GB2180466A - Technique for continuous performance of a reaction - Google Patents

Abstract

A technique for performing a selected reaction which comprises moving a continuous strip 2 having a substance which comprises a reactant or catalyst involved in the said reaction through a reactor vessel 1 in which the said reaction is effected, means 3 for moving the strip through the reactor vessel being enclosed therewithin. There is further provided a reactor comprising a reaction chamber containing a continuous strip having a substance comprising a reactant or catalyst immobilised thereon, and within the chamber means for moving the strip in a controlled manner through the chamber. In a modification (Fig. 2, not shown) the continuous strip is held fully within the reactor casing. Reagent solution may be passed through the reactor of either Fig. 1 or Fig. 2 via holes 5. <IMAGE>

Description

SPECIFICATION Technique for Continuous Performance of a Reaction The present invention relates to a technique for the continuous performance of a reaction.

When carrying out diverse e.g. chemical or biological reactions, valuable time and effort is wasted because the reactor system must periodically be shut down for renewal of the reactant or catalyst. In very small, laboratory-bench sized operations this renewal may be necessitated at very frequent intervals, with the result that more time may be spent cleaning delicate apparatus than performing the reaction. Therefore there is a need for a continuous small-scale reaction system wherein cleaning and renewal is not so frequently required.

The system should have a design such that there is good solid-liquid contact in order that there is optimum reactant or catalyst utilization. The system should also be strong enough to withstand continuous operation, and not be liable to excessive losses of liquid due to poor closures, "drag out" and similar effects.

According to the present invention there is provided a technique for performing a selected reaction which comprises moving a continuous strip having a substance which comprises a reactant or catalyst involved in the said reaction through a reactor vessel in which the said reaction is effected, means for movin#g the strip through the reactor vessel being enclosed therewithin.

By the term "reaction" used herein is meant any chemical, biological or immunochemical reaction whatsoever including, for example, organic or inorganic chemical reactions, enzyme catalysed reactions, antibody/antigen reactions, adsorbent reactions, whatever their purpose, e.g. preparative testing, diagnostic or assay purposes. The terms "reactant", "catalyst", "reactor" and "reactant material" should be construed accordingly.

The invention is not limited to isolated chemical substances but may involve the use of complex chemical and/or biological mixtures, living material or other systems where reactions occur or through the agency of which chemical reactions can be made to occur.

The term "continuous strip" is not to be limited to a strip of uniform dimension, nor need the whole strips bear immobilised catalyst or reactant. A system could be envisaged whereby only part of the continuous strip acts as a bearer for immobilised catalyst or reactant, the rest of the strip acting as a tether to draw the loaded strip through the system.

A suitable device for carrying out the proposed technique is a box holding a continuous strip randomly folded therein. The strip may be a woven tape of man-made or natural fibres. A nylon ribbon is preferred. A short length of the strip, which may be in the form of a woven tape, may be exposed outside the box. The tape is continuously moved through the box by a means such as a pair of tightly gripping gear-wheels, and may be drawn at the other end of the box through a spring-loaded gate.

In this manner the randomly folded pattern on the tape within the box is maintained and the folded mass of tape moves slowly through the box.

Thus, according to a further aspect of the invention, there is provided a reactor comprising a reaction chamber containing a continuous strip having a substance comprising a reactant or catalyst immobilised thereon, and within the chamber means for moving the strip in a controlled manner through the chamber.

According to the technique, the continuous strip may be used as the support for a reactant or catalyst involved in the desired reaction. This reactant or catalyst may be held in the weave of the tape in a suitable medium, such as a gel, or by direct chemical bonding. Various techniques are known in the art for bonding of reactants to fabric supports.

The folded tape in the box provides a firm support and presents a large and determinable area of the immobilized reactant or catalyst for reaction with a substrate solution flowing continuously through the box. The tape may also preferably be passed out from the reactor box so that it passes through one or more treatment vessels to regenerate or clean, as the case may be, the reactant or catalyst, ready for repassage through the first reactor box. This movement may be continuous. The reaction vessels may be in series or in parallel.

