WO2007110592A2

WO2007110592A2 - Fermentation process for the production of ethanol

Info

Publication number: WO2007110592A2
Application number: PCT/GB2007/001018
Authority: WO
Inventors: Neil Bookless; Brian Hartley; Namdar Baghaei-Yazdi; Muhammed Javed
Original assignee: Elsworth Ethanol Company Limited
Priority date: 2006-03-24
Filing date: 2007-03-21
Publication date: 2007-10-04
Also published as: WO2007110592A3

Abstract

A method of producing ethanol using a bacterial fermentation process is described. Fermentation is carried out using a first bacterium with desired ethanol producing pathway activity to produce ethanol. Process conditions are controlled to prevent saturation of pathway flux, or to restore pathway flux following saturation, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity. A micro-organism with improved fermentation properties is also described.

Description

Improved Fermentation Process

Summary

This invention relates to improved methods for the production of fermentation products, in particular ethanol, by micro-organisms. The invention also relates to improved microorganisms for the production of ethanol.

This invention relates to regulation of thermophile ethanol fermentation and enhancement of microbial ethanol production.

In one aspect, this invention relates to methods for stable industrial ethanol production from agro-industrial and municipal wastes, or any suitable biodegradable waste, using mutant thermophiles lacking L-lactate dehydrogenase activity.

In another aspect, this invention relates to the enhancement of microbial ethanol production from mixed sugars derived from the hydrolysis of biomass. In particular, the invention envisages a novel pathway for ethanol production by cloning an fdh gene (which encodes an NAD-linked formate dehydrogenase enzyme) into a microorganism that possesses a functional pfl gene (which encodes a pyruvate-formate lyase enzyme complex) but lacks lactate dehydrogenase activity.

Introduction

Many bacteria have the natural ability to metabolise simple sugars into a mixture of acidic and neutral fermentation products via the process of glycolysis. Glycolysis is, the series of enzymatic steps whereby the six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound pyruvate. The glycolytic pathways of many bacteria produce pyruvate as a common intermediate. Subsequent metabolism of pyruvate results in a net production of NADH and ATP as well as waste products commonly known as fermentation products. Under aerobic conditions, approximately 95% of the pyruvate produced from glycolysis is consumed in a number of short metabolic pathways which act to regenerate NAD⁺ via oxidative metabolism, where NADH is typically oxidised by donating hydrogen equivalents via a series of steps to oxygen, thereby forming water, an obligate requirement for continued glycolysis and ATP production.

Under anaerobic conditions, most ATP is generated via glycolysis. Additional ATP can also be regenerated during the production of organic acids such as acetate. NAD⁺ is regenerated from NADH during the reduction of organic substrates such as pyruvate or acetyl Co A. Therefore, the fermentation products of glycolysis and pyruvate metabolism include organic acids, such as lactate, formate and acetate as well as neutral products such as ethanol.

The majority of facultatively anaerobic bacteria do not produce high yields of ethanol either under aerobic or anaerobic conditions. Most facultative anaerobes metabolise pyruvate aerobically via pyruvate dehydrogenase (PDH)₁ the tricarboxylic acid (TCA) cycle, and the electron transport chain (ETC). Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via the pyruvate-formate-lyase (PFL) pathway to give formate and acetyl-Co A. Acetyl-Co A is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (AK) with the co-production of ATP, or reduced to ethanol via acetaldehyde dehydrogenase (Ac DH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re-oxidised to NAD⁺ by lactate dehydrogenase (LDH) during the reduction of pyruvate to lactate. NADH can also be re-oxidised by Ac DH and ADH during the reduction of acetyl-Co A to ethanol but this is a minor reaction in cells with a functional LDH. Theoretical yields of ethanol are therefore not achieved since most acetyl Co A is converted to acetate to regenerate ATP and excess NADH produced during glycolysis is oxidised by LDH.

Ethanologenic microorganisms, such as Zymomonas mobilis and yeast, are capable of a second type of anaerobic fermentation commonly referred to as alcoholic fermentation in which pyruvate is metabolised to acetaldehyde and CO₂ by pyruvate decarboxylase (PDC). Acetaldehyde is then reduced to ethanol by ADH regenerating NAD⁺. Alcoholic fermentation results in the metabolism of 1 molecule of glucose to two molecules of ethanol and two molecules of CO₂. DNA which encodes both of these enzymes in Z mobilis has been isolated, cloned and expressed recombinantly in hosts capable of producing high yields of ethanol via the synthetic route described above. For example; US 5,000,000 and Ingram et al. ((1997) Biotechnology and Bioengineering 58, Nos. 2 and 3) have shown that the genes encoding both PDC (pdc) and ADH (adh) from Z mobilis can be incorporated into a "pet" operon which can be used to transform Escherichia coli strains resulting in the production of recombinant E coli capable of co- expressing the Z mobilis pdc and adh. This results in the production of a synthetic pathway re-directing E coli central metabolism from pyruvate to ethanol during growth under both aerobic and anaerobic conditions. Similarly, US 5,554520 discloses that pdc and adh from Z mobilis can both be integrated via the use of a pet operon to produce Gram negative recombinant hosts, including Erwina, Klebsiella and Xanthomonas species, each of which expresses the heterologous genes of Z mobilis resulting in high yield production of ethanol via a synthetic pathway from pyruvate to ethanol.

An important improvement in the production of ethanol can be achieved with thermophilic microorganisms that operate at high temperature, because the conversion rate of carbohydrates into ethanol is much faster. For example, ethanol productivity in a thermophilic Bacillus can be up to ten-fold faster than a conventional yeast fermentation process which operates at 30⁰C. Consequently, a smaller production plant is required for a given volumetric productivity, thereby reducing plant construction costs. At high temperature, there is also a reduced risk of contamination in the fermenter from unwanted microorganisms, resulting in less downtime, increased plant productivity and a lower energy requirement for feedstock sterilisation. Moreover, since fermentation cooling is not required, operating costs are reduced further. The heat of fermentation helps to evaporate ethanol, which reduces the likelihood of growth inhibition from high ethanol concentrations, a common problem with most bacterial fermentations. Ethanol evaporation in the fermenter head space also facilitates product recovery.

Many micro-organisms contain a pyruvate-formate lyase (PFL) pathway that converts pyruvate into acetyl CoA + formate, (Figure 1A). Heterolactate fermentative microorganisms are one such class. These microorganisms first convert input sugars to pyruvate (generally by the EMP pathway of glycolysis), which then can take many routes to produce lactate, formate, acetate, ethanol and CO_∑, in various proportions, depending on the growth conditions.

