NL2006984C2

NL2006984C2 - A process for converting organic material.

Info

Publication number: NL2006984C2
Application number: NL2006984A
Authority: NL
Inventors: Adrie Straathof; Corjan Berg; Luuk Wielen
Original assignee: Univ Delft Tech
Priority date: 2011-06-22
Filing date: 2011-06-22
Publication date: 2013-01-02

Description

A Process for converting organic material FIELD OF THE INVENTION

The present invention is in the field of a process 5 for obtaining a reaction product suitable for use as a bio fuel, flavouring agent or fragrance and its use therefore.

BACKGROUND OF THE INVENTION

Bio-fuels have received increasing attention over 10 recent years in light of concerns over ever decreasing supplies of fossil fuels. The term bio-fuel may be applied to a wide range of compounds that are combustible and which are derived from living or recently living organisms (biomass).

The term bio-fuel refers to solid biomass, liquid fuels and 15 various biogases. Of particular interest in the present context are liquid fuels, and in particular bio alcohols and their alternatives, such as esters. The most common bioalcohols are ethanol, propanol and butanol; typically these are produced by fermenting sugars, starches, or cellulose in 20 the presence of microorganisms such as fungi, yeasts or bacteria. It remains a problem to obtain bio-fuels that are suitable for the range of applications to which fossil-fuel derived fuels are currently employed and which may be produced cheaply enough and in sufficient quantities.

25 Butanol and in particular 1-butanol is widely cited as a preferred bio alcohol since it may be used as an alternative to petrol with internal combustion engines without requiring that the engine be modified. It is not preferable to use ethanol directly with an unmodified engine since ethanol 30 e.g. readily takes up water from the atmosphere and if used can result in the engine becoming corroded. Ethanol may be used as a blend with conventional petrol of up to about 15 % w/w with unmodified engines.

1-butanol is obtainable through acetone-butanol-35 ethanol (ABE) fermentatation. ABE fermentation relates to a fermentation process for converting sugars into acetone, 1-butanol and ethanol. The process is anaerobic and is typically performed in the presence of a strain or strains of bacteria from the genus Clostridium, in particular Clostridium 2 acetobutylicum. A problem with ABE fermentation is that the achievable 1-butanol concentration is low since at 1-butanol concentrations of greater than 3% w/w (solute/solvent e.g. 3% w/w of 1-butanol in water is eguivalent to 30 g 1-butanol in 5 1000 g of water), growth and metabolism of most microorganisms are severely inhibited [S. Liu and N. Qureshi, New Biotechnology, 2009, 26(3/4), 117-121]. Conseguently, energy intensive separation is reguired in order to obtain a concentrated product of sufficient purity. In current 10 industrial 1-butanol production processes separation is by distillation [Y. Ni and Z. Sun, Applied Microbiology and Technology, 2009, 83, 415-423]. This involves heating the whole fermentation broth to around its boiling point. The fermentation broth is typically ~98 % water; water has a lower 15 boiling point than 1-butanol and must therefore be evaporated first; separation by distillation is any case an energy intensive process and is particularly so for separating 1-butanol from an aqueous mixture.

Alternative 1-butanol recovery methods have been 20 evaluated for their energy efficiency [A. Oudshoorn, L. A. M. van der Wielen and A. J. J. Straathof, Industrial and Engineering Chemistry Research, 2009, 48, 7325-7336] . However, to date all have proved to have problems, in particular with regards to their energy requirements. The energy requirements 25 for steam stripping and distillation have been estimated to be 66% of the combustion energy of the recovered 1-butanol while extraction and adsorption have the best energy efficiency with losses of 25 and 22%, respectively. Extractive ABE fermentations have been studied numerous times using organic 30 solvents [W. E. Barton and A. J. Daugalis, Applied

