Theses and dissertations (Applied Sciences)

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Adressing food and nutrition insecurity through the development and implementation of an agricultural and nutrition education programme on legumes for older farmers living in Mariannhill, KwaZulu-Natal, South Africa
(2020) Mkhize, Nkumbulo Xolile; Napier, Carin E.; Oldewage-Theron, Wilna; Duffy, Kevin Jan
Introduction Food and nutrition security can be influenced by various external and internal factors that contribute to a compromised wellbeing status amongst older farmers who are considered as a vulnerable group. This group faces diverse socio-economic and health challenges within an evolving food system. The complexity of this challenge persistently requires multisectoral intervention programmes, as reflected in the nature of this study. Aim and Objectives The aim of the study was to determine the levels of and address the food intake and nutrition insecurity status of elderly farmers. The main objective was to address the challenges that compromised the health of selected previously non-legume producing farmers by encouraging legume production and consumption amongst them. The findings of the study informed the proposal of appropriate strategies to develop an intervention programme intended to educate the identified elderly farmers and to promote their dietary diversification by means of legume production and consumption. Methodology The study employed a cross-sectional design. A baseline study was conducted that aimed at examining variable relationships using a mixed methods approach involving both qualitative and quantitative data collection techniques. A sample of 112 elderly farmers participated in the baseline study, followed by a case-controlled intervention programme that included preand post-intervention surveys. The measuring instruments included socio-demographic as well as health and medical surveys to elicit data such as blood pressure, blood indices for glucose and cholesterol due to dietary intake, household food security, legume knowledge, legume acceptability and agricultural practices. The data informed the findings of both the baseline and the intervention phases of the study. The baseline study involved a sample of 112 elderly farmers, whereas the intervention study involved two case controls (n=103) that comprised an experimental group (EG: n=53) and a control group (CG: n=50). Data were obtained for both groups by analysing blood indices, dietary intake, yields of legume production (EG only) and legume acceptability levels using statistical analyses of variance, significance and trends of pre- and post-intervention surveys over two years. A follow-up survey (FU) was conducted six months after the conclusion of the intervention phase and involved only the EG (n=32), to assess the impact of the intervention programme on their dietary intake and farming practices. The follow-up study thus compared the intervention data of the farmers’ consumption and production patterns with data obtained postintervention. During this latter study, only dietary intake and production levels were measured. All the data were analysed using IBM Statistical Package for Social Sciences (SPSS), Food Finder software, the Mann-Whitney U test, Analysis of Variance (ANOVA) and T-tests for statistical frequencies, variances and correlations. Results In the baseline study, 79.5% of the participants were women. The mean age of the participants was 63.3 ± 8.2 years. A diverse legume consumption was not observed as indicated by a low legume food group diversity score (FGDS) for 0-9 varieties with a mean of 3.5 (SD ± 1.70). Legumes were the least (19.0%) preferred crop planted by the farmers prior to the study. In the intervention study, the majority of participants were also women (73.6% in the EG and 66.0% in the CG). Of the six varieties of legumes under study, red kidney beans was the most preferred (141.60 kg/ha-1 SD ± 162.11). There was a high significance (p=0.001) amongst the six legume varieties produced, which implies that some legumes had a higher yield than others. A comparison between the genders indicated that the women produced 81.70 kg/ha-1 (SD ± 77.95), while the men produced 33.63 kg/ha-1 (SD ± 166.40). The higher yields produced by the women were significant (p=0.011). Legume consumption improved for the EG because the intervention programme resulted in a significantly (p=0.000) increased dietary diversity score (DDS) of legume intake with a mean (±SD) FGDS from 2.4 (SD ± 1.35) to 5.7 (SD ± 2.56) and a mean (±SD) Dietary Diversity Score (DDS) from 8.59 ± 0.74 to 8.23 ± 1.11, while that of the CG was 3.0 (SD ± 1.82). According to the post-intervention survey, the blood results of the EG indicated nutrient improvement at the 5.0% significance level for blood glucose (p=0.038) and cholesterol (p=0.008). The blood glucose levels of the EG were statistically significant (p=0.037) when this group was compared with the CG in both the pre-intervention and post-intervention surveys. Trend analyses were conducted and compared between those men and women who participated throughout the study (i.e. participation from the baseline to the post-intervention surveys). It was found that cholesterol (p=0.033) and Systolic Blood Pressure (SBP) (p=0.013) were statistically significant when the genders were compared across all phases of the study. Data obtained for the EG in the Follow Up (FU) study six months after the intervention study indicated that the production of legumes had declined (71.8%). This was because the legume intake with a mean (±SD) FGDS of 3.0 ± 2.2 indicated a significant (p=0.000) drop from 5.7 (SD ± 2.56) with DDS from 8.23 (± 1.11) to 8.81 (± 0.47) at p=0.005 significance as determined by the FU survey. It was also found that limited marketing of legumes had occurred, which suggests that the sustainability of legume production and consumption post-intervention by the elderly farmers was a challenge. However, 100.0% of the farmers indicated that they intended to continue their production and consumption of legumes, provided that the constant supply of legumes did not cease. Conclusions Evidence was obtained that the intervention project positively impacted the production and consumption of legumes and that the elderly farmers thus diversified their dietary intake for the better during this phase of the study. However, further investigations need to be conducted to explain the decline in consumption and production in the post-intervention period if support for a healthier lifestyle amongst elderly farmers is to be sustained. The technology to produce legumes with high yields to address food insecurity through more appropriate consumption patterns also needs to be explored in greater depth. Because the two streams of knowledge transfer that involved hands-on training of elderly farmers improved legume production and consumption patterns, this study established a sound foundation on which future studies and intervention strategies for food security amongst vulnerable groups may be built.
Anti-HIV activity of selected South African medicinal plants
(2013-09-17) Hurinanthan, Vashka; Odhav, Bharti; Parboosing, Raveen
South Africa has the largest number of people infected with HIV/AIDS. It also has more than 30 000 species of plants and many of these have a long tradition of medicinal use. It is highly likely that the treatment for HIV will come from this traditional knowledge. The need for effective preventative and therapeutic agents for HIV remains an urgent global priority. The aim of this study was to screen selected South African medicinal plants for anti-HIV activity and to identify and characterise an active compound from a plant that can be used for HIV treatment. The aqueous and methanolic extracts of the roots, leaves, flowers and stems of thirty eight plant species (108 extracts) were screened for anti-HIV activity. The plants which had anti-HIV activity were further screened for anti-reverse transcriptase activity. Thirty-two extracts exhibited varying degrees of anti-HIV activity. Cleome monophylla, Dichrostachys cinerea and Leonotis leonurus aqueous leaf extracts had anti-HIV-1 reverse transcriptase activity. The aqueous extracts of D. cinerea showed the best anti-HIV activity with a Selectivity Index of 43.5 and significant anti-HIV-1 reverse transcriptase activity. Crude phytochemical screening of D. cinerea showed that it had tannins, saponins, flavonoids and alkaloids but did not contain any phlobatannins, terpenoids, steroids or phenols. D. cinerea displayed a high degree of free radical scavenging activity with an IC50 of 25 μg/ml, therefore the anti-HIV activity could be attributed to the flavonoids present in the plant. Bio-guided fractionation was used to isolate and purify the active compound from the D. cinerea extract. Compounds were isolated by thin layer chromatography and were tested for anti-HIV-1 and anti-reverse transcriptase activity. From these results the active compound was identified, and purified using preparative TLC. The active compound was characterised by High Performance Liquid Chromatography, Ultraviolet-visible spectrophotometry, and Ultra Performance liquid chromatography coupled to MS/MS. Structural elucidation was performed using Nuclear Magnetic Resonance. From these results, it was deduced that the compound isolated from D. cinerea was a catechin. In this study we show that the catechins present in D. cinerea are responsible for the anti- HIV-I activity and inhibits the reverse transcriptase activity which is a key factor in the progression of HIV. Potentially, these results can be used to develop a new drug for the treatment of HIV or as a cost effective therapeutic agent in treating HIV-infected individuals with oxidative stress.
