Doctoral Degrees (Microbiology)

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Screening, optimization of process parameters and scale-up of native and recombinant thermophilic Xylanases and their application in chicken feed hydrolysis.
(2023) Dhaver, Priyashini.; Govinden, Roshini.; Sithole, Bruce.; Pletschke, Brett.
Lignocellulosic biomass is a renewable raw material that has gained industrial interest due to its abundance, low cost, and potential to mitigate greenhouse gas emissions. Biomass is treated with various microbial enzymes to produce desired products under ideal conditions. Thermophilic microorganisms are excellent sources of thermostable enzymes that can tolerate extreme conditions. Optimized xylanases can be produced through genetic engineering, and recombinant DNA techniques. The biotechnological potential of xylanases from thermophilic microorganisms is discussed and the ways they are being optimized and expressed for industrial applications. Monogastric animal farming relies on grain feedstocks with non-starch polysaccharides (NSP’s) and anti-nutritive factors that cause adverse effects like increased digesta viscosity and nutrient inaccessibility which leads to reduced feed conversion, energy metabolism, and growth. Exogenous enzymes have been used to reduce viscosity and increase nutrient absorption in poultry and pigs. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their ability to modulate the composition of the gut microbiota. This study aimed to isolate and screen potential xylanolytic fungi from soil and tree bark samples in South Africa and to determine their growth conditions for maximum xylanase production. The highest xylanase activity was produced by Trichoderma harzianum. The enzyme with a molecular weight of 72 kDa retained >70% activity after 4 h at pH 6.0 and 70°C. The study also identified multiple isoforms of xylanase, which could be beneficial for animal feed and biofuel industries. The xylanase was purified from the submerged culture and displayed maximum xylanolytic activity at pH 6.0 and 65°C and the enzyme was activated by Fe2+, Mg2+, and Zn2+. Enzyme production was then optimized for maximal xylanase production strain using the Plackett-Burman Design (PBD) and Box Behnken Design (BBD), screening, and optimization design strategies, respectively. Xylanase production was enhanced to 153.80 U/ml by BBD representing a 3.99-fold increase and a 2.24- fold increase, respectively compared to the preliminary one-factor-at-a-time (OFAT) activity of 68.7 U/ml. The experimental design effectively provided conditions for the production of an acidic enzyme based on pH and incubation time. This is an exciting prospect for the application of enzymes in animal feed improvement (pH 5.0). The Geobacillus stearothermophilus glycoside hydrolase family 10 xylanase, endoxylanase XT6, is a promising candidate for industrial application. This gene was cloned and expressed and the xylanase was applied as an additive to locally produced chicken feeds. Optimization of cell lysis and expression conditions led to enhanced recombinant XT6 xylanase production. The recombinant XT6 xylanase was purified using cobalt chromatography, resulting in a 43 kDa protein with 15.69-fold purity. Cellulose, hemicelluloses, and lignin are the primary sources of fermentable sugars in lignocellulosic feedstock. Carbohydrate-active enzymes which can help release functional compounds from the carbohydrate matrix, such as phenolics are used to modify polysaccharides for industrial purposes. However, it should be noted that the primary action of carbohydrate enzymes like cellulases or xylanases is specific to the carbohydrate structure and may involve the hydrolysis of polysaccharides like xylan or other complex carbohydrate-lignin compounds,rather than being directly responsible for the release of phenolic compounds. Corn, with its antioxidant potential, is used in animal feed production, thus improving its quality for animal feed supplementation is crucial. The crude and purified T. harzianum xylanases as well as the recombinant XT6 xylanase were applied to locally produced chicken feeds in an experimental BBD design to optimize hydrolysis to monosaccharides and XOS. The Response Surface Methodology (RSM) results showed that higher (8.05 U/ml) levels of reducing sugars were produced for the crude T. harzianum xylanase and starter feed, than on the grower feed (3.11 U/ml). Treatment with the purified T. harzianum and recombinant XT6 xylanases produced lower levels of reducing sugars with similar levels for both feed types of starter feed (2.81 U/ml) and (2.98 U/ml), compared to the grower feed (2.41 U/ml) and (2.62 U/ml), respectively. Profiling of the hydrolysis products by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed that the chicken feed enzymatic hydrolysates contained a range of monosaccharides (mannose, glucose and galactose) and XOS, with xylobiose being the predominant XOS. Toxicity studies showed that the higher dilutions of the feed enzymatichydrolysates were not toxic to HEK293 cells. Therefore, the T. harzianum and recombinant XT6 xylanase are appropriate for application in the feed industry to produce XOS. These results are promising for future studies and application in the poultry feed industry as additives. The novelty of this study was the identification and characterization of a thermostable xylanase from a South African T. harzianum isolate, the application of experimental design to optimize its production and as well as that of a recombinant XT6 xylanase. The recombinant XT6 xylanase exhibited high yields in bioreactor production with activity superior to that of a commercial xylanase preparation, further emphasizing its potential for commercialization through scalingup techniques and its industrial application. Moreover, the enzymes investigated in this researchhold promise for the production of prebiotics in animal feed applications.
