Theses and dissertations (Applied Sciences)

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The accumulation of heavy metals by aquatic plants
(2003) Maharaj, Saroja; Moodley, Kandasamy Govindsamy; Southway-Ajulu, F. A.; Baijnath, Himansu
The pollution of water bodies by heavy metals is a serious threat to humanity. The technique known as phytoremediation is used to clean up these polluted water bodies. The accumulation of heavy metals by aquatic plants is a safer, . cheaper and friendlier manner of cleaning the environment. The aquatic plants -studied in this project are A.sessilis, P.stratiotes, R.steudelii and T.capensis. The accumulation of heavy metals in aquatic plants growing in waste water treatment ponds was investigated. The water, sludge and plants were collected from five maturation ponds at the Northern Waste Water Treatment Works, Sea Cow Lake, Durban. The samples were analysed for Zn, Mn, Cr, Ni, Pb and Cu using ICP-MS. In general it was found that the concentrations of the targeted metals were much lower in the water (0.002 to 0.109 mg/I) compared to sediment/sludge (44 to 1543mg/kg dry wt) and plants (0.4 to 2246 mg/kg dry wt). These results show that water released into the river from the final maturation pond has metal concentrations well below the maximum limits set by international environmental control bodies. It also shows that sediments act as good sinks for metals and that plants do uptake metals to a significant extent. Of the four plants investigated it was found that }t.sessi[ir (leaves, roots and stems) and }A.sessilis (roots and stems) are relatively good collectors of Mn and Cu respectively. These findings are described in the thesis. The concentration of heavy metals in the stems, leaves and roots of the three plants were compared to ascertain if there were differences in the ability of the plant at different parts of the plant to bioaccumulate the six heavy metals studied.
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.
Anthropometric profile and food consumption patterns of an emerging middle income community in Umlazi
(2017) Memela, Sinenhlanhla Ntokozo; Napier, Carin E.
Introduction: The nutrition transition is a global phenomenon affected by developing economic prosperity. ‘Nutrition transition’ may be defined as changes in dietary patterns and nutrient intakes occurring in individuals, families, groups of people or whole populations when the food environment and other circumstances change. Post-1994 democratic elections in South Africa, economic laws called for the de-racialization of the economy. As a result of the annulment of the apartheid laws in the 1990s entrepreneurial opportunities became available for black people. Based on the 2011 Census, the middle class constituted 8.3 million (17%) out of the 51 million population in South Africa. Blacks made up 51%, whites 34%, coloureds 9% and Indians 6%. This is a vast difference from two decades prior to this when blacks made up only 3.3% of the population, with coloureds making up 9.1% and Indians 18.5%. In a number of Sub-Saharan African studies obesity has been shown to have a significant link to socio-economic status determined by access to water and electricity, smaller numbers of members residing in one household and increased amounts of money spent on food among other factors, which all promote a tendency to leading a more Westernised lifestyle. The main objective of the study was to determine the anthropometric status and analyse the food consumption of a community of emerging middle income status in Umlazi Township (Ward 84) outside Durban in the province of KwaZulu-Natal. Methods: This study had a sample size of n=250 adult men (n=56) and women (n=194) who were heads of households or primary caregivers above the age of 19 years old with households receiving a total household income of more than R100 000.00 per annum/ more than R8000.00 per month. The study was quantitative and descriptive in nature with different tools used to measure the sample population. These included waist circumference and height and weight measurement to determine BMI and WHtR. A socio-demographic questionnaire was completed where the multi-dimensional poverty index (MPI) was established; a food frequency questionnaire (FFQ) and three 24-hour food recall were also administered to all participants; one weekend day recall and two weekday recalls. Data were analysed on SPSS and nutrient analysis done through Food Finder Version 3 Software and compared to the World Health Organisation standards recommendations for optimal health. Results: The mean age of the group was 52 years old. Slightly more than half (51.6%; n=129) of the population was unemployed. Retired participants made up 27.2% (n=68) of the population. The majority (80%) of the households indicated that between one to three members contributed to the total household income. The MPI of the Ward 84 community amounted to 9.09. The prevalence of obesity was 37.59% (n=21) among men and 76.80% (n=149) for women. Women had a higher prevalence of risk factors linked to non-communicable diseases than men. Mineral and fat soluble vitamin intake was deficient; however protein and carbohydrates were consumed in excess with means of 187.70% and 111.43% respectively. Top 20 foods consumed lists indicated that little fruit or vegetables were consumed. The average food variety score was medium indicating the consumption of between >30 but <60 different foods across the total population. A positive relationship of statistical significance was found between food variety score and income (p=0.007). Conclusion: The Ward 84 community consisted of an ageing population with households mostly supported by another member’s income from within or outside the household. The adults had sufficient education to have secured promotions in the employment arena and could afford a lifestyle similar to that of those who had always been more privileged. Although the Umlazi residents benefited from good housing and infrastructure and appeared to have averted poverty, the prevalence of overnutrition malnutrition was high and linked with an excessive intake of macronutrients. Majority (70%) of the community of Ward 84 is ‘food secure’ as indicated by the number of participants’ households indicating never having a shortage of money to buy food. This correlates negatively with the high prevalence of overweight and obesity (BMI) (p=-0.029). According to literature a persistent issue in black communities is the acceptance of an overweight body image being perceived as ‘healthy’ and an indicator of affluence. This community appears to be in the 4th stage/ pattern of the nutrition transition. It appears that a ‘westernised diet’ and lifestyle is more accessible and is embraced by this urban community as described in various literature and seen in the top 20 foods lists compiled from the 24 hour recalls. There is some oversight regarding the nutrient quality of a varied diet and achieving energy balance with macronutrient intake linked to informed nutritional choices. Suggested interventions could include the dissemination of information regarding nutrition and practical suggestions to achieve energy balance. In addition, future research could investigate the prospects of introducing participation in urban agriculture, investigate the issue of physical activity, and foster an environment for more physical activity and increase availability and access to micronutrient dense foods and investigate the development of a body mass index specifically for the black African population.
