US 20140030778A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0030778 A1 SCHOLTEN et al. (43) Pub. Date: Jan. 30, 2014 (54) BACTERIAL STRAIN AND PROCESS FOR (30) Foreign Application Priority Data THE FERMENTATIVE PRODUCTION OF ORGANIC ACDS Aug. 14, 2008 (EP) .................................. O71145.742 (71) Applicant: BASF SE, LUDWIGSHAFEN (DE) Publication Classification (72) Inventors: EDZARD SCHOLTEN, MANNHEIM (51) Int. Cl. (DE): DIRK DAGELE, VOGTSBURG CI2P 7/46 (2006.01) (DE); STEPHAN HAEFNER, SPEYER CI2R I/OI (2006.01) (DE); HARTWIG SCHRODER, (52) U.S. Cl. NUSSLOCH (DE) CPC. CI2P 7/46 (2013.01); CI2R I/01 (2013.01) USPC ........................................ 435/145; 435/252.3 (73) Assignee: BASF SE, LUDWIGSHAFEN (DE) (57) ABSTRACT (21) Appl. No.: 14/045,096 The present invention relates to a novel bacterial strain des ignated DD1, which has the ability to produce organic acids, (22) Filed: Oct. 3, 2013 in particular Succinic acid (SA), which was originally isolated from bovine rumen, and is capable of utilizing glycerol as a Related U.S. Application Data carbon Source; and variant strains derived there from retain (62) Division of application No. 12/673,714, filed on Feb. ing said capability; as well as to methods of producing 16, 2010, now Pat. No. 8,574,875, filed as application organic acids, in particular Succinic acid by making use of No. PCT/EP2008/0067.14 on Aug. 14, 2008. said microorganism. Patent Application Publication Jan. 30, 2014 Sheet 1 of 9 US 2014/0030778 A1 Pasteurella festudinis CCUG 19802' Pasteurella pneumotropica NCTC 814 Haemophilus influenzae ATCC 3339t' Pasteureila muttocida ssp. muttockla NCTC 10322' Pasteurella canis ATCC 43326' solate DD "Mannheimia succiniciproducers MBEL 55E Mannheimia ruminalis CCUG 38470' Mannheimia glucosida CCUG 38457 Mannheinia haemolytica NCTC 9380 Mannheinia granulomatis AfCC 49244.' Mannheimia varigena CCUG 38482 Pasteureira battyae CCJG 2042 Haemophilus parahagmolyticus NCTC 84.79 Actinobacilius minor CCUG 38923' Pastaurella irehalosi NCTC 10641 Actinobacilus capsulatus CCUG 12396 Pasteureila aerogenes ATCC 27883' Pasieurella langaaensis ATCC 43328 Pasteurella anatis ATCC 43329' Pasteuralia gallinarum NCTC 11188' Eschercia cox-2 19. Fig.1 Patent Application Publication Jan. 30, 2014 Sheet 4 of 9 US 2014/0030778 A1 Fig.4 Patent Application Publication Jan. 30, 2014 Sheet 5 of 9 US 2014/0030778 A1 --30 gll -- 30 gill --50 gll -i- 50 gll -- 75 g/L -- 75 g/L 8 O 25 2O 1 5 O incubation time (h Fig.5 Patent Application Publication Jan. 30, 2014 Sheet 6 of 9 US 2014/0030778 A1 -e-34.5°Ciph 6.5 -o- 37°CpH 6.5 -a-39.5°CpH 6.5 -- 37°Ciph 6.0 -o- 37°CipH 6.5 -A-37°CipH 7.0 4. O 2 5 2 O 1 5 O incubation time (h) Fig.6 Patent Application Publication Jan. 30, 2014 Sheet 7 of 9 US 2014/0030778 A1 Es-SYSP a 5Y-A2Y15C -- 2Y-15C -e- 25C-e-25C 80 70 80 5 O 4. O 3. O O 8 6 24 32 40 48 incubation time hi Fig.7 Patent Application Publication Jan. 30, 2014 Sheet 8 of 9 US 2014/0030778 A1 ---or-5YSPEactic acid - - - - 5Y5P formic acid --5Y5Pacetic wuru Grrrr 5Y actic acid - - - - - 5Y formic acid re-A-5Y acetic acid incubation time gl.) Fig.8 - ---" --- O 2 3 4 5 8 7 8 9 10 11 12 13 14 15 inkubation time (h Fig.9 Patent Application Publication Jan. 30, 2014 Sheet 9 of 9 US 2014/0030778 A1 -glucose -O-succinic Aracetic H-formic --lactic 20 15 10 incubation time h Fig.10 US 2014/0030778 A1 Jan. 30, 2014 BACTERIAL STRAN AND PROCESS FOR Sumed than with common Sugars like glucose were achieved THE FERMENTATIVE PRODUCTION OF (Lee et al., 2001). However, the space time yield obtained ORGANIC ACDS with glycerol was substantially lower than with glucose (0.14 vs. 1.0 g SA/L h) and no crude glycerol was used. RELATED APPLICATIONS 0008. Only in a few cases anaerobic metabolisation of 0001. This application is a divisional of U.S. patent appli glycerol to fermentation products have been described. E. coli cation Ser. No. 12/673,714, filed Feb. 16, 2010, which is a is able to ferment glycerol under very specific conditions such National Stage filing under 35 U.S.C. S371 of PCT/EP2008/ 006714 filed Aug. 14, 2008, which claims priority to Euro as acidic pH, avoiding accumulation of the fermentation gas pean Patent Application No. 07114574.2, filed in Europe on hydrogen, and appropriate medium composition. (Dharmadi Aug. 17, 2008. The entire contents of each of the above et al 2006, Yazdani and Gonzalez 2007) Many microorgan applications are incorporated herein by reference in their isms are able to metabolize glycerol in the presence of exter entirety. nal electronacceptors (respiratory metabolism), few are able to do so fermentatively (i.e. in the absence of electron accep SUBMISSION OF SEQUENCE LISTING tors). The fermentative metabolism of glycerol has been stud 0002 The Sequence Listing associated with this applica ied in great detail in several species of the