Thesis (DTech (Environmental-health))--Cape Peninsula University of Technology, 2018. / The management of agricultural soil and its sustainable use, namely productivity, is paramount to the agricultural industry worldwide. Large-scale agricultural product producers and scientists emphasise using environmentally benign methods to increase agricultural production such as taking a green chemistry approach to agricultural activities and/or using cultivation techniques for the bio-augmentation of agricultural soil. Some of these agricultural products, such as cassava (Manihot esculenta), produce cyanogens which promote the infestation of a cyanogen-resistant microbial species known to produce mycotoxins during decomposition. Although cyanogens and mycotoxins are important components in the functioning of the earth system and agricultural soil, their cumulative effects can result in reduced soil productivity, hence degradation. Furthermore, the presence of mycotoxins in the environment and agricultural produce is hazardous to the environment, including the biotic communities in soil and humans. Therefore, an environmentally benign (green chemistry approach) method for the reduction of cyanogens and mycotoxins was proposed for this research study. The method investigated had to be applicable in-situ for the biodegradation of cyanogens and mycotoxins. Their reduction from decomposing cassava in cultivated soil, which can be used on a small and large scale, would mitigate deleterious effects of a less reported, unknown mycotoxins producer (fungal species), Cunninghamella bertholletiae (KT275316), found to be a free cyanide- (CN-) resistant isolate. The C. bertholletiae was isolated from decomposing cassava tubers and silt, subsequent to culturing on potato dextrose agar (PDA) and in an equivalent volume of nutrient broth (NB) containing KCN (4mg/40mL) at 30 °C for 120 hrs. The isolate demonstrated an ability to biodegrade CN- into NH3 and NO3. NH3 and NO3 are nitrogenous by-products produced when young cassava plants are cultivated in a controlled environment, with 80% of the initial CN- concentration being efficiently degraded to NH3/NO3 at a conversion rate of 77.5% and 72.5% (fungus from silt and cassava), respectively, within 120 hrs. From this research, it was observed that Sub-Saharan Africa is the largest contributor to the CN- load into the environment; from cassava cultivation as per FAO data. The quantity of CN- released was estimated at 0.025x10-3 to 6.71 ppq, with further increases of 60.5% being projected to be released into the environment by 2024. As such, it was hypothetically assumed that numerous species in cassava-cultivated soil become CN- resistant as they are exposed to CN- from decomposing cassava, becoming pathogenic thus antigonistic towards other biota in cassava-cultivated soil. Consequently, the pathogenicity of the isolate was investigated against organisms (n = 12) from cassava-cultivated soil. The isolate demonstrated inhibitory pathogenic activity against some soil bacterial communities such as Oligella ureolytica, Acinetobacter sp., Pseudomonas luteola and Sphingomonas paucimobilis. The isolate also demonstrated minor antagonistic effects against Myroides sp., Stenotrophomonas maltophilia, Candida lipolytica, Cryptococcus albidus and Rhodotorula sp.. Further research to identify extracellular metabolites produced by this organism, using a fermentation method was also carried out using a liquid state fermentation technique. 30 mL Erlenmeyer flasks containing 25 mL of NB/KCN (source of CN-) at 37 °C for 168 hrs, with a volume of (5 mL), extracts from the fermentation being filtered, centrifuged, mixed with chloroform for a liquid-liquid extraction procedure subsequent to a nitrogen-facilitated blow-down technique and reconstitution with 100% analytical grade methanol, for LC/MS-TOF 6230 analysis. The analysis revealed that the isolate was able to produce the mycotoxins/secondary metabolites, Fumonisin B1 (FB1) and Deoxynivalenol (DON). Though the isolate (KT275316) demonstrated the ability to biodegrade cyanide as well as produce mycotoxin, an environmentally benign strategy (green chemistry method) with a potential to biodegrade CN-/NH3/NO3/NO2 for the biodegradation of mycotoxins was evaluated, including the identification of biodegradation by-products post-biodegradation treatment. Thus, plant extracts from Nepenthes mirabilis were found to contain enzymes such as carboxylesterase, β-glucosidase, β-glucoronidase and phosphatidyl inositol phospholipase C (identified using both quantitative and qualitative methods). The plant extracts were used with treated samples from the fermentation and were subjected to biodegradation. Thus, resulting in biodegradation by-products such as Heptadecanone Octadecanamide, Octadecenal for FB1 and Tolmetin for DON, respectively. For future research, it is therefore recommended that plant extracts with similar properties to those observed for N. mirabilis extracts (juice) be sought for application of the proposed method.