In fully aerobic cells, the pyruvate is normally metabolised to H₂O and CO₂ via the pyruvate dehydrogenase (PDH) pathway, tri-carboxylic acid (TCA) cycle and the Electron Transport Chain (ETC). However, in many of these organisms, particularly thermophilic Bacilli, sugar uptake and glycolysis appear to be unregulated and, at high sugar concentrations, lactate is a dominant product even under aerobic conditions. This dominant lactate production suggests that the PDH flux has become saturated, and that the excess pyruvate is diverted into the lactate dehydrogenase pathway.

However, if the ldh gene (encoding the lactate dehydrogenase enzyme) is inactivated, lactate production stops and the excess pyruvate is diverted mainly into the (PFL) pathway, (Figure 1A). Moreover, at very high sugar concentrations and/or at acid pH, the PFL pathway flux also becomes saturated and the excess pyruvate is then diverted into an anaerobic (PDH) pathway, which yields only ethanol and CO₂, (Figure 1 B).

Hartley, B.S. (PCT/GB88/00411, published as WO 88/09379) observed that a mutant thermophilic Bacillus that lacks lactate dehydrogenase activity (strain LLD-15) can metabolise a wide range of sugars anaerobically by two pathways, (Figure 1A): • The well-known pyruvate-formate lyase (PFL) pathway, yielding: 1 mol. of acetate, 1 mol. of ethanol and 2 mol. of formate / mol. of glucose equivalent consumed. • A novel anaerobic pyruvate dehydrogenase (PDH) pathway, yielding 2 mol. of ethanol and 2 mol. of CO₂/ mol. of glucose equivalent consumed.

To obtain high ethanol yields the preferred conditions are, therefore, those that reduce flux through the PFL pathway and increase flux via the PDH pathway, (Hartley, B.S. and Shama, G. Proc. Roy. Soc. Lond. 321, 555-568 (1987)). Unfortunately, under such conditions the cells experience metabolic stress, with reduced ATP production, and a potential imbalance in NAD/NADH and CoA/acetyl CoA ratios, (Figure 1 C). Hence growth stops, pyruvate secretion is seen and wild type revertants or contaminants take over the fermentation.

In batch or continuous fermentations, at high sugars concentrations or at acid pH, PFL- flux declines and growth stops long before all sugars are consumed, but PDH flux continues, allowing high yields of ethanol, (Figure 1B), as required for industrial ethanol production. However, the moribund cells eventually die, requiring the addition of fresh viable cells, to maintain ethanol productivity.

To achieve commercially viable yields, PCT/GB88/00411 envisaged a two-stage continuous 'Closed System¹ fermentation, in which: i) sugars are fed continuously to an anaerobic ethanol production fermenter, with spent cells being removed continuously from the effluent broth, by centrifugation or membrane filtration; ii) ethanol is stripped continuously from the cell-free broth, with the residual sugars and by-products fed to an aerobic fermenter, in which they are converted into fresh cells, and iii) a proportion of the fresh cells are fed back to the production stage, to maintain cell viability; the rest being used as animal feed.

Although attractive in theory, the 'Closed System' was found to be impractical for two reasons: i) the ldh mutation in strain LLD-15 reverted rapidly to wild type, at high sugar concentrations, with undesirable lactate production taking over the production stage. ii) with high sugars feeds, even gene-deleted ldh strains were unstable, due to washout and / or takeover, by variants that produced less ethanol.

We conclude that sugars uptake and glycolytic flux are unregulated in such thermophiles, so both increase as external sugar concentrations rise. With unstable mutants, such as LLD-15, pyruvate secretion was seen, growth rates declined and takeovers occurred when sucrose uptake reached a critical level of 4 - 5 g/g cells per hour, (San Martin, R. et a/., J. Gen. Microbiol. 139. 1033-1040 (1993)). This suggests that the PDH-flux becomes saturated at this critical point so that: i). Pyruvate accumulates, NADH levels rise and NAD levels fall. ii). The PDH pathway flux begins to decline even further, because NAD is a co-substrate for pyruvate dehydrogenase, iii). This consequent catastrophic metabolic collapse leads to what one may call 'Redox

Death'.

There is a need, therefore, to provide improved methods of ethanol production.

Description of a first aspect of the Invention

According to the invention there is provided a method of producing a fermentation product using a bacterial fermentation process, which comprises: carrying out fermentation using a first bacterium with desired product producing pathway activity to produce the product; and controlling process conditions to prevent saturation of flux through the pathway, or to restore pathway flux following saturation, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity. Preferably the pathway flux is measured or monitored during the process so that process conditions can be controlled appropriately to prevent saturation of flux through the pathway, or to restore pathway flux following saturation.

There is also provided according to the invention a method of producing a fermentation product using a bacterial fermentation process, which comprises: carrying out fermentation using a first bacterium with desired product producing pathway activity to produce the product; measuring or monitoring flux through the pathway; and controlling process conditions in response to the result of the measurement or monitoring to prevent saturation of pathway flux, or to restore pathway flux following saturation, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity.

Preferably the fermentation product is ethanol. Preferably the ethanol pathway producing activity is an anaerobic pyruvate dehydrogenase (PDH) pathway activity.

Methods of the invention are advantageous because there is no requirement for addition of fresh viable cells, for partial cell recycle (i.e. removal of cells followed by their re- introduction after a period of aerobic growth), or for removal of fermentation medium during the fermentation process.

It is believed that preventing saturation of pathway flux, or restoring pathway flux following saturation, should maintain cells of the first bacterium in redox balance (or minimise loss of redox balance of cells of the first bacterium) during the fermentation process, thereby preventing (or minimising) onset of redox death of the cells. This is believed to reduce the risk of revertants or variants of the first bacterium being formed during the fermentation process, so that the fermentation process can be continued for as long as is desired. This in turn allows high yields of the fermentation product to be obtained.

There is also provided according to the invention a method of producing a fermentation product using a bacterial fermentation process, which comprises: carrying out fermentation using a first bacterium with desired product producing pathway activity to produce the product; and controlling process conditions to prevent or minimise loss of redox balance of cells of the first bacterium, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity. There is further provided according to the invention a method of producing a fermentation product using a bacterial fermentation process, which comprises: carrying out fermentation using a first bacterium with desired product producing pathway activity to produce the product; and controlling process conditions to prevent or minimise onset of redox death of cells of the first bacterium, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity.