Lactic acid production from Kraft waste-pretreated corn cobs in dairy wastewater using Lactobacillus plantarum ATCC 14917: process modelling and preliminary scale-up.
(2024) David, Anthea Naomi.; Gueguim Kana, Evariste Bosco.; Sukai, Yeshona.
Abstract available in PDF.
Evaluation of the larvicidal potential of Bacillus velezensis strain PHP1601 as a viable biological control agent against selected fly species = Ukuhlolwa kwamandla elarvicidal yeBacillus velezensis strain PHP1601 njenge-ejenti yokulawula yebhayoloji esebenzayo ngokumelene nezinhlobo zezimpukane ezikhethiwe
(2024) Ramesar, Danvir Rajesh.; Hunter, Charles Haig.
Flies are one of the most abundant and prevalent insect pests posing a growing threat to various sectors of the economy. In response to this, a study was undertaken to evaluate Bacillus spp. strain PHP1601 as a candidate biocontrol agent against Lucilia cuprina larvae as a proxy for fly species of biocontrol significance. The identity of PHP1601 was confirmed as B. velezensis using MLSA and species-specific PCR. Bioassays demonstrated a larvicidal effect of cell, endospore (102 – 1010 cells/endospores g -1 ) and cell-free supernatant (1 – 30% v w -1 ) treatments on second instar larvae of L. cuprina. Studies were directed to the larvicidal effect of extracellular compounds, namely lipopeptides. Crude lipopeptide extract (CLP) was acquired using organic extraction from Landy broth. Bioassays with CLP extract (5 – 1000 μg g -1 ) resulted in a dose-dependent larvicidal response. Lipopeptides in the CLP extract were purified by TLC and characterised using UPLC ESI-TOF MS. This indicated the presence of iturin, fengycin and surfactin homologues of which, the purified surfactin fraction (Rf 0.91) was the most larvicidal. Bioassays were repeated with commercial surfactin, confirming its larvicidal potency, exhibiting an LC50 of 9.87 μg g -1 at 240 h. Larvae scent choice tests using TSB and MG bioassay medium fermented by PHP1601 showed that resulting VOCs were attractive to fly larvae, which was considered a viable trait of a fly biocontrol agent. CG-MS of the VOCs produced indicated that ketones were the dominant VOC class and, presumably, the major contributor to this larvae attraction effect. Field performance evaluation using pig manure trials demonstrated successful inhibition of several fly species of agricultural and veterinary importance using endospore treatments (105 and 1010 endospores g -1 ) of PHP1601. qPCR and REP-PCR fingerprinting confirmed that PHP1601 could grow in the manure slurries and was amiable to recovery and monitoring. Zebrafish embryo toxicity bioassays of the CLP produced by PHP1601 indicated that they achieved an LC50 of 22.77 µg ml-1, which characterised these metabolites as slightly toxic. Genome mining detected no genes associated with pathogenicity or virulence and presented no apparent pathogenic threat. The investigation demonstrated that B. velezensis PHP1601 is a viable fly biocontrol