¹ Biotransformations ' is a generic term for production of a useful chemical from a feedstock via biological reaction, such as a catalysis step. Microorganisms are often able to catalyse complex chemical reactions at ambient conditions of temperature and pressure, and produce products of a greater purity than traditional catalytic routes. Considerable potential therefore exists to exploit these systems commercially. An example is the synthesis of enantiomerically pure chiral compounds, where the biological activity resides in a single enantiomer. This is of increasing importance to both the pharmaceutical and agroche ical industries.

One class of chiral intermediates that is of particular interest is the chiral epoxides which have a demonstrated utility in synthetic organic chemistry, and will readily undergo nucleophilic substitution. Although there are chemical methods for the synthesis of chiral epoxides these require allylic alcohols as starting compounds, and give rise to relatively expensive manufacturing routes. In contrast, as exemplified in patent applications (EPA 0166527, EPA 0256586) , icrobial systems have the ability to synthesise epoxides of high enantio eric purity from a wide range of substrates with a potential for considerable cost savings. In addition, the quantities and nature of the side products and wastes generated through biological processes may make them more favourable than traditional chemical

- 3 - and may even display harmful toxicity. There are many examples of synthetically useful biotransformations but there has been relatively little work on designing bioreactors and integrated bioreactor/separation techniques to exploit these biotransformations . Many biotransformation systems involve hydrophobic compounds, which makes their exploitation difficult in a conventional fer entor, hence, two reactor designs were the aqueous/organic two phase reactor and the immobilised cell reactor. However, both these reactors have a number of drawbacks; the two phase system has problems with downstream separation and a high power input for mixing, while the immobilised cell reactor suffers from mass transfer limitations.

A major problem associated with biotransformations at present is the scarcity of suitable large scale biological reactors. The precursors and products of the biotransformations are often organic materials which are sparingly soluble, and even toxic to microorganisms in high concentrations .

Various two-phase reactors have been proposed, in which one phase is aqueous and contains the biological culture (microorganisms) and the other is an organic phase, containing the biotransformation precursor (and eventually product) dissolved in a suitable organic solvent, or even in a pure form (for instance toluene might be the other liquid phase in a reactor used for converting toluene to cis-diol) . The biological culture is grown in the aqueous phase, and then the two phases are mixed. Biotransformation precursor diffuses from the organic phase into the aqueous phase, and

- 4 - is acted on by the microorganisms to form the biotransformation product. This product then has to be separated from the aqueous phase, this process often occurring because the product too has limited aqueous solubility and so diffuses back into the organic phase, which can then be extracted from the system. Problems arise with this sort of batch process due to the toxicity of the organic phase, which comes directly in contact with the biological phase, the contamination of the organic phase with biological materials and cell debris, which must be separated out, and the generally low productivities of the batch systems employed.

There may be solvent swelling of membrane. There is prior work in the use of porous membranes. The dissadvantage of using porous rather than dense phase membranes is the difficulty in controlling the interface which has to be done using pressure. What happens apparently is that the spaces in the membrane fill with organic phase but where the interface within the membrane is depends on the pressure across the membrane. If it was too high on the organic side the organic would pass into the aqueous and vice versa.

According to one aspect of this invention there is provided bioreactor apparatus suitable for use in liquid phase biotransformation of organic precursor to organic product which comprises a generally hollow housing having an inlet which can receive aqueous phase containing biologically active culture, and a length of at least one selectively permeable polymeric tubular membrane extending

- 5 - within the interior of said housing whereby the exterior surface of at least part of the length of the membrane(s) can contact said aqueous phase when present in the interior of said housing, membrane inlet means and membrane outlet means provided in the housing which can permit a flow of other liquid phase containing precursor through the interior of the membrane(s) , the housing further having generally liquid-tight sealing means in the proximity of said membrane inlet and outlet means effective to separate said aqueous phase from said other liquid phase in use of the apparatus. Broadly stated, a method according to a second aspect of effecting a liquid phase biotransformation of precursor to product comprises a step of contacting the aqueous phase with the organic phase across a selectively permeable polymeric membrane, preferably of dense phase homogeneous lipophyllic material, allowing permeation of precursor through that membrane, allowing reaction to take place whereby precursor is transformed into product in the aqueous biological phase, and said product permeating back into the organic phase through said membrane, optionally followed by product separation.