Loss of redox balance or onset of redox death may be measured or monitored during the process so that process conditions can be controlled appropriately to prevent or minimise loss of redox balance or onset of redox death.

Flux through the product producing pathway (or loss of redox balance or onset of redox death) may be measured in any suitable way. For example, a change in level or rate of production of a substance that accumulates or is used up as pathway flux becomes saturated may be used to measure or monitor pathway flux (or loss of redox balance, or onset of redox death of cells of the first bacterium). A change in rate of production of a product or by-product of the fermentation process may be used.

In particular, residual sugar level, redox potential or pyruvate level of the fermentation broth, or rate of carbon dioxide production can be used to measure or monitor pathway flux (or loss of redox balance, or onset of redox death of cells of the first bacterium). As anaerobic PDH pathway flux becomes saturated, pyruvate accumulates, NADH levels rise and NAD levels fall (it is believed that this will cause a change in the redox potential of the fermentation broth), the rate of carbon dioxide production (in particular, the rate of anaerobic carbon dioxide production) falls, and residual sugar levels increase.

In other embodiments of the invention, growth rate may be used as an indicator of pathway flux (or loss of redox balance, or onset of redox death of cells of the first bacterium). If during the fermentation process, growth of the first bacterium declines or ceases altogether, this may indicate that pathway flux is becoming or is saturated. Accordingly, a decline (or arrest) in growth rate may be used to measure or monitor pathway flux. Growth of the bacterium may be conveniently measured, for example, by measuring optical density of the fermentation mixture (or of a sample of the fermentation mixture) during the fermentation process. The second bacterium may be any bacterium that is different to the first bacterium. For example, the second bacterium may be a revertant or variant of the first bacterium. Undesired fermentation activity may be fermentation activity that produces the same fermentation product as the first bacterium but in lower yields, or a different fermentation product, under the conditions used for the fermentation process. Alternatively, the second bacterium may not have any fermentation activity.

The degree of aeration of the fermentation process and the rate of sugar feed provision are examples of process conditions that can be controlled to prevent saturation of pathway flux or to restore pathway flux following saturation. Accordingly, saturation of pathway flux may be prevented or pathway flux may be restored following saturation by controlling aeration of the fermentation process and/or sugar feed rate. This may be done by varying the level of aeration or sugar feed and/or by intermittent aeration or intermittent provision of sugar feed during the fermentation process.

It will be appreciated that control of aeration and/or sugar feed rate will also prevent or minimise loss of redox balance or redox death of cells of the first bacterium.

Aeration of the fermentation process may be controlled by episodic aeration of the fermentation process, or by regulating a level of continuous aeration of the fermentation process. If desired, sugar feed rate may be maintained substantially constant throughout the fermentation process.

It will be appreciated that excess aeration should be avoided, since this will adversely affect ethanol yield. Accordingly, aeration is preferably controlled so as to favour minimal viable bacterial cell growth with minimal aerobic sugar utilisation, thereby favouring optimisation of ethanol yield.

Alternatively, or additionally, sugar feed rate may be controlled during the fermentation process. It is preferred that sugar feed rate is regulated to be below sugar uptake of the resident cells. Sugar feed rate may be controlled by providing sugar feed intermittently during the fermentation process, or by regulating a level of continuous sugar feed provision during the fermentation process. According to some embodiments of the invention, provision of sugar feed may be stopped during an episode of aeration of the fermentation process. It will be appreciated that where intermittent aeration of the fermentation process is used, the fermentation process may proceed in cycles of anaerobic fermentation followed by aerobic growth. Alternatively, where continuous minimal aeration is provided during the fermentation process, this may allow aerobic growth of some bacterial cells in the fermentation at the same time as anaerobic fermentation is carried out by other bacterial cells.

It is desired to feed the bacterium during the fermentation process with sugar feed that contains as much sugar as possible to maximise ethanol yield, without allowing takeover of fermentation by a bacterium with undesired fermentation activity. It is preferred that the bacterium is fed during the fermentation process with sugar feed comprising at least 4% (w/v) sugar. However, it is believed that methods of the invention allow sugar feed approaching 20% (w/v) (for example, 10-20% (w/v)) or greater to be used.

The sugar feed may comprise a mixture of any suitable sugars. The sugar feed may comprise xylose, glucose, or arabinose, or preferably a mixture of xylose, glucose, and arabinose.

It will be appreciated that ethanol yield will be optimised if ethanol is produced by anaerobic pyruvate dehydrogenase (PDH) pathway activity. Accordingly, conditions are preferably optimised to favour production of ethanol by the first bacterium by anaerobic pyruvate dehydrogenase (PDH) pathway activity, whilst minimising saturation of pathway flux (and loss of redox balance and onset of redox death).

Optimal ethanol yield may be achieved throughout the fermentation process when anaerobic carbon dioxide production is maximised and aerobic carbon dioxide production is minimised. Accordingly, conditions may be adjusted during the fermentation process so as to favour maximisation of the ratio of anaerobically to aerobically produced carbon dioxide, whilst minimising saturation of pathway flux (and loss of redox balance and onset of redox death). In this way, optimisation of ethanol yield is favoured.

Sensors may be used to measure or monitor one or more indicators of saturation of pathway flux (or loss of redox balance or onset of redox death) during the fermentation process. The sensors may be feedback sensors that control process conditions in response to the result of the measurement or monitoring. For example, the sensors may measure or monitor one or more of the residual sugar level, the redox potential or pyruvate level of the fermentation broth, or the rate of carbon dioxide production. The sensors may control sugar feed rate and/or aeration of the fermentation process.

The fermentation process may be any suitable fermentation process, for example a continuous, fed-batch, or batch fermentation process. However, a continuous fermentation process is preferred.

Any suitable bacterium may be used for the fermentation process. However, it is preferable that the bacterium has certain characteristics which are desirable for its use in the fermentation process.

The bacterium is preferably selected for ability to grow on any suitable biodegradable waste (such as agro-industrial or munucipal waste, or any suitable cellulosic biomass), for example hexose and/or pentose sugars, and oligomers thereof, at thermophilic temperatures. The bacterium should preferably have no restriction system, thereby avoiding the need for in vivo methylation. The bacterium should preferably be stable to at least 3% ethanol. Preferably the bacterium should have the ability to utilise C3, C₅ and C₆ sugars (or their oligomers) as a substrate, including ceilobiose and starch. Pentoses are the major components of waste streams from paper-making or from pre-treatments of straw such as steam-explosion or dilute acid hydrolysis. There are also large volumes of solid food processing residues that are rich in hemicelluloses (for example, bran and shives from dry-milling, sugar cane bagasse, or oil seed processing residues) that can readily be converted to a mixture of sugars by dilute acid or alkali hydrolysis.

