2. Review of Literature - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/9472/6/06_chapter...

REVIEW OF LITERATURE . 9

2. Review of Literature

Foam in fermentation processes

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% (Van’t Riet and Tramper, 1991). Knowledge of the

fermentation conditions permits optimization of fermentation processes to

minimize factors that may cause foam such as oxygen starvation and nitrogen

limitation (Noble et al., 1994a). Varley et al., (2004) states that foam behaviour is

characterised in terms of foam frequency, a steady state conductance, liquid

distribution through the foam phase and foam formation rates. Neural

networks have been used to predict foam behaviour and recommend specific

action i.e., add or not add antifoam and adjust process conditions (Brown et al.,

2001).

Foams and emulsions are well known to scientists and the general public

alike because of their everyday occurrence (Prud’homme and Khan, 1995;

Weaire and Hutzler, 2000). Ghosh and Pirt, 1954 states that unless foaming is

suppressed it can have very adverse effects. The problems of foaming are

process specific but can be severe enough to require the installation of multiple

foam probes. More complex probes based on ultrasonic, electrical capacitance

and nuclear particle detectors have been developed, but all are somewhat


unreliable because they give spurious signals when they become fouled. Many

different designs of foam controllers are available but there is still a

requirement or foam detection and subsequent antifoam addition whatever

technique is used (Garry Montague, 1997).

Effects of fermentation conditions on foam formation

Vander Pol et al. (1993), Prins and Van’t Riet, (1987) describes that some

amount of foaming during fermentation is acceptable but excessive foaming

requires some type of control action. Vardar-Sukan, (1992) and Noble et al.

(1994a) points out that the foams in fermentations are likely derived from a

variety of excreted products or cell lysis products and not solely from

extracellular proteins. Vidyarthi et al. (2000) describes that the protein content of

growth medium has been cited as the most vulnerable factor for foaming.

Taticek et al. (1991) and Vardar-Sukan, (1992) describes that conditions

that affect the degree of foaming during fermentation include gas introduction,

medium composition, cell growth, metabolite formation, surface-active

substance formation and indirectly, vessel geometry. Fermentation operating

conditions strongly impact on the initiation and severity of foam formation.

High air flow rates, 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).


Foam generally is reduced by increasing back-pressure, decreasing

agitation and decreasing aeration during cultivation. Reducing airflow or

agitation rates to reduce foam usually adversely affects productivity (Vardar -

Sukan, 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 to 30 psig

(Pound-force per Square Inch Gauge) decreases foam during production of

itaconic acid by Aspergillus (Pfeifer et al., 1952). A little foam formation was

observed in steady state continuous culture with an air supply of 5.0 LPM in a

20 L fermentor where as of 0.5 LPM in a 3.0 L fermentor (Holme and Zacharias,

1965).

Gas flow rate: 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 (Van’t Riet and Tramper, 1991; Vardar-Sukan, 1992). 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.

Sparger orifice designs: Sparger orifice designs impact foaming by

affecting bubble size. For hybridoma cells cultivated in serum-containing

media, porous metal spargers (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: 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 build-up (Pandit, 1989).

Once foam has formed, however the 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.

Liquid properties: Foam formation is affected by the liquid 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 Van’t Riet, 1987). Liquid-phase surface tension directly affects

stable foam height (Pandit, 1989).

pH: Broth pH affects the action of antifoam agents (Vardar-Sukan, 1992;

Van’t 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 (Van’t Riet and Tramper,

1991). In one example, when broth pH decreases from 6 to 4 increased foaming

and foam stability about 10-fold for a medium containing 4-5% soybean meal

(Hall et al., 1973). 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 7.0 to 6.0 (Wongsamuth and Doran, 1994).

There should be no appreciable adverse effect on oxygen transfer by

antifoams (Vardar-Sukan, 1992; Stanbury and Whitaker, 1984, 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).

Temperatures of sterilization and during process: 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 millard

reactions between reducing sugars and amino acids, proteins and peptides

which enhance foam formation (Vardar-Sukan, 1992). Millard reaction products

increase with higher sterilization temperatures, longer sterilization times,

higher pre-sterilization and pH values (Kotsaridu et al., 1983a). Overall millard

reaction products can increase foaminess by a factor of 2100 during sterilization


of potato-protein liquor and glucose medium (Ghildyal et al., 1988).