Anticancer activity of ceratotheca triloba
(2016) Naicker, Leeann; Odhav, Bharti; Matsabisa, M.G.; Mohanlall, Viresh
Plants have provided a source of medicine from the beginning of human history and are the core of modern medicine. Moreover, plant based drug discovery has led to the development of various anticancer drugs (such as vincristine, vinblastine, etoposide, paclitaxel, camptothecin, topotecan and irinotecan). The use of botanical, phytochemical, biological and molecular techniques have facilitated the discovery of anthraquinones from Ceratotheca triloba that can inhibit the human topoisomerase II enzyme (target for anticancer drugs) and kill cancer cells. However, the C. triloba plant has not been extensively studied for its anticancer activity. Therefore, the aim of this study was to further investigate the anticancer activity of C. triloba and determine the classes of compounds that contributed towards its activity. In this study the leaf and root extracts were prepared by using hexane, DCM, hexane: DCM (1:1), methanol and/or water. These extracts were examined for their growth inhibitory potential on three cancer cell lines (A375 [melanoma], MDA-MB-231[breast] and WHCO1 [esophageal]) by using the MTT assay. Then, different mobile phases were prepared for optimizing the separation of the compounds of the active extract by TLC. Column chromatography was performed with the active extract by using five mobile phases (hexane : DCM [60 : 40, 40 : 60], DCM, DCM : ethyl acetate [90 : 10, 70 : 30, 60 : 40, 50 : 50, 50 : 60, 30: 60, 20 : 80], ethyl acetate and ethyl acetate: methanol [80 : 20, 70 : 30, 50 : 50]). The fractions collected from the column were examined for their growth inhibitory potential on two melanoma cell lines (A375 and UACC-62). The IC50 and TGI (total growth inhibition) values of the active fractions were determined. Also, the apoptosis inducing effects of the active fractions and standards (camptothecin and doxorubicin) were determined by using flow cytometer based assays (FITC annexin assay, PE active caspase 3 assay and BD MitoScreen assay). Subsequently, the chemical structures of the compounds that contributed towards the activity of these fractions were obtained by EI-LC-MS analysis. The results demonstrated that the hexane root extract exhibited the best percentage of growth inhibition (%GI) on all three cancer cell lines. The separation of the compounds of the hexane root extract was optimized on TLC plates by using different ratios of hexane and DCM. Column chromatography allowed for fractionation of this extract. Purified compounds were not obtained due to co-elution. Further research would have to be conducted to obtain purified compounds. This may involve the use of mini-column chromatography and PTLC. Overall a total of ten combined fractions were collected from the column. Four of these fractions (F2, F4, F5 and F8) displayed a high %GI on the A375 and UACC-62 cell lines. Moreover, fraction F4 was the most active fraction as it had the lowest IC50 (0.70 µg.ml-1 [A375] and 0.39 µg.ml-1 [UACC-62]) and TGI (12.50 µg.ml-1[A375] and 25 µg.ml-1 [UACC-62]) values in comparison to the other fractions. All four fractions induced depolarization of the mitochondria membrane potential (ΔΨ), caspase 3 activation, early apoptosis (phospholipid phosphatidylserine exposure) and/or late apoptosis in the melanoma cells. The results also revealed that fraction F4 (25 µg.ml-1) induced depolarization of the ΔΨ in a higher percentage of A375 (78.11%) and UACC-62 (87.4%) cells than the other fractions and standards. This fraction also induced caspase 3 activation in a high percentage of A375 (90.56%) and UACC-62 (96.78%) cells. Therefore fraction F4 was also the most active fraction in terms of apoptosis activity. Based on our results and literature findings we can deduce that the active fractions induced the intrinsic or extrinsic (type II) apoptosis pathway in the melanoma cells. Six classes of compounds were identified from the four active fractions. These were: benzothiophenones, benzopyranones, naphthoquinones, anthraquinones, androstanes and quinazolines. In conclusion, this is the first study that evaluated the growth inhibition potential of the leaf and root extracts of C. triloba on a panel of cancer cells. This research indicated that the hexane root extract displayed the best levels of cell growth inhibition. The active constituents of this extract were isolated into four fractions which elicited apoptosis inducing effects that promoted the extrinsic (type II) or intrinsic apoptosis pathway in the melanoma cells. Furthermore, fraction F4 contained the most active compounds from C. triloba as it had the lowest IC50 and TGI values (in comparison to the other fractions) and induced depolarization of the ΔΨ in the highest percentage of melanoma cells. It was confirmed that six classes of compounds were accountable for the anticancer activity of these fractions. Thus, the C. triloba plant is a rich source of anticancer compounds.
Antioxidant and anticancer properties of bioactive peptides from Lablab purpureus
(2023-05) Sipahli, Shivon; Mellem, John Jason
Cancer can be described as a non-communicable disease that develops from defective cells in the human body and grows uncontrollably. Globally in 2020, statistics revealed that the disease had affected approximately 19.3 million people. With about 51% of these cases resulting in death. Cancer treatments usually comprise surgery, chemotherapy, radiotherapy, or a combination of the three. Traditional therapies such as chemotherapy and radiotherapy drugs are effective at shrinking tumours. However, a key disadvantage is that these drugs are unable to distinguish between cancerous and healthy cells. Subsequently, the human body experiences many adverse side effects such as hair loss, vomiting, lowered immunity, and a general deterioration of health. Drug resistance and rejection are also major disadvantages of these traditional therapies. Alternative therapies are required to mitigate these drawbacks. The vital factor to consider for alternative treatments should be to selectively target cancer cells thereby alleviating the unwanted side effects. Compounds derived from non-toxic edible plants have shown to have bioactive potential. These plants are regarded as non-toxic to the human body therefore they would be able to target the tumour cells alone. Plant compounds also provide additional protection such as their antioxidant abilities and apoptotic potential. Evidence suggests that bioactive peptides derived from legumes can act as both anticancer agents and strong antioxidants. This study investigated the bioactive potential of peptides derived from Lablab purpureus. This investigation began by assessing the antioxidant capacity (2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic (ABTS), superoxide radical scavenging and Ferric Reducing Antioxidant Power (FRAP) assays) and antiproliferative abilities (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)) of L. purpureus protein isolate and hydrolysates (alcalase, trypsin and pepsin). The hydrolysate and fractions of interest were selected based on the MTT assay with the pepsin hydrolysate selected for further apoptotic studies (caspase-3 and -7, and annexin V-PI). Thereafter, the pepsin hydrolysate was fractionated by ultrafiltration (molecular weight cut-off: <1, 3, 5, 10, >10 kDa). The 3 kDa fraction was further fractionated by RP-HPLC. Five peaks appeared on the chromatogram, however, fraction 2 was selected, for apoptotic investigations (caspase -3 and -9, p53 and annexin V-PI). Antioxidant studies are a good measure of the isolate or hydrolysate's ability to perform as a bioactive compound. The 50% inhibitory concentration (IC50) observed for the respective antioxidant studies showed the radical scavenging ability of the isolate and hydrolysates to be 1.81-4.47 mg/mL (DPPH), 1.73-2.42 mg/mL (ABTS), 1.36-4.4 mg/mL (superoxide radical scavenging) and 19.20-21.94 mg/mL (FRAP). Anticancer activity was substantiated by the peptides' ability to induce apoptosis. The pepsin hydrolysate was selected using the MTT assay (IC50 values of A549, 119.6; MCF7, 9.80 and HEK293, 13.86 µg/mL). Pepsin hydrolysate inhibited cancerous cells (A549 and MCF-7) while causing minimal damage to healthy cells (HEK293). Thereafter apoptotic markers, caspase 3/7 and annexin V-PI were quantified. Visualisation of cells in different stages of apoptosis was investigated by Annexin V-PI staining quantified by flow cytometry. During early apoptosis; A549, 42%; MCF-7, 17%; HEK293, 34%. Caspase 3/7 assay verified that the pepsin hydrolysate caused an increase in apoptotic activity. Caspase-3 and -9 activity of cells, determined by ELISA showed that Fraction 2 treated cancer cells (A549 - 0.067 ng/mL, 21.966 ng/mL, and MCF-7 - 0.137 ng/mL, 0.205 ng/mL respectively) had a greater caspase concentration over camptothecin (A549 - 0.029 ng/mL, 20.486 ng/mL and MCF-7 - 0.051 ng/mL, 0.112 ng/mL respectively). Tumour suppressor protein, p53, acts as a protective mechanism by initiating apoptosis in ‘suspicious’ cells. The A549 cell line showed the greatest p53 expression compared to MCF-7 and HEK293. Increased p53 can regulate signalling pathways leading to targeted apoptosis. Finally, annexin V-PI confirmed that Fraction 2 did induce apoptosis in the cells (cells in early apoptosis, A549, 85%; MCF-7, 90%; HEK293, 94%). Results from this study have shown that peptides derived from L. purpureus (specifically fraction 2) have potential anticancer abilities which may be attributed to their antioxidant and apoptotic abilities.