It is preferable if the bacterium is transformable at a high frequency. The bacterium should preferably have a growth rate in continuous culture to support dilution rates of 0.3 h^'1 and above (typically 0.3 OD₆O₀).

The bacterium preferably comprises pyruvate-formate lyase (PFL) pathway activity. The bacterium preferably comprises anaerobic pyruvate dehydrogenase (PDH) pathway activity. Preferably the bacterium lacks lactate dehydrogenase activity.

The bacterium may be a spore-former or may not sporulate. The success of the fermentation process does not depend necessarily on the ability of the bacterium to sporulate, although in certain circumstances it may be preferable to have a sporulator, for example when it is desirable to use the bacterium as an animal feed-stock at the end of the fermentation process.

The bacterium is preferably a thermophile. Preferably the bacterium will grow in the temperature range of 40°C-85°C. Preferably, the bacterium will grow within the temperature range 50°C-70°C. It is also desirable that the bacterium grows in conditions of pH7.2 or below, in particular pH6.9-pH4.5.

The bacterium may be derived from a Bacillus spp. Examples of Bacillus species include Bacillus stearothermophilus, B. calvodex, B. caldotenax, B. thermoglucosidasius, B. coagulans, B. licheniformis, B. thermodenitrificans, and S. caldolyticus. In particular, it is preferred that the micro-organism is of the Geobacillus species, in particular Geobacillus thermoglucosidasius.

The bacterium may be derived from Bacillus stearothermophilus strain LLD-R (NCIB 12403), or from other bacteria described in WO 2006/131734, for example deposited under NCIMB no. 41277, 41278, 41279, 41280, 41281 modified to inactivate the endogenous lactate dehydrogenase gene, or described in WO 2006/117536, for example deposited under NCIMB Accession No. 41275. Alternatively, the bacterium may be derived from bacteria described in WO 02/29030 and WO 01/49865.

Preferably the bacterium is a facultative anaerobe. The bacterium may be a Gram positive bacterium.

If a thermophile is used in a method of the invention, the fermentation process may be carried out at elevated temperature. A suitable temperature is 50⁰C or above, for example 65-70⁰C, or even 70⁰C or above.

Preferably ethanol is removed continuously during the fermentation process to maintain ethanol concentration below the ethanol tolerance of the bacterium. If methods of the invention are carried out at elevated temperature, the ethanol produced can be removed as a vapour, for example by application of a mild vacuum.

There is also provided according to the invention a method of ethanol production by a thermophilic micro-organism in a batch, fed-batch, or continuous fermentation process, in which redox death of the cells carrying out fermentation is avoided by regulating the sugars feed rate, and/or by controlled minimal aeration.

The sugars feed may comprise at least 4% w/v mixed sugars (for example 10-20% w/v mixed sugars).

Sugars feed rate and/or aeration may be variable and/or intermittent. For example, sugars feed may be stopped during a series of brief episodes of aeration, or aeration may be continuously varied so as to maintain minimal viable cell growth with minimal aerobic sugars utilisation.

The sugars feed rate is preferably regulated to be below the sugars uptake of the resident cells.

Saturation of anaerobic pyruvate dehydrogenase (PDH) flux may be used as a signal of onset, or impending onset, of redox death. Accordingly, the sugars feed rate and/or the aeration level may be changed when PDH flux is, or is becoming saturated. In particular, the sugars feed rate may be reduced when PDH-flux is, or is becoming saturated.

In a preferred embodiment, the feed rate is cut off when PDH flux is or is becoming saturated, and a short pulse of aeration is switched on. In an alternative preferred embodiment, a constant feed rate is used, and a minimal supply of air is continuously varied to prevent saturation of PDH flux.

Secretion of pyruvate may be used as a signal of onset (or impending onset) of redox death. Feed rate is reduced, or aeration is begun if pyruvate levels in the fermentation broth rise above a minimal level.

Onset of redox death, or PDH flux, may be determined by measuring the level of pyruvate in the fermentation broth, the redox potential of the fermentation broth, the residual sugar level of the fermentation broth, or the rate of carbon dioxide production.

Sugars feed rate and/or minimal aeration is preferably controlled by sensors (preferably feedback sensors). The sensors preferably measure resident sugar concentration (or residual sugars level), broth redox potential, rate of carbon dioxide evolution (preferably rate of anaerobic carbon dioxide production), or broth pyruvate level. There is further provided according to the invention ethanol production by a thermophilic micro-organism in a batch, fed-batch, or continuous fermentation process of at least 4% w/v mixed sugars (for example 10-20% w/v mixed sugars), fed at rates controlled by feedback sensors so as to maintain the cells in redox balance.

Preferably the thermophilic organism is a mutant thermophile lacking L-lactate dehydrogenase activity (for example, a thermophilic Bacillus lacking lactate dehydrogenase activity).

Embodiments of the first aspect of the invention are described by way of example only below, and in the following examples, with reference to the accompanying Figure 1 which illustrates the effects of pH and sugars concentration on product yields in anaerobic pathways.

1. Regulation of sugar feed rates

We have seen that a simple and convenient way to avoid such 'Redox Death' in industrial fermentations will be to use batch, fed-batch or continuous fermenters in which the sugars feed rate is regulated to be below the sugars uptake of the resident cells. The feed rate may conveniently be controlled by on-line sensors that continuously measure either resident sugar concentrations, broth redox potential or rates of CO₂ evolution since, under anaerobic conditions, this will equal the PDH-flux. An example of such methods is described in Example 1 below.

2. Semi-aerobic fed-batch fermentations.

This simple protocol (i.e. as described in 1 above, and in Example 1) will maximise ethanol yields, but increasingly slower feed rates will reduce volumetric productivity. We have seen that a simple way to circumvent this reduced productivity will be to use controlled minimal aeration, to maintain cell viability at or below the 'critical point'. Excessive NADH will then be metabolised by the tricarboxylic acid cycle (TCA) (abbreviated as TCC in Figure 1D) and the electron transport chain (ETC), to provide an abundance of ATP, restoring NAD/NADH balance and reviving PDH flux (Figure 1D).