Microbiology and Biotechnology, 1992, 36(5), 632-639; W. J. Groot et al., Bioprocess Engineering, 1990, 5, 203-216] and ionic liquids [S. H. Ha, N. L. Mai and Y-N. Koo, Process Biochemistry, 2010, 45, 1899-1903]. However, all tested 35 extractants with a high 1-butanol partition coefficient have been found to be toxic towards Clostridium acetobutylicum. The highest partition coefficient for an extractant that is nontoxic for Clostridium acetobutylicum is oleic acid; oleic acid has a 1-butanol coefficient of 3.9 (Log P = 0.59): a value 3 that is still problematically low in terms of its potential for use as an extractant in a process that is to be commercially viable. From these data it can be assumed that the benefits of integrated 1-butanol extraction are limited 5 due to its poor extraction behaviour. Whilst, Li et al. [Q. Li et al., Applied Biochemistry and Biotechnology, 2010, 162, 2381-2386] used biodiesel as a 1-butanol extractant to improve biodiesel properties in terms of providing a decreased cold filter plugging point, a decreased acid number and an 10 increased cetane number, there is a strong European incentive for blending 10% bio-fuel with transport petrol and diesel by the year 2020 [H. -G. Pottering and P. Necas, Directive 2009/28/EC of the European Parliament and of the Council, of 23 April 2009], but this appears not to be attainable by 1-15 butanol extraction.

The present invention therefore relates to a process for obtaining a reaction product, suitable for use as a biofuel, which overcomes one or more of the above disadvantages, without jeopardizing functionality and advantages.

20

SUMMARY OF THE INVENTION

The present invention relates in a first aspect to a process for obtaining a reaction product, batch wise or continuously, that is suitable for use as a bio fuel, 25 flavouring agent or fragrance, comprising the steps: (i) providing organic material, such as biomass, such as ligno cellulose comprising biomass, or a hydrolysate thereof; (ii) providing one or more strains of microorganism; 30 (iii) forming a mixture of the organic material and one or more strains of microorganism in an aqueous medium; (iv) (a) converting the organic material into at least a first alcohol and at least a first carboxylic acid; and providing 0 to 10 % w/w, preferably 0 to 5 % w/w 35 and more preferably 0 to 3 weight percent of a second alcohol and/or 0 to 10 weight percent, preferably 0 to 5 % w/w and more preferably 0 to 3 % w/w of a second carboxylic acid; or 4 (b) converting the organic material into at least a first alcohol and providing 0.1 to 10 % w/w, preferably 0.1 to 5 % w/w and more preferably 0.1 to 3 % w/w of at least a first carboxylic acid; or 5 (c) converting the organic material into at least a first carboxylic acid and providing 0.1 to 10 weight percent, preferably 0.1 to 5 % w/w and more preferably 0.1 to 3 % w/w of at least a first alcohol; (v) forming at least a reaction product, such as an ester, 10 of (a) the first alcohol and the first and/or second carboxylic acid, or (b) of the first carboxylic acid and the first and/or second alcohol, 15 wherein the reaction product is more hydrophobic than the most hydrophobic of the components from which it is formed, preferably by producing or adding at least one catalyst, such as an enzyme, such as a cell-surface displayed lipase or an immobilized lipase; 20 (vi) separating said reaction product at least concomitantly with formation of the reaction product, preferably by extracting said reaction product with an extractant, and preferably directly using an apolar medium, such as a hydrophobic organic medium.

25 The present invention therefore relates to a process that comprises: providing organic material and one or more strains of microorganism; converting the organic material in the presence of a microorganism into at least a first alcohol and/or at least a first carboxylic acid; providing if 30 necessary a second alcohol or second carboxylic acid; reacting the first alcohol and the first and/or second carboxylic acid, or reacting the first carboxylic acid and the first and/or second alcohol, to form a reaction product that is more hydrophobic than the most hydrophobic of the first and/or 35 second alcohol and the first and/or second carboxylic acid, as appropriate; separating the reaction product.

Whilst the present invention was developed in the context of bio-fuels, it should be noted that the process of the invention is not limited to bio-fuels, but is similarly 5 applicable, for example, for the production of a reaction product, such as an ester, that is suitable for use as a flavouring agent or fragrance. Many of the advantages disclosed both above and in the remainder of the description 5 are similarly applicable.