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.
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.
Assessment of the anaerobic baffled reactor for treatment of vegetable oil effluent
(2001) Frost, Lee-Anne; Tivchev, G. N.
The vegetable oil industry produces effluent containing quantities of fat, oil, sodium, phosphates as well as other pollutants. Oils and greases tend to clog sewers and pumps, thus creating difficulties within the municipal wastewater treatment works. Physico-chemical treatment methods, such as (Dissolved Air Flotation) OAF, gravity separation and the use of coagulants have been attempted providing a considerable reduction in organic loading; however, discharge standards are still not met. Thus, biological treatment methods are being sought after. Aerobic treatment has been attempted however, shock loads cause problems while running such a process. The objective of this study was to assess the efficiency of anaerobic digestion to degrade Vegetable Oil Effluent (VOE) as well as the efficiency of the Anaerobic Baffled Reactor (ABR). Anaerobic digestion involves the breakdown of organic matter by the action of microorganisms in the absence of oxygen, producing methane-rich biogas. The VOE was characterized, providing significant information on its chemical composition. It was found that the effluent had high sulphate content as well as a high COD content. High sulpahte content of wastewaters have known to promote growth of Sulphate Reducing Bacteria (SRB), which utilize the same energy source as Methane Producing Bacteria (MPB) and therefore compete for the same energy source. Sulphate and lipid reduction pretreatment experiments were carried out, using barium chloride and gravitational separation respectively. The results obtained, showed that the use of barium chloride to reduce sulphate content in VOE was successful, with significant sulphate reduction. The lipid reduction experiments however, did not show any significant lipid reduction. Batch tests were conducted in serum bottles to assess the extent of biodegradation of the VOE in its raw state as well as with reduced sulpahte content. Methanogenic toxicity tests on the raw and pretreated VOE provided a range of toxicity results. These assays are relatively simple and inexpensive. Gas production was monitored to determine the rate and extent of biodegradation. The efficiency of digestion was assessed by COD reduction. Results indicated potential inhibition of the methanogenic bacteria responsible for methane production by the
Bioconversion of biodiesel-derived crude glycerol waste to 1,3 propanediol and gellan using adapted bacterial isolates
(2013-09-19) Raghunandan, Kerisha; Permaul, Kugen
The continual growth of the global biodiesel industry has resulted in a proportional increase in crude glycerol production. The by-production of glycerol waste during the manufacture of biodiesel has, with recent research, proven to hold use as a feedstock for the production of several commodity chemicals. The conversion of glycerol may be carried out by both chemical as well as biological means. The biological conversion of glycerol surpasses chemical conversion with respect to higher yield and selectivity, normal reaction conditions and the use of cheaper biological catalysts. Many microorganisms are known to convert glycerol to different value added products. This study involved the isolation of bacteria from soil and crude glycerol from a local biodiesel plant. Isolates were then used to convert crude glycerol supplemented with salts and a nitrogen source into commercially viable products. Isolates which successfully degraded glycerol were then identified via 16S PCR. A strain of Klebsiella pneumoniae, which is a known producer of 1,3-propanediol (1,3-PDO), was isolated from soil and two strains of Sphingomonas sp., which is a known gellan producer, was isolated from biodiesel waste. Gellan is an exopolysaccharide used in the food, cosmetic and pharmaceutical industries sold commercially as a product known as Gelrite or Gelzan while 1,3-PDO is an important component of fuels and polyesters (used widely in the petroleum industry) and is currently chemically produced. Using crude glycerol for producing 1,3-PDO is a good solution from an economic as well as ecological point of view. K. pneumoniae, Sphingomonas psueudosanguinis and Sphingomonas yabuuchiae were subjected to a series of shake flask fermentations in order to determine optimal growth conditions. This microoganism was able to successfully produce significant amounts of 1,3-PDO and lactic acid using crude glycerol (80 g/l), without pre-treatment (37 and 6.8 g/l respectively). S psueudosanguinis and S. yabuuchiae were both able to produce two of the highest amounts of gellan gum than that reported by other studies using crude glycerol (80 g/l) as a sole carbon source in a minimal medium (50.9 and 52.6 g/l respectively).
