WO2020211941A1 - Extreme thermophilic bacteria of the genus caldicellulosiruptor - Google Patents

Abstract

The technology provided herein relates to novel isolated cellulolytic extreme thermophilic bacterial cells belonging to the genus Caldicellulosiruptor, mutants thereof, isolated strains, microbial cultures and microbial compositions. The novel bacteria are in particular suitable for the production of fermentation products such as lactic acid from any carbon source, not limited to cellulosic but especially useful for converting cellulosic and ligniocellulosic biomass.

Description

EXTREME THERMOPHILIC BACTERIA OF THE GENUS

CALDICELLULOSIRUPTOR

FIELD OF THE DISCLOSURE

The present disclosure pertains to novel isolated cellulolytic extreme thermophilic bacterial cells belonging to the genus C aldicellulosiruptor, mutants thereof, isolated strains, microbial cultures and microbial compositions. The novel bacteria are in particular suitable for the production of fermentation products such as lactic acid from any carbon source, not limited to cellulosic but especially useful for converting cellulosic and ligniocellulosic biomass.

BACKGROUND

In general, fermentation products are produced by degradation of carbon-based biomass like starch-containing material into fermentable sugars by liquefaction and saccharification followed by conversion of the sugars directly or indirectly into the desired fermentation product using a fermenting organism.

However, the industry of producing fermentation products such as ethanol and lactic acid is facing the challenge of redirecting the production process from fermentation of relatively easily convertible but expensive starchy materials, to the complex but inexpensive cellulosic waste or lignocellulosic biomass such as plant biomass.

Unlike starch, which contains homogenous and easily hydrolysed polymers, lignocellulosic biomass contains variable amounts of cellulose, hemicellulose, lignin and small amounts of protein, pectin, wax and other organic compounds. Lignocellulosic biomass should be understood in its broadest sense, so that it apart from wood, agricultural residues, energy crops also comprises different types of waste from both industry and households. Cellulosic biomass is a vast poorly exploited resource, and in some cases a waste problem. However, hexoses from cellulose can be converted by yeast or other microorganisms to fuel ethanol for which there is a growing demand.

Typically, the first step in utilization of lignocellulosic biomass is a pre-treatment step, in order to fractionate the components of lignocellulosic material and increase their surface area.

The pre-treatment method most often used is steam pretreatment, a process comprising heating of the lignocellulosic material by steam injection to a temperature of 130 - 230°C. Prior to or during steam pretreatment, a catalyst like mineral or organic acid or a caustic agent facilitating disintegration of the biomass structure can be added optionally.

Another type of lignocellulose hydrolysis is acid hydrolysis, where the lignocellulosic material is subjected to an acid such as sulphuric acid whereby the sugar polymers cellulose and hemicellulose are partly or completely hydrolysed to their constituent sugar monomers and the structure of the biomass is destroyed facilitating access of hydrolytic enzymes in subsequent processing steps.

A further method is wet oxidation wherein the material is treated with oxygen at 150-185 °C. Either pretreatment can be followed by enzymatic hydrolysis to complete the release of sugar monomers. This pre-treatment step results in the hydrolysis of cellulose into glucose while hemicellulose is transformed into the pentoses xylose and arabinose and the hexoses glucose, mannose and galactose. Thus, in contrast to starch, the hydrolysis of lignocellulosic biomass results in the release of pentose sugars in addition to hexose sugars. This implies that useful fermenting organisms need to be able to convert both hexose and pentose sugars to desired fermentation products such as ethanol.

After the pre-treatment the lignocellulosic biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1 ) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g. glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose).

Each processing step can make the overall process more costly and, therefore, decrease the economic feasibility of producing biofuel or carbon-based chemicals from cellulosic biological material. Thus, there is a need to develop methods that reduce the number of processing steps needed to convert cellulosic biological material to biofuel and other commercially desirable materials.

The four biologically mediated transformations may occur in a single step in a process configuration called consolidated bioprocessing (CBP), which is distinguished from other less highly integrated configurations in that CBP does not involve a dedicated process step for cellulase and/or hemicellulase production. CBP offers the potential for higher efficiency than a processes requiring dedicated cellulase production in a distinct unit operation. Therefore, the availability of novel microorganisms for converting lignocellulosic biomass material to carbon-based chemicals would be highly advantageous.

SUMMARY OF THE DISCLOSURE

The present invention relates to novel microorganisms, and compositions useful for processing lignocellulosic biomass.

In a first aspect, embodiments of the disclosure provide novel isolated cellulolytic thermophilic bacterial cells belonging to the genus C aldicellulosiruptor, in particular capable of producing high levels of lactic acid from cellulosic biomass material like lignocellulosic biomass.

In one aspect, embodiments of this disclosure relate to an Caldicellulosiruptor sp. BluCon006, Caldicellulosiruptor sp. BluCon014 and Caldicellulosiruptor sp. BluCon016, which are listed in table 1 , are deposited on April 09th, 2019 under the accession numbers DSM 33095, DSM 33096 and DSM 33097 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), IhIioIΐbheΐGqbb 7B, 38124 Braunschweig (DE) by BluCon Biotech GmbH, Nattermannallee 1 , 50829 Cologne (DE).

In still another aspect the present invention relates to an isolated strain comprising a Caldicellulosiruptor sp. cell according to any of the preceding aspects.

In a further aspect, embodiments of this disclosure relate to microorganism of the strain Caldicellulosiruptor sp. BluCon006 deposited as DSM 33095, a microorganism derived therefrom or a Caldicellulosiruptor sp. BluCon006 homolog or mutant.

In a further aspect, embodiments of this disclosure relate to an isolated Caldicellulosiruptor sp. BluCon0006 deposited as DSM 33095, or mutants thereof retaining the properties.

In a further aspect, embodiments of this disclosure relate to microorganism of the strain Caldicellulosiruptor sp. BluCon014 deposited as DSM 33096, a microorganism derived therefrom or a Caldicellulosiruptor sp. BluCon014 homolog or mutant.

In a further aspect, embodiments of this disclosure relate to an isolated Caldicellulosiruptor sp. BluCon0014 deposited as DSM 33096, or mutants thereof retaining the properties.