Bailey and Ollis, (1977) describes that sterilization either in batch or in

continuous will potentially causes foaming of fermentation media. During

sterilization, foam stabilizing components form in soybean flour medium based

on millard reaction (Vardar-Sukan, 1992). Since the millard 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., 1983a). Other specific examples of these effects have been

reported, example-1: The foam stability of a medium containing 2% soybean

meal, 4% glucose and 0.5% CaCO3 increases 5-fold during a 90 minutes

sterilization at 125°C (Hall at al., 1973), example-2: The foaming coefficient of

molasses increases 2-fold for a sterilization temperature increase from 110 to

130°C (Berovic, 1992).

Foam level and persistence decreases as temperature increases

potentially (Vardar-Sukan, 1992; Prins and Van’t Riet, 1987). In some instances

foaming increases when broth is cooled while awaiting harvest. Temperature

increase results in decreased foam stability but enhance foaminess (Ghildyal et

al., 1988) since protein denaturation increases (Vardar -Sukan, 1992).


Foam controlling methods

There are different approaches and strategies related to foam controlling

in fermentations. Foam control is essential in a process as the foam adversely

affects different aspects including productivity (Ghildyal et al., 1988). Foaming

can be greatly reduced in the tubular fermentor as no air ever encountered the

impeller (Russell et al., 1974). A combined chemico-mechanical method of

implementation of foam control is the effective method (Viesturs et al., 1982). At

the beginning of the fermentation, the foam stabilization will be caused by the

proteins already present in the growing medium, where as at the end of

fermentation, by the proteins produced by the microorganisms (Hall et al.,

1973). Most fermentation media produce foam vigorously under stirring and

aeration conditions and require the addition of some type of antifoam agent or

the use of special mechanical foam breaking equipment to confine the foam to

the fermentor.

Physical methods

Physical methods of foam controlling are intended to use the ultrasound,

thermal or electrical treatment. The use of ultrasonic energy caused no

reduction in cell count (Dorsey, 1959), but later found that all physical methods

of foam controlling are of too costly and some times not suitable for

fermentations. Physical methods to prevent foam such as ultrasound, thermal

or electrical treatment can adversely affect cells (Vardar-Sukan, 1992).


Although sonic and ultrasonic energy are technically satisfactory as foam

breakers, their operating power costs are too high (Hass and Johnson, 1965).

Foam can be broken by passing it through heated grids or by irradiation with

alpha particles, but it was suggested that both the methods were not suitable for

use in fermentations (Kato and Kano, 1963).

Mechanical methods

Mechanical foam controlling methods are based on rapid pressure

change, shear force, compressive force and impact force either alone or in

combination which leads to the collapse of the bubbles (Goldberg and Rubin,

1967). The possible advantages that were achieved on one system may not be

attainable successfully on the other systems. There are controversial statements

in the literature about the mechanical foam controlling devices and methods.

The performance characteristics of tower fermentor with mechanical foam

control were determined (Takesono et al., 1992). The mechanical rupture of the

liquid films forming the foam is well established (Bikerman et al., 1953) and the

mechanical foam control system provides better not only in oxygen transfer

performance but also in power input economy (Yasukawa et al, 1991).

The mechanical foam breaking equipment has been described by

Edwards (1946), Gordon and Veldhuis (1953), Humfeld et al. (1952) and

Naucler (1948). 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). Common mechanical foam

breakers use rotating elements such as discs, bladed wheels, stirrers or a simple

bar attached to the agitator shaft above the liquid level (Solomons, 1969;

PharmaTec, 2004; Yasukawa et al., 1991; Takesono et al., 2001). They often are

complicated in design, have high running costs and also are unreliable (Prins

and Van’t 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, 1993). Mechanical methods of foam controlling

involve either external or internal foam breaking. Loginov, (1947) in their patent

describes that external foam breaker are the devices where the bulk liquid is

pumped out of the fermentor and sprayed onto the surface of the foam and

hence foam can be controlled. Internal foam breaking is the treating of foam

within the fermentor installed with the foam breaking devices.

Foam reduction in relation with the stirring was studied and the stirring

as foam disruption (SAFD) technique was developed using a 20 L bioreactor

having artificial media, which is said to be the major mechanism for the foam

disruption (Hoeks et al., 1997). Original mechanical techniques to prevent foam

formation in bioreactors have been elaborated from combined knowledge

involving the fluid dynamics of gas–liquid dispersion and interfacial processes


e.g. the stirring as foam disruption concept (Delvigne and Lecomte, 2010).