In a third aspect the invention provides a method for effecting a liquid phase biotransformation of organic precursor to organic product which comprises separating by selectively permeable polymeric sheet or tubular membrane(s) a supply of aqueous feedstock containing a biocatalyst such as biologically active culture or an enzyme effective to transform said precursor to said product, from a supply of other liquid phase containing said precursor whereby

- 6 - permeation of said precursor through said membrane(ε) into said aqueous phase occurs, followed by biologically effected reaction to form said product which then permeates back through said membrane(s) into said other liquid phase.

Whilst the biocatalyst can be a viable and growing cell mass, it does not have to be since an enzymatic biocatalyst could equally be used.

Preferably the or each membrane is a dense phase medium, more preferably dense phase homegeneous lipophyllic medium in which perforations are generally absent, tubular (for example a coil with a large surface area) and the method can conveniently be carried out using apparatus according to the said one aspect, or in apparatus adapted for batch processing.

The supply of aqueous feedstock may be a continuous flow or provided in batches which can be replaced or replenished as the biotransformation reaction proceeds. Similarly, the other liquid phase can be supplied in a continuous flow or in batches for replacement or replenishment. In one preferred embodiment of the method, both aqueous feedstock and other liquid are provided in batches, as in batch processing, with the liquids being physically separated by a barrier constituted by the membrane. A coiled, tubular membrane may work well.

The membrane may be hollow tubular with an inlet remotely and independently supplied from any inlet of the bioreactor. The membrane is preferably removable from the bioreactor by withdrawing it, without damage, and whilst liquid is still flowing therethrough to permit cleaning of

- 7 - debris or excess micro-organism growth on the exterior of that tubing.

The membrane material is preferably one which allows permeation of the biotransformation precursor and product at a rate higher than permeation by chloride ion, under the same conditions. For example the membrane may comprise silicone rubber, pvc, polyethylene, polypropylene and/or polysulphone and similar organic polymeric materials. In use, biological nutrients or enzyme activators can be added to the biological phase.

Oxygen (or air) can be added to the biological phase e.g. by bubbling in a supply of such gas at the base of the receptacle serving as the bioreactor (see e.g. Figures 11 and 12) . The organic phase can be supplied, if required, on a continuous basis to the bioreactor apparatus, or to the interior of the hollow tubular membrane, whichever may be used for that phase. In some embodiments the precursor may be an organic liquid in which case it is preferably dissolved in an organic solvent first.

As regards classes of biotransformation precursor the process may work on a variety of organic substrates e.g. alkanes, alkenes, ethers, alcohols, halogenated species to produce a range of chiral products epoxides, alcohols, ketones , diols, halogenated species, amino acids. A range of batch and continuous processing options are possible.

The system is preferably an organic phase/aqueous phase system with phase separation by the membrane. Chemical reactions i.e. non-biological may occur on one side of the membrane.

- 8 -

In order that the invention may be illustrated, more easily appreciated and readily carried into effect, preferred features and embodiments will now be described purely by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1, illustrates one kind of apparatus for evaluating suitability of any given polymeric tubular membrane,

Figure la illustrates the principles of the present biotransformations,

Figure 2, shows graphs demonstrating permeation of precursor and products through selectively permeable polymeric tubing,

Figure 3, shows schematically an embodiment of one preferred bioreactor system, in use,

Figure 4 shows schematically a simple form of bioreactor apparatus according to the first aspect,

Figure 5, shows a more complex structure of bioreactor apparatus than Figure 4,

Figure 6 is an enlarged cross sectional detail of membrane inlet or outlet means depicted in Figure 5,

Figure 7 shows accumulation of product in the organic phase,

Figure 8 shows fluctuation in the biomass concentration in the biomedium,

Figure 9 and 10 illustrate product formation in batch processing,

Figure 11 shows product formation during continuous processing,

- 9 -

Figure 11a shows an alternative arrangement using a gas/liquid phase bioreactor and independently removable membrane, and

Figure 12 shows an arrangement similar to Figure 11 but with reversal of the flows of aqueous and organic phases.