An advantage of converting (a) the first alcohol and the first and/or second carboxylic acid, or (b) the first carboxylic acid and the first and/or second alcohol, into a reaction product that is more hydrophobic than the first 10 and/or second alcohol and the first and/or second carboxylic acid is that it can be extracted more effectively than its substrates i.e. the first and/or second alcohol and the first and/or second carboxylic acid, as appropriate. As an example, it has been found that butyl butyrate formed in accordance 15 with the process of the invention from 1-butanol and butyric acid can be extracted more than three orders of magnitude more effectively than its substrates. As such, a major disadvantage of current ABE fermentation processes is overcome. The advantage similarly applies more generally to any process that 20 would otherwise require extraction of an alcohol or carboxylic acid. Through improving the effectiveness of an extraction, the commercial viability of any process wherein said extraction is used is similarly improved.

Thereby the present invention provides a solution to 25 one or more of the above mentioned problems.

In an example the present invention relates to a process, wherein the organic matter is biomass, preferably lignocellulosic biomass. Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, 30 and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin.

Lignocellulose biomass can be grouped into four main categories: agricultural residues (including corn stover and sugarcane bagasse), dedicated energy crops, wood residues 35 (including sawmill and paper mill discards), and municipal paper waste; all constitute cheap, readily available and renewable feedstocks.

In an example, the present invention relates to a process, wherein the organic matter is converted into a first 6 alcohol and/or a first carboxylic acid, preferably in a ratio of about 1:1. At least a second alcohol and/or at least a second carboxylic acid may be added additionally.

In an example, the present invention relates to a 5 process, wherein a mixture is formed of organic material and one or more strains of microorganism in an aqueous medium and wherein the organic material is converted into a first alcohol and/or a first carboxylic acid by fermentation. It is preferred that the first alcohol and the first carboxylic acid 10 are both obtained through fermentation. However, any process whereby organic matter is converted into an alcohol or carboxylic acid through the action of a microorganism would equally be completely in keeping with the spirit if the invention and the process as disclosed. In an alternative 15 example, it is equally envisaged to use a combination of two or more strains of microorganism, at least one of which produces alcohols and at least one of which produces carboxylic acids. It is noted that the second alcohol and second carboxylic acid may be obtained chemically, however it 20 is preferred that they are obtained from biomass.

In an example, the present invention relates to a process, wherein at least an ester of (a) a first alcohol and a first and/or second carboxylic acid, or (b) a first carboxylic acid and a first and/or second alcohol, is formed, 25 wherein the ester is more hydrophobic than the most hydrophobic of the reactants. Similarly to alcohols, esters have the advantage that they may be used directly as fuels.

In an example, the present invention relates to a process, wherein (a) a first alcohol and a first and/or second 30 carboxylic acid, or (b) a first carboxylic acid and a first and/or second alcohol, are converted into a reaction product, such as an ester, in the presence of an enzyme, preferably a lipase, more preferably a cell-surface displayed lipase. In an alternative example the lipase is an immobilized lipase. In a 35 further alternative example, it is equally envisageable that a microorganism be used that both converts organic matter into an alcohol and/or carboxylic acid, and that produces lipase. Immobilized lipases are preferred as they constitute a cost-effective catalyst.

7

In an example, the present invention relates to a process, wherein the extractant is a hydrophobic organic medium and wherein with the extractant provided on the aqueous medium, a proportion of the extractant and aqueous medium 5 remain as distinct phases. It should be noted that - separating said reaction product at least concomitantly with formation of the reaction product - is taken to mean that separation of the reaction product is performed for at least a portion of the time during which the reaction product is being 10 formed.

Advantages of the present description are detailed throughout the description.

