Fermentation Blues: Analyzing the Microbiota of Traditional Indigo Vat Dyeing in Hunan, China

Traditional indigo dyeing through anaerobic fermentation has recently gained worldwide attention in efforts to address concerns regarding the sustainability of industrial indigo dyeing and the impact of toxic reducing agents such as sodium dithionite (Na(2)S(2)O(4)) on human health and the ecological environment. Intriguingly, changes in the microbiota during indigo fermentation are known to potently affect the onset of indigo reduction, and thus elucidation of the microbial community transitions could help develop methods to control the initiation of indigo reduction. Here, we investigated the microbiota associated with the traditional indigo dyeing practiced in Hunan, China. Specifically, we identified the bacterial and fungal components of the microbiota at distinct stages in the indigo fermentation process by analyzing 16S rRNA gene and internal transcribed spacer sequences. Our analyses revealed two substantial changes in the microbiota during the traditional indigo fermentation process. The first change, which was probably caused by the introduction of Chinese liquor (featuring a high alcohol concentration), resulted in decreased bacterial diversity and increased proportions of Pseudomonas, Stenotrophomonas, and Bacillaceae family members. The second change, which could be attributed to the addition of specific plant species, led to an increase in the abundance of Alkalibacterium, Amphibacillus, the obligate anaerobe Turicibacter, the facultative anaerobe Enterococcus, and ZOR0006, as well as to a decrease in the pH and redox potential values. Our results indicate that the specific plant mixture included in the procedure here could be used as an effective additive to accelerate the initiation of indigo reduction during the fermentation process. To the best of our knowledge, this is the first report revealing the fungal diversity during the indigo fermentation process and, furthermore, showing that the fungal diversity has remained in transition despite the relatively stable bacterial diversity in the proper indigo fermentation process. Although traditional indigo fermentation in China is challenging to manage, we can benefit from local knowledge of the fermentation process, and understanding the scientific bases of traditional indigo fermentation will facilitate the development of environmentally friendly procedures. IMPORTANCE Chemical reducing agents included in modern indigo dyeing to initiate indigo reduction can be harmful to both human health and the environment. Given that traditional indigo dyeing involves natural fermentation in a dye vat using natural organic additives without the use of toxic chemicals and that changes in the microbiota during traditional indigo fermentation potently affect the onset of indigo reduction, elucidation of these microbial community transitions could help develop methods to control the initiation of indigo reduction. This study on the microbiota associated with the traditional indigo dyeing practiced in Hunan, China, has identified the bacterial and fungal communities at distinct stages of the indigo fermentation process. Notably, the addition of specific plant species might yield the desired microbial communities and appropriate fermentation conditions, which could be used as an effective additive to accelerate the initiation of indigo reduction. This study has also revealed the fungal diversity during the indigo fermentation process for the first time and shown that the fungal diversity has remained in transition despite the relatively stable bacterial diversity. Thus, this work provides new insights into the traditional indigo fermentation process used in China and substantially enhances current efforts devoted to designing environmentally friendly methods for industrial indigo dyeing.