for downstream processing, however. In contrast, little effect is observed when 10 mL/L silicone oil (Dow-Corning 200, 10 cSt) is added. For another complex medium [Medium 3 ) 37 g/L Stadex 60, 5 g/L soy flour (Thumb Oilseed, Ubly, MI), 7.9 g/L Hy-case (casein hydrolysate, Sheffield, Norwich, NY), 8.5 g/L yeast extract 106, 3.2 mL/L P2000], foam is greater during cooldown for 60 min vs 45 min sterilization hold times, consistent with more extensive Maillard reactions. In contrast to higher foam expected from greater Maillard degradation at higher temperatures, foam actually is lower during the sterilization hold time when the sterilization temperature is 124-126 C versus 122-124 C, presumably due to the higher vessel back-pressure of 1.3-1.35 kgf/cm2 required to attain the higher temperature. Table 9 summarizes foaming observations during batch sterilization for Medium 3. In another complex medium at the 600 L scale [Medium 4 ) 20 g/L Stadex 60, 3 g/L NZ amine A, 2 g/L yeast extract, 5.5 g/L cerelose, 2 mL/L P2000] foam is watery/soapy in nature, with about 6 in. appearing when sparger air is applied, disappearing when internals are applied, and then re-appearing when internals are shut-off. A few inches remain during the sterilization, there is no increase when sparger air is applied to end the sterilization, and foam decreases as the batch cools, possibly because the watery/soapy foam responds well to the increased back-pressure applied during cooldown. Increasing the PAG antifoam (2, 4, 8, and 16 mL/L) does not appreciably decrease foam; in fact foaming when air is applied to the medium containing 16 mL/L is 2- to 3-fold greater. In another complex medium at the 15,000 L scale [Medium 5 ) 6.5 g/L soy peptone SL (Marcor, Carlstadt, NJ), 6.5 g/L yeast extract 106, 5 g/L Hy-case SF, 2 mL/L P2000], foaming is not problematic during sterilization. However, a stable foam appears when the medium is cooled below 40 C, which does not decrease with higher back-pressure or agitation rate but rises about 6 in. when the airflow rate is raised from 1000 to 2000
Lpm. Interestingly, about 20-30 min after inoculation, the foam drops dramatically (about 16 in.). As is characteristic of stable foams, this foam is primarily sensitive to surface-active components believed present in the inoculum.
Summary
The desired overall characteristics of antifoams are summarized and presented as a compiled, comprehensive list of properties, from which the most critical attributes must be selected for the specific process of interest. Consequently, trends in antifoam usage remain as general guidelines, based on experiences of specific practitioners for their scale and cultures of interest. Despite the plethora of available antifoams, certain antifoam classes more or less consistently are used for specific types of cultivations. Recent data from screening of alternative antifoams at the shake flask and laboratory bioreactors also is presented, illustrating the process-specific nature of antifoam selection based on the desired process quantities for optimization. By far the majority of foaming and antifoam studies are conducted at the laboratory scale with significant attention seldom devoted to finding an adequate scale down model for foam generation and prevention exploration. Some studies addressing comparisons of antifoams and selection of medium components to minimize foam are conducted in ways that mimic large-scale performance. Until further studies are available, the strongest approach remains to reformulate media to minimize or omit foaming components for processes that cannot tolerate high foaming, high contamination rates, and/or high additions of antifoams.
Notation
a DO KLa OTR OUR surface area per unit volume, 1/cm dissolved oxygen, % sat volumetric mass transfer coefficient, h-1 oxygen transfer rate, mmol/L-h oxygen uptake rate, mmol/L-h
Abbreviations AAN AF AFA ALC BR CSL DF EO FCO FE added as needed antifoam antifoam agents alcohol bioreactor corn steep liquor defoamer ethylene oxide foam control agent fatty acid or fatty acid ester
782 NO OX PAG PDMS PEP pI PO PPG S SAFD SE SF UK XFO natural oil oxazolidine poly(alkylene glycol) poly(dimethylsiloxane)s polyester polyol protein isoelectric point propylene oxide poly(propylene glycol) silicone (100%) stirring as foam disruption silicone emulsion shake flask unknown X-foam

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Biotechnol. Prog.

2007, 23, 767784

Foam and Impact of Antifoams

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

Vardar-Sukan, 1992; Vant Riet and Tramper, 1991 Pandit, 1989

increases with higher rates

Hoeks et al., 1997,2003; Pandit, 1989

Prins and Vant Riet, 1987

Prins and Vant Riet, 1987; Gaden and Kevorkian, 1956

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

Chemical Antifoams (Defoamers)

Complex Polyhydric Alcohol/Silicone Polymer Sigma

Biotechnol. Prog., 2007, Vol. 23, No. 4

difunctional ethylene/propylene oxide (EO/PO) block copolymers

Biotechnol. Prog., 2007, Vol. 23, No. 4

additives in natural oils

commercial solvents commercial solvents ICI Bussetti/Jastbolaget Cognis/Henkel Lambent Henkel

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

5 mL/L 15 g/L 5 mL/L 0 mL/L 5 mL/L

SF 14 L BR SF Marcin et al., 1993

lipase production by Pseudomonas aeruginosa

Kamal et al., 1999

Blom et al., 1952

Burg et al., 1979 Stefaniak et al., 1946

antifungal production by Micromonospora

Sigmund and Hirsch, 1998

SE PAG PAG NO PAG S PAG/S SE PAG SE NO

1 g/L 2 g/L 1 g/L 5 g/L AAN

Koch et al., 1995

Vidyarthi et al., 2000

Biotechnol. Prog., 2007, Vol. 23, No. 4

reference Zhang et al., 1992

Biotechnol. Prog., 2007, Vol. 23, No. 4

e0.01% e0.1 mL/L 9 33 8 14 0 11 28 30 15 25 10 17 33 0 100

0.01<Xe0.1% 0.1<Xe1 mL/L 31 33 39 42 60 45 45 50 35 30 38 40 67 100 0

0.1<Xe0.2% 1<Xe2 mL/L 24 12 0 8 0 11 0 0 8 5 0 0 0 0 0

0.2<Xe1.0% 2<Xe10 mL/L 29 0 0 14 20 11 0 0 8 3 5 3 0 0 0

1.0<Xe2.0% 10<Xe20 mL/L 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0

>2% >20 mL/L 0 0 0 0 0 0 0 0 4 2 0 0 0 0 0

Biotechnol. Prog., 2007, Vol. 23, No. 4

Specific Foaming Situations

Biotechnol. Prog., 2007, Vol. 23, No. 4

normal 60 min normal

Biotechnol. Prog., 2007, Vol. 23, No. 4

References and Notes

Biotechnol. Prog., 2007, Vol. 23, No. 4

Biotechnol. Prog., 2007, Vol. 23, No. 4

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