Application of lipid extracted algae in feed and energy production
(2019) Ansari, Faiz Ahmad; Bux, Faizal; Gupta, Sanjay Kumar; Guldhe, Abhishek
Microalgae are well considered to be promising feedstocks for biodiesel production. Microalgae can be grown under different types of cultivation conditions and their biomass has tremendous potential to be used as biofuel feedstock and for other applications such as feed, food, cosmetics, pharmaceutical etc. Despite the many benefits and the significant development in the field of microalgal biodiesel production, there are several challenges including high cultivation cost and developing efficient downstream processing methods. The biomass production cost is high, which significantly hinders the use of microalgae as a feedstock. Most of the available literature is focused on upstream, single strain and single product strategy, where mainly algal lipids are used for biofuel production. Hence, for improving the sustainability of the algal biofuel production processes and related process economics, a multiple applications approach using integrated biorefinery and exploiting microalgae for environmental benefits is required. To explore the microalgal biorefinery concept it is vital to understand the various cultivation conditions and applications of biomass in different sectors. There are various strategies, which have potential to make algal biofuel technologies more economically feasible and environmentally sustainable. Use of alternative culture media, improving the biomass production and the efficiency of downstream processing (drying, cell disruption, lipid extraction etc.) algal biofuel technology economical. Utilizing lipid-extracted algae (LEA) for energy and aqua feed application will maximize overall economic return and will leave minimal residues as by-product. The major focus of this thesis was to utilize LEA as substrate for biomethane production and protein source in aquaculture feed. However, effect of preceding steps such as microalgae cultivation, biomass drying and cell disruption on major metabolites extraction was also studied. Microalgae were cultivated in different medium (domestic wastewater and BG11) and their biomass yields and biochemical composition (lipid, protein and carbohydrate) were compared. Different drying and cell disruption techniques were employed for lipid extraction and their effect on lipid, protein and carbohydrate yields were evaluated. The yield of major metabolites on whole cell and LEA were also compared. Suitable solvent systems were selected for optimum lipid extraction from wet and dry biomass with minimal toxic effect on LEA metabolites so that LEA can be further used for biomethane and aquaculture feed production. The choice of microalgae at large scale depends upon the number of factors such as their adaptability to large-scale cultivation, biomass production, major metabolites content, robustness towards the open system cultivation and contamination. In this study, S. obliquus and C. sorokiniana were cultivated in wastewater and BG11 medium at laboratory scale. Both strains are indigenous to KwaZulu-Natal. C. sorokiniana showed lower biomass and major metabolites (lipid, protein and carbohydrate) production at large scale compared to S. obliquus. Considering better adaptability to open cultivation, high biomass and metabolites yields, S. obliquus strain was selected for the LEA application study. Microalgae species, C. sorokiniana and S. obliquus were cultivated on BG11 and using different ratios of raw domestic wastewater and post-chlorinated wastewater as nutrient media. The cultivation of S. obliquus and C. sorokiniana showed biomass yield of 1.2-3.5 and 0.78-1.8 g L-1 in BG11 medium, respectively. While biomass yield observed in wastewater was 0.59-1.59 g L-1 for S. obliquus and 0.67-1.45 g L-1 for C. sorokiniana. The higher biomass yield in BG11 medium attributed to the higher nutrient contents in this medium compared to wastewater. The lipid contents for S. obliquus and C. sorokiniana were 20 and 16.5% dry cell weight (DCW), respectively when grown using BG11 medium. While increases in lipid contents of 26.25 and 29.4% DCW were found for S. obliquus and C. sorokiniana, respectively when cultivated using wastewater. Similarly, carbohydrate contents for S. obliquus and C. sorokiniana were 18 and 17% DCW, respectively for BG11 medium. Increased in carbohydrate contents of 25% for S. obliquus, 28.4% DCW for C. sorokiniana were observed for wastewater. Microalgae tend to accumulate more lipids and/or carbohydrates under nutrient stress condition. The nitrogen and phosphorus contents in wastewater are lower than BG11 medium, which were responsible for stressed condition for microalgae. With limited nutrients in wastewater compared to BG11 medium, growth of microalgae is also lower which resulted in lower protein content. Protein content for S. obliquus and C. sorokiniana in BG11 medium were 37.83-48.8 and 25-35.3% DCW, respectively. The protein contents for S. obliquus and C. sorokiniana in wastewater medium were 16.4-27.29 and 15.8-27.3% DCW, respectively. The biochemical composition depends upon the nutrient composition of the medium and cultivation conditions. The two selected microalgae have shown potential for nutrient removal while cultivated in wastewater. The removal efficiency by S. obliquus was found to be 76.13% for COD, 98.54% for nitrogen and 97.99% for phosphate. Microalgae C. sorokiniana cultivation in wastewater removed 69.38% COD, 86.93% nitrogen and 68.24% phosphates. Increased lipid accumulation in the cells was also recorded in stressed conditions due to low nutrient availability from wastewater. After harvesting of microalgae from culture media, the water content in thick algal slurry (>85% DCW) lowers the products recovery. To overcome this challenge drying and cell disruption are required to enhance the efficiency of lipid extraction. Where drying and cell disruption increase the viability of biomass for lipid extraction process. Three biomass-drying techniques viz. sun, oven and freeze-drying and four-cell disruption techniques viz. microwave, sonication, osmotic shock and autoclave disruption were studied for their effect on recovery of major metabolites from S. obliquus. Microalgae metabolites recovery from whole cell and LEA were analysed and compared. The results showed that after lipid extraction, LEA still contained comparable protein to whole algae biomass however, the carbohydrate concentration was reduced. Oven drying exhibited the highest recovery of all the major metabolites followed by freeze-drying; sun drying however, showed lower yields. Despite lower metabolites recovery sun-drying technique is preferable at large scale due to its easy application and cost-effective nature. The main drawback of sun drying technique is weather dependence and required longer period to dry. The microwave and autoclave microalgal cell disruption improved the lipid yield but loss of other compounds was observed. In osmotic shock treatment, due to poor cell disruption efficiency low lipid were obtained and comparably lower protein loss was noticed during lipid extraction. Lipid extraction is crucial step for microalgae biodiesel production. Solvent-assisted lipid extraction is widely used technique for lipid recovery from dry or wet algae biomass. In a biorefinery approach, it is vital to choose appropriate solvents for the optimum lipid extraction whilst having minimal effect on the remaining metabolites (protein and carbohydrates) in LEA. LEA could be used for energy generation or aquaculture feed applications. Six commonly used organic solvents/ solvent systems were used for lipid extraction from wet and dry biomass. The results showed that the lipid extraction efficiency depends strongly on types of biomass as well as solvent systems selected. Lipid extraction from wet algal biomass could reduce the processing steps and save energy incurred in drying. However, the water present in wet algal slurry acts as a barrier, which results in lower lipid yield compared to the dry biomass. The results revealed that among all six-selected solvents, chloroform: ethanol (1:1 v/v) was most effective if wet biomass used specifically for lipid purpose only. To explore the biorefinery concept, isopropanol/hexane composition is the most suitable solvent system because it is less toxic and resulted in high protein (20.07% DCW) and carbohydrate (22.87%) yields in LEA. For dry algal biomass, chloroform: methanol (2:1 v/v) is an appropriate solvent system if biomass used especially for lipid (19.25%) extraction. If LEA to be used for energy and/or aquaculture feed application, DCM: methanol was found to be a suitable solvent system, which gave 32.79% protein and 26.92% carbohydrate yield. Comparatively hexane has lower lipid recovery but shown higher protein and carbohydrate yield in LEA. Due to less toxic, easy to scale up and inexpensive, hexane is preferable as a solvent for lipid extraction if LEA is to be further utilized at large scale for energy or feed application. Anaerobic digestion (AD) of organic residues is well-researched technology for biomethane production. Whole microalgae and LEA has promising potential for biomethane production. The anaerobic sludge used as inoculum for microalgal biomass digestion. Biomethane production from whole algae and products extracted algae highly depends on sludge to algae biomass ratio for higher methane production. The extraction of metabolites also changes the biochemical composition of residual biomass, which can affect the biomethane production. It is vital to understand the effect of various product-extracted algae and as well as pre-treated algae on the biochemical methane potential. In order to compare biomethane potential, four types of biomass were selected namely sun dried powder algae (SDPA), mild heat-treated algae (MHTA), LEA (using hexane as lipid extracting solvent) and protein-extracted algae (PEA). The average methane (CH4) production rate was ~ 2.5 times higher for protein and lipid extracted algae than for whole algae SDPA and MHTA whilst the cumulative CH4 production was higher for pre-treated algae. Highest cumulative CH4 production (318.7mL CH4 g-1 VS) was found for MHTA followed by SDPA (307.4mL CH4 g-1 VS). The CH4/CO2 ratios of 1.5 and 0.7 were observed for MHTA and LEA, respectively. Outcome of this objective revealed that pre-treatment process disrupts the microalgae cell walls, exposing intracellular material and increasing the surface area. The product-extracted algae changes the elemental composition, which decreases the cumulative gas yield CH4/CO2 ratio. Presence of high nitrogen in the form of protein produces ammonia (NH3) which inhibits the methane production. Therefore, it is imperative to use PEA biomass to improve the methane production yield than the whole cell biomass. Due to escalating price and unstable supply of fish meal (FM), alternative protein sources are used in aqua feed, however these sources do not meet to the requirement. The use of less expensive protein source in aquaculture feed as alternative to FM is required. Microalgae are primary producers in the food chain as well as a natural food for fish. Microalgal biomass is comprised of proteins, lipids, carbohydrates, pigments and many other bioactive compounds. The microalgal proteins have an appropriate balance of all essential amino acids, while lipids are rich in polyunsaturated fatty acids (omega-3 fatty acids, EPA, DHA). Whole algae contain all required ingredients while LEA also contain protein, carbohydrates, vitamins, bioactive compounds even though most of the lipid soluble nutrients have been removed. Thus, microalgae have promising potential to be used in aquaculture feed. Aquaculture production continues to increase globally, to meet the aquaculture feed demand algae supplemented aquaculture feed will play an important role in providing good quality fish. In this study, approximately 200 kg of microalgal biomass was harvested for the feed application. Due to lower toxicity, ease of availability and ease of recovery from mixture, hexane was used as a lipid extracting solvent at pilot scale to generate LEA. The 44 weeks (from juvenile to finisher stage) feeding trials were conducted to evaluate the effect of whole and LEA supplementation of S. obliquus strain on growth performance, disease tolerance, feed utilization, physiological activity, and fillet biochemical composition of Nile tilapia (Oreochromis niloticus). In the first trial, fish were fed with an algae free diet (control) and four experimental diets (2.5, 5, 7.5 and 10 wt%) as protein source of dried S. obliquus. The study showed that microalgae could be used as a protein supplement in the Tilapia feed for enhancement of morphological characteristics and nutritional value. The 7.5% and 10% supplementation of whole algal biomass in tilapia feed showed significant improvement in weight and length of the fish compared to the control. The daily body weight gain was 0.25 g higher in experimental groups than the control. The hepatosomatic index percentage was also higher in fish feed when 7.5% whole algae was used in fish feed as a protein source. The results also showed that 7.5% and 10% have better specific growth rate (1.57 and 1.5%), daily body weight gain (1.1 and 0.86 g), overall body weight gain (427.16 and 331.48 g), protein assimilation (43.96 and 40.46%) higher than the control diet fed fish. The survival rate of fish were 100% at every inclusion level. In second trial (44 weeks), two supplementations (7.5 and 10 wt%) of LEA as protein source were used in Nile tilapia diets. Results showed 7.5% and 10% LEA supplemented feed shown better growth performance than control. The protein content were 42.2%, 41.3% and 36.1% in tilapia fed with 7.5%, 10% LEA and control feed, respectively. The body weight gain, tilapia fed with 7.5% LEA shown 357 g while 10% LEA and control have 331.78 g, and 330.08 gm, respectively. The application of whole and LEA of S. obliquus in tilapia feed, shown appropriate supplementation level for tilapia feed at demonstration scale. This thesis presents advances in knowledge in the field of microalgae biorefinery research for pilot scale operations. This research work has covered various aspects such as effect of drying, cell disruption and lipid extraction on whole and LEA metabolites yield. The extraction of lipid from wet and dry microalgal biomass using various solvent systems provides a new insight for the selection of appropriate solvent systems, which can be used for the large-scale lipid extraction. The study on LEA for biomethane production enhances the understanding about the effects of different pre-treatments and product extractions on biomethane production. The results revealed that the supplementation of whole cell and LEA using S. obliquus for tilapia feed is safe therefore, can be used as an alternative protein source. The findings of this study have both academic and industrial value.
