General characteristics of the microbial communities in static batch experiments
To examine the capabilities of dark fermentation microbial communities to convert lactate and acetate to butyrate, five independent static batch experiments in three replicates were performed. Each one was inoculated with the same community derived from hydrogen-producing packed-bed reactors described previously [18, 46]. The experiments provided different carbon sources as shown in Table 1: molasses (Experiment M), molasses supplemented with lactate (Experiment ML), molasses supplemented with lactate and acetate (Experiment MLA), sucrose supplemented with lactate and acetate (Experiment SLA) and lactate and acetate (Experiment LA). The batch experiments were maintained for 18 days and passaged every 3 days.
Bacterial growths measured by OD600nm of the digestive liquids after every passage are presented in Table 1. The results clearly show that sucrose stimulates bacterial growth. The densities were higher (OD600nm after every three days ≈ 2-3) when bacteria grew on the media containing sucrose (either from molasses or used a pure additive; Experiments M, ML, MLA and SLA) compared to Experiment LA when lactate and acetate were provided as an exclusive carbon source (OD600nm after every three days ≈ 1), 0.001 < p < 0.005 between LA group and any other group (Tukey's HSD test; Table 1, Additional file 1). Interestingly, in comparison to molasses and lactate alone (Experiment ML), addition of acetate in Experiment MLA increased bacterial growth on days 6, 9, and 12 (p < 0.05, Tukey's HSD test). Differences in bacterial growth were also found on day 9 between Experiments M and ML as well as between Experiments M and SLA (p < 0.05, Tukey's HSD test).
Biodiversity and microbial changes in all the experiments were analysed by sequencing of the 16S V4 amplicon profiling. Total of 119 samples were sequenced in one MiSeq run, and 7,431 ASVs were detected. After chimera identification and removal, 93.15% ASVs remained. 29 samples from an unrelated project were filtered out, and the remaining 90 samples were further analyzed. For detailed taxonomic assignments see Additional file 2. All negative controls for the V4 amplification by PCR (collection day 0 for each experiment) did not show any amplification and these controls were removed from analysis during the quality control steps due to insufficient number of reads. Alpha diversity analysis revealed that the microbial communities are moderately rich in taxa, and that communities grown in media supplemented with molasses only or molasses and lactate (Experiments M and ML) had the lowest diversity as compared to the inoculum alone or to other groups (Figure 1, Additional file 3). Taxonomic composition of each experimental microbial community (Figures 2 and 3) was analysed in the reference to its metabolites, i.e. non-gaseous fermentation products and pH of the digestive liquids (Figures 3 and 4, Table 2).
Analysis of metabolites and microbial community composition after the initial 3 days of fermentation
After the intial 3 days of fermentation, we found no statistically significant differences in the concentration of the analyzed non-gaseous fermentation products among the batch experiments where growth media contained sucrose (either as a component of molasses or pure sucrose; Experiments M, ML, MLA and SLA). The concentration of butyrate was low (<1 g/L; Table 2, Figure 4, Additional file 4). The main fermentation products were ethanol (5.5 – 6.7 g/L) and lactate. After the initial 3 days of fermentation with molasses only (Experiment M), lactate concentration was the lowest at 2.6 g/L. In the case of Experiments ML, MLA and SLA, the concentration of lactate in the digestive liquids (10.4 – 11.2 g/L) was the sum of that in the media and as a product of sucrose fermentation. Fermentation of sucrose (Experiments M, ML, MLA, and SLA) resulted in similar pH of the digestive liquids (4.0, 4.4, 4.6 and 4.6, respectively). Detailed data for each metabolite and time point are presented in Table 2 and Additional file 4.