candidate and constitutes the first report of a B. velezensis antagonist of Brachycera flies. Iqoqa: Izimpukane izinambuzane ezivamile kakhulu nezibanga ubungozi obukhulayo emikhakheni eyahlukahlukene kwezomnotho. Ukubhekana nalokhu, lolu cwaningo lwenziwa ukuhlola ingcindezi yeBacillus sp. i-PHP1601 njengesinye sezilawulo semvelo ukubhekana nezimpethu zeLucilia cuprina njengesimeleli sohlobo lokundizayo lokwengamela okuphilayo okubalulekile. Ubunjalo bePHP1601 batholakala njengeB. velezensis kusetshenziswa i-MLSA kanye nohlobo oluthile lwe-PCR. Izikali zokuphilayo zaveza ukukhephuka kwezimpethu kweseli i-endospore (102 – 1010 cells/endosporesꞏg-1) kanye nokubhekelelwa kwe-cell-free supernatant (1 – 30% vꞏw-1) esigabeni sesibili sokukhula kwezimpethu ze-L. cuprina. Ucwaningo lwalubhekiswe emitheleleni yokuphethuka kwezimpethu endaweni ezungeze amaseli, nokuyi-lipopeptides. Ukuhluzwa kwe-crude lipopeptide (CLP) kwatholakala ngokusebenzisa ukudonswa kwendabuko kwi-Landy broth. Izikali zokuphilayo nokukhanywa kwe-CLP (5 – 1000 μgꞏg-1) kwaveza ekuncikeni kokusetshenzwa kwempendulo enezimpethu. Ama-Lipopeptides ekukhanyweni kwe-CLP acwengisiswa nge-TLC futhi abukwa ngokusebenzisa i-UPLC ESI-TOF MS. Lokhu kwaveza ubukhona be-iturin, i-fengycin kanye ne- surfactin homologues nobukhona bengxenyana yesimanzisi esicwengekile (Rf 0.91) esasiphethula izimpethu kakhulu. Izikali zokuphilayo zaphindwa ngesimanzisi esikhulu sokwakha inzuzo, ukuqinisekisa ukukhuphuka kokuphethuka kwempethu, okuveza i-LC50 of 9.87 μgꞏg-1 at 240 h. Izikali zokuphilayo ngobungozi besiqalo se-Zebrafish zokukhanywa kwe-CLP kwaveza ukuthi kwakhipha i-LC50 of 22.77 µgꞏml-1, nekhomba ama-metabolites anobungozi obuncane. Ukukhiqiza ufuzo kwaveza ukuthi alukho ufuzo oluhlobene nobugciwane bofuzo noma ubungozi futhi kwaveza ukungabi bikho kokuhlobana kofuzo. Ukuhlola ukusebenza kwendawo kusetshenziswa umanyolo wezingulube kwaveza ukunqamuka okuyimpumelelo kwezinhlobo zezimpukane eziningi zezolimo kanye nokubaluleka kwezilwane kusetshenziswa ukusetshenzwa kwe-endospore (105 and 1010 endosporesꞏg-1) ye-PHP1601. I-qPCR kanye nokuhlotshaniswa kwe-REP-PCR kwavuma ukuthi i-PHP1601 ingakhula ezindaweni zikamanyolo futhi kwakuhlobene nokuthuthuka kanye nokuqapheleka. Iphunga lezimpethu lezikali ezikhethiwe laveza ukuthi ama-VOCs akhiqizwa esimuncwaneni se-PHP1601 eyenomthelela wezimpethu zezimpukane, nokwavela njengenjwayelo eshintshayo yesakhi sokwengamela ukuphila kwezimpukane. Ama-CG-MS e-VOCs akhiqizwa akhomba ukuthi ama-ketones ayewuhlobo olukhulu lwe-VOC futhi, okulindelekile, umthelela omkhulu womphumela wokumema izimpethu. Ukuhlola kwaveza ukuthi i-B. velezensis PHP1601 uwuhlobo olushintshayo lwesengamelo sezimpukane futhi kwakha umbiko wokuqala wokuphikisana kwezimpukane ze-Brachycera.
Phycoremediation of industrial and municipal domestic wastewaters with concomitant biomass propagation for bioenergy production.
(2022) Gumbi, S'fiso Thuthukani.; Olaniran, Ademola Olufolahan.