In a further aspect, embodiments of this disclosure relate to microorganism of the strain Caldicellulosiruptor sp. BluCon016 deposited as DSM 33097, a microorganism derived therefrom or a Caldicellulosiruptor sp. BluCon016 homolog or mutant.

In a further aspect, embodiments of this disclosure relate to an isolated Caldicellulosiruptor sp. BluCon0016 deposited as DSM 33097, or mutants thereof retaining the properties. In another aspect the present disclosure relates to a method of producing a fermentation product comprising culturing a cell according to the disclosure or a strain according to the disclosure under suitable conditions.

In still another aspect, embodiments of this disclosure relate to methods for converting a carbon based biomass like lignocellulosic and/or cellulosic biomass material, starch and/or hemicellulose to a carbon-based chemical, in particular lactic acid and/or a salt or an ester thereof, comprising the step of contacting the lignocellulosic and/or cellulosic biomass material with a microbial culture for a period of time at an initial temperature and an initial pH, thereby producing an amount of a carbon-based products, in particular lactic acid and/or a salt or an ester thereof; wherein the microbial culture comprises an extremely thermophilic microorganism of the genus Caldicellulosiruptor, in particular all microorganisms of the strain Caldicellulosiruptor sp. as listed in table 1 with their respective deposition numbers, microorganisms derived from either of these strains or mutants or homologues thereof, in particular mutants thereof retaining the properties.

In still another aspect, embodiments of this disclosure relate to methods of making lactic acid from a carbon-based biomass like lignocellulosic and/or cellulosic biomass material, wherein the method comprises combining a microbial culture and the biomass in a medium; and fermenting the biomass under conditions and for a time sufficient to produce lactic acid, a salt or an ester thereof, in a single step process as part of a consolidated bioprocessing (CBP) system, with a cell, strain, microbial culture and/or a microorganism according to the present disclosure under suitable conditions, in particular using mutants thereof retaining the properties.

In still another aspect, embodiments of this disclosure relate to methods of making both ethanol and lactic acid from biomass material, wherein the method comprises combining a microbial culture and the biomass in a medium; and fermenting the biomass under conditions and for a time sufficient to produce ethanol and lactic acid, a salt or an ester of the latter, in a single step process as part of a consolidated bioprocessing (CBP) system, with a cell, strain, microbial culture and/or a microorganism or mutants thereof retaining the properties according to the present disclosure under suitable conditions.

In still another aspect, embodiments of this disclosure relate to methods of making lactic acid from biomass material, wherein the method comprises combining a microbial culture and the biomass in a medium; and fermenting the biomass under conditions and for a time sufficient to produce ethanol and / or lactic acid, and/or acetic acid, a salt or an ester of the latter, in a single step process as part of a consolidated bioprocessing (CBP) system, with a cell, strain, microbial culture and/or a microorganism according to the present disclosure under suitable conditions.

Further, embodiments of this disclosure relate to compositions for converting carbon-based biomass material like lignocellulosic biomass or a microbial culture comprising a cell, strain or microorganism according to the present disclosure.

Further, embodiments of this disclosure relate to the use of a cell, strain, microorganism and/or a microbial culture according to the present disclosure for the production of lactic acid, a salt or an ester thereof or for the production of ethanol.

Before the disclosure is described in detail, it is to be understood that this disclosure is not limited to the particular component parts of the devices described or process steps of the methods described and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include singular and/or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

To provide a comprehensive disclosure without unduly lengthening the specification, the applicant hereby incorporates by reference each of the patents and patent applications cited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a 16S rDNA from Caldicellulosiruptor sp. BluCon006 cell.

FIG. 2 shows a 16S rDNA from Caldicellulosiruptor sp. BluCon014 cell.

FIG. 3 shows a 16S rDNA from Caldicellulosiruptor sp. BluCon016 cell.

FIG. 4 shows a 16S rDNA from Caldicellulosiruptor sp. DIB104C cell.

DETAILED DESCRIPTION OF THIS DISCLOSURE

The present disclosure relates to methods, microorganisms, and compositions useful for processing lignocellulosic biomass. The disclosure relates, in certain aspects, to microorganisms which are able to convert pretreated lignocellulosic biomass such as, for example pretreated miscanthus grass, to soluble products that can be used by the same or by another microorganism to produce an economically desirable product such as, for example, a carbon-based chemical, in particular lactic acid and/or a salt or an ester thereof.

The application of this technology has the potential to render production of carbon-based chemicals more economically feasible and to allow a broader range of microorganisms to utilize recalcitrant biomass. The use of cellulosic materials as sources of bioenergy is currently limited by typically requiring preprocessing of the cellulosic material. Such preprocessing methods can be expensive. Thus, methods that reduce dependence on preprocessing of cellulosic materials may have a dramatic impact on the economics of the use of recalcitrant biomass for carbon-based chemicals production. One challenge in converting biomass into fermentation products is the recalcitrance and heterogeneity of the biological material.

The present inventors have found microorganisms of the genus Caldicellulosiruptor which have a variety of advantageous properties for their use in the conversion of lignocellulosic biomass material to carbon-based chemicals, preferably to lactic acid and/or a salt or an ester thereof, preferably in a single step process as part of a consolidated bioprocessing (CBP) system.

In particular, these microorganisms are extremely thermophilic and show broad substrate specificities and high natural production of lactic acid. Moreover, ethanol and lactic acid fermentation at high temperatures, for example over 70 °C has many advantages over mesophilic fermentation. One advantage of thermophilic fermentation is the minimization of the problem of contamination in batch cultures, fed-batch cultures or continuous cultures, since only a few microorganisms are able to grow at such high temperatures in un-detoxified lignocellulose biomass material.

It is also an advantage that the cells, strains and microorganisms according to the present disclosure grow on pre-treated as well as on untreated lignocellulosic biomass material.

The isolated cells, strains, microorganisms, compositions and microbial cultures are capable of growing and producing fermentation products on very high dry-matter concentrations of lignocellulosic biomass material.

