Tetrodotoxin (TTX) is a potent neurotoxin isolated mainly from toxic puffer fish. To date, the TTX biosynthetic mechanism inside its hosts remains unresolved. Here, we hypothesize the TTX synthesis relies on the host gut microbiota, including the neglected non-culturable bacteria. In these studies, we collected the gut contents from 5 puffer fish species of the genus Takifugu including one suspected hybrid species for gut microbiota study by 16S rRNA amplicon metagenomics approach. Their gut samples were divided into toxic and non-toxic groups based on the TTX concentrations in the livers detected by LC-MS/MS. Bacterial diversity studies showed that gut microbiota structures were significantly different between toxic and non-toxic species. Vibrio and Cyanobacteria centered at the gut bacterial co-occurrence network, suggesting their importance in TTX biosynthesis. The results of PICRUSt2 metagenomic prediction and gene set enrichment analysis provided new support of arginine-precursor required in TTX biosynthesis. This is the first study to profile the gut microbiota in toxic and non-toxic puffer fish species by 16S rRNA amplicon metagenomic approach, defining significant microbial co-occurrence patterns in their gut environment. Our data supported the proposed biosynthesis of TTX inside the hosts by their gut bacterial symbionts using arginine as a precursor.

This study investigated the application of waste banana peels as feedstock for the production of ethanol fuel as a second-generation biofuel. First, acid pretreatment followed by enzymatic hydrolysis converted both the cellulosic and hemicellulosic biomass of the peels into fermentable sugars, which eventually produced ethanol by yeast fermentation. The optimal conditions for the production of ethanol fuel were determined by orthogonal experimental design method. The results showed that 100 g of fresh banana would produce 31.4 g of banana peel which could be turned into 2.8 g dried peel powder. Under optimal conditions of acid pretreatment with 0.2% tartaric acid, enzymatic hydrolysis by cellulase and yeast fermentation, 115 mg of ethanol (95% purity) could be recovered by distillation from the fermentation broth, which was approximately 4% by weight of the dried peel powder. This study concluded that banana fruit, after the removal of peels for consumption or food processing, the discarded peels will no longer pose an organic waste problem to the environment if they could be recollected and converted into value-added products like ethanol fuel. We also demonstrated that tartaric acid, an organic acid used in the acid pretreatment, for the first time to our knowledge, outperformed the traditional sulfuric acid used commonly in other studies.