The utilization of microalgae has been endorsed as a great source of biofuel generation and wastewater reclamation without any adverse effects. Microalgae have high growth rates, efficient photosynthesis process and biomass productivity which serve as an economic advantage. Microalgae can be used for the dual purpose of biodiesel production and wastewater treatment due to their ability to sequester organic pollutants such as nitrogen and phosphorus in wastewater. Thus, the aim of this study was to bioprospect for indigenous hyper lipid producing indigenous microalgal strain for biofuel production and wastewater treatment. Different water samples were collected from diverse aquatic habitats, including freshwater, brackish and marine water in KwaZulu-Natal, South Africa. Eight indigenous microalgal strains were isolated and screened for biomass accumulation and lipid yield using Nile red fluorescence microscopy screening and gravimetric analysis. The strains were identified based on their morphological characteristics and 18S rRNA gene sequence analysis to belong to the genera Chlorella, Neochloris and Chlamydomonas. They showed high lipid yield ranging from 14 ± 6.2 to 38 ± 8.8% dcw, proving to be a good feedstock for biodiesel production. Of the eight isolated microalgae, Chlorella sp. T4 was selected for further analysis based on the growth kinetic, lipid productivity and fatty acid profiles. The strain was subjected to different cultivation conditions to enhance lipid productivity by varying nitrogen and phosphorus concentration. A significant decrease in biomass accumulation and low quantum efficiency of photosystem (Fv/Fm) value was observed under nitrogen and phosphorus limiting conditions. The lowest biomass yield of 0.58 ± 0.03 g L-1 was found in nitrogen limiting medium (0.75 g L-1). High lipid productivity of 15.54 ± 0.7 mg L-1 d-1 was obtained under nitrogen limiting condition which was 1.37- fold higher than phosphorus limiting (0.02 g L-1) condition after 21 days. Nutrient stress caused an increase in the expression of Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta (accD), ketoacylACP synthase-1 (KAS-1), omega-6 desaturase (ω-6 FAD) and omega-3 desaturase (ω-3 FAD) genes responsible for lipid biosynthesis. Whereas a decrease in Ribulose bisphosphate carboxylase large chain (rbcL) gene expression level was noted due to nutrient stress lowering the photosynthetic rate. Fatty acid methyl esters produced - under nutrient limiting conditions were found to be suitable for the production of high-quality biodiesel with enhanced oxidative stability and cold flow properties. The ability of Chlorella sp. T4 to utilize the different nutrient-rich environments and remove nutrients from poultry and dairy wastewater was investigated to ascertain its possible use for the sustainable and low-cost treatment of wastewater. Chlorella sp. T4 showed high nitrogen and phosphorus removal efficiency of 85 to 95% and 35 to 93%, respectively. This was followed by a high biomass yield of 1.28 g L-1 and 0.85 g L1 obtained using raw poultry and dairy sludge wastewater, respectively. The biomass contained significant iv amounts of lipids (16.2–25.7 % dry wt.), carbohydrates (20.7–33.1 % dry wt.), and proteins (24.5–34.6 % dry wt.), regardless of the wastewater type. Biodiesel properties of lipids extracted from the cell grown in poultry and dairy wastewater complied with most of the international standards by ASTM D6751 and EN 14,214. Based on the lipid productivity and fatty acid profile, sludge dairy wastewater was used for biomass propagation for simultaneous bioethanol and biodiesel production. Optimization of cell disruption and extraction techniques resulted into high lipid and sugar recovery efficiency. Through acid hydrolysis using sulphuric acid, 2.14 g L-1 of sugar was recover from Chlorella sp. T4 biomass and fermented to ethanol (0.81 g L-1) using Saccharomyces cerevisiae. High lipid content of 21.7 ± 0.5% was recovered from the residual biomass after hydrolysis and converted into biodiesel via transesterification. The biodiesel produced from the residual biomass meets most of the standards specified by ASTM D6751 and EN 14214. In conclusion, hyper lipid producing microalgal strain Chlorella sp. T4 isolated from KwaZulu-Natal, South Africa showed potential for biofuel production after proper optimization of growth conditions. The potential of Chlorella sp. T4 to utilize different wastewater high in nutrient concentration confirm potential application during large scale cultivation for biofuel production to address energy crisis and water shortage.
Biodegradation of pentachlorophenol by Bacillus tropicus isolated from activated sludge of a wastewater treatment plant in Durban, South Africa.
(2021) Aregebesola, Oladipupo Abiodun.; Olaniran, Ademola Olufolahan.; Mokoena, Mduduzi Paulos.
Abstract available in PDF.
Polyhydroxyalkanoate production by Bacillus thuringiensis: an aspect of biorefining pulp and paper mill sludge.