In the present context the term " lignocellulosic biomass material" is intended to designate a untreated lignocellulosic biomass and/or a lignocellulosic biomass which has been subjected to a pretreatment step whereby lignocellulosic material has been at least partially separated into cellulose, hemicellulose and lignin thereby having increased the surface area and / or accessibilitv of the material. The lianocellulosic material mav tvDicallv be derived from Dlant material, such as straw, hay, perennial grass, garden refuse, comminuted wood, fruit hulls and seed hulls.

The pretreatment method most often used is steam pretreatment, a process comprising heating of the lignocellulosic material by steam injection to a temperature of 130-230 degrees centigrade with or without subsequent sudden release of pressure. Prior to or during steam pretreatment, a catalyst like a mineral or organic acid or a caustic agent facilitating disintegration of the biomass structure can be added optionally. Catalysts often used for such a pretreatment include but are not limited to sulphuric acid, sulphurous acid, hydrochloric acid, acetic acid, lactic acid, sodium hydroxide (caustic soda), potassium hydroxide, calcium hydroxide (lime), ammonia or the respective salts or anhydrides of any of these agents.

Such steam pretreatment step may or may not be preceded by another treatment step including cooking of the biomass in water or steaming of the biomass at temperatures of 100 - 200 °C with or without the addition of a suitable catalyst like a mineral or organic acid or a caustic agent facilitating disintegration of the biomass structure. In between the cooking step and the subsequent steam pretreatment step one or more liquid-solid-separation and washing steps can be introduced to remove solubilized biomass components in order to reduce or prevent formation of inhibitors during the subsequent steam pretreatment step. Inhibitors formed during heat or steam pretreatment include but are not limited to furfural formed from monomeric pentose sugars, hydroxymethylfurfural formed from monomeric hexose sugars, acetic acid, levulinic acid, phenols and phenol derivatives.

Another type of lignocellulose hydrolysis is acid hydrolysis, where the lignocellulosic material is subjected to an acid such as sulfuric acid or sulfurous acid whereby the sugar polymers cellulose and hemicellulose are partly or completely hydrolysed to their constituent sugar monomers. A third method is wet oxidation wherein the material is treated with oxygen at 150-

185 degrees centigrade. The pretreatments can be followed by enzymatic hydrolysis to complete the release of sugar monomers. This pre-treatment step results in the hydrolysis of cellulose into glucose while hemicellulose is transformed into the pentoses xylose and arabinose and the hexoses glucose, mannose and galactose. The pretreatment step may in certain embodiments be supplemented with treatment resulting in further hydrolysis of the cellulose and hemicellulose. The purpose of such an additional hydrolysis treatment is to hydrolyze oligosaccharide and possibly polysaccharide species produced during the acid hydrolysis, wet oxidation, or steam pretreatment of cellulose and/or hemicellulose origin to form fermentable sugars (e.g. glucose, xylose and possibly other monosaccharides). Such further treatments may be either chemical or enzymatic. Chemical hydrolysis is typically achieved bv treatment with an acid such as treatment with aaueous sulDhuric acid or hydrochloric acid, at a temperature in the range of about 100-150 degrees centigrade. Enzymatic hydrolysis is typically performed by treatment with one or more appropriate carbohydrase enzymes such as cellulases, glucosidases and hemicellulases including xylanases.

It has been found that the microorganisms according to the present disclosure can grow efficiently on various types of pretreated and untreated biomass (e.g. wood incl. poplar, spruce and cotton wood; various types of grasses and grass residues incl. miscanthus, wheat straw, sugarcane bagasse, corn stalks, corn cobs, whole corn plants, sweet sorghum).

As used herein "efficient" growth refers to growth in which cells may be cultivated to a specified density within a specified time.

The microorganisms according to the present disclosure can grow efficiently on crystalline cellulose. The main product when grown on untreated biomass substrates was L-lactate.

The microorganisms according to the present disclosure also can grow efficiently on spent biomass— insoluble material that remains after a culture has grown to late stationary phase (e.g., greater than 10⁸ cells/mL) on untreated biomass.

Furthermore, the microorganisms according to the present disclosure grew efficiently on both the soluble and insoluble materials obtained after heat-treating the biomass.

The microorganisms according to the invention are anaerobic thermophile bacteria, and they are capable of growing at high temperatures even at or above 70 degrees centigrade. The fact that the strains are capable of operating at this high temperature is of high importance in the conversion of the cellulosic or lignocellulosic material into fermentation products. The conversion rate of carbohydrates into e.g. lactic acid and/or ethanol is much faster when conducted at high temperatures. For example, the volumetric ethanol productivity of a thermophilic Bacillus is up to ten-fold higher than a conventional yeast fermentation process which operates at 30 degrees centigrade Consequently, a smaller production plant is required for a given plant capacity, thereby reducing plant construction costs. As also mentioned previously, the high temperature reduces the risk of contamination from other microorganisms, resulting in less downtime and increased plant productivity. The high operation temperature may also facilitate the subsequent recovery of the resulting fermentation products.

Lignocellulosic biomass material and lignocellulose hydrolysates contain inhibitors such as furfural. Dhenols and carboxvlic acids which can Dotentiallv inhibit the fermentina oraanism. Therefore, it is an advantage of the microorganisms according to the present disclosure that they are tolerant to these inhibitors.

The microorganisms according to the present disclosure are novel species of the genus Caldicellulosiruptor or novel subspecies of Caldicellulosiruptor saccharolyticus.

For example, the genus Caldicellulosiruptor includes different species of extremely thermophilic (growth at temperature significantly above 70 °C) cellulolytic and hemicellulolytic strictly anaerobic nonsporeforming bacteria. The first bacterium of this genus, Caldicellulosiruptor saccharolyticus strain Tp8T (DSM 8903) has a temperature optimum of 70 °C and was isolated from a thermal spring in New Zealand (Rainey et al. 1994; Sissons et al. 1987). It hydrolyses a variety of polymeric carbohydrates with the production of acetate, lactate and trace amounts of ethanol (Donnison et al. 1988). Phylogenetic analysis showed that it constitutes a novel lineage within the Bacillus/Clostridium subphylum of the Gram-positive bacteria (Rainey et al. 1994).