However, excessive aeration must be avoided, as this will reduce ethanol yield. Feedback controls; such as residual sugars concentration, redox potential or rates of

CO₂ production can again be used to detect the critical point, prompting further controlled aeration. This control could be episodic, by cutting off sugars feed briefly, when rates of CO₂ production begin to decline, and by resuming feed when cell growth returns. Alternatively, continuous minimal aeration could be used to maintain the residual sugars level, or the redox potential of the broth, at or below the critical point. Examples of such methods are described in Examples and 2 and 3 below.

Alternatively, batch or continuous fermentations could be carried out.

3. Continuous monitoring of pyruvate production. Secretion of pyruvate is an infallible signal of the onset of 'redox death' (Figure 1C). Continuous monitoring of pyruvate levels in the broth may, therefore, be used as an alternative feedback control in any of the above examples. An example of such methods is described in Example 4 below.

Benefits of the first aspect of the Invention

These techniques will offer advantages, for example, for any high temperature fermentation, performed using an organism that uses all biomass-derived sugars extremely rapidly (or an organism that uses biomass derived sugars). It circumvents the main disadvantage of the Hartley process, 'redox death' at high sugar concentrations, allowing feeds for example approaching 20% w/v (or in excess of 10% w/v) sugars to be used, as with yeasts.

Although most thermophiles have an ethanol tolerance below 4% w/v, the excess ethanol may be removed continuously, during fermentation carried out at elevated temperature (65-70°C, for example). Standard fermenters adapted to mild vacuum may then be used, rather than the complex Closed System envisaged in Hartley (1988).

Another considerable advantage of fermentations that utilise these inventions is that even spontaneous unstable ldh mutants, such as strain LLD-15, may be used, as well as more stable gene-deleted varieties constructed by genetic engineering. The wild-type takeovers, seen in continuous cultures by San Martin et al. (1993), will be avoided because growth is limited by these methods, throughout the fed-batch or continuous (or batch) fermentations, meaning revertants or contaminant strains do not have time to replace the high density of resident process cells. Description of a second aspect of the Invention

Various fermentation protocols have been devised to avoid or minimize the problem of metabolic co-factor imbalance (e.g. PCT/GB88/00411 , 1988; and the first aspect of the invention described above). An alternative solution is envisaged according to a second aspect of the invention.

There are two classes of formate dehydrogenase, one (encoded by the fdhF gene) converts formate into CO₂ + H₂ and is typical of enterobacteriae such as E. coli. Another (encoded by the fdhi gene) converts formate + NAD into CO₂ + NADH₂ and is present in many facultative anaerobes. Berrios-Rivera et al (Metabolic Engineering 4, 217-219 (2002)) replaced the fdhF gene in E. coli with a yeast fdhλ gene and found that the reduced anaerobic products such as ethanol, lactate and succinate increased relative to oxidised products such as acetate.

If the fdM gene is introduced and expressed in strains that lack lactate dehydrogenase activity, but have a strong PFL pathway - such as those described above, it is believed that a novel highly productive ethanol-producing pathway is created. Since, at low sugar concentrations and in conditions approaching neutral pH (7.4), the PFL pathway predominates, allowing cells to grow most vigorously, by means of this invention, cells will continue to grow vigorously even at high sugar concentrations, since the additional NADH supplied by the FDH pathway will be utilised to restore redox balance, with most of the pyruvate being converted to ethanol and CO₂ (Figure 2).

If input sugar uptake were to rise even further, the PFL-FDH pathway flux may itself become saturated. In this case the residual pyruvate will be metabolised to ethanol and CO₂, by the overflow anaerobic PDH pathway, without risk of metabolic imbalance. Therefore, even at very high sugar concentrations, the only products will be ethanol and CO₂, as in traditional yeast fermentations that operate by the well-known pyruvate decarboxylase pathway.

According to a second aspect of the invention there is provided a micro-organism that essentially lacks lactate dehydrogenase activity, but comprises an heterologous gene encoding an NAD-linked formate dehydrogenase. A lack of lactate dehydrogenase activity helps to prevent the breakdown of pyruvate into lactate, and therefore promotes (under appropriate conditions) the breakdown of pyruvate into ethanol.

The micro-organism may lack lactate dehydrogenase activity because a lactate dehydrogenase (Idh) gene of the micro-organism has been disrupted thereby preventing expression of functional lactate dehydrogenase from the Idh gene.

It is preferred that the lactate dehydrogenase gene is disrupted by a deletion within or of the gene.

The nucleic acid sequence for lactate dehydrogenase is now known. Using this sequence, it is possible for the skilled person to target the lactate dehydrogenase gene to achieve inactivation of the gene through different mechanisms. It is preferred if the lactate dehydrogenase gene is inactivated either by the insertion of a transposon, or, preferably, by the deletion of the gene sequence or a portion of the gene sequence.

Deletion is preferred, as this avoids the difficulty of reactivation of the gene sequence which is often experienced when transposon inactivation is used. In a preferred embodiment, the lactate dehydrogenase gene is inactivated by the integration of a temperature- sensitive plasmid (for example, plasmid pUBI9O-ldh), which achieves natural homologous recombination or integration between the plasmid and the chromosome of the micro-organism. Chromosomal integrants can be selected for on the basis of their resistance to an antibacterial agent (for example, kanamycin). The integration into the lactate dehydrogenase gene may occur by a single cross-over recombination event or by a double (or more) cross-over recombination event.

Examples of modified micro-organisms with inactivated lactate dehydrogenase genes are described in WO 2006/131734 and WO 2006/117546.

According to other embodiments, the micro-organism may comprise a functional lactate dehydrogenase gene, but lack lactate dehydrogenase activity because expression of the gene has been disrupted, or the lactate dehydrogenase may have been inactivated or inhibited. The term "heterologous gene" is used herein to mean a gene of different origin than the host micro-organism, in particular derived from a different species (for example a thermophile).

An example of an heterologous gene encoding an NAD-linked formate dehydrogenase is an fdh1 gene. An fdh1 gene from any suitable species may be used. An example is an fdh1 gene obtained from a methylotrophic bacterium Pseudomonas sp. 101. The gene may encode a functional equivalent of an NAD-linked formate dehydrogenase. Functional equivalents of NAD-linked formate dehydrogenase include expression products of insertion or deletion mutants of natural genes encoding NAD-linked formate dehydrogenase. Preferably the NAD-linked formate dehydrogenase is thermostable (for example in the temperature range 40-85⁰C or above, or 50-70⁰C).