The basis of this invention is a membrane which is used to separate the biological and organic phases of the process. This membrane is permeable to the biotransformation precursor and product, but relatively impermeable to the aqueous solutions, the salts and biological material it contains. Thus, the bioreactor is separated into two zones; a process zone which contains the biotransformation precursor at the membrane inlet and the product at the membrane outlet (the precursor could be dissolved in water, a suitable solvent such as decanol, or even a pure phase, such as toluene) ; and a biological zone, in which the biotrans ormation is performed by a suitable culture under suitable conditions.

The principal processes involved in the present embodiment of bioreactor are shown in Figure la. The biocatalyst is maintained at its optimal conditions (pH, temperature, dissolved oxygen, etc.) in the biomedium. The reactant is transported to the membrane through the organic phase, where it diffuses through the membrane to the biomedium/membrane interface due to a favourable partition coefficient. Here the reactant diffuses in the biomedium and is transported to the biocatalyst where it is enzymatically converted into the desired product. The product molecule is then transported back to the membrane,

- 10 - and diffuses through the membrane into the organic phase, again due to a favourable partition coefficient. This results in a solvent phase containing the bulk organic solvent, small quantities of the reactant and the product, which only requires simple unit operations such as distillation to recover the product. Alternatively, the organic phase could simply be pure reactant, without any dilution with another organic solvent, if the product is easily extracted from the biomedium into the reactant.

It is possible to use silicone rubber tubing, such as an alkylsiloxane silicone rubber membrane of which polydi ethylsiloxane is an effective example. The internal diameter and wall thickness may be determined by routine experiment. Polydimethylsiloxane silicone rubber is a good example of a "dense phase' selectively permeable membrane where pores are absent, even when viewed by electron microscope.

It is also possible to use polyvinylchloride as the selectively permeable membrane.

Figure 1 shows an apparatus used to test the permeability of potentially useful membranes to allyl phenyl ether (APE) (biotrans ormation precursor) , and its corresponding epoxide (biotransformation product) .

A flask 1 containing aqueous epoxide 2 is linked via teflon tubing 3 and peristaltic pump 4 to another flask 5 containing aqueous APE 6 and stirred by magnetic stirrer 9. Within flask 5 there is a partially coiled length of dimethylpolysiloxane rubber tubular membrane 7, connected to supply line 3 and return line 8. The two aqueous solutions,

- 11 - one containing epoxide and the other containing APE are separated by the use of the silicone rubber membrane. Figure 2 shows the results from this experiment. In less than 300 minutes equilibrium has been reached and the concentrations of both chemicals are approximately equal on both sides of the membrane. Similar tests performed with decanol as the solvent for the APE side showed that it is feasible to use an organic solvent on the process side (APE side) which can contain much greater APE concentrations, over 500 mM of APE. Concentration of the APE in the inlet side can be increased and therefore the epoxide concentration in the outlet stream through appropriate choice of solvents. Many of the chemicals with the potential to be produced through biotransformations (styrenes, cis-diols, epoxides) are sparingly soluble due to their hydrophobic nature but can still travel across these types of membranes.