DETAILED DESCRIPTION OF THE INVENTION 15 The present invention relates in a first aspect to a process for obtaining a reaction product, batch wise or continuously, that is suitable for use as a bio fuel, flavouring agent or fragrance, comprising the steps: (i) providing organic material, such as biomass, such as 20 ligno cellulose comprising biomass, or a hydrolysate thereof; (ii) providing one or more strains of microorganism; (iii) forming a mixture of the organic material and one or more strains of microorganism in an aqueous medium; 25 (iv) (a) converting the organic material into at least a first alcohol and at least a first carboxylic acid; and providing 0 to 10 % w/w, preferably 0 to 5 % w/w and more preferably 0 to 3 weight percent of a second alcohol and/or 0 to 10 weight percent, 30 preferably 0 to 5 % w/w and more preferably 0 to 3 % w/w of a second carboxylic acid; or (b) converting the organic material into at least a first alcohol and providing 0.1 to 10 % w/w, preferably 0.1 to 5 % w/w and more preferably 0.1 to 3 % w/w of at 35 least a first carboxylic acid; or (c) converting the organic material into at least a first carboxylic acid and providing 0.1 to 10 weight percent, preferably 0.1 to 5 % w/w and more preferably 0.1 to 3 % w/w of at least a first alcohol; 8 (v) forming at least a reaction product, such as an ester, of (a) the first alcohol and the first and/or second carboxylic acid, or 5 (b) of the first carboxylic acid and the first and/or second alcohol, wherein the reaction product is more hydrophobic than the most hydrophobic of the components from which it is formed, preferably by producing or adding at least one catalyst, such 10 as an enzyme, such as a cell-surface displayed lipase or an immobilized lipase; (vi) separating said reaction product at least concomitantly with formation of the reaction product, preferably by extracting said reaction product with an extractant, 15 and preferably directly using an apolar medium, such as a hydrophobic organic medium.

In an example, the present invention relates to a process, wherein a Log P (extractant/water) of the reaction product is greater than a Log P (extractant/water) of the 20 first alcohol and/or of the first carboxylic acid, said Log P (extractant/water) of the reaction product being at least 0.5 log units, preferably at least 1 log unit, more preferably at least 2 log units, most preferably at least 3 log units greater than the most hydrophobic of the first alcohol and the 25 first carboxylic acid. P is a partition coefficient i.e. the ratio of concentrations of a compound e.g. the reaction product, in the two phases, of a mixture of two immiscible solvents e.g. the extractant and water, at equilibrium, measured at T = 25 °C and at a pH at which the carboxylic acid 30 is not ionized. Through using an extractant with as high a Log P (extractant/water) as possible, loss of solvent through mixing is minimised and costs are reduced.

In an example, the present invention relates to a process, wherein at least steps (iv) to (vi) are performed in 35 a single reactor, preferably at least steps (iii) to (vi) and most preferably steps (i) to (vi) .

Accordingly, it is preferred that the steps are performed such that an equilibrium exists between: converting the organic material in the presence of a microorganism into 9 at least a first alcohol and/or a first carboxylic acid; reacting the first alcohol and the first and/or second carboxylic acid, or reacting the first carboxylic acid and the first and/or second alcohol, to form a reaction product that 5 is more hydrophobic than the most hydrophobic of the first and/or second alcohol and the first and/or second carboxylic acid, as appropriate; extracting the reaction product. Particular advantages are that the aqueous concentration of the at least one alcohol or carboxylic acid is prevented from 10 reaching a level at which it would prove toxic for the microorganism. Furthermore, alternative more energy intensive and hence expensive techniques for obtaining bio-fuel are avoided.

In a preferred example of the invention, the 15 extraction phase containing the reaction product is useable directly as a bio-fuel; alternatively, the first alcohol and/or the first and/or second carboxylic acid, or the first carboxylic acid and the first and/or second alcohol are reobtained through an additional reaction step subsequent to 20 extraction, such as, for example hydrolysis.

In an example, the present invention relates to a process, wherein the at least one strain of microorganism comprises at least one of bacteria, yeasts and fungi.

In an example, the present invention relates to a 25 process, wherein the at least one microorganism is a bacteria belonging to the genus Clostridium.