After the initial 3 days of fermentation, communities grown on the media containing molasses (Experiments M, ML, and MLA) were composed primarily of Lactobacillus (38.3% ± 15.6, 34.7% ± 4.4, 38.3% ± 5.1, respectively), Fructobacillus (26.1% ± 17.6, 27.8% ± 12.0, 21.5% ± 5.8, respectively), Bifidobacterium (12.2% ± 3.0, 20.1% ± 7.8, 24.7% ± 1.4, respectively), Leuconostoc (20.7% ± 3.3, 11.7% ± 3.2, 10.6% ± 3.9, respectively), with a smaller proportion of Clostridium sensu stricto 1 (0.6% ± 0.5, 3.3% ± 2.2, 6.7% ± 1.2, respectively). Compared to Experiments M, ML and MLA, microbial community grown with pure sucrose (Experiment SLA) showed lower contribution of Bifidobacterium (4.0 % ± 3.4; p = 0.036, p = 0.05, and p = 0.003, respectively), Lactobacillus (6.5% ± 5.8; p = 0.057, p = 0.003, p = 0.03, respectively), and Leuconostoc (2.6% ± 1.3; p = 0.005, p = 0.025, p = 0.062, respectively), and the community became dominated by Clostridium sensu stricto 1 (51.1% ± 7.5; p = 0.007, p = 0.005, p = 0.008, respectively). However, at this stage of fermentation, this genus did not appear to correlate with butyrate production. The relative abundance of Fructobacillus (28.9% ± 3.2) in SLA community was comparable to the communities in M, ML, and MLA (p = 0.81, p = 0.89, p = 0.14, respectively) (Figures 2 and 3, Additional file 2).
The dynamics of the fermenation process were followed over four additional passages until 18 days post-inoculation and showed considerable differences between experimental groups. These are discussed in detail in the following sections.
Dynamics of fermentation processes with molasses only (Experiment M)
When molasses were fermented without exogenous SFCAs, pH of the digestive liquids after six days dropped below 4 and remained in the 3.6 – 3.9 range (Table 2, Figure 4, Additional file 4). During the whole experiment, the main non-gaseous fermentation products were ethanol and lactate. Lactate was the only metabolite that significantly changed over time (ANOVA, p = 0.000015), with a gradual increase from day 3 to day 18 (2.6, 3.9, 5.2, 6.3, 5.2 and 5.6 g/L, respectively). The results of the detailed statistical comparisons are presented in Table 2 and Additional file 1. Between 6-18 days, the concentrations of ethanol remained relatively stable at 5.6, 3.8, 3.1, 4.1 and 4.9 g/L. The concentrations of butyrate and acetate were low (≤1 g/L) throughout the experiment (Table 2, Figure 4).
The overall biodeivrsity was low compared to the inoculum and showed changes over time, although without a clear trend (Figure 1). Microbial community was dominated by Lactobacillus (54.2% ± 7.8, 69.8% ± 5.3, 71.7 ± 11.6, 61.3 ± 11.9 and 72.6 ± 12.0, respectively after 6, 9, 12, 15 and 18 days), with Bifidobacterium as the second most abundant genus (11.3% ± 3.7, 15.7 % ± 6.0, 19.5% ± 9.6, 10.1% ± 2.2 and 19.7% ± 7.9, respectively after 6, 9, 12, 15 and 18 days) (Figure 2). The Leuconostoc genus was a significant component of the microbial community on day 3 and 6 (20.7% ± 3.3 and 25.8 % ± 9.7, respectively), but its relative abundance started to decline on day 9 and onward to eventually constitute a minor genus (10.1% ± 8.1 on day 6, 2.1% ± 1.5 on day 6, and < 1% on days 15 and 18). The relative abundance of the genera Clostridia sensu stricto was generally low (5-6%), among them Clostridium sensu stricto 12 dominated (4.8% ± 3.8%, 5.3 % ± 2.8 %, 3.8% ± 1.3%, 4.6% ± 0.5% and 3.5% ± 1.3%, respectively after 6, 9, 12, 15 and 18 days).
