Methods to produce isobutyric acid and / or isobutanol

Abstract

The present invention relates to methods to produce isobutyric acid and / or isobutanol by converting a carbohydrate such as e.g. glucose and / or lactic acid using a microorganism. More in particular, the present invention discloses the usage of the bacterium Clostridium luticellarii in order to convert a carbohydrate and / or lactic acid into isobutyric acid and / or isobutanol and presents the process sequence. The latter compounds can be applied in animal feed and food, as flavor ingredient, as pharmaceutical compound, as plastic precursor.

Description

[0001] Methods to produce isobutyric acid and / or isobutanol

[0002] Technical Field of the invention

[0003] The present invention relates to methods to produce isobutyric acid and / or isobutanol by converting a carbohydrate such as e.g. glucose and / or lactic acid using a microorganism. More in particular, the present invention discloses the usage of the bacterium Clostridium luticellarii in order to convert a carbohydrate and / or lactic acid into isobutyric acid and / or isobutanol and presents the process sequence. The latter compounds can be applied in animal feed and food, as flavor ingredient, as pharmaceutical compound, as plastic precursor etc...

[0004] Background art

[0005] Isobutyric acid is a branched four-carbon monocarboxylic acid with a global market of 180 million USD and ample applications in animal feed & food, as flavor ingredients, pharmaceutical compounds, plastics as precursor for methyl methacrylate (MMA), etc.

[0006] Currently, isobutyric acid is produced via an acid-catalysed Koch carbonylation of petrochemically derived propylene under high pressures of 140-200 bar. Besides the usage of non-renewable feedstocks, this process uses carbon monoxide, hydrogen fluoride, and boron fluoride, all harmful to human health and the environment. There is not yet a commercial route to produce bio-based isobutyric acid.

[0007] Recently, the production of isobutyric acid has been reported from methanol+CO2[1] and / or CO2+H2[2] at concentrations of up to 5 g / L using the acetogen Clostridium luticellarii. While CO2is an abundant and renewable resource, it is not without challenges. It is a gaseous substrate that requires high-liquid mass transfer processes. The cost to activate it either directly (H2as electron donor) or indirectly (hydrogenation to methanol) makes a potential isobutyric acid production route economically challenging and constrained by the intermittent and unpredictable supply of surplus renewable energy.

[0008] The presence of isobutyric acid in mixed culture systems fed with lactic acid or glucose has recently been reported up to respectively 3.0 and 3.4 g / L [3,4]. Since little is known of the lactic acid metabolism of acetogens, and H2and CO2was present in the aforementioned studies, the isobutyric acid production in those systems has been hypothesized to be derived from H2+CO2consumption. Isolates have been found and described that produce isobutyric acid up to 2.2 g / L from lactic acid [5].

[0009] Other approaches for isobutyric acid production rely on genetically modified microorganisms to convert glucose to isobutyric acid. This has been done for Escherichia coli, producing up to 11.7 g / L of isobutyric acid through a synthetic metabolic pathway [6]. In another approach, researchers overexpressed a natural isobutyric acid synthesis pathway in Pseudomonas putida while knocking out subsequent degradation pathways to produce up to 2.3 g / L of isobutyric acid [7].

[0010] There is -however- still a need to design cost-competitive methods to efficiently produce isobutyric acid and / or isobutanol starting from inexpensive materials such as organic waste which contains carbohydrates.

[0011] Brief description of figures

[0012] Figure 1. Growth (A) and product spectrum (B) of Clostridium luticellarii growing on various combinations of lactic and acetic acid concentrations in Experiment 1. Explanation of conditions: LA, lactic acid; AA, acetic acid; numbers correspond to mM concentrations. Values in subplot B are calculated as absolute amounts of net consumed / produced electron equivalents (eeq.) in mol eeq.

[0013] Figure 2. Production organic acids and consumption of substrates by Clostridium luticellarii in Experiment 2. Values are calculated as absolute amounts of net consumed / produced electron equivalents (eeq.) in mol eeq.

[0014] Figure 3. Production organic acids and consumption of substrates (in g / L) by Clostridium luticellarii in Experiment 3

[0015] Figure 4. Average production (+) of organic acids and consumption (-) of glucose by Clostridium luticellarii at 50 mM glucose and 0 mM acetic acid (A00), 25 mM acetic acid (A25), and 50 mM acetic acid (A50) acetic acid.