According to the present disclosure, the microorganisms produce lactic acid and show several features that distinguish them from currently used microorganisms: (i) high yield and low product inhibition, (ii) simultaneous utilization of lignocellolytic biomass material and/or sugars, and (iii) growth at elevated temperatures. The microorganisms according to the present disclosure are robust thermophile organisms with a decreased risk of contamination. They efficiently convert an extraordinarily wide range of biomass components to carbon-based chemicals like lactic acid.

As mentioned above, in one aspect, the present disclosure relates to an isolated cell comprising a 16S rDNA sequence selected from the group consisting of: SEQ ID NO 1 , SEQ ID NO 2 and SEQ ID NO 3, and a combination of any thereof.

In one aspect, the present disclosure pertains to an isolated Caldicellulosiruptor sp. cell having a 16S rDNA sequence at least 99, at least 99,3, at least 99,5, at least, 99,7, at least 99,9, at least 99,99 percent identical to either sequence SEQ ID NO 1 , SEQ ID NO 2 and/or SEQ ID NO 3 or a combination thereof.

Each independently an embodiment of the invention is an isolated cell which is Caldicellulosiruptor sp. BluCon006 (DSMZ Accession number 33095,), an isolated cell which is Caldicellulosiruptor sp. BluCon014 (DSMZ Accession number 33096), an isolated cell which is Caldicellulosiruptor sp. BluCon016 (DSMZ Accession number 33097), cells derived from either, mutants or a homolog of either, in particular of mutants thereof retaining the properties of BluCon006, BluCon014 and/or BluCon016.

As used herein "mutant" or“homolog” means a microorganism derived from the cells or strains according to the present disclosure, which are altered due to a mutation. A mutation is a change produced in cellular DNA, which can be spontaneous, caused by an environmental factor or errors in DNA replication, or induced by physical or chemical conditions. The processes of mutation included in this and indented subclasses are processes directed to production of essentially random changes to the DNA of the microorganism including incorporation of exogenous DNA. All mutants of the microorganisms comprise the advantages of being extreme thermophile (growing and fermenting at temperatures above 70°C) and are capable of fermenting lignocellulosic biomass to lactic acid, in particular to L-lactic acid . In an advantageous embodiment, mutants of the microorganisms according to the present disclosure have in a DNA-DNA hybridization assay, a DNA-DNA relatedness of at least 80%, preferably at least 90%, at least 95%, more preferred at least 98%, most preferred at least 99%, and most preferred at least 99,9% with one of the isolated bacterial strains Caldicellulosiruptor sp. BluCon006, BluCon014 and/or BluCon016. In particular, the mutants of BluCon006, BluCon014 and/or BluCon016 retaining the properties of the deposited strains BluCon006, BluCon014 and/or BluCon016.

The invention is based on the isolated bacterial strains Caldicellulosiruptor sp. BluCon006, BluCon014 and/or BluCon016 that contain 16S rDNA sequences at least 99 to 100%, preferably 99,5 to 99.99, more preferably at least 99,99 percent identical to the respective sequences SEQ ID NO 1 , SEQ ID NO 2 and/or SEQ ID NO 3.

Table 1

The strains listed in table 1 have been deposited in accordance with the terms of the Budapest

Treaty on April 9, 2019 with DSMZ - Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH, Inhoffenstr. 7B, 38124 Braunschweig, Germany, under the respectively indicated DSMZ accession numbers and deposition dates, respectively, by BluCon Biotech

GmbH, Nattermannallee 1 , 50829 Cologne (DE). The microorganisms of the species Caldicellulosiruptor sp. according to the present disclosure in particular refer to a microorganism which belongs to the genus Caldicellulosiruptor and which preferably has one or more of the following characteristics: a) it is a microorganism of the genus Caldicellulosiruptor ;

b) in a DNA-DNA hybridization assay, it shows a DNA-DNA relatedness of at least 70%, preferably at least 90%, at least 95%, more preferred at least 98%, most preferred at least 99% with either Caldicellulosiruptor sp. strain listed in table 1 with their respective accession numbers; and/or

c) it displays a level of 16S rDNA gene sequence similarity of at least 98%, preferably at least 99% or at least 99,5%, more preferably 100% with either either Caldicellulosiruptor sp. strain listed in table 1 with their respective accession numbers; and/or

d) it is capable of surviving in high temperature conditions above 75 °C e) it is capable of surviving in high temperature conditions above 70 °C, and or f) it is a Gram-positive bacterium.

Preferably, at least two or at least three, and more preferred all of the above defined criteria a) to f) are fulfilled.

In an advantageous embodiment, the microorganisms according to the present disclosure in particular refer to a microorganism which belongs to the genus Caldicellulosiruptor and which preferably has one or more of the following characteristics: a) It is a microorganism of the genus Caldicellulosiruptor

b) it is a microorganism of the species Caldicellulosiruptor saccharolyticus;

c) in a DNA-DNA hybridization assay, it shows a DNA-DNA relatedness of at least 80%, preferably at least 90%, at least 95%, more preferred at least 98%, most preferred at least 99%, and most preferred at least 99,9% with one of the strains of table 1 ; and/or

d) it displays a level of 16S rDNA gene sequence similarity of at least 98%, preferably at least 99%, at least 99,5% or at least 99,7%, more preferably 99,99% with one of the strains listed in table 1 ; and/or

e) it is capable of surviving and/or growing and/or producing a fermentation product selected from the group consisting of acids and alcohols at temperature conditions above 70 °C, in particular of above 72 °C. Preferably, at least two or at least three, and more preferred all of the above defined criteria a) to e) are fulfilled.

The term "DNA-DNA relatedness" in particularly refers to the percentage similarity of the genomic or entire DNA of two microorganisms as measured by the DNA-DNA hybridization / renaturation assay according to De Ley et al. (1970) Eur. J. Biochem. 12, 133-142 or Hub et al. (1983) Syst. Appl. Microbiol. 4, 184-192. In particular, the DNA-DNA hybridization assay preferably is performed by the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) Identification Service.