Application of selected ionic liquids for different separation problems : liquid-liquid equilibria and activity coefficients at infinite dilution
(2021) Kabane, Bakusele; Redhi, G. G.
This work focusses on the application and pre-screening of selected ILs for different industrial separation problems based on limiting activity coefficients at infinite dilution, and liquid-liquid equilibrium data at different temperatures. The selected ionic liquids for pre-screening based on activity coefficients at infinite dilution data include (1,3- dimethyimidazolium dimethylphosphate, trioctylmethylammonium chloride, trihexyltetradecylphosphonium dicyanamide, 2,3-dihydroxypropyl-N-methyl-2- oxopyrrolidinium chloride, 2,3-epoxypropyl methyl-2-oxopyrrolidinium chloride) and deep eutectic solvent (1-butyl-3-methylimidazolium chloride + glycerol) at 1:2 molar ratio. These ionic liquids were tested in 33 solutes (alkanes, alkenes, alkynes, alcohols, tetrahydrofuran, ketones, aromatic hydrocarbons, thiophene, acetonitrile) and water at T = (313 – 343) K and at p = 101 kPa. The use of ionic liquid as a stationary phase on the column loading ranged between (30 – 36) % by mass. Thermodynamic functions at infinite dilution such as (Gibbs free energy, , entropy term, Tref and partial molar enthalpy, ) were also computed to further elucidate the types of intermolecular interactions existing between solutes and the investigated ionic liquids. The separation potential of the ionic liquids was determined from the selectivity ( ), and capacity ( ), values. Different ionic liquids (1-ethyl-3-methylimidazolium tetrafluoroborate, 1-ethyl-3-methylimidazolim dicyanamide, 1,3-dimethylimidazolium dimethylphosphate) were also tested in the separation of azeotropic mixture (ethyl acetate/hexane) and in the desulfurization (thiophene/hexadecane or octane) process. The extraction process was conducted at varying temperatures, T = (308 and 298) K and at p = 101 kPa. The results were compared to previously published ionic liquids involving thermodynamic data found in the literature. The separation capacity was based on the calculated values of selectivities (S) and distribution ratio (β). The data was successfully correlated using the thermodynamic non-random two liquids (NRTL). From this study, it was found that the investigated ionic liquids showed some possible application for selected industrial separation problems.
Application of thermostable a-Amylase from Thermomyces lanuginosus ATCC 58157 to nutritionally enhance starch based food
(2006) Padayachee, Thiriloshani
In Sub-Saharan Africa there is an urgent need to sustain and improve the quality of its food resources. Poverty eradication features high on the agenda of a number of world health organisations, while the number of underweight children in Africa continues to increase (Pellet, 1996). Providing nutritionally enhanced foods to the poor will help towards achieving this objective. Protein-energy malnutrition has been identified as one of the most important problems facing Africa, with maize as the staple diet (Nkama et al., 1995). However, a combination of several factors limits availability and the nutritional quality of maize. During starvation, energy and protein intakes decrease by 20-30%, with most of the children in Africa having an average protein intake of only 20 g per day (Igbedioh, 1996). Energy availability also affects protein utilization because of interrelationships of protein and energy metabolism (Elwyn, 1993). The diets of inhabitants in developing regions depend mainly on cereals (maize) for both protein and dietary energy which lacks indispensable amino acids, minerals, vitamins and carbohydrates. In light of these growing concerns an attempt was made to devise a scientific strategy to combat the nutritional shortfalls of maize meal. A multidisciplinary and concerted approach was followed within this project aimed at designing an improved thermostable amylase and applying the enzyme to nutritionally enhance maize meal. It was envisaged that the manipulation of maize meal, by the application of enzyme technology will improve the nutritional status of this staple food. The consequences is that an alternate solution for the eradication of an ailing, poverty stricken and malnourished African population is achievable. It is possible that the boundaries defining the limits of life will extend to even greater extremes through the application of novel technologies.
Assessment of heavy metals and pathogens removal from municipal wastewater using a constructed rhizofiltration system
(2018) Odinga, Christine Akinyi; Swalaha, Feroz Mahomed; Bux, Faizal; Otieno, Fredrick Alfred O.
Wastewater discharged from municipal treatment plants contain a mixture of organic contaminants, trace metals, enteric pathogens, viruses, and inorganic materials. The presence of such pollutants in wastewater poses a huge challenge to the choice and applications of the preferred treatment method. Conventional treatment methods are inefficient in the removal of some environmentally toxic pollutants and pathogens. This study evaluated the effectiveness of a constructed rhizofiltration system in the removal of heavy metals and enteric pathogens from municipal wastewater. The study was conducted at an eThekwini municipal wastewater treatment plant in Kingsburgh - Durban in the province of KwaZulu-Natal. The pilot-scale rhizofiltration unit included three different layers of substrates consisting of medium stones, coarse gravel and fine sand. The system had one section planted with Phragmites australis and Kyllinga nemoralis while the other section was unplanted and acted as the reference section. Influent and effluent, plant tissue and sediment from the rhizofilter were sampled bi-monthly for a period of two years and assessed for the presence and removal of selected enteric pathogens, trace heavy metals and changes in physicochemical and biological parameters using standard methods. Antibacterial potential of the two experimental plants was determined by the agar-well diffusion method using plant root exudates exposed to selected pathogenic bacteria. Observation of details of plant morphology, distribution and assessment of the metals attachment onto the various plant tissues was determined using images from scanning electron microscopy (SEM). The Langmuir model was used to assess the heavy metal adsorption of the plants. There was an increase in pH from 6.95 pH units to 7.55 pH units in the planted and 6.72 to 7.23 pH units in the reference sections. There was an average reduction in biochemical oxygen demand (BOD) by 79% and chemical oxygen demand (COD) by 75%. Suspended solids were reduced by 86% in the planted section and 59.8% in the reference section, Electrical conductivity was reduced by 7.7% in the planted section and 0.83% in the reference section, Total Dissolved Solids was reduced by 11.5% in the planted section and 3.5% in the reference section, temperature was reduced by 11.9% in the planted section and 1.2% in the reference section, while dissolved oxygen was raised by 10% in the planted section and 5% in the reference section. Turbidity was reduced by 9.7 NTU in the planted section and 9.1 NTU in the reference section, while alkalinity was reduced by 46.3% in planted and 45.5% on reference sections of the rhizofilter. There was a significant reduction in organic loading in the system which was statistically significant (phosphorous, p = 0.029; ammonia, p = 0.03).These average reductions and increases were observed after the system was fully established. The results indicate a comparatively better removal efficiency in the planted than the reference sections of the system. Considering the entire rhizofilter, heavy metals were accumulated at varying percentages of 96.69% on planted and 48.98% in reference sections for cadmium. Chromium was 81% and 24%, Copper was 23.4% and 1.1%, Nickel was 72% and 46.5, Lead was 63% and 31%, while Zinc was 76% and 84% in the planted and reference section of the rhizofilter respectively. The planted section had a much higher removal efficiency as compared to the reference section of the rhizofilter. The macrophytes were found to display some metals binding potential according to observations from SEM and EDX analysis. Significant amounts of Cu deposits were recorded on the roots of K. nemoralis at 0.31wt% with a peak at 0.6cps/eV than on P. australis which was at 0.31wt% with a peak at 0.6cps/eV. Further, higher deposits of Ni at 0.01 wt% with peak at 0.5 cps/eV and 0.0 wt% with peak at 0.2 cps/eV, Pb at 0.22 wt% with peak at 0.2 cps/eV and 0.21wt% with peak at 0.2 cps/eV were recorded on the roots of K. nemoralis and P. australis respectively. Kyllinga nemoralis was found to have greater metals adsorptive capabilities than P. australis. The planted and reference sections had varied removal capacities of between 45% and 98% for the various pathogens detected in the influent wastewater. For example, the concentration of coliphage was reduced by 94.6% in the planted section and 93.6% in the reference section, Candida spp. removal was 64.7% in the planted section and 62.5% in the reference section. Escherichia coli was reduced by 65%- 85% while Salmonella spp. was removed by 94% in the planted section compared to 78% in the reference section. Ascaris lumbricoides was reduced by 77% in the planted section and 53% in the reference section. Accordingly, higher pathogens reduction was achieved in the planted section as compared to the reference section of the rhizofilter. Root exudates from Kyllinga nemoralis were found to display a wider zone of growth inhibition at 9.97±0.19 mm compared to P. australis which had a zone of 8.63 ± 0.22 mm when exposed to cultured colonies of Escherichia coli.