[0016] Figure 5. Production organic acids and consumption of substrates by Clostridium luticellarii in Experiment 4. Values are calculated as absolute amounts of net consumed / produced electron equivalents (eeq.) in mol eeq.

[0017] Figure 6. Production organic acids and consumption of substrates by Clostridium luticellarii in Experiment 5

[0018] Figure 7. Production organic acids and consumption of substrates by Clostridium luticellarii in Experiment 6 Figure 8. Production of organic acids and consumption of substrates by Clostridium luticellarii in Experiment 7. Values are calculated as absolute amounts of net consumed / produced electron equivalents (eeq.) in mol eeq.

[0019] Figure 9. Production of organic acids and consumption of substrates by Clostridium luticellari in Experiment 8. Explanation of condition: LA, lactic acid; F, formic acid; numbers correspond to the mM concentrations of formic acid. Values are calculated as absolute amounts of net consumed / produced electron equivalents (eeq.) in mol eeq.

[0020] Description of invention

[0021] The present invention relates to an organic material containing carbohydrates which is pretreated to either release the carbohydrates in solution or fermented to lactic acid. The presence of (for example a concentration of 3-6 g / L) acetic acid after pretreatment is an advantageous embodiment for further isobutyric acid conversion. This process can be carried out by either pure or mixed cultures of lactic acid bacteria, at temperatures 20-60°C and pH 3.5-8. The carbohydrate-rich stream or fermentation broth containing lactic acid is further filtered to remove potential cells and / or particles. The filtered broth is then fed to a reactor containing Clostridium luticellarii as culture (either pure or in abundances > 20%, based on DNA sequencing) at -for example- a temperature of 37°C (possible growth range of 20-40 °C) and pH 6.5 (possible growth range of pH 4-8). With the terms 'abundances > 20%' is meant that in a bacterial culture more than 20, 30, 40, 50, 60, 70, 80, 90 or 95% are bacteria belonging to the species Clostridium luticellarii he bioreactor can be operated in batch, fed-batch or in continuous.

[0022] In other words, the current invention thus involves a production method for isobutyric acid and / or isobutanol from organic substrates, such as but not limited to the organic fraction of municipal solid waste, which at least consists of the following steps:

[0023] (i) a conditioning step during which the organic substrate is prepared for isobutyric acid and / or isobutanol production. This step comprises the addition of water to the organic substrate to achieve a desired dry matter content required for the following process steps, and the addition of an acid or base to achieve the desired pH for the next step. This step is optional when dealing with liquid streams.

[0024] (ii) a pre-fermentation step, which can be either operated as a batch, fed-batch, or continuous process, for the conversion of the easily biodegradable organic matter in the substrate to lactic acid and preferably acetic acid . To achieve this conversion, an external microbiome might be used as an inoculum to initiate this fermentation step, and / or be added as a seed culture during this fermentation step. Carbohydrates such as mono and di-saccharides may also be released in solution during this process and / or through the addition of hydrolytic enzymes. The pH of this process is maintained by dosing acid or base. Alternatively, this step can be skipped when using an organic substrate in which carbohydrates (i.e. monosaccharides, disaccharides) are already in solution.

[0025] (iii) a separation step to separate the recalcitrant solid organic matter from the liquid fraction. Separation of both fractions can be done by and is not limited to sieving, extruding, filtration, etc. The solid fraction can be sent to anaerobic digestion while the lactic acid and acetic acid rich liquid fraction will be used for the isobutyric acid production in the secondary fermentation step (iv). This step is also optional for liquid streams.

[0026] (iv) a (secondary) fermentation step for isobutyric acid and / or isobutanol production, by which the carbohydrate- or LA-rich solution is contacted with a culture of Clostridium luticellarii and acid / base dosing for pH control. After isobutyric acid and / or isobutanol production; these compounds can be either extracted (vii) followed by their transformation to bioproducts (viii) and bioproduct separation (ix); or can be in-situ transformed to bioproducts (v) followed by bioproduct separation (vi)

[0027] The present invention thus in first instance relates to the usage of a strain belonging to the bacterial species Clostridium luticellarii to convert lactic acid and / or a carbohydrate such as glucose into isobutyric acid and / or isobutanol. With the term carbohydrate are meant any short-chain saccharides (e.g. monosaccharides, disaccharides) or commonly known as simple sugars, such as glucose, fructose, xylose, arabinose, galactose, rhamnose, palatinose, fucose, (iso)maltose, lactose, or a mixture thereof. The bacterial species Clostridium luticellarii has been previously described [1,2,8] and can be obtained from any culture collection (e.g. German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany)) or isolated from anaerobic environments or bioreactors (isolate has > 97% 16S rRNA gene sequence identity with the DSMZ deposited type strain). The compounds lactic acid, carbohydrates such as glucose, fructose, xylose, arabinose, galactose, rhamnose, palatinose, fucose, (iso)maltose, lactose, isobutyric acid and isobutanol are all well-known in the art.