The term "16S rDNA gene sequence similarity" in particular refers to the percentage of identical nucleotides between a region of the nucleic acid sequence of the 16S ribosomal RNA (rDNA) gene of a first microorganism and the corresponding region of the nucleic acid sequence of the 16S rDNA gene of a second microorganism. Preferably, the region comprises at least 100 consecutive nucleotides, more preferably at least 200 consecutive nucleotides, at least 300 consecutive nucleotides or at least 400 consecutive nucleotides, most preferably about 480 consecutive nucleotides.

The strains according to the present disclosure have the potential to be capable of producing a number of different fermentation products, including acids, alcohols, ketones and hydrogen. In one embodiment, the alcohol is selected from ethanol, butanol, propanol, methanol, propanediol and butanediol. In a further embodiment the acid is lactic acid, propionic acid, acetic acid, succinic acid, butyric acid or formic acid and the ketone is acetone.

In an advantageous embodiment, the strains according to the present disclosure have the potential to be capable of producing a high amount of lactic acid and/or a salt or an ester thereof. In particular, the strains according to the present disclosure produce a high amount of lactic acid and/or a salt or an ester thereof after 60h of cultivation and/or fermentation time in particular after 90 h of cultivation and/or fermentation time.

The Caldicellulosiruptor sp. strains according to the present disclosure have several highly advantageous characteristics needed for the conversion of lignocellulosic biomass material.

Thus, these base strains possess all the genetic machinery for the hydrolysis of cellulose and hemicelluloses and for the conversion of both pentose and hexose sugars to various fermentation products such as lactic acid and ethanol. As will be apparent from the below examples, the examination of the complete 16S rDNA sequence showed that the closely related strains may all be related to Caldicellulosiruptor saccharolyticus although the 16S rDNA seauences mav Dlace them in a seoarate subsDecies or even a different sDecies In a preferred embodiment, the Caldicellulosiruptor sp. microorganism is

a) Caldicellulosiruptor sp. BluCon006, deposited on April 09, 2019 under the accession number DSM 33095 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), IhIioIΐbheΐGqbb 7B, 38124 Braunschweig (DE) by BluCon Biotech GmbH, Nattermannallee 1 , 50829 Cologne (DE), b) a microorganism derived from Caldicellulosiruptor sp. BluCon006 or

c) a Caldicellulosiruptor sp. BluCon006 mutant retaining the properties of BluCon006.

In another preferred embodiment, the Caldicellulosiruptor sp. microorganism is

a) Caldicellulosiruptor sp. BluCon014, deposited on April 09, 2019 under the accession number DSM 33096 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), IhIioIΐbhbΐGqbb 7B, 38124 Braunschweig (DE) by BluCon Biotech GmbH, Nattermannallee 1 , 50829 Cologne (DE), b) a microorganism derived from Caldicellulosiruptor sp. BluCon014 or

c) a Caldicellulosiruptor sp. BluCon014mutant retaining the properties of BluCon014.

In another preferred embodiment, the Caldicellulosiruptor sp. microorganism is

a) Caldicellulosiruptor sp. BluCon016, deposited on April 09, 2019 under the accession number DSM 33097 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), IhIioIΐbhbΐGqbb 7B, 38124 Braunschweig (DE) by BluCon Biotech GmbH, Nattermannallee 1 , 50829 Cologne (DE), b) a microorganism derived from Caldicellulosiruptor sp. BluCon016 or

c) a Caldicellulosiruptor sp. BluCon016 mutant retaining the properties of BluCon016.

All strains listed above and in table 1 belong to the genus Caldicellulosiruptor and are strictly anaerobic, non-sporeforming, non-motile, gram-positive bacteria. Cells are straight rods 0.4- 0.5 pm by 2.0-4.0 pm, occuring both singly and in pairs. After 7 days incubation at 72 °C on solid medium with agar and cellulose as substrate both strains form circular milky colonies of 0.5 - 1 mm in diameter. Clearing zones around the colonies are produced indicating cellulose degradation.

The term "a microorganism" as used herein may refer to only one unicellular organism as well as to numerous single unicellular organisms. For example, the term "a microorganism of the genus Caldicellulosiruptor³' may refer to one single Caldicellulosiruptor bacterial cell of the genus Caldicellulosiruptor as well as to multiple bacterial cells of the genus Caldicellulosiruptor. The terms "a strain of the genus Caldicellulosiruptor³' and "a Caldicellulosiruptor cell" are used synonymously herein. In general, the term "a microorganism" refers to numerous cells. In particular, said term refers to at least 10³ cells, preferably at least 10⁴ cells, at least 10⁵ or at least 10⁶ cells.

As mentioned above lignocellolytic biomass according to the present disclosure can be but is not limited to grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, Napier grass, sugar-methoding residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, pressmud from sugar beet, cotton stalk, banana leaves, oil palm residues and lignocellulosic biomass material obtained through processing of food plants. In advantageous embodiments, the lignocellulosic biomass material is hardwood and/or softwood, preferably poplar wood. In advantageous embodiments, the lignocellulosic biomass material is a grass or perennial grass, preferably miscanthus.

In advantageous embodiments, the lignocellulosic biomass material is subjected to mechanical, thermochemical, and/or biochemical pretreatment. The lignocellulosic biomass material could be exposed to steam treatment. In further embodiments, the lignocellulosic biomass material is pretreated with mechanical comminution and a subsequent treatment with lactic acid, acetic acid, sulfuric acid or sulfurous acid or their respective salts or anhydrides under heat and pressure with or without a sudden release of pressure. In another embodiment, the lignocellulosic biomass material is pretreated with mechanical comminution and a subsequent treatment with either sodium hydroxide, ammonium hydroxide, calcium hydroxide or potassium hydroxide under heat and pressure with or without a sudden release of pressure.

