WO2012035420A1

WO2012035420A1 - Clostridium beijerinckii dsm 23638 and its use in the production of butanol

Info

Publication number: WO2012035420A1
Application number: PCT/IB2011/002166
Authority: WO
Inventors: Daniele Bianchi; Francesca De Ferra; Luca Paolo Serbolisca
Original assignee: Eni S.P.A.
Priority date: 2010-09-16
Filing date: 2011-09-15
Publication date: 2012-03-22
Also published as: ITMI20101686A1; IT1405689B1

Abstract

The present invention relates to a new strain of Clostridium beijerinckii (DSM 23638) and its relative use in the production of biobutanol through fermentation. The present invention also relates to the use of said new strain of Clostridium beijerinckii DSM 23638 in the production of biobutanol through fermentation starting from derivatives obtained from the treatment of renewable sources such as, for example, biomasses, in particular, lignocellulosic biomasses, or algal biomasses.

Description

CLOSTRIDIUM BEIJERINCKII DSM 23638 AND ITS USE IN THE PRODUCTION OF BUTANOL

The present invention relates to a new strain of 5 Clostridium beijerinckii DSM 23638 and its relative use in the production of biobutanol through fermentation.

The present invention also relates to the use of said new strain of Clostridium beijerinckii DSM 23638 in the production of biobutanol through fermentation0 starting from derivatives obtained from the treatment of renewable sources such as, for example, biomasses, in particular, lignocellulosic biomasses, or algal biomasses .

Butanol is a chemical compound of great industrial5 interest, especially for the petrochemical industry.

It can be produced chemically through oxo-synthesis based on propylene and is commonly used as a base for paints and solvents or as an intermediate for the production of biopolymers . In particular, two-thirds of0 the production of butanol is at present absorbed in the synthesis of butyl acetate and butyl methacrylate .

It has been produced on a wide scale ever since the first World War, first of all as a by-product of the fermentation of acetone-butanol-ethanol (ABE) for the5 production of acetone, and subsequently for the solvent market. Numerous companies have used the ABE process scheme and developed fermentative processes using their own selection strains. The commercial production was interrupted due to the competitive production costs of butanol on the part of the petrochemical industry and also due to the competitive use of biomasses (corn starch or molasses sugars) as food for animals.

The overall market (about 3M of tons/year in 2007) is constantly growing at a rate of about 3% per year.

Interest in the development of bio-derivatives as energy source for transportation, or as pure products or additives for fuels has grown considerably in recent years. This has led to a significant increase in the production and commercialization of bioethanol through the fermentation of sugars deriving from biomasses. It was subsequently discovered that other alcohols or liquid bio-derivative products could also be advantageously used as additives for fuels. In particular, molecules such as butanol having longer carbonaceous chains than ethanol and consequently a higher energy content and lower hygroscopy, have proved to be promising candidates.

New production technologies of biobutanol have therefore been reproposed or developed, based on the good characteristics of the product as bio-additive for fuels, in particular with respect to bioethanol (i.e. lower vapour pressure, higher miscibility with diesel fuels and gas oil, lower solubility in water) .

In recent years, the ABE fermentation process has aroused great attention from an economic and environmental point of view, due to its potential use for the production of oxygenated products (such as butanol, acetone and their derivatives) from renewable sources or biomasses. ABE fermentation, which has been one of the most widely studied anaerobic fermentative processes, essentially consists in the fermentation of carbohydrates in acetone, butanol and ethanol due to solventogenic Clostridia (i.e. Clostridium acetobutylicum, Clostridium beijerinckii) .

A mutant strain of Clostridium acetobutylicum

{Clostridium acetobutylicum ATCC 55025) , for example, which can be advantageously used in ABE fermentation due to the good butanol yields, is known in the state of the art (Patent US 5,192,673). International patent application W098/51813 (Blasheck et al . ) describes a process for the preparation of acetone, butanol and ethanol through a fermentation process which uses a mutant strain of Clostridium beijerinckii NCIMB 8052 (Clostridium beijerinckii BAlOl) .

On the basis of what is indicated above, the need is evident for new strains of solventogenic Clostridia and relative fermentation processes more selectively directed towards production with high butanol yields starting from renewable sources such as, for example, lignocellulosic biomasses or algal biomasses.

The Applicant has now isolated a wild-type strain of Clostridium beijerinckii (filed on May 17, 2010 at the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen - with the filing number DSM 23638) , which is capable of giving high yields of butanol in an ABE fermentation process also using derivatives obtained from the treatment of renewable sources such as, for example, biomasses, in particular, lignocellulosic biomasses, or algal biomasses.

In particular, the metabolic activity of the strain

Clostridium beijerinckii DSM 23638 favours the production of butanol with respect to that of ethanol and acetone.

The use of the wild-type strain Clostridium beijerinckii DSM 23638 appears to be advantageous, for example, with respect to the mutant strain Clostridium acetobutylicum ATCC 55025 for the production of butanol having higher butanol yields with respect to glucose (0.272 with respect to 0.227 g/g) and preferably using a less rich culture medium with respect to that used in US 5,192,673.

It has also been found that by using the strain Clostridium beijerinckii DSM 23638, the relative ratio between butanol and the other two fermentation products acetone and ethanol is more than doubly unbalanced in favour of butanol with respect to Clostridium beijerinckii NCIMB 8052 (1:5:0.1 instead of 1:2:0.2 for acetone : butanol : ethanol) , thus resulting in a mixture of products in which the butanol is predominant with respect to the by-products (Table 6) , thus favouring the purification from the by-products.

Finally, with respect to the distribution profiles of the ABE fermentation products typical of the strains Clostridium acetobutylicum DSM792 (3:6:1) and Clostridium beijerinckii 8052 (1:2,1:0,2), the strain according to the invention accumulates acetone, butanol and ethanol in the relative proportion of 1:5:0.1. This distribution is advantageous due to the preferential accumulation of butanol with respect to other solvent by-products. The sequencing of the genes encoding the enzymatic activities responsible for direct conversion into ABE products has allowed significant differences to be identified with respect to the corresponding genes of the strain Clostridium beijerinckii 8052 and the strain Clostridium acetobutylicum DSM792. These differences in the amino acid composition of the enzymes have to be correlated directly to both the different metabolic activity and to the different metabolic profiles.

Furthermore, the strain is particularly stable with respect to the vitality and the fermentation capacity. The repeated use of the same microbial culture, moreover, maintained in the presence of oxygen, in water, at 4°C, proves to have the same enzymatic performance for long periods of time (at least a year) . The strain is also much less sensitive to the presence of oxygen during the pre-culture and culture phases with respect to other well-characterized Clostridia strains (data not provided) .

The halophilic nature of the isolated strain which is capable of growing and producing butanol also under high salinity conditions (e.g., 45 g/1) should also be observed, together with the capacity of producing butanol at pH > 5, thus demonstrating both a considerable versatility of use and also a substantial tolerance in the presence of unfavourable culture conditions .