[0028] In other words, the present invention discloses that the bacterial species Clostridium luticellarii is capable to ferment or convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol.

[0029] Furthermore, the present invention relates to a process to convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol, comprising: providing lactic acid and / or carbohydrates, providing a strain belonging to the bacterial species Clostridium luticellarii, and allowing the conversion of said lactic acid and / or said carbohydrates into said isobutyric acid and / or isobutanol by said strain belonging to the bacterial species Clostridium luticellarii.

[0030] More specifically, the present invention relates to a process to convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol as described above further comprising: providing lactic acid and / or carbohydrates, which may further comprise at least one of the following steps: (i) the addition of water, nutrients, pH adjustment solutions, and / or hydrolytic enzymes to said lactic acid and / or carbohydrates in order to obtain a solution of said lactic acid and / or carbohydrates ; (ii) the fermentation of said solution in batch, fed-batch, or continuous process in order to produce lactic acid and optionally acetic acid , via the addition of a lactic acid microbial culture or microbiome; ill) maintaining the pH of said solution by dosing acid or base; and (iv) separating the solid and liquid fractions to remove cells / organic material via sieving, extruding and / or filtration. converting said lactic acid and / or said carbohydrate into said isobutyric acid and / or said isobutanol via a bioprocess, further comprising at least one of the following steps: (i) addition of a strain belonging to the bacterial species Clostridium luticellarii or a culture containing at least 20% of the bacterial species Clostridium luticellarii to the solution containing lactic acid and / or carbohydrates; ii) addition of nutrients, acetic acid and / or pH adjustment solutions; (iii) fermentation of lactic acid and / or carbohydrates to isobutyric acid and / or said isobutanol in a batch, a fed-batch or a continuous process; recovering said isobutyric acid and / or isobutanol produced via either the separation of the cells from the broth and extraction followed by their transformation to bioproducts, or, their in-situ transformation to bioproducts followed by bioproduct separation.

[0031] In a further embodiment, the present invention relates to a process as described above wherein said process further comprises the optional provision of acetic acid, a well-known compound in the art.

[0032] The present invention further relates to a process as described above wherein the concentration of said lactic acid in the fermentation broth equals or is lower than 600 mM lactic acid. The terms 'equal or lower than 600 mM lactic acid' relate to the finding that Clostridium luticellarii does not grow on lactic acid concentrations above 600 mM, and refers to concentrations ranging between 600 and 0 mM, more specifically between 600-550 mM, or 550-500 mM, or 500-450 mM, or 450-400 mM, or 400-350 mM, or 350-300 mM, or 300-350 mM, or 350-300 mM, or 300-250 mM, or 250-200 mM, or 200-150 mM, or 150- 100 mM, or 100-50 mM, or 50-0 mM. The presence of acetic acid may be required to enable growth at lactic acid concentrations above 50 mM.

[0033] The present invention further relates to a process as described above wherein said process further comprises the provision of methanol, formate, hydrogen gas and / or carbon monoxide. Methanol, formate, hydrogen gas and carbon monoxide are compounds well-known in the art. The provision of any of the aforementioned compounds (i.e. methanol, formate, hydrogen gas and / or carbon monoxide) alone or in combination, will supply additional reducing equivalents for the conversion and can result in higher production and selectivity towards isobutyric acid and / or isobutanol.

[0034] Moreover, the present invention relates to a process as described above wherein said process can be carried out in various reactor configurations such as batch, fed-batch or continuous, with or without the additional supply of acetic acid.

