Biochemistry and Microbiology

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The antimicrobial activity of five food spices when tested against various gram-positive –and gram-negative microorganisms
(2008) Seepersad, Kashimee; Basson, A.K.; Djarova, T.; Shandu, J.S.
The discovery of antibiotics by Alexander Fleming in the early nineteen hundreds not only created an enormous breakthrough in medical treatment but along with it introduced the emergence of new and now what is considered an ever increasing number of multi-drug resistant pathogens. Like antibiotics, herbs and spices have been used traditionally by many, for the treatment of various aliments ranging from stomach indigestion, lesions of the skin to beauty therapy. At present it is estimated that about 80% of the world population rely on botanical preparations as medicines to meet their health needs as opposed to treatment by conventional medicine with spices creating a shelf of its own in the global medical cabinet. In this study, the antimicrobial potential of five spices (commonly known as ginger, cinnamon, turmeric, nutmeg and chilli) was analysed against various Gram positive- and Gram negative microorganisms namely, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Salmonella spp, Shigella spp and Staphylococcus aureus. Analysis of the results of sensitivity tests (disc and agar well diffusion assays) indicated each of the microorganisms to be completely inhibited, intermediately inhibited or completely resistant towards a particular spice extract. The formation of zones of inhibition present where inhibition had occurred indicated that the spice tested was effective as an antimicrobial agent when screened. Zones of absolute inhibition (greater than fifteen millimetres in diameter) were obtained during positive agar well and disc diffusion assaying with neomycin used as the antimicrobial agent of choice. Inhibition zones observed to be in the upper limit range (pertaining to the study) of 20 mm – 27 mm in diameter. Comparative studies using the test spices indicated that chilli, turmeric, nutmeg, cinnamon and ginger each demonstrated zones of inhibition within this limit at one or more laboratory testing. Chilli was the most active antimicrobial agent when tested and in some instances demonstrated antimicrobial effectiveness greater than that exhibited by the positive control neomycin. Turmeric, nutmeg, cinnamon and ginger however each demonstrated inhibition within the same range as that of neomycin. The observations of such inhibition amongst the spices were comparatively significant and demonstrated the potential use of these spices as antimicrobial agents with an efficacy that can be compared to that of the already recognized and widely used antibiotic, neomycin. The minimum inhibitory concentration (MIC) was successfully determined for each of the spice extracts. The reactions observed during MIC determination were confirmatory of the antimicrobial activity present in the extracts of each spice. Analyses of the results conclude that the active compounds present in the selected spices were effective against certain microbial species. This observation demonstrated that spice can and may be used in the treatment of bacterial infections. This could in the future be an alternative treatment to antibiotics for one or all of the microbial species investigated and in so doing allow the healing powers of spices to be acknowledged.
Antimicrobial activity testing of traditionally used plants for treating wounds and sores at Ongoye area KwaZulu-Natal, South Africa
(2009) Mthethwa, Ntombeziningi Shirley; de Wet, H.; Basson, A.K.
This study focused on the investigation of plants used for the treatment of wounds and sores by local people living around the Ongoye forest, KwaZulu-Natal. An ethnobotanical survey was conducted in eighty homesteads in this area. The ethnobotanical data revealed that 33 plant species were used in treating sores and wounds, but only 15 plant species were collected from the wild and homesteads and three plant species were bought from a muthi market. According to the ethnobotanical information Hypericum aethiopicum (unsukumbili) was the most used plant for treating sores and wounds in this area. The survey showed that women (62.5%) possessed more knowledge than the men (37.5%) who were interviewed at the homesteads regarding the medicinal uses of plants. Acetone, methanol, cold and hot water extracts from the different plant parts (bark, leaves, stems and the whole plant) were done on 18 species. These plants species are: Acanthospermum australe, Acorus calamus, Albizia adianthifolia, Baccharoides adoensis, Clerodendrum hirsutum, Combretum erythrophyllum, Faurea saligna, Gerbera ambigua, Gunnera perpensa, Hypericum aethiopicum, Hypoxis hemerocallidea, Lippia javanica, Pentanisia prunelloides, Sclerocarya birrea, Solanum aculeastrum, Trichilia dregeana, Warburgia salutaris, Ziziphus mucronata. The above-mentioned plants were screened for antibacterial activity against the following bacteria strains: Bacillus subtilis (6051), Escherichia coli (7751, U1405s, U16406, U16403), Klebsiella pneumoniae (13883), Staphylococcus aureus (12600, P5020, P4790, T1266), ‘Salmonella spp., Shigella flexneri and Shigella sonnei’. The antibacterial activities were determined by disk-diffusion, agar-well diffusion, minimum inhibitory concentrations (MIC) and bio-autographic methods. The plant extracts were also screened for the following phytochemicals: alkaloids, flavonoids, saponins, anthraquinones, cardiac glycosides and tannins. The following plants were the most effective against the micro-organisms tested: Gunnera perpensa, Hypericum aethiopicum, Hypoxis hemerocallidea, Lippia javanica, Pentanisia prunelloides, Trichilia dregeana and Warburgia salutaris. The bio-autographic results showed several compounds separated on the TLC with activity against the test organism, Staphylococcus aureus (ATCC2600). This study thus lends some support to traditional knowledge and may serve as a basis for selecting the most active medicinal plants to use in traditional medicine practices in the future.