The strain Clostridium beijerinckii DSM 23638 is capable of fermentatively converting the main C5 and C6 sugars (i.e. glucose, arabinose, xylose) which derive from the hydrolysis of lignocellulosic biomasses into ABE products, with an efficiency comparable to the use of glucose.

Finally, the use of the strain according to the present invention also offers an advantage of an economic nature. The strain Clostridium beijerinckii DSM 23638 is in fact capable of effectively using, as a source of nutrients, the fraction rich in amino acids (hereafter indicated as "algal extract") which derives from a production process of bio-oil from algal biomasses, in particular from algal biomasses obtained from the cultivation of microalgae, by thermal treatment of said algal biomasses, instead of costly products such as, for example yeast extract, peptone, or similar products rich in nitrogen and microelements, normally used. This extract, can therefore be advantageously used, preferably at an appropriate dilution (0.5 g/1 - 4 g/1) , instead of the above costly products thus allowing a reduction in the production costs of butanol .

An object of the present invention therefore relates to a solventogenic wild-type strain of Clostridium beijerinckii having the filing number DSM 23638 (filed at the DSMZ on May 17, 2010) .

The present invention also relates to a fermentation process for the production of acetone, butanol and ethanol, which comprises the following phases :

i) anaerobic culture of the strain Clostridium beijerinckii having the filing number DSM 23638 in a culture medium comprising:

a) a carbon source in an amount ranging from 5 g/1 to 80 g/1, preferably ranging from 10 g/1 to 50 g/1, selected from carbohydrates deriving from the hydrolysis of a lignocellulosic biomass and/or of lignocellulose derivatives, glucose, starch, or mixtures thereof;

b) a source of amino acids in an amount ranging from 0.5 g/1 to 5 g/1, preferably ranging from 1 g/1 to 2 g/1, selected from algal extract, yeast extract, tryptone, peptone, or mixtures thereof;

for a period ranging from 24 hours to 96 hours, at a temperature ranging from 24 °C to 30°C, at a pH ranging from 7 to 4.8;

ii) fermentation of the strain Clostridium beijerinckii having the filing number DSM 23638 in a culture medium comprising :

c) a phosphate buffer in a concentration ranging from 3 g/1 to 9 g/1;

iii) recovery of the butanol, acetone and ethanol obtained.

According to a preferred embodiment of the present invention, the algal extract optionally present in the culture medium of phase i) and ii) , can be obtained by subjecting an algal biomass, in particular an algal biomass obtained from the cultivation of at least one microalga, to a thermal treatment, obtaining an oily fraction which can be sent for treatments suitable for producing biofuels and an aqueous fraction rich in amino acids (i.e. algal extract). Said thermal treatment can be carried out using operative conditions known in the art, as described, for example, in international patent application WO 2010/046115.

In a preferred embodiment of the present invention, the microalga used is the sea microalga Nannochloropsis.

According to a further embodiment of the fermentation process according to the present invention, when algal extract and yeast extract are used in a mixture as source of amino acids in phase ii) , the amount of each extract used ranges from 1 g/1 to 4 g/1.

According to a further embodiment of the fermentation process according to the present invention, said process can also comprise a phase iv) for the removal of the butanol from the culture medium by means of gas stripping.

In a further embodiment of the fermentation process according to the present invention, said process can comprise a phase v) for the treatment of the butanol obtained by membrane pervaporation or by catalytic systems based on zeolites, for the conversion of the butanol into esters or ethers insoluble in the culture medium.

A further object of the present invention relates to the use of the strain Clostridium beijerinckii having the filing number DSM 23638 for the production of biobutanol on a growth and fermentation substrate comprising carbohydrates deriving from the hydrolysis of a lignocellulosic biomass and/or of lignocellulose derivatives, wherein said substrate can comprise an algal extract as amino acid source. According to a preferred embodiment of the present invention, the algal extract can be obtained by subjecting said algal biomass, in particular an algal biomass obtained from the cultivation of a microalga, to a thermal treatment, obtaining an oily fraction which can be sent for treatments suitable for producing biofuels, and an aqueous fraction rich in amino acids (i.e. algal extract) .

According to a preferred embodiment of the present invention, said microalga is the sea microalga Nannochloropsis .

The present invention also relates to the use of the strain Clostridium beijerinckii having the filing number DSM 23638 for the production of butanol by means of acetone-butanol -ethanol fermentation on a growth and fermentation substrate comprising glucose as carbon source and yeast extract or peptone as amino acids source .

Finally, the present invention relates to the use of an algal extract as growth substrate of solventogenic strains belonging to the Clostridium genus in an acetone-butanol-ethanol fermentation process .

Said strain is preferably Clostridium beijerinckii DSM 23638 (filed at the DSMZ on May 17, 2010) .

In an embodiment of the present invention, said algal extract can be obtained by subjecting said algal biomass, in particular an algal biomass obtained from the cultivation of a microalga, to thermal treatment, obtaining an oily fraction which can be sent for treatments suitable for producing biofuels and an aqueous fraction rich in amino acids (i.e. algal extract) . According to a preferred embodiment of the present invention, said microalga is the sea microalga Nannochloropsis.

The present invention will now be described for illustrative but non- limiting purposes, according to its preferred embodiments, with particular reference to the enclosed figures, in which:

Figure 1 shows a map of the genes encoding the ABE enzymatic pattern from the strain of Clostridium beijerinckii DSM 23638 according to the invention and the differences in the nucleotide and amino acid sequence with respect to the corresponding genes of the reference strains Clostridium acetobutylicu ATCC 824 (sequence with Accession number AE001437.1) and Clostridium beijerinckii NCIMB 8052 (sequence with Accession number AF547210) ;

Figures 2A-2C show a comparative graph between the production of butanol (ppm) (Figure 2A) , acetic acid and ethanol (ppm) (Figure 2C) and the consumption of glucose and xylose (ppm) (Figure 2B) among the cultures of Clostridium beijerinckii DSM 23638 cultivated in the presence of a mixture of sugars deriving from the hydrolysis of a lignocellulosic biomass (LC) and in the presence of glucose (Ref.);

Figures 3A and 3B show the increase in the production of butanol (ppm) of the strain Clostridium beijerinckii DSM 23638 with an increase in the concentration of phosphate buffer ( H₂P0₄) in the culture medium called CM (Figure 3A) and the consequent increase in the final pH of the cultures (Figure 3B) ;

Figures 4A and 4B show the comparison between the production of butanol (ppm) of the strain Clostridium beijerinckii DSM 23638 in the presence of different concentrations of yeast extract (Y.E.) in the culture medium (1 g/1 and 2 g/1) (Figure 4A) and the comparison between the final pH of the cultures (Figure 4B) ;

Figure 5A shows the trend of the production of butanol (ppm) with an increase in the salinity [increasing concentrations of phosphate buffer (KH₂P0₄) and sodium chloride (NaCl)]; whereas Figure 5B shows the accumulation of acetic acid in the culture mediums containing an amount of phosphate buffer (KH₂P0₄) higher than 6 g/1 ;

