Microbiome and Biocatalytic Bacteria in Monkey Cup (Nepenthes Pitcher) Digestive Fluid

Abstract

Tropical carnivorous plant, Nepenthes, locally known as “monkey cup”, utilises its pitcher as a passive trap to capture insects. It then secretes enzymes into the pitcher fluid to digest the insects for nutrients acquisition. However, little is known about the microbiota and their activity in its pitcher fluid. Eighteen bacteria phyla were detected from the metagenome study in the Nepenthes pitcher fluid. Proteobacteria, Bacteroidetes and Actinobacteria are the dominant phyla in the Nepenthes pitcher fluid. We also performed culturomics approach by isolating 18 bacteria from the Nepenthes pitcher fluid. Most of the bacterial isolates possess chitinolytic, proteolytic, amylolytic, and cellulolytic and xylanolytic activities. Fifteen putative chitinase genes were identified from the whole genome analysis on the genomes of the 18 bacteria isolated from Nepenthes pitcher fluid and expressed for chitinase assay. Of these, six clones possessed chitinase activity. In conclusion, our metagenome result shows that the Nepenthes pitcher fluid contains vast bacterial diversity and the culturomic studies confirmed the presence of biocatalytic bacteria within the Nepenthes pitcher juice which may act in symbiosis for the turn over of insects trapped in the Nepenthes pitcher fluid.

Integrated Hydrolysis of Mixed Agro-Waste for a Second Generation Biorefinery Using Nepenthes mirabilis Pod Digestive Fluids

“…These processes are necessary for an effective and efficient way to resolve challenges associated with, (1) delignification, (2) cellulolysis and (3) the production of recalcitrant residual inhibiting by-products from the biomass hydrolysis process, all of which have an effect on other downstream processes [10].To achieve this, process integration is required to improve and reduce some of the stages in the hydrolysis of biomass, i.e., using a suitable single pot multi-reaction process for biodelignification, cellulolysis and the reduction of inhibitory by-products from the feedstock. For such a strategy to succeed, digestive enzymes, such as those produced by Nepenthes mirabilis, which have been previously associated with delignification and cellulolysis [11] are required, with the potential to biodegrade delignification inhibitors.The Nepenthes plants are carnivorous and use specialised pitfall traps like the pitcher plant, which traps insects and decomposes organisms that are perceived to be indigestible as they consist of fibrous chitin [12]. Most pitcher plant species produce an acidic fluid, with a pH ranging from 1.5 to 6 depending on the species [13].…”

“…Although numerous studies have been conducted to profile the constituents of such acidic fluids, there is minimal information on the pitcher plant's acidic fluid (extract) usability in novel processes [14], and their ability to facilitate the biodegradation of lignin, including the conversion of holocelluloses in biomass. However, recent studies have addressed some previously unknown information, indicating that a diverse and complex enzymatic cocktail does exist in N. mirabilis pods, with a high concentration of digestive/hydrolytic enzymes [11]. Chan et al [11] reported that the biodegradation of the insects includes complex chitinolytic, proteolytic, amylolytic, cellulolytic and xylanolytic enzymes.Generally, N. mirabilis digestive fluids, i.e., from the pitcher plants 'monkey cup', have been reported to contain digestive enzymes which are capable of biodegrading complex and polymeric molecules, such as glycan, starch and elemental metallic species, even under anaerobic conditions [11].…”

“…However, recent studies have addressed some previously unknown information, indicating that a diverse and complex enzymatic cocktail does exist in N. mirabilis pods, with a high concentration of digestive/hydrolytic enzymes [11]. Chan et al [11] reported that the biodegradation of the insects includes complex chitinolytic, proteolytic, amylolytic, cellulolytic and xylanolytic enzymes.Generally, N. mirabilis digestive fluids, i.e., from the pitcher plants 'monkey cup', have been reported to contain digestive enzymes which are capable of biodegrading complex and polymeric molecules, such as glycan, starch and elemental metallic species, even under anaerobic conditions [11]. The application of such digestive enzymes in a biorefinery can minimise energy requirements, plant footprint and the use of hazardous chemical compounds while reducing production of fermentation inhibitors.…”

“…To achieve this, process integration is required to improve and reduce some of the stages in the hydrolysis of biomass, i.e., using a suitable single pot multi-reaction process for biodelignification, cellulolysis and the reduction of inhibitory by-products from the feedstock. For such a strategy to succeed, digestive enzymes, such as those produced by Nepenthes mirabilis, which have been previously associated with delignification and cellulolysis [11] are required, with the potential to biodegrade delignification inhibitors.…”

“…However, recent studies have addressed some previously unknown information, indicating that a diverse and complex enzymatic cocktail does exist in N. mirabilis pods, with a high concentration of digestive/hydrolytic enzymes [11]. Chan et al [11] reported that the biodegradation of the insects includes complex chitinolytic, proteolytic, amylolytic, cellulolytic and xylanolytic enzymes.…”