Lipase Membrane Reactor For Continuos Hydrolysis of Tallow Previous

Fat splitting is the industrial hydrolysis of fats and oils to produce fatty acids and glycerol. Fatty acids are then used to make a variety of products like soaps, detergents, surfactants, cosmetics, plasticizers,coatings and lubricants which find diverse applications in virtually all categories of industry and consumer products. Because of its low price and small percentage of poly-unsaturated fatty acids, tallow is the major raw material for fat splitting. Conversion of fatty chemicals is the highest value added use of tallow. Improved fat splitting technology could increase the value and utilization of tallow.

The best available technology for fat splitting is a thick walled stainless steel tower approximately 20 meters high and one meter in diameter. The tower is maintained at approximately 250 degree C and 50 atm by the injection of high pressure steam. High pressure pumps introduce melted fat to the bottom of the tower and water to the top. The tower has heat heat exchange sections at the top and bottom. Between these, the fat and water form a continuos phase in the heat exchange where complete hydrolyis takes place in approximately three hours. Continuos countercurrent flow is maintained by the density difference between the two phases in the heat exchange sections. The fatty acids, black in color, float to the top where they are drawn off and distilled. The unreacted water containing approximately 20% glycerol is removed from the bottom of the tower and also distilled to produce pure glycerol.

The use of lipases to carry out industrial hydrolysis of tallow has a number of advantages. The heat required is much less, only enough to melt the tallow, about 50 degree C , so the consumption of fossil fuels to make steam and stainless steel is much less. Also because of the low temparature, there is less degradation of unsaturated fatty acids, so pure, natural fatty acids can be obtained without distillation, even from highly unsaturated oils. Because of their nutritional value, undegraded poly-unsaturated fatty acids may be important to preserve in the production of food additives such as mono and di-glycerides. Finally, depending on the specificity of the lipase and the raw material , partial hydrolysis could yield a concentrated or purified mixture of fatty acids and/or partial glycerides with unique properties not found in bulk fatty acids from total hydrolysis of tallow.

In the past, commercialization of enzymatic fat splitting has been prevented by the high price of lipase and by the need for a vigourously stirred fine emulsions to achieve maximum activity. Energy costs for producing, stirring and then separating the emulsion are high. One solution to the problem of high energy cost is to immobilize the enzyme on a solid support so that it can be used repeatedly or continuosly for a long time. Lipase is normally active only at an oil/water interface. Although lipases have been found to be active in a single phase containing solvent, oil and a small amount of water , these sovent systems are mainly used for interesterification reactions or synthesis. When an excess of water is needed to drive the reaction towards complete hydrolysis, a two phase, oil/water system is preferred. However, when lipases are immobilized on a solid support, the number of phases become three, two liquid and one solid. Because reaction ocuurs only where all the three phases meet, a very different reactor design problem is created. If the immobilized enzyme is stirred in an emulsion the support particles tend to become coated with oil, and when the water soaking the porous particles is used up, the reaction slows down or stops. When an emulsion is pumped through a packed bed of immobilized lipase, it tends to separate and change so that the enzyme contacts oil or water but not both. Again the activity is short lived.

Solution to the severe reactor design problem in the hydrolysis of tallow with immobilized lipase was to use a membrane. Theoretically, a very thin membrane could immobilize the lipase at the oil/water interface. Oil and water could be made to flow past on either side. The hydrolysis would take place, and the products would be swept away. There would be effecient and long lived activity and no emulsion would be required. Actual ultrafiltration or microfiltration membranes have thickness many thousand times the diameter of an enzyme molecule, so the location of the oil/water interface relative to the enzyme within the membrane as well as the method of immobilization must be considered. At least one and perhaps both of the reactants and their corresponding aqueous or oily products must diffuse through the pores of the membrane to reach the interface. The stability, not only of the enzyme but also of the interface is important. In the case of tallow, a thermostable enzyme is required. Finally, the cost of the membrane is important and will depend on how long the immobilized activity lasts and whether the membranes can be cleaned and reloaded with the enzyme, and if so how many times.

These considerations were the subject of research at Eastern Regional Center, Agricultural Reasearch Service, U.S. department of Agriculture Philadelphia, Pensylvania and are summarized as follows:

Lipase Assay:

Their method consisted of two continuos stirred reactors(CSTR's) in series. The first CSTR was optimized for hydrolysis by lipase. It was fed by known fixed flows of ordinary olive oil and buffered lipase solution of unknown activity to be determined. The reactor was immersed in a constant temparature bath at 37 degree C. The reactor was small (less than 5mL) and was rapidly stirred with a powerful magnetic stirrer. The reacting mixture produced in the first CSTR passed to the second CSTR which was also small, magnetically stirred and containing the pH electrode. The reference portion of the electrode was filled with methanol saturated with KCl to prevent clogging of the reference junction with insoluble soaps. Ethanol was pumped into the second CSTR to stop the hydrolysis and facilitate stirring. The pH-stat and automatic buret added standard 0.1NaOH, titrating the fatty acids to pH 10.4. After 20-30 minutes, the system came to equilibrum and the rate of addition was recorded. From this rate, the measured rates of titratable acidity entering with the olive oil and enzyme solution were substracted to give the rate of acid formation. The enzyme activity in the first CSTR expressed as m moles/min of fatty acid produced was divided by the measured volume of lipase solution in the first CSTR to obtain the lipase concentration in units/mL, one unit being defined as the amount of lipase that produces one m mole/min of fatty acid under assay conditions.

