s c # c #f» -jc sjc s|c ^jc -^c } ? c sjc

FIELD OF THE INVENTION

The present invention relates to the use of biological components of anaerobic microorganisms, in particular cultures or enzymes of anaerobic bacteria, as agents for the capture and sequestration of carbon dioxide by reaction with an appropriate organic substrate. The products resulting from this reaction are organic molecules usable as raw materials in industries, such as food, biomedical, cosmetics and zootechnical industries. STATE OF THE ART

The current consensus of the scientific community is that the atmospheric concentration of C0 ₂ will continue to increase in a way that is directly proportional to the consumption of fossil fuels, with significant consequences for the global climate. Clearly, the optimal scenario for the future entails a transition to renewable energy, but, alternatively or in interim strategies, sequestration or attenuation are deemed to be the main objectives to mitigate the risk of greenhouse-induced climate changes.

The object of the present invention, therefore, is to provide a process for abating carbon dioxide.

SUMMARY OF THE INVENTION

The above indicated object has been attained by a process able to capture carbon dioxide and convert it into organic compounds usable as industrial raw materials. Hence, the invention pertains to a process for roducing at least one organic compound of formula (I):

R is H or alkaline metal,

Ri is -H, -OH, -O-alkyl, -O-CO-alkyl, -NH ₂ , =0, -NH-CO-alkyl, -NH-alkyl, -SH, -S- alkyl, where alkyl is linear or branched C1-C3,

R ₂ is -H, -OH, -NH ₂ , linear or branched C1-C6 alkyl, or linear or branched C1-C6 alkyl substituted with at least one -OH group or -NH ₂ group,

provided that Ri and R ₂ are not simultaneously -H, and R ₂ is not present, if Ri is =0,

comprising the steps of:

i) providing at least one bacterium of the taxonomic Order Thermotogales comprising pyruvate: ferredoxin oxidoreductase (PFOR), ferredoxin, acetyl-coenzyme A synthetase, and coenzyme A;

ii) adding an organic substrate;

iii) adding C0 ₂ ;

iv) reacting the resulting mixture under anaerobic conditions for at least 6 hours; and v) separating the at least one organic compound of formula (I) thus obtained.

According to another aspect, the present invention concerns a plant for implementing said process, comprising:

- at least one reactor, provided with inlet for feeding C0 ₂ , inlet for feeding the organic substrate, outlet for collecting the reaction products, and outlet for collecting gas;

- at least one device for separating the reaction products; and

- at least one container for collecting the at least one organic compound of formula (I) separated.

In the present invention, when using the term:

- "organic substrate", what is meant is one or more compounds, preferably formic acid or salts thereof, acetic acid or salts thereof, propionic acid and salts thereof, butyric acid or salts thereof, said one or more compounds being provided in pure form or as an in situ metabolic product of simple organic matrices, such as monosaccharides, disaccharides, polysaccharides, proteins, or of complex organic matrices, such as byproducts or waste materials of the food processing industry, of agriculture, or algal biomasses; and

"C0 ₂ " or "carbon dioxide", what is meant is that said component may be in gaseous form, pure or mixed with other gases, or in the form of an inorganic salt, in solid form or in solution, or in an organic matrix able to release it.

BRIEF DESCRIPTION OF THE FIGURES

The characteristics and advantages of the present invention will be apparent from the following detailed description, as well as the working examples provided for illustrative and non limiting purposes, and the attached Figures wherein:

Figure 1 shows the sequence listing of the tetrameric protein PFOR; - Figure 2 shows the metabolic pathway that, in the bacterium T neapolitana, glucose follows in the presence of C0 ₂ ;

- Figure 3 shows (a) the metabolic pathway that, in the bacterium T. neapolitana, glucose follows in the presence of C0 ₂ and (b) the corresponding reactions of sequestration of [ ¹³ C]-C0 ₂ which take place along said pathway; bold line = bond between two paired carbons;

- Figure 4 shows the gel SDS-PAGE (10%) of the Western Blot test of the expression of the PFOR enzyme in cultures of T. neapolitana grown in standard conditions and fed at regular intervals with N ₂ (TnN) or C0 ₂ (TnC);

- Figure 5a shows the glucose consumption rate in cultures of 71 neapolitana in the presence of glucose 25mM and fed with N ₂ or C0 ₂ ;

Figure 5b shows the hydrogen (H ₂ ) yield after 24 hours and after 48 hours of the cultures of T. neapolitana in the presence of glucose 25mM and fed with N ₂ (TnN) or C0 ₂ (7nC);

- Figure 5c shows the acetic acid and lactic acid yield after 24 hours and after 48 hours of the cultures of T. neapolitana in the presence of glucose 25mM and fed with N ₂ (TnN) or C0 ₂ (TnC);

- Figure 5d shows the yield of the products: acetic acid, lactic acid and hydrogen, expressed in terms of the product mol/glucose mol ratio of the cultures of T. neapolitana in the presence of glucose 25mM and fed with N ₂ (TnN), C0 ₂ (TnC) or

N ₂ (TnN) + NaHC0 ₃ 14mM;

Figure 6 shows a schematic view of an embodiment of the plant of the invention; 1 = feeding inlet of the substrate for the reaction with carbon dioxide; 2 = feeding inlet of carbon dioxide, either gaseous or in solution; 3 = hydrogen outlet; 4 = reactor; 5 = product outlet;

- Figure 7 shows the ¹³ C-NMR spectra of the culture medium of T neapolitana after feeding with ¹ C0 ₂ or NaH ¹ C0 ₃ . (a) fed with ¹³ C0 ₂ (TnC); (b) fed with 14 mM NaH ¹³ C0 ₃ and N ₂ ; (c) control. * = marked carbon; bold line = bond between two paired carbons; and

