This invention relates to a process and particularly, although not exclusively, relates to a fermentation process for producing a biomass and/or ethanol using a filamentous fungus, for example, Fusarium venenatum.

It is known, for example from EP0341878A, US5198362, W091/17669 and EP062084A to use fermentation processes to produce a proteinaceous composition comprising a filamentous fungus, for example Fusarium venenatum.

It is also known from WO2016/063053 A1 to co-produce a filamentous fungus (or “mycoprotein”) and ethanol from carbohydrate feedstock material, for example cereals. Fusarium venenatum produced may be used as a proteinaceous composition and the Fusarium venenatum may also be used to ferment, under anaerobic conditions, a carbohydrate feedstock to produce ethanol. However, it is found that, over time, during the process described, the rate of fermentation reduces and may stop altogether. It may then, disadvantageously, take some time for the process to be returned to production of desired products (e.g. filamentous fungus or ethanol) at commercially acceptable rates. It is an object of the present invention to address the above described problems.

According to a first aspect of the invention, there is provided a method of producing ethanol in the presence of fungi from the genus Fusarium (herein referred to as “Fusarium fungus”), for example Fusarium venenatum, the method comprising:

(i) preparing a culture medium which includes Fusarium fungus, for example Fusarium venenatum;

(ii) providing conditions for production of ethanol in the culture medium;

(iii) producing ethanol in the culture medium;

(iv) removing ethanol from the culture medium;

(v) continuing the production of ethanol in the culture medium after removal of ethanol from the culture medium.

Said Fusarium venenatum is suitably Fusarium venenatum A3/5 (formerly classified as Fusarium graminearum) (IMI 145425; ATCC PTA-2684 deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA.) as described for example in W096/21361 (Zeneca) and WQ95/23843 (Zeneca). After initial preparation of said culture medium referred to in step (i), a fungus ratio defined as the wt% of Fusarium fungus, for example Fusarium venenatum (on a dry weight basis) divided by the total weight of all fungus (on a dry weight basis) in the culture medium may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1. That is, preferably, the only fungus introduced into the fermentation broth in step (i) is a Fusarium fungus, for example Fusarium venenatum.

Preferably, in the culture medium referred to in step (ii) said fungus ratio may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1. That is, preferably, the only fungus present in step (ii) is a Fusarium fungus, for example Fusarium venenatum.

Preferably, in the culture medium referred to in step (iii), said fungus ratio may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1. That is, preferably, the only fungus present in step (iii) is a Fusarium fungus, for example Fusarium venenatum.

Preferably, in the culture medium referred to in step (iv), said fungus ratio may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1. That is, preferably, the only fungus present in step (iv) is a Fusarium fungus, for example Fusarium venenatum.

Preferably, in the culture medium referred to in step (v), said fungus ratio may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1. That is, preferably, the only fungus present in step (v) is a Fusarium fungus, for example Fusarium venenatum.

Preferably, in the entire method of producing ethanol (i.e. suitably from step (i) to step (v)), the fungus ratio may be at least 0.50, at least 0.80, at least 0.90 or at least 0.99. Said fungus ratio is preferably 1 . That is, preferably, the only fungus present in the entire method of producing ethanol step is a Fusarium fungus, for example Fusarium venenatum.

A single Fusarium fungus, for example Fusarium venenatum, is preferably used in steps (i) to (v).

Preferably, the only fungus used in the method of the first aspect to produce ethanol and/or the only fungus present in the culture medium is Fusarium venenatum. Said culture medium prepared in step (i) preferably includes a carbon source and sources of other nutrients for growth of the Fusarium fungus, for example Fusarium venenatum and/or production of ethanol.

In the culture medium, the carbon source may be any suitable carbon source, for example starch, starch containing materials, or products of their hydrolysis, e.g. glucose, sucrose, sucrose containing materials or hydrolysed sucrose, i.e. invert sugars, or mixtures thereof. Thus, the carbon source may comprise hydrolysed potato, molasses, glucose, maltose, hydrolysed bean starch or cassava. Alternative carbon sources, such as carbon sources of animal origin e.g. milk whey, may also be used.