For measurement of the activity of immobilized lipase, an entirely different system is used. The activity of immobilized lipase is also expressed as m moles/min, but this activity must not be confused with the units of lipase. Units of lipase are equivalent to m moles/min only under the standard assay conditions described above. In immobilized enzyme reactors the actual m moles/min are often much less than the units of enzyme immobilized. The ratio of the actual activity to the units immobilized is the effectiveness factor. It is usually less than one because of diffusional limitations and also because some portions of the immobilized lipase may be inactive becuase it is not located near the oil/water interface.

Lipase Production By Fermentation

For complete hydrolysis of tallow they preferred to use a lipase that would hydrolyze all the ester bonds in tallow triglycerides equally. They also wanted a lipase that was thermostable at 50 degree C , (the approximate minimum temparature at which tallow remains liquid) and one which was available at a reasonable cost. They selected a lipase from the filamentous fungus Thermomyces(formerly Humicola) lanuginosus.

They found that the culture was shear sensitive. In this fermentation, rapid mixing caused cell damage, and although the fermentation time increased from 3 days to 7 days when agitation speed was decreased, the yield of the lipase in the crude filtrate increased from less than 50 units/mL to 250units/mL or more. They therefore used a 70-liter fermentor with an oversized impellor diameter (8 inch), so that the culture could be mixed at a low shear rate.

They also found that the stability of various crude preparations of this lipase in hot solutions could be greatly increased by the addition of a tiny amount of the enzyme inhibitor, p-chloromercuribenzoic acid(PCMB). At 50 degree C, the half life of one partially purified preparation increased from 0.9 day to 111 days when 20 mg/L PCMB was added. It was necessary to keep that solution at that temparature for almost five months, sampling and assaying lipase activity several times a week in order to measure such a long half life. At 57 degree C, PCMB increased the half life from 7.8 hrs to 250 hrs. PCMB reacts with free sulphydryl groups in proteins.It did not affect the lipase activity directly, but by inhibiting a sulphydryl protease that was produced by T.lanuginosus along with the lipase. Proteolytic activity in the enzyme preparations was not a problem after immobilization.

The extreme thermostabilty of the this lipase in solution was only observed in the absence of a substrate. When the enzyme solution was mixed in an emulsion with tallow at 50 degree C and centrifuged, the enzyme collected in a thin layer between the enzyme free, clear oil and aqueous layers and it was unrecoverable. Thus the lipase was inactivated 100 times more faster by contact and reaction with substrate in a rapidly stirred emulsion than in a substrate free buffer solution at the same temparature.

The yield of fermentation was significantly increased by the addition of PCMB to the fermentor shortly before harvest. Without PCMB, the lipase activity in the fermentor increased rapidly towards the end of the fermentation, reached a maximum and then declined immediately and just as rapidly. Thus the timing of the harvest was critical, and a delay of only one hour in cooling the fermentor down from 45 degree C to stop the fermentation resulted in significant loss of activity. They added 20mg/L PCMB to the fermentor when the activity reached half of its expected maximum. The activity continued to increase in the presence of PCMB, reached a higher maximum than without PCMB and stayed at that maximum for a much longer time.

Lipase Immobilization

The original conception of the lipase membrane reactor was to immobilize the enzyme at the oil/water interface. The method of immobilization would be simple adsorption from the aqueous phase to the oil/water interface. This adsorption was known to be quite strong and becuase the interface was stationary, the lipase would be immobilized. Flow of the separate oil and water phases was to be across the membrane surface and not through the membrane surface. This could be accomplished by using a hydrophobic membrane and a slightly positive pressure on the aqueous side, or by using a hydrophilic membrane and maintaining a slightly positive pressure on the oil side. In either case as long as the largest pore through the membrane was small enough, the surface tension at the oil/water interface would be enough to prevent flow through the pores. The membrane holder or housing would have to be provide with four separate openings, an inlet and an outlet for each side of the membrane, to allow continuos flow across both membrane surfaces. Some commercially available membrane configurations such as spiral wound units typically have only one connection to the downstream or permeate side and are therefore not suitable for this appication, but others such as hollow fibre or flat plate units can easily be adapted.