Centrifugal foam breakers are suitable for use during the vinegar

fermentation or the production of bakers yeast (Ebner et al., 1967). Hartmann

whistle consist of a jet that can be operated with a minimum of 40 psig (Pound-

force per Square Inch Gauge) by consuming the free air at 2-4 feet cube/min to

control the foam, demonstrated on growth of Serratia marcescens (Dorsey, 1959).

The air and entrained foam were exhausted through a convergent nozzle type

foam breaker where the sudden acceleration is created when velocity reached

100-300 ft/sec. The broken foam fell down to the fermentor and the air was

allowed to exhaust (Phillips et al., 1960). The nozzle foam breakers are reported

to be unsuitable for mold fermentations or for culture broths with particulate

materials (Solomons, 1969).

Rapidly rotating discs works on shearing principle, not specifically

suitable for fermentors, in which foam to be destroyed is directed onto the

surface of a rapidly rotating disc (Goldberg and Rubin, 1967). Mechanical foam

breaking rotating disk (MFRD) installed in a stirred draft-tube bioreactor and

the foam breaking characteristics of MFRDs in relation to the air sparge rate and

the impeller speed was investigated (Ohkawa et al., 1994).

Blade paddles with slits used for foam controlling has given significant

reduced power consumption in a stirred vessel compared with the conventional

foam breakers (Deshpande and Barigou, 1999). Rotating paddles or vanes can


be attached to the stirrer shaft to control the foam in a fermentor (Steel et al.,

1955). The effectiveness of an inverted hollow spinning cone (IHSC) for

controlling foam and liquid hold-up is demonstrated in pilot scale recombinant

Bacillus fermentation with the characteristically challenging foam and hold-up

issues. The cone rotates on the existing agitator shaft at the normal production

operating level (Stocks et al., 2004).

Foam breaking and oxygen transfer coefficient are improved with an

agitation impeller and foam breaking impeller mounted on the same shaft

demonstrated on the stirred tank fermentor containing low viscosity liquids

(Takesono et al., 2005). A cyclone separator consists of an entry pipe for flow of

foam, a separator for liquid and gases, reverse circulation pipe for return of the

liquid to the fermentor and an outlet pipe for discharge of gases (Hass, 1965).

Chemical methods

Chemical methods are designed to break the foam by adding chemical

agents usually called as either antifoam or defoamer. 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 (Van’t Riet and Tramper, 1991; Van’t 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). Foaming later in the fermentation

process has been found easier to control using antifoams (Hastings, 1954).

Antifoams are often divided into two categories depending whether they

are based on soluble or insoluble oils (Lee and Tynan, 1988). The soluble

antifoams are often based on polyethyleneoxyde or polypropylene oxide

moieties where as the insoluble antifoams are based on insoluble oils such as

polydimethyl siloxane or mineral oil which are most usually formulated with

hydrophobic particles, which help the small antifoam droplet to enter the

solution surface (Wasan and Christiano, 1997; Denkov, 2004). Antifoams used

in plant cell culture are sometimes used to control formation of crusts that

prevent broth circulation and silicon antifoam toxicity varies according to the

type of plant culture (Bond et al., 1987).


Characteristics of antifoaming agents

The most universal characteristic of any defoamer is the fact that it is

surface active but highly insoluble in water (Mini-Encyclopaedia of paper

making, Wet–End Chemistry). The antifoaming agent should cause foam

bubbles to coalesce and break to the point where they are large enough. The

foaming occurs only if any solution possesses surface elasticity. Therefore any

chemical antifoaming agent must eliminate surface elasticity (Kitchener and

Cooper, 1959). The silicon defoamers are not satisfactory in mold fermentations

because the mold itself inactivates the silicons though they are most suitable for

bacterial fermentations at alkaline pH and yeast fermentations (Fink et al., 1976).

Moderate action and poor lasting effects of defoaming were observed

when octadecanol in cold-pressed lard oil used as a defoaming agent

(Deindoerfer and Gaden, 1955). Simethicones are a complex mixture of high

molecular weight polydimethylesiloxane oligomers such as dimethicon with

particulate silicon dioxide added (Moore et al., 2002). Since often antifoams are

added on demand, initial instantaneous action is highly desirable (Vardar-

Sukan, 1992) to achieve fast foam-breaking (Stanbury and Whitaker, 1984; Hall

et al., 1973; Vardar-Sukan, 1992) or knock down (Berovic, 1992; Solomons, 1967).