[0035] Examples

[0036] Experiment 1: Production of isobutyric acid from lactic and acetic acid mixtures by Clostridium luticellarii

[0037] Clostridium luticellarii was obtained as has been previously described [1,2,8] and grown in batch in serum bottles, in four different lactic acid and acetic acid concentrations: a) Lactic acid (50 mM); b) Lactic acid (50 mM) + acetic acid (25 mM); c) Lactic acid (200 mM); d) Lactic acid (200 mM) + acetic acid (100 mM). Experiments were performed with a basal medium. The basal medium contained (per 1 L): 0.36 g NH4H2PO4; 0.133 g CaCI2; 0.325 g MgCI2.6H2O; 0.197 g MgSO4.7H2O; 0.149 KCI; 19.524 g 2-(N-morpholino) ethanesulfonic acid (MES) buffer; and 0.001 g resazurine. The basal medium was boiled, cooled down under continuous sparging of N2and dispensed in 120 mL penicillin bottles under a N2atmosphere. Subsequently the headspace of the bottles was flushed with N2 / CO2(90 / 10 v / v%) for at least 15 min and bottles were pressurized to 150 kPa absolute and autoclaved. Lactic acid and acetic acid were added from separately autoclaved stock solutions in the respective concentrations. Before inoculation, vitamins and trace elements (Tables 1, 2 and 3), 30 mM NaHCO3, and 0.4 mM titanium (III) citrate (reducing agent) were added to the basal medium from anaerobic filter sterilized concentrated stock solutions and pH was set to 6.5 with sterile 2 M NaOH and / or 2 M HCI. All experiments were carried out by incubating bottles statically at 37 °C. Right after inoculation, and at regular time intervals, the headspace pressure was measured with a tensiometer (GMH 3111 equipped with a 603310 MSD 2.5 BAE sensor, Greisinger) and needle and liquid samples (1.0 mL) were taken for immediate pH and OD measurements. Remaining liquid samples were filtered (0.20 pm) and stored at -18 °C until HPLC analysis. Carboxylic acids (Cl, C2, C3, C4, C5, C6 including C4, C5 and C6 isoforms) and alcohols (ethanol, isobutanol, 1-butanol) were analysed by HPLC (LC-2030C Plus Prominence-© series, Shimadzu) equipped with an Aminex HPX-87H column (300 x 7.8 mm, BioRad) coupled with a Micro-Guard cartridge. Carboxylic acids and alcohols were quantified using a UV detector (UV / Vis SPD-40, Shimadzu) and a refractive index detector (RID-20A, Shimadzu), respectively. 20 pL of sample was eluted with 5mM H2SO4and 1% acetonitrile at a flow rate of 0.45 mL min-1. The column oven temperature was set at 30 °C and the UV and RID cell temperatures were set at 40 °C. OD was measured on 250 pL aliquots at 620 nm in 96 well plates using a plate reader (Infinite M200 PRO, Tecan).

[0038] Table 1 - Trace element stock solution composition (lOOOx concentrated).

[0039] Component Concentration (g L'1)

[0040] ZnSO47H2O 1.00

[0041] MnCI2-4H2O 0.30

[0042] H3BO33.00

[0043] COCI2-6H2O 2.00

[0044] CuCI2- 2H2O 0.10

[0045] NiCI2-6H2O 0.20

[0046] Na2MoO4 2H2O 0.30

[0047] Na2SeO30.10

[0048] FeSO4-7H2O 20.98 Table 2 - Vitamin stock solution composition (lOOOx concentrated).

[0049] Component Concentration (g L'1)

[0050] Biotin (B7) 0.106

[0051] Folic acid (B9) 0.005

[0052] Pyridoxal-HCI (B6) 0.0025 Lipoic acid 0.015

[0053] Riboflavin (B2) 0.0125

[0054] Thiamine-HCI (Bl) 0.266

[0055] Ca-D-pantothenate (B5) 0.413

[0056] 4(P)-aminobenzoic acid (B10) 0.0125

[0057] Nicotinic acid (B3) 0.0125

[0058] Table 3 - Elevated vitamin B12 stock solution composition (3000x concentrated).

[0059] Component Concentration (g L'1)

[0060] Cyanocobalamin (B12) 0.390 Results (Figs 1 A and B) show that Clostridium luticellarii grows on 50 mM lactic acid with or without acetic acid, but growth at 200 mM lactic acid is only significant when acetic acid is supplied. Lactic acid is converted to a mixture of acetic, butyric and isobutyric acid. Acetic acid addition increases the substrate consumption rates and the yields of butyric and isobutyric acid production, thus it is beneficial for isobutyric acid production and selectivity, but not essential at low substrate concentrations.