Sufficient amounts of an assimilable carbon source together with other essential growth elements such as nitrogen, sulphur, phosphorus and trace elements may be maintained in the culture medium so that growth of the microorganism is not limited by any of these nutrients. In addition to the nutrients discussed above the presence of one or more vitamins such as biotin may be included in the culture medium.

The culture medium is suitably a fermentation broth.

The culture medium or fermentation broth may be as described in WO2016/063053.

Said carbon source may comprise a carbohydrate feedstock. The carbohydrate feedstock may be a mixed or single feedstock. Preferably the feedstock comprises one or more cereal materials. Typically said one or more cereal materials may include wheat, maize (corn), barley, rice, sorghum, buckwheat, oats, rye and the like. The cereal may be of food grade quality or may be material which is no longer suitable for human consumption. One of the aforementioned examples may be used, or mixtures comprising two or more types of cereals may be employed.

The one or more cereal materials may be subjected to a milling, grinding and/or cutting process, suitably prior to step (i) of the method, in order to break the cereal material down into smaller fragments and also potentially to release some of the proteins, sugars and other materials which may be present in the cereal. The broken-down material may be mixed with water and the pH adjusted as necessary, in order to provide the culture medium . Any starch which may be present in the culture medium may be subjected to hydrolysation or partial hydrolysation by employing one or more of gelatinization, liquefaction and/or saccharification. Starch is found in nature as insoluble, non-dispersible granules resistant to enzymic breakdown. Gelatinisation is the swelling of the starch granule in the presence of heat and water. At this point, the starch or ground cereal slurry thickens considerably and would be difficult to process if an alpha-amylase were not added to partially hydrolyse the starch to dextrins. A dextrin containing solution is generally much more fluid or liquefied. The alpha-amylase serves to reduce the viscosity of the solution and also to produce a lower molecular size substrate. A lower molecular size substrate molecule is desired for the efficient action of glucoamylase which hydrolyses the dextrins to glucose.

Enzymes such as alpha-amylase and glucoamylase may be added in order to break down or hydrolyse the starch which is present. The alpha-amylase is a bacterial thermostable endo- amylase. It hydrolyses alpha 1 ,4- bonds at random points in the starch molecule to rapidly reduce the viscosity of gelatinised starch solutions. This enzyme is a metal ion-containing protein and requires a small amount of calcium ion during use for maximum activity and stability. The enzyme cannot hydrolyse alpha 1 ,6- bonds but can bypass these branch points in amylopectin. The product of the reaction is dextrins - short glucose chains, and small amounts of glucose and maltose. Glucoamylase, produced by fungi, is an exo-amylase. It hydrolyses the maltose and dextrins from the non-reducing end of the molecule. Glucoamylase hydrolyses both alpha 1 ,4- and alpha 1 ,6- bonds to completely degrade the dextrins to glucose. The enzyme is optimally active at pH 3.5-4.5. Typically, alpha-amylase may be added at a concentration of 0.25 - 1.5% w/w of the solid material and glucoamylase may be added at a concentration of 0.25 - 3% w/w of the solid material. Following the enzyme digestions, the fermentation broth may be subjected to a heat treatment in order to destroy the enzymes and kill any bacteria which may be present and which could interfere with subsequent process steps. However, the addition of enzymes, adjustment of pH and heating and cooling which is necessary adds to the cost dramatically and can therefore be undesirable. Fusarium venenatum can undergo fermentation with unhydrolysed cereal grain starch solution.

Step (ii) of the method may include limiting and/or controlling the supply of oxygen to the culture medium. Preferably, in step (ii), conditions are provided for anaerobic fermentation, suitably whereby ethanol is produced as a product, preferably a major product, of the fermentation process, suitably as described in step (iii).