(2021) Singh, Sarisha.; Govinden, Roshini.; Sithole, Bishop Bruce.; Lekha, Prabashni.; Permaul, Kugenthiren.
The substantial success of plastic as a material is owed to its unparalleled designs with unique properties and proved versatility in an extensive range of applications. Unfortunately, the reliance on single-use plastic commodities consequently results in the incorrect disposal and accumulation of this waste at staggering rates in our environment and landfill sites. In this regard, there is a vested interest in replacing petrochemical plastics with natural, biodegradable plastics (bioplastics). Of the many natural polymers available, microbially synthesized polyhydroxyalkanoates (PHAs) have gained popularity. Eco-friendly PHA-based bioplastics are characteristically as robust and as durable as their oil-based equivalents. Pulp and paper mill sludge (PPMS) is another solid waste stream that is predominantly disposed of via landfilling. The environmentally hazardous gases and leachate emitted from PPMS together with limited landfill space availability and the implementation of strict waste management legislation may not make landfilling practicable in the future. However, this carbohydrate-rich biomass has favorable traits that make it applicable as a feedstock for microbial biomass and PHA production. Hence, in the interest of addressing the issues mentioned above, this study aimed to beneficiate PPMS into PHAs by applying it as the sole feedstock for microbial cell proliferation and subsequent PHA production. Presently, to the best of the author’s knowledge, there are no reports on PHA production as a route for valorization of PPMS from South African pulp and paper mills. Thus, the novelty of the present study is marked by the unique ways of incorporating PPMS as a low-cost substrate as well as the various fermentative strategies navigated to enhance both microbial cell biomass and PHA productivity. In the present study, it was established that Bacillus thuringiensis had promising PHA-producing capability. The strain synthesized a copolymer and terpolymer using untreated (raw) neutral semi-sulphite chemical pulping and cardboard recycling mill (NSSC-CR) and prehydrolysis kraft and kraft pulping mill (PHKK) PPMS in a consolidated bioprocessing fermentation. A separate hydrolysis and fermentation strategy was pursued whereby a glucose-rich hydrolyzate was obtained from enzymolysis of PPMS and subsequently utilized in a cyclic fed-batch fermentation (CFBF) strategy to obtained enhanced yields of cell biomass and PHAs. Response surface methodology (RSM) was first implemented to optimize the conditions for enzymatic saccharification of de-ashed PHKK PPMS. The optimized variables were; pH 4.89; 51°C; hydrolysis time 22.9 h; 30 U/g β-glucosidase and 60 U/g cellulase; and 6.4% of dried de-ashed PPMS fiber resulting in a hydrolyzate comprising of 48.27% glucose. Thereafter, CFBF was pursued where the glucose-rich hydrolyzate was employed as the sole carbon source for cell proliferation and PHA production. The statistically optimized fermentation conditions to obtain high cell density biomass (OD600 of 2.42) were; 8.77 g L-1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised of three cycles and after the third cyclic event, maximum cell biomass (20.99 g L-1) and PHA concentration (14.28 g L-1) were achieved. This cyclic strategy yielded an almost 3-fold increase in biomass concentration and a 4-fold increase in PHA concentration compared with batch fermentation. The properties of the synthesized PHAs were similar to commercial polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) and also displayed slightly higher thermostability and lower crystallinity compared with commercial PHB and PHBV. This is the first report detailing the proof of concept of using PPMS from South African pulp and paper making mills for cell biomass and PHA production by B. thuringiensis. In addition, this study reports on the practicality and novelty of utilizing PPMS either in its raw, untreated state or as enzymatically saccharified glucose-rich hydrolyzate as cheap substrates applicable for both cell biomass and PHA production using different fermentation strategies. Finally, to the best of our knowledge, this is also the first report that has successfully applied B. thuringiensis in a CFBF strategy coupled with glucose-rich hydrolyzate as the sole carbon source for the production of high cell density biomass and enhanced PHA production. From this study, it is intended that innovative insights and prospective solutions to valorizing pulp and paper mill sludge are provided, whilst simultaneously generating a value-added product.
Chlorine dioxide and ozone facilitated disinfection of selected bacteria in aqueous systems.
(2018) Ofori, Isaac.; Jonnalagadda, Sreekantha Babu.; Lin, Johnson.
