GB2535920A - Method for enhancement of enzyme activity - Google Patents

Abstract

The use of microwave energy to enhance the production of compounds (e.g. ethanol) by microbes (e.g. yeast), specifically where microbial growth is fuelled by an energy source, such as a biomass whose degradation is enhanced by microwave energy. The microwave energy has a frequency of 0.3 GHz to 16 GHz and a power from 1 to 100 watts. The application also relates to the use of microwave energy to enhance the activity of enzymatic reactions, such as enhancing the activity of enzymes involved in the degradation of cellulose to glucose and intermediates thereto. The application further relates to a microwave reactor vessel suitable for conducting such enhancement of enzyme activity and such fuelling of microbial growth.

Description

Method for enhancement of enzyme activity The present invention relates to the use of microwave energy to enhance the activity of enzymatic reactions, such as enhancing the activity of enzymes involved in the degradation of cellulose to glucose and intermediates thereto. The invention further relates to the use of microwave energy to enhance the production of compounds by microbes, such as where microbial growth is fuelled by an energy source, for example, a biomass, whose degradation is enhanced by microwave energy. The invention further relates to a microwave reactor vessel suitable for conducting such enhancement of enzyme activity and such fuelling of microbial growth.

Microwave irradiation has become an effective tool in synthetic organic chemistry, dramatically increasing reaction rates and yields. In contrast, much less work has been done investigating the effects of microware radiation on enzymatic reactions and there is conflicting data as to whether microwave radiation has any direct effect on enzymatic reactions.

Cellulose is the most abundant renewable energy resource on earth. Complete hydrolysis of cellulose yields easily fermentable saccharides. When the saccharides are biologically converted into other products, such as ethanol, they can provide economic environmental and strategic benefits on a large scale. Therefore, there is a great interest in understanding the process of enzymatic cellulose degradation and its industrial application.

Through the development of biotechnology, nearly every field of biotechnology has focused on the principle of generating greater product formation either directly from light or from a saccharide intermediate. This has been seen in blue biotechnology through aquaculture with the development of marine species with the increased expression of oils; green biotechnology with the development of C4 photosynthetic pathway in C3 autotrophs and transgenic and crossbred crops for increased polysaccharide content; red biotechnology through the increased expression of medicinal products and white biotechnology through the increased production of industrially commercial volumes of synthetic materials. With the development of biotechnology, the use of biological processing has enhanced the number of compounds available from the saccharide carbon sources. With the development of both the white and red biotechnology streams, the exploitation of microorganisms has led to the production of increasingly sophisticated biopharmaceuticals and biosynthetic materials far removed from the original biochemical pathway of the source microorganisms. Therefore, there is an ongoing need for improvements to increase the efficiency of production in biotechnological processes.

We have found that microwaves can increase the activity of enzymes involved in the metabolism of compounds, for example, in aqueous conditions, such as in the degradation of cellulose, i.e. increasing the rate of degradation of the cellulose glucose polymer to shorter length glucose polymers and to oligomer, dimer and mono-saccharides. We have also found that microwaves can affect the yield of an enzymatic reaction increasing the yield of products in addition to enhancing the rate of the enzymatic reaction.

According to a first aspect of the invention there is provided a method of enhancing the activity of an enzyme in a composition comprising an enzyme and a substrate said method comprising the provision of microwave radiation to said composition with sufficient microwave energy to enhance the activity of the enzyme.

We have found that microwaves can enhance the activity of an enzymatic reaction at lower microwaves energies that previously recognised using a waveguide configured to deliver microwave energy to the enzyme.

According to a further aspect of the invention there is provided a method of enhancing the activity of an enzyme said method comprising irradiating a composition comprising the enzyme and a substrate with microwave radiation delivered via a waveguide, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

When the frequency required is about 2.45 GHz the waveguide is substantially rectangular in shape and has cross sectional dimensions of about 86 by about 43 mm. The length of the waveguide is not critical to the method. Tn general a length between about 10cm to about 100cm would be suitable but the skilled person would be able to select an appropriate length of waveguide suited to a particular microwave apparatus design.

Therefore, in a further embodiment of the invention there is provided a method of the invention wherein the waveguide has cross sectional dimensions of about 86 mm by about 43 mm and the frequency is about 2.45 GHz.

If a different frequency of microwaves is required a different cross section of waveguide would be required dimensioned according to the frequency. the skilled person, knowing the cross sectional dimensions for 2.45 GHz would be able make a waveguide suitably dimensioned to the frequency required. Therefore, in a further embodiment of the invention there is provided a method of the invention wherein the frequency is other than about 2.45GHz and the cross section is dimensioned according to the frequency required relative to cross sectional dimensions of 86 by 43 mm for 2.45 GHz.

The effect of the microwave radiation is believed to be independent of any microwave heating effect on the composition; therefore in some embodiments it is advantageous to keep the reaction mixture at a relatively constant temperature, i.e. to keep the reaction mixture under isothermal control.

According to a further embodiment of the invention there is provided a method of enhancing the activity of an enzyme said method comprising irradiating a composition comprising the enzyme and a substrate with microwave radiation delivered via a vviaveguidc, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in composition rather than other components of the composition, and wherein the method is carried out under substantially isothermal conditions.

According to a further embodiment of the invention there is provided a method of enhancing the activity of an enzyme said method comprising the irradiation of a composition comprising an enzyme and substrate solution with microwave radiation.

For the avoidance of doubt the substrate is any compound whose chemical transformation is catalysed by an enzyme.

An enzyme for use in a method of the invention is any enzyme which shows an enhancement of activity and/or an enhancement of the yield of products when exposed to microwave radiation. Example of enzymes include: cellulase, hem icellul ase, amylase, xylanase, pectinase and chitinase, a and P-glucosidase, glucoamylase, sucrase, isomaltase, lactase, invertasc and maltase.

In one embodiment the enzyme is a cellulase.

According to a further aspect of the invention there is provided a method of enhancing the activity of a cellulase, said method comprising the provision of microwave radiation.

According to a further aspect of the invention there is provided a method of enhancing the activity of a cellulase, said method comprising the irradiation of a composition comprising the cellulase and a substrate with microwave radiation delivered via a waveguide, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the wavcguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

According to a further embodiment of the invention there is provided a method of enhancing the activity of a cellulase said method comprising the irradiation of a cellulase and substrate solution with microwaves.

In general, when the enzyme is a cellulase, the substrate comprises cellulose, but could comprise any carbohydrate polymer, including polymer, oligomer and dimer, which can be cleaved by a cellulase. The substrate could also be a biomass.

According to a further embodiment of the invention there is provided a method of enhancing the degradation of cellulose by a cellulase said method comprising the irradiation of said cellulase with microwaves.