In advantageous embodiments, the lignocellulosic biomass material is pretreated with mechanical comminution and subsequent exposure to a multi-step combined pretreatment process. Such multi-step combined pretreatment may include a treatment step consisting of cooking in water or steaming of the lignocellulosic biomass material at a temperature of 100 -

200 °C for a period of time in between 5 and 120 min. Suitable catalysts including but not limited to lactic acid, acetic acid, sulfuric acid, sulfurous acid, sodium hydroxide, ammonium hydroxide, calcium hydroxide or potassium hydroxide or their respective salts or anhydrides may or may not be added to the process. The process may further include a step comprising a liquid-solid separation operation, e.g. filtration, separation, centrifugation or a combination thereof, separating the process fluid containing partially or fully hydrolyzed and solubilized constituents of the lianocellulosic biomass material from the remainina insoluble Darts of the lignocellulosic biomass. The process may further include a step comprising washing of the remaining lignocellulosic biomass material. The solid material separated from solubilized biomass constituents may then be treated in a second step with steam under heat and pressure with or without a sudden release of pressure at a temperature of 150 - 250 °C for a period of time in between 1 and 15 min. In order to increase pretreatement effectiveness, a suitable catalyst including but not limited to lactic acid, acetic acid, sulfuric acid, sulfurous acid, sodium hydroxide, ammonium hydroxide, calcium hydroxide or potassium hydroxide or their respective salts or anhydrides may be added also to the second step.

In advantageous embodiments, the lignocellulosic biomass is milled before converted into biofuels like ethanol and/or carbon-based chemicals like lactic acid. In one embodiment, the lignocellulosic biomass is pretreated biomass from Populus sp, preferably pretreated with steam pretreatment or multi-step combined pretreatment. In another embodiment, the lignocellulosic biomass is pretreated biomass from any perennial grass, e.g. Miscanthus sp., preferably treated with steam pretreatment or multi-step combined pretreatment.

In advantageous embodiments the cells, strains, microorganisms may be modified in order to obtain mutants or derivatives with improved characteristics. Thus, in one embodiment there is provided a bacterial strain according to the disclosure, wherein one or more genes have been inserted, deleted or substantially inactivated. The variant or mutant is typically capable of growing in a medium comprising a lignocellulosic biomass material.

In another embodiment, there is provided a process for preparing variants or mutants of the microorganisms according to the present disclosure, wherein one or more genes are inserted, deleted or substantially inactivated as described herein.

In some embodiments one or more additional genes are inserting into the strains according to the present disclosure. Thus, in order to improve the yield of the specific fermentation product, it may be beneficial to insert one or more genes encoding a polysaccharase into the strain according to the invention. Hence, in specific embodiments there is provided a strain and a process according to the invention wherein one or more genes encoding a polysaccharase which is selected from cellulases (such as EC 3.2.1.4); beta-glucanases, including glucan-1 ,3 beta-glucosidases (exo-1 ,3 beta-glucanases, such as EC 3.2.1.58), 1 ,4-beta- cellobiohydrolases (such as EC 3.2.1 .91 ) and endo-l,3(4)-beta-glucanases (such as EC

3.2.1 .6); xylanases, including endo-l,4-beta-xylanases (such as EC 3.2.1.8) and xylan 1 ,4- beta-xylosidases (such as EC 3.2.1.37); pectinases (such as EC 3.2.1 .15); alpha- glucuronidases, alpha-L-arabinofuranosidases (such as EC 3.2.1 .55), acetylesterases (such as EC 3.1 .1.-1. acetvlxvlanesterases (such as EC 3.1 .1 .721. alDha-amvlases (such as EC 3.2.1.1 ), beta-amylases (such as EC 3.2.1.2), glucoamylases (such as EC 3.2.1.3), pullulanases (such as EC 3.2.1 .41 ), beta-glucanases (such as EC 3.2.1 .73), hemicellulases, arabinosidases, mannanases including mannan endo-l,4-beta-mannosidases (such as EC 3.2.1 .78) and mannan endo-l,6-alpha-mannosidases (such as EC 3.2.1.101 ), pectin hydrolases, polygalacturonases (such as EC 3.2.1.15), exopolygalacturonases (such as EC 3.2.1 .67) and pectate lyases (such as EC 4.2.2.10), are inserted.

In accordance with the present disclosure, a method of producing a fermentation product comprising culturing a strain according to the invention under suitable conditions is also provided.

The strains according to the disclosure are strictly anaerobic microorganisms, and hence it is preferred that the fermentation product is produced by a fermentation process performed under strictly anaerobic conditions. Additionally, the strain according to invention is an extremely thermophillic microorganism, and therefore the process may perform optimally, when it is operated at temperature in the range of about 40-95 degrees centigrade, such as the range of about 50-90 degrees centigrade, including the range of about 60-85 degrees centigrade, such as the range of about 65-75 degrees centigrade

For the production of certain fermentation products, it may be useful to select a specific fermentation process, such as batch fermentation process, including a fed-batch process or a continuous fermentation process. Also, it may be useful to select a fermentation reactor such as a stirred vessel reactor, an immobilized cell reactor, a fluidized bed reactor or a membrane bioreactor.

In accordance with the invention, the method is useful for the production of a wide range of fermentation products including acids, alcohols, ketones and hydrogen. Thus fermentation products such as ethanol, butanol, propanol, methanol, propanediol, butanediol, lactic acid, propionic acid, acetic acid, succinic acid, butyric acid, formic acid and acetone may be produced in accordance with the disclosure.

The expression "comprise", as used herein, besides its literal meaning also includes and specifically refers to the expressions "consist essentially of" and "consist of". Thus, the expression "comprise" refers to embodiments wherein the subject-matter which "comprises" specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which "comprises" specifically listed elements may and/or indeed does encompass further elements. Likewise, the expression "have" is to be understood as the expression "comprise", also including and specifically referring to the expressions "consist essentially of and "consist of".

The following methods and examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.

Methods and Examples

In the following examples, materials and methods of the present disclosure are provided including the determination of the properties of the microbial strains according to the present disclosure. It should be understood that these examples are for illustrative purpose only and are not to be construed as limiting this disclosure in any manner.

Description of Caldicellulosiruptor sp. strain BluCon006, Caldicellulosiruptor sp. strain BluCon014 and Caldicellulosiruptor sp. strain BluCon016

Caldicellulosiruptor sp. BluCon006, Caldicellulosiruptor sp. BluCon014 and Caldicellulosiruptor sp. BluCon016, which are listed in table 1 and table 2, are deposited on April 09^th, 2019 under the accession numbers DSM 33095, DSM 33096 and DSM 33097 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), IhIioIΐbheΐGqbb 7B, 38124 Braunschweig (DE) by BluCon Biotech GmbH, Nattermannallee 1 , 50829 Cologne (DE).