In an example, the present invention relates to a process, wherein the at least one strain of microorganism comprises yeasts such as Saccharomyces cerevisiae, and/or 30 bacteria such as Pseudomonas, Lactobacillaceae, lactococcii and Escherichia coli.

In an example, the present invention relates to a process, wherein the first and/or second alcohol is selected from C1-C20 alcohols, preferably a C4-C20 alcohol, such as 35 ethanol, propanol, butanol, pentanol and/or oleylalcohol and/or wherein the first and/or second carboxylic acid is selected from C1-C20 carboxylic acids, preferably a C4-C20 carboxylic acid, such as acetic acid, propionic acid, butyric acid, valeric acid and oleic acid.

10

In an example, the present invention relates to a process, wherein the ester comprises from 6-40 carbon atoms, preferably from 8-25 carbon atoms, such as from 12-20 carbon atoms .

5 In an example, the present invention relates to a process, wherein the apolar medium is selected from alkanes, preferably C4-C20 alkanes, alkenes, aromatics, gas oil, diesel, gasoline and petrol. Extraction directly into diesel, gasoline or petrol is particularly advantages wherein a mixture of the 10 reaction product and diesel, gasoline or petrol has desirable properties, or where the presence of a percentage of reaction product enhances the properties of the diesel, gasoline or petrol, for example through increasing its octane rating.

In an example, the present invention relates to a 15 process, wherein the process further comprises a step of providing additives, such as nitrogen and/or nitrogen containing compounds, such as sugar, such as glucose. Such additives can be advantageous, for example to improve or restore the efficiency of at least one of the steps of the 20 process.

In an example, the present invention relates to a process, wherein the second alcohol or second carboxylic acid is added in order to substantially balance the ratio of (a) the first alcohol and the first and/or second carboxylic acid, 25 or (b) of the first carboxylic acid and the first and/or second alcohol. In general, it is preferred that the ratio of alcohol to carboxylic acid is close to 1:1 in order to maximise production of a reaction product. Under certain circumstances other ratios such as 1:2 or 2:1 may be optimal. 30 In an example, the present invention relates to a process, wherein the process further comprises at least one of the steps (a) monitoring the pH of the mixture of the organic material and one or more strains of microorganism in aqueous 35 medium for at least a portion of the duration of the process (b) adjusting a control parameter, and (c) adding at least one species chosen from: a group comprising acids and bases, a group comprising alcohols and carboxylic acids, 11 a group comprising additives, e.g. N-releasing compounds such as nitrogen, and a sugar.

In an example, the present invention relates to a process, wherein first a substantial part of the first 5 carboxylic acid is formed and thereafter the first alcohol, preferably until a pH is increased to 7.

In an example, the present invention relates to a process wherein at least steps (iv) - (vi) are performed at a temperature of 37°C. Biological processes, such as those 10 involving microorganisms and enzymes typically operate optimally at a temperature of around 20-60°C.

In an example, the present invention relates to a process, wherein steps (iv) - (vi) are performed between a pH of 3 and a pH of 7. In particular with regards to the 15 production of esters, such as butyl butyrate, from (a) a first alcohol and a first and/or second carboxylic acid, or (b) of a first carboxylic acid and a first and/or second, such as 1-butanol and butyric acid, wherein the reaction is catalysed in the presence an enzyme, such as a lipase, the selectivity of 20 conversion of the reactants into the reaction product is favoured by an acid pH, preferably between pH 4 and 7, more preferably between a pH of 4 and 6, and most preferably between a pH of 4 and 5.

The present invention relates in a second aspect to 25 the use of a reaction product such as an ester, prepared in the process of at least claim 1, as a flavouring agent, fragrance and/or a bio-fuel.

EXAMPLES

30 The invention is further detailed by the accompanying figures, which are exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling 35 within the scope of protection, defined by the present claims .

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FIGURES

The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying figures.

5 Figure 1 shows equilibria involved in an esterification reaction between 1-butanol and butyric acid with concomitant extraction of butyl butyrate.

Figures 2A and B show results of Clostridium acetobutylicum fed-batch fermentation coupled with extraction 10 and esterification.