According to a further embodiment of the invention there is provided a method of enhancing the degradation of cellulose by a cellulase said method comprising the irradiation of a composition comprising the cellulase and cellulose with microwave radiation.

In our experiments pre-irradiation of the cellulose has no effect on the rate the pre-irradiated cellulose is digested by cellulase. Therefore, in another embodiment of the invention the cellulose is not pre-irradiated with microwave radiation prior to enhancing the activity of cellulase using microwave radiation.

In general the composition is any aqueous composition, for example, it may be a buffered solution, but could be any solution in which an enzyme, such as cellulase, is enzymatically active, including suitable organic solvents and suitable mixtures of organic solvents and aqueous solvents. Suitable organic solvents include glycerol, ethylene glycol and formamide, aqueous mixtures of organic alcohols for example, up to 25% ethanol, sec for example Iyer and Ananthanaarayan (2008), Process Biochemistry 43, 1019-1032 incorporated herein by reference. A solution could also comprise cellulase enzymes incorporated into micelles, for example reverse micelles composed of Triton X100, sodium bis(2-ethylhexyl)sulphosuccinate, cetyltrimethyl ammonium bromide or mixtures thereof, for example see Chen et al (2006) Bioch m B ophys Acta 1764, 1029-1035 incorporated herein by reference.

Enzymatic activity is dependent on temperature, therefore methods of the invention comprise methods conducted at any temperature where an enzyme, such as cellulase is enzymatically active. In one embodiment the temperature is in the range 0°C to 99°C. In a further embodiment the temperature is the range about 1°C to about 80°C, such as about 1°C to about 70°C, such as about 1°C to about 60°C, such as about 10°C to about 60°C, such as about 20°C to about 60°C, such as about 30°C to about 60°C, such as about 40°C to about 60°C, for example, about 50°C. In a further embodiment the temperature is in the range about 30°C to about 50°C or about 35°C to about 45°C.

Cellulase from different sources can have different active temperature ranges. In general cellulases arc enzymatically active in the range about 1°C to about 70°C, such as about 40°C to about 60°C, for example, about 50°C. Examples of sources of cellulases include Trichodertna reesei and Aspergillus niger.

Microwaves can be delivered at a range of frequencies. In methods of the inventions the frequency of microwaves used is any frequency in the range 0.3 GHz to 16 GHz which enhances enzymatic activity.

Microwaves can be delivered at a range of power levels. In methods of the inventions the power level of the microwaves used is any power level, within the range about 1 W to about 100W, which enhances enzymatic activity.

We have found that there is not a simple linear relationship between the power of the microwave energy delivered and the enhancement of enzymatic activity even at power levels below that which would induce enzymes denaturation. In methods of the invention, power levels are any power which enhances enzymatic activity, for example about 1W to about 100W, such as about 1W to about 95W, about 1W to about 90W, about 1W to about 85W, about 1W to about 80W, about 1W to about 75W, about 1W to about 70W, about 1W to about 65W, about 1W to about 90W, about 25W to about 75W, about 40W to about 60W or about 45W to about 55W. Power levels may also comprise about 1W to about 50W, about 20W to about 40W and about 20W to about 30W.

We have also found that there is a power optimum for the relationship between microwave energy and the enhancement of enzymatic activity. As the microwave energy is increased from 0 watts the enhancement of enzymatic activity peaks at a low microwave energy after which there is a decrease in enzymatic activity as the microwave energy is increased further. his power optimum is expected to vary for different enzymes. The c skilled man would be able to titrate the increase in microwave power to identify the power optimum for any enzyme to optimise the enhancement of activity for a given enzymatic reaction.

Suitable microwave frequency levels are frequencies which enhances enzymatic activity. In general microwave frequencies would be in the range from about 1 to about 16GHz, for example from about 1 to about 10 GHz. Frequencies may also be in the range from about 1 to about 5 GHz, about 1 to about 4 GHz and about 2 to about 3 GHz, for example from about 2.5 GHz such as about 2.45 GHz.

We have found that microwave irradiation can enhance the generation of commercially valuable compounds by microbes, particularly where a biomass is provided to the microbes.

According to a second aspect of the invention there is provided a method for the production of a compound by a microbe characterised in that the production of the compound is enhanced by the application of microwave radiation.

According to a further embodiment of the invention there is provided a method for the production of a compound by a microbe in a culture medium characterised in that the production of the compound is enhanced by irradiation of the culture medium with microwave radiation delivered by a waveguide, wherein said microwave radiation has: (0 a frequency from 0.3 GHz to 16 GHz; and (i) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

In a further embodiment of the second aspect of the invention the method further comprises the provision of an energy source, such as a biomass.

The enhancement of compound production may be via the enhancement of the activity of the enzymes involved in the production of the compound, enhancement of the degradation of a biomass to a carbon source used by the microbes, enhancement of the activity of other enzymes present in the microbes, for examples enzymes involved in respiration, or any combination of these. Therefore, in one embodiment of the invention the enhancement of the production of said compound is via enhancement of the activity of an enzyme which generates said compound or an intermediate in the generation of said compound. In a further embodiment of the invention the enhancement of the production of said compound is via enhancement of the degradation of a biomass. In a yet further embodiment of the invention the enhancement of the production of said compound is via enhancement of the activity of an enzyme which generates said compound or an intermediate in the generation of said compound and the enhancement of the degradation of a biomass. In a yet further embodiment of the invention the enhancement of the production of said compound is via enhancement of the activity of an enzyme involved in respiration.

The microbes may be cultured in a suitable culture medium in a suitable fermentor to produce a compound. Any suitable fermentor may be used, which can incorporate a microwave source, including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, a plug fermentor, a packed bed fermentor, a fluidised bed fermentor or any combination thereof. Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986; El-Mansi, Fermentation Microbiology and Biotechnology, second edition, Taylor and Francis, London, 2007; Stanbury et al., Principles of Fermentation Technology, second edition, Butterworth Heinemann, 2003, each of which is incorporated herein by reference).

Consideration must be given to appropriate fermentation medium, pll, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the microbe, the fermentation, and the process. The culture medium used is not critical, but it must support growth of the microbe used and promote biomass degradation and the biosynthetic pathway(s) necessary to produce the desired compound or compounds. Suitable culture media would be familiar to the skilled man. A conventional culture medium may be used, including, but not limited to, complex media containing organic nitrogen sources such as yeast extract or peptone and optionally at least one fermentable carbon source; minimal media; and defined media. A suitable nitrogen source, such as an ammonium salt, yeast extract or peptone, minerals, salts. cofactors, buffers and other components, would be familiar to those skilled in the art (Bailey et al., supra). Suitable conditions for the extractive fermentation depend on the particular microbe used and may be readily determined by one skilled in the art using routine experimentation. In general the carbon source would be provided by a biomass.