Considering its projected elapsed 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; Hall et al., 1973; Vardar-Sukan,

1992; Berovic, 1992), as well as readily transferable to and dispersible in

fermentation broth (Stanbury and Whitaker; 1984; Hall et al., 1973). Antifoams

should be low cost and effective at low concentrations (Vardar-Sukan, 1992;

Berovic, 1992; Stanbury and Whitaker, 1984; Hall et al., 1973). In some cases, a

balance is explored between selection of a costlier which is more effective but

less utilized antifoam and selection of a cheaper which is less effective but more

highly utilized antifoam (Corbett, 1985).

No single antifoam can possess all of the ideal and desirable

characteristics, thus a compromise often is needed (Vardar-Sukan, 1992).

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

minimum volume required and maximum yield of product or absence of any

microbial activity (Duitschaever et al., 1988). Antifoams must have properties

that permit them to function as antifoams, be used with living cells and not to

interfere with electrodes such as pH or DO sensors (Vardar-Sukan, 1992).

Consequently, silicone-based antifoams contain finely divided solids,

specifically silica, 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).

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 and 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). Foams also can be stabilized by these

surfactants (Jenkins et al., 1993).

Metabolites as antifoaming agents

The onset of substantial cell growth reduces foaming, suggesting

metabolism modifies the composition of surface active 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) 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 avoids

changes in fermentor operating conditions and reduces antifoam additions

(Ishizuka et al., 1989).

Biological and metabolic aspects of antifoaming agents

The presence of antifoam can change the growth rate and even

morphology of cells (Noble et al., 1994b; PharmaTec, 2004), also negatively

affects cell density and 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).

Mechanism of action of antifoaming agents

Antifoams act via four steps: (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 (Vardar-Sukan, 1992).

Strategies for the usage of antifoaming agent

The main concern of the antifoam usage is to use correct dosage. Too

little defoamer addition may mean that inefficient foam control and too much

defoamer often adversely affects the process. 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 the

amount of antifoam added, the interval between antifoam additions to permit

mixing and for the antifoam to take effect and 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 seconds continuously then alternately adds antifoam for 30 seconds and mixes

without antifoam being added for 30 seconds, repeating this cycle until foam is

not detected for 5 seconds 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 fermentor back pressure) or antifoam reservoirs can be

weighed continuously. The other strategies reported by Bungay et al. (1960) for

the usage of antifoams are the addition of antifoaming agent by using

distribution devices.

Antifoam distribution methods and devices

Straight pipe entry of antifoam into the fermentor is the earliest method

reported. The distribution devices can improve efficiency over straight pipeline

entry of antifoam (Bungay et al., 1960).

Distribution through air flow is a method where the antifoam can be

introduced into the main air flow being sparged into the fermentor (Stefaniak et

al., 1946b).


Distribution onto the rotating disc is a method where more complete

coverage of the surface of the foam can be obtained if the antifoam is

discharged onto a rotating disc fitted to the stirrer shaft of a fermentor (Nelson

et al., 1956).

A spray nozzle is a distribution device that can be used to introduce the

antifoam into the fermentor, but the antifoam is wasted as the entrainment of

fine antifoam droplets in the exhaust air (Bungay et al., 1960).

Wick devices are designed in such a way that wicking material is

supported on pipes of wire mesh and soaked with oil as required by pumping

the oil through the connected tubing. The foam level rides just below the wicks

and oil are drawn into the foam in small amounts. This minimizes excess oil

being consumed. It was reported that the oil usage reductions of 25 to 50

percent with this wicks or with carefully adjusted continuous feed of oil. In this

method a constant foam level was achieved (Bungay et al., 1960).

Disc feeder is a device that was designed to place within a fermentor.

This is a modification of a drum feeder. But it was reported that during

sterilization the device traps moisture which collects under the oil and floats

out when the oil is not needed. For closed system work where contamination is

a problem, feeders of this type present problems which outweigh their possible

advantages (Bungay et al., 1960).


Blow-pot system gives exact shot of oil by forcing the contents of a small

chamber into the fermentor with air pressure. But also there are both

advantages and disadvantages of the delivery systems of antifoam into the

fermentor (Bungay et al., 1960).