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 biodiesel from Litsea glutinosa
(2014-08-08) Perumal, Alicia Ann; Odhav, Bharti
Global warming is a major concern to the world’s population. It is caused by greenhouse gases that result from the burning of fossil fuel. The fossil fuel reserves are rapidly depleting as the needs and wants of man in the world increases. Biodiesel is one of the solutions proposed to remedy this environmental crisis facing the world today. The aim of this study was to characterise the biodiesel that can be produced from the oil of Litsea glutinosa by transesterification. Biodiesel can be used in a diesel engine without modification and be produced from many different natural renewable oil sources such as algae, plants and kitchen waste material. Jatropha curcas has been identified as a potential producer of oil for biodiesel. The biodiesel properties of Jatropha curcas meet the required American Society for Testing and Materials (ASTM) standards. The fruit of Jatropha curcas contains 40.0% lipids. The oil has a saponification number of 202.6 and an iodine value of 93.0. However Jatropha curcas cannot be grown in South Africa because it is a highly invasive plant. Cetane number is the most important parameter of biodiesel. The higher the cetane value, the better the quality of the biodiesel. Oil from Jatropha curcas has a cetane number of 57.1. An alternative is the oil from Litsea glutinosa, which is found as a naturalised free forest along the South African coastline, and is also found in many Asian countries. It has many medicinal properties, however, it is not edible and hence its use for biodiesel does not add to the debate of fuel versus food production. The cetane number of oil from Litsea glutinosa is 64.79, which is ideal for ignition, and the fruit with 61.29% lipids can yield valuable quantities of biodiesel. Thus, the aim of the research was to determine the potential of Litsea glutinosa as a source of biodiesel. Furthermore, to maintain a sustainable source, Litsea glutinosa was micropropagated, and transformation of Litsea glutinosa was attempted for hairy root cultures. The Clevenger apparatus was used to extract fatty acids from dried crushed fruit of Litsea glutinosa. Fatty acids were converted to fatty acid methyl esters by transesterification. Transesterification was conducted in the presence of nitrogen and the reaction was catalysed with a mixture of methanol and sodium hydroxide (NaOH). The ratio 1 : 3 of oil to catalyst mixture was used for optimum transesterification to ensure a forward reaction and it was transferred to a separating funnel to allow the glycerol and fatty acid methyl esters to separate. GC-MS was used to determine the fatty acids. The iodine number, saponification number, acid value, viscosity, kinematic viscosity, density, specific gravity, thermostability, distillation point and sulphur content were determined. The seeds of Litsea glutinosa were germinated and tissue culture callus was produced from the seeds and leaves. The leaves and stems were used to produce hairy root cultures by inoculating them with Agrobacterium rhizogenes. Litsea glutinosa yielded 61% oil, which included 47 fatty acids in the fruit and 24 fatty acids in the seeds. The fatty acid profile of the oils indicated that the predominant fatty acids present were those that are essential for good quality biodiesel. The dominant fatty acids found in the fruit were 65.4% 9-octadecenoic acid and 13.6% hexadecanoic acid. The dominating fatty acids found in the seeds contained 36.3% 9-octadecenoic acid, 13.9%, hexadecanoic acid and 39.1%, dodecanoic acid. The iodine value was 6.3. The saponification value was 274. The acid value was 0.45 mg KOH. g-1. The viscosity was 22.48 mm2. s-1 and the kinematic viscosity was 23.84 mm2. s-1. The density was 942.69 kg. m-3 and the specific gravity was found to be 0.9 g. cm-3. The distillation temperature ranged between 52.2°C to 610.2°C. The sulphur content was found to be 383 µg. ml-1. These characteristics indicate that Litsea glutinosa can be used as a source of biodiesel, because the properties meet the required ASTM standards. However, the production of biodiesel from Litsea glutinosa has not been commercialised because the production of fuel is dependent on the fruit of the plant, which is seasonal. To overcome this, a part of this study investigated micropragation of Litsea glutinosa and transformation of Litsea glutinosa by Agrobacterium rhizogenes into hairy roots and attempts where made to determine whether fatty acid could be produced by these techniques. Callus cultures were grown on MS media and McCowns woody plant media supplemented with 1 ml BAP and 1 ml 2,4-D per 1 L of media. Callus cultures were obtained in the light. However, Litsea glutinosa resisted transformation by Agrobacterium rhizogenes.
Cloning, characterization and directed evolution of a xylosidase from Aspergillus niger
(2016) Khan, Bibi Khadija; Permaul, Kugen; Singh, Suren
β-xylosidases catalyse the hydrolyses of xylooligosaccharides into the monosaccharide sugar, xylose. In this study we report the production of xylose under different conditions in Pichia pastoris and Saccharomyces. cerevisiae, and its conversion to bioethanol using Pichia stipitis. The aim of this study was to change the characteristics of the A. niger 90196 β-xylosidase through random mutagenesis and increase expression under the control of different promoter systems in yeasts P. pastoris and S. cerevisiae. The recombinant library created through random mutagenesis was screened for changes in activity and subsequently pH and temperature stability. One variant showed an increase in enzyme expression, thermostability, and a change in amino acid sequence at residue 226. The enzyme was then cloned, expressed and characterized in P. pastoris GS115 and S. cerevisiae. β-xylosidase was constitutively expressed in P. pastoris using the GAP promoter and the inducible AOX promoter. In S. cerevisiae the enzyme was expressed using the constitutive PGK promoter and inducible ADH2 promoter systems. Enzyme functionality with the different expression systems was compared in both hosts. The GAP system was identified as the highest-producing system in P. pastoris, yielding 70 U/ml after 72 hours, followed by the PGK system in S. cerevisiae, with 8 U/ml. A 12% SDS-PAGE gel revealed a major protein band with an estimated molecular mass of 120 kDA, and the zymogram analysis revealed that this band is a fluorescent band under UV illumination, indicating enzyme activity. Stability characteristics was determined by expressing the enzyme at different pH and temperatures. Under the control of the GAP promoter in P. pastoris, enzyme activity peaked at pH4 while retaining 80% activity between pH 3 – 5. Highest activity of 70 U/ml xylosidase was recorded at 60ºC. Due to the high enzyme production in P. pastoris, the co-expression of this enzyme with a fungal xylanase was evaluated. The xylanase gene from Thermomyces lanuginosus was cloned with the GAP promoter system and expressed together with the β-xylosidase recombinant in P. pastoris. Enzyme activities of the co-expressed recombinant revealed a decrease in enzyme activity levels. The co-expressed xylanase production decreased by 26% from 136 U/ml to 100 U/ml while the xylosidase expression decreased 86% from 70 U/ml to 10 U/ml. The xylose produced from the hydrolysis of birchwood xylan was quantified by HPLC. The monosaccharide sugar was used in a separate saccharification and fermentation strategy by P. stipitis to produce bioethanol, quantified by gas chromatography. Bioethanol production peaked at 72 h producing 0.7% bioethanol from 10 g/l xylose. In conclusion a β-xylosidase from Aspergillus niger was successfully expressed in P. pastoris and was found to express large quantities of xylosidase, that has not been achieved in any prior research to date. The enzyme was also successfully co-expressed with a Thermomyces xylanase and is now capable of bioethanol production through xylan hydrolysis. This highlights potential use in industrial applications in an effort to reduce the world dependence on petroleum and fossil fuels. However the technical challenges associated with commercialization of bioethanol production are still significant.