DETAILED DESCRIPTION OF THE DRAWINGS / FIGURES

Figure 1 shows equilibria involved in an esterification reaction between 1-butanol and butyric acid 15 with concomitant extraction of butyl butyrate.

With regards to the abbreviations used: I = aqueous phase e.g. the aqueous Clostridium medium and lipase II = organic phase e.g. hexadecane 20 Biomass = e.g. glucose B“ = butyrate BH = butyric acid BuOH = butanol

BuB = butyl butyrate subscripts org and aq. refer to the 25 species being in the organic or aqueous phase respectively.

The aqueous phase comprises Clostridium bacteria and a lipase.

Reaction of butanol and butyric acid to form butyl butyrate and water is an equilibrium. Similarly, distribution of these species between the aqueous phase e.g. the aqueous 30 Clostridium medium and lipase, and the organic phase e.g.

hexadecane, are equilibria. The position of these equilibria depends on the hydrophobicities of the species, e.g. butanol, butyric acid and butyl butyrate, defined in terms of their Log P values.

35 Partition coefficients for each of 1-butanol, butyric acid and butyl butyrate in a hexadecane/aqueous system are given in Table 1.

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Table 1: Partition coefficients of solutes in _hexadecane/agueous system (T = 25 °C) ._

hexadecane/aqueous Log P

solute partition coefficient, P

1-butanol 0.44 ± 0.04 -0.36 butyric acid 0.13 ± 0.06 -0.87 butyl butyrate 340 ± 103 2.53

As is evident from a comparison of the Log P 5 (hexadecane/aqueous) values in Table 1, the Cs-ester butyl butyrate is several orders of magnitude more hydrophobic than its substrates i.e. 1-butanol and butyric acid (2.53, cf. - 0.36 and -0.87 respectively). Therefore, partition of butyl butyrate between the aqueous and organic phases lies towards 10 the organic phase, whereas the substrates butanol and butyric acid remain mostly in the aqueous phase. By separating, by extraction, the reaction product butyl butyrate from its substrates, the equilibrium corresponding with reaction of butanol and butyric acid to form butyl butyrate and water, is 15 shifted to the right i.e. towards butyl butyrate production. This is further advantageous since through continuously extracting butyl butyrate the concentration of substrates such as butanol and butyric acid is prevented from reaching a level, toxic or highly inhibiting for the alcohol and/or 20 carboxylic acid forming microorganism(s).

Figures 2A and B show results of Clostridium acetobutylicum fed-batch fermentation coupled with extraction and esterification.

With respect to Fig. 2A:

25 Left axis = concentration (g/1) right axis = pH horizontal axis = time (h) no symbol = pH

triangle = total dissociated and undissociated butyric acid 30 (g/L) cross = cell dry weight (g/L) diamond = butanol in aqueous phase (g/L)

With respect to Fig. 2B:

Left axis = concentration (g/1) 14 right axis = biomass e.g. glucose concentration (g/1) horizontal axis = time (h) diamond = butylbutyrate in hexadecane phase (g/L) square = butyrate in aqueous phase (g/L) 5 cross = butyric acid in aqueous phase (g/L) triangle = glucose in aqueous phase (g/L)

Prior to inoculation, a fermentation vessel was charged with 50 ml overnight incubated shake-flask content comprising Clostridium acetobutylicum, strain ATCC824 10 (obtained from CBS-KNAW Fungal Diversity Biocentre;

Netherlands). To this was added 1.5 1 of culture medium and 250 ml, 02-free, n-hexadecane. The culture medium contained glucose at a concentration of 40 g/1, which, for test purposes, was used as feedstock. The fermentation mix was 15 buffered with CaC03 at a concentration of 1 g/1 and was maintained under anaerobic conditions through continuous sparging with N2. The fed-batch fermentation was performed with a feed phase that contained butyric acid and glucose as substrates for 1-butanol production and ester formation.