The effect of seasonal variation on nutrient removal from municipal wastewater using a constructed wetland microcosm
(University of Zululand, 2018) Zulu, Ndumiso Clement Talente; Mthembu, M.S.; Basson, A.K.
South Africa is facing a severe water scarcity due to the exponential deterioration of natural water bodies such as dams and lakes. The deterioration of natural waterways is fuelled by anthropogenic activities that produce bulk amount of wastewater. The haphazard disposal of nutrient-rich wastewater from household, industries and institutions may lead to the occurrence of eutrophication which impair the integrity and quality of water in natural water bodies. This has resulted in the urgent need for the development and implementation of new innovative green technology for wastewater treatment. Constructed wetlands have proven to be an ideal alternative technology for wastewater treatment. This is because they are environmentally friendly and economically sustainable treatment systems. In addition, these systems have a potential of reducing contaminants to acceptable levels that pose no threat to human and environmental health. Despite these advantages, their application is still challenging in some parts of the world. This is due to the limited information about the seasonal performance of these systems and poor understanding of the influence of environmental parameters in pollutant assimilation. This study delineates the effect of seasonal variation in microbial community structures in wetland microcosm. In addition, this study also investigated the seasonal effect of physiochemical parameters on nutrient assimilation in these systems. The constructed wetland microcosms were setup at the Empangeni (University of Zululand), and was divided into planted (planted with Amaranthus hybridus and Bidens pilosa) and unplanted (reference) section. These systems were operated in warm and cold seasons for one month. The physiochemical parameters (dissolved oxygen, pH and temperature) were monitored. The removal efficiency of chemical oxygen demand, ammonia, nitrite, nitrate and vi phosphorus were measured pre- and post-treatment using spectrophotometric methods. The spectrophotometer was used with commercial kits (Merck) following manufacturers protocol. Nutrient removal was seasonal and varying degree of nutrient removals were observed in planted and reference section of the wetland microcosms. The highest reduction efficiencies were obtained in warm than cold seasons. In warm season, the highest removals were 97%, 95%, 90%, 70% and 74% for ammonia, nitrite, nitrate, phosphorus and COD in planted section, while in reference section the removals were 69%, 69%, 82%, 57% and 59% for ammonia, nitrite, nitrate, phosphorus and COD respectively. In cold season, the removals were 60%, 73%, 65%, 68% and 64% for ammonia, nitrite, nitrate, phosphorus and COD in planted section, while in reference section the removals were 42%, 64%, 50%, 46% and 50% ammonia, nitrite, nitrate, phosphorus and COD respectively. The increase in physiochemical parameters was directly proportional to nutrient reduction in the microcosms. The correlation of physiochemical parameters with the nutrients removal ranged from very poor (temperature (0.11≤r≤0.95), moderate negative (COD (-0.44≤r≤0.94) (pH (-0.45≤r≤0.89) and to a very strong positive correlation (DO (-0.72≤r≤0.89). Based on the discharge limits of nutrients, the effluent for nitrite, nitrate and phosphorus were within the discharge limit while ammonia did not meet the discharge standards in both seasons as per South Africa’s Department of Water and Sanitation. Microbial community structure and diversity occurred in the microcosms. However, their occurrence was seasonal with warm season showing high abundance than the cold season. Furthermore, the planted sections showed high microbial abundance and diversity than the reference sections. This indicated that macrophytes supported the growth, diversity and activity of microorganisms within these systems. This was supported by the high removal of nutrients in the planted sections than in the reference sections. Nitrosomonas and Nitrobacter were the most dominant nitrifiers in the microcosms in both seasons while Thauera, Pseudomonas and vii Acidovorax were the dominant denitrifiers in the both warm and cold seasons. Phosphate accumulating microorganisms were dominated by Accumulibacter in warm season and Bacillus in cold season.