Fermentation methods include batch, fed-batch, continuous or semi-continuous perfusion Fermentors suitable for use in methods of the invention included disposable fermentors, for example bags which can be inserted into resilient vessels such as glass or stainless steel vessels. Further disposable fermentors include systems based on wave agitation for example see US Patent 6,544,788 and International patent application WO 2000/66706, incorporated herein by reference.

According to a further embodiment of the second aspect of invention there is provided a method of producing a compound comprising: (i) fermenting a culture medium comprising at least one microbe capable of producing said compound; and (ii) optionally, isolating said compound from said culture medium, characterised in that said culture medium is irradiated with microwaves.

According to a further embodiment of the second aspect of invention there is provided a method of producing a compound comprising: (i) fermenting a culture medium comprising at least one microbe capable of producing said compound; and (ii) optionally, isolating said compound from said culture medium, characterised in that the culture medium is irradiated with microwaves, wherein said microwavc radiation is delivered via a waveguide and has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwavc source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

According to a further embodiment of the second aspect of invention there is provided a method of producing a compound comprising: (i) fermenting a culture medium comprising an energy source, for example a biomass, and at least one microbe capable of producing said compound; and (ii) optionally, isolating said compound from said culture medium.

characterised in that said culture medium is irradiated with microwaves.

According to a farther embodiment of the invention there is provided a method for the production of a compound by a microbe characterised in that the production of the compound is enhanced by the application of microwavc radiation, comprising: (i) fermenting a culture medium comprising an energy source and at least one microbe capable of producing said compound; (ii) irradiating said culture medium with microwave radiation delivered via a waveguide, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition; and (iii) optionally, isolating said compound from said culture medium.

According to a further embodiment of the second aspect of invention there is provided a method of producing a compound comprising: (i) providing an energy source, for example a biomass; (ii) providing a microbe capable ofproducing a compound; (iii) providing a culture medium, comprising said biomass and said microbe; (iv) fermenting said culture medium to produce said compound; and (v) optionally, isolating said compound from said culture medium.

characterised in that said culture medium is irradiated with microwaves.

According to a further embodiment of the second aspect of invention there is provided a method of producing a compound comprising: (i) providing an energy source, for example a biomass; (ii) providing a microbe capable of producing a compound; (iii) providing a culture medium, comprising said biomass and said microbe; (iv) fermenting said culture medium to produce said compound; and (v) optionally, isolating said compound from said culture medium. characterised in that said culture medium is irradiated with microwaves, wherein said microwave radiation is delivered via a waveguide and has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

In embodiments of the invention comprising a biomass, the microbe may be able to degrade said biomass to provide glucose or may require degradation of the biomass to comprise glucose or shorter length glucose polymers before being able to use the biomass as an energy source. Therefore, in a further embodiment of the second aspect of the invention, there is provided a method of producing a compound comprising: (i) providing a biomass; (ii) providing a composition capable of degrading said biomass, for example a composition comprising a cellulose; (iii) incubating said biomass with said composition; (iv) providing a microbe capable of producing a compound (v) fermenting a culture medium comprising said microbe and said degraded biomass to produce a compound (vi) optionally, isolating said compound from said culture medium; characterised in that microwave radiation is provided during degradation of the biomass or during production of the compound or both.

According to a further embodiment of the second aspect of the invention, there is provided a method of producing a compound comprising: (i) providing a biomass; (ii) providing a composition capable of degrading said biomass, for example a composition comprising a cellulose; (iii) incubating said biomass with said composition; (iv) providing a microbe capable of producing a compound; (v) fermenting a culture medium comprising said microbe and said degraded biomass to produce a compound (vi) optionally, isolating said compound from said culture medium; characterised in that microwave radiation is provided during degradation of the biomass or during production of the compound or both, wherein said microwave radiation is delivered via a waveguide and has: (i) a frequency from 0.3 GHz to 16 GHz; and (ii) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition.

It should be understood that in methods of the invention where the biomass is not degraded by the microbe, the biomass, biomass degrading composition and microbe may be mixed together at the start of the method or the biomass and degrading composition may be mixed and biomass degradation allowed to begin before addition of the microbe. In a further embodiment biomass degradation and compound production can be conducted in separate steps such that a degraded biomass comprising an energy source, such as glucose or an intermediate in the degradation of cellulose is provide to the microbe. In such separate processes microwave radiation can be provided at just one or more than one stage of the process.

The biomass may be pre-treated before enzymatic degradation to increase the efficiency of the cellulose degradation. Pre-treatment comprises breaking the lignin seal, solubilising hemicellulose and/or disrupting the crystalline structure of cellulose. Various pre-treatment options would be familiar to the skilled man to fractionate. solubilise, hydrolyse and separate cellulose, hemicellulose and lignin components. These include steam explosion, acid treatment, alkaline treatment, treatment with SO,, treatment with hydrogen peroxide, ammonia fibre explosion and organic solvent treatments. In one embodiment there is no pre-treatment of the biomass with microwave radiation. In a further embodiment there is no pre-treatment of the biomass.

According to a further embodiment of the second aspect of the invention there is provided a microwave irradiated biomass.

According to a further embodiment of the second aspect of the invention there is provided a microwave irradiated culture media comprising a biomass and/or breakdown product thereof.

According to a further embodiment of the second aspect of the invention there is provided a microwave irradiated culture media comprising a biomass and/or breakdown product thereof and one or more microbes.

According to a further embodiment of the invention there is provided a compound obtained by a process of the second aspect of the invention.

It should be noted that in the first and second aspects of the invention the microwave radiation can be provided as a continuous signal for continuous power delivery or as a pulsed signal for delivering power averaged over a number of pulses.

In a further embodiment there is provided a microwave apparatus suitable for fermenting said microbes to produce said compound and/or enzyme catalysis. Therefore, in a third aspect of the invention there is provided a microwave apparatus for enhancing enzymatic activity comprising: (i) a reactor vessel; (ii) a continuous microwave source for the provision of microwaves; (iii) a means for controlling the power of said microwaves; (iv) a means for controlling the frequency of said microwaves; and (v) a means for controlling the temperature of the reactor vessel.

In a further embodiment of the third aspect of the invention there is provided a microwave apparatus for fermentation comprising: (i) a reactor vessel; (ii) a continuous microwave source for the provision of microwaves; (iii) a means for controlling the power of said microwaves; (iv) a means for controlling the frequency of said microwaves; and (v) a means for controlling the temperature of the reactor vessel.