Assessment of microalgal ACCase and rbcl gene expression as a function of nutrient and metal stress
(2017) Singh, Poonam; Bux, Faizal; Kumari, Sheena K.; Guldhe, Abhishek
Microalgae are considered to be a potential feedstock for biodiesel production. However, the main concern with regard to the large scale microalgal biodiesel production process is its competence and economic viability. The commercial realization of microalgal biodiesel production requires substantial impetus towards development of efficient strategies to improve lipid yields upstream. Nitrogen (N) and phosphorus (P) stress during cultivation are the widely used lipid accumulation strategies for microalgae. However, these individual nutrient stress strategies are associated with compromised biomass productivity which hampers overall lipid productivity. Lipid enhancement strategies based on light, temperature and CO2 are associated with technological barriers for scale up and incur additional cost. Thus, the main aim of this study was to develop an integrated, easily applicable and scalable lipid enhancement strategy based on nutrients and metals such as N, P, iron (Fe), magnesium (Mg), calcium (Ca) and EDTA stress for selected indigenous microalgal strains. The effect of metal concentrations individually and in combination on microalgal lipids and biomass production is a scarcely exploited area. In this study, a novel approach involving individual as well as combined metals and EDTA stress under N and P limited conditions for lipid enhancement in microalgae was investigated. Microalgal growth physiology, photosynthetic performance, biochemical composition (lipid, carbohydrate and protein) and expression of selected key genes involved in photosynthesis (rbcL) and fatty acid biosynthesis (accD) were studied both under selected individual and combined stress conditions. Out of seven microalgal isolates obtained during the initial isolation and screening process, two strains were selected for lipid enhancement study based on their growth rates, biomass yields, lipid content and lipid productivities. The strains were later identified as Acutodesmus obliquus and Chlorella sorokiniana based on both morphological characteristics and phylogenetical analysis. The selected strains were thereafter subjected to different cultivation conditions involving varying metal, EDTA and nutrient stress conditions. A significant increase in lipid productivity was observed when the concentrations of Fe, Mg and EDTA were increased and Ca was decreased to degree in the N and P stress BG11 medium. For A. obliquus, a highest lipid productivity of 80.23 mgL-1d-1 was achieved with the developed strategy under limited N (750 mg L-1) condition which was 2.18 fold higher than BG11 medium and 1.89 fold higher than N limited condition alone. Similarly, for C. sorokiniana, highest lipid productivity of 77.03 mgL-1d-1 was achieved with the developed strategy under limited N (500 mgL-1) and P (10 mgL-1) which was 2.67 fold higher than BG11 medium and 2.35 fold higher than N and P limited condition alone. For both the microalgal strains, Fe was the most significant trace metal affecting their lipid productivity. These above observations were further confirmed through photosynthetic performance analysis and gene expression studies. At mid log phase, 6.38 and 5.15 fold increases in the expression levels of rbcL gene were observed under combined stress (OCMS+OE) as compared to the control (BG11) condition in A. obliquus and C. sorokiniana respectively. This also resulted in an increased expression level of accD gene involved in lipid biosynthesis to 10.25 fold and 9.79 fold in A. obliquus and C. sorokiniana respectively at late log phase. The results from expression studies of rbcL and accD genes were in compliance with biomass yields, photosynthetic performance, protein yield and lipid productivities for both the strains under different cultivating conditions. The universal applicability of the above strategy was confirmed by applying it to five other microalgae strains isolated in this study which resulted in considerable increase in their overall lipid productivity under optimized conditions. Attempts were made to scale up the lab scale study to open circular pond (3000L) cultivation for A. obliquus. Results showed a 2.08 fold increase in lipid productivity under optimized conditions compared to the control, which emphasizes the scalability of the developed strategy even under uncontrolled conditions. In conclusion, the developed combined metal and EDTA stress strategy not only assisted in alleviating the biomass productivity but also enhanced the lipid accumulation which resulted in overall increased lipid productivity under N and P limited condition. Furthermore, the improved carbohydrate and protein productivities observed with the developed lipid enhancement strategy make it suitable for biorefinery approach with multiple products. An improvement in lipid profile and high biodiesel conversion were also observed with this universally applicable and scalable lipid enhancement strategy confirming their potential applicability during large scale cultivation for biodiesel production.
An assessment of the conformational profile of bombesin and its mammalian analogues using computational chemistry methods
(2011) Sharma, Parul; Bisetty, Krishna
Understanding the dynamics and mechanism of protein folding continues to be one of the central problems in molecular biology. Peptide folding experiments characterize the dynamics and molecular mechanisms of the early events of protein folding. However, generally the highly flexible nature of peptides makes their bioactive conformation assessment reasonably difficult as peptides fold at very fast rates experimentally, requiring probing on the nanosecond time resolution. On the other hand, determining the bioactive conformation of biological peptides is a requirement for the design of peptidomimetics in computer-aided drug design. Peptides offer a unique opportunity to bridge the gap between theoretical and experimental understanding of protein folding. Therefore, the present work focuses on the exploration of the conformational space of biologically active neuropeptides with the aim of characterizing their conformational profile. Specifically, bombesin, neuromedin B (NMB) and neuromedin C (NMC), have been chosen for the current investigations. These peptides are widely distributed in the gastrointestinal tract, spinal cord and brain, and are known to elicit various physiological effects, including inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. These peptides act as a growth factor in a wide range of tumours including carcinomas of the pancreas, stomach, breast, prostate, and colon. This work is intended to get some insight into the performance of different procedures used to explore the configurational space to provide an adequate atomic description of these systems. Different methodological studies involving utilization of molecular dynamics (MD), multicanonical replica exchange molecular dynamics (REMD) and simulate annealing (SA) are undertaken to explore the folding characteristics and thermodynamics of these neuropeptides. MD and REMD calculations on bombesin peptide have revealed its dual conformational behaviour never discovered before and is described in chapter 3. These results explain the known structure-activity studies and open the door to the understanding of the affinity of this peptide to two different receptors: BB1 and BB2. In the case of NMC, REMD calculations are carried out in explicit and implicit solvents, using the Generalized Born (GB) surface area, and are then complemented with two additional MD simulations performed using Langevin and Berendsen thermostats. The results obtained clearly reveal that REMD, performed under explicit solvent conditions, is more efficient and samples preferentially folded conformations with a higher content of  and γ turns. Moreover, these results show good agreement with the experimental results supporting the role of two -turns for its biological action, as reported in the literature. Finally, the results obtained from MD, REMD and SA calculations on NMB reveal that the peptide has a tendency to adopt both turns and helices suggesting its two different receptor recognizing and binding conformations during its biological action. Hence, the present work provides comprehensive information about the conformational preferences of neuropeptides which could lead to a better understanding of their native conformations for future investigations and point the way towards developing their new antagonists.
Bioactive compounds from South African plants against Mycobacterium tuberculosis
(2016) Singh, Alveera; Odhav, Bharti; Coovadia, Yacoob
Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna.
Biochemical characterization of selected carbohydrases from Beauveria bassiana and their potential applications
(2021-07) Amobonye, Ayodeji Emmanuel; Pillai, Santhosh Kumar Kuttan; Singh, Suren
Different 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.
Biodiesel production from microalgae by enzymatic transesterification
(2015) Guldhe, Abhishek; Bux, Faizal; Permaul, Kugen; Mutanda, Taurai
Main focus of this study is to investigate the enzymatic-conversion of microalgal lipids to biodiesel. However, preceding steps before conversion such as drying of microalgal biomass and extraction of lipids were also studied. Downstream processing of microalgae has several challenges and there is very little literature available in this area. S. obliquus was grown in the pilot scale open pond cultivation system for biomass production. Different techniques were studied for biomass drying and extraction of lipids from harvested microalgal biomass. Effect of these drying and extraction techniques on lipid yield and quality was assessed. Energy consumption and economic evaluation was also studied. Enzymatic conversion of microalgal lipids by extracellular and whole cell lipase application was investigated. For both applications, free and immobilized lipases from different sources were screened and selected based on biodiesel conversion. Process parameters were optimized using chosen extracellular and whole cell lipases; also step-wise methanol addition was studied to improve the biodiesel conversion. Immobilized lipase was studied for its reuse. Final biodiesel was characterized for its fuel properties and compared with the specifications given by international standards. Enzymatic conversion of microalgal lipids was compared with the conventional homogeneous acid-catalyzed conversion. Enzymatic conversion and chemical conversion were techno-economically investigated based on process cost, energy consumption and processing steps. Freeze drying was the most efficient technique, however at large scale economical sun drying could also be selected as possible drying step. Microwave assisted lipid extraction performed better compared to sonication technique. Immobilized P. fluorescens lipase in extracellular application and A. niger lipase in whole cell application showed superior biodiesel conversion. The extracellular immobilized P. fluorescens lipase showed better biodiesel conversion and yields than the immobilized A. niger whole cell lipase. Both the enzyme catalysts showed lower biodiesel conversion compared to conventional chemical catalyst and higher processing cost. However, techno-economic analysis showed that, the reuse potential of immobilized lipases can significantly improve the economics. Fewer purification steps, less wastewater generation and minimal energy input are the benefits of enzymatic route of biodiesel conversion. Microalgae as a feedstock and lipase as a catalyst for conversion makes overall biodiesel production process environmentally-friendly. Data from this study has academic as well as industrial significance. Conclusions from this study form the basis for greener and sustainable scaling-up of microalgal biodiesel production process.
