Effects of I. tinctoria LP on early fermentation phase
Samples of the early fermentation phase of Experiment 1 were observed to determine the effect of I. tinctoria LP on the bacterial community. Results of dyeing and changes in pH and ORP are shown in Figure 1 and Supplementally Figure 1. There were evident differences in terms of fluid dyeing between I. tinctoria LP treatment and the control after day 5. The dyeing intensity of I. tinctoria LP slightly decreased after the pH increased at day 10. The effects of this sudden change especially in the control were reflected in the ORP curves. These changes persisted from days 10–21. No increase in ORP was observed in I. tinctoria LP treatment during the foregoing time period. Nevertheless, its dyeing intensity transiently decreased (Fig. 1, Supplementally Figure 1).
Firmicutes was the dominant phylum in all samples (including facultative, obligate and aerotolerant anaerobes) followed by Proteobacteria and Actinobacteria (Fig. 1). The microbiota detected in both I. tinctoria LP (LP1 and LP2) in natural state are shown in Supplementary Fig. 2. The estimated CFU for I. tinctoria LP and sukumo are shown in Supplementary Table 1. The bacterial community profiles in both I. tinctoria LP in raw state dose not correlates with those of the fluid with sukumo at day 1. Hence, the I. tinctoria LP bacterial flora did not markedly influence the bacterial flora profile in the broth. This finding corroborated with the results of the estimated bacterial counts for I. tinctoria LP compared against those for sukumo (Supplementary Table 1). The ORP-lowering effect of I. tinctoria LP might be explained by endogenous aerobic bacterial oxygen consumption. Thus, the aerobic bacterial colony count was estimated and maximized using the neutral pH medium. The expected neutralophilic bacterial count for I. tinctoria LP in prepared fermentation fluid was ~0.9% that for the sukumo. Wood ash extract treatment at 26 °C for 30 min lowered the I. tinctoria LP bacterial count. In contrast, wood ash extract treatment even at 55 °C for 30 min had no effect on the sukumo bacterial count. There were more Bacillaceae in fluid where I. tinctoria LP was added than in the control at day 1. Samples from day 1 showed that the bacterial community had four major operational taxonomic units (OTUs) related to Bacillus cohnii, Bacillus taeanensis, Alkaliphilus oremlandii, and Mogibacterium neglectum. All other taxa except Mogibacterium neglectum were more abundant in I. tinctoria LP treatment than the control. All five OTUs decreased in both I. tinctoria LP treatment and the control over next 5 d. However, the sum of the decrease in I. tinctoria LP (50%) was greater than the control (36.2%). The control treatment exhibited relatively higher bacterial diversity on day 1. The proportion of taxa grouped as “others” (˂ 1.5%) was 21.8 % higher than I. tinctoria LP. The control contained more Proteinivoraceae than I. tinctoria LP at day 5. In I. tinctoria LP treated batch, an OTU related to Alkalihalobacillus alkalinitrilicus was highly active by day 5. In Experiment 1, there were comparatively more microorganisms such as Tissierellaceae in I. tinctoria LP treated batch after day 10. In addition, an OTU related to Polygonibacillus indicireducens appeared in I. tinctoria LP after 58 d.
At fermentation onset, there was a decrease in numbers of facultative anaerobes. However, a subsequent increase in the ratio of facultative anaerobes to absolute anaerobes modulated staining intensity. The ratio of facultative anaerobes (F) to obligate anaerobes (O) (designated F/O), the total abundance of facultative anaerobes (F) and obligate anaerobes (O) (designated F + O), and their correlation with dyeing intensity are plotted in the Fig. 2. Nevertheless, the strength of this relationship fluctuated because of the differences in the constituent microorganisms and time lag until the effects reflected in the dyeing intensity. In most case, F/O increased with dyeing intensity.
Diversity decreased on days 5 and 25 according to the observed changes in the OTUs at the sample depth of 18512 (Figure 3). Relatively fewer OTUs on day 1 of the I. tinctoria LP treatment indicated that I. tinctoria LP addition affected the microbiota. The diversity decreased on day 5 coinciding with changes in redox potential. The diversity decreased on day 25 was explained by depletion of the readily assimilated substrates which coincided with the increase in redox potential and decrease in dyeing intensity in I. tinctoria LP (Fig. 2 and Supplementary Fig. 1).