A comparative study of supercritical fluid extraction and accelerated solvent extraction of lipophilic compounds from lignocellulosic biomass
(2022-09) Khanyile Andile; Sithole, B.B.; Paul, Vimla; Andrew, Jerome Edward
Lipophilic compounds are non-structural, heterogeneous compounds rich in terpenes, sterols, fatty acids, hydrocarbons, and glycerides. They have found widespread uses in different industries, such as the pharmaceutical, medical, cosmetic and nutraceutical sectors. They are typically extracted from wood using traditional techniques such as solvent extraction hydro- and steam- distillation. However, these techniques have several drawbacks such as long extraction times, high energy consumption, extensive solvent use and degradation of thermosensitive compounds, which are highly volatile. In this study, supercritical fluid extraction (SFE) and accelerated solvent extraction (ASE) were evaluated to extract lipophilic compounds from lignocellulosic biomass such as pinewood sawdust and Cannabis Sativa L. Their advantages of using low amounts of solvent, short extraction times and high selectivity allow them to be used as an alternative extraction technique to traditional methods. Moreover, SFE uses carbon dioxide, which is safe, cheap and readily available, and it does not alter the structure of the compounds. In contrast, ASE uses elevated temperatures and high pressures to prevent the evaporation of highly volatile compounds. In order to solve challenges from both an economic and an environmental perspective, the interaction of process conditions on lipophilic compounds extraction efficiency was modelled and optimized using Response Surface Methodology (RSM) and BoxBehnken design (BBD). The extraction variables optimized for pinewood sawdust compounds were, SFE: co-solvent (ethanol) flow rate (1-2 ml/min), carbon dioxide (CO2) flow rate (1-3 ml/min), Temperature (40-60 °C) and pressure (200-300 bar), and for ASE: static time (10-15 mins), static cycle (1-3) and temperature (80-160 °C). The process parameters were optimized, and the experimental data was modelled using RSM for statistical analysis of the BBD extraction process. The experimental data's quadratic polynomial models gave a coefficient of determination (R2 ) of 0.87 and 0.80 for ASE and SFE, respectively. The optimum conditions of ASE were temperature (160 °C), static time (12.5 mins), and static cycle (1), which resulted in a maximum yield of 4.2%. The optimum SFE conditions were temperature (50 °C), pressure (300 bar), CO2 flow rate (3.2 ml/min), and a 2 ml/min co-solvent (ethanol) flow rate that yielded 2.5% lipophilic compounds. The extraction efficiency of pinewood sawdust lipophilic compounds with ASE was higher compared to the SFE. Although ASE uses high temperatures that may degrade thermolabile compounds, the short extraction times may work in their favor since the extracts are not exposed to high temperatures for long periods. SFE uses low temperatures and long extraction times compared to ASE. Several properties affect the extraction efficiency, such as volatility, dissolving power, solubility, and fluid density of the extracting solvent. The extraction efficiency of lipophilic compounds by SFE may be affected by the supercritical fluid's solubility and differences in densities at different pressures. In ASE, the high yields were influenced by the high polarity of the solvent mixture and temperature with a short extraction time. The extraction variables optimized using RSM for Cannabis Sativa L. for SFE were pressure (200-300 bar), co-solvent (ethanol) flow rate (1-2 ml/min) and CO2 flow rate (1-2 ml/min). The R2 was determined to be 0.9108. The optimum conditions were 300 bar pressure, 1 ml/min co-solvent (ethanol) flowrate, and 2 ml/min CO2 flowrate, which gave a maximum yield of 88%. The high efficiency observed was brought by the increase in the flow rate of CO2 at high pressures, which reduces the mass transfer resistance, while the cosolvent enhanced the solvating power of CO2. The ASE had a high extraction efficiency for the pinewood sawdust lipophilic compounds. However, the method's selectivity was very low according to the results obtained by pyrolysis gas chromatography-mass spectrometry (Py-GC/MS). The thermosensitive compounds, such as terpenes, decreased from 2.01% to 1.69% upon the addition of Tetramethylammonium hydroxide (TMAH). The initial concentration of terpenes was 7.21% in pinewood sawdust by SFE. Upon the addition of TMAH, the concentration of terpenes of the pinewood sawdust decreased to undetectable levels. The initial concentration of the terpenes of Cannabis Sativa L. was 14.29% and decreased in the presence of TMAH to 0.39%. The Fourier Transform Infrared Spectroscopy (FTIR) confirmed the presence of lipophilic compounds functional groups and a fingerprint region of lipophilic compounds of pinewood sawdust and Cannabis Sativa L. Thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) showed high thermal stability (250 – 400 ℃). This research demonstrated the ability of SFE to extract lipophilic compounds from pinewood sawdust Cannabis Sativa L.