Dynamics of fermentation processes with molasses supplemented with lactate (ML)
After the addition of lactate, pH of the digestive liquids after the first passage (days 6-18) remained in the range 4.4–4.6. The concentrations of ethanol decreased steadily (ANOVA, p = 0.004) from 6.7 g/L on day 3 to 3.4, 2.7 and 2.8 g/L on days 9, 15 and 18, respectively. Concentration of lactate varied over time (ANOVA, p = 0.006). It peaked on day 6 at 12 g/L and decreased to 6.3 and 6.8 g/L on days 15 and 18, respectively. The concentration of butyrate gradually increased (ANOVA, p = 0.006) from 0.03 g/L on day 3 to 3.2 g/L on days 15 and 18. The concentration of acetate remained low and steady (≤1 g/L) throughout the experiment (Table 2, Figure 2, Additional files 1 and 4).
Supplementation of molasses with lactate as a source of carbon overall did not change the richness of the bacterial community, which remained similar to that in Experiment M, with molasses as a sole source of carbon. After inoculation, we observed a transient drop of richness until day 12 followed by the restored number ASVs to the original level on day 15 follwed by a not statistically significant decline on day 18 (Figure 1). Similar to the Experiment M, the microbial communities were dominated with Lactobacillus (49.9% ± 5.1, 67.7% ± 15.9, 74.5% ± 9.7, 55.1% ± 8.4 and 56.7% ± 7.1, after 6, 9, 12, 15 and 18 days, respectively) and Bifidobacterium (11.4% ± 0.8, 13.9% ± 5.6, 15.7% ± 6.0, 22.8% ± 2.3 and 24.7% ± 6.2, after 6, 9, 12, 15 and 18 days, respecively). The relative contribution of the Leuconostoc genus decreased over time (12.5% ± 6.3%, 7.4% ± 7.0% and <1%, on day 6, 9 days and on and beyond day 12; p = 0.016, Kruskall-Walllis test). The Fructobacillus genus followed a similar pattern (13.6% ± 12.8%, 4.7% ± 5.7% and <1%, on day 6, 9 days and on and beyond day 12; p = 0.015, Kruskall-Walllis test). Relative abundance of Clostridium sensu stricto 12 genus showed a slight decline from day 3 to 6, followed by recovery and modest expansion (11.9% ± 13.8%, 6.1% ± 2.7%, 8.5% ± 4.1%, 15.2% ± 5.4% and 13.4% ± 3.1%, on day 6, 9, 12, 15 and 18, respecitively; p = 0.06, Kruskall-Walllis test). Corresponding with increased butyrate synthesis on days 15 and 18, the microbial community showed relative expansion of Lactobacillus, Clostridium sensu stricto 12 and Bidifidobacterium genera (Figure 2). Among minor genera, contribution of Caproiciproducens increased to 1.3% ± 0.9% and 3.2% ± 1.2%, after 15 and 18 days, respectively (p = 0.011, Kruskall-Walllis test).
Dynamics of fermentation processes with molasses or sucrose supplemented with lactate (MLA and SLA)
In the MLA and SLA Experiments, the media contained molasses as a source or sucrose or pure sucrose, both supplemented with lactate and acetate. These two experiments are described together due to similar tendencies observed, which reflects the dominant efect of lactate/acetate supplementation over the source of sucrose (Figures 3 and 4, Table 2, Additional files 1 and 4). In the MLA Experiment, the pH of the digestive liquids changed from 4.6 on day 3 to 5.4 and 5.5 on days 6 and 9 (p = 0.0002, p = 0.0002, respectively; Tukey's HSD test). In the SLA Experiment, the pH changed from 4.6 on day 3 to 6.2 and 5.8 on days 6 and 9 (p=0.002, p = 0.02, respectively; Tukey's HSD test). In the MLA Experiment, the pH increase was associated with increased butyrate concentration, from 0.2 g/L on day 3 to 7.1 and 6.5 g/L on days 6 and9 (p = 0.002, p = 0.004, respectively; Tukey's HSD test). the SLA Experiment, the concentration of butyrate increased from 0.9 g/L on day 3 days to 7.0 g/L and 8.0 g/L on days 6 and 9 (p = 0.0004, p = 0.0003, respectively; Tukey's HSD test). During longer fermentation (days 12, 15 and 18), butyrate remained an abundant fermentation product and pH was maintained at ca. 5. On day 6 and onward, ethanol concentration decreased and it became a minor metabolite compared to samples collected on day 3 in either MLA or SLA Experiment (p = 0.0002, Tukey's HSD test) .