The relative difference between I. tinctoria LP and the control in terms of their microbiota were estimated by principal coordinate analysis (PCoA) using a Bray-Curtis dissimilarity matrix (Figure 4). The microbiota of I. tinctoria LP rapidly changed until day 10. However, the rate of change declined after day 10. In contrast, 58 d were required to reach the final stage in the control. There were three stages in both I. tinctoria LP and the control. However, the duration of each phase varied between treatments. Thus, I. tinctoria LP addition may affect both the bacterial community composition and the rate of change in the microbiota.
Effects of I. tinctoriaLP on the middle and late fermentation phases
Samples from Experiments 2 and 3 were used to determine the effects of I. tinctoria LP no the microbiota in the middle and later fermentation phases. The effects of I. tinctoria LP on the sukumo fermentation in terms of fabric dyeing and pH and ORP changes are shown in Fig 5 and Supplementary Figs. 3 and 4. I. tinctoria LP addition was correlated with an early, steep decline in ORP (near -600 mV) compared with the control. This finding was true for Experiments 1–3 inclusive (Supplementary Figs. 1, 3, and 4). I. tinctoria LP addition during initial sukumo fluid preparation hastened the onset of indigo reduction compared with the control (Experiment 3) (Supplementary Fig. 4). In contrast, despite of I. tinctoria LP on the ORP at the start of fermentation, there was no marked difference in dyeing intensity for Experiment 2. A possible reason is that unlike the other experiments in Experiment 2, neither I. tinctoria LP nor the control reached pH ≥ 10.7 in the initial phase. The ORP for both I. tinctoria LP and the control) in Experiment 2 were lower than those in Experiment 1 and 3 for most of the fermentation period.
Both the control and I. tinctoria LP in Experiment 2 had similar amounts of OTUs related to Erysipleothrix inopinata (93.2–93.4% similarity). This taxon predominated but its abundance decreased with increasing fluid age. On days 50 and 94, I. tinctoria LP and the control had similar quantities of two OTUs identified with Pseudomonas spp. In Experiment 2, OTUs related to Proteinivorax tanatarenses (93.1–93.8% similarity) predominated in the control but not I. tinctoria LP samples. The latter had relatively higher numbers of an OTU related to Tissierella creatinini (94.5% similarity) compared with the control. An OTU related to indigo reducing P. indicireducens was also detected.
Experiment 3 showed that decrease in the amounts of obligate anaerobes and increase in the numbers of facultative anaerobes were similar for both I. tinctoria LP and the control by day 142. However, there were abundant OTUs related to P. indicireducens in I. tinctoria LP (97.7–98.6%) and OTUs related to Alkalihalobacillus hemicellulosilyticus (98.1–99.3%) in the control. Tissierellacea usually predominates compared to Proteinivoraceae in I. tinctoria LP. Nevertheless, this difference was not observed in Experiment 3.
In Experiment 2, the ratio of facultative anaerobes (F) to absolute anaerobes (O) (F/O) was nearly constant for the control. In contrast, F/O varied with fermentation time for I. tinctoria LP. Changes in F/O were not correlated dyeing intensity for I. tinctoria LP in Experiment 2. However, both I. tinctoria LP and the control presented with the highest dyeing intensities (Figs. 6A–6B). In contrast, changes in F/O varied with dyeing intensity in Experiment 3 (Figs. 6C–6D).
The changes in OTU number at the 18512-sampling depth (Supplementary Fig. 5) in Experiment 2 indicated a decrease in diversity with fermentation time for I. tinctoria LP added fluid but not the control. In Experiment 3, the observed OTU more rapidly increased with fermentation time for I. tinctoria LP than the control. PCoA by the Bray-Curtis dissimilarity matrix showed a distinct separation between I. tinctoria LP and the control for in Experiments 2 and 3 (Supplementary Figs. 6A–6B). The microbiota changed faster in I. tinctoria LP than the control in Experiment 2, but the opposite was true for Experiment 3.