The substrate flowing through the reactor vessel is therefore presented with a constantly renewed immobilized matrix, the strip providing for good solid-liquid contact. This system obviates the need to shut down the reactor repeatedly to renew the saturated reactant or catalyst. The reactor vessel may preferably be provided with an inlet and an outlet for passage of reaction materials into and out of the reaction chamber.

The system also has the advantage that the circulation speed of the strip can be modified to cope with a wide range of substrate flow rates.

The technique has been found to be particularly suited to the isolation of small quantities of highvalue biological products or for diagnostic or analytical procedures. According to a preferred aspect of the invention, biologically-active materials such as enzymes may be immobilized on to the strip and passed through a reactor containing a suitable substrate for the enzyme. As the enzyme emerges from the substrate medium, the enzyme is washed to remove the reaction products and fouling materials. The technique may be carried out also on immobilised co-enzymes.

The technique has also proved useful for an immobilized specific absorption system whereby crystals of a synthetic zeolite bonded to a ribbon are used to adsorb ethanol selectively from an aqueous solution. The ethanol can then be removed from the bonded zeolite and the system continuously reused.

In a further aspect, the technique has proved useful in continuous fermentation with cells immobilised onto the continuous strip. This bioreactor uses only one reactor vessel, although passage into further vessels could be envisaged for, e.g. washing the immobilised cells. The strip is circulated through the reactor vessel with e.g. yeast cells immobilised thereon, whilst a nutrient, e.g.

glucose solution is circulated through the reactor, causing cell growth and fermentation. A reaction which combines e.g. cells and adsorbents may be effected.

The modular reactor containing a continuous strip may be of use in reactions wherein it is desired to change over the reactant or catalyst. The complete reaction system may be designed so that the reactor vessel is used as a 'cassette'. Such a system would allow for quick changeover of reactants obviating the need to drain a whole system to change the reactant or catalyst.

The accompanying drawings show reactor vessels used in accordance with the present invention.

Figure 1 shows a vessel comprising a reactor casing 1 in the form of a rectangular box. A randomly folded pleated ribbon 2 is partly stored therein. The ribbon is driven through the box between two geared wheels 3 which can themselves be driven by a small electric motor. The ribbon is kept taught as it passes in to and out of the box by spring loading of the said gear wheels and a leaf spring 4. Reagent solution is passed through the reaction vessel via two holes 5.

The ribbon 2 has a reactant or catalyst immobilised thereon. This reacts with the liquid reagent in the vessel. The ribbon is drawn through the box, and the reactant or catalyst then passes on to one or a plurality of regeneration stages. Such stages may comprise identical reaction boxes such as that presently decribed. Eventually the regenerated catalyst or reactant bonded to the ribbon reenters the original reaction vessel.

Figure 2 shows a reactor wherein the continuous strip is held fully within the reactor casing. The randomly folded pleated ribbon 2 is moved through the box between the geared wheels 3, whilst reagent solution passes through the reaction vessel via two holes 5.

The following non-limiting Examples describe uses of the technique according to the present invention.

EXAMPLE 1 Immobilized Enzyme Reactor Lactase from Aspergillus niger was obtained from the Sigma Chemical Co. as a suspension containing 21 units/ml (1 unit hydrolyzes 1 p-mole o nitrophenyl-,3-D-galactoside ("ONPG) per minute at pH 4 and 25 C, ref. J. Bio. Chem., 244 (1969), 2970).