1 ^

- Figure 8 shows the C-NMR spectra of the culture medium of T. neapolitana after feeding with 14 mM NaH CO and different organic substrates. Growing was carried out under standard conditions with insufflation of N ₂ . Spectra enlargements indicated the paired ¹³ C carbons, (a) 28 mM 2- ¹³ C glucose and 14 mM NaH ¹³ C0 ₃ ; (b) 14 mM 2- ¹³ C sodium pyruvate and 14 mM NaH ¹³ C0 ₃ ; (c) 14 mM 1- ¹³ C sodium acetate and 14 mM NaH ^l3 C0 _3; (d) control (28 mM glucose and 14 mM NaH ¹³ C0 ₃ ). - Figure 9 shows the synthesis of lactic acid in cultures of Thermotoga neapolitana kept in nitrogen or carbon dioxide atmosphere. In accordance with the mechanism known as Dark Fermentation, glucose metabolism leads to the theoretical synthesis of four moles of hydrogen and two moles of acetic acid (black arrows). The increase of carbon dioxide in the reactor causes the recycling of the acetate (red arrows) with consequent synthesis of lactic acid.

DETAILED DESCRIPTION OF THE INVENTION

Hence, the invention concerns a process for producing at least one organic compound of formula (I):

RK

^ C H- COOR

^{¾ }} (I)

wherein

R is H or alkaline metal,

Ri is -H, -OH, -O-alkyl, -O-CO-alkyl, -NH ₂ , =0, -NH-CO-alkyl, -NH-alkyl, -SH, -S- alkyl, where alkyl is linear or branched C1-C3,

R ₂ is -H, -OH, -NH ₂ , linear or branched C1-C6 alkyl, or linear or branched C1-C6 alkyl substituted with at least one -OH group or -NH ₂ group,

provided that Ri and R ₂ are not simultaneously -H, and

R ₂ is not present, if R] is =0,

comprising the steps of:

i) providing at least one bacterium of the taxonomic Order Thermotogales comprising pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin, acetyl-coenzyme A synthetase, and coenzyme A;

ii) adding an organic substrate;

iii) adding C0 ₂ ;

iv) reacting the resulting mixture under anaerobic conditions for at least 6 hours; and v) separating the at least one organic compound of formula (I) thus obtained.

It was surprisingly found that the cells of said at least one bacterium of the taxonomic Order Thermotogales, thermophilic and heterotrophic, act as micro-reactors able to catalyze the reaction between organic substrate and carbon dioxide to produce the organic compound of formula (I), which has low molecular weight, in particular lactic acid, through a direct process without fixation of C0 ₂ in the cellular biomass. In fact, C0 ₂ is, for all intents and purposes, captured and used as a reactant in the synthesis of the compound having formula (I), without entering into cell growth mechanisms that are typical, for example, of autotrophic bacteria.

Preferably, said at least one bacterium of the taxonomic Order Thermotogales is a bacterium of the genus Thermotoga, such as T. neapolitana, T. naphtophila, T. petrophila, T. maritima, T subterranea, T. elfii, T. lettingae, T. thermarum, or Thermosipho, such as T. geodei, T. melanesiensis, T. japonicus, T. africanus.

These bacteria are able to produce hydrogen by anaerobic and non-photosynthetic fermentation (dark fermentation) of carbohydrates. In particular, as shall be seen in the following Examples, a production of H ₂ was observed that was very close to the theoretical value of 4 molecules of H ₂ per molecule of glucose that enters into the glycolytic process. Total hydrogen production corresponds to approximately 0.25-0.50 m ³ of hydrogen gas per kilogram of glucose. The fermentative synthesis of hydrogen is associated with the release of two molecules of acetic acid and two of carbon dioxide per glucose molecule.

In a preferred embodiment, said at least one bacterium of the taxonomic Order Thermotogales, is of the species Thermotoga neapolitana. T. neapolitana may be isolated from marine sources of sea water.

Preferably, the bacteria Thermotogales are provided in an aqueous medium, such as sea water or water enriched with mineral salts, vitamins and similar nutrients.

According to a particularly preferred embodiment, said PFOR is a tetrameric protein having access numbers NCBI YP_002534223.1 (SEQ ID NO.l), YP_002534222.1 (SEQ ID NO.2), YP_002534225.1 (SEQ ID NO.3), YP_002534224.1 (SEQ ID NO.4) and it is encoded by the genetic cluster porBACD of the bacterium Thermotoga neapolitana (Figure

1):

Subunit porA (SEQ ID NO.l): YP_002534223.1

MKMER WER VA VTGAEA VANAMRQIEPD WAA YPITPQTPIVEYFARFVAD

GIVKTEMIP VESEHSAMSA WGAAAA GARAMTA TSANGLALMHEIVYIAASY RLPIVMP WNRALSGPINIHCDHSDAMAERDSG WIQLFAETNQEA YDFTILA VRLAEHSD VRLP VMVNLDGFILSHG VEP VEFYPDDL VKKFVGELKPMYPLL GTEHPVTWGPLDLYDYYFEHKRQQIEAMENVKKVFPEIAKEFEETFGRKY WFVEPYKLDDAEHVMVALGSTNSTIKYWDELREEGHKVGALKIWMFRPF PKEQLQELLNGRKSVIVLDRA VSFGAEAPLYEA VKSSL YE VAARPSLGSYVYG LGGRDIKPEHIRKAFEDAINGNLIADEQRYLGLRE