In a further embodiment there is provided a microwave apparatus for irradiating a composition comprising an enzyme and a substrate with microwaves comprising: (i) a reactor vessel for in use containing said composition; (ii) a microwave source for the provision of microwaves; and (iii) a waveguide arranged with the microwave source to in use deliver energy therefrom to the reactor vessel; characterised in that the wavcguidc is configured such that in use with a composition comprising an enzyme energy provided to the waveguide by the microwave source is substantially absorbed by the enzyme in the reactor vessel rather than other components of the composition in the reactor vessel.

In a further embodiment there is provided a microwave apparatus for irradiating a composition comprising an enzyme and a substrate with microwaves comprising: (i) a reactor vessel for in use containing said composition; (ii) a continuous microwave source for the provision of microwaves; (iii) a means for controlling the power of said microwaves; (iv) a means for controlling the frequency of said microwaves; (v) a wavcguidc arranged with the microwave source to in use deliver energy therefrom to the reactor vessel; and (vi) optionally a means for controlling the temperature of the reactor vessel; and characterised in that the wavcguidc is configured such that in use with a composition comprising an enzyme energy provided to the waveguide by the microwave source is substantially absorbed by the enzyme in the reactor vessel rather than other components of the composition in the reactor vessel.

The wave guide has any cross section such that when the microwave radiation is supplied to a composition comprising an enzyme and a substrate the energy is substantially absorbed by the enzyme rather than other components of the composition. When the microwave radiation has a frequency of 2.45 GHz the waveguide is a substantially rectangular and has cross sectional dimensions of about 86 by about 43 mm. In a further embodiment when the frequency is other than 2.45 GHz the cross section is dimensioned according to the frequency required relative to cross sectional dimensions of 86 by 43 mm for 2.45 GHz.

The length of the waveguide is not critical to the operation of the waveguide. In one embodiment the waveguide has a length between about 10cm to about 100cm.

Thc reactor vessel comprises any vessel which allows transmission of microwave radiation to the contents. The materials for such vessels would be well known to the skilled man, for example glass, for example borosilicatc glass, quartz, or plastic, such as TeflonTM. Such reactor vessels may consist of one piece or several pieces, for example the vessel may disassemble for easier cleaning or my allow use of a disposable inner, such as a sterile disposable inner sleeve. The volume of the reactor is any volume required for the experiments being performed. In one embodiment the reaction vessel has a volume of about 2 litres. In one embodiment the reactor vessel is cylindrical in cross section.

The microwave source may deliver microwave radiation from one direction or concurrently from a number of directions. When delivering the microwave radiation concurrently from a number of directions, the microwave source may comprise one microwave generator or a number of microwave generators arranged as a series around the vessel. Such series of microwave generators would be expected to allow a more uniform delivery of microwave radiation.

The microwave apparatus comprises a control means for controlling the power of the microwave signal to reduce or increase the energy supplied to the reactor vessel.

The control means may be configured to provide the microwave radiation having a power from about 1W to about 300W, for example from about 1W to about 150W.

Thc power consumption of the microwave may be optimised by tuning of the microwave source to give minimum reflected power.

The microwave apparatus comprises a control means for controlling the frequency of the microwave signal to reduce or increase the frequency of the microwave radiation supplied to the reactor vessel.

The control means may be configured to provide the microwave radiation having a frequency from about 0 GHz to about 16 GHz. Frequencies may also be in the range about 0 to about 10 GHz, about 0 to about 5 GHz, about I to about 5 GHz, about I to about 4 GHz and about 2 to about 3 GHz. In one embodiment the frequency is about 2.45 GHz. In a further embodiment the frequency is 2.45 GHz The means for controlling the temperature of the contents of the vessel may be internal to the vessel or external to the vessel for example an internal submerged heating/cooling clement or an internal tube, such as a coil, through which a heated or cooled liquid is circulated, or an external jacket such as a water jacket, or other heated or cooled liquid, filled jacket.

The apparatus may further comprise a mixing means. The mixing means may be internal to the vessel, for example, an internal stirrer or external such as via shaking or other form of agitation.

The apparatus may further comprise a shield means, to reduce or prevent extraneous microwave radiation being delivered other than to the reactor vessel. The shield means comprises any means which protects the user from extraneous microwave radiation. Such means may comprise stainless steel, lead or any other suitable metal. Such shielding means would be well known to the skilled man.

The apparatus may further comprise one or more probes to measure the physical parameters of the material in the reactor vessel. Such probes include a p11 sensor, a dissolved oxygen sensor and a temperature sensor. Such probes would be familiar to the skilled man. The apparatus may further comprise one or more ports for delivering materials, such as reaction components or gases, to the reactor vessel and/or removing material from the reactor vessel.

The apparatus may further comprise a waveguide for conveying the microwave radiation to the reactor vessel. In general the waveguide comprises a linear structure that conveys the microwaves between the microwave source and the reactor vessel, such as a hollow metal pipe.

The term 'about' when used in this specification refers to a tolerance of ±5%, of the stated value, i.e. about 50% encompasses any value in the range 45% to 55%, i.e. about 50°C encompasses any value in the range 47.5% to 52.5%. In further embodiments 'about' refers to a tolerance of +2%, +0.5%, +0.2% or 0.1% of the stated value.

The term 'aqueous' refers to the conditions where water is present as the solvent and conditions where water is present with other solvents. In one embodiment 'aqueous' refers to conditions where water is the only solvent used. In a further embodiment 'aqueous' refers to conditions where water is used as the solvent but small amounts of other solvents may be present, for example, from the addition of components to the reaction medium, for example, where the water is >90% of the solvent present, for example >95%, >98% or >99°A of the solvent present.

The term 'biomass' means any material which can be used as a food source for a microbe. In general biomass would comprise cellulose, which is composed of 1,4 beta-linked glucose molecules. Biomass typically also contains other components of plant fibre such as lignin, starch, hemi-cellulose and pectin, or other materials. Examples of biomass include hay, grass, paper, cotton, and wood. Further examples, of biomass include corn stover, wheat straw, rice straw, rice hulls, baggasse fibre, news print, cotton gin trash, black locust, douglas fir, hybrid poplar, eucalyptus, pine, switch grass and generic plant cell wall fibres.

The term cellulase' means any enzyme involved in the degradation of cellulose to oligo-di-and mono-saccharides' i.e. any enzyme which cleaves cellulose to a shorter chain carbohydrate intermediate or directly to glucose. This phrase further comprises any enzyme involved in the further cleavage of any intermediate in the degradation of cellulose to glucose. Cellulase comprise exocellulases, endocellulases, cellobiase (f3-gluosidase), oxidative cellulases and cellulose phosphorylases. Examples of such enzymes include cellobiohydrolase (for example cellobiohydrolase I and cellobiohydrolase II), endoglucanase and beta-glucosidase. It should be understood that the word cellulase may relate to one enzyme or a mixture of enzymes capable of degrading cellulose or degradation products of cellulose.

