Actinobacillus succinogenes

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Actinobacillus succinogenes

Taxonomy

• Eubacteria (Kingdom)
• Bacteria (Domain)
• Proteobacteria (Phylum)
• Gamma Proteobacteria (Class)
• Pasteurellales (Order)
• Pasteurellaceae ( Family)
• Actinobacillus (Genus)
• succinogenes (species)

Description

Actinobacillus succinogenes is a chemoorganotroph removed from bovine rumen. Single cells have been measured 0.63 by 0.63 by 1.21 micrometers, but they vary in shape between rod and coccobacillus (McKinlay, et. al., 2007; Guettler et. al., 1999). Colonies formed on TSB agar with CO₂ are circular, grey, and translucent. It is facultatively anaerobic, unlike many of the organisms found in the rumen that were obligate anaerobes. In actively growing broth cultures the cells are often interspersed, giving a 'Morse code' form, and chains are also common (Guettler et. al., 1999). It grew between 6.0 and 7.4 pH (Van der Werf et. al., 1997). Although it grew fine in air, Actinobacillus succinogenes is capnophilic and grows better at increased CO2 concetrations. Cells grown in CO₂ were the least pleomorphic, cells produced at less CO₂, were swollen and pleomorphic, and cells produced without CO₂ were unhealthy looking and pleomorphic (Guettler et. al., 1999). They are non-motile, non-spore-forming, gram-negative, and fermentative. Actinobacillus succinogenes is mesophilic because it grew well around 37 degrees Celsius and did not grow at all at 20 or 40 degrees Celsius. In an enrichment culture containing corn sheep liquor and MgCO₃, it produced succinic acid, acetic acid, formic acid, and other byproducts detected by HPLC (Guettler et. al., 1999). Glycogen, lipid, protein, and RNA levels, account for about 86% of the dry cell weight of Actinobacillus succinogenes (McKinlay et. al., 2007).

Metabolism and Waste Exchange

Actinobacillus succinogenes fermented glucose to produce the major products succinate, acetate, and formate, and ethanol as a minor product (Van der Werf et. al., 1997). A. succinogenes doesn’t have a full Krebs cycle (McKinlay et. al., 2005). It was not very difficult to please Actinobacillus succinogenes because it grew well without NAD or other special growth factors, and it grew rapidly on blood-free media. It can use many sugars, and it accumulates high concentrations of succinic acid (Guettler et. al., 1999). CO₂ and N₂ atmospheres were tested using Oxoid jars. It converted nitrate it nitrite. The formation of succinate requires the fixation of 1 mol of CO₂ per mol of succinate produced, so as the amount of CO₂ was increased the amount of succinate produced increased. When CO₂ levels were reduced more ethanol was formed (Van der Werf et. al., 1997). Yeast extract also increased growth (Guettler et. al., 1999). Succinate is produced during log phase (McKinlay et. al., 2005). PEPCK is the main CO₂ fixing enzyme in the A. succinogenes succinate production pathway, and when overexpressed succinate production was increased (Van der Werf et. al., 1997; Kim et. al., 2004). The glyoxylate cycle, the oxidative pentose phosphate pathway, and the Entner–Doudoroff pathway are a few of the pathways that have been described in detail (McKinlay et. al., 2007). The two main metabolic branches are the succinate producing branch and the formate, acetate, and ethanol producing branch. The growth rate of A. succinogenes was 1.3 to 1.4 times higher at a 25 mM bicarbonate concentration because both energy producing metabolic branches were stimulated (McKinlay et. al., 2005). No L- or D-lactate was detected. pH did not appear to affect the concentrations of fermentation products, but cell growth increased at lower pH (Van der Werf et. al., 1997). A. succinogenes uses the phosphoenolpyruvate (PEP) carboxykinase pathway to produce succinate. When hydrogen is added Actinobacillus succinogenes can use its’ hydrogenase to generate low-redox-potential electrons, so more phosphoenolpyruvate flows to oxaloacetate than to pyruvate. Oxaloacetate steers fermentation towards succinate production rather than lactacte, formate, acetate, or ethanol production (Zeikus et. al., 1999) The use of D-mannitol, D-sorbitol, and D-arabitol, carbohydrates reduced more then hydrogen, caused the formation of higher amounts succinate and ethanol (Van der Werf et. al., 1997).

