Faculty of Applied Sciences
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Item A comparative study of supercritical fluid extraction and accelerated solvent extraction of lipophilic compounds from lignocellulosic biomass(2022-09) Khanyile Andile; Sithole, B.B.; Paul, Vimla; Andrew, Jerome EdwardLipophilic compounds are non-structural, heterogeneous compounds rich in terpenes, sterols, fatty acids, hydrocarbons, and glycerides. They have found widespread uses in different industries, such as the pharmaceutical, medical, cosmetic and nutraceutical sectors. They are typically extracted from wood using traditional techniques such as solvent extraction hydro- and steam- distillation. However, these techniques have several drawbacks such as long extraction times, high energy consumption, extensive solvent use and degradation of thermosensitive compounds, which are highly volatile. In this study, supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE) were evaluated to extract lipophilic compounds from lignocellulosic biomass such as pinewood sawdust and Cannabis Sativa L. Their advantages of using low amounts of solvent, short extraction times and high selectivity allow them to be used as an alternative extraction technique to traditional methods. Moreover, SFE uses carbon dioxide, which is safe, cheap and readily available, and it does not alter the structure of the compounds. In contrast, ASE uses elevated temperatures and high pressures to prevent the evaporation of highly volatile compounds. In order to solve challenges from both an economic and an environmental perspective, the interaction of process conditions on lipophilic compounds extraction efficiency was modelled and optimized using Response Surface Methodology (RSM) and BoxBehnken design (BBD). The extraction variables optimized for pinewood sawdust compounds were, SFE: co-solvent (ethanol) flow rate (1-2 ml/min), carbon dioxide (CO2) flow rate (1-3 ml/min), Temperature (40-60 °C) and pressure (200-300 bar), and for ASE: static time (10-15 mins), static cycle (1-3) and temperature (80-160 °C). The process parameters were optimized, and the experimental data was modelled using RSM for statistical analysis of the BBD extraction process. The experimental data's quadratic polynomial models gave a coefficient of determination (R2 ) of 0.87 and 0.80 for ASE and SFE, respectively. The optimum conditions of ASE were temperature (160 °C), static time (12.5 mins), and static cycle (1), which resulted in a maximum yield of 4.2%. The optimum SFE conditions were temperature (50 °C), pressure (300 bar), CO2 flow rate (3.2 ml/min), and a 2 ml/min co-solvent (ethanol) flow rate that yielded 2.5% lipophilic compounds. The extraction efficiency of pinewood sawdust lipophilic compounds with ASE was higher compared to the SFE. Although ASE uses high temperatures that may degrade thermolabile compounds, the short extraction times may work in their favor since the extracts are not exposed to high temperatures for long periods. SFE uses low temperatures and long extraction times compared to ASE. Several properties affect the extraction efficiency, such as volatility, dissolving power, solubility, and fluid density of the extracting solvent. The extraction efficiency of lipophilic compounds by SFE may be affected by the supercritical fluid's solubility and differences in densities at different pressures. In ASE, the high yields were influenced by the high polarity of the solvent mixture and temperature with a short extraction time. The extraction variables optimized using RSM for Cannabis Sativa L. for SFE were pressure (200-300 bar), co-solvent (ethanol) flow rate (1-2 ml/min) and CO2 flow rate (1-2 ml/min). The R2 was determined to be 0.9108. The optimum conditions were 300 bar pressure, 1 ml/min co-solvent (ethanol) flowrate, and 2 ml/min CO2 flowrate, which gave a maximum yield of 88%. The high efficiency observed was brought by the increase in the flow rate of CO2 at high pressures, which reduces the mass transfer resistance, while the cosolvent enhanced the solvating power of CO2. The ASE had a high extraction efficiency for the pinewood sawdust lipophilic compounds. However, the method's selectivity was very low according to the results obtained by pyrolysis gas chromatography-mass spectrometry (Py-GC/MS). The thermosensitive compounds, such as terpenes, decreased