Figures 6A-6E show the comparison between the production of butanol, acetone, ethanol and acetic acid (ppm) on the part of the strain Clostridium beijerinckii DSM 23638 (TC02) and of the collection strains Clostridium beijerinckii DSM791 and Clostridium acetobutylicum DSM792;

Figure 7 shows the comparison between the production kinetics of butanol (ppm) of the strain Clostridium beijerinckii DSM 23638 in a bioreactor and in two different bottle systems (with 30 ml and 60 ml of culture medium called CM) ;

Figure 8 shows the trend of the pH registered in the bioreactor of the culture of Clostridium beijerinckii DSM 23638 during the first 96 hours of fermentation;

Figure 9 shows the production of butanol on the part of cultures of Clostridium beijerinckii DSM 23638 in a bioreactor starting from two different pH values, i.e. pH 6.9 and pH 7.5, with or without (free) pH control; Figure 10 shows the production of acetone on the part of cultures of Clostridium beijerinckii DSM 23638 in a bioreactor starting from two different pH values, i.e. pH 6.9 and pH 7.5, with or without (free) pH control ;

Figure 11 shows the accumulation of acetic acid in the culture medium of Clostridium beijerinckii DSM 23638 in a bioreactor starting from two different pH values, i.e. pH 6.9 and pH 7.5, with or without (free) pH control;

Figure 12 shows the accumulation of butyric acid in the culture medium of Clostridium beijerinckii DSM 23638 in a bioreactor starting from two different pH values, i.e. pH 6.9 and pH 7.5, with or without (free) pH control;

Figure 13 shows the production of butanol on the part of cultures of Clostridium beijerinckii DSM 23638 in a bioreactor maintaining the pH of the culture medium constant (i.e. pH 6.8 and pH 5.3);

Figure 14 shows the accumulation of organic acids

(acetic acid and butyric acid) in the culture medium of Clostridium beijerinckii DSM 23638 in a bioreactor maintaining the pH of the culture medium constant (i.e. pH 6.8 and pH 5.3);

Figure 15 shows the trend of the consumption of glucose (ppm) in the various tests carried out starting from two different pH values, i.e. pH 6.9 and pH 7.5, with or without (free) pH control, or maintaining the pH of the culture medium constant (6.8 and 5.5);

Figure 16 shows the trend of the production of butanol and consumption of glucose in a fermentation process of Clostridium beijerinckii DSM 23638 in a bioreactor on a culture medium called CM2 with removal of the butanol by means of gas stripping, administering gas in continuous (F17) ;

Figure 17 shows the trend of the consumption of glucose, the glucose present in the culture medium and the concentration of butanol, in a fermentation process of Clostridium beijerinckii DSM 23638 on a culture medium called CM2 with removal of the butanol by means of gas stripping, administering gas in continuous (F17) ;

Figure 18 shows the trend of the accumulation of organic acids with time in the fermentation process of Clostridium beijerinckii DSM 23638 on a culture medium called CM2 with removal of the butanol by means of gas stripping, administering gas in continuous (F17) ;

Figure 19 shows the trend of the growth of the microbial biomass measured by means of spectrophotometry (optical density (DO) at 600 nm) during the fermentation process of Clostridium heijerinckii DSM 23638 on a culture medium called CM2 with removal of the butanol by means of gas stripping, administering gas in continuous (F17) ;

Figure 20 shows the nucleotide sequences of the ABE genes of the operons 1 and 2 from Clostridium heijerinckii DSM 23638;

Figure 21 shows the alignment at a protein level of the ABE genes from Clostridium heijerinckii DSM 23638 with the corresponding genes from Clostridium acetobutylicum and Clostridium heijerinckii NCIMB 8052.

Some embodiment examples of the ABE fermentation process with the strain Clostridium heijerinckii DSM 23638 are provided hereunder for illustrative and non- limiting purposes of · the present invention, together with some comparative examples on the production yields of butanol with respect to mutant and non-mutant strains of solventogenic Clostridia known in the state of the art .

EXAMPLE 1 : Screening of solventogenic strains and identification of the strain Clostridium heijerinckii DSM 23638

Procuring of solventogenic strains

Bacterial strains were first procured, which were capable of transforming lignocellulosic biomasses and/or their sugar derivatives into alcohols alternative to ethanol or other compounds which can be used as building blocks for the synthesis of bio- additives for fuels. For this purpose, the most important commercial strains were purchased at the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) European collection centre and in particular, the following strains were purchased:

Clostridium acetobutylicum, strain DSM792, producer of acetone, butanol, ethanol (ABE) , acetic acid and butyric acid;

Clostridium beijerinckii , strain DSM791, producer of ABE, organic acids and, in some strains, isobutanol ;

Clostridium tyrobutyricum, strain DSM2637, producer of acetic acid and butyric acid.

Formulation of culture mediums

The first phase of the actual experimental activity was focused on the preparation of methods for the cultivation, preservation of the microorganisms of interest .

On the basis of data available in literature, two types of mediums were essentially taken into consideration: a "rich" -type medium, complete with all the components, organic and inorganic, essential for the growth and development of the microbial biomass; and a medium of the "semisynthetic" type, prevalently saline-based with yeast extract alone as organic component in addition to glucose used as the main carbon source .

Among the rich mediums tested, the one which provided the best results in terms of yield of microbial biomass, preservability of the cells and production of solvents is called RCM ("Reinforced Clostridial Medium") and has the following composition:

Table 1

A culture medium of the RCM type described above has the advantage of being much less expensive with respect to other "rich" -type mediums. Among the semisynthetic mediums formulated in the laboratory, the one which guaranteed the best growth (i.e. the best yield of microbial biomass) and the best productivity of products of interest is the culture medium called CM ("Clostridial Medium"), whose composition is obtained by assembling the concentrated solutions described hereunder:

KH₂P0₄ 150 g/1 (50X - 3 g/1 final) , pH adjusted to a value of 6.5 with NaOH in drops;

- glucose 500 g/1 (50X /10X; 10 g/1 - 50 g/1 final) ; yeast extract (Y.E.) 100 g/1 (100X; 1 g/1 final); MgCl₂.6H₂0 100 g/1 (500X; 0.2 g/1 final);

NH₄C1 100 g/1 (50X; 2 g/1 final) ;

solution of oligoelements 500X indicated in Table 2;

solution of vitamins 100X indicated in Table 2.

Table 2

(*) The concentrated solution of oligo-elements was prepared at a pH > 0.5 by means of HC1.

The composition of the culture medium called CM was then modified following results of subsequent experimentations which will be described hereunder.

Cultural conditions

The cultivation of the anaerobic strains was carried out in different fermentation systems:

anaerobic bottles having different volumes, closed by a butyl rubber stopper and sealed by a metal ferrule; the bottles, sterilely filled with the culture medium of interest, were then insufflated with pure nitrogen into the medium, using a sterile needle, for a time sufficient for completely eliminating any trace of dissolved oxygen; the bottles thus prepared were transferred to an anaerobic hood, where the inoculation was carried out by introducing, with a sterile syringe, a suitable aliquot (about 3% - 6%) of cellular suspension coming from previous microbial cultures;

a 100 ml three- inlet reactor for the collection of the biomass and the optional addition of nutrients;

1-litre fermentation vessels equipped with direct control on the main parameters such as temperature, biomass stirring, pH and dissolved oxygen.