In the method, the concentration of ethanol in the culture medium is preferably assessed and/or analysed. It may be assessed and/or analysed at least 2, at least 5 or at least 10 times, for example over a time in which fermentation, for example ethanol production, is undertaken. It may be assessed and/or analysed at least 2, at least 5 or at least 10 times over a period of 50 hours or over a period of 25 hours. It is preferably assessed and/or analysed during steps (iii) to (v) of the method. It may be assessed and/or analysed at least once over a period of 5 hour. It is preferably assessed and/or analysed at least twice or at least four times over a period of 20 hours or at least 10 hours. The culture medium is preferably sampled. It may be sampled continuously, for example over a period of at least 30 minutes or at least 2 hours or at least 5 hours; or preferably over substantially the entirety of steps (ii) to (v) of the method. Alternatively, sampling could be periodic, for example at least once every 2 hours or at least once every 1 hour. Such periodic sampling may continue for a period of at least 10 or at least 50 hours. The culture medium is preferably assessed at least prior to step (iv). Samples of the culture medium may be assessed and/or analysed to determine the concentration of ethanol in the culture medium. A chromatographic technique, such as GC, may be used. The culture medium may be assessed to ensure the ethanol concentration does not exceed a predetermined level, suitably for the majority (preferably the entire) fermentation process.

In the method, the concentration of ethanol in the culture medium is preferably controlled so the concentration is less than 10g/litre (i.e. less than 10g of ethanol per litre of culture medium).

Preferably, from the start of step (ii) and for at least 1 hour, 10 hours or 50 hours thereafter, the concentration of ethanol in the culture medium is controlled to be less than 10g/litre, preferably less than 5g/l itre, more preferably less than 3g/litre, especially less than 2g/litre.

Preferably from the start of step (i) and for at least 1 hour, 10 hours or 50 hours thereafter, the concentration of ethanol in the culture medium is controlled to be less than 10g/litre, preferably less than 5g/l itre, more preferably less than 3g/litre, especially less than 2g/litre.

Preferably from the start of step (iii) and for at least 1 hour, 10 hours or 50 hours thereafter, the concentration of ethanol in the culture medium is controlled to be less than 10g/litre, preferably less than 5g/l itre, more preferably less than 3g/litre, especially less than 2g/litre.

In step (iii), the temperature at which the process is conducted may be selected so as to produce acceptable yields, and conversion ratios. Typical temperatures are within the range 25 to 34 °C.

Similarly, the pH at which the process is conducted is preferably kept within a range at which maximum growth is exhibited for a particular microorganism. Typically, the pH is within the range 5.0 and 8.0, and more usually about a pH of 6.

In step (iv), ethanol is preferably removed from the culture medium before the concentration of ethanol in the culture medium exceeds 10g/litre, before it exceeds 5g/litre, before it exceeds 3g/litre, and, especially, before it exceeds 2g/litre.

In a first embodiment, removal of ethanol from the culture medium may comprise removal of culture medium from a vessel containing culture medium, in which ethanol is produced by fermentation, to produce “removed culture medium”, and treatment of the removed culture medium to reduce the concentration of ethanol therein.

In said first embodiment, said removed culture medium is suitably treated outside said vessel to reduce the concentration of ethanol therein. Preferably, after such treatment, treated culture medium is returned to the vessel containing culture medium in which fermentation is undertaken. Thus, culture medium is suitably circulated from and back to said vessel. During such circulation the culture medium is treated to remove ethanol. Such treatment may comprise a physical separation process. It may comprise vaporization of the ethanol from the culture medium and/or a process wherein separation is dependent on the molecular size and/or chemical properties of ethanol relative to other components of the culture medium. An example where vaporization is used may be a pervaporation process. An example where separation is dependent on molecular size and/or chemical properties of the ethanol may comprise an adsorption process.

In the process of the first embodiment, culture medium may be passed through an assembly in which the separation process is undertaken. Downstream of the assembly, the ethanol may be isolated and/or culture medium returned to the vessel.

In a second embodiment, a fluid comprising ethanol may be selectively removed from the culture medium, suitably so the fluid removed has a different composition and/or has a high concentration of ethanol relative to the concentration of ethanol in the culture medium. For example, a vacuum may be applied to an outlet of the vessel containing culture medium and/or ethanol and water vapour removed from the vessel, such as in a vacuum fermentation process. Alternatively, an anaerobic gas may be introduced into the vessel containing the culture medium to evaporate the ethanol, in a vacuum stripping process.