from 2.01% to 1.69% upon the addition of Tetramethylammonium hydroxide (TMAH). The initial concentration of terpenes was 7.21% in pinewood sawdust by SFE. Upon the addition of TMAH, the concentration of terpenes of the pinewood sawdust decreased to undetectable levels. The initial concentration of the terpenes of Cannabis Sativa L. was 14.29% and decreased in the presence of TMAH to 0.39%. The Fourier Transform Infrared Spectroscopy (FTIR) confirmed the presence of lipophilic compounds functional groups and a fingerprint region of lipophilic compounds of pinewood sawdust and Cannabis Sativa L. Thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) showed high thermal stability (250 – 400 ℃). This research demonstrated the ability of SFE to extract lipophilic compounds from pinewood sawdust Cannabis Sativa L.Item Biochemical characterization of selected carbohydrases from Beauveria bassiana and their potential applications(2021-07) Amobonye, Ayodeji Emmanuel; Pillai, Santhosh Kumar Kuttan; Singh, SurenDifferent filamentous fungi have continued to attract scientific interests as novel sources of enzymes and other important bioproducts. Beauveria bassiana, a well-known entomopathogenic fungi has long been valued for its biotechnological application as a biocontrol agent in its entomopathogenic state and as a plant-growth promoter in its endophytic state. The fungus has also been proven to be safe for human health, as studies have shown B. bassiana strains to be non-pathogenic to humans, other animals and plants. Furthermore, its ability to utilize various agro-residues for its growth and the concomitant production of important bioproducts have been well demonstrated. However, despite all of these, there has been no appreciable attempt at exploring this remarkable fungus for the production of industrially important enzymes, especially in its saprophytic state. Recently, a filamentous fungus was isolated in its endophytic state from onion leaves, in our laboratory. It was confirmed by rDNA ITS sequencing to be a B. bassiana strain and was subsequently designated as B. bassiana SAN01. Preliminary experiments revealed the remarkable ability of this novel strain to utilize lignocellulosic biomass for its metabolism while secreting various biomass-degrading enzymes in the process. Hence, carbohydrases from B. bassiana SAN01 were considered worthy of investigation because of the established safety of the source organism, as well as the probable low production cost of the enzymes using readily available plant biomass. Besides, it was also observed that there has been no significant investigation into the biochemical properties of lignocellulolytic enzymes from B. bassiana, which has probably hindered their industrial applicability. Hence, this Ph.D. research was focused on investigating the production, the biochemical properties, as well as the potential applicability of selected biomass-degrading enzymes, viz., amylase, cellulase (endoglucanase), pectinase (polygalacturonase) and xylanase from B. bassiana SAN01. To achieve these, the phylogenetic relationship of the fungal strain was established, and its carbon utilization profile was annotated using phenotypic microarray technology. Furthermore, to understand the dynamics surrounding its lignocellulosic biomass utilization and its carbohydrase-production capabilities, comparative transcriptomics analysis was carried out B. bassiana SAN01 under three different simulated conditions i.e., endophytic, fermentation and lab control conditions. In addition, to fully explore the carbohydrase production potential of the fungus, the production of the selected carbohydrases was optimized using response surface methodology; subsequently, all the selected enzymes were purified to enhance the evaluation of their biochemical properties as well as their potential industrial applications. The proclivity of B. bassiana SAN01 for polyols, pentoses, N-acetyl-D-glucosamine and some other carbon sources was demonstrated by the phenotype microarray profiling. While the comparative genome-wide transcriptome analyses revealed a clear distinction between the fungus under the different trophic conditions investigated. It was observed that 4-5% of the 10,365 B. bassiana SAN01 genes were differentially expressed between these conditions, and a significant proportion of the genes were