[0061] Experiment 2: Effect of lactic acid concentration on growth of Clostridium luticellarii

[0062] Clostridium luticellarii was grown in batch in serum bottles, at four different lactic acid concentrations: a) Lactic acid (200 mM); b) Lactic acid (400 mM); Lactic acid (600 mM); b) Lactic acid (800 mM). Acetic acid was always added at a constant 100 mM. Medium composition and preparation, except for the concentrations of lactic and acetic acid, sampling, and sample analysis were identical to Experiment 1.

[0063] Results (Fig 2) show that Clostridium luticellarii was not able to grow at concentrations of 400 mM or above. Experiment 3: Production of isobutyric acid from glucose by Clostridium luticellarii

[0064] Clostridium luticellarii was grown in batch in serum bottles at a concentration of 10 g / L of glucose. Acetic acid was initially present at a concentration of 0.2 g / L. Medium composition and preparation, with exclusion of the addition of lactic and acetic acid, sampling, and sample analysis were identical to Experiment 1. Glucose was added from a filter sterilized stock solution before inoculation.

[0065] Results (Fig 3) show that Clostridium luticellarii can produce isobutyric acid from glucose, reaching final isobutyric acid concentrations around 1 g / L.

[0066] In another series of batch tests, Clostridium luticellarii was grown in batch in serum bottles at a glucose concentration of 50 mM with a variable acetic acid concentration of 0 mM, 25 mM and 50 mM to screen the influence of the addition of acetic acid on the glucose fermentation. Medium composition and preparation, with exclusion of the addition of lactic and acetic acid, sampling, and sample analysis were identical to Experiment 1. Glucose was added from a filter sterilized stock solution before inoculation.

[0067] The results (Fig 4) show that the addition of acetic acid shifts the product spectrum towards C4-organic acids, from 46% at 0 mM, to 60% at 25 mM, to 63% at 50 mM acetic acid. The ratio of the C4-organic acids, i.e. butyric and iso-butyric acid, is not influenced by the acetic acid addition.

[0068] Experiment 4: Production of isobutyric acid from lactic acid and methanol or hydrogen by Clostridium luticellarii

[0069] Clostridium luticellarii was grown in batch in serum bottles containing 50 mM lactic acid and addition of either H2(by pressurizing the headspace with filter sterilized H2 / CO280 / 20 v / v% to 150 kPa absolute after autoclaving) or addition of 50 mM methanol. Medium composition and preparation sampling, and sample analysis were identical to Experiment 1. Acetic acid was not added as a co-substrate. Methanol was added after inoculation from a filter sterilized stock solution.

[0070] Results (Fig 5) show that Clostridium luticellarii's growth was enhanced by the addition of either methanol or H2. The addition of methanol increased concentrations of products and resulted in higher selectivity of butyric and isobutyric acid. Experiment 5: Production of isobutyric acid from lactic acid in a batch bioreactor

[0071] Clostridium luticellarii was grown in 2 L batch reactors (Eppendorf BioFlo 120, Germany) with medium containing 150 mM lactic acid and 90 mM acetic acid. The reactors (Eppendorf BioFlo 120, Germany) had a total volume of 2 L and were equipped with a direct-drive stirrer (set to 50 rpm), a microsparger, pH sensor (Hamilton EasyFerm Plus, Switzerland), condenser and heating blanket. Sterile gas filters (Merck Millex, Germany) were installed before the sparger and after the condenser to shield the reactor from contamination. A water lock was installed after the condenser to prevent oxygen from entering the headspace. To enable gas sampling, a septum was installed between the condenser and water lock. Basal medium was prepared according to the composition detailed in experiment 1, but without MES buffer. Lactic and acetic acid were added to the medium in the reactor and the reactor was autoclaved. Vitamins and trace elements (Tables 1, 2 and 3), 30 mM NaHCO3, 0.4 mM titanium (III) citrate and 1 g L1yeast extract was added to the reactor from filter sterilized stock solutions via a rubber septum in the headplate of the reactor. The reactor was flushed with N2 / CO2 (90 / 10 v / v%) for at least one hour before inoculation. Inoculation was done with a 10% (v / v%) inoculum grown on 200 mM lactic acid and 100 mM acetic acid. The pH was controlled at 6.50 throughout the experiment by dosing sterile 2 M NaOH and 2 M HCI. Liquid samples were taken every 1-2 days. Liquid samples were taken through an aseptic sampling port (Avantor, USA) and used for direct OD62o and pH measurements, after which they were filtered over 0.2 pm Chromafil Xtra syringe filters and stored at -20 °C until IC / HPLC analysis. Sample analysis was performed as described in Experiment 1.