Five units of lactase were immobilized on to 900 mm nylon ribbon, 12.5 mm wide, using Hornby's method (Sundaram & Hornby, 1970), FEBS Letters, 10(5), 325327). In a modification of the lactase assay, a 225-mm length of the ribbon was agitated in a shake-flask with ONPG in buffer pH 4 at 25 C and showed an activity equivalent to 0.2 units, indicating an immobilization effectiveness of 4%. In a similar test using 8 g/litre lactose in buffer, pH at 25 C, lactose was hydrolised at a rate of 0.05 micro-mole/minute, and in water at 250C at 0.04 micro-mole/minute.

For a trial using the continuous ribbon, 15 units of lactase were used with 17.5 m of continuous ribbon, which was circulated through the reactor at 33 mm/s, giving a ribbon residence time of 9 minutes.

200 ml of 0.01 M ONPG in buffer was circulated through the reactor at room temperature at 101 ml/minute, giving a liquid residence time of about 2 minutes. Over a period of 7 hours, the system gave a fairly constant hydrolysis rate of about 0.01 micromoles ONPG/minute. This demonstrated that, apart from the loss of enzyme activity incurred in immobilization, the enzyme activity was maintianed for a protracted period at ambient temperature while the ribbon was circulated continuously.

Shake4lasktestswith skimmed milk, using free enzyme and ribbon-immobilized enzyme, indicated severe loss of activity. Some activity was recovered by removing the lengths of ribbon and washing first in distilled water and then in 0.1 M phosphate buffer pH 8 before returning them to the shake-flasks. This indicated that for lactose hydrolysis in a fouling medium such as milk, continual washing of the immobilized enzyme is required. The continuous ribbon system is well suited to the incorporation of one or more washing stages between leaving and re-entering the reactor.

EXAMPLE 2 Immobilized Specific Adsorption System Crystals of a synthetic zeolite, about 1 m in diameter, were immobilized on to a continuous nylon ribbon 9.7 m long, using Hornby's method as for lactase. The zeolite is known to adsorb ethanol selectively from aqueous solution, and spent fermentation liquor containing about 3% ethanol was contacted with the ribbon using the continuous system, which showed a rapid loss of ethanol. The ribbon was washed in water and buffer and dried overnight in an incubator at 80 C. The test was repeated using 5 gil ethanol in water solution, whose concentration fell to less than 3 g/l in the first hour.A comparative test using zeolite-free ribbon showed a steady fall in ethanol concentration, due to evaporation, but from a comparison of the tests, an ethanol loss of about 0.75 g/l was attributable to the zeolite, which corresponded to the adsorption capacity of the zeolite probably immobilized.

Electron micrographs of portions of the ribbon take after the tests showed that the zeolite crystals were genuinely bonded to the nylon filaments and not merely entrapped in the weave of the fabric. This demonstrated that the zeolite remained immobilized after several hours' circulation through the device, washing, drying, further circulation, washing and drying, and that the adsorption capacity is renewable by washing and drying.

As with the immobilized enzyme system, the continuous ribbon system is well suited to the incorporation of resuscitation stages between leaving and re-entering the reactor.

EXAMPLE 3 Immobilized Microbial Cell Reactor The micro-organism used was brewer's yeast, Saccharomyces cerevisiae, grown in shake-Flasks in a standard yeast growth-medium and harvested at maximum biomass yield. The yeast was centrifuged, re-suspended in distilled water and centrifuged again.

The washed yeast was immobilized on to the ribbon by calcium alginate precipitation; 10 grammes (wet weight) of washed, centrifuged yeast was mixed into 100 ml of a well-mixed 2% w/v solution of sodium alginate (BDH Chemicals, Poole, Dorset). A continuous nylon ribbon, 10 mm wide, 5 mm long, was sterilised by boiling in water for one hour, dried and loaded into the reactor. The suspension of yeast in sodium alginate solution was fed into the device, and the ribbon circulated several times to give good contact between the alginate and the nylon. The algin'ate-soaked ribbon was then fed into a bath of 0.2 M calcium chloride solution from the device, which proved to be a convenient means of dispensing the slippery ribbon into the precipitating bath. Care was taken that the ribbon did not become tangled.When all the ribbon was in the precipitating bath, the bath was covered and kept overnight in a refrigerator at 5 C. It was calculated that the residual sodium chloride concentration, formed by exchange between the sodium alginate and the calcium chloride, in the precipitating bath was approximately optimal for that organism (Salihon etal., (1983), Biochem. Soc.