Design and operation of a laboratory scale photobioreactor for the cultivation of microalgae
(2011) Bhola, Virthie; Bux, Faizal
Due to greenhouse gas emissions from fossil fuel usage, the impending threat of global climate change has increased. The need for an alternative energy feedstock that is not in direct competition to food production has drawn the focus to microalgae. Research suggests that future advances in microalgal mass culture will require closed systems as most microalgal species of interest thrive in highly selective environments. A high lipid producing microalga, identified as Chlorella vulgaris was isolated from a freshwater pond. To appraise the biofuel potential of the isolated strain, the growth kinetics, pyroletic characteristics and photosynthetic efficiency of the Chlorella sp was evaluated in vitro. The optimised preliminary conditions for higher biomass yield of the selected strain were at 4% CO2, 0.5 g l-1 NaNO3 and 0.04 g l-1 PO4, respectively. Pulse amplitude modulation results indicated that C. vulgaris could withstand a light intensity ranging from 150-350 μmol photons m-2s-1. The pyrolitic studies under inert atmosphere at different heating rates of 15, 30, 40 and 50 ºC min-1 from ambient temperature to 800 oC showed that the overall final weight loss recorded for the four different heating rates was in the range of 78.9 to 81%. A tubular photobioreactor was then designed and utilised for biomass and lipid optimisation. The suspension of microalgae was circulated by a pump and propelled to give a sufficiently turbulent flow periodically through the illuminated part and the dark part of the photobioreactor. Microalgal density was determined daily using a Spectrophotometer. Spectrophotometric determinations of biomass were periodically verified by dry cell weight measurements. Results suggest that the optimal NaNO3 concentration for cell growth in the reactor was around 7.5 g l-1, yielding maximum biomass of 2.09 g l-1 on day 16. This was a significant 2.2 fold increase in biomass (p < 0.005) when compared to results achieved at the lowest NaNO3 cycle (of 3.8 g l-1), which yielded a biomass value of 0.95 g l-1 at an OD of 1.178. Lipid accumulation experiments revealed that the microalga did not accumulate significant amounts of lipids when NaNO3 concentrations in the reactor were beyond 1.5 g l-1 (p > 0.005). The largest lipid fraction occurred when the NaNO3 concentration in the medium was 0.5 g l-1. Results suggest that the optimal trade-off between maximising biomass and lipid content occurs at 0.9 g l-1 NaNO3 among the tested conditions within the photobioreactor. Gas chromatograms showed that even though a greater number of known lipids were produced in Run 8, the total lipid percentage was much lower when compared to Runs 9-13. For maximal biomass and lipid from C. vulgaris, it is therefore crucial to optimise nutritional parameters such as NaNO3. However, suitable growth conditions for C. vulgaris in a tubular photobioreactor calls for innovative technological breakthroughs and therefore work is ongoing globally to address this.
Determination of the relationship between epiphytes and selected filamentous bacteria in activated sludge
(2016) Conco, Thobela; Bux, Faizal; Sheena Kumari, S.K.; Stenström, Thor-Axel
Activated sludge (AS) flocs are paramount in biological treatment of wastewater, are comprised of microbial consortia with organic and inorganic material bound together by extra polymeric substances (EPS). The filamentous bacteria play a vital role in the floc formation process by providing the necessary structural support. Presence of epiphytic attachment on selected filamentous bacteria is a commonly occurring phenomenon in activated sludge samples. Different theories have been proposed to describe this phenomenon; however, not much research has been carried out to explore the profundity of the attachment. In this study, an attempt has been made to elucidate the intrinsic nature of the epiphytic attachment between the bacterial rods and filamentous bacteria based on microscopic (morphological and structural) analysis. Characterization of these epiphytes were performed using fluorescence in situ hybridization (FISH) at group level using Alpha, Beta and Gamma Proteo-bacterial probes. Morphological characteristics of filament hosts and the bacterial rods at the interface region was assessed using scanning electron microscopy (SEM). The SEM micrographs indicated that the attachment was facilitated by more than the EPS layer. Further ultrastructural examination using transmission electron microscopy (TEM) indicated a possible cell-to-cell interaction between epiphytes and the selected filaments. Fibrillar structures resembling amyloid-like proteins were observed within the filament cell targeted by the epiphytes. An interaction was apparent between the amyloid like proteins and the epiphytes as exhibited by the direction of fibrillar structures pointing towards the approaching epiphytes. Common bacterial appendages such as pili and fimbria were absent at the interface and further noted was the presence of cell membrane extensions on the epiphytic bacteria protruding towards the targeted filamentous cell. The sheath of host filaments however, remained intact and unpenetrated, during colonization. Amyloid-like fibrils at interface may potentially play the role of attachment sites for the attaching epiphytes, as attachment facilitating appendages were not visualized.