The biodiversity measured by richness index in both MLA and SLA Experiments increased over time (Figure 1). The microbial communities (Figures 2 and 3) associated with the highest butyrate production in Experiments MLA and SLA, were dominated by Bifidobacterium (16-30% for both Experiments), Clostridium sensu stricto 12 (20-30% for both Experiments), Lactobacillus (40-50% for Experiment MLA and 20-30% for Experiment SLA), Prevotella (up to 6% in Experiment MLA and above 15% in Experiment SLA on day 12 and 18), and Caproiciproducens. In the MLA and SLA Experiments, we detected a higher contribution of Prevotella in comparison to the other culture conditions. In the MLA Experiment, the contribution of the Leuconostoc and Fructobacillus genera decreased over time, from 6.6% ± 2.2 and 5.3% ± 0.4 after 6 days, respectively, to below 1% from day 9 onward. In the SLA Experiment, Leuconostoc was a minor genus whereas Fructobacillus also decreased in time (23.2% ± 2.5, 18.0% ± 4.7, 5.1% ± 1.8, 1.2% ± 0.3 and 2.1% ± 2.9) on days 6, 9, 12, 15 and 18, respectively (p=0.007 and p=0.009 for Leuconostoc and Fructobacillus, respectively; Kruskall-Wallis test). In both experiments, we observed an increasing contribution of Caproiciproducens genus (MLA: <1%, 2.1% ± 1.8%, 5.4% ± 0.6%, 4.6% ± 1.8%, on days 9, 12, 15, 18, respectively; SLA: <1%, 3.4% ± 2.4%, 7.9% ± 5.2%, 2.3% ± 2.0%, 5.0% ± 1.6%, on days 6, 9, 12, 15 and 18, respectively; p=0.02 for either genus, Kruskall-Wallis test).
Clustering analysis of each experiment revealed that in some individual experimental replicates differed from the other counterparts and were more similar to those from other experiments. For example, the replicate B after the 3rd passage (9 days) from the Experiment MLA grouped with the samples collected after the 3rd passage (9 days) from the Experiment ML. Other examples are replicates from the Experiment SLA after the 4th, 5th and 6th passages, respectively, 12, 15 and 18 days (Figure 3). We had no logical explanation and thus no reason to exclude these replicates from analysis.
Dynamics of fermentation of lactate and acetate as the main carbon sources (LA)
A distinct scenario occured for Experiment LA when the source of carbon was limited to lactate and acetate (Table 2 and Additional files 1 and 4). The pH of the digestive liquids was maintained at approximately 7, and the lowest reached value of 6.6 was observed after the first three days. Since the second passage (after 6 days), lactate was efficiently utilized (90-100%) and the dominant fermentation product, butyrate, was maintained at similar level during the whole experiment at around 4 g/L (Table 2). Acetate was detected as the second component (1 – 2 g/L) of the digestive liquids, wheareas propionate (0.2 – 0.4 g/L) and ethanol (0.05 – 0.2 g/L) were detected as minor products.
