Anaerobic Fermentation of Glycerol in Paenibacillus macerans: Metabolic Pathways and Environmental Determinants

Paenibacillus macerans is one of the species with the broadest metabolic capabilities in the genus Paenibacillus, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several P. macerans strains are capable of anaerobic fermentation of glycerol. One of these strains, P. macerans N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h⁻¹ and was chosen for further characterization. The use of U-¹³C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO₂-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in P. macerans N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by P. macerans N234A are of paramount importance for the production of fuels and chemicals.Although many microorganisms can metabolize glycerol in the presence of external electron acceptors (respiratory metabolism), few are able to do so fermentatively (i.e., in the absence of electron acceptors). Fermentative metabolism of glycerol has been reported in species of the genera Klebsiella, Citrobacter, Enterobacter, Clostridium, Lactobacillus, Bacillus, Propionibacterium, and Anaerobiospirillum but has been studied more extensively in a few species of the family Enterobacteriaceae, namely, Citrobacter freundii and Klebsiella pneumoniae (6, 9). Glycerol fermentation in these organisms is mediated by a two-branch pathway, which results in the synthesis of the glycolytic intermediate dihydroxyacetone (DHA) phosphate (DHAP) and the fermentation product 1,3-propanediol (1,3-PDO) (Fig. ​(Fig.1A)1A) (6). In the oxidative branch, glycerol is dehydrogenated to DHA by a type I NAD-linked glycerol dehydrogenase (glyDH). DHA is then phosphorylated by ATP- or phosphoenolpyruvate (PEP)-dependent DHA kinases (DHAKs) to generate DHAP. In the parallel reductive branch, glycerol is dehydrated by glycerol dehydratase, and 3-hydroxypropionaldehyde (3-HPA) is formed. 3-HPA is then reduced to the major fermentation product 1,3-PDO by an NADH-linked 1,3-PDO dehydrogenase (1,3-PDODH), thereby regenerating NAD⁺ (Fig. ​(Fig.1A).1A). Organisms that lack the capacity to synthesize 1,3-PDO have been deemed unable to utilize glycerol in a fermentative manner (6, 9, 10). The metabolism of glycerol in these organisms is thought to require an electron acceptor and takes place through a respiratory pathway that involves a glycerol kinase and two respiratory (aerobic and anaerobic) glycerol-3-phosphate dehydrogenases (G3PDHs) (6, 7, 24, 29, 35, 38) (Fig. ​(Fig.1B).1B). A recent development in this area is the finding that Escherichia coli, an organism that is unable to produce 1,3-PDO, can indeed ferment glycerol in the absence of external electron acceptors (15, 26). In this model, synthesis of the fermentation products 1,2-PDO and ethanol enables glycerol fermentation by facilitating redox balance and ATP generation, respectively (Fig. ​(Fig.1C)1C) (15). A type II glyDH and a PEP-dependent DHAK mediate the conversion of glycerol to glycolytic intermediates. glyDH also catalyzes the last step in the synthesis of the key fermentation product 1,2-PDO (Fig. ​(Fig.1C1C).Open in a separate windowFIG. 1.Glycerol metabolism in bacteria. (A) 1,3-PDO model for the fermentative utilization of glycerol. (B) Respiratory metabolism of glycerol (i.e., metabolism in the presence of an electron acceptor). (C) 1,2-PDO-ethanol model for the fermentative utilization of glycerol. Dashed lines indicate multiple steps. glyD, glycerol dehydratase; glyDH-I, type I glyDH; GK, glycerol kinase; ae-G3PDH, aerobic G3PDH; an-G3PDH, anaerobic G3PDH; QH₂, reduced quinone; glyDH-II, type II glyDH; FHL, formate hydrogen lyase; ADH, alcohol/acetaldehyde dehydrogenase.Paenibacillus macerans, previously called Bacillus macerans and Bacillus acetoethylicum, is a gram-positive, spore-forming bacterium belonging to the genus Paenibacillus (17) that is capable of fermentative metabolism of hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose (33, 39, 40, 41). Glycerol, however, is considered a nonfermentable carbon source for P. macerans. The “nonfermentable status” of glycerol has been used to determine whether certain electron acceptors, such as fumarate, trimethylamine N-oxide, nitrate, and nitrite, can mediate anaerobic respiration in this organism (34).In this study we found that several P. macerans strains are able to ferment glycerol in the absence of external electron acceptors. The fermentation of glycerol by one of these strains, P. macerans N234A, occurred at high metabolic rates and in the absence of an active 1,3-PDO pathway. Therefore, the environmental and metabolic determinants of glycerol fermentation in P. macerans N234A were investigated.