Ethanologenesis from glycerol by the gut acetogen Blautia schinkii.

The human gut is an anoxic environment that harbours a multitude of microorganisms that not only contribute to food digestion. The microbiome is also involved in malfunctions such as diseases, inflammation processes or development of obesity, but it is also involved in processes that increase the human well-being. Both, the good and the bad, are mediated by fermentation end products of bacterial metabolism, among others. However, despite a steadily growing knowledge of 'who lives out there', little in known of 'what do they do out there'. The genus Blautia is commonly found in the gut and associated with human well-being, but the exploration of their metabolic potential has just started. We demonstrate that B. schinkii grows on glycerol by producing acetate and ethanol. Transcriptome studies and biochemical analyses revealed a glycerol dehydrogenase and dihydroxyacetone kinase that funnel the substrate into glycolysis. Consequently, cells also grew on dihydroxyacetone. Cells could be adapted to grow at high (up to 1.5 M) glycerol concentrations but then only ethanol was formed. Ethanol production from glycerol is not only of relevance for the human host but also for potential bioindustrial production of bioethanol from waste glycerol.

Trischler R ，Poehlein A ，Daniel R ，Müller V ... -  《-》

3-Hydroxypropionate production from myo-inositol by the gut acetogen Blautia schinkii.

Species of the genus Blautia are not only abundant in the human gut but also contribute to human well-being. Our study demonstrates that the gut acetogen Blautia schinkii can grow on myo-inositol. We identified the pathway of myo-inositol degradation through a combination of physiological and biochemical studies, genome-wide expression profiling and homology searches. Initially, myo-inositol is oxidized to 2-keto-myo-inositol. This compound is then metabolized by a series of enzymes - a dehydratase, hydrolase, isomerase and kinase - to form 2-deoxy-5-keto-d-gluconic acid 6-phosphate. This intermediate is split by an aldolase into malonate semialdehyde and dihydroxyacetone phosphate, which is an intermediate of the Embden-Meyerhof-Parnas pathway. This pathway leads to the production of pyruvate and, subsequently, acetate. Concurrently, malonate semialdehyde is reduced to 3-hydroxypropionate (3-HP). The genes responsible for myo-inositol degradation are clustered on the genome, except for the gene encoding the aldolase. We identified the putative aldolase Fba_3 and 3-HP dehydrogenase Adh1 encoding genes bioinformatically and verified them biochemically using enzyme assays with heterologously produced and purified protein. The major fermentation end products were 3-HP and acetate, produced in similar amounts. The production of the unusual fermentation end product 3-HP is significant not only for human health but also for the potential bioindustrial production of this highly desired compound.

Trischler R ，Rustler SM ，Poehlein A ，Daniel R ，Breitenbach M ，Helfrich EJN ，Müller V ... -  《-》

Glycerol fermentation by (open) mixed cultures: a chemostat study.

Glycerol is an important byproduct of bioethanol and biodiesel production processes. This study aims to evaluate its potential application in mixed culture fermentation processes to produce bulk chemicals. Two chemostat reactors were operated in parallel, one fed with glycerol and the other with glucose. Both reactors operated at a pH of 8 and a dilution rate of 0.1 h(-1). Glycerol was mainly converted into ethanol and formate. When operated under substrate limiting conditions, 60% of the substrate carbon was converted into ethanol and formate in a 1:1 ratio. This product spectrum showed sensitivity to the substrate concentration, which partly shifted towards 1,3-propanediol and acetate in a 2:1 ratio at increasing substrate concentrations. Glucose fermentation mainly generated acetate, ethanol and butyrate. At higher substrate concentrations, acetate and ethanol were the dominant products. Co-fermentations of glucose-glycerol were performed with both mixed cultures, previously cultivated on glucose and on glycerol. The product spectrum of the two experiments was very similar: the main products were ethanol and butyrate (38% and 34% of the COD converted, respectively). The product spectrum obtained for glucose and glycerol fermentation could be explained based on the general metabolic pathways found for fermentative microorganisms and on the metabolic constraints: maximization of the ATP production rate and balancing the reducing equivalents involved.

Temudo MF ，Poldermans R ，Kleerebezem R ，van Loosdrecht MC ... -  《-》

Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production.

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable 'coproduct'. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2-propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol-fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h-1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l-1 ) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell-free extracts demonstrated that glycerol is degraded via glyceraldehyde-3-phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.

Patil Y ，Junghare M ，Müller N 《Microbial Biotechnology》

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli.

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.

Cintolesi A ，Clomburg JM ，Rigou V ，Zygourakis K ，Gonzalez R ... -  《-》