Subunit porB (SEQ ID NO.2): YP 002534222.1

MPVNIKQLAQEFDKKEIGITQGHRLCPGCGAPITVKFVMMIARHLGYEPW GLA TGCLEVSTTIYPYTA WSVPYIHNAFENVAA TMSGVETA YRALKNKGKIPE DKKYAFIAFGGDGGTYDIGLQSLSGMLERGHKVLYVLYDNEGYMNTGNQR SGSTPPGSDTTTAP VGKKLPGKVQLKKNIVE1VAAHENVYAA TAALSEPMDF FAKVEKALNFDGPSFLA VLSPCVRFWR VNDDKTVEISKLA VETKYWPL YE V ERGVYR VTRKPRQFKP VEEFLRA QGRFRKLLSRPDAKEIIDELQEYVDRR W ERLLALEEATKDKPIR

Subunit porC (SEQ ID NO.3): YP_002534225.1

MCKEVQSMP VAKKYFEIR WHGRA GQGAKSASQMLAEAALEAGKFVQAFP EYGAERTGAPMRAFNRIGDEYIRVRSAIENPDVVWIDETLLSPAIVEGLSED GILL VNTVKDFEFVRQKTGFNGKICWDA TDIALQEIKRGIPNTPMLGAL VR VTGWPLEVIEKRIEKMFGKKFPQEWEANKRALRRGYEEVKCSE

Subunit porD (SEQ ID NO.4): YP_002534224.1

MSLKSWKEIPIGGVIDKPGTAREYKTGTWRVMRPILHKEKCIDCMFCWLYC PDQAIVQEGGIMKGFNYDYCKGCGLCANVCPKQAIEMRPETEFLGEEG

In particular, said at least one bacterium, mainly thanks to the presence of PFOR, and of other reducing agents such as reduced ferredoxin, NADH and NADPH, is able to capture and sequester carbon dioxide, advantageously promoting the synthesis of the at least one organic compound of formula (I), which can then be further utilized as an industrial raw material, for example in the food, biomedical, cosmetic or zootechnical industries.

As will be shown herein below, the supply of C(¾ directly influences the main parameters for the production of said at least one organic compound of formula (I) when the reaction mixture is allowed to react as such.

Advantageously, the carbon dioxide may be an exhaust gas from industrial facilities or from thermoelectric power plants, thereby contributing to the abatement of atmospheric pollution.

The carbon dioxide can be added, for example in the form of exhaust gas, at the same time, in order to heat the reaction environment, preferably to temperatures between 60 and 80°C. Therefore, according to the invention, the organic substrate, within the bacterial cells, reacts with C0 ₂ to give the at least one organic compound of formula (I), by direct synthesis when, in the presence of Coenzyme A and acetyl-coenzyme A synthetase, the organic substrate is formic acid or salts thereof, acetic acid or salts thereof, propionic acid and salts thereof, butyric acid or salts thereof, in pure form or passing through the formation of a keto-acid intermediate compound, when the organic substrate is a simple or complex organic matrix comprising a metabolic precursor of formic acid or salts thereof, acetic acid or salts thereof, propionic acid and salts thereof, butyric acid or salts thereof (Figure 3).

In step iv) of the process of the invention, the mixture is allowed to react for at least 6 hours, thus obtaining the at least one organic compound of formula (I).

Preferably, in step iv) of the process, the mixture is allowed to react for 12 to 72 hours, more preferably 20 to 50 hours.

Preferably, said organic substrate comprises a compound selected from formic acid or salts thereof, acetic acid or salts thereof, propionic acid and salts thereof, butyric acid or salts thereof, or at least a metabolic precursor thereof, i.e. a monosaccharide, a disaccharide, a polysaccharide, or mixtures thereof.

The term "monosaccharide", means a simple carbohydrate, i.e. one that cannot be decomposed by hydrolysis. Non-substituted monosaccharides have the general formula (CH ₂ 0) _n (with n=3, 4, 5, ...). The number of carbon atoms ranges from 3 to 9 for monosaccharides with higher molecular weight, but the most important ones are pentoses (arabinose, ribose, xylose, etc.) and hexoses (glucose, fructose, galactose etc.).

The term "disaccharide" means an acetal formed by two monosaccharides bonded by an acetal bond commonly called O-glycosidic bond. The most important disaccharides are: cellobiose, two molecules of D-glucose with 1-4 β glycosidic bond; maltose, two molecules of a-D-glucose with 1-4 a glycosidic bond; lactose, one molecule of D-glucose and one of D-galactose with 1-4 β glycosidic bond; saccharose, one molecule of glucose and one of fructose with 1-2 a glycosidic bond; trehalose, two molecules of glucose with 1,1 a glycosidic bond.

The term "polysaccharide" means compounds formed by at least 3 monosaccharides. Among the most important are cyclodextrin, melitose, fructo-oligosaccharides, agarose, glycogen, dextran, starch, cellulose, hemicellulose, pectin, amylopectin, amylose, chitin, callose, laminarin, mannan, fucoidan, galactomannan, inulin, dextrin, maltodextrin, glycans and xylans.

Preferably, said organic substrate comprises acetic acid or salts thereof or a metabolic precursor thereof, such as a monosaccharide; more preferably, said at least one metabolic precursor is glucose.

Preferably, said at least one organic compound has formula (I), wherein:

R is H or alkaline metal,

Ri is -H, -OH, -O-alkyl, -NH ₂ , -NH- alkyl, -SH, -S- alkyl, where alkyl is linear or branched C1-C3,

R ₂ is -H, -OH, -NH ₂ , linear or branched C1 -C5 alkyl, or linear or branched C1 -C5 alkyl substituted with at least one -OH group or -NH ₂ group,

provided that Ri and R ₂ are not simultaneously -H.