Characterisation of Opuntia phenolic extracts and enzymatic modification of selected compunds
(2019) Aruwa, Christiana Eleojo; Kudanga, Tukayi; Amoo, Stephen O.
Opuntia species are utilised as local medicinal interventions for chronic diseases and as food sources. The phytochemical profile varies within and across Opuntia species and has been related to differences in cultivar and geographical location. Macromolecular antioxidant (MA) fractions are also largely ignored from most conventional extractive processes compared to the well-known extractable polyphenol fractions. This study characterised subtropical spineless cladode, fruit pulp and peel extracts and selected phenolic compounds for enzymatic modification using a laccase from Trametes pubescens. MA extracts were also characterised in comparison with extractable fractions. The effects of drying methods and extraction solvent on extract yields and bioactivities were also studied. Extracts were assayed for phenolic content and antioxidant activities were determined using standard 2,2’-diphenyl-1-picrylhydrazyl (DPPH), 2,2,-azinobis3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and ferric reducing antioxidant power (FRAP) assays. Antimicrobial activities and mode of antibacterial action were assessed against type-bacterial cultures. Minimum inhibitory concentration (MIC) values were recorded for the extracts and compounds. Compound profiling was achieved using liquid chromatography-time of flight-mass spectrometry (LC-TOF/MS) in negative ionisation mode. Antibacterial and antioxidant activities were higher in MA, hydrolysed and hydroalcoholic cladode and fruit extracts than in aqueous fractions. Ethanolic, methanolic and hexane extracts of freeze-dried Opuntia cladode, MA and peel samples showed higher total phenolic content, and in vitro antioxidant and antimicrobial activities than the oven-dried extracts. Cladode extracts inhibited growth of both Gram-positive and Gram-negative microorganisms (MIC range of 25 to 250 mg/mL). Likewise, fruit extracts inhibited both Gram-positive and Gram- negative microorganisms (MIC range of 2.5 to 18.75 mg/mL). Cladode and fruit extract profiles showed the presence of mainly phenolic acids and flavonoid derivatives. Isovitexin 7-O- xyloside-2"-O-glucoside, polyhydroxypregnane glycoside and neohancoside C in Opuntia cladode, and pinellic acid in Opuntia fruit were identified for the first time in this study. Some compounds, however, remained unidentified. Thereafter, selected Opuntia cladode and fruit phenolic compounds (isorhamnetin and luteolin) were used for enzymatic (laccase) transformation after preliminary screening reactions. Laccase-catalysed oxidation of luteolin in a monophasic system containing sodium acetate buffer (pH 5.0) and ethanol (50%, v/v) as co- solvent, resulted in the production of a dimer (m/z 569, M=570). Using a similar approach, oxidative coupling of isorhamnetin produced two main products, IP1 which was a dimer (m/z 629, M=630) and IP2 (m/z 457, M=458) which was most likely a result of coupling of an oxidative cleavage product and the isorhamnetin monomer. Dimers showed up to two-fold improvement in antioxidant and antimicrobial activities, compared to their respective substrates. The synthesised products showed a bactericidal mode of action as demonstrated by time-kill and bacterial cell integrity assays. The bactericidal action was further confirmed by scanning electron microscopy (SEM) which showed that treatment of bacterial cells with the synthesised compounds resulted in deformed, pitted, broken or fragmented cells, indicating strong bactericidal action. In conclusion, this study showed that Opuntia fruit pulp, peel and cladode extractable and MA extracts have potential as sources of phenolic compounds with antioxidant and antimicrobial activities. Laccase catalysis has potential to transform the phenolic compounds into coupling products with higher biological activities. The synthesised products have potential for application in the food, nutraceutical and other relevant industries.
Characterization and application of bambara groundnut starch-lipid complexes
(2017) Oyeyinka, Samson Adeoye; Amonsou, Eric Oscar; Singh, Suren
Bambara groundnut (Vigna subterranea) is an indigenous underutilised leguminous crop to Africa. It is a good source of protein and carbohydrate including starch. Bambara groundnut is a traditional crop grown mainly for subsistence in Southern Africa. Bambara groundnut has the advantage of being drought tolerant and can thrive in hot temperatures and poor soil conditions. Therefore, it has great potential as an alternative crop to soya bean and peanuts for cultivation and utilisation. Bambara groundnut starch can potentially be used for various industrial applications. However, native starches are not suitable for most industrial applications, hence the need for modification. Bambara groundnut starch has been previously modified using physical and chemical modification methods. Natural alternatives such as the use of lipids are being sought to modify starches due to the associated risk with chemically modified starch. In this research, Bambara groundnut starch was modified with lipids to improve functional properties, utilisation and application. Specifically, the physicochemical properties of native Bambara groundnut starch obtained from five Bambara groundnut genotypes and three landraces (maroon, brown and cream) were determined. Bambara groundnut starch was modified with lipids (palmitic acid, stearic acid, oleic acid, linoleic acid and lysophosphatidylcholine) and the physicochemical properties of the modified starch were investigated. Further, the influence of high-pressure homogenization on complexation of Bambara groundnut starch with lipids was assessed in comparison with maize and potato starches. Lastly, an application of modified Bambara groundnut starch in biofilm production was also studied. Bambara groundnut landraces generally showed higher amylose contents (approx. 33%) than the genotypes (approx. 28%). Differences were observed in the crystalline patterns of these starches. Bambara groundnut genotypes exhibited the C-type-crystallinity, while the landraces showed the unusual A-type pattern. In terms of functionality, landrace starches showed better swelling than the genotypes. Subsequent studies on modification used maroon Bambara groundnut starch since the amylose content was higher than other landraces and there was a consistent supply of the grains during the period of the study. Generally, Bambara groundnut starch showed higher complexing ability with all the lipids than maize and potato reference samples. These differences in complexing ability among the starches could be due to the variation in amylose contents (Bambara groundnut starch: 31.5%, maize: 22.5% and potato: 24.6%). Fatty acids complexed better with Bambara groundnut starch than lysophosphatidylcholine, which could be due to the structural differences in comparison with the lysophosphatidylcholine molecule. The number of fatty acid in the glycerol backbone and the additional steric hindrance of the polar phosphatidic acid group in the lysophosphatidylcholine may have reduced its complexing ability. Among the fatty acids, palmitic acid complexed better than stearic and the unsaturated fatty acids, possibly due to its short chain length compared to other fatty acids. Bambara groundnut starch showed reduced peak and setback viscosities in the presence of stearic acid, linoleic acid and lysophosphatidylcholine, suggesting the formation of V-amylose complex. Bambara groundnut starch pasted with lipids displayed reduced gelling ability compared to their unmodified counterparts. XRD studies of freeze-dried paste revealed peaks at 2Ѳ = 7.4, 12.9 and 19.9o confirming the formation V-amylose complexes in Bambara groundnut starch. Modification of Bambara groundnut starch with lipids resulted in reduced digestibility. High-pressure homogenization significantly increased the complexing ability of Bambara groundnut starch with lipids. Homogenized Bambara groundnut starch-lipid complexes generally exhibited higher complex index than their unhomogenized counterparts. The higher complexing ability could be attributed to the effect of high-pressure which may have enhanced greater dispersion of lipids in the starch-water system. X-ray diffraction studies also revealed the formation of higher complexes as shown by high intensities at peaks (2Ѳ= 7.4, 12.9 and 19.9o) corresponding to V-amylose complexes. Bambara groundnut starch-lipid complexes displayed significantly higher melting temperatures (95.74-103.82oC) compared to native uncomplexed starch (77.32oC). Homogenized Bambara groundnut starch complexes were non-gelling while the unhomogenized types produced weak gels, with G′ ˃ G″ in the range of 0.1- 10 Hz. Complexation of Bambara groundnut starch with lipids using high-pressure homogenization may be employed in the production of modified starch with non-gelling properties and higher thermal stability suitable for certain industrial application, such as fat replacers in mayonnaise, frozen foods and desserts for a better mouth feel. The physicochemical and mechanical properties of biofilm prepared from Bambara groundnut starch modified with stearic acid at varying concentrations of 0, 2, 4, 6, 7 or 10% were further studied. By SEM, Bambara groundnut starch films containing stearic acid (˃ 2%) showed a progressively rough surface compared to those with 2% stearic acid and the control. The addition of 2% stearic acid to Bambara groundnut starch film reduced water vapour permeability by approximately 17%. However, mechanical properties of starch films were generally negatively affected by stearic acid. Bambara groundnut starch film may be modified with 2% stearic acid for improved water vapour permeability and thermal stability with minimal effect on tensile strength.