found to be involved in lignocellulose deconstruction. The annotation of CAZymes from the B. bassiana SAN01 transcriptome under fermentation (saprophytic) conditions confirmed the upregulation of biomass-degrading enzymes such as amylases, cellulases, chitinases, glucanases, laccases, lignases, pectinases and xylanases. The subsequent optimization of the production parameters of B. bassiana SAN01 amylase, endoglucanase, polygalacturonase and xylanase led to heightened yields of 34.82 UmL-1, 23.03 UmL-1, 51.05 UmL-1, and 1061 UmL-1, respectively. These were estimated to be 1.79-, 1.35-, 1.87- and 3.44- folds higher than unoptimized production levels and are also the highest ever production levels recorded for these enzymes from any B. bassiana strain. Further in the study, the xylanase from B. bassiana SAN01 was purified to homogeneity while the other three enzymes were partially purified. The purified xylanase was demonstrated to have a molecular mass of ~37 kDa and performed optimally at pH 6.0 and 45oC. However, the optimum pH of the partially purified amylase, endoglucanase, and polygalacturonase were found to be pHs 6.0, 6.0 and 7.0, while the optimum temperatures were observed to be 35oC, 35oC and 45oC, respectively. Consequently, the purified B. bassiana SAN01 xylanase was demonstrated to be effective in deinking wastepaper with an optimized deinking rate of 106.72% relative to the control. In addition, the partially purified amylase-polygalacturonase from B. bassiana SAN01 was demonstrated to adequately clarify pear juice with a 1.37-fold improvement in clarity recorded under optimal conditions. Furthermore, results also showed that the enzymatic- assisted juice clarification was without any detrimental effect on some quality parameters of the juice. In the same vein, crude endoglucanase-xylanase from the fungus was shown to significantly hydrolyze sugarcane bagasse, releasing ~20% reducing sugars under optimal conditions. Finally, to gain insights into the structure-function relationship of B. bassiana carbohydrases, the structural properties of B. bassiana chitinases and xylanases were elucidated for the first time using computational techniques. The in silico prediction revealed that the enzymes were generally hydrophilic, thermostable, negatively charged and extracellularly secreted. The modelled tertiary structures of B. bassiana chitinase and xylanase were validated by the presence of ~ 90% of their amino acid residues in the Ramachandran plot’s favoured region. The findings from this study have thus created a strong framework for the prospective utilization of B. bassiana and its carbohydrases in alternative biotechnological processes.Item Valorisation of bambara and cowpea haulms for bioethanol production(2020) Okuofu, Somiame Itseme; Pillai, Santhosh Kumar KuttanBambara and cowpea are important pulses grown in semi-arid South Africa due to their balanced nutrient profile and drought resilient capacity. The haulm is the lignocellulosic residue obtained after grain harvest and are rich in carbohydrates. However, these haulms are underutilised and under researched. The aim of the study, therefore, was to investigate the potential to valorise bambara haulms (BGH) and cowpea haulms (CH) to bioethanol which is the most promising biofuel with commercial prospects currently. The structural and chemical composition of BGH and CH was elucidated using techniques such as compositional analysis, XRD, FTIR, ICP-AES, and SEM. Results indicated a volatile matter and fixed carbon mass fraction of 77.70% and 13.15% (w/w) in BGH and 76.16% and 16.26% (w/w) in CH respectively. The polysaccharides make up the largest fraction (51%), followed by extractives (> 20%), while the lignin in BGH (12%) and CH (10%) was low. X-ray diffraction pattern showed a higher percentage of amorphous regions in BGH (78%) than CH (56%). CH was then subjected to dilute acid pretreatment (DAP) to enhance biosugar production for bioethanol fermentation. The effects of operational factors for DAP including temperature, time, and acid concentration on sugar yield and inhibitor formation was investigated and optimised using response surface methodology (RSM). The solid recovered after DAP was subjected to prehydrolysis with simultaneous saccharification and fermentation (PSSF). In addition, the pretreatment hydrolysate was detoxified