Trans., ii, 401). The ribbon was reloaded into the reactor and circulated at 10 mm/s, while a 5% w/v glucose solution was circulated through the device, to remove excess alginate from the ribbon, for several hours. The ribbon in the reactor was again kept refrigerated overnight.

In a preliminary trial, a glucose solution was circulated through the stationary ribbon, and the glucose concentration was reduced from 10.9% to 9% in four hours. Examination of the ribbon with an optical microscope showed that the nylon fibres were coated with alginate and that yeast cells were adhering in clusters to the coating. A high proportion of the yeast cells were budding, indicating that the yeast was viable and the glucose removal was the result of microbial growth. The ribbon in the reactor was kept refrigerated for several days.

The ribbon was circulated through the reactor at a speed of 10 mm/s, and glucose solution, initially 10% w/v, was circulated through the device at 0.75 ml/s. The system was operated in this manner for six hours. After an initial lag, the glucose concentration was reduced at a steady rate of 0.5% per hour over this period. Liquid hold-up in the device was 33 ml, giving a mean hydraulic residence time in the reactor of 44s, the total liquid volume used being 250 ml. The organism was growing aerobically in this experiment, aeration being effected by running the reactor output into an open buffer vessel, holding most of the circulating solution, from which the circulating pump took its input. The system was run at ambient temperature, which was 19 C. The system was operated hygienically rather than aseptically, but could be engineered for aseptic operation.

This work demonstrates that the device can be used successfully for continuous fermentation using immobilized cells, in principle animal and plant cells as well as microbial cells. The immobilization technique used did not interfere with the operation of the device, and the cells remained active after several hours circulation and intermittent refrigeration.

The glucose assay method used was the modified Keston procedure (Keston, A.S. (1956), ACS, 129th Mtg., Dallas, April, 310), marketed in kit form by Sigma Chemicals, Poole, Dorset, from whom the glucose itself was obtained.

Claims (12)

1. A technique for performing a selected reaction which comprises moving a continuous strip having a substance which comprises a reactant or catalyst involved in the said reaction through a reactor vessel in which the said reaction is effected, means for moving the strip through the reactor vessel being enclosed therewithin.

2. A technique as claimed in claim 1 wherein the continuous strip is completely within the reactor vessel.

3. A technique as claimed in claim 1 or claim 2 which further comprises passing the strip out from the reactor vessel into and through a second vessel to regenerate or clean, as the case may be, the reactant or catalyst ready for repassage through the first vessel.

4. A technique as claimed in claim 3 and performed continuously.

5. A technique as claimed in any one of claims 1 to 4 wherein the substance is an enzyme or a coenzyme.

6. A technique as claimed in any one of claims 1 to 4wherein the substance comprises cells.

7. A technique as claimed in claim 6 wherein the substance comprises bacterial, yeast, plant, fungal, animal or human cells.

8. A technique as claimed in claim 6 or claim 7 wherein a solution, e.g. a glucose solution, is passed through the reactor vessel for fermentation purposes.

9. A technique as claimed in claim 1 substantially as hereinbefore described in any one of the Examples.

10. A reactor comprising a reaction chamber containing a continuous strip having a substance comprising a reactant or catalyst immobilised thereon, and within the chamber means for moving the strip in a controlled manner through the chamber.

11. A reactor as claimed in claim 10 also provided with an inlet and an outlet for passage of reaction materials into and out of the chamber.

12. A reactor substantially as hereinbefore described with reference to and as illustrated in Figure 1 or Figure 2 of the accompanying drawings.