More preferably, said at least one organic compound has formula (I), wherein:

R is H or alkaline metal,

Ri is -H, -OH, -O-alkyl,, where alkyl is linear or branched C1-C3,

R ₂ is linear or branched C1 -C3 alkyl, or linear or branched C1 -C3 alkyl substituted with at least one -OH group or -NH ₂ group,

In a preferred embodiment, said at least one organic compound of formula (I) is lactic acid. According to a preferred embodiment, the process of the invention comprises the steps of: i) providing bacterium Thermotoga neapolitana wherein pyruvate:ferredoxin oxidoreductase (PFOR) is encoded by the genetic cluster porBACD;

ii) adding an organic substrate comprising acetic acid or salts thereof, or glucose, and obtaining acetyl-CoA;

iii) adding C0 ₂ ;

iv) reacting the resulting mixture in anaerobic, non-photosynthetic conditions for at least 6 hours; and

v) separating lactic acid thus obtained.

Said glucose can be in pure form or in organic matrices that contain it.

It should be understood that all the possible combinations of the preferred aspects of the process, as described above, are also similarly preferred, and hence described.

As illustrated in Figure 2, T. neapolitana has a new biochemical pathway for the direct synthesis of pyruvate from acetyl coenzyme A (Ac-CoA) and carbon dioxide (C0 ₂ ). The pathway is activated by elevated levels of C0 ₂ and it represents the first case of carbon dioxide sequestration by coupling with an exogenous substrate (i.e., the organic substrate employed in the present process) to form a final product, i.e., organic compound of formula (I), in particular, lactic acid, directly released outside the cells. This process corresponds to a type of fermentative and heterotrophic assimilation.

As shall be better illustrated by the following Example, in this case, the use of acetic acid and C0 ₂ is in molar agreement with the synthesis of lactic acid as a result of the substitution of the oxidative decarboxylation of the pyruvate with a NADH-dependent reduction of the pyruvate to lactic acid. However, the process shows an apparently inconsistent carbon balance, which is due to the ability of the anaerobic culture to sequester carbon dioxide through mechanisms that are not yet altogether clear.

It should be pointed out that, as shown in Figures 2 and 3, the production of H ₂ by dark fermentation is associated to the release of organic acid, such as acetic acid, in the culture medium as final products of the metabolic transformation of the sugars. The accumulation of these metabolites inhibits the synthesis of hydrogen, but the process is easily restored by adjusting the pH, thus allowing to obtain the complete consumption of the sugars. Moreover, the high partial pressure of ¾ tends to inhibit the production of H ₂ , deflecting the metabolic pathway towards derivatives of pyruvic acid such as lactic acid or alanine. This inhibitory effect was surprisingly overcome, in the present invention, by feeding C0 ₂ into the reaction environment. In this way, it was observed that the synthesis of hydrogen and acetic acid frequently occurs simultaneously with the production of other organic acids, among which lactic acid (LA) is by far the most abundant. Analysis of the cultures grown in the presence of N ₂ alone, as a control, or with C0 ₂ showed that glucose consumption and the growth rate were substantially different, as described in the Example that follows. In fact, cells grown with C0 ₂ exhibited significantly higher growth rates and glucose consumption (Figure 5a). At the same time, an increase in lactic acid was also observed, but no reduction in hydrogen yield. This was systematically due to the synthesis of the new pool of pyruvate deriving from acetate recycling (Figure 2). The overall process includes carboxylation of the acetate by carbon dioxide to give pyruvate and then lactic acid, according to the following reaction scheme:

CH ₃ C0 ₂ H + C0 ₂ > CH ₃ COC0 ₂ H * CH ₃ CHOHC0 ₂ H acetic acid pyruvic acid lactic acid

Bicarbonate or other carbonated salts are possible alternatives to the feeding of pure C0 ₂ , although lower efficiency is observed in the specific case of PFOR encoded by the genetic cluster porBACD of the bacterium Thermotoga neapolitana, probably because this bacterium does not possess specific mechanisms for carbon dioxide concentration in cells and hence it is possible for C0 ₂ in pure form to be able to penetrate into the cells through passive mechanisms that instead are more difficult in the case of bicarbonates and carbonates.

According to a further aspect, the present invention concerns a plant for implementing the aforementioned process, comprising:

- at least one reactor, provided with inlet for feeding C0 ₂ , inlet for feeding the organic substrate, outlet for collecting the reaction products, and outlet for collecting gas;

- at least one device for separating the reaction products; and

- at least one container for collecting the at least one organic compound of formula (I) separated.

In Figure 6, an embodiment of said plant is schematically shown.

It should to be understood that all the aspects identified as preferred and advantageous for the process of the invention are to be also, analogously, deemed preferred and advantageous for the production plant and the related uses.

Working examples of the present invention are provided for illustrative purposes.

Example 1.

Production of lactic acid by the process of the present invention

Microorganisms, media and culture conditions - An anaerobic culture of bacterium Thermotoga neapolitana (DSM 4359) was maintained in Tn medium supplemented with 25 mM (0.5% wt/v) of glucose and 0.4% (wt/v) of yeast extract/tryptone. The Tn medium contained (g/L): NaCl 10; KC1 0.1 ; MgCl ₂ · 6 H ₂ 0 0.2; NH ₄ C1 1.0; K ₂ HP0 ₄ 0.3; KH ₂ P0 ₄ 0.3; CaCl ₂ · 2 H ₂ 0 0.1 ; cysteine-HCl 1.0; yeast extract 2.0; tryptone 2.0; glucose 5.0; resazurin (redox marker) 0.001; 1 L distilled H ₂ 0. Before sterilization, the pH was adjusted to 8.0 with 1 M NaOH at room temperature. After sterilization, the medium was then supplemented with 10 mL/L of both vitamins (sterilized by filtration) that solutions of trace elements (as per the commercial product 'Medium 141. METHANOGENIUM MEDIUM' by DSMZ). The excess oxygen was removed by heating the batch reactors, at the same time making oxygen-free gas flowing in them until the solution became colorless. The bacteria (25 mL) had grown overnight at 80°C without stirring and subsequently used to inoculate (1.5 mL, 6% v/v) flasks closed with fluid-tight stoppers (total volume 120 mL). The bacterium T neapolitana had also grown in a 3.8 liter fermenter with a working volume of 1 liter and a head space of 2.8 liters. The cultures were maintained under continuous stirring at 250 rpm at 80°C in the medium described above. The cultures were regularly fed with 30 ml/min of C0 ₂ or N ₂ (control). The fermenters were inoculated with a culture in logarithmic phase (6% v/v) and transferred from the flasks prepared as described above. The pH of the culture was adjusted to 7.5 with 1 M NaOH at ambient temperature and fed with N ₂ , unless otherwise specified. For experiments with bicarbonate, sodium salt was added (5 mM, 14 mM, 20 mM, 40 mM, 60 mM) under standard conditions (25 mM glucose and 0.4% wt/v yeast extract/tryptone) and the cultures were fed with N ₂ . Cell growth was determined by optical density (OD) at 540 ran (UV/Vis DU 730 spectrometer, Beckman Coulter). All culture transfers and samplings were carried out with sterile syringes and needles.