and fermented to ethanol using cocultures of Saccharomyces cerevisiae BY4743 and Scheffersomyces stipitis wild type (PsY633). A total ethanol titre of 15.67 g/L was obtained corresponding to 75% conversion efficiency. On the other hand, BGH was subjected to deep eutectic solvent (DES) pretreatment. Five deep eutectic solvents were prepared and screened for their effectiveness in improving enzymatic sugar yield. This was achieved by pretreating BGH with each DES followed by a 48 h enzymatic saccharification. Choline chloride – lactic acid (ChCl-LA) treatment provided the most promising result and was further optimised by investigating the effect of different temperatures and time on cellulose loss and enzymatic sugar yield. ChCl-LA pretreatment at 100°C for 1 h was observed to be the best condition for maximum sugar recovery. The hydrolysate thus obtained was concentrated and fermented for 72 h with S. cerevisiae BY4743. A maximum ethanol yield of 11.57 g/L was obtained. From the results, it is evident that bambara and cowpea haulm are promising substrates for bioethanol production. Dilute acid hydrolysis was shown to be effective in the pretreatment of CH with over 85% of the theoretical sugar recoverable for conversion to bioethanol. In addition, deep eutectic solvents are effective media for breaking the recalcitrance in BGH to achieve high sugar yield for conversion to bioethanol. However, further studies are required to reduce cellulose loss during pretreatment to improve bioethanol yield.Item Production of oligosaccharides from lignocellulosic biomass(2020) Arumugam, Nanthakumar; Pillai, Santosh Kumar Kuttan; Singh, SurenLignocellulosic biomass is the most abundant plant material present on earth which is primarily composed of cellulose, hemicellulose and lignin. The composition of lignocellulosic biomass varies depending on the type of plant material and the conditions at which the plant grow. Exploration of lignocellulose for the production of value-added compounds including all types of platform chemicals, biofuels and bioactive compounds is gaining momentum. However, extensive research needs to the carried out to minimize the cost of production to make the processing of this biomass more viable. In the last two decades, several agricultural biomass types have been studied to facilitate the production of biochemicals and biofuels at a low cost. Biomass such as peanut shells, bambara, cowpea and sorghum are some of the indigenous crops of South Africa that are yet to be explored for value addition. Therefore, this study was designed to characterize the underutilized agro-residues such as peanut shell, bambara, cowpea and sorghum biomass for the enzymatic production of prebiotic xylooligosaccharides (XOS) and their application.Item Selective extraction of lignin from lignocellulosic biomas using ionic liquids(2016) Mkhize, Thandeka, Y.; Deenadayalu, Nirmala; Reddy, P.Globally there is a drive for the use of renewable materials for the production of biofuels or high-end value chemicals. The current production of chemicals from crude oil refining is unsustainable and leads to global warming effects. Biomass is the most attractive renewable energy source for biofuel or fine chemical production. Sugarcane bagasse is a by-product of the sugar milling industry and is abundantly available. In this study lignin was sequentially extracted using ionic liquids. The ionic liquids (ILs) 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]) and triethylammonium hydrogen sulfate ([HNEt3][HSO4]) were used to fractionate the sugarcane bagasse. The pre-treatment of sugarcane bagasse was carried out at different temperatures ranging from 90 - 150 0C and reaction times ranging from 1 - 24 h in a convection oven at a 10 % biomass loading. Both ILs were able to dissolve the raw bagasse samples at 120 0C with [Emim][OAc] giving a lignin maxima of 28.8 % and a low pulp yield of 57 % after 12 h; [HNEt3][HSO4] gave a lignin recovery of 17.2 % and low pulp yield of 58.5 % after 6 h. Regenerated lignin was obtained by adding ethanol/ water to the mixture followed by vacuum filtration. The regenerated pulp materials were characterized by Scanning Electron Microscope (SEM) to study the morphology; Fourier Transform Infrared Spectroscopy (FTIR) to study the characteristic bands and thermal analysis to study the thermal stability.