By the term 'compound' we mean any compound which can be produced by a microbe and which has utility in a human activity, for example as an industrial product or intermediate or a research tool. Compounds include small organic molecules (molecular weight below 1000KDa), large organic molecules (molecular weight greater than 999 KDa) and macromolecules such as peptides, polypeptides, proteins and nucleic acids. It should be understood that the word compound relates to one compound with utility or a mixture of compounds with utility such that a method of the invention could produce one or more useful compounds.

The phrase 'isothermal control' refers to a system in which the temperature remains relatively constant. This typically occurs when a system is under internal or external temperature control and the system is able to continually adjust the temperature to maintain a relatively constant temperature. Temperature control may be via any method which keeps the temperature relatively constant, for example, externally such as via a thermal jacket or internally such as via a coil or loop within the reaction mixture. The isothermal control may be ±5°C, ±2°C, ±1°C or ±0.5°C.

By the term 'microbe' we mean any cellular system capable of producing a compound. Such microbes include bacteria (gram positive or gram negative), yeast or mammalian cell lines. Such microbes also include any artificial organism capable of producing a compound. Microbes may have their natural genotype, they may be transfected with a gene or genes which allow the microbe to produce a compound or any other genetic modification different from the natural genotype. Suitable microbes include: members of the genera, Zyntontonas, Escherichia, Salmonella, Rhodococcus, Pseudotnonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paetzibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Klityverontyces, Issatchenk-ia, Saccharomyces and Tricoderma. For example hosts include: Escherichia colt, Alcaligenes eutrophus, Bacillus licheniformis, Puenibacillus macerans, Rhodococcus erythropolis, Pseudomonas Lactobacillus plantarum, Enterococcus laecium, Enterococcus gallinariutn, Enterococcus faecalis, Pediococcus pentosaceus, Pediococcus acidilactici, Bacillus subtilis, Saccharontyces cerevisiae and Tricoderma reesei and virdi.

The term microwave radiation' refers to the provision of microwaves. Microwaves are electromagnetic waves with frequencies between 300 MHz (0.3 GHz) and 300 GHz. In general, for applications of the present invention microwaves in the range about 1 Gllz to about 16 GHz would be most suitable, for example about 2 to about 10 GHz, about 2 to about 8 GHz or about 2 GHz to about 5GHz. In one embodiment the frequency of the microwave radiation is about 2.45 GHz.

The terms 'peptide' and polypeptide' refers to a polymer of amino acids. The amino acids forming said polymer arc selected from naturally occurring amino acids and non-naturally occurring amino acids. Such peptides may include modifications, such as glycosylation.

For the avoidance of doubt where this specification refers to enhancing the activity of an enzyme this refers to enhancing the rate of conversion of substrate to product by the enzyme and/or increasing the yield of product from the reaction catalysed by the enzyme.

The application will now be exemplified by the following non-limiting examples, in which the following abbreviations are used: BGL -13-glucosidase; CBH -cellobiohydrolase; CBH 1 -cellobiohydrolase L CBH II -Cellobiohydrolase II; EGL -cndoglucanasc; HPLC -high performance liquid chromatography YDP -Yeast Extract, Dextrose, Peptone And in which: Figure 1: shows a schematic of the microwave reactor vessel used in the studies.

Figure 2: shows the enzymatic degradation of cellulose to glucose via cellobiose, cellotriose and cellotctraosc via the action of endoglucanase (EGL), cellobiohydrolasc I (CBH 1) and cellobiohydrolase 11 (CBH 11), and beta-glucosidase (BGL). 2.1, 2.2 and 2.3 refer to Examples 1, 2 and 3 respectively.

Figure 2a is related to Figure 2. Figure 2 concludes with glucose production whilst Figure 2a shows the additional steps of biomass destruction, through hydrolysis and into glucose metabolism to the fermentation end points of biomass and product formation (e.g. ethanol). In the figure: Section I -Partial hydrolysis by CBH/EGL digestion of cellulose to the predominant product cellobiose; Section 2 -Complete hydrolysis by CB11/EGL and BGL digestion of cellulose to the end product glucose; Section 3 -Partial hydrolysis by BGL digestion of cellobiose to the end product glucose; Section 4 -Fermentation of glucose to ethanol and biomass; Section 5 -Consolidation of complete hydrolysis by CBH/EGL and BGL to the intermediate product of glucose with simultaneous fermentation by an ethanogen to ethanol and biomass; and Section 6 -Complete, direct, native fermentation by a fungal strain of lignocellulosic biomass to fungal biomass.

Sections 1 to 5 have been observed in the experiments performed. Section is the expected process during fungal fermentation.

Figure 3: shows the effect of microwave radiation on the degradation of cellulose to cellobiose in the presence of CBH and EGL wherein the x axis is time in hours and the y axis is concentration of cellobiose in grams/litre. The lines relate to different microwave energies wherein (1) relates to OW, (2) relates to 50W, (3) relates to 100W and (4) relates to 150W.

Figure 4: shows the effect of microwave radiation on the degradation of cellulose to glucose in the presence of CBH and EGL wherein the x axis is time in hours and the y axis is concentration of cellobiose in grams/litre. The lines relate to different microwave energies wherein (1) relates to OW, (2) relates to 50W, (3) relates to 100W and (4) relates to 150W.

Figure 5: shows the percentage saccharification after microwave radiation of cellulose to glucose in the presence of CBH and EGL wherein the x axis is time in hours and the y axis is percentage saccharification. The lines relate to different microwave energies wherein (1) relate to OW, (2) relate to 50W, (3) relate to 100W and (4) relate to 150W.

Figures 5a: shows the relationship between power and the enhancement of enzyme activity of CBH arid EGL as measured by formation of glucose and the percentage saccharification. In the figure the x axis is microwave power in Watts, the left y axis is the rate of formation of glucose in Ohre/hour and the right y axis is percentage saccharification per hour. In the figure the diamonds relate glucose and the squares relate to the percentage saccharification.

Figure 6: shows the effect of microwave radiation on the degradation of cellulose to cellobiosc in the presence of CBII, LGL and BGL wherein the x axis is time in hours and the y axis is concentration of cellobiose in grams/litre. The lines relate to different microwave energies OW, 12W, 25W, 50W and 75W. In the figures the five lines are superimposed on each other.

Figure 7: shows the effect of microwave radiation on the degradation of cellulose to glucose in the presence of CBH, EGL and BGL wherein the x axis is time in hours and the y axis is concentration of glucose in grams/litre. The lines relate to different microwave energies wherein (1) relates to OW, (2) relates to 12W, (3) relate to 25W, (4) relate to 50W and (5) relates to 75W.