A biosystem of the con iguration shown in Figure 3 can be employed to produce a continuous product flow from a biotransformation process. A traditional, primary bioreactor 10 equipped with stirrer 11, gas inlet 12 and gas outlet 13 can be linked through a recycle to bioreactor apparatus 14 according to the first aspect. The aqueous medium 17 containing biological culture (effective to transform at least some precursor into product) flows over the outer surface of the tubular membranes 7 and is recycled via supply line 3, pump 4 and return line 8. The other liquid phase 18, containing the biotrans ormation precursor, either dissolved in aqueous solution, an organic solvent, or

- 12 - present as a pure phase, can flow into the tubing at inlet 15. The precursor diffuses from the process side into the biological phase, where it is acted on by the culture to produce the desired product. This product then diffuses back across the membranes 7 into the process 19 stream, and is collected via outlet 16 as a stream containing product and possibly unreacted precursor. Such a system offers a range of advantages over the two phase reactors outlined above.

The outlet process stream contains no contamination from the biological phase, the (often toxic) process stream is kept out of contact with the biological phase, and a high cell density can be maintained in the biological phase, thus greatly enhancing reaction rates. The almost completely enclosed biological side should be relatively more easy to maintain aseptic. Another advantage is the possibility for continuous operation, and thus high productivities, that are made easily possible by a system such as shown in Figure 3. The primary bioreactor 10 can have a carefully controlled pH, temperature, dissolved oxygen and contain the biological culture performing biotransformation. The secondary bioreactor 14, remote from primary bioreactor 10, is in accordance with the first aspect of the invention. It includes membrane inlet means 15 to allow a flow of the process stream (other liquid phase) in, which contains the organic product to be transformed in a solvent or other liquid medium, which is aqueous and/or organic, or even a pure phase. After passing through the membrane module 14, which is a shell and tube module containing a plurality of

- 13 - silicone rubber tubes, of which the exterior surface only contacts the aqueous phase containing biologically active culture, the flow exits through membrane outlet means 16, also containing the product.

Figure 4 shows a bioreactor apparatus in simple form in accordance with the first aspect. A rigid housing 20 has inlet 21 for aqueous phase containing biological culture and an outlet 22 therefor. A single silicone rubber tube passes within the interior of the housing. To prevent collapse of the sidewalls of the tubing at the junction between housing and tubing, the interior of the tubing is reinforced with teflon or other rigid hollow plastic collars 23. Sealing means, such as O-rings 24, abut the membrane inlet means 15 and outlet means 16 which are projecting extensions of the tubular membrane 7 at the exterior surface of the tubing. In place of collars 23, the length of tubular membrane which may be straight, partly coiled or tightly coiled could extend between internally projecting glass or other inert rigid hollow tubes which are themselves held in place by holed rubber bungs (not shown) . In such case the glass tubes act as membrane inlet and outlet means. Although the single piece of silicone rubber tubing is shown straight and elongated, a coiled arrangement can be used providing access by the culture to a much greater surface area of the exterior of the tubular membrane.

A more sophisticated bioreactor in accordance with the first aspect is shown in Figure 5 and a detail thereof in Figure 6. This enclosed arrangement permits removal of a bundle of tubular membranes for cleaning or replacement. A

- 14 - rigid housing 20 has inlet 21 and outlet 22 for aqueous phase containing biological culture. Membrane inlet 15 for receiving a flow 18 of other liquid phase containing precursor and membrane outlet 16 through which processed liquid 19 passes are detachable from the housing 20. They are held in position by spaced flanges 25 (see Figure 6) which also abut and compress O-rings 24 to make a liquid tight separation of the two respective phases. A bundle of tubular membranes 7 is fastened between the portions 23a which are in the nature of purpose cast silicone rubber end pieces cemented to glass tubes.

The system shown in Figure 3 has but one bioreactor apparatus 14 according to the first aspect. Several could be utilised, all supplied in parallel from the primary bioreactor 10.

- 15 - Examp le 1

Materials and Methods

Micro-organism Pseudomonas oleovorans TF4-1L (ATCC 29347) was obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A) , and maintained on Nutrient Broth (Oxoid, Basingstoke, Hants) agar plates.