Chlorination is the most commonly used disinfection technology for the control of pathogenic microorganisms in drinking water or wastewater treatment. However, the reactions of chlorine with natural organic matter in water have been found to produce harmful by-products including trihalomethanes, haloacetic acids, and haloacetonitriles. Regulations on these deleterious disinfection by-products keep increasing and have consequently focussed considerable attention on the use of alternative chemical disinfectants. The design and operation of an efficient disinfection system at a water treatment facility aim at providing adequate control of microbial threats and simultaneously satisfying regulatory requirements on by-products. Achieving these require an in-depth understanding of the inactivation kinetics of the disinfectant on potential target organisms. In this study, the microbial inactivation kinetics of chlorine dioxide (ClO2) as an alternative chemical disinfectant for water treatment was monitored on two Gram-negative bacterial species: Escherichia coli (ATCC 35218) and Pseudomonas aeruginosa (ATCC 27853) and a gram-positive: Staphylococcus aureus (ATCC29313) under varied conditions of disinfectant concentration, pH, temperature and bacterial density in an oxidant demand free water. Further studies were conducted to investigate the effect of ClO2 on bacterial outer cell membrane permeability, the cytoplasmic membrane integrity, inhibition of intracellular enzyme activity and changes in cell morphology by TEM to elucidate the bactericidal mechanism of action of ClO2. In addition, autochnous bacteria from urban wastewater were exposed to chlorine dioxide and the susceptibilities monitored and compared by a culture-dependent heterotrophic plate count technique and culture-independent 16S rRNA gene-directed polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE). Furthermore, the influence of four organic solvents commonly discharged from industrial lines into wastewater systems, namely, ethanol, methanol, ethyl acetate and dimethyl sulfoxide (DMSO) on ozone absorption, stability and consequent inactivation of Escherichia coli (ATCC 25218) and Staphylococcus aureus (29213) in water were also examined. Chlorine dioxide showed strong and rapid disinfection capabilities at relatively lower dosages with significant influences by pH and temperature. However, the efficiency generally appeared unaffected by changes in bacterial density. The PCR- DGGE technique showed that 1.0 mg/L was sufficient to inactivate three predominant bacterial species from an urban wastewateridentified as Arcobacter suis F41, Pseudomonas sp strain QBA5 and Pseudomonas sp B-AS- 44, whereas a significant population of other species such as Pseudomonas sp CCI2E was observed to presumably remain viable to 5.0 mg/L chlorine dioxide whilst the heterotrophic plate count method indicated complete elimination of bacteria at 3.0 mg/L. ClO2 was not found to inactivate bacteria by inflicting gross morphological damages to the cell wall, but instead, increases the permeability of the outer cell membrane, disrupts the integrity of the inner cytoplasmic membrane which leads to the efflux of intracellular contents of the cell and hence, resulting in the overall cell death. The presence of ethyl acetate and DMSO were observed to significantly enhance ozone absorption and stability in water with a consequent increase in bacteria inactivation efficiency whilst methanol-containing water rather accelerated the decomposition of ozone. The findings herein provide further knowledge to enhance the disinfection operations at a water treatment plant when ClO2 or O3 is applied.
Bacterivory of three freshwater ciliates isolated from Blackbarough Spruit in KwaZulu-Natal, South Africa.
(2021) Bulannga, Rendani Bridghette.; Schmidt, Stefan.
Abstract available in PDF.
Diversity of quorum sensing pherotypes amongst ecotypes of plant-associated Bacillus subtilis sensu lato isolates.
(2021) Tredgold, Heather Rayne.; Hewer, Raymond.; Hunter, Charles Haig.