Preservation

The strains were grown in 30 ml of culture medium called RCM or CM, under the conditions previously described.

Aliquots of about 1.4 ml of microbial culture were collected, during the growth phase, or in stationary phase (not less than 96 hours of culture) , and introduced into 2 ml "cryovials" containing 400 μΐ of pure (99%) glycerol. The glycerinates were kept in a freezer at -80°C. It was observed that the glycerinates prepared from microbial cultures in stationary phase (at the end of the growth) , are normally more vital with respect to those prepared from microbial cultures with cells in active growth.

Analytical methods

The following analytical techniques were used for measuring the consumption kinetics of the substrates and the contemporaneous development of the fermentation products :

- HPLC-RI: analysis of the consumption of the carbonaceous substrates (glucose and xylose) , the production of organic acids (acetic acid, butyric acid) and solvents (ABE) ;

- GC-FID (head-space) ·. control of the solvents produced during the different fermentation phases

(ABE) ;

- spectrophotometer: measurement of the absorbance at 600 nm for the determination of the optical density (OD) of the microbial biomass.

Preliminary results on the collection strains

The first tests on the collection strains showed a poor solventogenic productivity of the Clostridium acetobutylicum DSM792 strain, with values of butanol produced in the culture medium called RC of about 1.5 g/1 and about 0.7 g/1 in the culture medium called CM with the addition of 10 g/1 of glucose.

The value of butanol produced in the culture medium called CM was subsequently significantly enhanced by discontinuous additions of glucose to said culture medium, until a maximum value of about 8 g/1 final was reached. It should be specified, however, that the experimentation carried out using Clostridium acetobutylicum DSM792 was always subject to a considerable variability between duplicated tests, often exceeding a deviation of 100% from the production values .

An experimentation aimed at identifying the possible factors responsible for this variability revealed an inhibiting effect on the part of traces of atmospheric oxygen which can be introduced into the fermentation system during the perforation phases of the septum which seals the culture (inoculation, addition of nutrients, collection of the culture medium for the analysis of the products) which should guarantee the condition of complete anaerobiosis . The preparation of a second series of experiments carried out inside an anaerobic chamber, completely without oxygen, revealed that the variability of the butanol production values can be reduced, with percentage deviations of the ratio between standard deviation and production average not higher than 5%. As far as the organic acids are concerned, concentrations of about 2.5 g/1 of acetic acid and 2 g/1 of butyric acid were obtained in both the culture medium called RCM, and in the culture medium called CM, with a much higher reproducibility with respect to that obtained on butanol .

Peaks of 6 g/1 - 7 g/1 of butyric acid, produced by discontinuous additions of nutrients to the culture medium, were measured on the acidogenic strain Clostridium tyrobutirycum DSM 2637.

Isolation of new strains of interest

The activity was then focalized on the isolation of new anaerobic strains from samples of lignocellulosic biomasses and soil. The objective of the research was to isolate, in pure culture, new strains belonging to the Clostridium species capable of efficiently fermenting glucose into products of interest.

The activity aimed at isolating new bacterial anaerobic strains of potential interest led to the identification of numerous different species, all belonging to the Clostridium genus. Among those isolated, the strain Clostridium beijerinckii DSM 23638 (TC02) showed an improved solventogenic metabolism in culture medium called CM with respect to that of the collection strain Clostridium beijerinckii DSM792 object of previous studies. The strain Clostridium beijerinckii DSM 23638 (TC02) was classified as Clostridium beijerinckii by means of genetic techniques based on the comparison of the sequence of 16S rDNA with those indicated on the public data bank (Figure 21) . Table 3 below indicates the most significant strains isolated and their fermentation products starting from glucose as carbonaceous substrate.

Glue: glucose; Ac: Acetic acid; Buti (+Acet) : Butyrric + acetone; Lat: Lactic; Acet: Acetone; EtOH: Ethanol; ButOH: Butanol

Out of 10 fermentations carried out in batch starting from 10 g/1 of glucose as carbon source, the strain Clostridium beijerinckii DSM 23638 (TC02) showed an average production of 830 ppm of butanol produced and a standard deviation of 56.1 (6.8% with respect to the average) against an average production (out of 15 tests) of the collection strain Clostridium beijerinckii DSM792 of 680 ppm and a standard deviation of 72.8 (10.6% with respect to the average). It was therefore demonstrated that the strain Clostridium beijerinckii DSM 23638 (TC02) was potentially more productive, stable and effective in the glucose/butanol conversion with respect to the reference strain Clostridium beijerinckii DSM792.

In the tests carried out in fed-batch, the strain DSM 23638 (TC02) accumulated a maximum amount of butanol equal to 7 g/1, a value comparable to the best values obtained with the reference strain Clostridium beijerinckii DSM792.

Genetic characterization of the strain Clostridium Beijerinckii DSM 23638

The genes homologous to the ABE genes known in literature by Clostridium acetobutylicum (Accession number AE001437.1) and by Clostridium Beijerinckii NCIB8052 (international patent application W098/51813, Accession number AF547210) , were amplified by means of PCR with primers designed on the basis of sequences known by these strains. Two homologous operons were characterized with the primers designed on the basis of the sequences of Clostridium Beijerinckii NCIB8052, one of which (operon 1) is considered as being that which encodes the enzymes active in the synthesis of butanol.

Figure 1 shows a map of the genes sequenced and the respective table indicates the differences in the nucleotide and amino acid sequence of the ABE sequences from Clostridium Beijerinckii DSM 23638 (TC02) with respect to the corresponding genes of Clostridium Acetobutylicum and Clostridium Beijerinckii NCIB8052. The sequences are indicated in Figure 20 together with the alignments on a protein level with the corresponding genes from Clostridium Beijerinkii NCIB8052 (Figure 21) .

EXAMPLE 2 : Culture tests of Clostridium Beijerinckii DSM 23638 (TC02) on a renewable substrate

In order to verify the possibility of using Clostridium Beijerinckii DSM 23638 (TC02) in a production process based on the consumption of renewable sources, the fermentation performances in batch were compared in the presence of two different carbonaceous substrates : glucose and a mixture of sugars obtained from the hydrolysis of a lignocellulosic biomass carried out as described hereunder .

10 g of ground conifer flour (particle-size < 0.2 mm) were added to a solution of 1 g of 2- naphthalenesulfonic acid in 100 ml of water. The mixture is kept under stirring in an autoclave at 200°C for 4 hours .

After cooling, the solid phase is separated by filtration and dried obtaining 2.5 g of lignin with a purity >95%, corresponding to a yield of lignin >95 (the lignin content of the starting biomass is equal to 25% by weight) .

The aqueous solution is extracted with 300 ml of a toluene/n-butanol solution 3:1 by volume. The organic phase is then separated and the solvent is evaporated at reduced pressure, obtaining 0.97 g of solid residue consisting of 2-naphthalenesulfonic acid (recovery yield = 97%) .