Development and optimization of technology for the extraction and conversion of micro algal lipids to biodiesel
(2015) Ramluckan, Krishan; Moodley, Kandasamy Govindsamy; Bux, Faizal
Fossil fuel reserves have been diminishing worldwide thus making them very scarce in the long term. These fuel sources and their by-products which are used commercially tend to produce large quantities of emissions. Some of them are believed to be toxic to flora and fauna. It is primarily for this reason that researchers worldwide have begun to seek out alternative sources of environmentally safe fuel. Biodiesel from algae is one of these sources that have been examined over the last few decades. Biodiesel has been produced from other plant-based material and waste oils in countries like America and Japan. However, the use of food based crops for biodiesel production has been challenged as it has an impact on food production on an international scale. Algae have only recently been investigated for their feasibility for biodiesel production on a large scale. The aim of this study was to investigate and develop technologies for biodiesel production from algae. The species of algae chosen were chlorella sp and scenedesmus sp., since they are indigeneous to Kwazulu Natal in South Africa. Samples were obtained from a local raceway pond and prepared for analysis. Drying protocols used freeze, oven and sun drying for initial preparation of the samples for analysis. Sun drying was the least energy intensive but most time consuming. At laboratory scale, oven drying was chosen as the best alternative. Lipid extraction methods investigated were the separating funnel method, the soxhlet method, microwave assisted extraction (MAE) and the expeller press. Thirteen solvents covering a range of polarities were used with the extraction methods to determine the efficiency of the solvent with these methods. Optimization of the MAE method was conducted using both the one factor at a time (OFAT) method and a design of experiment (DOE) statistical method. The shelf life of algal biomass was determined by ageing the samples for approximately three months. Direct and in-situ transesterification of lipid extracts to produce biodiesel was investigated using both acid and base catalysis. Qualitative and quantitative analyses were conducted using Fourier transform infra-red (FTIR) and gas chromatography (GC). Chemical and physical characterization of the biodiesel produced from the algal lipid extracts were compared to both local and international standard specifications for biodiesel. In terms of extraction efficiency, it was found that soxhlet and microwave assisted extraction methods were almost equally good. This was proved by the MAE method yielding an average of 10.0% lipids for chloroform, ethanol and hexane after 30 mL of solvent was used in an extraction time of 10 minutes, while the soxhlet method yielded 10.36% lipids using an extraction volume of 100 mL of solvent with an extraction time of 3 hours. Chloroform, ethanol and hexane were more efficient than the other ten solvents used. This was shown by these three solvents producing lipid quantities between 10% to 11% while all the other solvents produced lipid quantities between 2 and 10 %. The best extraction efficiency was achieved by the binary solvent mixture made up of chloroform and ethanol in a 1:1 ratio. Under the conditions optimized, this solvent ratio yielded a lipid content of 11.76%. The methods chosen and optimized for extraction are very efficient, but the actual cost of production of biodiesel need to be determined. Physical methods like the expeller press are not feasible for extraction of the type of biomass produced unless algae are pelletized to improve extraction. This will impact on the cost of producing biodiesel. The transesterification protocols investigated show that the base catalysis produced biodiesel with a ratio of saturates to unsaturates conducive to a good fuel product. The direct esterification method in this study proved to be better than the in-situ method for biodiesel production. The in-situ method was also more labour intensive. Chromatography was found to be a fast and efficient method for qualitative and quantitative determination of biodiesel. Characterization tests showed that the quality of biodiesel produced was satisfactory. It also showed that the methods used in this study were feasible for the satisfactory production of biodiesel which meets local and international specifications.
Development of methods for the separation and characterization of natural organic matter in dam water
(2015-01-15) Sobantu, Pinkie; Chetty, Deenadayalan Kisten; Moodley, Kandasamy Govindsamy
This project arose out the need for a simple method to analyse NOM on a routine basis. Water samples were obtained from the Vaal dam, which is one of the dams used by a hydroelectric power station. Analysis was preceded by separation of NOM into the humic and non-humic portions. The humic portion was separated into two fractions by employing a non-ionic resin (DAX-8) to separate humic acid from fulvic acid. High performance size exclusion chromatography (HPSEC), equipped with an Ultraviolet( UV) detector and an Evaporative Light Scattering (ELS) detector connected in series, was used to obtain molecular weight distribution information and the concentration levels of the two acids. Mixed standards of polyethylene oxide/glycol were employed to calibrate the selected column. Suwanee River humic acid standard was used as a certified reference material. The molecular weight distributions (MWDs) of the isolated fractions of humic and fulvic acids were determined with ELSD detection as weight-average (Mw), number-average (Mn) and polydispersity (ρ) of individual NOM fractions. The Mw/Mn ratio was found to be less than 1.5 in all the fractions, indicating that they have a low and narrow size fraction. An increase in Mn and Mw values, with increasing wavelength for all three humic substances (HS) examined was observed. The HS, isolated from the dam water, was found to be about the same molecular weight as the International Humic Acid Standard (IIHSS). For the fulvic acid standard, the molecular weight was estimated to be around 7500 Da. Characterization of NOM was done to assist in the identification of the species present in the water. FTIR-ATR was used to as a characterization tool to identify the functional groups in the structure of the humic and fulvic acid respectively present in the Vaal Dam. Analysis of the infrared (IR) spectra indicated that the humic acids of the Vaal dam have phenolic hydroxyl groups, hydroxyl groups, conjugated double bond of aromatic family (C=C), and free carboxyl groups. The isolation method has proved to be applicable and reliable for dam water samples and showed to successfully separate the humic substances from water and further separate the humic substances into its hydrophobic acids, namely, humic and fulvic acids. It can be concluded that the Eskom Vaal dam composes of humic substance which shows that the technique alone gives a very good indication of the characteristics of water. The HPSEC method used, equipped with UV and ELSD was able to identify the molecular weight range of NOM present in source water as it confirmed that the Eskom Vaal dam contains humic substances as humic acid and fulvic acid and these pose a health concern as they can form disinfectant byproducts in the course of water treatment with chemicals. FTIR characterization was successful as important functional groups were clearly assigned. Lastly, the use of the TOC and DOC values to calculate SUVA was also a good tool to indicate the organic content in water. It is recommended to use larger amounts of water must be processed to obtain useful quantities of the humic and fulvic acid fractions.