Ecologically-adapted populations, or ecotypes, are species forms that are functionally adapted to particular habitat niches. Bacterial ecotypes are challenging the significance and implications of ecological adaptation in terms of prokaryote taxonomy, community ecology, and biocontrol applications. Plant-associated members of the Bacillus subtilis sensu lato group—in particular B. subtilis and B. velezensis—are ecologically specialised to perform numerous functions that are beneficial to plant and soil health. Underpinning these beneficial activities is the ability to colonise plant surfaces by biofilm formation. Biofilms are a result of co-ordinated social behaviour amongst microorganisms. In the B. subtilis sensu lato this sociality is governed by the ComQXPA quorum-sensing cassette, which uses the ComX pheromone for intercellular communication leading social behaviours like biofilm formation. This peptide pheromone contains a post-translational modification on a conserved tryptophan residue. This modification is highly variable between populations, resulting in discrete ComX variants which form communication groupings known as pherotypes. The limitation of communication to within a pherotype may constitute a form of ecological adaptation designed to protect the products of co-operative behaviour and restrict their benefit to the producing population. The present study aimed to explore ecotypes and pherotypes amongst a subset of plantassociated B. subtilis and B. velezensis isolates. These isolates originated from phylloplane and rhizosphere samples from seven crop species grown across the KwaZulu-Natal province, South Africa, and had demonstrated biocontrol potential in previous studies. An exploratory study set out to apply in silico approaches to determine gene-sequence-based variation amongst representative strains of the B. subtilis sensu lato available in the GenBank database. Nine housekeeping gene targets (viz., 16S rRNA, cheA, dnaJ, groEL, gyrA, gyrB, polC, purH, and rpoB) were evaluated for suitability to resolve clustering of closely-related B. subtilis sensu lato. Four of these gene sequences (viz., 16S rRNA, dnaJ, gyrA, and rpoB) were identified as candidates for a Multilocus Sequence Analysis (MLSA) scheme to distinguish between members of the B. subtilis sensu lato group. Putative pherotype variation amongst these reference strains was explored in silico using comQXP gene sequence data. The suitability of a comQXP PCR-RFLP protocol with potential for rapid pherotyping amongst B. subtilis sensu lato was evaluated in silico using simulated comQXP amplicons. This necessitated the design of a PCR primer set targeting the quorum-sensing gene region of B. velezensis. Four restriction enzyme candidates namely, BtsCI, Fnu4HI, Cac8I, and Hpy166II, were identified for further study. Ecotyping amongst the B. subtilis and B. velezensis isolates was carried out using a four-gene (viz., 16S rRNA, dnaJ, gyrA, and rpoB) MLSA. This concatenated sequence dataset was applied to ecotype simulation (ES) analysis to corroborate putative ecotype sub-clusters in the MLSA phylogeny. Two DNA fingerprinting approaches—Repetitive Element Palindromic PCR (Rep-PCR) and Random Amplified Polymorphic DNA PCR (RAPD-PCR)—were also evaluated for their potential to identify putative ecotypes within the isolate subset. This investigation of pherotypes amongst the putative ecotype groupings examined isolate comQ sequence data as well as the comQXP PCR-RFLP, and also applied a srfA-LacZ reporter gene assay to examine isolate stimulation of seven known pherotype tester strains (viz., 168, RO-B- 2, RO-C-2, RO-E-2, RO-FF-1, RO-H-1, and RS-D-2). The MLSA of isolate gene sequences presented distinct sequence clusters suggestive of ecotype populations amongst the two Bacillus species which were corroborated by ES analysis. The MLSA and ES determined two putative ecotypes within the B. subtilis isolates, and six within the B. velezensis isolates. Ecotype groupings were found to contain isolates from different crop species and locations, and four B. velezensis ecotypes were distinct from included B. subtilis sensu lato reference strains. PCR fingerprinting identified strain-level variances amongst the isolates, and were able to differentiate plant-associated B. velezensis from closelyrelated B. amyloliquefaciens, but did not define groupings entirely consistent with the ES and MLSA phylotypes. The MLSA, ES, and PCR fingerprinting delineated a group of five isolates (viz., B81, CT-R67, bnd136, bng221, and sqo271) to be grouped with reference strain Bacillus sp. JS in the MLSA. This grouping is of interest as Bacillus sp. JS is a strain demonstrating biocontrol capability and formed a clade distinct to B. subtilis sensu stricto in gene sequence phylogenies. This Bacillus sp. JS grouping was further confirmed by ES to constitute a single ecotype, and both Rep-PCR and RAPD-PCR OPG-16 distinguished this grouping based on fingerprint profiles. Investigation of reference strain variation in the comQ gene sequence determined significant levels of sequence variation amongst reference strains evaluated in silico, and