Enterobacteriaceae tion is filed in electronic format via EFS-Web and hereby family, Such as Citrobacter freundii and Klebsiella pneumo incorporated by reference into the specification in its entirety. niae. Dissimilation of glycerol in these organisms is strictly The name of the text file containing the Sequence Listing is linked to their capacity to synthesize the highly reduced prod Sequence Listing PF60072 3. The size of the text file is 32 uct 1,3-propanediol (1,3-PDO) (Dharmadi et al 2006). The conversion of glycerol into Succinic acid using Anaerobio KB, and the text file was created on Oct. 1, 2013. spirillum succiniciproducens has been reported (Lee et al. DESCRIPTION 2001). This study demonstrated that succinic acid could be produced with little formation of by-product acetic acid by 0003. The present invention relates to a novel bacterial strain designated DD1, which has the ability to produce using glycerol as a carbon Source, thus facilitating purifica organic acids, in particular Succinic acid (SA), which was tion of succinic acid. The highest yield was obtained by originally isolated from bovine rumen, and is capable of intermittently feeding glycerol and yeast extract, a strategy utilizing glycerol as a carbon source; and variant strains that resulted in the production of about 19 g/L of succinic derived there from retaining said capability; as well as to acid. It was noted, however, that unidentified nutritional com methods of producing organic acids, in particular Succinic ponents present in yeast extract were needed for glycerol acid by making use of said microorganism. fermentation to take place. 0009 Carboxylation reactions of oxaloacetate catalyzed BACKGROUND by the enzymes phopshoenolpyruvate carboxylase (PEPC), 0004. The fermentative production of succinic acid (SA) phopshoenolpyruvate carboxykinase (PEPCK) and pyruvate from biomass has already drawn much attention because said carboxylase (PycA) are utilizing HCO as a source of CO acid represents an important constituent of synthetic resins or (Peters-Wendisch, PG et al). Therefore hydrogencarbonate is a source of further valuable low-molecular chemical com sources such as NaHCO, KHCO. NHHCO and so on can pounds, in particular tetrahydrofuran (THF), 1,4-butanediol be applied to fermentation and cultivation media to improve (BDO), gamma-butyrolactone (GBL) and pyrrolidones (WO the availability of HCO, in the metabolisations of substrates A-2006/066839). to Succinic acid. The production of succinic acid from glucose 0005. A SA-producing bacterium isolated from bovine has not been found to be dependent on the addition of HCO rumen was described by Lee etal (2002a). The bacterium is a non-motile, non-spore-forming, mesophilic and capnophilic in the prior art so far. gram-negative rod or coccobacillus. Phylogenetic analysis 0010 Biomass production by anaerobic organisms is lim based on the 16S rRNA sequence and physiological analysis ited by the amount of ATP produced from fermentative path indicated that the strain belongs to genus Mannheimia as a ways. Biomass yield of glycerol in anaerobic organisms is novel species, and has been named Mannheimia succinicipro lower than of saccharides, like hexoses such as glucose, fruc ducens MBEL55E. Under 100% CO, conditions, it grows tose, pentoses such as Xylose arabinose or disaccharides Such well in the pH range of 6.0–7.5 and produces succinic acid, as sucrose or maltose (Lee et al. 2001, Dharmadi 2007). acetic acid and formic acid at a constant ratio of 2:1:1. When M. succiniciproducens MBEL55E was cultured anaerobi 0011 Saccharides, however, theoretically can be con cally under CO-saturation with glucose as carbon source, Verted to Succinic acid with a significantly lower yield than 19.8 g/L of glucose were consumed and 13.3 g/L of SA were glycerol due to the lower reduction state of saccharides com produced in 7.5h of incubation. pared to the polyol glycerol. The combination of saccharides 0006. A significant drawback of said organism is, how with glycerol have been found to function in an Succinic acid ever, its inability to metabolize glycerol, which, as a constitu producing anaerobic organisms (Lee et al. 2001), however ent of triacylglycerols (TAGs), becomes readily available e. without reaching Succinic acid titers beyond 28 g/l. g. as by-product in the transesterification reaction of Biodie 0012. There is, therefore, a need for further bacterial sel production (Dharmadi et al., 2006). strains, which have the ability to produce organic acids, in 0007. The fermentative production of succinic acid from particular SA, from glycerol. In particular, such strains glycerol has been described in the scientific literature (Lee et should produce said acids with high productivity from glyc al., 2001; Dharmadi et al., 2006) and with glycerol higher erol, especially if crude glycerol e.g.