Directed evolution of B-xylanase from Thermomyces lanugtnosus
(2000) Stephens, Dawn Elizabeth; Permaul, Kugen; Prior, A.; Singh, Suren
Most natural enzymes may be unsuitable for biotechnological processes since they have evolved over millions of years to acquire their specific biological functions. Such enzymes are often genetically altered to suit the rigours of industrial processes. Directed evolution is one such strategy and makes use of iterative rounds of random mutagenesis, screening and recombination to enhance the existing properties of enzymes. Thermomyces lanuginosus is a thermophilic fungus that produces high levels of a thermostable xylanase. The xylanase gene from T lanuginosus DSM 5826 (xynA) was functionally expressed in E. coli as a LacZ-fusion protein (Schlacher et al., 1996) and later crystallized (Gruber et al., 1998). In this study, it was undertaken to improve the thermo stability and catalytic activity of xynA using error-prone PCR with different concentrations of MnCh. The first step prior to mutagenesis was to determine the levels of xylanase that could be attained by the wild type XynA, both in the presence and absence of an inducer. IPTG, a lactose analogue, was used since xynA was expressed with a lac promoter. High amounts of IPTG were found to adversely affect xylanase production, whilst a low amount (0.1 mM) enhanced xylanase production. This amount was used to later induce xylanase production by the variants obtained after mutagenesis. IPTG was found to increase the rate and production of xylanase. After random mutagenesis of xynA, transformed colonies were first selected for xylanase production on 0.4% Remazol Brilliant Blue xylan and then screened at different temperatures for improved stability and activity. After the first round of screening, four variants, viz., IB5, IB7, IBLl and ID2, showed slight improvement in both stability and activity and were subjected to further mutagenesis, using low concentrations of MnCh. Three variants, viz., 2B7-1O, 2B7-6 and 2BIl-16, with markedly enhanced stability, were obtained. Variant 2B7-10 exhibited a five fold higher activity (3430 nkat/ug total protein) than the wild type XynA (657 nkatl ug total protein). It retained 71% of its activity after treatment at 80°C for 60 min and had a t1/2of 215 min at 70°C, which is higher than that attained by XynA. Long-term thermo stability screening at 70, 80, 90 and 100°C revealed that variants 2B7-6 and 2B11-16 were, however, the most stable enzymes generated in this study, although their activities were lower or almost comparable with their parents. Sequence analysis of variant ID2 revealed 4 amino acid substitutions within the a-helix of the protein. This region was strongly conserved with the more stable variant xylanases generated in this study. The most profound mutation seen with variant 2B7-10 was the disruption of the disulphide bridge. Most of the mutants obtained in this study displayed a trade-off between stability and activity, the exception being mutant 2B7-10. Currently, DNA shuffling techniques are being used to recombine these traits in a single xylanase.
Enhancement of biohydrogen production from the aquatic weed Pistia stratiotes through a dark fermentation process
(2019) Mthethwa, Nonsikelelo Precios; Pillai, Santhosh Kumar Kuttan; Bux, Faizal; Kiambi, Sammy Lewis
Aquatic weeds are well known for their fast growth rate and high carbohydrate content that can be easily hydrolysed into fermentable sugars. This study was aimed at the utilization of an indigenous aquatic weed, Pistia stratiotes for biohydrogen production through the dark fermentation process. Characterization of the biomass, effect of pre–treatment methods on biomass hydrolysis, effect of reactor operational conditions and type of inoculum on enhancing hydrogen production potential of P. stratiotes was assessed. Physical and chemical pre–treatments were employed on P. stratiotes biomass to increase digestibility and to achieve high conversion rates of fermentable sugars. The highest sugar yield of 139± 0.8 mg/g was obtained when the oven dried biomass was subjected to H2SO4 (2.5%) pre– treatment followed by autoclaving at 121°C for 30 min. Biohydrogen production under different operational conditions was thereafter optimized using One–factor–at–a–time (OFAT) batch experiments in 120 mL serum bottles. A maximum hydrogen yield (HY) of 2.46 ± 0.14 mol-H2/mol-glucose (3.51 ± 0.20 mg-H2/g-dry weight) and 2.75 ± 0.07 mL h-1 hydrogen production rate was observed under optimized conditions (pH 5.5, Temp 35°C, S/X: 1.0 g-COD/g-VSS and HRT 8 h). The organic mass balance (92 – 96%) and electron– equivalent balance (92 – 98%) further indicated the reliability of the obtained fermentation data. Assessment of microbial activity was achieved using molecular techniques such as quantitative polymerase chain reaction (qPCR) targeting both 16s rRNA (of Clostridium spp., Bacillus spp., and Enterobacter spp.) and the functional hydrogenase gene (hydA). The highest gene activity of hydrogenase was noted at pH of 5.5 with 2.53×104 copies/ng-DNA compared to low pH: 4.5 (6.95 × 103 copies/ng-DNA) and high pH: 8.5 (7.77×103 copies/ng- DNA). A similar trend was also observed for the species containing a highly active hydrogenase (i.e. Clostridium spp., Bacillus spp., and Enterobacter spp.). During the optimum reactor conditions, three hydrogen producing bacterial strains Bacillus cereus and Enterobacter cloacae were successfully isolated. These isolates were used as inoculums for the pure culture studies and achieved HYs of 2.2, 1.10 and 1.97 mol-H2/mol-glucose respectively under optimized fermentation conditions. However, the thermally treated mixed culture displayed a marginally higher HY (2.46 mol-H2/mol-glucose) compared to the pure culture used alone. Furthermore, the cost estimation indicated a potential and economically feasible for biotransformation of P. stratiotes to hydrogen energy. In conclusion, the results from this study has revealed the potential of employing P. stratiotes biomass for biohydrogen production. The results also indicated the importance of employing suitable pre–treatment methods, operating conditions as well as inoculum types for enhanced hydrogen production using P. stratiotes.