Antifoam leaching device is a rubber composition contains an antifoam

compound that leaches out of the rubber composition when placed in contact

with a functional fluid. The antifoam compound is delivered into a functional

fluid such as an automatic transmission fluid (ATF) or engine oil by contacting

the functional fluid. ATFs, engine oils and other functional fluids generally

contain detergent and similar additives that tend to produce foam if air is

entrained into the fluid. Additional impurities are produced in the fluid over

time, some of which may contribute to a foaming tendency in the functional

fluid (Chapaton et al., 2006).

Formation of foaming should be prevented by the addition of

antifoaming agent at regular intervals which were sufficiently short to inhibit

foaming completely through out the intervening periods by that takes less

antifoam than the other but only suitable for continuous culture where the

agitation and air flow are continuous (Pirt and Callow, 1958). The method of

using antifoam has an important significance along the selection of the most

suitable solvents, emulsifiers, choice of storage conditions, sterilization and

methods of introducing the antifoams into the fermentor (Soifer et al., 1967).


Methods for metering of antifoams

Antifoam metering pumps which may be of various types such as a gear

pump, reciprocating pump, centrifugal pump, diaphragm check-valve pump,

flexible liner pump, peristaltic pump (Evans and Hall, 1971; Solomons, 1969).

The metering and addition of the antifoam are affected by timed delivery

through a valve (for example: solenoid valves), a drip or sight feeder, a drum

feeder placed under the fermentor, the blow pot system or a pressurized oil

system with needle valve and on-off delivery.

Dosages of antifoaming agents

The additions of the antifoaming agent in small increments depressed

the oxygen absorption rates to a lesser extent but the usage of silicon

compounds resulted in the 10-fold increase in oxygen transfer rate (Phillips et

al., 1960; Chain and Gualandi, 1954). No stable or long-termed effect of

antifoaming agent dosing on the biological foam occurrence was observed in

lab scale sequencing batch reactors by using eight antifoaming agents (Iveta

Ruzickova et al., 2005).

Foam controllers and circuit designs for the addition of antifoams

Circuits for the addition of antifoams into a fermentor were described by

Dworschack et al. (1954), Nelson et al (1956), Fuld and Dunn, (1958). An

electronic foam controller which operates at low electrode voltage that can be

constructed with the components readily available from radio and electric


supply companies (Pfeifer and Heger, 1957). A definite increment of antifoam

is added to a fermentor each time an insulated electrode is contacted by the

foam (Stefaniak et al., 1946a). An electronic interface controller was described

that can be used as a foam controller (Hersh et al., 1938). An electronic foam

controller actuated by two electrodes and functioning in a manner such that the

foam is maintained at or below the upper electrode tip (Echevarria, 1955).

An automatic addition kit for bench scale fermentors was designed with

a one minute delay time between each addition of antifoam by using a circuit

controlled by a timer making one revolution in 60 seconds. There was a 0-5

second’s control of the “ON” cycle for opening the solenoid valve to admit

antifoam of 1-2 drops which proved to be reliable and used less antifoam than

the manual addition (Bartholomew and Koslow, 1957). Pfeifer and Heger,

(1957) described about the electronic foam controllers and stated that they were

quite satisfactory but some difficulties were experienced with those

fermentations that produce uncontrollable foam, in such case foam rose to the

top, left residue on the probe and shorted it. In such case they suggested to

provide a spray of sterile liquid to wash the probe by that to remove the residue

from the probe.

Foam generation methods

In laboratory studies, foam can be generated in different ways (Wilde,

2000; Domingo et al, 1992). Several air incorporation systems can be tested like


sparging, whipping, shaking or pouring. The synthetic system made of bovine

serum albumin can be used for screening of foam controlling agents (Sie and

Schugerl, 1983).

Foam 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 between volume of foam and volumetric flow rate of gas or

the height of foam and 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 and Schugerl, 1979). Foaminess is

a measure of foaming capacity which is 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 flow rates to increase foaming for solutions with low

foaminess is hampered by difficulty detecting the foam or liquid interface when

high air flow rates are present (Edwards et al., 1982). Other relevant measures to

characterize foaming include the liquid volume held in the foam (foam volume

or liquid volume before foaming), volumetric foam overflow rate and foam

volume decrease over time (Wongsamuth and Doran, 1994; Abdullah et al.,

2000).