The aqueous phase after extraction has a pH equal to 6 and contains the following mixture of sugars: glucose 68.1%, xylose 13.6%, arabinose 4.5%, cellobiose 2.2% and mannose 1.1%.

The strain Clostridium Beijerinckii DSM 23638 (TC02) was cultivated in a culture medium called CM to which 10 g/1 of the mixture of sugars obtained as described above had been added. For comparative purposes, said strain was cultivated in a culture medium called CM to which 10 g/1 of glucose had been added .

Microbial cultures were carried out in triplicate in 55 ml sealed bottles containing 30 ml of medium deaerated with nitrogen and incubated at 30°C.

Samples were taken from said microbial cultures at different times for the quantitative determination of the fermentation products obtained and consumption of the carbon sources .

Table 4 below illustrates the average production values of butanol, ethanol and acetic acid on the part of the culture containing the mixture of sugars obtained as described above (LC) as sole carbon source with respect to the reference culture (Ref.) containing glucose. The consumption values of the main carbon sources are also indicated. Table 4

T: time; DO: optical density; Glue : glucose; Xyl: Xylose; Ac: Acetic acid; EtOH: Ethanol; ButOH: Butanol

The graphs of Figures 2A-2C show a comparison between the production of butanol (Figure 2A) , acetic acid and ethanol (Figure 2C) and the consumption of glucose and xylose (Figure 2B) between the cultures of the strain Clostridium Beijerinckii DSM 23638 (TC02) cultivated in the presence of the mixture of sugars obtained as described above (LC) and in the presence of glucose (Ref) .

These results clearly demonstrate that the strain Clostridium Beijerinckii DSM 23638 (TC02), cultivated in the presence of the mixture of sugars obtained as described above (LC) , is capable of giving performances identical to those obtained when cultivated in the presence of glucose (Ref) . EXAMPLE 3 : Optimization of the culture medium

After completing the first series of experiments in batch and fed-batch, the possibility of improving the ABE fermentation performances was verified by modifying the main components of the culture medium called CM.

The components considered were:

the buffer system;

the salinity;

the amount of organic component in the medium provided in the form of yeast extract (Y.E.) .

The strain Clostridium beijerinckii DSM 23638 (TC02) was used as strain and the initial pH was regulated from 6.5 to 6.8 with the buffer system (KH₂P0₄/NaOH) .

The microbial cultures were carried out in batch in

55 ml anaerobic bottles with 30 ml of culture medium having a starting glucose content of 50 g/1 (in triplicate) . On the basis of the formulation of the culture medium called CM it was decided to first verify the effect of scalar concentrations of the phosphate buffer (KH₂P0₄) in the culture medium, starting from an initial value of 1.5 g/1 (half of the amount of the phosphate buffer (KH₂P0₄) present in the culture medium called CM) up to a concentration of 6 g/1 (double the amount of the phosphate buffer (KH₂P0₄) present in the culture medium called CM) .

The analyses were carried out at fermentation times of over 100 hours, after which no further solventogenic activity is observed on the part of microbial cells grown in batch. The results are indicated in Figures 3A and 3B, where the increase in the production of butanol is evident (and other fermentation products - data not shown) with an increase in the concentration of the phosphate buffer (KH₂P0₄) (Figure 3A) and, consequently, the final pH of the microbial cultures (Figure 3B) .

The effect of the increase in the concentration of yeast extract (Y.E.) in the culture medium, was subsequently verified. In particular, the production of butanol in the presence of 1 g/1 and 2 g/1 of yeast extract (Y.E.) in the culture medium was compared, maintaining the concentration of the phosphate buffer (KH₂P0₄) at 6 g/1. The results showed a clear stimulation (about 35%) of the production of butanol in the sample to which 2 g/1 of yeast extract (Y.E.) had been added with respect to that containing 1 g/1 of yeast extract (Y.E.) (Figure 4A) . The value of the final pH also differs significantly, remaining at a value of 5.3 (Figure 4B) thus confirming the hypothesis according to which the solventogenesis can also take place at pH values higher than 5. On the basis of these results, the effect of the concentration of phosphate buffer (KH₂P0 ) and salinity in general was further examined. Cultures were prepared in bottles, combining different concentrations of phosphate buffer (KH₂P0₄) and of sodium chloride (NaCl) in order to compare the solventogenic and acidogenic response in relation to the different saline concentrations (empirically indicated on the axis of the abscissa as sum of phosphate buffer (KH₂P0₄) and of sodium chloride (NaCl) .

The graph of Figure 5A shows how the maximum, production of butanol has been reached at concentrations of the phosphate buffer (KH₂P0₄) /sodium chloride (NaCl) system ranging from 6 g/1 to 12 g/1 with yeast extract (Y.E.) equal to 2 g/1. The halophilic nature of the strain Clostridium beijerinckii DSM 23638 (TC02) , capable of growing and producing butanol also at a high salinity (45 g/1) , should also be noted.

The graph of Figure 5B shows a definite increase in the acetic acid accumulated in the culture mediums containing quantities of the phosphate buffer (KH₂P0₄) /sodium chloride (NaCl) system higher than 6 g/i.

The indications obtained from this series of experiments led to the definition of a new culture medium called CM2, in which the concentration of the phosphate buffer (KH₂P0₄) /sodium chloride (NaCl) system and of yeast extract (Y.E.) are doubled with respect to the culture medium called CM (final from 3 g/1 and 1 g/1 to 6 g/1 and 2 g/1 respectively) and the initial fermentation pH is brought from 6.5 to 6.8 by means of sodium hydroxide (NaOH) .

Evaluation of renewable components of the culture medium

Considering the high costs of the yeast extract, mainly used in fermentations on a small scale or for the production of molecules with a very high added value, it was decided to carry out a verification on the possibility of using other low-cost organic components in the culture medium. The study was concentrated on extracts deriving from algal biomass (i.e. algal extract). In this particular case, an algal extract was used, obtained by subjecting an algal biomass obtained from the cultivation of Nannochloropsis microalgae to thermal treatment obtaining an oily fraction and an aqueous fraction rich in amino acids (i.e. algal extract) . This thermal treatment was carried out as described in the Example provided in international patent application WO 2010/046115 included herein as reference.

The strain Clostridium beijerinckii DSM 23638 (TC02) was cultivated in a culture medium called CM to which glucose (40 g/1) had been added as carbon source and with variable amounts (from 0 g/1 to 2 g/1) of yeast extract (Y.E.), of algal extract obtained as described above, or a mixture of yeast extract (Y.E.) and algal extract.

Samplings were carried out from said microbial cultures, carried out triply in 55 ml sealed bottles containing 30 ml of culture medium called CM, at the end of the growth, for quantitatively determining the main fermentation products obtained.

Table 5 illustrates the average values of the production of butanol, ethanol, acetone and acetic acid on the part of the various microbial cultures.

Table 5

Upon comparing the data relating to the production of butanol with 1 g/1 of yeast extract (Y.E.) or of algal extract, no significant differences can be observed .