Nutrient media 20 g L of monosodiu glutamate monohydrate was used as the sole carbon and energy source (1 , 7-octadiene and 1 , 2-epoxy-7-octene are not metabolised) . The nutrient salts included 1.4 mg L ^_1 of ZnS0 ₄ .7H ₂ 0 and 0.28 mg L ^-1 of NiCl ₂ .6H ₂ 0.

Biomass concentration measurements Samples of the biomedium were diluted by 1 part in 9 with high purity reverse osmosis water, and the absorbance of the mixture measured at 660 mm in a Philips PU8625 UV/Vis spectrophoto eter. The absorbance of biomass was correlated to actual dry cell mass concentrations using Sartorius MA 30 moisture analyser.

Assay of 1 , 2-epoxy-7-octene 100 μl of organic phase was mixed with 100 μl of a 0.1% v.v 1-octanol in hexadecane internal standard mixture and 600 μl of hexadecane (as a diluent) on a vortex mixer for 10 seconds. 1 μl of this mixture was injected directly onto a SGE BP1 capillary column, 0.53 mm i.d, 1 urn thickness column on a Perkin-Elmer Auto System FID GC with Hewlett-Packard 3390A integrator.

- 16 - The ratio of the epoxide peak area to the internal standard peak area was compared with a standard curve to obtain the actual epoxide concentration.

Membrane Bioreactor for Biotransformations The bioreactor consisted of a 1.75 L working volume CSTR connected to a membrane module of 0.05 surface area. The biomedium contained the growing Pseudomonas oleovorans culture, and the organic phase of 0.47 L contained approximately 32 g L ^-1 of 1, 7-octadιene in n-hexadecane . Recirculation of the biomedium to the membrane was started 24 hours after the culture was inoculated.

Chemicals n-Hexadecane (99+%) was obtained from Sigma Chemicals (Poole, Dorset) , and 1 , 7-octadιene (98+%) , 1,2- epoxy-7-octene (97+%) and 1-octanol (99+%) were obtained from Aldπch Chemicals (Gillingham, Dorset) .

The bioreactor of the type shown in Figure 3 was continuously run for a period of over 100 hours at a dilution rate of 0.1 h . As can be seen in Figure 7, during this period 1, 2-epoxy-7-octene was produced and accumulated in the organic phase at an approximately linear rate of 2.8 mg (L or . phase) ^-1 h ^~ . Figure 8 shows that over the course of the example, the biomass concentration increased slowly to an end value of 2.5 g (L biomedium)- as steady state was approached . Over the course of the example the average biomass concentration was approximately 1.5 g dry wt. (L biomedium)-, which implies that the

- 17 - specific activity of the cells over the period of interest was 0.5mg (g dry wt) ^-1 h ¹ . The linear rate of accumulation of the epoxide suggests that the epoxidation rate was independent of the biomass concentration over this period. This situation could have been caused by the mass transfer of 1, 7-octadιene across the membrane being the rate limiting step, as opposed to the level of activity and expression of the -hydroxylase enzyme system that must be induced to perform the epoxidation. Thus, increasing the rate of mass transfer of 1 , 7-octadιene should increase the rate of production and accumulation of 1 , 2-epoxy-7-octene. This will increase the volumetric productivity of the reactor system and the specific productivity of the bacterial cells, and lead to a even more economic production system.