Figure 8: shows the percentage saccharification after microwave radiation of cellulose to glucose in the presence of CBH, EGL and BGL wherein the x axis is time in hours and the y axis is percentage saccharification. The lines relate to different microwave energies wherein (1) relates to OW, (2) relates to 12W, (3) relates to 25W, (4) relates to 50W and (5) relates to 75W.

Figure 8a: shows the relationship between power and the enhancement of enzyme activity of CBH, EGL and BGL as measured by formation of glucose and the percentage saccharification. In the figure the x axis is microwave power in watts, the left y axis is the rate of formation of glucose in g/litreihour and the right y axis is percentage saccharification per hour. In the figure the diamonds relate to glucose and the squares relate to the percentage saccharification.

Figure 9: shows the effect of microwave radiation on the degradation of cellulose to cellobiose in the presence of BGL wherein the x axis is time in minutes and the y axis is concentration of cellobiose in grams/litre. The lines relate to different microwave energies OW, 12W, 25W, 50W and 75W. The line denoted by diamonds is OW, the line denoted by circles is 25W and the other lines are superimposed upon one another.

Figure 10: shows the effect of microwave radiation on the degradation of cellulose to glucose in the presence of BGL wherein the x axis is time in minutes and the y axis is concentration of glucose in grams/litre. The lines relate to different microwave energies wherein (1) relates to OW, (2) relates to 12W, (3) relate to 25W, (4) relates to 50W and (5) relates to 75W.

Figure 11: shows the percentage saccharification after microwave radiation of cellulose to glucose in the presence of BGL wherein the x axis is time in minutes and the y axis is percentage saccharification. The lines relate to different microwave energies wherein (1) relate to OW, (2) relates to 12W, (3) relates to 25W, (4) relates to 50W and (5) relates to 75W.

Figure 12 shows the effect of microwave radiation on the rate of microbial fermentation wherein the x-axis relates to microwave power in watts, the left y axis relates to the ethanol production in grams/litre/hours and the right y axis relates to the uptake of glucose in grams/litre/hour. In the graph triangles relate to the microbial growth rate, diamonds relates to the production of ethanol and squares relates to the uptake of glucose.

Material and Methods (1) Microwave reactor Experiments were conducted in a bespoke bench top research reactor with a total volume of 2 litres. The reactor comprised the following components: 21 fermentation stirred tank reactor made of borosilicate glass vessel with stainless steel top plate (Metrohm-Aplikon) 24v DC geared agitation motor (PI systems) 0-30 V peak 2 Amp variable power supply (RS Components E30/1) 12mm dissolved oxygen probe (Broadly James Technologies) 12mm fermentation autoclavable pH probe (Broadly James Technologies) Coaxial launching cable (Saricm) A microwave source selected from: (a) 0-200 Watt variable frequency (0-10GHz) solid state microwave generator (Sariem); or (b) 0-200 Watt fixed frequency (2.45 GHz) microwave generator (Microtron) 2.45 GHz S band stub tuning section 2.45 GHz S band launching section The vessel was a borosilicate glass vessel with stainless steel top plate, incorporating probe ports for a dissolved oxygen (DO) probe, a pH probe, a triple port and a condensing gas exhaust port. A bottom draw sample tube, a top draw sample tube (flush), a top draw sample tube (protruding), a submersed thermal regulation loop and a bottom (central) sparger tube were also incorporated. Agitation was through a central agitation shaft with three Rushton turbines placed at equal distance up the length of the shaft. Above the top plate the agitator shaft was coupled to a 24v DC geared motor (PI Systems) through the use of a modified coupling. To ensure agitation with the insoluble cellulose, agitation was set at 300 rpm for complete mixing without aeration and foam formation. Temperature control was carried out through the use of a circulating water bath pumping water through the heat exchanger loop within the reactor. In the event of the heating effect being less than the heat loss from the system, the heat exchanger automatically compensated using the same heating/cooling circuit.

The reactor vessel was placed within a metal cavity where the bioreactor top plate is used as the microwave cavities top face, with a microwave launching section (with tuning stubs) attached by a milled flat surface bisecting the circumference of the cavity. The microwave sources used were a Microtron 200 series unit and a Sairem 200W solid state continuous wave microwave generator with connection through the use of a co-axial cable. The biorcactor is placed within the cavity with the tuning stubs used to minimise reflected power, and the reactor turned in its seating position to ensure minimal reflected power. Negligible microwave leakage was observed.

(if) Reactor substrates Glucose -Used as the sole carbon source in microwave irradiated fermentation studies.

Cellulose -To remove bias of lignin content and feedstock variation, a standardised cellulosic feedstock was used. a-cellulose (Sigma-Aldrich C8002) comprising 131-4 linked glucose chains of approximate fibre length of 4000-6000 units in length was used, where the bulk substrate is mixed prior to experimentation to prevent substrate batch variability.

Cellobiose -In reactions investigating the enzymatic rate reaction of BGL, cellobiose (s gmaA ldrich C7252) was used as the sole substrate.

Enzymes -All of the reactions investigated made use of cellulases (Sigma-Aldrich C8546) from Trichoderma reesei strain ATCC26921, and 13-glucosidase (Sigma-Aldrich C6105, synonym Novozyme 188) from Aspergillus niger. All materials were stored in accordance to supplier's recommendations.

(iii) Microwave Reactor Conditions -cellulose hydrolysis by cellulose enzymes Experiments were conducted in the microwave reactor at a volume of I litre in 0.IM sodium citrate buffer (pH 4.8). To this was added: (i) 2000 of Tetracycline (10mg/mL in 70% ethanol) (Sigma-Aldrich 87128); 200u1 Cycloheximide (10mg/mE in distilled water) (Sigma-Aldrich 01810); and (iii) 10g/L a-cellulose.

The temperature was maintained at 50°C using the heat exchanger.

(iv) Microwave Reactor Condition -Saccharomyces fermentation (anaerobic) Experiments were conducted in the microwave reactor at a volume of 1 litre in 5% YDP media, pH 4.5 -5. No aeration was supplied, and agitation was set at 150rpm. To this was added: (i) 10m1 Saccharomyces cerevisiae with a biomass OD value at 600nm <0.8; or (ii) 2g Saccharomyces cerevisiae in freeze dried form. The temperature was maintained at 30 °C using the heat exchanger throughout the experimental time frame.