Effects of Carbon, Nitrogen, Particle size and moisture on Oyster Mushroom Production in KwaZulu Natal - Cedara
(University of Zululand, 2018) Tembe, Nkosinathi Jacob; Mthembu, M.S.; Basson, A.K.
Mushrooms are liked for their delicious flavour, high protein content and absence of cholesterol. Farmers require guidance in how they can manipulate the current existing mushroom production methods to fit in their cheaper, local ready available raw materials. Mushroom production can be optimized only if its substrate specific requirements are known and well maintained. Different potential sources of carbon and nitrogen such as wheat bran, teff, lucerne, bagasse, maize, sawdust, juncao and hominy chop were analysed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES Analysis). Carbon and nitrogen content was used to set up five carbon/nitrogen (C/N) ratios (15:1, 25:1, 35:1, 70:1 and 100:1). Milling of the raw materials was done in three phases which were 8 mm, 12 mm and 25 mm using different sieves of the multi-purpose milling machine. The raw materials were mixed and the water was added to achieve 65% moisture. The raw materials were bagged and pasteurized at 900C for 14 hours. The substrates were inoculated with Pleurotus ostreatus spawn produced at Department of Agriculture and rural Development (DARD). The rate of the mycelium growth and contamination was monitored and recorded. When the substrates were fully colonised, they were cut on top and randomly placed on the wall and irrigated daily. Fresh mushrooms of each mushroom pack were harvested and weighed. The optimum substrate requirements were determined based on best C/N ratio, particle size, biological efficiency and contamination rate. The highest biological efficiency was achieved with the following substrate composition 31.3% bagasse, 6.3% wheat bran, 12.5% maize, 12.5% sawdust, 12.5% hominy chop, 12.5% juncao, 6.3% lurcerne and 6.3% teff with particle size of 8 mm and C/N ratio of 35:1. There are many agro-waste that can be successful used in production of oyster mushrooms, but its combination to form a required optimum C/N ratio is very important. This study showed that the particle size also played a crucial role in mycelium growth. The study concluded that particle size of the raw material when using plastic bag (150x300 mm) must be at least between 8 and 12 mm and C/N ratio should above 25:1 and below 35:1.
Microbiological evaluation of the Mhlathuze River in KwaZulu-Natal
(2004) Mthembu, Nompumelelo; Lin, J.; Basson, A.K.
High levels of faecal coiifonns pose a treat to the health of the rural community that uses the river water directly for domestic use without treatment. The microbiological, physical and chemical analysis of the Mhiathuze River was investigated over a twenty-one months period. Five water samples were collected along the Mhiathuze River and analysed to monitor the indicator bacteria pollution with changing seasonal patterns. Surface water temperarure and rainfall during the period of study appeared to be some of the factors affecting the increased bacterial counts. Elevated levels of indicator microorganisms (both faecal and total coiifonns) and heterotrophic plate count bacteria were observed from March 1998 to November 1999. Bacteria isolated from the river included Escherichia coli, Pseudomonas spp., Enterobacter spp.r Serraiia spp,, Klebsiella spp. and Aeromonas hydrophila. The average monthly pH values ranged between 6.5 and 8.5. The turbidity, dissolved oxygen, hardness, ortho- and total phosphates values obtained did not show any major changes that would call for caution. A polymerase chain reaction (PCR) method was used to amplify E6S rRNA and phoP gene fragments from the isolated bacteria and directly from the water resource. Annealing temperature was adjusted to set up the optimum conditions of the PCR mixture. Performing serial dilutions of DNA carried out the sensitivity of detection for PCR products. It was deduced that amplifications with phoP and 16S rRNA primers were visualised up to 10"' and 10"° ug of DNA, respectively. Multiplex PCR with the two primers generated an amplification product of approximately 755 bp for all environmental isolated used and an additional 299 bp product for E. coll C. freundii and C. ctversus. Rsa I and Hinf I restriction enzymes were also used for double digestion ofE.coli and P. vuigaris.