The data relating to the addition of 2 g/1, show maximum values of butanol produced using yeast extract (Y.E.) alone, followed by the mixture of 1 g/1 of yeast extract (Y.E.) and 1 g/1 of algal extract, and 2 g/1 of algal extract, with an inhibition higher than 20%. The inhibitory effect of high concentrations of algal extract is confirmed by the samples containing 4 g/1 of alga extract in which the production of butanol and the growth rate of the microbial biomass are less than half the value reached with 2 g/1 of yeast extract (Y.E.). Without being tied to any interpretative theory, a toxic effect due to the thermal treatment of the algal extract can be assumed, which could lead to the formation of new undesxred organic compounds.

It can therefore be concluded that the addition of algal extract represents a valid and economic alternative to yeast extract.

Comparison between the strain Clostridium beijerinckii DSM 23638 (TC02) and the collection strains

30 ml microbial cultures of culture medium called

CM were prepared, to which 40 g/1 of glucose had been added, in order to compare the amount of fermentation products obtained from the following three solventogenic strains:, Clostridium beijerinckii DSM

23638 (TC02) , Clostridium beijerinckii DSM791 and

Clostridium acetobutylicum DSM792.

The samples, prepared in triplicate, were incubated at 30°C, for 10 days.

The results confirmed what had already been observed from the preliminary tests :

much higher levels of butanol produced for the strain Clostridium beijerinckii DSM 23638 (TC02) with respect to the two reference strains (i.e. Clostridium beijerinckii DSM791 and Clostridium acetobutylicum DSM792) ; in particular, amounts 4 times higher were obtained with respect to the strain Clostridium acetobutylicum DSM792 and 1.5 times with respect to the strain Clostridium beijerinckii DSM7 1;

the strain Clostridium beijerinckii DSM791 proves to be a better acetone producer (1.6 times with respect to the strain Clostridium beijerinckii DSM23638 and 18 times with respect to the strain Clostridium acetobutylicum DSM792;

negligible quantities of ethanol produced (a few hundreds of ppm for each strain) ;

evident predominance of the strain Clostridium acetobutylicum DSM792 with respect to the organic acids produced, in particular acetic acid which is measured in an amount 3 times higher than that produced by the strain Clostridium acetobutylicum DSM 23638 (TC02) .

The graphs relating to the above experiment are shown in Figure 6, whereas the table indicates the statistical data relating to the samples in triplicate from which can be seen the variability characterizing the performances of the strain Clostridium acetobutylicum DSM792 with respect to that demonstrated by the two strains Clolstridium beijerinckii [i.e. Clostridium beijerinckii DSM791 and Clostridium beijerinckii DSM23638 (TC02)] already observed.

Toxicity of butanol with respect to the strain Clostridium beijerinckii DSM 23638 (TC02)

During the alcohol fermentations, the growth of the microbial biomass and the formation of metabolites is significantly influenced by the toxicity exerted by the alcohols produced. In the case of ABE fermentations, the toxic effect of butanol is mainly due to its destructive action on the bacterial membrane whose normal functionality is altered.

The concentration limit of butanol over which the metabolism of Clostridium beijerinckii DSM 23638 (TC02) was completely inhibited was consequently investigated. For this purpose, a series of microbial cultures in bottles were inoculated, containing culture medium called CM2, 10 g/l of glucose and scalar concentrations of butanol, starting from 1 g/l up to 15 g/l.

The data obtained show that starting from a concentration of 9 g/l - 10 g/l of butanol in the culture medium, no growth of the microbial biomass or accumulation of fermentation products can be observed. Fermentations in batch of the strain Clostridium beijerinckii DSM 23638 (TC02) in a bioreactor

Once the best culture medium had been defined for the strain Clostridium beijerinckii DSM 23638 [i.e the culture medium called (CM2 ) ] , the scaling-up of the fermentation system was carried out, passing from 30 ml of the cultures in anaerobic bottles to a litre of the bioreactor (Lafors - Infors HT) . The microbial culture in the fermenter offers a wider experimental margin as it allows a direct control of the main culture parameters such as stirring of the microbial biomass, H, dissolved oxygen and temperature. The starting values selected for this series of experimentations are: stirring 100 rpm, temperature 27.5°C, initial pH 6.8, starting glucose ranging from 35 g/1 to 50 g/1 and the total absence of dissolved oxygen in the culture medium by the insufflation of sterile nitrogen before the inoculation (carried out with 30 ml of culture medium grown for 72 hours in culture medium called CM2 to which 10 g/1 of glucose had been added) .

A comparison between the production of butanol in the bioreactor with respect to that obtained under the same conditions in two different systems in bottles (containing 30 ml or 60 ml of culture medium) showed a completely comparable production kinetics of butanol (Figure 7) .

The graph shown in Figure 8 indicates the typical pH trend, registered in the bioreactor of the microbial culture during the first 96 hours of fermentation.

The study then continued on two main lines:

studying the effect of the pH control of the culture medium on the productivity of the strain Clostridium beijerinckii DSM 23638 (TC02) ;

studying the effect of the removal of the butanol produced by the culture medium by means of gas stripping .

pH Control

Fermentation in a bioreactor allows controlled pH values to be maintained by the action of an automated peristaltic pump capable of adding acids or bases to the culture medium.

In the first series of tests, production kinetics of ABE fermentation products were compared, in culture mediums based on the culture medium called CM2 to which 50 g/1 of glucose had been added as carbon source starting from two different pH values: 6.9 and 7.5 (in duplicate). The two batch tests, one at pH 7.5 and one a pH 6.8 did not undergo any type of control (→free) . In a further two fermentations at a starting pH of 6.9, the pH was controlled so as to reach pH values of 6.0 and 5.5.

Figure 9 shows the results relating to the different fermentations. The maximum values, both in terms of kinetics and of total production, were obtained in fermentations in which the starting pH was fixed at 7.5 and was not subjected to any type of subsequent control. In this case, the butanol produced reached values of 10.5 g/1 in about 70 hours with productivity peaks equal to 0.27 gxl^xh^"1. As demonstrated by previous experimentations, the concentration close to 10 g/1 of butanol in the culture medium represents an insuperable limit in a closed batch system as it is the tolerance limit to the toxicity of butanol exerted on the strain Clostridium beijerinckii DSM 23638 (TC02) . The levels of butanol produced are significantly lower if the starting pH of the microbial culture has a value of 6.9 without subsequent controls.

By regulating the drop in pH values to 5.5 and 6.0, the production of butanol passes from 6.5 g/1 (obtained without any correction) to 8 g/1 and 9 g/1 respectively, followed by a considerable increase in the organic acids produced (see graphs shown in Figures 11 and 12 relating to the trend of the production of acetic acid and butyric acid) .

The same trend observed for butanol can be found for that relating to acetone (see Figure 10) . The maximum production value is fixed at around 2.6 g/1 with a productivity of 80 mgxl^xh^"1.