The initial richness of the LA community was higher compared to other experiments condition and did not significantly changed over time (Figure 1). Taxonomic analysis to some extent reflected this result (Figure 2). The genera Clostridium sensu stricto constituted on average 45-50%. Clostridium sensu stricto 12 increased over time (0.5% ± 0.3%, 5.7% ± 4.9%, 28.4% ± 12.6%, 28.2% ± 2.3%, 29.8% ± 8.1% and 34.2% ± 1.4%, respectively after 3, 6, 9, 12, 15 and 18 days, Kruskall-Wallis test p = 0.034) whereas Clostridium sensu stricto 1 decreased (41.9% ± 0.6%, 21.9% ± 0.8%, 14.0% ± 3.0%, 8.9% ± 2.3%, 7.9% ± 3.1% and 6.0% ± 1.4%, respectively after 3, 6, 9, 12, 15 and 18 days, Kruskall-Wallis test p = 0.012) in time. Clostridium sensu stricto 1 was a dominant genus after the first 3 days when conversion of lacate to butyrate was on the lowest level (Figures 3 and 4, Table 2). Relative abundance of Clostridium sensu stricto 13 was also high, but maintained at a similar level over time (6.8% ± 1.5%, 13.9% ± 3.5%, 7.2% ± 1.5%, 7.9% ± 1.6%, 6.1% ± 2.1% and 6.3% ± 1.1%, respectively after 3, 6, 9, 12, 15 and 18 days). Compared to the other experiments, Lactobacillus (9.2% ± 4.7%, 3.1% ± 2.4%, 2.1% ± 2.0%, respectively, after 3, 6, 9 days and <1% since the 12th day), Fructobacillus (1.0% ± 0.2% after 3 days and and <1% since the 6th day), Bifidobacterium (3.5% ± 0.4% after 3 days and and <1% since the 6th day) and Leuconostoc (1.6% ± 0.4% after 3 days and and <1% since the 6th day) were significantly reduced. Contribution of the following genera increased: Terrisporobacter (5-10%), Sutterella (5-10%), Paraclostridium (up to 10%), Lachnoclostridium (up to 10%), Escherichia (up to 5%) and Dialister (4-6%).
Summary of the static batch experiments and redundancy analysis
Detailed statistical comparison of the pH and metabolite (ethanol, butyrate, propionate, and lactate) formation between Experiments M, ML, MLA, LA and SLA are depicted in Additional file 1. For simplicity, in this section we focus on the results from day 6 and 9. The pH values were significantly different among all experiments (0.001 > p > 0.0002, Tukey's HSD test; Additional file 1) with the lowest pH recorded in Experiment M (molasses only; Table 2). Butyrate synthesis in Experiments M and ML was significantly lower than in Experiments MLA, SLA and LA (0.02 > p > 0.0004, Tukey's HSD test; Additional file 1, Table 2). A reverse tendency was observed for ethanol production which was higher in Experiments M and ML compared to MLA, SLA and LA (0.05 > p > 0.0005, Tukey's HSD test). Detected lactate concentrations in the digestive liquids from Experiments ML, MLA, SLA and LA, where lactate was added to the media, clearly show more efficient utilization of lactate in Experiments MLA, SLA and LA as compared to Experiment ML (0.04 > p > 0.0004, Tukey's HSD test; Additional files 1 and 4, Table 2).
To integrate the targeted metabolomic data with the analyses of sample biodiversity, we performed redundancy analysis (RDA), a direct gradient analysis technique which summarises linear relationships between components of response variables that are "redundant" with (i.e. "explained" by) a set of explanatory variables. The results of RDA analysis and the correlation between the fermentation products, pH of the digestive liquids and the dominant bacterial genera in respective experiment are presented in Figure 5. The following positive correlations were observed: Clostridium sensu_stricto_12 with butyrate and pH (Experiments ML, MLA, SLA); Fructobacillus and Leuconostoc with lactate and ethanol (Experiments ML); Fructobacillus,Leuconostoc and Clostridium sensu_stricto_1 with ethanol (Experiment MLA); Clostridium sensu_stricto_1 with lactate and ethanol (Experiment SLA) or with lactate and acetate (Experiment LA); Bifidobacterium with acetate (Experiments M and ML) or acetate and lactate (Experiment MLA); Fructobacillus with ethanol and pH; Lactobacillus and lactate (Experiment M); collection day with butyrate and pH (Experiment LA). It is noteworthy that Lactobacillus correlated with lactate only in Experiment M.