Methods for the preparation and incorporation of heterologous genes into micro- organisms are known, for example in Ingram et al, Biotech & BioEng, 1998; 58 (2+3): 204-214, and US 5,916,787, the content of each being incorporated herein by reference. The heterologous gene may be introduced in a plasmid (preferably a self-replicating plasmid) or integrated into the chromosome, as will be appreciated by the skilled person.

According to a preferred embodiment the heterologous gene may be integrated within a gene of the micro-organism encoding lactate dehydrogenase (Idh) thereby preventing expression of functional lactate dehydrogenase from the Idh gene.

The heterologous gene may be operatively linked to its own promoter or to a host promoter.

The micro-organism is suitably a bacterium, preferably a Gram-positive bacterium. Preferably the micro-organism is a facultative anaerobe.

The micro-organism is preferably a thermophile. Preferably the micro-organism will grow in the temperature range of 40°C-85°C or higher. Preferably, the micro-organism will grow within the temperature range 50°C-70°C. It is also desirable that the microorganism grows in conditions approaching neutral pH, or of pH7.2 or below, in particular pH6.9-pH4.5. A micro-organism of the invention may be derived from a Bacillus spp. Examples of Bacillus species include Bacillus stearothermophilus, B. calvodex, B. caldotenax, B. thermoglucosidasius, B. coagulans, B. licheniformis, B. thermodenitrificans, and B. caldolyticus. In particular, it is preferred that the micro-organism is of the Geobacillus species, in particular Geobacillus thermoglucosidasius.

The micro-organism may be derived from Bacillus stearothermophilus strain LLD-R (NCIB 12403), or from other bacteria described in WO 2006/131734, for example deposited under NCIMB no. 41277, 41278, 41279, 41280, 41281 modified to inactivate the endogenous lactate dehydrogenase gene, or described in WO 2006/117536, for example deposited under NCIMB Accession No. 41275. Alternatively, the bacterium may be derived from bacteria described in WO 02/29030 and WO 01/49865.

It is preferable that a micro-organism of the invention has certain desirable characteristics which permit the micro-organism to be used in a fermentation process.

The micro-organism is preferably selected for ability to grow on any suitable biodegradable waste (such as agro-industrial or municipal waste, or any suitable cellulosic biomass), for example hexose and/or pentose sugars, and oligomers thereof, at thermophilic temperatures. The micro-organism should preferably have no restriction system, thereby avoiding the need for in vivo methylation. The micro-organism should preferably be stable to at least 3% ethanol. Preferably the micro-organism should have the ability to utilise C₃, Cs and C₆ sugars (or their oligomers) as a substrate, including cellobiose and starch. It is preferable if the micro-organism is transformable at a high frequency. The micro-organism should preferably have a growth rate in continuous culture to support dilution rates of 0.3 h^"1 and above (typically 0.3 OD₆oo)-

The micro-organism preferably comprises pyruvate-formate lyase (PFL) pathway activity. The micro-organism preferably comprises pyruvate dehydrogenase (PDH) pathway activity.

The micro-organism may be a spore-former or may not sporulate. The success of the fermentation process does not depend necessarily on the ability of the micro-organism to sporulate, although in certain circumstances it may be preferable to have a sporulator, for example when it is desirable to use the micro-organism as an animal feed-stock at the end of the fermentation process. There is also provided according to the invention a method of producing ethanol, which comprises culturing a micro-organism of the second aspect of the invention under conditions for production of ethanol by the micro-organism.

If the micro-organism is a thermophile, the method may be operated at elevated temperature. A suitable elevated temperature is 50⁰C or above, for example 65-70⁰C, or. even 70⁰C or above.

A micro-organism of the invention may be cultured under conventional culture conditions, depending on the micro-organism chosen. The choice of substrates, temperature, pH and other growth conditions can be selected based on known culture requirements, for example see W001 /149865 and WO01 /85966, the content of each being incorporated herein by reference.

The micro-organism may be cultured under conditions that favour ethanol production by PFL-FDH pathway activity (for example, low to high sugar concentrations, and conditions approaching neutral pH, for example pH 7.4). Alternatively, the micro-organism may be cultured under conditions that favour ethanol production by PFL-FDH pathway activity and PDH pathway activity (for example, high to very high sugar concentrations, and conditions approaching neutral pH, for example pH 7.4).

Although the micro-organism of the second aspect of the invention is designed to allow production of ethanol at high sugar concentrations without onset of redox death, there may be circumstances in which it is appropriate to culture the micro-organism using a method according to the first aspect of the invention. This may be appropriate, for example, when using sugar feed with extremely high sugar concentrations (for example, greater than 20% (w/v)).

It has been appreciated that methods of the invention may be used to improve production of other fermentation products. An example is lactic acid. Bacterial strains able to ferment sugar to produce lactic acid are known to the skilled person (for example, see WO 03/008601). Benefits of the second aspect of the Invention.

Almost all bio-ethanol is currently made from glucose, maltose or sucrose derived from cereal starch, sugar cane or sugar beet, which have value as a foodstuff. Celluloses and hemicelluloses form a major part of agricultural by-products and could, in principle, be a huge source of low-cost, renewable bio-ethanol. However, it is difficult and expensive to derive fermentable sugars from cellulose. In contrast, hemicelluloses are almost as abundant as cellulose and are easy to hydrolyse, but yield a mixture of mainly pentose sugars that yeasts cannot ferment.

For this reason, Hartley (Patent PCT/ GB 8800411) proposed production of ethanol by mutants of a thermophilic Bacillus, which very rapidly ferments all of the sugars derived from biomass, at temperatures up to 70°C. As discussed above, optimum ethanol yield is achieved only by stressed and moribund cells, however, strains constructed according to the present invention will not suffer this problem, since optimal ethanol yield and volumetric productivity will be achieved under optimal growth conditions. Therefore, any microorganism incorporating this invention, may be simply applied to any traditional or novel fermenter configuration, e.g. batch, fed-batch or continuous. Whilst the modified microorganisms will posses a lower tolerance to ethanol than yeasts, ethanol may be simply removed from the process continuously, during the fermentation at 70°C (for example), by the application of a mild vacuum. Moreover, batch fermentations are expected to be 5-10 times faster than typical yeast fermentations.

Embodiments of the first aspect of the invention are described in the following examples.