Chemical analyses - Acetic acid and lactic acid in cultures of T. neapolitana were determined by means of 1H-NMR with spectrophotometer at 600 MHz (Bruker Avance 400) equipped with Cryoprobe®, using triethylamine hydrochloride (TMA) 3.8 M as an internal standard. The measures of the gases (H ₂ and C0 ₂ ) were carried out by means of gas-chromatography on an instrument (Focus GC, Thermo Scientific) equipped with a thermal conductivity detector (TCD), integrated to a 3 m packed column (HayeSep Q), and N ₂ as gas carrier and reference. The samples were quantified by means of calibration curves made through pure gases. In the culture medium, dissolved C0 ₂ and bicarbonate were quantified by TDC gas-chromatography after acidification with HC1 6N (2 mL). Glucose concentration was determined with the method of dinitrosalicylic acid calibrated on the standard 2 g/L glucose solution. Protein concentration was determined by Bradford test, using a bovine serum albumin solution (BSA, 1 g/L) as a standard. The measurements were carried out on the supernatants after centrifugation of microbial cultures at 10,000 rpm for 30 min at 5°C.

Carbon 13 ( ¹³ C) experiments - Under standard conditions, the anaerobic cultures of T. neapolitana in 25 ml of Tn medium had been bubbled with a flow of nitrogen that carried ¹³ C0 ₂ deriving from the reaction of ¹³ C marked barium carbonate (Ba ¹³ C0 ₃ ) with HC1 IN. A glucose-free medium was used for experiments with 25 mM 2- ¹³ C glucose or 25 mM 2- ¹³ C glucose/14 mM NaH ^l3 C0 ₃ . The other experiments were carried out in 120-ml sealed flasks under standard conditions with whole medium supplemented with one of the following components: (4.7 mM, 14 mM and 58.3 mM) NaH ¹³ C0 ₃ ; (1.5 mM, 3.5 mM, 7 mM, 14 mM and 28 mM) 2- ¹³ C- sodium pyruvate/14 mM NaH ¹³ C0 ₃ ; (10 mM and 20 mM) l- ¹³ C-sodium acetate/14 mM NaH ¹³ C0 ₃ . The incubations were carried out in triplicate at 80°C for 72 h with regular feeding of N ₂ . Sterile NaOH 1M was used to buffer the medium at pH 7.5. After centrifuging, the fermentation products were analyzed directly in samples of the medium by means of NMR (1H, ¹³ C- e ¹³ C- ¹³ C COSY). The supernatants from 0.5 ml culture samples were diluted with 1 % D ₂ 0/CD ₃ OD (1 :1 v/v). Hydrogen was measured from the head space of the sealed flasks by TCD gas-chromatography. The marked compounds were supplied by the Cambridge Isotope Laboratories (USA).

Western Blot Analysis - Under standard medium conditions, the bacteria were grown in 3L fermenters for 72h. The cultures were regularly fed with 5 mL C0 ₂ for 10 minutes every 6 h. The culture means containing an equivalent cell density were collected at every interval, i.e. 6h, 12h, 24h, 30h, 48h and 72h. The cells were gathered by centrifuging at 10,000 rpm for 30 min at 5°C. The samples were suspended in a lysis buffer (50 mM Tris- HC1 pH 8.0, 5 mM phenylmefhanesulfonyl fluoride) and homogenized by ultrasonication (20 KHz, 8 cycles of 5 min) (Ultrasonic Processor XL). After centrifuging at 3000 rpm at 5°C for 10 min, the opalescent supernatant was clarified by centrifuging at 20,000 rpm for 1 h at 5°C and the resulting material was dialyzed for a long time against 50 mM Tris-HCl pH 8.0 at the same temperature before being loaded onto SDS-PAGE gel (10% acrylamide in separator gel, 5% acrylamide in packing gel, Tris-glycine (pH 8.3) - 1.0 g/L SDS as running buffer). The molecular weight of 250-10 kDa were calculated by means of Precision Plus Protein WesternC Standards (Bio-Rad). The protein bands were colored with 0.1% Coomassie brilliant blue R-250 and de-colored with a mixture of distilled water/methanol/acetic acid (5:4:1 v/v/v). The proteins were transferred by electroblotting on a PVDF Hybond-P membrane (Amersham) and measured using a primary antibody produced in rabbit (1 :10000) (PRIMM Sri, Milano, Italy) which recognizes the porA subunit of the PFOR (149 - 359 aa) of T. neapolitana (access number NCBI YP_002534223.1), as shown in Figure 1 (underlined sequence portion). The linked antibody was detected by anti-rabbit IgG (whole molecule)-alkaline phosphatase (Sigma Aldrich) as secondary antibody (1 :15000) and detected by means of 50 mg/mL 5-bromo-4- chloro-3-indolyl phosphate (BCIP) and 50 mg/mL nitrotetrazolium blue chloride (NBT)

(Sigma Aldrich). The blots were repeated three times.

