Nepenthes mirabilis Fractionated Pitcher Fluid Use for Mixed Agro-Waste Pretreatment: Advocacy for Non-Chemical Use in Biorefineries

Abstract

This study determined whether it is feasible to pretreat mixed agro-waste of different particle sizes using the pitcher fluid of Nepenthes mirabilis (N. mirabilis), which is known to digest leaf litter due to the enzyme cocktail contained in the fluid. This is due to the need for the holocellulolysis (a source of fermentable sugars) of mixed agro-waste to produce fermentable hydrolysates. The pitcher fluid was fractionated (<3 kDa, ˃3 kDa, <10 kDa, ˃10 kDa) and slurrified with the mixed agro-waste, i.e., 25% (w/w) for each waste—orange peels, apple peels, maize cobs, grape pomace, and oak plant leaf litter of various particle sizes, i.e., >75 µm x < 106 µm and >106 µm. The process of producing a high concentration of total reducible sugars (TRSs) with the lowest production of total phenolic compounds (TPCs) was determined to be a particle size of >106 µm, pretreatment for 72 h, and an enzyme fraction of <10 kDa, whereby 97 g/L of TRSs were produced with a significantly lower TPCs load (1 g/L). Furthermore, the <10 kDa showed preferable physico-chemical properties, with the highest reduction-oxidation potential including acidity. Several enzymes, i.e., β-1,3-Glucanase, Putative peroxidase 27, Thaumatin-like protein, among others, were identified in the <10 kDa fraction, i.e., enzymes known to perform various functions in plant-based waste. Therefore, there is a need for the renewable energy industry to consider solely using pitcher fluids to pretreat mixed agro-waste for fermentable hydrolysates’ production, which can be used as liquid feedstock for the bioenergy and/or biorefinery industries for environmental pollution reduction.

Nepenthes mirabilis Pitcher Fluid Functionality for Agro-Waste Pre-Treatment: Effect of pH, Temperature, Trace Element Solution and the Pore Size of the Waste

“…Overall, preparation of fermentable hydrolysates, pre-treatment of waste feedstock, pH, temperature, and to some extent, trace elements in varying proportions can produce hydrolysates that enhance a fermenter's performance and, thus, its efficacy. Some researchers suggest that when you have lignocellulosic waste, a pH of 2 and a temperature of 33 • C provide adequate conditions for pre-treatment [2,5,7]. Nepenthes mirabilis pitcher fluid with a pH range of 2 to 2.9 under ambient temperature conditions (from 15 to 25 • C or up to 30 • C) can satisfy these requirements.…”

“…When the pre-treatment of agro-waste is performed using pitcher fluids containing a cocktail of enzymes, such as those from Nepenthes spp., it can be advantageous for processes that focus on eco-friendliness without using chemicals. It was further suggested that pre-treated agro-waste at lower pH improves the solubilisation of hemicellulose [7][8][9].…”

“…Since it was determined that N. mirabilis pitcher fluids contain enzymes, such as β-1,3-Glucanase, putative peroxidase 27, and Thaumatin-like protein, among others [7], their functionality must be determined by focusing on pH, temperature, and co-factors in the form of trace elements. Some enzymes were determined to be stimulated by Ca 2+ , Mg 2+ , and Mn 2+ [10,11].…”

“…Another aspect that needs to be assessed is the relation between the porosity of the MAW and the enzyme fraction (<10 kDa) determined to be suitable for the pre-treatment of the milled mixed agro-waste used in this study [7]. This is because if the pore size of the milled agro-waste is large enough in comparison to the size of the enzymes, the enzymes in the pitcher fluid can adequately be embedded in the pores of the agro-waste.…”

“…Therefore, the aim of this study was to (1) assess the influence of pH, temperature, and trace element solution on the pre-treatment of the milled agro-waste using N. mirabilis pitcher fluids and (2) to assess the link between the porosity of the used agro-waste to the fraction (<10 kDa) of the pitcher fluid enzymes. Previously, it was determined that a 72 h holding time, a particle size of >106 µm, and an enzyme fraction of <10 kDa resulted in the highest production of total reducible sugars, while total phenolic compounds were low using N mirabilis as a sole pre-treatment agent [7]. Therefore, these conditions were adopted for this study, specifically assessing TRSs in hydrolysates produced from the pre-treatment of agro-waste.…”