(v) HPLC Analysis of Breakdown Products For the determination of oligomer, dimer and mono-saccharidc constituents, HPLC was employed with the use of a REZEX ROA column (Phenomenex), 300mm length by 5mm ID at 65°C. Mobile phase (4mM sulphuric acid, flow rate: 0.6mIlmin) was used with refractive index (RI) detection on a Perkin Elmer 200 Series system with a sample time of 30 minutes. All data was recorded on the proprietary Perkin Elmer TotalChrom software. A 200 Series autosampler with pettier sample rack (4°C) is used for automatic sample loading. Injection volumes were 10(11 with 500 being used where sample threshold was low. Standard curves were used for peak identification and quantification with citrate used as the internal standard for sample standardisation.

Standard curves for HPLC analysis were constructed using analytical grade reagents. Standard curves for xylose (0.01-0.10), glucose (0.50-10g/1), cellobiose (0.025-100), cellotriose (0.01-20) and cellotetarose (0.01-20) were used with at least 6 data point per curve. As all curves represent a linear relationship, the LINEST function (Microsoft ExcelTM) was used for determination of gradient and intercept for each reagent in conjunction with the coefficient of determination (R2) value. Standard curves are conducted for each exchange and replenishment of mobile phase.

(vi) Fermentation tracking Fermentation tracking was conducted using CO2 evolution, pH monitoring and DO2 monitoring. All parameters were controlled through the use of a bespoke Broadley Technologies fermentation controller.

Example 1: Investigation of variable microwave power on cellobiose and glucose accumulation from Cellulose hydrolysis with CBH/EGL in the absence of BGL The effect of 2.45 GHz microwave radiation was investigated in the range 0 -150W.

Experiment 1 was carried out in a 1 litre volume in citrate buffer containing tetracycline and cycloheximide as described above. The reactor was maintained at 50°C for 12-16 hours to ensure temperature equilibrium. 10 g cellulose was then added to give a final concentration of 10g/L. The reaction was started by the addition of 40 FPU/g cellulose of CBH and EGL. Samples were taken at T=0 through to 160 hours at hourly time points for the first ten hours, and then every other hour for each subsequent working day. Each sample was quenched by boiling for 10 minutes, centrifuged (15 minutes at 3400rpm (Sanyo MSE microfuge)) then filtered through a 0.2fm HPLC grade filter and stored on ice prior to HPLC analysis.

For variation of microwave power, the microwave generator at a set power output was switched on prior to enzyme addition and the thermal input adjusted to give the bulk temperature of 50°C. Once established and stabilised, minimal adjustment was required with temperature variation ±0.2°C. In general the apparatus was set up and left to stabilise overnight.

In this experiment an increase in saccharificat ion was observed with a non-linear response to microwave radiation with most enhancement observed at 50W (see Figure 5 and 5a). Interestingly, although with the inclusion of CBH and EGL, the reaction would have been expected to stop at cellobiose, further degradation to glucose was observed (sec Figures 3 and 4). One possibility is that this represents enhancement of the activity of a contaminating BGL activity in the enzyme preparation used in the experiment. However, repeating the studies with the inclusion of vanillin, an inhibitor of BGL, showed the same results, suggesting that the generation of glucose was not due to a contaminating BGL activity.

Table 1 below shows the initial rates of reaction (average over the first 2 hour time period) for the hydrolysis of cellulose by EGL/CBH to cellobiose and glucose and for the percentage saccharification.

Microwave Glucose Cellobiose power Watts Gradient le Difference Gradient It Difference (41/41[T]) (%) (dP/d[1]) (%) 0 0.131 0.924 100 0.066 0.926 100 0.206 0.899 198.3 0.258 0.903 389.9 0.155 0.953 118.3 0.248 0.874 374.4 0.096 0.901 73.7 0.115 0.938 174.0 Microwave % saccharification power Watts Gradient 12.2 Difference (dP'd[T]) (%) 0 0.026 0.893 100 0.080 0.964 300.8 0.056 0.988 210.5 0.020 0.978 75.6 Table 1. Summary of initial rates for CBH/EGL on cellulose with varying microwave irradiation energies Example 2: Investigation of variable microwave power on cellobiose and glucose accumulation from Cellulose hydrolysis with CBH/EGL and BGL The effect of 2.45 GHz microwave radiation was investigated in the range 0 -75W.

Experiment 2 was carried out in a 1 litre volume in citrate buffer containing tetracycline and cycloheximide as described above. 10 g cellulose was added to give a final concentration of 10g/L. The reactor was maintained at 50°C for 12-16 hours to ensure temperature equilibrium.

For variation of microwave power, the microwave generator at a set power output was switched on prior to enzyme addition and the thermal input adjusted to give the bulk temperature of 50°C. Once established and stabilised, minimal adjustment was required with temperature variation +0.2°C. In general the apparatus was set up and left to stabilise overnight.

The reaction was started by the addition of 40 FPU/g cellulose of CBH and EGL, I ml of BGL preparation at 250units,/g pNPGLiml. Samples are taken at T=0 through to 160 hours at hourly time points for the first ten hours, and then every other hour for each subsequent working day. Each sample is quenched by boiling for 10 minutes, centrifuged (15 minutes at 3400rpm) then filtered through a 0.2um HPLC grade filter and stored on ice prior to HPLC analysis.

In this experiment, as expected, all the cellulose was converted to glucose with no significant build up of cellobiose (see Figure 6). As before an increase in saccharification was observed with the accumulation of glucose (see Figures 7 and 8). Following on from Example 1, in this example a narrower range of microwave power levels was investigate and an optimum enhancement for glucose accumulation at 25W was observed (see Figure 8a).

Table 2 below shows the initial rates of reaction (average over the first 2 hour time period) for the hydrolysis of cellulose by EGL,/CBH + BGL to glucose and for the percentage saccharification.

Microwave Glucose Percentage saccharification power Gradient R2 Difference Yield* Difference Gradient It Difference Watts (d[PP11T) (%) (F11) % (%/hr) (%) 0 0.318 0.95 100 6.2 100 0.0289 0.955 100 12 0.454 0.96 143 7.7 124.2 0.0375 0.985 129.7 0.525 0.96 165.2 8.9 143.5 0.0477 0.965 165 0.491 0.95 154.4 7.4 119.6 0.0414 0.956 143.2 0.464 0.96 146 7.8 125.8 0.0402 0.962 139.1 Table 2. Summary of initial rates for CBH/EGL + BGL on cellulose with varying microwave irradiation energies *yield determined by extrapolation of data from curve fitting to a suitable point of negligible rate change.

Example 3: Investigation of variable microwave power on glucose accumulation from Cellobiose hydrolysis with BGL The effect of 2.45 GHz microwave radiation was investigated in the range 0 -75W.

Experiment 3 was carried out in a 1 litre volume in citrate buffer containing tetracycline and cycloheximide as described above. 10 g cellobiose was added to give a final concentration of IOWL. The reactor was maintained at 50°C for 12-16 hours to ensure temperature equilibrium.