In a third embodiment, the ethanol concentration in the culture medium in a vessel in which fermentation is undertaken may be reduced by removing a volume (eg 10 to 90vol% or 20 to 50 vol%) of culture medium from the fermentation vessel; introducing fresh culture medium into the vessel, suitably to replace the volume removed; and mixing the fresh culture medium with culture medium remaining in the vessel. Thus the concentration of ethanol in the vessel will be reduced. The culture medium removed from the fermentation vessel may be treated to concentrate the ethanol. By way of example, if the culture medium in the fermentation vessel has reached an ethanol concentration of 10g/litre, 90% of the culture medium may be removed and replaced with fresh, unfermented medium, thereby the reduce the ethanol concentration down to 1 g/litre.

In any of the processes described, ethanol is preferably collected and/or concentrated. Removal of the ethanol in step (iv) may be continuous or discontinuous. Preferably, during such removal ethanol continues to be produced, as described in step (v), suitably by fermentation involving Fusarium venenatum in the same vessel from which the ethanol is removed.

Preferably in step (iii), the Fusarium venenatum produces the ethanol. Preferably, the Fusarium venenatum produces at least 50 wt%, at least 75 wt%, at least 90 wt%, preferably suitably all of the ethanol produced in the process over a period of at least 1 hour, at least 5 hours, at least 50 hours or, especially, throughout the entire process wherein ethanol is produced.

The method may include an additional step (EX) which comprises aerobic fermentation by said Fusarium fungus, for example Fusarium venenatum. Such fermentation may produce a proteinaceous material comprising said Fusarium fungus, for example Fusarium venenatum which may be harvested as a foodstuff.

In the method, step (EX) preferably precedes step (ii). In step (EX) oxygen is preferably continuously supplied to the culture medium to effect said aerobic fermentation. In addition, during the method, additional culture medium and/or any component needed for fermentation of said Fusarium fungus, for example Fusarium venenatum, may be supplied to a vessel in which step (EX) (and step (iii) is carried out). Culture medium may be removed, preferably continuously, from the vessel. The removed culture medium may then be treated to separate the Fusarium venenatum therefrom. The removed culture medium and/or Fusarium fungus, for example Fusarium venenatum, may be heat treated, filtered and/or the resulting filter cake formulated into a state suitable for use as a foodstuff.

The method described may include an integrated method for producing Fusarium venenatum (or “mycoprotein”) and ethanol as described in WO2016/063053. Thus, a feedstock for producing Fusarium venenatum may include common ingredients as used to produce ethanol. In a preferred embodiment, the only significant difference in the feedstock and/or culture medium may be the level of oxygen introduced into the medium to effect aerobic or anaerobic fermentation. Is such a method aerobic fermentation is preferably carried out initially; and subsequently anaerobic fermentation to produce ethanol is undertaken.

Use of the method described is believed to be advantageous over the method described in WO2016/063053. The method may be used to convert a relatively low grade or low value carbonaceous material into Fusarium venenatum (or mycoprotein) which can be used for human food; ethanol which can be used as a biofuel; and spent material, such as Dried Distillers Grain with Insolubles (DDGS) which can be used as animal feed.

Preferably a vessel in the method is undertaken may have a volume of at least 10m ³ , preferably at least 100m ³ . The vessel may have a height of at least 5m or at least 10m. The vessel may contain at least 10000kg or at least 100000kg of culture medium.

In a second aspect there is provided apparatus for carrying out the method of the first aspect, the apparatus comprising:

(a) a vessel in which a culture medium is provided;

(b) means for controlling the supply of oxygen to the culture medium;

(c) means for assessing the concentration of ethanol in the culture medium;

(d) means for preferentially removing ethanol from the culture medium whilst fermentation is continuously undertaken in the culture medium.

The apparatus may include recirculating means for removing culture medium from the vessel, treating the culture medium to remove ethanol therefrom and returning treated culture medium to the vessel.

Any feature of any aspect of any invention or embodiment described herein may be combined with any feature of any aspect of any other invention described herein mutatis mutandis. Specific embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

Figure 1 is a diagram of apparatus for use in a pervaporation process; Figure 2 is a diagram of apparatus for use in a vacuum fermentation process;

Figure 3 is a diagram of apparatus for use in vacuum stripping; and

Figure 4 is a diagram of apparatus for use in adsorption of ethanol.