As previously mentioned, pH control during the fermentation phase leads to an increase in the amount of organic acids in the culture medium. Without this control, the organic acids accumulated during the first 24 hours - 36 hours of fermentation tend to almost completely disappear from the culture medium whereas in microbial cultures in which the pH has been maintained at pH values of 5.5 and 6.0, the levels of the acids have a significant consistency (4 g/1 and 5.5 g/1 for acetic and peaks higher than 2.5 g/1 for butyric acid) . Figures 11 and 12 show the curves relating to the accumulation of acetic acid and butyric acid produced.

Tests carried out maintaining the pH of the culture medium constant (at values of 6.8 and 5.3) starting from the inoculation phase, showed a low solventogenic activity in favour of a consistent production of organic acids (as shown in Figures 13 and 14) . From the graphs of Figures 13 and 14, an almost identical production kinetics and accumulation level of butanol in the two microbial cultures (up to a maximum value of about 3.5 g/1) can in fact be observed, whereas a higher amount of organic acids is observed in the microbial culture maintained at a pH of 6.8 (with a maximum peak of butyric acid of almost 8 g/1 and acetic acid around 6 g/1) with respect to those obtained at pH 5.3.

As last important fermentation parameter in Figure 15 is shown the trend of glucose consumption in the various fermentation tests carried out. The maximum transformation rate is about 1 gxl^xh^"1 (referring to the tests carried out with a pH of 7.5 → free) and the total consumption remains, for most of the tests, at around 40 g/1.

In conclusion to this series of experiments, it can be affirmed that not only does the initial pH of the microbial culture established on the basis of the initial formulation of the culture medium have an important influence on the trend of the solventogenesis , of the acidogenesis and of the consumption of sugar substrate, but also the direct control on the pH has a potentially strong impact on the trend of the formation of fermentation products . With respect to the productivity of butanol and the conversion yields of glucose, the best conditions found are those in which the pH of the microbial culture starts from a value of 7.5 without undergoing further controls imposed from the outside. In this case, the strain Clostridium beijerinckii DSM 23638 (TC02) is capable of producing ABE products with kinetics and at levels completely comparable to the best data which can be found in literature.

Table 6 shows a comparison between the best data obtained from fermentations in batch and those published on the part of the research group of Blasheck and Qureshi of the University of Illinois. This group has for some time been dedicated to studying a strain of Clostridium beijerinckii called NCIMB8052, phylogenetically very close to the strain object of the present invention, from which the mutant strain BA101 has been developed, which has become the object of the most recent patent applications (i.e. 098/51813) and publications .

Table 6

It can be seen how the most important values such as productivity and glucose conversion yields are completely comparable to those obtained using the strain Clostridium beijerinckii DSM 23638 (TC02) according to the present invention. The highest values of accumulated butanol at the end of the growth, found on the mutant strain Clostridium beijerinckii BA101, can refer to a high tolerance level to butanol conferred by the mutation itself, whereas the strain Clostridium beijerinckii DSM 23638 (TC02) does not have any mutation and allows higher butanol levels to be obtained with respect to those obtained with the wild- type strain Clostridium beijerinckii NCIMB8052.

Table 7 below compares the batch fermentation results obtained by Blasheck et al . , with respect to those obtained by combining batch, fed-batch and continuous techniques, with gas stripping by insufflation of gas into the culture medium in order to eliminate the butanol.

Table 7

'Batch" "Batch" "Batch" + 'Feed-batch'

"stripping" NCIMB +

BA101 "stripping" stripping in continuous

BA101 BA101 BA101

8052 mutant

A g/1 4.4 8.6 6.9 77.7 204

B g/1 9.2 18.6 - 1 1.6 16.4 151.7 251

E g/1 0.9 0.3 0.3 3.4 5.1

Tot ABE 14.5 27.5 23.6 232.8 460.4

Glucose 87% 95% 100% 100% N/A used

Productivity

g/l/hr 0.1 1 0.38 0.61 1.16 0.91 It can be seen that the removal of butanol significantly increases the productivity also in the case of batch fermentations. The amount of butanol produced, moreover, passes from 16.4 g/1 obtained in batch to 150 in the case of fed-batch and to 250 in the tests carried out in continuous. Productions of this type are capable of significantly reducing the separation costs of the butanol produced by the aqueous phase making the whole ABE process appealing from an economic point of view. Then it was considered to apply these techniques to the strain Clostridium beijerinckii DSM 23638 (TC02) .

EXAMPLE 4 : Removal of butanol by means of gas stripping A fermentation in a culture medium called CM2 to which 50 g/1 of glucose had been added, starting pH 7.5 without control, was carried out for 70 hours, in a 1 litre bioreactor until about 9 g/1 of butanol produced had been reached. Sterile nitrogen was then insufflated by means of a sparger with a flow of 0.5 wm (litres per minute) (gas stripping) in order to remove the butanol present in the culture medium. After 24 hours of gas stripping, the amount of butanol had dropped from 9 g/1 to a subtoxic concentration of about 5 g/1. The organic nutrients which had been consumed during the first batch phase were added to the culture medium until a glucose concentration of 50 g/1 and yeast extract (Y.E.) of 2 g/1 had been re-established. After a further 72 hours of fermentation, the concentration of butanol dropped to 1.5 g/1 without observing, however, any further consumption of glucose (Figure 16) .

In spite of the almost total removal of the butanol produced and the addition of all the essential nutrients, the microbial culture did not show, however, any recovery of the cell metabolism. Hypotheses on the reason for this lack of reactivation of the microbial activity were the following:

irreversible degeneration of the microbial cells exposed to the toxic effect of butanol;

- presence of other organic products inhibiting the cell metabolism and not identified by means of the analytical methods applied;

presence of further limiting factors in the exhausted medium.

In order to clarify these hypotheses, the culture medium of the above fermentation (F10) was removed and the microbial cells sterilely collected by centrifugation. Part of the culture medium thus obtained was treated with activated carbon in order to eliminate possible undesired organic compounds produced during the fermentation. The culture medium was then filtered with a filter having a pore diameter equal to 0.22 μπι in order to carry out a complete sterilization.

Microbial cultures in bottles containing 30 ml culture medium (F10) were then prepared, in duplicate, according to the following scheme:

- non-treated medium (TQ) (final pH 5.2),

- medium treated with activated carbon (final pH 5.2),

- non-treated medium to which 6 g/1 of phosphate buffer (KH₂P0₄) at pH 7.8 had been added (final pH 6.2);

- medium treated with filtered activated carbon to which 6 g/1 of phosphate buffer (KH₂P0₄) at pH 7.8 had been added (final pH 6.2)

The bottles were degassed with a sterile flow of nitrogen and inoculated with the microbial biomass obtained from the culture medium (F10) previously collected by centrifugation as described above. After 96 hours of fermentation at 28 °C, a sampling was taken for the analytical determination of the butanol produced .

Finally, a series of fermentation experiments were carried out in fed-batch associated with gas stripping.

Among the most significant, the fermentation (F17) is, described hereunder.

Fermentation (F17)

Culture medium called CM2 , was added to the bioreactor, at pH 8 (without control) , to which 55 g/1 of glucose had been added.