Antifoam efficiency test methods

There are various antifoam test methods to test the foam controlling

efficiency in specific foaming conditions. The effectiveness of antifoam 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

(Bryant, 1970).

Sparge test is a method to produces a qualitative evaluation of foam

controlling efficiency, where a specific foaming solution is sparged with air to

produce foam. The foam controlling efficiency of a material is evaluated by

measuring its effect on the foam height and the foam collapse time after a

specified sparging time.

Wrist-Action shaker test is a method that gives a relative measure of

defoaming performance, where the measured amount of sample is added to a

surfactant solution. The mixture is shaken and the time (in seconds) required

for the foam to collapse is recorded.

Recirculation pump test is a method to produces a qualitative

evaluation of foam controlling efficiency, where a specific foaming solution is

subjected to conditions like agitation and shearing. The defoaming efficiency of

a material is evaluated by determining the amount of time required to produce

a specified foam height while recirculating a solution through a closed loop.


The foam controlling agents which involves in the culture metabolism

should be avoided (Howe, 1978). Study on foam reducing effect of several

antifoaming agents on BSA solutions under standard conditions were

performed and measured the surface tension of the solutions and found no

relation between foaminess and surface tension, also found that the polyethers

and silicon compounds are the most efficient (Sie and Schugerl, 1983).

A simple laboratory test for the effective screening of foam control agents

on a synthetic fermentation broth (prepared by adding both proteins and

microorganisms) were developed. Mimicking the fermentation broth is a

simulation and can be used for the selection of appropriate antifoams (Etoc et

al., 2007).

The foam formation and foam collapse of four different fermentation

media were compared in the presence and absence of natural oils in a simulated

study and found that optimum effective natural oil concentration resulting in

optimum foam suppression did not always corresponds with the optimum for

foam collapse, not the optimum for maximum foam collapse rate (Vardar-

Sukan, 1991). A simple model can be used which simulates foam growth as

functions of defoamer concentration, air hold-up, reactor volume and air flow

rate (Pelton, 2002).

The toxicity of different antifoaming agents upon Aspergillus niger was

tested in petri dishes. Their effect on the decrease of the respiration ability of the


test organism Aspergillus niger was tested in a warburg apparatus. Various

antifoaming agents were tested as pure substances, as emulsions in seed oil and

as mixtures of different antifoaming agents in cylindrical vessels with sinter

glass discs under similar conditions as in the fermentor (Berovic and

Cimerman, 1979).

The efficiency of the antifoams in foam controlling generated during

paper making operations were invented where the diluted pulp mill black

liquor is used as the foaming medium which is re-circulated from a calibrated

reservoir (in centimetres) via a pump and is returned back to the reservoir. This

action agitates the medium which in turn causes foam. A known amount of the

defoamer is introduced into the test cell before the pump is turned on. The

calibration of the test cell ranges from 0 to 295 cm. A longer time required for

the foam to reach a certain level indicates a better defoamer (Nguyen and

Hendricks, 1997).

The stability of foam formed during fermentation is decisively affected

by the nature of the nutrient media used. In froth flotation models, the foam

formation time (characteristic of the tendency to foam) and foam subsistence

time (characteristic of the stability of foams formed) have been studied earlier

by Laszlo Szarka and Karoly Magyar, (1968).

Two procedures for foam determination based on the method of gas

sparging were applied in samples with different foam capacities. First


procedure comprises of maximum height, stability height and stability time.

The second procedure comprises of foam expansion, foam stability and

Bikerman coefficient (Magda Gallart et al., 1997).

Sterilization of antifoams

Wet sterilization: In moist conditions microorganisms germinate and

grow vegetatively. It is relatively easy to kill them in a wet environment. A

laboratory autoclave often is set up for thirty minutes at one atmosphere of

pressure or 121°C. When solids are present, e.g., soy meal in the culture

medium, the exposure time should be increased to allow heat to penetrate into

any solid clumps. Dry sterilization: Absence of moisture encourages the

formation of bacterial endospores, which means of preserving the organisms.

These spores are roughly 1000 times more resistant to heat than vegetative cells.

The sterilization of such materials as glass pipettes uses not a steam autoclave

but an oven. Typical conditions are several hours at 200°C. Antifoam oil is

essentially dry and should be sterilized either at higher temperatures or for very

prolonged periods in a steam autoclave.