Examples

Example 1

The anaerobic fermenter contains a small volume of concentrated thermophile cells grown aerobically to late exponential phase, on 2% w/w sugars, at a temperature of 40⁰C or above (suitably 65° -70⁰C). It is fed with 10 - 12% w/w sugars at a variable controlled rate, so that the residual sugars concentration remains below 1 -2 % w/w.

The rate of CO₂ production increases as cell growth increases and then begins to slow when the 'critical point' is reached. At that point, the feed rate is gradually reduced until the rate of CO₂ production stabilises. When the fermenter is full, or when all of the sugars are utilised, the cells are separated from the broth; ethanol is stripped from the supernatant by distillation under mild vacuum. The residual sugars and acid by-products are used to make a fresh inoculum for the next batch or fed-batch fermentation, or recycled to the front-end of a continuous process, either continuously or in controlled doses.

Example 2

In a batch, fed-batch or continuous fermentation, such as described in Example 1 , the feed-rate is reduced or cut off when the critical point is reached and a short pulse of aeration is switched on (or introduced), to allow cell growth sufficient to reduce resident sugar levels below the 'critical point'. Anaerobic growth is then resumed at the original feed rate. In this way a series of mini-fed-batch fermentations will be performed in the same fermenter, until it is full, or throughout the continuous process.

Example 3 In a batch, fed-batch or continuous fermentation, such as described in Example 1, with a constant feed rate, a minimal supply of air is continuously varied to maintain a constant redox potential of the broth (and by inference the internal NAD/NADH ratio) below the 'critical point' until the fermenter is full, or throughout the continuous process.

Example 4

A batch or fed batch fermenter or continuous process, as in Example 1 , is automatically sampled on-line and pyruvate levels in the samples are automatically assayed by a lactate dehydrogenase-linked spectrophotometric assay. A rise above a minimal level will be the signal to reduce feed rates or to begin aeration.

Example 5

Pulsed Thermophilic Fed-Batch Fermentations

The aim of this protocol is to produce high yields of ethanol from a 10% w/v mixture of sugars as found in biomass hydrolysates.

A 1.5 L fermenter is equipped with membrane cell recycle and on-line CO₂ analysis, controlled at 65°, pH 6.5 with N₂ sparging (20 ml/min) and stirred at 400 rpm. The feedstock (a 10% w/v mixture of xylose (60%), glucose (30%) and arabinose (10%) in 0.2 % TYE/salts medium adjusted to pH 6.5) is fed to the fermenter, via a peristaltic pump, from a 5L holding vessel at 25 ml/h.

The inoculum is a 2 litre shake-flask culture of strain LLD-18 grown aerobically at 50°, pH 7, overnight on 2% sugars /0.2% tryptone / 0.2% YE. Optical density is measured and samples taken for HPLC.

Samples analysis: CeIi density (D₂6o), viable cell counts, and HPLC for products and residual sugars.

To optimise successful control of episodic aeration and/or feed rates the following protocol can be used:

1. Add 1.2 L of inoculum to the fermenter and concentrate by membrane recycle to 400 ml. The desired biomass is 4-5g cells/L. Add more inoculum and reconcentrate to 400 ml to achieve this.

2. Begin sugars feed and take duplicate 2 hourly samples for analysis.

3. After an initial lag phase, rates of CO₂ evolution should continue to rise until cell growth ceases and then remain constant until cell death begins. If and when CO₂ evolution begins to decline, stop sugars feed and sparge with air instead of nitrogen for 2-3 h. CO₂ evolution will then rise until all or most residual sugars are used up. Then anaerobic products including ethanol will be used to create fresh aerobic cells.

4. Begin anaerobic feed again and continue until the fermenter is full.

5. Wash out contents and add fresh inoculum as in step 1. 6. In this way a series of serial fed-batch fermentations can be carried out in the same fermenter with cells that are progressing through stages from aerobic to semi-aerobic, anaerobic, and moribund. Rates of product formation can then be linked to each of these physiological states.

When satisfactory sets of data are collected and analysed, this protocol can be varied to study increased feed rates (increased volumetric productivity) and lower pH (lower cell viability but increased ethanol yield).

A further variable will be to sparge with a mixture of 5% air/95% N₂ throughout the run.

Claims

1. A method of producing ethanol using a batch, fed-batch, or continuous bacterial fermentation process, which comprises: carrying out fermentation using a first bacterium with desired ethanol producing pathway activity to produce ethanol; measuring or monitoring flux through the pathway; and controlling process conditions in response to the result of the measurement or monitoring to prevent saturation of pathway flux, or to restore pathway flux following saturation, thereby preventing or reducing the risk of takeover of fermentation by a second bacterium with undesired fermentation activity.

2. A method according to claim 1 , in which pathway flux is measured or monitored by measuring or monitoring residual sugar level, redox potential or pyruvate level of the fermentation broth, or rate of carbon dioxide production.

3. A method according to claim 1 or 2, in which the ethanol producing pathway activity is anaerobic pyruvate dehydrogenase (PDH) pathway activity.

4. A method according to any preceding claim, in which pathway flux is measured or monitored by measuring growth of cells of the first bacterium.

5. A method according to any preceding claim, in which controlled aeration of the fermentation process is used to prevent saturation of pathway flux, or to restore pathway flux following saturation.

6. A method according to claim 5, in which the controlled aeration comprises episodic aeration of the fermentation process.

7. A method according to claim 5, in which the controlled aeration comprises regulating a level of continuous aeration of the fermentation process.

8. A method according to any of claims 5 to 7, in which aeration is controlled so as to favour minimal viable bacterial cell growth with minimal aerobic sugar utilisation.

9. A method according to any of claims 5 to 8, in which sugar feed rate is maintained substantially constant throughout the fermentation process.

10. A method according to any of claims 1 to 8, in which control of sugar feed rate is used to prevent saturation of pathway flux, or to restore pathway flux following saturation.

11. A method according to claim 10, in which sugar feed rate is controlled by regulating a level of continuous sugar feed provision during the fermentation process.

12. A method according to claim 10, in which sugar feed rate is controlled by providing sugar feed intermittently during the fermentation process.

13. A method according to claim 12, in which provision of sugar feed is stopped during an episode of aeration of the fermentation process.

14. A method according to any preceding claim, in which the bacterium is fed during the fermentation process with sugar feed comprising at least 4% (w/v) sugar.

15. A method according to claim 14, in which the sugar feed comprises 10-20% (w/v) sugar.

16. A method according to any preceding claim, in which conditions are optimised to favour ethanol production by anaerobic pyruvate dehydrogenase (PDH) pathway activity.

