Cell factories converting lactate and acetate to butyrate: Clostridium butyricum and microbial communities from dark fermentation bioreactors

Abstract

BackgroundInteractions between microorganisms during specific steps of anaerobic digestion determine metabolic pathways in bioreactors and consequently the efficiency of fermentation processes. This study focuses on conversion of lactate and acetate to butyrate by bacteria of dark fermentation. The recently recognized flavin-based electron bifurcation as a mode of energy coupling by anaerobes increases our knowledge of anaerobic lactate oxidation and butyrate formation.ResultsMicrobial communities from dark fermentation bioreactors or pure culture of Clostridium butyricum are able to convert lactate and acetate to butyrate in batch experiments. The ability of C. butyricum to transform lactate and acetate to butyrate was shown for the first time, with ethanol identified as an additional end product of this process. A search for genes encoding EtfAB complexes and their gene neighbourhood in C. butyricum and other bacteria capable of lactate and acetate conversion to butyrate as well as butyrate-producers only and the lactate oxidiser Acetobacterium woodii, revealed that the Etf complexes involved in (i) lactate oxidation and (ii) butyrate synthesis, form separate clusters. There is a more extent similarity between Etf subunits that are involved in lactate oxidation in various species (e.g. A. woodii and C. butyricum) than between the different etf gene products within the same species of butyrate producers. A scheme for the metabolic pathway of lactate and acetate transformation to butyrate in C. butyricum was constructed.ConclusionsStudies on the conversion of lactate and acetate to butyrate by microbial communities from dark fermentation bioreactors or Clostridium butyricum suggest that a phenomenon analogous to cross-feeding of lactate in gastrointestinal tract also occurs in hydrogen-yielding reactors. A scheme of lactate and acetate transformation pathway is proposed, based on the example of C. butyricum, which employs flavin-based electron bifurcation. This process utilizes electron-transferring flavoprotein (Etf) complexes specific for (i) lactate oxidation and (ii) butyrate formation. Phylogenetic analysis revealed that such complexes are encoded in the genomes of other bacteria capable of lactate and acetate conversion to butyrate. These findings contribute significantly to our understanding of the metabolic pathways and symbiotic interactions between bacteria during the acidogenic step of anaerobic digestion.

Dynamics of Dark Fermentation Microbial Communities in the Light of Lactate and Butyrate Production

“…Our previous work demonstrated that dark fermentation microbial communities fed molasses under mesophilic conditions are able to convert lactate and acetate to butyrate in batch experiments [18]. Here we propose a logical continuation and extension of the previously published studies aimed at (i) recognition of biodiversity and dynamics of dark fermentation microbial communities able and unable to convert lactate and acetate to butyrate and (ii) de nition of the conditions for the process of transformation.…”

“…Cross-feeding of lactate was also observed in reduced microbial communities composed of two components: butyrateproducing Clostridium beijerinckii and lactate-producer Yokenella regensburgei [26]. Furthermore, pure cultures of Clostridium acetobutylicum [27], Butyribacterium methylotrophicum [28], Clostridium diolis [29], Clostridium buttyricum [18] and Clostridium tyrobutyricum [30,31] anaerobically grown in media with acetate and lactate as exclusive carbon sources produced carbon dioxide, hydrogen and butyrate.…”

“…Tests on the transformation of lactate and acetate to butyrate were conducted in static batch experiments, analogous to those described previously [18], in 250-ml Erlenmayer asks for 18 days in a Vinyl Anaerobic Chamber (Coy Laboratory Products, Inc.) without shaking at 30°C. Five-milliliter samples of microbial community taken from the dark fermentation hydrogen producing packed bed reactor described previously was used as inoculum.…”

“…The end product, butyrate, is a crucial molecule necessary in maintaining gut health, homeostasis, and serving as an energy source for the colonic epithelial cells [14][15][16][17]. Cross-feeding of lactate is also observed in dark fermentation bioreactors during fermentative conversion of organic substrates to biohydrogen both in mesophilic [18][19][20][21][22][23] and termophilic conditions [24,25].…”

Unravelling lactate-acetate conversion to butyrate by intestinalAnaerobutyricumandAnaerostipesspecies

“…Evidence for a molecular mechanism of conversion of lactate to acetate 76 was first demonstrated in the acetogenic model organism Acetobacterium woodii, where for 77 every molecules of lactate converted to acetate only 1.5 molecules of ATP were generated 78 (Weghoff et al, 2015). This mechanism, so-called electron confurcation, was suggested to be 79 wide spread in other anaerobic lactate utilizers and hypothesized to be present in intestinal 80 butyrogenic bacteria (Weghoff et al, 2015;Louis and Flint, 2017;Detman et al, 2019). 81 Therefore, using a proteogenomic approach we aimed to investigate whether intestinal bacteria 82 also employ this electron confurcation to convert lactate to butyrate with a focus on A.…”