For variation of microwave power, the microwave generator at a set power output was switched on prior to enzyme addition and the thermal input adjusted to give the bulk temperature of 50°C. Once established and stabilised, minimal adjustment was required with temperature variation +0.2°C. In general the apparatus was set up and left to stabilise overnight.

The reaction was started by the addition of 40 FPU/g, cellulose of CBH and EGL, lml of BGL preparation at 250units/g pNPGIIMI. Samples are taken at T=0 through to 160 hours at hourly time points for the first ten hours, and then every other hour for each subsequent working day. Each sample is quenched by boiling for 10 minutes, centrifuged (15 minutes at 3400rpm) then filtered through a 0.2um HPLC grade filter and stored on ice prior to HPLC analysis.

In this experiment the rate of reaction was very fast so it was only possible to discern slight variation in rate of reaction of the breakdown of cellobiose to glucose. However, it was possible to discern some enhancement of cellobiose degradation to glucose in response to microwave radiation.

Microwave power Glucose Watts Gradient 11' Difference (dP/d[T]) (%) 0 0.341 0.984 100 12 0.597 0.990 173.1 0.509 0.900 149.3 0.497 1.000 145.8 0.576 0.999 168.9 Table 3. Summary of initial rates for BGL on cellobiose with varying microwave irradiation energies Example 4; Investigation of the effect of microwave irradiation on the rate of microbial fermentation.

The effect of 2.45 GHz microwave radiation was investigated in the range 0 -75W. Experiment 7 was carried out in a 1 litre volume in 5% YDP. The reactor was maintained at 30°C for 12-16 hours to ensure temperature equilibrium.

For variation of microwave power, the microwave generator at a set power output was switched on prior to enzyme addition and the thermal input adjusted to give the bulk temperature of 30°C. Once established and stabilised, minimal adjustment was required with temperature variation ±0.2°C. In general the apparatus was set up and left to stabilise overnight.

The reaction was started by the addition of inoculum as previously described. Samples are taken at T=0 through to 50 hours with samples taken every hour for the first 8 hours (lag phase) and every hour from T = 18 to T = 36hrs to cover the exponential phase. Each sample was centrifuged (15 minutes at 3400rpm) then filtered through a 0.2tim HPLC grade filter and stored on ice prior to HPLC analysis. Cell counting was conducted for density calibration.

In this experiment an increase in glucose uptake was observed with a non-linear response to microwave radiation with most enhancement observed at 12W (see Figure 12 and Table 4). Cellular growth was not shown to have marked deviation to the thermal negative control. Ethanol production rate was shown to have significant increase at 12W in comparison to higher microwave densities and the thermal negative control (Table 5).

Microwave powc Glucose uptake rate (exponential phase) Watts Gradient 0.998 I)ifference 0 tailihr) (% to 0) -4.12 0 -4.28 0.998 100.00 12 -12.33 0.992 288.14 -7.37 0.999 172.30 -3.55 0.999 82.92 Table 4. Glucose uptake rate by microwave power Microwave power Ethanol production (exponential phase) Watts Gradient (gdthr) Difference (% to 0) 0 0.34 0.999 100 0 (repeat) 0.37 0.994 100 12 0.58 0.998 158.2 0.60 0.9969 162.5 1.00 0.9981 271.3 Table 5. Ethanol production rate by microwave power

Claims (15)

1. Claims 1. A method for the production of a compound by a microbe characterised in that the production of the compound is enhanced by irradiation with microwave radiation, comprising: (i) fermenting a culture medium comprising an energy source and at least one microbe capable of producing said compound; (H) irradiating said culture medium with microwave radiation delivered via a waveguide, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (H) a power from about 1 to about 100 Watts, and characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition; and (Hi) optionally isolating said compound from said culture medium.

   A method according to Claim 1 wherein the energy source is a biomass.

   A method according to Claim 1 or Claim
2. 2 wherein the biomass or energy source is selected from corn stover, wheat straw, rice straw, rice hulls, baggasse fibre, news print, cotton gin trash, black locust, douglas fir, hybrid poplar, eucalyptus, pine, switch grass and generic plant cell wall fibres.

   A method according to any one of Claims 1 to
3. 3 wherein the compound is selected from a small organic molecule, a large organic molecule, a peptide, a polypeptide, a protein and a nucleic acid.

   A method of enhancing the activity of an enzyme said method comprising irradiating a composition comprising the enzyme and a substrate with microwave radiation delivered via a waveguide, wherein said microwave radiation has: (i) a frequency from 0.3 GHz to 16 GHz; and (H) a power from about 1 to about 100 Watts, characterised in that the waveguide is configured such that in use with the composition energy provided to the waveguide by a microwave source is substantially absorbed by the enzyme in the composition rather than other components of the composition. 2. 3.
4. 4.
5. 5.
6. 6. A method according to Claim 5 wherein the waveguide has cross sectional dimensions of about 86 mm by about 43 mm and the frequency is about 2.45 GHz.
7. 7. A method according to Claim 5 wherein the frequency is other than about 2.45GHz and the cross section is dimensioned according to the frequency required relative to cross sectional dimensions of 86 by 43 mm for 2.45 GHz.
8. 8. A method according to any one of claims 5 to 7 wherein the composition is an aqueous composition.
9. 9. A method according to any one of claims 5 to 8 wherein the method is carried out under substantially isothermal conditions.
10. 10. A method according to any one of claims 5 to 9 wherein the enzyme is selected from cellulase, hemicellulase, amylase, xylanase, pectinase and chitinase, a and pglucosidase, glucoamylase, sucrase, isomaltase, lactase, invertase and maltase.
11. 11. A method according to any one of claims 5 to 10 wherein the enzyme is a cellulase.
12. 12. A method according to any one of the preceding claims wherein the microwave radiation is provided at a frequency in the range of about 1 GHz to about 5 GHz.
13. 13 A method according to any one of the preceding claims wherein the microwave radiation is provided at a power level in the range about 25 to about 75W.
14. 14. A microwave apparatus for irradiating a composition comprising an enzyme and a substrate with microwaves comprising: (i) a reactor vessel for in use containing said composition; (H) a microwave source for the provision of microwaves; and (iii) a waveguide arranged with the microwave source to in use deliver energy therefrom to the reactor vessel; characterised in that the waveguide is configured such that in use with a composition comprising an enzyme energy provided to the waveguide by the microwave source is substantially absorbed by the enzyme in the reactor vessel rather than other components of the composition in the reactor vessel.
15. 15. A microwave apparatus according to Claim 14 wherein the microwaves have a frequency of 2.45 GHz and the waveguide has cross sectional dimensions of 86 mm by 43 mm.