Detman et al. Microbiome (2021) 9:158 https://doi.org/10.1186/s40168-021-01105-x RESEARCH Open Access Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production Anna Detman1, Daniel Laubitz2, Aleksandra Chojnacka1,3, Pawel R. Kiela2, Agnieszka Salamon4, Albert Barberán5, Yongjian Chen5, Fei Yang5, Mieczysław K. Błaszczyk3 and Anna Sikora1* Abstract Background: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. Results: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0–6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. Conclusions: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation. * Correspondence: [email protected] 1Institute of Biochemistry and Biophysics – Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Detman et al. Microbiome (2021) 9:158 Page 2 of 21 Keywords: Dark fermentation, Lactic acid bacteria, Microbial communities, Nutritional interactions, Lactate, Acetate, Butyrate Background bioreactors inhibits hydrogen production due to sub- Anaerobic digestion (AD) is a complex and multistep strate competition and/or excretion of bacteriocins that conversion of biomass to methane and carbon dioxide inhibit the growth of other bacteria. In homolactic fer- resulting from the metabolic activity and nutritional in- mentation, two molecules of pyruvate formed during teractions between many groups of microorganisms. It glycolysis are converted to lactate; in heterolactic fer- involves four main stages: hydrolysis of polymeric or- mentation, one molecule of pyruvate is converted to lac- ganic matter to monomers, acidogenesis, acetogenesis tate and the other to ethanol and carbon dioxide [8]. and methanogenesis [1–3]. This study focuses on the Substrate competition includes the replacement of processes during acidogenesis when the products of hy- hydrogen fermentation by lactic acid or ethanol fermen- drolysis are converted to non-gaseous short-chain fatty tation. A decrease in hydrogen production is observed acids (SCFAs), alcohols, aldehydes and the gases, carbon with a simultaneous increase of lactic acid and ethanol dioxide and hydrogen [4]. The dominant end-products concentrations among non-gaseous fermentation prod- of the fermentation process determine the type of fer- ucts [9–13]. mentation. A part of acidogenesis, hydrogen-yielding fer- On the other hand, cross-feeding of lactate involves mentations (dark fermentation, DF) are considered to be the conversion of lactate and acetate to butyrate, hydro- one of the most attractive alternative biological methods gen and carbon dioxide. It is a syntrophic nutritional of hydrogen (biohydrogen) production. The main types interaction recognized between lactate- and acetate- of hydrogen-yielding fermentation under mesophilic producing bacteria and butyrate producers. This conditions, especially from carbohydrate degradation, phenomenon of metabolic interactions between different are acetic/butyric acid fermentation (Clostridium-type bacterial groups was described in the gut of many ani- fermentation) and mixed-acid fermentation (Enterobac- mals including in the human gut. The end product, bu- teriaceae-type fermentation) [3, 5, 6]. Hydrogen can be tyrate, is a crucial molecule necessary in maintaining gut also produced during the transformation of products health and homeostasis and serves as an energy source other fermentation types. Fermentative biohydrogen pro- for the colonic epithelial cells [14–17]. Cross-feeding of duction offers the additional advantage of potentially lactate is also observed in DF bioreactors during the fer- using various waste streams from different industries as mentative conversion of organic substrates to biohydro- feedstock such as the sugar beet industry. Optimization gen in both mesophilic [18–23] and thermophilic of biohydrogen yield during acidogenesis is challenging conditions [24, 25]. and requires a better understanding of the microbial The studies on fermentation of agave bagasse, tequila community (MC) dynamics in bioreactors and their vinasse and wastewater from nixtamalization supplied metabolic substrate conversion along hydrogen- data supporting the thesis that cross-feeding of lactate is promoting pathways. In multispecies microbial commu- significant in the MCs of DF bioreactors. The authors nities, nutrient utilization is a complex process and fre- postulated that the conversion of lactate and acetate to quently involves competition and symbiotic cross- butyrate is the main pathway of biohydrogen production feeding (syntrophy) [7]. The former is when two or more [19–23]. A specific succession of bacteria was observed groups of microorganisms compete for a substrate that in batch experiments. In the first stage, the substrate usually leads to a temporary increase in the relative was processed to acetate and lactate, which were trans- abundance of one interacting partner over the other. formed to butyrate and hydrogen in the second stage. The latter is when the metabolic products yielded by The pH was an important factor ensuring balance and one microbe constitute energy resources or nutrients syntrophy between lactate and butyrate producers [19– supporting growth for another one. Therefore, the ana- 23]. Studies on thermophilic DF of sugarcane vinasse lysis of nutrient metabolism in fermentative processes also showed lactate as the primary substrate for biohy- should integrate the dynamics of MC composition with drogen production and the relevance of pH in this metabolic nutrient conversion. process [24, 25]. Cross-feeding of lactate was also ob- Lactic acid bacteria (LAB) are ubiquitous in the envir- served in reduced MCs composed of two components: onment; they accompany the plant biomass to anaerobic butyrate-producing Clostridium beijerinckii and lactate- bioreactors and constitute a relevant component of producer Yokenella regensburgei [26] or butyrate- acidogenic microbial communities (MCs). It is com- producing Clostridium butyricum and lactate-producer monly believed that the development of LAB in Sporolactobacillus vineae [27]. Furthermore, pure Detman et al. Microbiome