RESULTS

It was observed that, when the reaction mixture is allowed to react as such, the feeding of C0 ₂ on distinct cultures of T, neapolitana (TnC) directly influences the main growth parameters, e.g. growth rate, sugar consumption, ¾ production rate and yield.

Instead, when the reaction mixture is buffered with NaOH, the feeding of C0 ₂ on distinct cultures of T. neapolitana (TnC) leads to the acceleration of the fermentation process with the complete consumption of the sugar in the first 24 hours, whereas after 48 hours no substantial differences were observed between the buffered mixtures and the non-buffered mixtures, (see Figures 5a and 5b)

On the basis of sugar consumption, the hydrogen production of the cultures fed with C0 ₂ was substantially completed in 24 hours, although total production was independent of culture conditions (1612 ml in TnC and 1699 ml in TnN after 48 h). The concentrations of NaHC0 ₃ (from 5 to 20 mM) gave the same results without apparent variations in glucose consumption and hydrogen production compared to the TnN cultures that were buffered with NaOH IN. This suggested that the effect on the fermentation process was not due to the direct metabolism of C0 ₂ but rather to the capability of this gas or of its inorganic salts to maintain the average pH (between 6.0 and 6.5) at more suitable conditions for cell vitality. Also remarkable was the fact that greater quantities of sodium bicarbonate (40 mM and more) negatively influenced bacterial growth, thus reducing the metabolic processes. Since pyruvate was obtained by glycolysis from the fermentation of the sugar in T. neapolitana, introducing glucose marked with ¹³ C, a rapid and complete marking of the pyruvate and of the other glycolysis intermediates was consequently observed. (Figure 3). Hydrogen was produced by non-photosynthetic fermentation (dark fermentation (DF)) by means of ferredoxinrNADH-dependent hydrogenase, which transfers electrons to the protons releasing molecular hydrogen. The process was associated to the conversion, which produces NADH, of pyruvate into acetic acid (AA), so the levels of acetic acid were linearly proportional to hydrogen production. On the other hand, the process was negatively influenced by other metabolic reactions that reduced the availability of pyruvate or NADH (e.g. the fermentation of the sugar to lactic acid (LA)). As shown in Figure 5c, the fermentation of acetic acid was comparable in the cultures in TnN and TnC and it was substantially in agreement with the volumetric production of H ₂ . Moreover, the feeding of C0 ₂ (TnC) caused a drastic increase in the levels of lactic acid that, in fact, were found to be approximately five times higher than those in nitrogen atmosphere (T nN). The analysis of the synthesis of the fermentation products in relation to sugar consumption (yield of the products) showed that the yields of acetic acid and hydrogen had not changed in either of the two evaluated systems (Table 1 below). However, the LA/ A A yield ratio increased threefold (from 0.27 to 0.66) from TnN to TnC, as a consequence of a sharp increase in the production of lactic acid promoted by C0 ₂ independently of the fermentation of acetic acid.

Table 1. Production of organic acid from cultures of T. neapolitana supplemented with glucose and fed with N ₂ (TnN) or C0 ₂ (TnC)

T neapolitana tolerates sodium bicarbonate (NaHC0 ₃ ) in concentrations between 5 mM and 20 mM, with 14 mM being the preferred concentration to produce AA (18.2 ± 6.4 mM), LA (18.2 ± 0.4 mM) and ¾ (886 mL/h). Above 20 mM, the salt significantly diminished bacterial fermentation that was nearly suppressed at concentrations above 40 mM (Tables 2 and 3 below). The addition of 14 mM NaHC0 ₃ induced an increase of lactic fermentation compared to the standard nN conditions, but at the same time it reduced the evolution of hydrogen. However, the boost on LA production due to NaHC0 ₃ was significantly lesser than the one observed with the feeding of C0 ₂ (24.9 ± 9.1 mM). In spite of the evident variation of the biochemical synthesis of hydrogen, and lactic acid in different experimental conditions (Figure 5d), analysis of the conversion of glucose revealed that the yields of AA (1.75 ± 0.13 with NaHC0 ₃₎ 1.82 ± 0.09 with C0 _2, 1.92 ± 0.21 in standard conditions) were not substantially influenced by the bicarbonate 14 mM. Table 2. Production of H ₂ from fermentation of glucose 25 mM through cultures of T. neapolitana fed with N ₂ and supplemented with sodium bicarbonate or 14 mM sodium bicarbonate / carbonic anhydrase (TnN+ CA). Culture Sugar consumption H ₂ production rate, H ₂ production yield,

% mL/L/h mol H ₂ /mol glu

24 h 48 h 24 h 48 h 24 h 48 h

7/«N + 14 mM NaHC0 ₃ 56.2 ± 6.3 90.6 ± 2.7 30.1 ± 2.4 18.5 ± 0.9 2.0 ± 0.07 1.7 ± 0.07

7«N + 20 mM NaHCO ₃ 53.9 ± 0.3 82.8 ± 3.5 16.2 ± 5.2 14.5 ± 5.8 1.1 ± 0.3 1.0 ± 0.2

Γ«Ν + 40 mM NaHC0 ₃ 10.0 ± 2.1 22.0 ± 1.9 16.1 ± 2.7 8.3 ± 1.3 2.9 ± 0.4 2.7 ± 0.6

7«N + CA 31.7 ± 3.7 75.0 ± 6.4 31.0 ± 3.1 25.3 ± 2.9 2.9 ± 0.3 2.0 ± 0.05

Table 3. Production of organic acids from fermentation of glucose 25 mM through cultures of T. neapolitana fed with N ₂ and supplemented with sodium bicarbonate or 14 mM sodium bicarbonate / carbonic anhydrase (TnN + CA).