Thesis (Doctor of Engineering in Chemical Engineering)--Cape Peninsula University of Technology, 2018. / Lignocellulosic biomass (agro-waste) has been recommended as the most promising feedstock for the production of bioalcohols, in the biofuel industry. Furthermore, agro-waste is well-known as the most abundant organic matter in the agricultural and forestry product processing industry. However, the challenge with utilizing agro-waste as a feedstock is its highly recalcitrant structure, which limits hydrolysis to convert the holocelluloses into fermentable sugars. Conventional pre-treatment methods such as dilute acid, alkaline, thermal, hot water and enzymatic, have been used in previous studies. The challenge with these conventional methods is the generation of residual toxicants during the pretreatment process, which inhibits a high bioalcohol yield, by reducing the microbial populations’ (fermenter) ability to be metabolically proficient during fermentation. Numerous studies have been developed to improve the engineered strains, which have shown to have an ability to reduce the inhibition and toxicity of the bioalcohols produced or by-products produced during pre-treatment, while enhancing the bioalcohol production. In the present study (chapter 5), evaluation of common conventional methods for the pretreatment of the mixed agro-waste, i.e. (˃45µm to <100µm) constituted by Citrus sinensis, Malus domestica peels, corn cobs from Zea mays and Quercus robur (oak) yard waste without a pre-rinsing step at a ratio of 1:1 at 25% (w/w) for each waste material, was undertaken, focusing on hot water pre treatment followed by dilute acid (H2SO4) pre-treatment. To further pretreat the mixed agro-waste residue, cellulases were used to further hydrolyse the pre-treated agro-waste in a single pot (batch) multi-reaction process. The TRS concentration of 0.12, 1.43 and 3.22 g/L was achieved with hot water, dilute acid and cellulases hydrolysis as sequential pretreatment steps, respectively, in a single pot multi-reaction system. Furthermore, a commercial strain was used to ascertain low (C1 to C3) and high carbon content (C4+) bioalcohol production under aerobic conditions. Multiple bioproducts were obtained within 48 to 72 h, including bioethanol and 1-Butanol, 3-methyl, which were major products for this study. However, undesirable bio-compounds such as phenolics, were detected post fermentation. Since multiple process units characterised by chemical usage and high energy intensivity have been utilized to overcome delignification and cellulolysis, a sustainable, environmental benign pretreatment process was proposed using N. mirabilis “monkey cup” fluids (extracts) to also reduce fermenter inhibitors from the delignification of mixed agrowaste; a process with minimal thermo physical chemical inputs for which a single pot multi-reaction system strategy was used. Nepenthes mirabilis extracts shown to have ligninolytic, cellulolytic and xylanolytic activities, were used as an enzyme cocktail to pretreat mixed agro-waste, subsequent to the furtherance of TRS production from the agro-waste, by further using cellulase for further hydrolysis. N. mirabilis pod extracts were determined to contained carboxylesterases (529.41±30.50 U/L), β-glucosidases (251.94±11.48 U/L) and xylanases (36.09±18.04 U/L), constituting an enzymatic cocktail with a significant potential for the reduction in total residual phenolic compounds (TRPCs). Furthermore, the results indicated that maximum concentration of TRS obtainable was 310±5.19 mg/L within 168 h, while the TRPCs were reduced from 6.25±0.18 to 4.26 ±0.09 mg/L, which was lower than that observed when conventional methods were used. Overall N. mirabilis extracts were demonstrated to have an ability to support biocatalytic processes for the conversion of agro-waste to produce fermentable TRS in a single unit facilitating multiple reactions with minimised interference with cellulase hydrolysis. Therefore, the digestive enzymes in N. mirabilis pods can be used in an integrated system for a second generation biorefinery.