Genome Structure

Its’ DNA is circular with a length of 2,319,663 nt (McKinlay et. al., 2007). DNA G + C content is approximately 45 mol % (Guettler et. al., 1999). The current count of known and putative peptidases in A. succinogenes is 70, and it contains 23 non-peptidase homologues (Rawlings et. al., 2008). They found that it is positive for catalase, oxidase, alkaline phosphatase and B-galactosidase (Guettler et. al., 1999). A. succinogenes differed only slightly from Bisaard Taxon 6 and Bisaard Taxon 10, and their 16S rRNA was roughly 5% different (Guettler et. al., 1999).

Significance

A. succinogenes has produced succinic acid at a concentration of about 110 g/l. (Zeikus et. al., 1999). If Genetic engineering techniques were used to make the possible theoretical succinate yield of two moles of succinate from one glucose and two CO₂, then succinate production could be cheaper and more environmentally friendly. For example, it can be used to produce food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to stimulate animal and plant growth. Succinic acid can be used to produce 1,4-butanediol, tetrahydrofuran, c-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters used in various industrial products in massive amounts. These materials are used to make clothing, carpets, nylon, and polyesters. They are also used in manufacturing plastics. (Zeikus et. al., 1999). In succinate fermentation, CO2 is fixed into succinate during glucose fermentation, which can therefore contribute to a cleaner environment. There are research designs for further improving the succinate fermentation economics. (Zeikus et. al., 1999). It is a moderate osmophile, likes high sugar concentrations, and has high tolerance to succinate salts, which is crucial to process requirements for succinate recovery (Zeikus et. al., 1999). Electrically reduced neutral red could be used as the reducing power instead of hydrogen. Even with hydrogen present it was still the soul source of reducing power, some mutant strains could not even grow without electrically reduced neutral red (Park et. al., 1999). Proton translocation is dependent on electrically reduced neutral red and/or H₂ as the electron donor and a sugar that can be converted to fumarate. The rate of fumarate production was twice as high with purified membranes and electrically reduced neutral red, and succinate production was inhibited with purified membranes and H₂ only. They are allowed to harness this energy because electrically reduced neutral red appears to replace menaquinone, a part of the membrane-bound fumarate reductase complex (Park and Zeikus, 1999).

References

• Guettler, M. V., Rumler, D., and Jain, M.K. 1999. Actinobacillus succinogenes sp. nov., a novel succinic-acid-producing strain from the bovine rumen. International Journal of Systematic Bacteriology. 49. 207-216.
• Kim, P., M. Laivenieks, C. Vieille, and J.G. Zeikus. 2004. Effect of overexpression of Actinobacillus succinogenes phosphoenolpyruvate carboxykinase on succinate production in Escherichia coli. Applied and Environmental Microbiology. 70. 1238-1241.
• McKinlay, J.B., Shachar-Hill, Y., Zeikus, J.G., and C. Vieille. 2007. Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13 C-labeled metabolic product isotopomers. Metabolic Engineering. 9. 177-192.
• McKinlay, J. B., Zeikus, J. G., and Vieille, C. 2005. Insights into Actinobacillus succinogenes fermentative metabolism in a chemically defined growth medium. Applied and Environmental Microbiology. 71. 6651-6656.
• Park, D. H., Laivenieks, M., Guettler, M. V., Jain, M. K., and Zeikus, J. G. 1999. Microbial utilization of electrically reduced neutral red as the sole electron donor for growth and metabolite production. Applied and Environmental Microbiology. 65. 2912-2917.
• Park, D. H., and Zeikus, J. G. 1999. Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation. Journal of Bacteriology. 181. 2403-2410.
• Rawlings, N.D., Morton, F.R., Kok, C.Y., Kong, J.,and Barrett, A.J. 2008. MEROPS: the peptidase database. Nucleic Acids Res 36, D320-D325 available at http://merops.sanger.ac.uk/cgi-bin/speccards?sp=sp004095&type=peptidase.
• Van der Werf, M. J., Guettler, M. V., Jain, M. K., and Zeikus, J. G. 1997. Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Archives of Microbiology. 167. 332-342.
• Zeikus, J. G., Jain, M.K., Elankovan, P. 1999. Biotechnology of succinic acid production and markets for derived industrial products. Applied and Environmental Microbiology. 51. 545-552.