Culture Acetic Acid (mM) Lactic Acid (mM) Acetic Acid (mM)/

Lactic Acid (mM)

24 h 48 h 24 h 48 h 24 h 48 h

7¾N + NaHC0 ₃ 14 mM 24.6 ± 3.2 32.1 ± 4.9 11.7 ± 0.7 18.2 ± 0.4 2.1 1.8

Γ«Ν + NaHC0 ₃ 20 mM 28.8 ± 6.0 39.4 ± 1.2 5.5 ± 3.7 9.8 ± 4.7 6.3 4.6

7¾N + NaHC0 ₃ 40 mM 12.5 ± 1.2 13.0 ± 4.3 0.7 ± 1.7 0.7 ± 1.5 16.7 18.6

ThN+BCA 23.9 42.4 0.7 3.0 33.5 14.2

The absorption of carbon dioxide in the cells was tested by bubbling, in the bacterial culture, a flow of ¹³ C0 ₂ generated by the reaction of ¹³ C marked barium carbonate (Ba ¹³ C0 ₃ ) with HC1 1M. Nitrogen was used as a gas carrier. The ¹³ C NMR spectra of the fermentation broth of these cultures showed the clear incorporation of carbon dioxide marked at the C-1 (183.3 ppm) of lactic acid (see NMR in Figure 7a, whilst Figure 7c shows the NMR of the control). The specific enrichment was further corroborated by HMBC experiments that showed the correlation of the signal of the ¹³ C at 183.3 ppm with the methyl and methylene protons of lactic acid (respectively at 1.33 and 4.09 ppm). Similarly, the growth of T. neapolitana on glucose supplemented with C marked sodium bicarbonate (NaH ¹³ C0 ₃ ) 14 mM, as expected, led to the specific production of 1- ¹³ C lactic acid (see NMR in Figure 7b). Glycolysis splits glucose in the middle, converting the carbon positions 1, 2, and 3 (and 6, 5, and 4) respectively into methyl, carbonyl, and carboxyl carbons of the pyruvate. Consequently, in view of the predominance of the Embden-Meyerhof pathway, the fermentation of 2- ¹³ C ₂ -glucose produced only 1 - ^,3 C ₂ -

13

acetate and 2- C-lactate in T. neapolitana under standard conditions. By contrast, the growth of the thermophilic bacteria on Tn supplemented with 25 mM 2- C-glucose and 14 mM NaH ¹³ C0 ₃ gave the expected 1- ¹³ C acetate, together with 1,2- ¹³ C ₂ lactate (see NMR in Figure 8a). The synthesis of this latter compound was unequivocal because the vicinal carbons at 69.3 ppm (C-2) and 183.3 ppm (C-l) of lactic acid resonated as doublets (J = 54.6 Hz) by homonuclear coupling. This proved that the bond between C-l and C2 of lactic acid was newly formed by reaction of the carbon dioxide and of a C ₂ unit deriving from glucose, similarly acetate or its equivalent form acetyl coenzyme A. No isotopic scrambling phenomena were observed in the fermentation products of T. neapolitana, nor was there any marking of other carbons of acetate and lactate, produced by the bacterium. Consistently with the marking of the lactic acid, a double marking was also found at CI (176.5 ppm, J = 54.6) and C-2 (51.5 ppm, J = 54.6 Hz) of alanine. Since this amino acid derives from the direct transamination of pyruvate, the overall results indicated the specific loss of the carbonyl carbon of the pyruvate presumably in the exchange reaction with the carbon dioxide present in the environment.

The cells of T. neapolitana were also able to capture 2- ¹³ C-pyruvate added to the Tn medium. In fact, the metabolism of this precursor led to l- ¹³ C-acetate and to a lesser extent to 2- ¹³ C-lactate. On the other hand, experiments with 2- ¹³ C-pyruvate 28 mM and NaH ¹³ C0 ₃ 14 mM obtained doubly enriched lactic acid and alanine (see NMR in Figure 8b), which was the same result obtained with the experiments whereby marked glucose and carbon dioxide were fed, as described above. The metabolism of the pyruvate was, in fact, surprising in a heterotrophic bacterium although it could be due to the partial restoration of the endogenous pool of this acid by the outer substrate. However, the synthesis of 1,2- ¹³ C ₂ lactic acid necessarily required the decarboxylation of the pyruvate to acetyl-CoA followed by the reductive transfer of carbon dioxide to this latter substrate and the new synthesis of 1,2- ¹³ C ₂ pyruvic acid (see Figure 3). In vitro, the reaction is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR). The use of reduced ferredoxin, whose potential redox is close to that of pyruvate as an oxidant, has the potential to make the coupling reaction possible, although PFOR is known only in reverse reactions from pyruvate to acetyl-CoA and C0 ₂ in physiological conditions. To test the synthetic function of PFOR in T. neapolitana, the bacterium had been grown under standard conditions on glucose supplemented with 1- ¹³ C acetate 20 mM and NaH ^I3 C0 ₃ 14 mM. The NMR analysis of the culture medium revealed the diagnostic doublets (J= 54.6) at 183.3 and 69.3 ppm for C-l and C-2 of the doubly marked lactic acid (see NMR in Figure 8c). This proved the coupling of acetate and C0 ₂ and the synthesis of lactic acid from the reduction of a transient pool of newly produced pyruvate or biochemically equivalent forms. The PFOR of T. neapolitana is a heterotetrameric complex consisting of four peptide chains called porA, porB, porC and porD (NCBI database accession numbers: YP_002534223.1, YP_002534222.1, YP_002534225.1, YP_002534224.1). porA, porB, porC, and porD are clustered in the genome of T. neapolitana and show high homology to genes having similar functions in other members of the order Thermotogales. For the sake of practicality, reference is expressly made to the reaction of the pyruvate synthase (I¾PS), but enzyme is generically referred to as PFOR. To verify the variation of 7 zPS during bacterial growth and in relation to the presence of C0 ₂ , PFOR levels were analyzed in cultures of T. neapolitana by means of polyclonal antibodies against the antigenic peptide 149 - 359 (AS) of porA of the tetrameric complex. In accordance with the recognized catalytic function of effecting the decarboxylation of pyruvate to acetyl-CoA, it was found that the signal of PFOR disappeared with the consumption of glucose in cultures under N ₂ after 36 h (Figure 4). This result was in line with a positive feedback of glucose on the expression of the protein. On the contrary, the immunoblots of the cell extracts from cultures under C0 ₂ clearly showed a persistent presence of PFOR throughout the experiment (72 h) in spite of the rapid consumption of sugar after only 24 h. No significant differences were observed in the levels of the protein before and after this point, thus suggesting that C0 ₂ stimulates the ex novo expression of PFOR, but this regulation is not correlated to glucose metabolism and the expression of PFOR is independently induced by the two factors.