Effects of natural oils on the accessories of fermentor

The control valves and metering devices of the fermentor are reported to

become clogged by the carbonaceous compounds or greasy sediments formed

during microbial decomposition of the natural oils. Also the diaphragm and


gaskets of the fermentor system are softened by the oil and it is even necessary

to keep the storage vessel and pipe lines warm in order to reduce viscosity and

to aid the flow of the oil (Bungay et al., 1960).

Oils increase the bubble size of foams, making them less stable. Their low

hydrophobicity limits their spreading or dispersion abilities and their high

viscosity limits their antifoaming capabilities (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 or inhibiting foam) as well as foam

collapse (eliminating or 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).

Effects of antifoaming agents other than the foam controlling

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). Most antifoaming agents have their other

effects like decreasing (Deindoerfer and Gaden, 1955; Pirt and Callow, 1958;

Ebner et al., 1967; Phillips et al., 1960; Phillips and Johnson, 1961) or increasing

oxygen transfer rates (Phillips et al., 1960; Zandi and Turner, 1970).


From earlier reports it was noted that oxygen 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). Cabral et al. (1985) studied the effect of several biocompatible chemical

antifoaming agents on the performance of ultra filtration membranes for yeast

cell concentration and reported a decreased flux rate of water solution as well

as the suspension of yeast cells and the increased cumulative fouling of the

membrane.

The amount of mass transfer reduction by antifoam varies depending on

the fermentation class (example: for yeast fermentation, 25% decrease in mass

transfer; for penicillin fermentation, 50-70% decrease in mass transfer) (Chain et

al., 1966). The effects of antifoams are the changes in physical properties of the

culture broth, deterioration of the operational characteristics of the fermentor

and a marked decline in fermentor performance (Deindoerfer and Gaden, 1955;

Dawson, 1961; Yagi and Yoshida, 1974). Antifoams can show a serious effect on

fermentations such as in white vinegar production which is very sensitive to

oxygen and in bakers yeast production which requires highly aeration rates. It

is essential to overcome or avoid the lowering of oxygen transfer rate by the

antifoam agent. Defoamer with a lesser effect on oxygen transfer rates should

be used in such cases (Solomons, 1967; Ebner et al., 1967).


The presence of antifoam causes difficulty in the extraction and

purification of the target product, formation of difficult emulsions in aqueous

solvent and the necessity for extra separation stages for the removal of

defoamer from the product makes the process more expensive (Solomons, 1967;

Ebner et al., 1967; Evans and Hall, 1971).

Effect of antifoams on foam behaviour of biological media

The foam behaviour of biological media in the presence and absence of

antifoaming agent and at different temperatures was studied and concluded

that the foam formation in biological media is because of protein denaturation

and by millard reactions (Kotsaridu et al., 1983b). Soybean oil have shown a

minor effect on mass transfer rate and bubble coalescence along with decreased

foam formation in a sodium caseinate - water solution in bubble column (Van’t

Riet et al., 1984).Decreased yield was observed when polypropylene glycol-2025

used to control foam in the exopolysaccharide synthesising culture of

Acremonium persicinum and Epicoccum purpurascens except Aureobasidium

pullulans. But no inhibition was noticed when silicone based compounds were

used to control the foam during the cultivation of Acremonium persicinum

(Stasinopoulos et al., 1988).

The addition of minute quantities of silicone based antifoam lead to

reduction in both hydrodynamic and mass transfer characteristics of all

reactors. New simple empirical correlations were determined for the reactors


design parameters like gas hold up, liquid circulation velocity and volumetric

mass transfer coefficient. It was also found that there is no discernible relation

of antifoam concentration with scale-up of airlift reactors for all the design

parameters (Waheed, 1999). The oil spreading on foam facilitates the entry of

mixed antifoam globules and the subsequent bridging and rupture of the foam

films occurs (Denkov et al., 2002).

Foam controlling methods that obviates the requirement of antifoam

Foam can be controlled by a process that obviates the requirement for

antifoam agents or other foam control methods where, the air and foam in the

head space are continuously withdrawn, entrained in the intake side of a self

priming pump and reintroduced into the bulk of the process liquid medium.

The headspace may be enriched with oxygen or other gases (Worthington et al.,

1967).