Evaluation of biohydrogen production potential of sugarcane bagasse using activated sludge in a dark fermentation process
(2016) Reddy, Karen; Bux, Faizal; Kuttun Pillai, Sheena Kumari; Gupta, Sanjay Kumar
Anaerobic dark fermentation is an efficient biological process to produce hydrogen from waste material. In South Africa, this technology has not been explored adequately to extract energy from biological wastes. Within the KwaZulu Natal region of South Africa, the sugar industry is a prominent venture that produces mass quantities of sugarcane bagasse amongst other waste products. This by-product can be an ideal source of substrate for biohydrogen generation. In this study, sugarcane bagasse was used as the main substrate for biohydrogen production by anaerobic fermentation using sewage sludge as the inoculum. Different pre-treatment methods were employed to maximize the release of fermentable sugars from the lignocellulosic biomass. Among the different pre-treatment methods employed, the maximum sugar yield (294.4 mg/g) was achieved with 0.25% H2SO4 for 60 minutes at 121°C. Prior to inoculation, the sewage sludge was also subjected to thermal pre-treatment to eliminate methanogens. Thermal pre-treatment of inoculum sludge for 30 min was effective in eliminating methanogens. Fluorescence in situ hybridization was used to positively identify the hydrogen producing bacteria present before and after treatment. The pre-treated substrate and inoculum was integrated into a dark fermentation process to further optimize the effect of pH, substrate to biomass, iron and magnetite nanoparticles on hydrogen production. The maximum hydrogen production (1.2 mol/mol glucose) was achieved at a pH range of 5-6, a substrate to biomass ratio of 3.5, and iron and magnetite nanoparticle concentration of 200 mg/L. Microbial analysis using quantitative polymerase chain reaction has confirmed the dominance of Clostridium spp. in the reactor. The highest hydrogenase gene activity (number of copies of hydrogenase gene expression/ng DNA) was recorded in the reactor supplemented with magnetite nanoparticles with lowest being in the raw sludge. There was a direct positive correlation between the hydrogenase gene copy number and the hydrogen yield obtained at different reactor conditions. Scanning electron microscopy was a useful to visually analyse the interaction of microorganisms with activated sludge. This study highlights the significance of anaerobic microorganisms from waste sludge being able to utilize agricultural waste material to produce biohydrogen which could be further scaled up for continuous hydrogen production. In addition, statistical tools used to predict the possible sugar (Design of experiments) and hydrogen yields (Gompertz model) produced would be helpful in saving time during full-scale operation of biohydrogen producing reactors.
Evaluation of chitosan–coated magnetic nanoparticle-immobilized thermostable hemicellulases for enhanced saccharification and production of bioethanol
(2022-09) Mdlaka, Sibongile Patience; Singh, Suren; Puri, Adarsh Kumar
Enhancing the efficiency of saccharification of pentose and hexose sugars present in lignocellulosic biomass is a major bottleneck for industrial bioethanol production. This problem can be addressed by a concerted effort combining nanotechnology, enzymology and fermentation technology. Functionalized chitosan-coated magnetic nanoparticles (CCMNPs) were prepared and used for co-immobilization of purified xylan hydrolysing xylanase and xylosidase from the thermophilic fungus Thermomyces lanuginosus SSBP for the release of xylose. Stability studies revealed that immobilized enzymes were more stable than free enzymes over a wide range of pH (4.0 – 7.0) and temperature (40 – 90 °C) for xylanase and 30 – 80 °C for xylosidase. The optimum activity of the co-immobilized enzymes shifted slightly as compared to the free enzymes, with coimmobilized xylanase and xylosidase showing optimum activity at pH 6.5 and 6.0, respectively. The study showed sustained production of xylose as the major fermentable sugar under repeated batch and fed-batch saccharification of lignocellulosic biomass. Statistical optimization of saccharification of 1% xylan using response surface methodology indicated the enhanced release of xylose at 50 °C, pH 7.0 and enzyme dose of 60 U/mL xylanase and 30 U/mL xylosidase. Finally, liberated xylose was fermented with Scheffersomyces stipitis to yield bioethanol.
Expression of anti-HIV peptides in tobacco cell culture systems
(2009) Moodley, Nadine; Odhav, Bharti; Chikwamba, Rachel
Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.