Nepenthes mirabilis plant digestive enzymes for semi-deligni-olocellulolysis of mixed agro-waste

Abstract

Environmental concerns regarding the dumping of agro-waste by agro-food processing industries in the environment are a major concern for human health in developing countries. Several of these industries are found in the Western Cape Province, South Africa, and the disposal of these agro-wastes is a challenge, as most of the agro-wastes are disposed of in landfills. A new approach is being proposed, where repurposing of the waste can be implemented cost-effectively, using appropriate technologies such as pretreatment of the waste for the extraction of value-added products. This approach has become a new promising strategy and a sustainable solution for the effective utilisation of this waste. Initially, this study started by developing a proof concept for semi-deligni-holocellulolysis, a term developed to describe agro-waste pretreatment as a partial delignification and holocellulolysis process. The current understanding is that ligninolysis and subsequent holocellulolysis occur. However, this study proposes that agro-waste pretreatment is a simultaneous semi-lignino-holocellulolysis, particularly for milled lignocellulosic waste. The study further proposes that digestive enzyme cocktails from the monkey cups of Nepenthes mirabilis and some plant exudates can be used entirely to perform agro-waste pretreatment for the extraction of fermentable hydrolysates focusing on total reducible sugars (TRSs) while minimising total phenolic compounds (TPCs). Generally, plant exudates and enzyme cocktails can be used, as they contain very useful constituents that can be harnessed for bio-waste and lignocellulosic waste pretreatment. Nepenthes mirabilis (N. mirabilis) pitcher fluids were identified as suitable to pretreat mixed agro-waste while reducing phenolic compounds in the hydrolysate. In this instance, different particle sizes, i.e., 106μm, >75μm x<106μm were pretreated with N. mirabilis pitcher fluid, revealing the efficacy of the pitcher fluid to pretreat the agro-waste, imparting a green chemistry approach for the pretreatment of agro-waste. For the pretreatment of the mixed agro-waste using N. mirabilis pitcher fluid, the pitcher fluid was fractionated (<3 kDa, >3 kDa, <10 kDa, >10 kDa) and slurried with the mixed agro-waste, i.e., 20% (w/w) for each wasteorange 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 produced a high concentration of total reducible sugars (TRSs) with the lowest production of total phenolic compounds (TPCs), with a particle size of >106 μm, pretreatment for 72 h, and an enzyme fraction of <10 kDa being identified as the best pretreatment conditions, 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, and thaumatin-like protein, among others, were identified in the <10 kDa fraction, i.e., enzymes known to perform various functions in plant-based waste. Furthermore, when assessing the pretreatment of the agro-waste using – under different pH, temperature, and co-factor (trace element) solution conditions – a pH of 2 and lower than ambient temperature conditions was determined to be suitable. The supplementation of the pretreatment slurry showed a minimal effect. Additionally, the correlation between the milled mixed agro-waste porosity and <10 kDa fraction pitcher fluid efficacy was determined. With an average pore size of 2.84 nm (28.4 Å) for the > 106 μm particle size of the agro-waste, the <10 kDa fraction pitcher fluid containing the identified enzymes can be embedded into the pores of the agro-waste. To further provide researchers with a platform to develop the study further, an artificial neural network assessment was done, which also provided a way to simulate the data obtained in this thesis. Overall, the ANN structure used was 1-5-2-2 (0.54 at epoch 3) with experimental run 12 (> 106 μm x> 3 kDa) being identified as having a potential to perform similarly to experimental run 14 (> 106 μm x< 10 kDa), i.e. an experimental run with the highest production of TRS’s. This study supported and demonstrated that agro-waste pretreatment can be facilitated using an environmentally friendly approach solely using biological means, i.e., using fractionated N. mirabilispitcher fluids. For such a process to be understood and applied on an industrial scale, an interdisciplinary approach encompassing environmental health, process engineering, and microbiology is required.