Characterization and modification of Bambara groundnut globulin fractions for the enhancement of functional properties
(2023-05) Alabi, Opeyemi Olaitan; Amonsou, Eric Oscar
There is a growing interest in the utilization of leguminous grain proteins for food and industrial applications. Bambara groundnut is a xerophyte pulse grain and a potential source of protein that can replace soybean protein, a trusted and widely used food ingredient in the food industry. However, the use of Bambara groundnut proteins including the subunits (legumin and vicilin) is limited in food applications. The understanding of the composition of Bambara groundnut proteins at the subunit level is vital to unlocking their potential and facilitating utilization. In this study, Bambara groundnut globulin is characterized in terms of the structures, composition, and physicochemical properties at the subunit level, and then modified using atmospheric plasma and enzymatic hydrolysis. Bambara globulin consisted of about 70% vicilin, whilst legumin protein was found in relatively low quantity. Gel electrophoresis revealed three major protein bands in globulin similar to vicilin with predominant β-sheet structures. The presence of a disulfide bond was also revealed in legumin. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components. Fluorescence and hydrophobicity data suggested a folded structure for the legumin fraction dominated by the helical secondary structure compared to the vicilin fraction. Bambara proteins contain an appreciable amount of methionine that is even higher than the FAO/WHO recommended value. Bambara vicilin had the highest amount of negatively and positively charged amino acids compared to globulin and legumin. This coincides with its high solubility profile (approximately 82% at pH 3.5). The least gelation concentration (LGC) significantly increased in the order of globulin (8%) < legumin (18%) < vicilin (20%) at pH 7. Bambara groundnut proteins formed weakly structured gels as indicated by the frequencydependent behaviours of both the storage (Gʹ) and loss (Gʺ) moduli with a difference of lesser than 1 log cycle. The highest Gʹ of vicilin gel indicated more firmness of the gel compared to the gel formed by globulin and legumin. The sol-gel transition temperatures increased in the order of globulin (40℃) < legumin (50℃) < vicilin (80℃). The Gʹ and Gʺ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet-like structure compared to globulin and legumin. Emulsifying stability of the proteins significantly differed (p < 0.05) at pH 7. The foaming capacity of the vicilin fraction was significantly (p < 0.05) higher than that of the storage protein at pH 3, 7, and 9. Atmospheric cold plasma-activated water (PAW) and enzymatic modification of Bambara groundnut globulin were further assessed. The cold plasma treatment resulted in the loss of the helical structure of Bambara globulin. The plasma treatment increased the hydrophobicity of Bambara globulin indicating an unfolded structure that was also reflected in the observed redshift in fluorescence intensity. No major changes were observed in gel electrophoresis, protein surface charge, and solubility profiles, except for about a 20% reduction in the glutamic acid content of the amino acid profile. Bambara globulin had reduced emulsifying capacity after treatment with PAW. However, foaming capacities were significantly better and stable at up to 15 mg protein/mL. Hydrolysates produced from Bambara groundnut globulin and vicilin, respectively using a combination of pepsin and pancreatin were investigated for ACE and renin inhibitory activities. The hydrophobic amino acid residues in both globulin and vicilin hydrolysates are high, improving the entry of their peptides into target organs via hydrophobic associations. Surface hydrophobicity increased significantly (p<0.05) with an increase in peptide size from <1 to <3 kDa with that of vicilin hydrolysate and membrane fractions having the highest values. The low molecular weight peptide (<1 kDa) membrane fractions from globulin at 1 mg/mL exhibited significantly higher (p<0.05) invitro ACE inhibitory activities compared to vicilin hydrolysate and its fractions. However, higher molecular peptide fraction (<3 kDa) favoured renin inhibitory activity at the same concentration. Vicilin is the major protein fraction of Bambara groundnut globulin. Bambara groundnut globulin was stabilized by disulfide linkages from the legumin, a minor fraction of the storage protein. Bambara globulin and its subfractions formed a weakly structured gel with the dominance of an elastic structure. The dominancy of the β-sheet structure in vicilin protein and the high crosslink density of the vicilin gel could be related to the firmness of the vicilin gel. The variations in the gel points of Bambara globulin and the subfractions were linked to the differences in their amino acid and subunit composition, the thermal unfolding properties of the protein fractions, and the presence of disulfide linkages. Modification of Bambara groundnut globulin using cold plasma-activated water treatment and enzymatic hydrolysis, respectively increased the hydrophobicity of the protein and influenced the emulsifying and foaming properties and the invitro angiotensin-converting enzyme (ACE) and renin inhibitory activities. Therefore, Bambara groundnut globulin could be a potential functional ingredient in the food system. The low molecular weight peptide (<1 kDa and <3 kDa) membrane fractions from globulin have the potential to serve as functional bioactive peptides against hypertension.
Chemoprotective action of natural products on cultured human epithelial cells exposed to aflatoxin B1
(2005) Reddy, Lalini; Odhav, Bharti
Previous studies indicate that a mutation in the non-oncogenic p53 gene is epidemiologically linked to human HCC (Ozturk, 1991; Chan et al., 2003). Hsu et al. (1991) found this link in Chinese, South African and Asian patients and Hollstein et al. (1993) found the same gene mutation in Taiwanese patients. The incidence of these aberrations is reported to be about 20- 50% in HCC’s (Kishimoto et al., 1997). There is sufficient evidence to indicate that carotenoids in addition to their well known antioxidant properties (Paiva and Russel, 1999), also affect intercellular communication, immune responses, neoplastic transformations and growth control, and cellular levels of enzymes that detoxify carcinogens (Zhang et al., 1991; Brockman et al., 1992; Pryor et al., 2000). To date studies carried out have used the rat (Foote et al., 1970; Gradelet et al., 1998) and the mule duckling model (Cheng et al., 2001) to show the protective effect of these carotenoids against AFB1 exposure. Of the well known carotenoids, lycopene and beta- carotene occur in abundance in fruits and vegetables and are safe for human consumption. Aflatoxin B1 frequently induces mutations of the p53 gene which is linked to HCC. Although there is much evidence from epidemiological studies linking the beneficial aspects of carotenoids to the prevention of cancer, the cellular and molecular mechanisms need to be understood in order to implement large scale intervention strategies to prevent AFB1 induced carcinoma. The use of chemical or dietary interventions to alter the susceptibility of humans to the actions of carcinogens and to block, retard or reverse carcinogenesis is an emerging chemoprotective strategy for disease prevention (Abdulla and Gruber, 2000; Kensler et al., 2003; Bingham and Riboli, 2004). Chemoprotection by natural products involves maintaining cellular integrity, preventing DNA alterations, activation of p53 suppressor protein and apoptosis. The aim of this study was thus to investigate the cellular and molecular mechanisms by which beta-carotene and lycopene may prevent the AFB1-induced toxic changes in human hepatocytes. In order to achieve this aim, the following objectives were set out: i. To optimise an in vitro system for the evaluation of AFB1 damage to cultured hepatocytes. ii. To determine the biochemical protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by measuring the mitochondrial activity, cell viability and ROS levels using appropriate enzyme assays and flow cytometry. iii. To determine the cellular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by studying the morphological changes at the structural and ultrastructural levels using phase contrast light and electron microscopy respectively. iv. To determine the molecular protection offered by beta-carotene and lycopene to AFB1-exposed hepatocytes, by detecting apoptotic bodies as genomic markers and measuring the levels of p53 protein and AFB1-N7-guanine adducts produced.
Cloning, expression, characterization and application of cyanase from a thermophilic fungus Thermomyces lanuginosus SSBP
(2018) Ranjan, Bibhuti; Singh, Suren; Pillai, Santhosh Kumar Kuttan; Permaul, Kugen
Rapid industrialization and proliferative development of chemical and mining industries have resulted in increased global pollution and environment deterioration, due to the release of numerous toxic substances. This has extreme relevance in the South African context due to the high amount of cyanide used by local mines in comparison to that utilized globally. This has created the need for the development of novel approaches viz., using microbial enzymes for its remediation because of lower process times, lower energy requirements, and their cost-effective, nontoxic and eco-friendly characteristics. From previous work in our lab, the whole genome sequencing and secretome analysis of the industrially-important fungus Thermomyces lanuginosus SSBP revealed the presence of a cyanate hydratase gene and enzyme, respectively. Cyanate hydratase detoxifies cyanate in a bicarbonate-dependent reaction to produce ammonia and carbon dioxide. The cyanate hydratase gene (Tl-Cyn) from this fungus was therefore cloned, overexpressed, purified, characterized and its potential in cyanate detoxification has also been evaluated. The recombinant cyanate hydratase (rTl-Cyn) showed high catalytic efficiency, suggesting that it could be used for bioremediation applications. Though, cyanate hydratase catalyzes the decomposition of cyanate, the requirement of bicarbonate is a major drawback for its effective utilization in large-scale applications. Hence, a novel strategy was developed to limit the bicarbonate requirement in cyanate remediation, by the combinatorial use of two recombinant enzymes viz., cyanate hydratase (rTl-Cyn) and carbonic anhydrase (rTl-CA) from T. lanuginosus. This integrative approach resulted in the complete degradation of cyanate using 80% less bicarbonate, compared to the cyanate hydratase alone. In addition, co-immobilization of these recombinant enzymes onto magnetic nanoparticles and evaluation of their potential in bio-remediation of cyanurated wastes together with their reusability resulted in more than 80% of cyanate detoxification in wastewater samples after 10 cycles. Another novel strategy was also developed for the simultaneous removal of heavy metals and cyanate from synthetic wastewater samples, by immobilizing the rTl-Cyn on magnetic multi- walled carbon nanotubes (m-MWCNT-rTl-Cyn). The m-MWCNT-rTl-Cyn simultaneously reduced the concentration of chromium (Cr), iron (Fe), lead (Pb) and copper (Cu) by 39.31, 35.53, 34.48 and 29.63%, respectively, as well as the concentration of cyanate by ≥85%. The crystal structure of Tl-Cyn in complex with inhibitors malonate or formate at 2.2 Å resolution was solved for the first time to elucidate the molecular mechanism of cyanate hydratase action. This structure enabled the creation of a mutant enzyme with ~1.3-fold enhanced catalytic activity as compared to the wild-type Tl-Cyn. In addition, the active site region of Tl-Cyn was found to be highly conserved among fungal cyanases. Information from the 3D structure could enabled the creation of novel fungal cyanases, which may have potential for biotechnological applications, biotransformation and bioremediation.