As a synthesis of our observations, two main scenarios for microbial communities fermenting sucrose-containing media (Experiments M, ML, MLA, SLA) can be proposed: (i) Low pH of the digestive liquids (<4) is associated with lactate and ethanol as the main non-gaseous fermentation products. Under such condition, the production of butyrate is very low. Microbial communities are dominated with LAB (especially Lactobacillus) and lactate- and acetate-producer Bifidobacterium. Contribution of Clostridium is very low. This scenario is best illustrated by Experiment M and to some extent by Experiment ML (till the 12th day). (ii) In the second scenario, ilustrated by Experiments MLA and SLA, butyrate dominates among the non-gaseous fermentation products and the pH of the fermentation process is in the range 5-6. Lactate and ethanol are the minor products. The Clostridium genus constitutes at least 25% of the microbial community.
Samples collected late (on days 15 and 18) in Experiment ML indicate an intermediate state between both scenarios. In these conditions, lactate is still the dominant fermentation product, concentration of ethanol decreases, while butyrate production increases and pH of the digestive liquids reaches 4.5-4.6. This corresponds with a higher contribution of Clostridium in the microbial communities. In all scenarios, propionate remains a minor product during the experiments, a decreasing contribution of Fructobacillus is observed over time, and Lactobacillus remains to be an abundant genus. Butyrate formation is related to pH increase, higher contribution of Clostridia (e.g. Clostridium sensu stricto 12) in the microbial community and increase of biodiversity that is the especialy proiminent in Experiment LA.
Carbon balance in the selected static batch experiments
We have previously described an approximate balance of carbon during the fermentation of lactate and acetate to butyrate by Clostridium butyricum and proposed a model of lactate/acetate conversion to butyrate [18]. To illustrate metabolic transformations in the batch experiments performed in this study, the approximate millimolar balance of carbon for the selected data from Experiments LA and SLA (as shown in Figure 4) was calculated and presented in Table 3. The selection criterion was butyrate concentration in the digestive liquids, low on day 3 and high on day 6 in both experiments. The carbon balances are based on the concentrations of sucrose (Experiment SLA only), acetate, lactate, propionate, butyrate and ethanol in the media and the digestive liquids. The calculations take into account (i) concentrations of the remaining non-fermented sucrose in the digestive liquids (~3 millimoles of carbon) which were subtracted from the initial amount of sucrose in the media; (ii) the concentrations of the yeast extract-derived butyrate (18 millimoles of carbon) and propionate (3 millimoles of carbon) in the media that were subtracted from the butyrate and propionate detected in the fermentation products.