[0072] The results (Fig 6) in a bioreactor validate the observations in serum bottles, with production of 3.6 g / L butyric acid and 3.4 g / L isobutyric acid. Yields of 0.20 g C2 / g lactate, 0.23 g C4 / g Lactate and 0.21 g iC4 / g Lactate can be calculated.

[0073] Experiment 6: Production of isobutyric acid and isobutanol from lactic acid in a fed-batch bioreactor

[0074] Clostridium luticellarii was grown in two 2 L replicate bioreactors described in Experiment 5. Reactors were started as in Experiment 5, but with a lower volume of 1 L. The fed batch phase was started when all lactic acid was consumed. Lactic acid was fed from a 100 g / L stock solution until 20 g / L of lactic acid was observed, after which feeding was stopped. Feeding was resumed at constant rate after lactic acid dropped below 10 g / L. Reactors were fed until ~2 L working volume was reached. The feed solution consisted of basal medium with the composition described in Experiment 1, but without MES buffer. The feed solution was autoclaved and filter sterilized vitamins and trace elements (Tables 1, 2 and 3), 30 mM NaHCO3, 0.4 mM titanium (III) citrate (reducing agent) and 3.33 g L-1yeast extract were added from filter sterilized stock solutions. Subsequently the medium was sparged with filter sterilized N2 / CO2(90 / 10 v / v%) for at least 30 minutes before attaching it aseptically to the reactor. Sampling and sample analysis was performed as described in Experiment s.

[0075] A fed-batch configuration allowed to increase the overall process productivity and isobutyric acid selectivity. Final isobutyric acid concentrations were 12 and 14 g / L for replicate 1 and 2 respectively. Isobutanol production was also observed in both reactors, and it started when lactic acid concentrations reached 10 g / L and accumulated up to 0.6 g / L. Replicate two also produced butanol, but concentrations were below quantification limit for replicate 1 (Fig 7)

[0076] Experiment 7: Production of isobutyric acid from glucose, fructose and maltose by Clostridium luticellarii

[0077] Clostridium luticellarii was grown in batch serum bottles at a concentration of 10 g / L of different sugars i:e glucose, fructose and maltose. Medium composition and preparation, with exclusion of the addition of lactic and acetic acid, sampling, and sample analysis were identical to Experiment 1. Sugars were added from a filter sterilized stock solution before inoculation.

[0078] Results (Fig 8) show that Clostridium luticellarii can produce isobutyric acid from glucose, fructose and maltose, reaching the final concentrations of 1.06 g / L, 0.92 g / L, and 0.25 g / L respectively.

[0079] Experiment 8: Production of isobutyric acid from lactic and formic acid mixtures by Clostridium luticellarii

[0080] Clostridium luticellarii was grown in batch serum bottles at a constant lactic acid concentration of 200 mM with varying formic acid concentrations of 50 mM and 100 mM. Medium composition, preparation, and sample analysis were identical to Experiment 1, Lactic acid and acetic acid were added to the medium at concentrations of 200 mM and 100 mM. Formic acid was added from a filter sterilized stock solution before inoculation. Results (Fig 9) show that Clostridium luticellarii was able to produce isobutyric acid from mixture of formic and lactic acid but the addition of formic acid resulted in higher selectivity of acetic acid.

[0081] References

[0082] [1] C. Petrognani, N. Boon, R. Ganigue, Production of isobutyric acid from methanol by Clostridium luticellarii., Green Chem. (2020). https: / / doi.org / 10.1039 / D0GC02700F.

[0083] [2] Q. Marien, A. Regueira, R. Ganigue, Steerable isobutyric and butyric acid production from CO2 and H2 by Clostridium luticellarii, Microb. Biotechnol. (2023) 1751-7915.14321. https: / / doi.org / 10.llll / 1751-7915.14321.

[0084] [3] K. Sabbe, L. D'Haen, N. Boon, R. Ganigue, Predicting the performance of chain elongating microbiomes through flow cytometric fingerprinting, Water Res. 243 (2023) 120323. https: / / doi.Org / 10.1016 / j.watres.2023.120323.

[0085] [4] Q. Marien, P. Candry, E. Hendriks, J.M. Carvajal-Arroyo, R. Ganigue, Substrate loading and nutrient composition steer caproic acid production and biofilm aggregation in high-rate granular reactors, J. Environ. Chem. Eng. 10 (2022) 107727. https: / / doi.Org / 10.1016 / j.jece.2022.107727.