Description of Caldicellulosiruptor sp. DIB104C

Caldicellulosiruptor sp. DIB104C listed in Table 2 was deposited on March 15, 2012 under the accession number DSM 25774 according to the requirements of the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Inhoffenst^e 7B, 38124 Braunschweig (DE) by DIREVO Industrial Biotechnology GmbH, Nattermannallee 1 , 50829 Cologne (DE).

Table 2. Survey of Description of Caldicellulosiruptor sp

Example 1 : Cultivation of seed culture

All procedures for enrichment and isolation of the strains listed in table 2 employed anaerobic technique for strictly anaerobic bacteria (Hungate 1969). The strains listed in table 2 were cultivated at 70 °C with crystalline cellulose as substrate. The cells are cultured under strictly anaerobic conditions applying the following basic medium:

All ingredients except L-cysteine are dissolved in deionized water and the medium is flushed with nitrogen gas (purity 99,999%) for 20 min at room temperature and the pH-value is adjusted to 7.0 at room temperature with 5 M NaOH. Then a sterile stock solution of L-cysteine, which has been filtered into a nitrogen containing serum flasks, is added to the medium. The medium is then dispensed into serum flasks under nitrogen atmosphere and the vessels are tightly sealed. After autoclaving at 121 °C for 20 min pH-value should be in between 7.0 and 7.2. Subsequent to autoclaving, cultures are inoculated by injection of a seed culture through the seal septum and inoculated in an incubator at 70 °C for 24 to 48 hours.

Example 2: HPLC

Sugars and fermentation products were quantified by HPLC-RI using a Prominence LC-20AD HPLC (company Shimadzu) fitted with a Rezex ROA Organic Acid H+ (Phenomenex). The analytes were separated isocratically with 2.5 mM H₂SO₄ and at 65 °C.

Example 3: Fermentation

Batch experiments with strains listed in table 2, were performed by cultivation on the following fermentation medium with addition of 80 g/L of microcrystalline cellulose:

All ingredients are dissolved in deionized water and added into 2L fermentation vessels with stirrers and pH and temperature control (company BBI). The pH-value should be in between 6.8 and 7.0 (adjusted with NaOH or HCL).

After autoclaving at 121 °C for20 min the medium is flushed with nitrogen gas (purity 99,999%) for 20 min at room temperature to remove excess oxygen before L-cysteine is added as described above.

Temperature is controlled to 62 to 75°C and the pH-value is controlled to 5.8 to 7.2 throughout the fermentation by 5M NaOH. The fermentation is started by addition of a seed culture prepared as described in example 1. The results are presented in table 3. Table 3. Lactic acid, acetic acid and ethanol from microcrystalline cellulose by the strains listed in table 2 at different cultivation times.

The results of the HPLC analysis as described in example 2 show that BluCon006, BluCon014 and BluCon016 produce higher lactic acid concentrations and lower acetic acid and ethanol concentrations in the fermentation compared to the reference strain, DIB104C (see Table 3). This makes the fermentative production of L-lactic acid from cellulolytic substrates with BluCon006, BluCon014 and BluCon016 commercially more attractive compared to the process with DIB104C.

LIST OF ADDITIONAL REFRENCES:

Rainey FA, Donnison AM, Janssen PH, Saul D, Rodrigo A, Bergquist PL, Daniel RM, Stackebrandt E, Morgan HW. (1994) Description of Caldicellulosiruptor saccharolyticus gen. nov., sp. nov: an obligately anaerobic, extremely thermophilic, cellulolytic bacterium. FEMS Microbiol Lett. 120:263-266.

Sissons CH, Sharrock KR, Daniel RM, Morgan HW. (1987) Isolation of cellulolytic anaerobic extreme thermophiles from New Zealand thermal sites. Appl Environ Microbiol. 53:832-838.

Donnison AM, Brockelsby CM, Morgan HW, Daniel RM. (1989) The degradation of lignocellulosics by extremely thermophilic microorganisms. Biotechnol Bioeng. 33:1495-1499.

Hungate RE. (1969) A roll tube method for cultivation of strict anaerobes. In: Methods in Microbiology Eds. Norris JR and Ribbons DW. pp 118-132. New York: Academic Press.

Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD. (2003) Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 13:3497- 3500.

Kumar S, Tamura K, Jakobsen IB, Nei M. (2001 ) MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 17:1244-1245.

Claims

Claims

1. An isolated strain of the genus Caldicellulosiruptor sp, wherein the strain is selected from the group consisting of Caldicellulosiruptor sp. BluCon006 (DSMZ Accession number 33095), Caldicellulosiruptor sp. BluCon014 (DSMZ Accession number 33096) and Caldicellulosiruptor sp. BluCon016 (DSMZ Accession number 33097), microorganism derived therefrom, progenies or mutants thereof, wherein the mutants thereof retaining the properties of BluCon006, BluCon014 and/or BluCon016.

2. The isolated strain according to claim 1 , wherein the cell is capable of growing in a medium comprising biomass material having a carbon source and a nitrogen source, wherein the carbon source can be lignocellulosic biomass, cellulose, hemicellulose, starch, C5 and/or C6 sugars.

3. The isolated strain according to claim 2, wherein the lignocellulosic biomass material is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-methoding residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, cotton stalks, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, and softwood.

4. The isolated strain according to any one of claims 2 to 3, wherein the biomass like lignocellulosic biomass material is subjected to mechanical, thermochemical, and/or biochemical pretreatment.

5. The isolated strain according to claim 4, wherein pretreating the lignocellulosic biomass material comprises exposing the lignocellulosic biomass to steam treatment.

6. The isolated strain of claim 5, wherein pretreating the lignocellulosic biomass material comprises mechanical comminution and a subsequent treatment with sulfurous acid or its anhydride under heat and pressure with a sudden release of pressure.