Performance of three oyster mushroom species grown on maize stalk supplemented with wheat bran and maize flour
(University of Zululand, 2017) Mkhize, Senzosenkosi Surprise; Zharare, G.E.; Basson, A.K.
The process of mushroom cultivation adds value to the economy, environmental restoration and food security (provision) worldwide. However, there is paucity of information to the mushroom growers about the strategy of improving yield and production of mushrooms at minimal costs with reduced contamination rate. Therefore, understanding the amendments, supplements, and pre-treatment requirements for the local substrate to make them optimally suitable for mushroom cultivation is an essential step towards developing ways of improving yield and production at minimal costs with reduced contamination. The study was conducted to determine or evaluate the performance of three selected Pleurotus mushroom species (Pleurotus ostreatus, P. pulmonarius, and P. salmoneostramineus) grown on maize stalk which was supplemented with varying increasing levels of maize flour (MF) and wheat bran (WB) supplements. The results obtained in this study indicated that the mushroom species were not highly affected by contamination. However, there were levels of supplementation that resulted in higher rates of contamination, such as 20% WB (75% rate) for P. pulmonarius and 20% MF (75% rate) for P. salmoneostramineus. The overall results showed that all the mushroom species were affected by the addition of both supplements into the maize stalk substrate. It was observed that the higher levels of supplementation resulted in lower mycelial growth rate (MGR) and days to full colonisation of substrate were delayed whereby the lower levels of supplementation (WB and MF) resulted in significantly faster mycelial growth rate and shorter colonisation period. The time it took for mushrooms to start pinning (TP) was shorter within the first flush for all the mushroom species compared to the second and third flushes. Although, higher levels of supplementation negatively affected the MGR and days to full colonisation, there were however, some advantages of high supplementation. The higher levels of supplementation improved the productivity (biological efficiency and yield) for all mushroom strains. The biological efficiency (BE) and yield increased up to certain levels of higher supplementation after which they both decreased or dropped after reaching peak. In addition, a higher rate of contamination was observed under supplement levels beyond the optimum level. It can be recommended that for quick production, low supplementation or no supplementation may be required. This action may save production costs, nonetheless for an improved productivity (BE and yield), high levels of supplementation should be used (12% WB and 14% MF for all mushrooms, and for P. salmoneostramineus 18% WB is also recommended). However, supplementing beyond certain limits will result in a decrease in productivity due to contamination.
Production and characterisation of bioflocculant produced by bacterial isolates from Richards Bay harbour, Kwazulu Natal
(University of Zululand, 2019) Akapo, Charles Sewanu Oluwaseun; Basson, A.K.; Ntombela, Z.G.
Bioflocculants are safe and biodegradable extracellular polymers produced by microorganisms during their growth. In comparison with conventionally used flocculants, bioflocculants have the advantage of being safe (no known toxic effects), biodegradable and harmless to the environment. In this study, halophile novel bacteria was isolated from Richards Bay Harbour, Kwazulu-Natal, Republic of South Africa and screened for bioflocculant producing potential using a production medium at 30 oC for 72 hours with a shaking speed of 160 rpm. Kaolin clay suspension was used as test material. Microorganisms with bioflocculant-producing potential were further identified using 16S rRNA molecular sequencing method. Among the identified isolates, one with flocculating activity above 70% was used for bioflocculant production. Bacillus atrophaeus culture conditions were optimised for improved flocculating activity as well as the production yield. To obtain an optimum culture medium condition, the parameters such as carbon and nitrogen (energy) sources, inoculum size, metal ions, cultivation time, initial pH of the medium, incubation temperature, shaking speed and fermentation time were investigated. The bioflocculant was extracted using ice-cold ethanol and purified with the mixture of chloroform and butanol (5:7 v/v). The purified bioflocculant was characterized using FTIR, scanning electron microscopy (SEM) equipped with element analyser and TG analyser. The bioflocculant was further analysed for its chemical composition. The purified bioflocculant BA-CGB was also assessed for antimicrobial activity properties and further applied on Vulindlela domestic wastewater and Mzingazi river water. Among the screened isolates, three of them with more than 70% flocculating activities were identified through 16S rRNA as Bacillus safensis, Bacillus pumilus xxi and Bacillus atrophaeus. A bioflocculant yield of 3.165 g was recovered for B. atrophaeus after 96 hours of incubation from 1 liter of fermentation broth in the presence of 4% inoculum size, glucose, ammonium chloride, at pH 9 and temperature of 35 oC as well as shaking speed of 110 rpm stimulated by Ba2+ . The bioflocculant BA-CGB produced, showed a strong thermal stability character as managed to attain more than 60% flocculating activity at 121 °C for 15 min, with 0.4 mg/ml dosage size and Ca2+ as a stimulating agent. The flocculating activity of purified BA-CGB was greatly influenced by a wide pH range (3-12). BA-CGB is amorphous in structure, composed of carbohydrates (65%), uronic acid (25%) and trace proteins (10%). Functional groups include hydroxyl, carboxyl, amine, amide and elements such as carbon and oxygen among others, were present in the molecular chain of bioflocculant BA-CGB. No antimicrobial activity was observed with bioflocculant BA-CGB. 0.4 mg/ml dosage size of a bioflocculant was more effective in flocculating wastewater, improving COD, BOD and removing nitrates, phosphates as well as sulphate from Mzingazi riverwater and Vulindlela domestic wastewater. This bioflocculant holds a promising future to replace in-use chemical flocculants in wastewater treatment
The study of rehabilation of hydrocarbon contaminated soil using bio-remedial microbes
(2006) Shandu, Jabulani Siyabonga; Basson, A.K.; Kelbe, B.E.