20 For interpretation of the results it is important to note that it has been established that ABE-producing Clostridia possess two distinct characteristic phases in their catabolic pathway, being the acidogenic and solventogenic phase. Typically, during acidogenesis, it has been found that 25 cell growth is exponential and products are acetic and butyric acid along with ATP. Undissociated acids can diffuse passively through the membrane causing the pH gradient to collapse. At high butyric acid concentrations this results in a low ATP/ADP ratio. However, it has been found that this does not 30 completely stop substrate assimilation and cell metabolism. Once a critical undissociated butyric acid concentration is reached (~ 1.4 g/L), cells shift to solventogenesis. Then, cell growth enters the stationary phase, the organic acids are reutilized, and acetone, 1-butanol and ethanol are produced.

35 Between t=0 and t=9h, the pH dropped by a few (1-2) pH units from about 6.6 to about 5.0. The time period from t=0 to t=9h corresponds to the acidogenic phase; at about t=9h, the solventogenic phase begins. The solventogenic phase is accompanied by an increase in pH. At about t=16h, 2500 U of 15 lipase (3.31 g CaLB ilmmobead-150, Chiralvision; Leiden, The Netherlands) was added, and pH control was switched on. The pH was set at 5.2 using a feed (containing 160 g/L butyric acid and 80 g/L glucose). It should be noted that, due to 5 thermodynamic constraints, only undissociated butyric acid (pKa = 4.82, see equation 1) will react with 1-butanol in aqueous media. It has been found experimentally that it is favorable to perform this reaction at low pH from an esterification point of view. Following addition of lipase, 10 the 1-butanol production rate was at its highest level at 23 h, being 1.03 g.L-1.h-1. Furthermore, 1-butanol yields on glucose were 0.56, 0.41 and 0.45 g/g between 19 and 23 h.

These values were close to, or even higher than the theoretical maximum of 0.41 g BuOH/g glucose if only glucose 15 would be used, indicating that 1-butanol was also produced from added butyric acid. The present invention acknowledges the advantages hereof.

During the time-interval of 17 to 25 h the average butyl butyrate production rate was 0.43 g.L_1.h_1. The 20 esterification stopped at butyl butyrate concentrations of 4.9 g/L in the organic phase. Fresh lipase (1 g) was added to the fermentation broth at about t=50 h to check whether previously added enzyme was inactivated. However, since no additional butyl butyrate was formed after 50 h it can be assumed that 25 esterification equilibrium had been reached. With further optimization, butyl butyrate concentrations of up to about 15 g/1 in organic phase should be achievable. Butyl butyrate hexadecane/fermentation broth partitioning did not change after 48 h, remaining between 474 and 492. This indicates that 30 extraction equilibrium for butyl butyrate was reached. 1-Butanol concentrations did not reach levels such that they would inhibit or become toxic for the Clostridium bacteria. In this example, wild-type Clostridium acetobutylicum was used; non-sporulating Clostrium acetobutylicum might be able to 35 produce butanol for longer periods of time and thereby extend the time-window in which Clostridia produce 1-butanol.

It should be appreciated that for commercial application it may be preferable to use continuous extraction in order to further favour production of butyl butyrate. Whilst extraction 16 is a preferred example, any technique that results in separation of butyl butyrate from its substrates would similarbe in keeping with the spirit of the invention.