Metagenomic analysis of the selected microbial communities
For better understanding of the dynamics of the microbial communities and explanation of the observed differences in their metabolic activity, we selected samples from the static batch experiments designated as MLA-3-AC, MLA-9-AC, LA-3-BC, LA-18-AB (summarized in Table 4) and subjected them to shotgun metagenomics analysis. MLA-3-AC is derived from the MLA Experiment (pooled replicates A and C) after the first passage (day 3), when the main non-gaseous fermentation products were lactate and ethanol, concentration of butyrate was very low (Figure 4, Additional file 4). Sample MLA-9AC is also derived from the MLA Experiment (pooled replicates A and C) but after the third passage (day 9), when lactate was efficiently utilized and the main fermentation product was butyrate (Figure 4, Additional file 4). Sample LA-3-BC comes from the LA Experiment (pooled replicates B and C) after first passage (day 3) when lactate was partially metabolized (Figure 4, Additional file 4). Sample LA-18-AB comes from the LA Experiment (pooled replicates A and B) after the sixth passage (day 18) when lactate was efficiently utilized and the main fermentation product was butyrate (Figure 4, Additional file 4). A total of 34,545,964 to 78,144,622 reads per sample was obtained. Taxonomic composition of the microbial communities on the level of phylum, class, family and genus are presented in Additional file 5. For detailed taxonomic assignments see Additional file 6. Metagenomic analysis confirmed the results obtained by 16S rRNA sequencing. The goal of this analysis was to identify species potentially responsible for sucrose, acetate and lactate utilization. However, due to limitations of the approach we chose, we limited the data interpretation to two aspects. Since the MLA community produces initially (on day 3) large quantity of lactate, the first goal was to identify the putative main lactate producers from sucrose (molasses) fermentation. The species more highly represented in MLA3 vs LA3 communities (>2-fold higher in MLA3, > 0.02% abundance in MLA3) were selected and 72 species that may be involved in fermentation of sucrose to lactate were identified (Figure 6, Additional file 7). They were the Lactobacillus, Leuconostoc, Bifidobacterium, Weissella, Enterococcus, Gardnerella, Pediococcus, Oenococcus, Peptoaerobacter species. The top species were Lactobacillus uvarum (7-fold higher in MLA3, =11.3%), L. brevis (7-fold higher in MLA3, =1.7%), Leuconostoc fallax (22-fold higher in MLA3, =8.9%), L. mesenteroides (5-fold higher in MLA3, =5.8%), Bifidobacterium crudilactis (8-fold higher in MLA3, =10.5%) and B. subtile (10-fold higher in MLA3, =6.4%).
The second aspect of the analysis was a comparison of LA_3 vs. LA_18 and MLA_3 vs. MLA_9 microbial communities to find lactate and acetate utilizers and butyrate producers (Figure 6, Additional file 7). The species more highly represented in MLA9 vs MLA3 communities (>2-fold higher in MLA9, > 0.02% abundance in MLA9) were selected and 52 species were identified (Figure 6, Additional file 7), They were mostly the Clostridium, Prevotella, as well as Lactobacillus, Dakarella and Bacillus species. The top species was C. tyrobutyricum (64.5-fold higher in MLA9, =11.3%). Interestingly, in comparison to MLA-3-AC in the sample MLA-9-AC a decreased contribution of Leuconostoc (below 1%) was observed whereas Lactobacillus (L. uvarum 12.4 %, L. brevis 4.4 %) and Bifidobacterium (B. crudilactis 3.3 % and B. subtile 12.9%) were still top species.
The species more highly represented in LA18 vs LA3 communities (>2-fold higher in LA18, > 0.02% abundance in LA18) were selected and 48 species were identified (Figure 6, Additional file 7), They were mostly the Clostridium, Terrisporobacter as well as Romboutsia, Shigella, Aerocolum, Gottschalkia, Klebsiella and Lactococcus species. The top species were Clostridium tyrobutyricum (106-fold higher in LA18, =28.6%) and Terrisporobacter glycolicus (7.2-fold higher in LA18, =4.1%) suggesting that these species contributed to butyrate synthesis. Interestingly, In the LA-3-BC sample (low butyrate formation) the top species were Clostridium sulfidigenes (7%) and Clostridium beijerinckii (4%). The former maintained at the level 4.8% whereas the latter dropped to 0.3% in the LA-18-AB sample.
Finally, the common species between MLA9 and LA18 communities were found. All of them were the Clostridium species (the most abundant C. tyrobutyricum and minor C. coskatii, C. kluyveri, C. ljungdahlii, C. ragsdalei, C. arbusti, C. estertheticum, Clostridium sp. DMHC 10, C. pasteurianum, C. carboxidivorans and C. acetobutylicum) (Figure 6, Additional file 7). There are putatively the most involved in butyrate formation independent on the growth medium (MLA or LA).