Example 2

Figures 9-11 show data for the biotransformation of 1,7- octadiene to 1 , 2-epoxy-7-octene by growing Pseudomonas oleovorans (ATCC 29347) cells. The data shown in Figures 9 and 10 were obtained for batch growth in shake flasks, and the data shown in Figure 11 was obtained during one week of growth in a continuous stirred tank bioreactor connected to a membrane module. A 'shake' flask is obtainable by clamping the tubing at the dotted line in Figure 1 leading into the flask 5. The membrane tube containing the solvent is clamped at both exits. Alkene from the solvent phase diffuses across the membrane and is epoxidated in the biomedium. The epoxide diffuses back across the membrane

- 18 - into the solvent phase contained within the tubular membrane. The aqueous biomedium contained living cells of pseudomonas oleovorans. The data shown in Figures 9 and 10 refer to batch growth in shake flasks. in both cases, the data obtained from the membrane systems was compared with data for a two-phase (aqueous/organic) system containing bacteria from the same inoculum. Two types of membrane were compared with the two-phase system, pvc and silicone rubber. The experiment was carried out by pre-swelling the membrane tubing with pure 1 , 7-octadιene, clamping the ends of the tube with metal clamps, and completely filling the tube with 1, 7-octadιene. The tubing was then suspended in the bacterial suspension and incubated. Samples were taken and analysed regularly. The data in Figures 9 and 10 show the accumulation of 1, 2-epoxy-7-octene in each of the three systems. The difference in concentration levels m the two graphs may be explained by differences in the total volume of organic phase, variations in the incubation conditions, or more likely, variation in the inocula.

Figure 11 shows the accumulation of 1, 2-epoxy-7-octene in a silicone rubber membrane module connected to a continuous stirred tank bioreactor. The relatively low concentrations of 1 , 2-epoxy-7-octene that accumulate are due to a large dilution factor in the membrane module. The concentration of 1 , 7-octadιene in the membrane module was 20 g/L, and the remainder of the organic solvent phase was n-hexadecane. The data shown in the graph vas obtained over the first week of growth.

- 19 - The membrane bioreactor for biotransformations described can be used to provide higher volumetric productivities and easier downstream processing for such biotransformations than current technologies afford.

The bioreactor uses a dense phase membrane to separate an aqueous and organic phase and to control the transport of molecules between the phases. Many biotransformations involve poorly water soluble reactants and products. Thus, in the bioreactor the reactant is delivered through the membrane from the organic solvent phase to the aqueous biomedium, where it is transformed, and the product is back- extracted through the membrane into the organic solvent. The dense phase membranes used allow the permeation of small organic molecules, but not ionic species and large bio oleculeε. Thus, the organic solvent phase contains only small organic molecules (e.g. solvent, reactant and product) and the product can be purified relatively easily using conventional separation techniques (e.g. distillation) .

The biotransformations of 1 , 7-octadiene to l,2-epoxy-7- octene by growing Pseudomonas oleovorans (ATCC 29347) cells was exemplified to demonstrate the feasibility and operability of the present bioreactor in chiral biotransformations. The use of pvc and silicone rubber as dense phase, unperforated membrane materials has been described. It was found that the -hydroxylase system of Pseudomonas oleovorans could be induced at sub-saturation concentrations of 1 , 7-octadiene, which allows

- 20 - biotransformations to take place in the membrane bioreactor. Biological and chemical hydrolysis of 1, 2-epoxy-7-octene are not significant at the temperature and pH conditions used for growth, and gas stripping and evaporation were found to be the principal causes of reduction in product yield.

Figures 11 and 12 shows arrangements of apparatus which differ from the described modular bioreactor apparatus such as shown in Figure 5. They are easier and less costly to construct whilst still permitting good control over the conditions pertaining in the biological phase. Essentially they are 'open' bioreactors in the sense that a gas/liquid interface is present with the gas being either the open atmosphere or another atmosphere i.e. the gas layer immediately above the maintained level of liquid medium in these 'open' bioreactors.

These arrangements have the advantage that the membrane, which is coiled tubular can be withdrawn for easy cleaning or replacement. For cleaning, the flow of liquid phase through the tubing need not be distrubed.

Figure 11 shows the aqueous biocatalytic medium in the receptacle with organic phase flowing through the membrane. The supply of phases to both 'sides' of the apparatus can be batchwise or continuous. In Figure 12 the flows of liquid phases are reversed. The key components m these drawings have been identified by appropriate brief keywords as present in the drawings.