The study investigated the feasibility of bioremediation as a treatment option for chronically diesel-oH-poHuted soil at petroleum and gas depot of Oilco (a company that is a division of Shell) situated at the east side of Empangeni which is in the Northern KwaZulu-Natal province, South Africa. To examine the efficiency of bioaugmentation, the contaminated site was treated with microbes, (previously isolated from the diesel-contaminated soil) to a depth of ±1, 2 meters, ±5 meters wide and 2 meters in length, plus the woodshavim^ as their nutrient source. The effectiveness of bioremediation was observed over a period of 11 weeks and samples were taken at 15-day intervals. Over that period of 11 weeks, the changes in hydrocarbon concentrations were monitored in the soil and soil leachate and the accompanying changes in the soil microbial counts and activity. A significant reduction in the diesel-oil level could be achieved. The BTEX method was used in GCMS to check for changes in TPH. Prior to GCMS analysis the soil texture was analyzed using the Particle Size Determination method and the soil was observed to be sandy-loam (Day, 1995). For checking the soil microbial counts and activity the following groups of microbes were observed Aerobic Total Counts, Nitrofyers, Nitrosojyers and Free-living nitrogen fixing bacteria (Chan^L aL, 1993). The four groups of microbial counts were used as a biological parameter, and there was a correlation between each other as well as with the residua] hydrocarbon concentration, indicating the importance of biodegradation. The effect of biostimulation of the indigenous soil microorganisms declined with time during the study.
Synthesis and application of a grafted flocculant produced from a chemical combination of a bioflocculant TKT and acrylamide (AM)
(University of Zululand, 2019) Masuku, Sicelo Keny; Basson, A.K.; Simonis, J.J.; Maliehe, T.S.
An increase in pollution from mining, industries and agriculture has decreased the quality of fresh water in South Africa. Contaminated water is unsafe for animals to drink and for plants to absorb. Synthetic flocculants are extensively used in water and wastewater treatment due to their effectiveness and low cost. These flocculants consist of a flexible chain of high molecular weight to create a bigger surface area for biochemical reactions to take place and are documented to flocculate well in small amounts. However, these flocculants are known to be carcinogenic, neurotoxic and hazardous to the environment. Moreover, natural polymers (bioflocculants) are not toxic and are biodegradable. Nevertheless, these flocculants need to be suitably controlled because biodegradability reduces their shelf-life. Through grafting synthetic polymers onto a backbone of natural polymers, the best properties of synthetic and natural flocculants are merged. When grafted copolymers are successful, the dangling grafted chains have easy access to contaminants in wastewater. In literature it has been reported that grafted polysaccharides provide efficient, shear stable and biodegradable flocculants. This study aimed at synthesizing and applying a grafted bioflocculant produced from marine Alcaligenes faecalis HCB2 to wastewater. Extraction and purification of the bioflocculant was obtained through using the solvent extraction method. Chains of polyacrylamide (PAM) were grafted onto the backbone of bioflocculant TKT extracted from Alcaligenes faecalis HCB2 using a microwave initiated method. Various grades of TKT-g-PAM were synthesized and the one with the highest percentage grafting was used throughout this study. Physicochemical analyses of the grafted bioflocculant (TKT-g-PAM) were attained by scanning electron microscopy (SEM), Zetasizer Nano, Thermogravimetric Analyzer (TGA) and Fourier Transform Infrared (IR) Spectrophotometry. A diphenyl tetrazolium bromide (MTT) assay was used to determine the biosafety of the grafted bioflocculant. The effect of dosage size on flocculating activity was evaluated. The pH and thermal stability of TKT-g-PAM were assessed spectrophotometrically. The removal efficiencies of TKT-g-PAM on chemical oxygen demand (COD), biological oxygen demand (BOD), nitrogen and sulfur on wastewater from Tendele Coal Mine were performed using different test kits. x | P a g e The bioflocculant yield obtained after extraction and purification was 4.0 g/l. TKT-g-PAM6 was the preferred grade with the highest percentage of 62% and a weight of 0.8 g. The images provided by SEM revealed changes in bioflocculant structure from a blocky to a granular structure after grafting. This confirmed that the grafting of PAM chains was successful on the TKT backbone. The zeta potential of grafted bioflocculant (-12.6±0.1) and bioflocculant (-17.1±0.65) had a negative charge. IR observations indicated hydroxyl, carboxyl and amine groups. TKT-g-PAM6 showed less cytotoxic effect on HEK293 cells (retained 82% of cell viability after it was treated in high concentration) while CaCO2 cells (retained 64% of cell viability after it was treated in high concentration). TKT-g-PAM6 was cation and pH dependent and optimally flocculated kaolin clay suspension at a dosage of 0.2 mg/ml with 74% of flocculating activity in the presence of Ba2+. TKT-g-PAM6 was more stable at acidic pH 3-5 with the highest flocculating activity (93%) observed at pH 3. TKT-g-PAM6 was thermostable and retained more than 90% of its flocculating activity after being heated at 100 °C for 1 hour. The pyrolysis properties of the grafted bioflocculant confirmed their thermal stabilities, the IDT has been found to 215 °C (29% weight loss) and FDT to be 571 °C (36% weight loss). Flocculating efficiencies were: 87% on COD, 92% BOD, 91% N and 93% S, respectively. TKT-g-PAM6 was effective and has the potential to be used in wastewater treatment.
Synthesis, characterization and application of polyacrylamide grafted bioflocculants (TMT-1-g-PAM 2 and TST-1-g-PAM 3)
(University of Zululand, 2018) Ngema, Siyanda Senzo; Basson, A.K.; Maliehe, T.S.; Simonis, J.J.
Water pollution is one of the major problems in the world. It contributes to water scarcity. Grafted bioflocculants have been found to have excellent wastewater remediation capabilities. Therefore, they have become one of the main research focal points in recent times as flocculants. The high efficacy is owed to their unique characteristics, which include having a branched structure and a high molecular weight. Therefore, the aim of this research was to synthesise, characterize and apply polyacrylamide grafted bioflocculants to wastewater treatment. Bioflocculants (TMT-1 and TST-1) produced by Bacillus pumilus JX860616 and a consortium of Bacillus pumilus JX860616 and Bacillus subtilis CSM5, respectively, were grafted with acrylamide chains using a microwave initiated method. To optimize the synthesis of the grafted bioflocculants, irradiation time and acrylamide concentration were varied. Optimum grades (TMT-1-g-PAM 2 and TST-1-g-PAM 3) were then characterized by intrinsic viscosity, elemental analysis, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD) and thermogravimetric analysis (TGA). Furthermore, these optimum grades’ biosafety (on human embryonic kidney 293 cells, HEK 293) were assessed by 3-(4,5-dimethylthiazol-2-)-2,5- diphenyl tetrazolium bromide (MTT) assay. The grafted bioflocculants were also screened for antibacterial activity, using micro-dilution assay. Their biodegradation was investigated by a composting method. The effects of dosage size, cations, pH, temperature and salinity on the flocculating activities of the grafted bioflocculants were also evaluated, spectrophotometrically. The flocculation mechanism was determined by evaluating zeta potentials using Zetasizer Nano. Spectrophotometric cell tests were utilized to assess COD, BOD, N, P and S removal on domestic and coal mine wastewater by the grafted bioflocculants. The optimum grade (TMT-1-g-PAM 2) was obtained when a concentration of 2.5 g of acrylamide was used for grafting on TMT-1 at irradiation time of 3 min.; while TST-1-g-PAM 3 was synthesized by using 7.5 g of acrylamide at irradiation time of 3 min. Changes observed in intrinsic viscosity, elemental analysis, SEM, FTIR, XRD and TGA confirmed that grafting was successful. HEK 293 cells displayed high viability (75% in TMT-1-g-PAM 2 and 85% in TST-1-g-PAM 3) when exposed to high concentrations (i.e. 200 μg/ml) of the grafted bioflocculants. Both grafted bioflocculants did not show any antibacterial activity against the tested species. The grafted bioflocculants were