Example 2.

Capture and sequestration of carbon dioxide and production of lactic acid

The ability of Thermotoga neapolitana to absorb exogenous acetate was tested by administering 2 uCi of [2- ¹⁴ C]- sodium acetate to bacteria held in 25 ml cultures and grown in sealed 120 ml bottles containing 28 mm (125 mg) of glucose. After the addition of the inoculum at 6 % (v/v), the cultures were incubated at 80°C and pH was buffered to 7.5 with NaOH (1M). After 48 hours and regular sparging with C0 ₂ , the bacterial cells were collected by centrifuging at 13,000 rpm for 10 min. Each pellet was resuspended in 10 g/L of NaCl and washed three times with the same solution before lyophilization. Supernatant (0,5 mL), wash solutions (1 ml) and cell pellets (20 mg of dry weight /ml MeOH) were diluted with OptiPhase "Hisafe" 2 (Perkin Elmer) to a final volume of 10 ml and radioactivity was measured with a scintillation analyzer (Tri - Carb 2100 TR, Perkin Elmer). As shown in Table 4 below, the measurements indicated that less than 5% of radioactivity (4980±446 dpm/mg of biomass) was actually incorporated in the biomass of the bacterial cells, thus confirming that the reductive carboxylation mechanism did not entail an actual fixation of the substrate in the biomass.

Table 4. Carbon assimilation in the biomasses of Thermotoga neapolitana grown in nitrogen atmosphere (7¾N2) or C0 ₂ atmosphere (TnCOl)

Growth Administered Recovered Percentage of conditions of radioactivity radioactivity incorporation the bacterial

strain α dpm %

[2- ^I4 C]-sodium Tri l 2'354xl0 ³ 48'095±4'364 5.0 acetate TnCOl 2'354xl0 ³ 48'801±2'882 5.1

As illustrated in Figure 9 for the synthesis of lactic acid, the mechanism for the synthesis of organic compounds of formula (I) in bacterium Thermotoga neapolitana implies the capture and sequestration of environmental carbon dioxide in the compound of formula (I). To estimate the quantitative efficiency of the process, 14 mM NaH ¹³ C0 ₃ and 20 mM of 1- ¹³ C-sodium acetate were added to cultures of T. neapolitana in standard conditions. The bacteria were kept for 3 days at 80°C. After centrifuging, the organic acids were purified from the culture medium with a GracePure™ anion-exchange column. The column was washed with 2% (w/v) of NaOH (10 mL) and then equilibrated in H ₂ 0 (10 mL). After elution with 10 ml of bidistilled H ₂ 0, the organic acids were recovered by means of a solution of 1M HC1 (1.5 mL) and H ₂ 0 (1.5 mL). The fraction containing organic acids was extracted three times with equal quantities of ethyl acetate. After evaporation of the organic solvent, the upper fractions were dissolved in methanol at a final concentration of 10 mg/ml and directly analyzed with a LC-MS equipped with a quadrupole-TOF analyzer using a source with electrospray ionization (ESI) in negative ion mode.

The analysis of the ratio between the masses of the isotopologues of the lactic acid produced by the cultures showed the following composition: M ^" = m/z 88.9657 for ¹² CH ₃ ¹² CH(OH) ¹² C0 ₂ ^" , 67.7%; (M+1V = m/z 89.9701 for ¹² CH ₃ ¹³ CH(OH) ¹² C0 ₂ ^" and ¹² CH ₃ ¹² CH(OH) ¹³ C0 ₂ \ 24.9%; (M+2V = m/z 90.9755 for ¹² CH ₃ ¹³ CH(OH) ¹³ C0 ₂ -, 7.4%). This composition indicated the presence of approximately 0.05 mmol of doubly marked product out of a total of 0.637 mmol of total lactic acid produced by the bacterium. Considering that the cultures were administered 0.35 mmol of marked sodium bicarbonate, the measurement established that nearly 15% of the inorganic carbon had been captured and sequestered in the lactic acid. Obviously, this value established only the quantity of lactic acid produced starting from acetate and C0 ₂ that were marked and bonded directly to form the doubly marked metabolite (peak M+2), but it underestimated the actual contribution of the exogenous precursors because it did not take into consideration the union of the marked products with unmarked acetate and C0 ₂ . As a good approximation, this latter combination is responsible for the mass of lactic acid with value M+1. Since this isotopologue of lactic acid corresponded to approximately 25% of the entire quantity of organic acid produced by the bacterial cultures, it can be estimated that approximately 40% of the marked bicarbonate administered to the cultures was captured and sequestered in lactic acid through the process of the present invention.