Claims

A method for obtaining a reaction product, batch or continuous, suitable for use as a biofuel, flavoring or fragrance, comprising the steps of: (i) providing organic material such as biomass, such as lignocellulose biomass, or a hydrolyzate thereof; (ii) providing one or more strains of microorganisms; (iii) forming a mixture of the organic material 10 and one or more strains of microorganisms in an aqueous medium; (iv) (a) converting the organic material into at least a first alcohol and at least a first carboxylic acid; and providing 0-10% w / w, preferably 0-5% w / w and more preferably 0-3% by weight of a second alcohol and / or 0-10% by weight, preferably 0-5% w / w and more preferably 0-3% w / w of a second carboxylic acid; or (b) converting the organic material into at least a first alcohol and providing 0.1-10% w / w, more preferably 0.1-5% w / w and most preferably 0.1- 3% w / w of at least a first carboxylic acid; or (c) converting the organic material into at least a first carboxylic acid and providing 0.1-10% by weight, more preferably 0.1-5% w / w and even more preferably 0.1-3% w / w of at least a first alcohol; (v) forming at least one reaction product, such as an ester, from (a) the first alcohol and the first and / or second carboxylic acid, or (b) from the first carboxylic acid and the first and / or second alcohol wherein the reaction product is more hydrophobic than most hydrophobic of the components from which it is formed, preferably by producing or administering at least one catalyst, such as an enzyme, such as a cell surface shown lipase or an immobilized lipase; (Vi) separating the reaction product at least during the formation of the reaction product, preferably by extracting the reaction product with an extraction agent, and preferably directly using an apolar medium, such as a hydrophobic organic medium.

A method according to claim 1, wherein a Log P (extractant / water) of the reaction product is greater than a Log P (extractant / water) of the first alcohol and / or of the first carboxylic acid, wherein the Log P (extractant / water) 10 of the reaction product is at least 0.5 log units, more preferably at least 1 log unit, even more preferably at least 2 log units, most preferably at least 3 log units, greater than most hydrophobic of the first alcohol and first carboxylic acid.

3. Process according to at least one of the preceding claims, wherein at least steps (iv) to (vi) are carried out in a single reactor, preferably at least steps (iii) to (vi) and most preferably steps (i) to (vi).

4. A method according to at least one of the preceding claims, wherein at least one strain of microorganisms comprises at least one of bacteria, yeasts and fungi.

The method according to at least one of the preceding claims, wherein the at least one microorganism is a bacterium belonging to the genus Clostridium.

6. Process according to at least one of the preceding claims, wherein the first and / or second alcohol are selected from C 1 -C 20 alcohols, preferably a C 4 -C 20 alcohol, such as ethanol, propanol, butanol, pentanol, and / or olyl alcohol and / or wherein the first and / or second carboxylic acid is selected from C 1 -C 20 carboxylic acids, preferably C 4 -C 20 carboxylic acid, such as acetic acid, propionic acid, butyric acid, valeric acid, and oleic acid.

The method according to at least one of the preceding claims, wherein the ester comprises from 4 to 40 carbon atoms, preferably from 6 to 25 carbon atoms, such as from 8 to 20 carbon atoms.

8. Method according to at least one of the preceding claims, wherein the non-polar medium is selected from alkanes, preferably C 4 -C 20 alkanes, olefins, aromatics, gas oil, diesel, gasoline, and combinations thereof.

The method of at least one of the preceding claims, wherein the method further comprises the step of providing additives such as nitrogen, sugar, such as glucose. «• b - 19 -

A method according to at least one of the preceding claims, wherein the second alcohol or the second carboxylic acid is added to the ratio of (a) the first alcohol and the first and / or second carboxylic acid, or (b) of the first carboxylic acid and the first and / or second alcohol.

11. A method according to at least one of the preceding claims, wherein the method further comprises at least one of the steps (a) of monitoring the pH of the mixture of the organic material and one or more strains of microorganisms in the aqueous medium for at least a portion of the duration of the process, (b) adjusting a control parameter, and (c) adding at least one species selected from: a group consisting of acids and bases, a group comprising alcohols and carboxylic acids, a group comprising additives, for example N-release compounds such as nitrogen, and a sugar.

12. A method according to at least one of the preceding claims, wherein first a substantial part of the first carboxylic acid is formed and then the first alcohol, preferably to a pH of 6-8, such as about 7.

A method according to at least one of the preceding claims, wherein at least steps (iv) - (vi) are carried out at a temperature of 20-60 ° C, such as at approximately 37 ° C.

A method according to at least one of the preceding claims, wherein steps (iv) - (vi) are performed between a pH of 3 and a pH of 7.

15. Use of a reaction product such as an ester prepared in the process for at least claim 1, as a flavoring agent, fragrance and / or a biofuel. 2006 9 84