biodegradable. The flocculants’ dosage size of 0.2 mg/ml possessed flocculating activities of xiv 81% for TMT-1-g-PAM 2 and 90% for TST-1-g-PAM 3. Furthermore, the grafted bioflocculants showed stability over a wide pH range (3–11), displaying flocculating activities above 75%. They also demonstrated thermal stability, both giving flocculating activity of 81% at 100 °C. Moreover, they were able to flocculate saline water (35 g/l NaCl), giving flocculating activity of 63% (TMT-1-g-PAM 2) and 64% (TST-1-g-PAM 3). There was a decrease in zeta potentials with the presence of cations (Ca2+; TMT-1-g-PAM 2 and Ba2+; TST-1-g-PAM 3), implying that flocculation could be due to double layer compression by cations, chemical reactions and bridging mechanisms. The removal of COD, BOD, N and P, in domestic wastewater, by the graft copolymer TMT-1-g-PAM 2 reached 98%, 54%, 53%, and 57% respectively. Whereas, the removal of those parameters by TST-1-g-PAM 3 was as follows: 98%; COD, 73%; BOD, 74%; N and 17%; P. In the case of coal mine wastewater, the removal efficiency by TMT-1-g- PAM 2 was: 98%; COD, 93%; BOD, 59%; N and 83%; S. For TST-1-g-PAM 3, the removal efficiency from coal mine wastewater achieved the following results: 95%; COD, 62%; BOD, 89%; N and 57%; S. Considering the unique properties – such as many functional groups, high stability, biosafety and removal efficiencies of water pollutants – both flocculants attract a potential industrial applicability.
The usefulness of multiple antibiotic resistance (MAR) indexing technique in differentiating fecal coliform bacteria from different sources
(2008) Mthembu, Mathews Simon; Djarova, T.; Biyela, P.T.; Basson, A.K.
Pollution of water sources with human fecal matter and associated intestinal pathogens poses a great risk to public health. Fecal contamination of water is not the only problem to communities that consume untreated water. The extent of the microbial contamination of water sources also needs to be considered when designing treatment regimes for the production of potable water. The more polluted the source of drinking water is, the more extensive and expensive treatment regimes have to be used to produce microbial risk-free water. For decades fecal coliform counts have been used as indicators of fecal contamination and the potential presence of intestinal pathogens in surface waters. However, fecal coliforms fail to provide information about the source of fecal contamination. Knowing the source of fecal contamination is vital in managing this problem in surface waters. This study explored the use of two techniques, multiple antibiotic resistance (MAR) indexing and caffeine detection as means of differentiating E. coli isolates from various sources. A total of 322 E. coli were isolated from domestic and wild animals as well as human sewage by using conventional culture methods. Standard chemical and biochemical tests were used to identify these isolates. All isolates were assayed against a battery of 10 antibiotics using the micro-dilution method. The results obtained were used to generate antibiotic resistance profiles which in turn were used to statistically group the isolates into different subsets. Caffeine detection by Thin Layer Chromatography (TLC) was used to differentiate between human and non-human derived E. coli isolates. The correct classification rate was 78% when MAR indexing was used and 50% when using caffeine detection. Sixty percent of E. coli from humans were correctly classified and 95.5% of E. coli from animals were correctly classified as non-humans sources respectively. The results of this study underscore the validity of MAR indexing as a method of bacterial source tracking. MAR indexing has great discriminatory power without the complexities and the high costs often associated with established genotype-based methods. Caffeine detection indicated an average classification rate (50%). With further research, caffeine detection may give another option for source tracking when genotyping methods are limited by either costs or lack of expertise. The use of combined techniques may provide a much more reliable and cost-effective option for bacterial source tracking when each technique used provide similar results.

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