17. A method according to any preceding claim, in which conditions are adjusted during the fermentation process so as to favour maximisation of the ratio of anaerobically to aerobically produced carbon dioxide, thereby favouring maximisation of ethanol yield.

18. A method according to any preceding claim, in which ethanol is removed continuously during the fermentation process to maintain ethanol concentration below the ethanol tolerance of the first bacterium.

19. A method according to any preceding claim, in which the fermentation process is a continuous fermentation process.

20. A method according to any preceding claim, in which the first bacterium is a facultative anaerobe.

21. A method according to any preceding claim, in which the first bacterium is a Gram positive bacterium.

22. A method according to any preceding claim, in which the first bacterium lacks lactate dehydrogenase activity.

23. A method according to any preceding claim, in which the first bacterium is a thermophile.

24. A method according to claim 23, in which the first bacterium is a Bacillus sp.

25. A method according to claim 24, in which the first bacterium is a Bacillus stearothermophilus.

26. A method according to claim 25, in which the bacterium is derived from Bacillus stearothermophilus strain LLD-R (NCIB 12403).

27. Ethanol production by a thermophilic micro-organism in a batch, fed-batch, or continuous fermentation process, in which redox death of cells of the micro-organism carrying out fermentation is avoided or minimised by regulating the sugars feed rate, or by controlled minimal aeration.

28. A method according to claim 27, in which saturation of anaerobic pyruvate dehydrogenase (PDH) flux is used as a signal of onset of redox death.

29. A method according to claim 28, in which the sugars feed rate or the aeration level is changed when PDH flux is or is becoming saturated.

30. A method according to claim 28 or 29, in which the feed rate is reduced when PDH-flux is saturated.

31. A method according to claim 28 or 29, in which the feed rate is cut off when PDH flux is saturated, and a short pulse of aeration is switched on.

32. A method according to claim 28, in which a constant feed rate is used, and a minimal supply of air is continuously varied to prevent saturation of PDH flux.

33. A method according to claim 27, in which secretion of pyruvate is used as a signal of onset of redox death.

34. A method according to claim 33, in which feed rate is reduced, or aeration is begun if pyruvate levels in the fermentation broth rise above a minimal level.

35. A method according to any of claims 27 to 32, in which onset of redox death, or PDH flux is determined by measuring the level of pyruvate in the fermentation broth, the redox potential of the fermentation broth, the residual sugar level of the fermentation broth, or the rate of carbon dioxide production.

36. Ethanol production by a thermophilic micro-organism in a fed-batch, or continuous fermentation process of 10-20% w/v mixed sugars, fed at rates controlled by feedback sensors so as to maintain the cells in redox balance.

37. Fermentations according to Claim 36 in which ethanol is removed continuously from the broth so as to reduce ethanol concentration below the ethanol tolerance of the organism.

38. Fermentations according to claims 36 or 37 in which the organism is a thermophilic Bacillus lacking lactate dehydrogenase activity,

39. Fermentations according to claims 36 to 38 in which the organism is derived from Bacillus stearothermophilus strain LLD-R (NCIB 12403)

40. Fermentations according to claims 36 to 39 in which the sensors measure rates of anaerobic CO₂ production and/or broth redox potential and/or residual sugars level and/or broth pyruvate levels.

41. Fermentations according to Claim 40 in which the sensors control sugars feed rate and/or a minimal aeration rate.

42. Fermentations according to Claim 40 in which the sugars feed is stopped during a series of brief episodes of aeration.

43. Fermentations according to Claim 40 in which the aeration is continuously varied 5 so as to maintain minimal viable cell growth with minimal aerobic sugars utilisation.

44. A micro-organism that lacks lactate dehydrogenase activity, but comprises an heterologous gene encoding an NAD-linked formate dehydrogenase.

10 45. A micro-organism according to claim 44, wherein the heterologous gene is an fdh1 gene.

46. A micro-organism according to claim 44 or 45, in which a lactate dehydrogenase (Idh) gene has been inactivated thereby preventing expression of functional lactate

15 dehydrogenase from the Idh gene.

47. A micro-organism according to any of claims 44 to 46, in which the heterologous gene is integrated into a chromosome of the micro-organism.

0 48. A micro-organism according to any of claims 44 to 47, in which the heterologous gene is operatively linked to its own promoter or to a promoter of the micro-organism.

49. A micro-organism according to any of claims 44 to 48, in which the heterologous gene is integrated within a gene of the micro-organism encoding lactate dehydrogenase 5 (Idh) thereby preventing expression of functional lactate dehydrogenase from the Idh gene.

50. A micro-organism according to any of claims 44 to 48, in which the heterologous gene is part of a plasmid. 0

51. A micro-organism according to claim 50, in which the plasmid is a self-replicating plasmid.

52. A micro-organism according to any of claims 44 to 51 which has pyruvate- 5 formate lyase (PFL) pathway activity.

53. A micro-organism according to any of claims 44 to 52 which is a bacterium.

54. A micro-organism according to any of claims 44 to 53 which is a thermophile.

55. A micro-organism according to claim 54 which is a Bacillus species.

56. A micro-organism according to claim 55 which is derived from Bacillus stearothermophilus LLD-R (NCIB 12403).

57. A method of producing ethanol, which comprises culturing a micro-organism according to any of claims 44 to 56 under conditions for production of ethanol by the micro-organism.

58. Ethanol production by a microorganism that lacks lactate dehydrogenase activity but contains an active pyruvate-formate lyase activity (PFL) pathway and expresses a cloned fdhλ gene encoding an NAD-linked formate dehydrogenase.

59. A method or microorganism according to claim 58 that can utilize a wide range of hexoses, pentoses and oligosaccharides derived from hydrolysis of biomass,

60. A method or microorganism according to claim 59 that is a thermophilic Bacillus.

61. A method or microorganism according to claim 60 that is derived from Bacillus stearothermophilus LLD-R (NCIB 12403).

62. A method according to claim 58 wherein the cloning is performed through a replicative or non-replicative plasmid vehicle.

63. A method according to claim 58 wherein the fdthλ gene is expressed in the host microorganism by a self-replicating plasmid.

64. A method according to claim 58 wherein the fdhλ gene is integrated into the host genome and is expressed either from its own promoter or from a host promoter.

65. A method according to claim 64 wherein the fdfti gene is integrated within the host ldh gene, so as to inactivate the latter.