7. The isolated strain according to any one of the preceding claims, which is capable of producing a fermentation product selected from the group consisting of an acid, an alcohol and hydrogen.

8. The isolated strain according to claim 7, wherein the acid is selected from the group consisting of lactic acid, propionic acid, acetic acid, succinic acid, malic acid, butyric acid and formic acid.

9. The isolated strain according to claim 7, wherein the acid is lactic acid like L-lactic acid and/or D-lactic acid or a salt or ester thereof.

10. The isolated strain according to any one of the preceding claims, wherein one or more genes have been inserted, deleted or substantially inactivated.

1 1. The isolated strain according to any of the preceding claims, which is BluCon006 (DSMZ Accession number 33095) or a mutant thereof, wherein the mutant thereof retaining the properties of BluCon006.

12. The isolated strain according to any of the preceding claims, which is BluCon014 (DSMZ Accession number 33096) or a mutant thereof, wherein the mutant thereof retaining the properties of BluCon014.

13. The isolated strain according to any of the preceding claims, which is BluCon016 (DSMZ Accession number 33097) or a mutant thereof, wherein the mutant thereof retaining the properties of BluCon016.

14. The according to any of the preceding claims, having one or more of the following characteristics: a) It is a microorganism of the genus Caldicellulosiruptor

b) it is a microorganism of the species Caldicellulosiruptor saccharolyticus c) in a DNA-DNA hybridization assay, it shows a DNA-DNA relatedness of at least 80%, preferably at least 90%, at least 95%, more preferred at least 98%, most preferred at least 99%, and most preferred at least 99,9% with one of the strains of claim 1 and have the same properties of one of the strains; and/or d) it displays a level of 16S rDNA gene sequence similarity of at least 98%, preferably at least 99%, at least 99,5% or at least 99,7%, more preferably 99,99% with one of the strains listed in claim 1 and have the same properties of one of the strains ; and/or e) it is capable of surviving and/or growing and/or producing a lactic acid or a salt or ester thereof at temperature conditions above 70 °C, in particular of above 72 °C.

15. A composition for converting lignocellulosic biomass comprising a strain according to any one of claims 1 to 14.

16. A microbial culture comprising a strain according to any one of claims 1 to 14.

17. Use of a microbial culture according to claim 16 for the production of lactic acid, a salt or an ester thereof, in particular to L-lactic acid and/or D-lactic acid or a salt or ester thereof..

18. A method for converting biomass material to a carboxylic acid comprising the step of contacting the biomass material with a microbial culture for a period of time at an initial temperature and an initial pH, thereby producing an amount of a carboxylic acid; wherein the microbial culture comprises an extremely thermophilic bacteria strain of the genus Caldicellulosiruptor, wherein the strain is selected from the group consisting of Caldicellulosiruptor sp. BluCon006 (DSMZ Accession number 33095), Caldicellulosiruptor sp. BluCon014 (DSMZ Accession number 33096) and Caldicellulosiruptor sp. BluCon016 (DSMZ Accession number 33097), microorganism derived therefrom, progenies or mutants thereof, wherein the mutants thereof retaining the properties of BluCon006, BluCon014 and/or BluCon016, and wherein the biomass material is converted in a single step process as part of a consolidated bioprocessing (CBP) system.

19. The method according to claim 18, wherein the period of time is 10 hours to 300 hours.

20. The method according to any one of claims 18 to 19, wherein the period of time is 50 hours to 200 hours, 80 hours to 160 hours.

21. The method according to any one of claims 18 to 20, wherein the initial temperature is in the range between 55 °C and 80 °C, optionally between 72 °C and 78 °C, in particular 70 °C.

22. The method according to any one of claims 18 to 21 , wherein the initial pH is between 5 and 9, optionally between 6 and 8.

23. The method according to any one of claims 18 to 22, wherein the carboxylic acid is lactic acid and/or salts or esters thereof, in particular L-lactic acid and/or D-lactic acid or a salt or ester thereof.

24. The method according to any one of claims 18 to 23, wherein the biomass material is lignocellulosic biomass material is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-methoding residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, and softwood, pressmud from sugar beet, cotton stalk, banana leaves, and lignocellulosic biomass material obtained through processing of food plants.

25. The method according to any one of claims 18 to 24, wherein said lignocellulosic biomass material is selected from the group consisting of corn stover, sugarcane bagasse, cotton stalks, switchgrass and.

26. The method according to any one of claims 18 to 25, wherein said lignocellulosic biomass material is a grass or perennial grass, optionally miscanthus.

27. The method according to any one of claims 18 to 26, wherein said lignocellulosic biomass material is subjected to mechanical, thermochemical, and/or biochemical pretreatment.

28. The method according to claim 27, wherein pretreating the lignocellulosic biomass material comprises exposing the lignocellulosic biomass to steam treatment.

29. The method according to claim 27, wherein pretreating the lignocellulosic biomass material comprises mechanical commination and a subsequent treatment with sulfurous acid or its anhydride under heat and pressure with a sudden release of pressure.

30. The method according to claim 27, wherein pretreating the lignocellulosic biomass comprises milling the lignocellulosic biomass.

31. The method according to any one of claims 27 to 29, wherein the lignocellulosic biomass material is pretreated in addition with enzymes, preferably cellulose and hemicellulose degrading enzymes.

32. The method according to claim 27 comprising: pretreating lignocellulosic biomass material, wherein the pretreatment is selected from the group consisting of: exposing the lignocellulosic biomass material to a steam treatment, mechanical comminution and a subsequent treatment of the lignocellulosic biomass material with sulfurous acid or its anhydride under heat and pressure with a sudden release of pressure, milling the lignocellulosic biomass material, exposing the lignocellulosic biomass material to cellulose and hemicellulose degrading enzymes, and contacting the lignocellulosic biomass material with a microbial culture for a period of time at an initial temperature and an initial pH, thereby producing an amount of a carboxylic acid, wherein the microbial culture comprises Caldicellulosiruptor sp. DIB004C, deposited as DSM 25177, and wherein the lignocellulosic biomass material is converted in a single step process as part of a consolidated bioprocessing (CBP) system.