In the figures, the same or similar parts are annotated with the same reference numerals.

A fermentation process, for example as described in EP0341878A, US5198362, WQ91/17669, EP062084A or WO 2016/063053 may be used to produce a biomass comprising Fusarium venenatum from a carbohydrate feedstock. Production of the biomass suitably involves an aerobic fermentation in the presence of a controlled amount of oxygen. The fermentation process may be modified by reducing the level of oxygen, to favour production of ethanol and/or to favour anaerobic fermentation. To optimise the process, the concentration of ethanol in the fermentation broth may be monitored by periodic (eg every 30 minutes) sampling and assessment by GC. The broth may be assessed to ensure the ethanol concentration does not exceed a predetermined level for the majority (preferably the entire) fermentation process. The broth may be processed intermittently or preferably continuously (to minimise shock to the fungus) to remove ethanol from the broth and maintain the ethanol concentration in the broth below the predetermined level.

In one embodiment, the process may include a two-step fermentation as described in WO2016/063053. In particular, reference is made to “Experiment 4” on page 18 and page 25, line 28 to page 31 , line 10 of WO 2016/063053, the content of which is incorporated herein by reference.

In contrast, however, to the process and/or apparatus described in WO2016/063053, in a preferred embodiment, the ethanol concentration in the fermentation broth is monitored and the concentration is maintained below a predetermined level (eg below 10g/I, preferably below 5g/l, more preferably below 1 g/l or below 0.5 g/l) by removal of ethanol from the broth.

Various processes may be integrated with the fermentation process to maintain the ethanol concentration in the fermentation broth at an acceptable concentration, as discussed further below.

Referring to Figure 1 , a pervaporation process may be used. A stirred reactor 2 comprises a fermentation broth. The broth is circulated via pipes 4, 6 and recycling pump 8 to a membrane module 10. Module 10 is arranged so ethanol permeates through a relatively hydrophobic membrane of the module. The vapour is evaporated downstream and the vapour passes (reference numeral 12 relates) to a cold trap 14. The upstream side of the membrane is at ambient pressure and a vacuum is applied to the downstream side of the membrane by vacuum pump 16. Liquid ethanol can be led away from the apparatus as represented by numeral 18. In the process, the membrane acts as a selective barrier between two phases: the liquid-phase fermentation broth and the vapour-phase ethanol permeate. The broth is recycled, after passing through the module 10 to the reactor 2.

Referring to Figure 2, a vacuum fermentation process may be used. In the process, ethanol is continuously recovered via outlet 22 from the fermentation broth in reactor 2 by application of vacuum pressure via pump 20. Ethanol/water vapour 24 is led from the reactor 2 where it is chilled via chiller 26 to produce a condensate at 28. Essentially, ethanol evaporates at the fermentation temperature and is subsequently condensed using chiller 26. During the vacuum fermentation, the ethanol concentration in the broth can be controlled at a predetermined level.

Referring to Figure 3, a gas stripping process may be used. Gas stripping involves separation by removal of ethanol by the dissolution into a gas passing from container 30 through the fermentation broth. In this process, an anaerobic gas such as the carbon dioxide or nitrogen is recycled through the reactor 2 and evaporates the ethanol. The gas/ethanol mixture (reference numeral 32 relates) is recovered from the gas stream via downstream condensers 34, 36. Ethanol 38, 40 is trapped and gas 42 passes out of the apparatus.

Referring to Figure 4, an adsorption process may be used. Fermentation broth may be recycled via a module 60 comprising a porous adsorbent, with the pore size of the absorbent being similar to the molecular size of ethanol. In this process, the fermentation broth passes through the module 60 comprising a bed packed with the adsorbent, and the effluent is recycled again to the fermentation broth in reactor 2. The process may involve periodic desorption from the adsorbent to recover a concentrated ethanol solution and regenerate the adsorbent.

Thus, by controlling the concentration of ethanol in the broth, the fermentation process can be run more efficiently. In preferred embodiments, the process may be run for at least 100 hours whilst maintaining the ethanol concentration below the amounts referred to above.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.