After inoculation with Clostridium beijerinckii DSM

23638, the microbial culture was subjected to fermentation at 27.5°C, maintained under stirring at 100 rpm, with no gas flow until the first growth phases. After 29 hours of fermentation, once an optical density (DO) of 4.9 and a subtoxic concentration of butanol equal to 1.5 g/1 had been reached, the butanol was removed from the culture medium by means of gas stripping with nitrogen at a flow-rate of 2 vvm (litres/ minute) and a stirring of 300 rpm.

About 80 ml of water, lost as a result of the gas stripping, were added daily, in order to bring the volume of the culture medium back to 1 litre. After a further 71 hours of fermentation, after reaching a glucose value equal to 15 g/1 and a pH of 5.8, a first mixture of nutrients consisting of 25 g/1 of glucose, 2 g/1 of yeast extract (Y.E.), oligoelements (see Table 1) , vitamins (see Table 1) and a further 6 g/1 of buffer phosphate at pH 7.8 in order to increase the pH of the culture medium to a value of 6.7, were added to the bioreactor. The glucose consumption kinetics (about 0.7 gxl^xh^"1) proceeded without variations for a further

24 hours approximately and then suddenly diminished (0.16 gxl^xtT¹) during the subsequent hours. After 174 hours of fermentation, when the glucose level had decreased to less than 10 g/1, a second addition of nutrients was carried out, this time omitting the oligoelements and buffer phosphate (as the pH had not undergone relevant changes) . The glucose consumption maintained its constant rate and at 238 hours from the beginning of the fermentation, when a pH of 5.7 had been reached, a further 6 g/1 of buffer phosphate and 2 g/1 of ammonium chloride (NH₄C1) were added (in order to test possible limitations of nitrogen sources) . The cell metabolism continued its activity for a further 100 hours, even if slowly, with a final glucose consumption of 92 g/1, a value which is about double with respect to that obtained from the bests tests carried out in batch with no gas stripping.

Figure 17 illustrates the trend of glucose consumed, glucose present in the culture medium and the butanol concentration (secondary axis of the ordinates) .

The graph of Figure 18 shows the trend of the accumulation of the organic acids with time. The levels of acetic acid and of butyric acid reached an average value of about 1.5 g/1 after the first addition of nutrients and buffer phosphate (time (T) = 73 hours) subsequently further increasing following the second addition of nutrients and buffer phosphate (time (T) = 238 hours) . Values higher than 5 g/1 obtained in the batch fermentations at controlled pH were not observed, even if a non-measurable loss of volatile organic acids as a result of the gas stripping, cannot be excluded.

Finally, the graph of Figure 19 shows the growth of the microbial biomass [spectrophotometrically measured as optical density (OD) at 600 nm] .

The experimental data obtained, compared to the most recent works appearing in literature, show that there is the concrete possibility of developing an ABE fermentation process based on the strain Clostridium beijerinckii DSM 23638, which is economically advantageous .

Claims

1. A solventogenic wild-type strain of Clostridium beijerinckii having the filing number DSM 23638 (filed at the DSMZ on May 17, 2010) .

2. A fermentation process for the production of butanol which comprises the following phases :

a) a carbon source in an amount ranging from 5 g/1 to 80 g/1, selected from carbohydrates deriving from the hydrolysis of a lignocellulosic biomass and/or of lignocellulose derivatives, glucose, starch, or mixtures thereof;

b) an amino acids source in an amount ranging from 0.5 g/1 to 5 g/1, preferably ranging from 1 g/1 to 2 g/1, selected from algal extract, yeast extract, tryptone, peptone, or mixtures thereof;

ii) fermentation of the strain Clostridium beijerinckii having the filing number DSM 23638 in a culture medium comprising:

a) a carbon source in an amount ranging from 5 g/1 to 80 g/1, selected from carbohydrates deriving from the hydrolysis of a lignocellulosic biomass and/or of lignocellulose derivatives, glucose, starch, or mixtures thereof; b) an amino acids source in an amount ranging from 0.5 g/1 to 5 g/1, preferably ranging from 1 g/1 to 2 g/1, selected from algal extract, yeast extract, tryptone, peptone, or mixtures thereof;

c) a phosphate buffer in a concentration ranging from 3 g/1 to 9 g/1;

for a period ranging from 24 hours to 96 hours, at a temperature ranging from 24°C to 30°C, at a pH ranging from 7 to 4.8;

iii) recovery of the butanol, acetone and ethanol obtained.

3. The process according to claim 2, wherein said carbon source is present in an amount ranging from 10 g/1 to 50 g/1, and said amino acids source is present in an amount ranging from 1 g/1 to 2 g/1.

4. The process according to anyone of the claims 2 or 3, wherein said algal extract optionally present in the culture medium of phase i) and ii) is obtained by subjecting an algal biomass to thermal treatment, obtaining an oily fraction and an aqueous fraction rich in amino acids which forms said algal extract.

5. The process according to claim 4, wherein said algal biomass is obtained by the cultivation of at least one microalga.

6. The process according to claim 5, wherein said microalga is the sea microalga Nannochloropsis .

7. The process according to anyone of the claims 2-6 wherein when algal extract and yeast extract are used in a mixture as amino acids source in phase ii) , the amount used ranges from 1 g/1 to 4 g/1.

8. The process according to anyone of the claims 2-7 also comprising a phase iv) for the removal of the butanol from the medium by means of gas stripping.

9. The process according to anyone of the claims 2-8 also comprising a phase v) for the treatment of the butanol obtained by membrane pervaporation or by catalytic systems based on zeolites, for the conversion of the aqueous butanol into esters or ethers insoluble in the culture medium.

10. Use of the strain according to claim 1, for the production of biobutanol on a growth and fermentation substrate comprising carbohydrates deriving from the hydrolysis of a lignocellulosic biomass and/or of lignocellulose derivatives, wherein said substrate can comprise an algal extract as amino acids source.

11. Use according to claim 10, wherein said algal extract is obtained by subjecting an algal biomass to a thermal treatment, obtaining an oily fraction and an aqueous fraction rich in amino acids which forms said algal extract.

12. Use according to claim 11, wherein said algal biomass is obtained by the cultivation of at least one microalga .

13. Use according to anyone of the claims 11-12, wherein said microalga is the sea microalga

Nannochloropsis .

14. Use of the strain according to claim 1, for the production of butanol by means of acetone-butanol- ethanol fermentation on a growth and fermentation substrate comprising glucose as carbon source and yeast extract or peptone as amino acids source.

15. Use of an algal extract which can be obtained by subjecting an algal biomass to a thermal treatment, obtaining an oily fraction and an aqueous fraction rich in amino acids which forms said algal extract, as growth substrate of solventogenic strains belonging to the Clostridium genus in an acetone-butanol-ethanol fermentation process.

16. Use according to claim 15, wherein said algal biomass is obtained by the cultivation of at least one microalga .

17. Use according to anyone of claims 15-16, wherein said microalga is the sea microalga Nannochloropsis .

18. Use according to anyone of claims 15-17, wherein said strain is Clostridium beijerinckii DS 23638

(filed at the DSMZ on May 17, 2010) .