Combined methods of foam controlling

Foam breakers used in conjunction with antifoam addition can reduce

antifoam addition by 33-50% (Yamashita, 1972). The effectiveness of mechanical

and ultrasonic vibrations in destabilizing foams is shown to be governed by

vibrational amplitude, frequency and foam structure established in bubble

columns and stirred vessels (Barigou, 2000). The influence of different

surfactants on process of foam breaking in aqueous solutions was investigated

in an experimental study and a physical model for the mechanisms of a


mechanical foam breaker was developed (Gutwald and Mersmann, 1996).

Open celled polyurethane foam

Open celled polyurethane foam (PUF) is a three dimensional network of

interwoven strands and inter connected air cells. There is no connecting

membrane between strands. Random orientation of strands and air cells

minimizes possibility of open flow through channels (Design News Report,

1964). Technically it is a polymer consisting of a chain of organic units joined by

urethane (carbamate) links (Polyurethane Foam Association, 1991). PUFs are

chemically inert and not regulated for carcinogenicity. So far no exposure limits

have been established by any regulatory organization. PUFs start melting at

240°C (464°F) (Health alert, 2004). Based on texture open celled PUFs are of two

types namely flexible and rigid.

Properties and specifications of open celled PUFs

Open celled polyurethane foam is made by a process that provides an

open mesh like skeletal structure containing a high percentage of void space. As

a result, the material is extremely porous and permeable, has a low resistance to

flow and is capable of entrapping or holding large amounts of liquid and solid

matter (Design News Report, 1964). The strength of PUF depends upon the

parameters like porosity and density. Porosity is measured in pores per lineal

inch (ppi) where as the density is measured in pounds per cubic foot (pcf) or in

metric terms, kilogram per cubic meter (kg/m3) (Design News Report, 1964).


Uses of open celled PUFs

Open celled PUFs are widely used in different applications. They were

extensively used in air and liquid filtrations, the flexible foams are behind the

upholstery fabrics in commercial and domestic furniture. The rigid foams are

inside the metal and plastic walls of most refrigerators and freezers or behind

paper, metal and other surface materials in the case of thermal insulation panels

in the construction sector. The usage of PUF in the garment industry is

increasing, for example: in lining the cups of brassieres. These are also used for

moldings which include door frames, columns, balusters, window headers,

pediments, medallions and rosettes.

PUF is also used in the concrete construction industry to create foam

liners which serve as a mold for concrete, creating a variety of textures and art.

The PUFs are used in many forms, for use in insulation, sound deadening,

flotation, industrial coatings and packing material. The main reason for these

many uses of PUFs is that they adhere to most surfaces and automatically fill

voids. Flexible open celled polyurethane foam is a recyclable product.

Polyurethane Foam Association (www.pfa.org/intouch/index.html).

Polymeric foams are used in cushions, packing and structural materials

(Gibson and Ashby, 1999). Glass, ceramic and metal foams can also be made

and find an increasing number of new applications (Ashby et al., 2000). In

addition, mineral processing utilizes foam to separate valuable products by


flotation. Finally, foams enter geophysical studies of the mechanics of volcanic

eruptions.

Open celled PUFs were suggested to be the best solid supporting

material for the submerged fermentation (Zhu et al., 1994; John et al., 2007) and

used in the immobilization of cells (Guisan et al., 2006). PUFs can be coated with

the defoaming agent and used for the defoaming of blood in an extracorporeal

circulation (Sevastianov, 2007). PUFs were used in the biomedical industry to

prepare pressure sensitive foam coated with polypyrrol which exhibits a piezo

resistive reaction that can be used in the wearable sensing (Dunne et al., 2005).

PUF serve well as a sampling and selection medium for particles (Aitken et al.,

1993; Vincent et al., 1993; Chen et al., 1998; Kenny et al., 1998; Page et al., 2000;

Mohlmann et al., 2002). PUFs were also used in monitoring of some specific

range of aerosols (Kenny et al., 2001). PUF can be coated with the silver

nanoparticles which exhibit antibacterial property that can be used as a

drinking water filter (Prashant and Pradeep, 2005).

Immobilization of Pleurotus ostreatus 1804 on PUF cubes showed

enhanced laccase expression in rapid fermentation time compared to free

mycelia fermentation (Krishna Prasad et al., 2006). Enhanced secondary

metabolite production (gluconic acid, vinegar and lignolytic enzymes) was

reported because of immobilization on PUF (Mukhopadhyaya et al., 2005;

DeOry et al., 2004; Nakamura et al., 1999).

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