[0086] [5] B. Liu, D. Popp, N. Muller, H. Strauber, H. Harms, S. Kleinsteuber, Three Novel Clostridia Isolates Produce n-Caproate and iso-Butyrate from Lactate: Comparative Genomics of Chain-Elongating Bacteria, Microorganisms 8 (2020) 1970. https: / / doi.org / 10.3390 / microorganisms8121970.

[0087] [6] K. Zhang, A.P. Woodruff, M. Xiong, J. Zhou, Y.K. Dhande, A Synthetic Metabolic Pathway for Production of the Platform Chemical Isobutyric Acid, ChemSusChem 4 (2011) 1068-1070. https: / / doi.Org / 10.1002 / cssc.201100045.

[0088] [7] K. Lang, J. Zierow, K. Buehler, A. Schmid, Metabolic engineering of Pseudomonas sp. strain VLB120 as platform biocatalyst for the production of isobutyric acid and other secondary metabolites, Microb. Cell Factories 13 (2014) 2. https: / / doi.org / 10.1186 / 1475-2859-13-2.

[0089] [8] Q. Wang, C.-D. Wang, C.-H. Li, J.-G. Li, Q. Chen, Y.-Z. Li, Clostridium luticellarii sp. nov., isolated from a mud cellar used for producing strong aromatic liquors, Int. J. Syst. Evol. Microbiol. 65 (2015) 4730- 4733. https: / / doi.Org / 10.1099 / ijsem.0.000641.

Claims

Claims1. Use of a strain belonging to the bacterial species Clostridium luticellarii to convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol.

2. Use of a strain belonging to the bacterial species Clostridium luticellarii according to claim 1, wherein said carbohydrate is chosen from the list consisting of: glucose, fructose, xylose, arabinose, galactose, rhamnose, palatinose, fucose, (iso)maltose, lactose, or a mixture of said carbohydrates.

3. A process to convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol comprising: -providing lactic acid and / or carbohydrates, - providing a strain belonging to the bacterial species Clostridium luticellari and allowing the conversion of said lactic acid and / or carbohydrate into said isobutyric acid and / or said isobutanol by said strain belonging to the bacterial species Clostridium luticellari.

4. A process to convert lactic acid and / or carbohydrates into isobutyric acid and / or isobutanol according to claim 3 further comprising: providing lactic acid and / or carbohydrates, which may further comprise at least one of the following steps: (i) the addition of water, nutrients, pH adjustment solutions, and / or hydrolytic enzymes to said lactic acid and / or carbohydrates in order to obtain a solution of said lactic acid and / or carbohydrates; (ii) the fermentation of said solution in batch, fed-batch, or continuous process in order to produce lactic acid and optionally acetic acid , via the addition of a lactic acid microbial culture or microbiome; ill) maintaining the pH of said solution by dosing acid or base; and (iv) separating the solid and liquid fractions to remove cells / organic material via sieving, extruding and / or filtration. converting the said lactic acid and / or said carbohydrate into said isobutyric acid and / or said isobutanol via a bioprocess, further comprising at least one of the following steps: (i) addition of a strain belonging to the bacterial species Clostridium luticellarii or a culture containing at least 20% of the bacterial species Clostridium luticellarii, to the solution containing lactic acid and / or carbohydrates; (ii) addition of nutrients, acetic acid and / or pH adjustment solutions; (iii) fermentation of the lactic acid and / or carbohydrates to isobutyric acid and / or said isobutanol in a batch, a fed-batch or a continuous process;Recovering the said isobutyric acid and / or isobutanol produced via either the separation of the cells from the broth and extraction followed by their transformation to bioproducts, or their in-situ transformation to bioproducts followed by product separation.

5. A process according to claim 3, wherein said process further comprises the provision of acetic acid.

6. A process according to claims 3-5 wherein the concentration of said lactic acid concentration in the reactor shall be maintained below 600 mM.

7. A process according to claims 3-6 wherein acetic acid is provided at lactic acid concentrations in the reactor above 50 mM.

8. A process according to claim 3-7 wherein said process further comprises the provision of methanol, formate, hydrogen gas and / or carbon monoxide.

9. A process according to claim 3-8 wherein said process can be carried out in various reactor configurations such as batch, fed-batch or continuous, with or without the additional supply of acetic acid.

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