A combined metagenomics and metatranscriptomics approach to assess the occurrence and reduction of pathogenic bacteria in municipal wastewater treatment plants
(2023-05) Conco, Thobela; Bux, Faizal; Kumari, Sheena; Stenström, Thor-Axel; Ismail, Arshad
The emergence and spread of pathogens, antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG) through insufficiently treated effluents from wastewater treatment plants (WWTP) pose a risk to human health and the environment. The present study focused on assessing the occurrence, prevalence and fate of dominant pathogenic bacteria, ARGs and mobile genetic element (MGE) in different WWTPs in Durban, Kwa-Zulu Natal, South Africa. The samples were taken from three wastewater treatment plants with different configurations, including trickling filter (TF), biological nutrient removal (BNR), and conventional activated sludge processes (CAS). Total genomic DNA and RNA were extracted from the samples for metagenomic and tracriptomic analysis. A total of 23 pathogenic bacterial genera, including enteric and emerging opportunistic pathogens, were detected in the samples. Acinetobacter spp. and Aeromonas spp. were the predominant pathogens in influent metagenomes, while Escherichia coli and Acinetobacter spp. dominated influent transcripts. Based on Shannon-Wiener indices, the diversity of bacteria increased from influents to final effluents in two treatment plants. ARGsthat confer resistance to aminoglycosides, beta-lactamases, tetracycline and sulfonamides were abundant in both influent and effluent samples. Results further exposed that MGE-ARG associations were the main drivers of ARG persistence to final effluents. This included 5 plasmids: R338-R151 (sulI), pRH-1238 (strB), pPM91 (aadA), pRH-1238 (aadA4-5), pRH-1238 (sulII); two class 1 integrons (aadA and arr) and 1 transposon Tn4351 (tetX). In transcripts, the MGE-ARG associations showed two plasmids: pRH-1238 (aadA) and pPM91 (aadA) and one hybrid plasmid R338-R151 (sulI). The study investigated the potential impact of operational parameters (dissolved oxygen (DO), total suspended solids (TSS), pH and temperature) on selected bacterial pathogens (Aeromonas spp, Acinetobacter spp., Pseudomonas aeruginosa and Klebsiella pneumoniae) and their fates at different stages of the three WWTPs. Principal component analysis (PCA) showed that temperature, DO, and pH were the most relevant factors influencing pathogen abundance. Among the studied pathogens, Acinetobacter spp. was the most prevalent in the influent samples, followed by Aeromonas spp. As for the aeration samples, Aeromonas spp. was dominant in WWTP1 (CAS configuration) and WWTP2 (BNR configuration), while Acinetobacter spp dominated in WWTP3 (BNR configuration). Acinetobacter spp., Aeromonas spp., and Pseudomonas aeruginosa were the dominant ones in secondary effluents, with their dominance varying across the sampling period. In the final treated effluent, Acinetobacterspp., Aeromonas spp., and P. aeruginosa were dominant, with their dominance varying from sample to sample. Additionally, free living amoebas (FLA) were also investigated for their contribution to the propagation and persistence of pathogens in secondary and final effluents. Using the conventional isolation technique, FLAs were isolated from different samples. The internalized bacteria and ARGs were further identified using metagenomic analysis. Metagenomic profiles identified nine species belonging to Acanthamoeba and two species belonging to Entamoeba. A. castellini was the most prevalent dominant species detected in effluent and final effluent samples of all three WWTPs. P. aeruginosa, S. maltophilia, A. spanius, C. testosteroni, and E. cloacae were the most dominant bacterial endosymbionts detected. Among these, S. maltophilia and P. stutzeri were detected in FLAs isolated from the final treated effluents indicating their prevalence in the chlorinated effluents. The presence of ARGs within FLAs were also ascertained. Genes conferring resistance to aminoglycosides (aadA); trimethoprim (dfrA15 and dfrA5); sulfonamides (Sul1 and SulII), macrolides (msrA, mphC); rifamycin (Arr); quinolones (qnrE1) and tetracyclines (TetA and TetG). SulI, dfrA5, AadA, dfrA15, SulII, TetA, TetG and qnrE1 were among the resistance genes that persisted into final effluents. The results of this study have contributed significantly to our current understanding of pathogens, particularly the dominant pathogens and the role of FLAs in the dispersal of pathogens and ARGs into the environment via WWTPs. The study also indicatesthat the conventionally treated effluents may still contain human pathogens, ARGs, and MGEs, which may contribute to the propagation of emerging pathogens and antibiotic resistance in the receiving environment.
Composition and functional bioactive properties of bambara groundnut protein and hydrolysates
(2016) Arise, Abimbola Kemisola; Amonsou, Eric Oscar; Ijabadeniyi, Oluwatosin Ademola
Bambara groundnut (Vigna substerranea) is an indigenous legume of African origin which is currently experiencing a low level utilisation. It is tolerant to drought and can grow under poor soil conditions in which other lucrative crops such as groundnut cannot grow. Bambara is a good source of protein comparable to that of cowpea and slightly lower than soya bean. In order to assess the potential use of bambara protein as a functional ingredient in food systems and as an important ingredient for the formulation of therapeutic product, the knowledge of its protein composition, structure and functionality becomes important. The main goal of this thesis was to determine the composition and bioactive properties of bambara protein and its hydrolysates. Specifically, a comparative study was carried out on the protein content, yield and functional properties of protein concentrates prepared from three different bambara landraces using different extraction methods (Salt solubilisation and Acid precipitation). There was no significant difference in protein content, yield and functional properties of the landraces. However, the method of extraction had an influence on their physicochemical and functional properties. Acid precipitation produced bambara protein concentrates with high protein content and yield (79% and 52% respectively) when compared to salt solubilisation (protein content - 57% and yield - 25%). Protein concentrates prepared through salt solubilisation method exhibited better functional properties in terms of water absorption capacity, oil absorption capacity, foaming capacity, foaming stability and emulsion activities when compared to concentrates obtained through acid precipitation. Furthermore, the composition of bambara proteins produced through isoelectric precipitation was determined. SDS PAGE revealed four major bands; a broad band at 55 kDa which was analysed to be vicilin, two medium bands at 62 kDa and 80 kDa and a high molecular weight (HMW) protein at 141 kDa. Further investigation of bambara protein revealed vicilin (55 kDa) with two sub units as the major protein in bambara and this was also confirmed by the proteomic map. The proteomic map revealed acidic amino acids as the major protein of bambara which is characteristic of vicilin, the map also showed that there were differences in the number of spots across the landraces with 77 spots matching each other. Circular dichroism spectroscopy exhibited reductions in α-helix, and β-pleated sheet conformations as pH varies. In addition, the tertiary structures as observed from the near-UV CD spectra were also influenced by shifts in pH conditions. Differential scanning calorimetry thermograms showed two endothermic peaks at around 67 and 81oC respectively. These can be attributed to thermal denaturation of vicilin and the HMW protein. Subsequent studies used isolates from red bambara since the composition of the landraces were similar. Bambara protein isolate was subjected to enzymatic hydrolysis using three proteases (alcalase, pepsin and trypsin) to produce various bambara protein hydrolysates (BPHs). BPHs were investigated for antioxidant and antihypertensive activities. The in vitro structural and functional characteristics of bambara protein and its enzymatic protein hydrolysate revealed that bambara groundnut possessed antioxidant properties against a variety of physiologically relevant free radicals. High surface hydrophobicity and the molecular size of the peptide seem to be important for scavenging of hydroxyl radicals, ferric reducing power and metal chelation. BPHs and peptide fractions were able to scavenge DPPH radicals with greater affinity for smaller size. Less than 1 and 1-3 kDa pepsin fraction was able to scavenge DPPH radical more than glutathione, BPHs and its fractions scavenge ABTS•+ three folds than the isolate. Scavenging of superoxide radicals was generally weak except for 5-10 kDa peptide fractions. All BPHs inhibited linolenic acid oxidation with greater affinity for the lower molecular size peptide. BPHs showed potential antihypertensive properties because of the in vitro inhibition of activities of angiotensin converting enzyme (ACE) and renin inhibition. The molecular size had significant effect on the ACE inhibitory properties with low molecular weight peptide (<1 kDa) fractions exhibiting significantly higher (p<0.05) inhibitory activities. However, enzyme type had synergistic effects on renin inhibition with alcalase hydrolysate showing highest inhibition at 59% when compared to other hydrolysates and their membrane fractions. The fractions with <1 and 1-3 kDa peptides showed a higher potential as antihypertensive and antioxidant peptides. Based on this study, incorporation of bambara protein isolate as an ingredient may be useful for the manufacture of high quality food products. Likewise, the bambara protein hydrolysates, especially the <1 kDa and 1-3 kDa fraction represent a potential source of bioactive peptides in formulating functional foods and nutraceuticals.