showed divergence in some strains from known pherotype counterparts. Furthermore, sequence clusters were resolved in B. velezensis that suggested unique pherotype variants amongst reference strains, and showed comQ similarity amongst the five isolates related to Bacillus sp. JS. This trend was observed for the comQ phylogeny applying isolate and pherotype sequences, which resolved two distinct clusters each amongst the B. velezensis and B. subtilis isolates, with only two isolates (viz., bnd134 and bnd156) grouping with known pherotypes. The B. velezensis clades demonstrated significant comQ sequence variance from the pherotype reference strains based on clustering distances in the phylogeny. Of the four enzyme candidates identified for potential use in a comQXP PCR-RFLP protocol, two were found to be applicable: BtsCI resolved the grouping of the Bacillus sp. JS clade, while Fnu4HI was found to distinguish between more closely-related isolates in B. velezensis. The profile groupings for both of these enzymes demonstrated most of the isolates to be distinct from known reference pherotype strains, and the technique proved able to corroborate groupings in the comQ phylogeny. The pherotyping srfA-LacZ reporter gene assay failed to support the pherotype groupings defined by the comQ phylogeny, but demonstrated that the environmental isolates were capable of eliciting substantial responses in the pherotype tester strains, particularly amongst the B. velezensis isolates. Eight isolates did not elicit significant responses in any of the tester strains, while most matched to multiple pherotypes on the basis of tester strain response to conditioned medium from isolates. These findings demonstrated that putative ecotype variation was present amongst the B. subtilis and B. velezensis isolates that were resolvable by MLSA, and that these ecotype groups comprised isolates from different crop types and location sites. Some of these ecotypes bore only distant relation to B. subtilis sensu lato reference strain counterparts. The investigation of PCR fingerprinting methods for ecotyping purposes found that the primer sets applied were not able to consistently corroborate putative ecotype groupings defined by MLSA and ES. Pherotyping investigations demonstrated that there was evidence of gene-sequence-based pherotype variation amongst the isolates within these putative ecotype groupings. The pherotype clades resolved with the comQ phylogeny and with comQXP PCR-RFLP were not corroborated by srfA-LacZ reporter gene pherotyping assays, but the data suggested that assay optimisation to suit environmental strains is warranted in future studies. This study presents the first instance of ecotyping and pherotyping amongst plant-beneficial B. velezensis. The B. velezensis isolates were largely distinct in ecotype and pherotype from the plant-associated model strain FZB42, which further promotes that these isolates demonstrate localised adaptation. The Bacillus sp. JS-related isolate grouping is of interest as these isolates comprised a single putative ecotype resolvable by MLSA, ES, and Rep-PCR and RAPD-PCR OPG-16. This grouping was distantly-related to B. subtilis sensu stricto, and this is interesting from a taxonomic standpoint, as is the fact that that Bacillus sp. JS is reported to have plantbeneficial capabilities. The existence of uniquely South African plant-associated B. subtilis sensu lato ecotypes is valuable in agricultural management approaches targeting beneficial microbes, as these organisms likely possess adaptations allowing them to compete with extant bacterial communities and thrive within the South African agricultural climate.
Impact of nanobiocatalysts on Saccharomyces cerevisiae metabolism for ethanol production: process optimization, kinetic studies and preliminary scale-up.
(2021) Sanusi, Adeyemi Isaac.; Gueguim Kana, Evariste Bosco.
Abstract available in PDF.
Production of lipopeptide biosurfactant by Paenibacillus sp. D9: basic and applied aspects.
(2019) Jimoh, Abdullahi Adekilekun.; Lin, Johnson.
Abstract available in PDF.
Study on Fusarium toxins contamination of cereal grains in Jiangsu Province, China.
(2018) Fang, Ji.; Mokoena, Mduduzi Paulos.; Olaniran, Ademola Olufolahan.; Shi, J.
Jiangsu Province is located in the transition zone from subtropical to warm temperate zone, with the characteristics of monsoon climate. The province's natural ecological conditions are suitable for the production of winter wheat and rice. In this region, the warm and humid climate is very suitable for the plant epidemics such as Fusarium head blight (FHB). Fusarium mycotoxins are secondary metabolites produced by Fusarium species; and can cause acute and chronic toxic effects on the body and are a threat to humans and animal health. Therefore, knowledge about the prevalence of FHB-producing Fusarium species, incidence of deoxynivalenol, zearalenone and fumonisins as well as possible influencing factors is imperative for preventing influx of contaminated grains into food supply chain. The present study focuses on the occurrence of Fusarium species coupled with contamination levels of Fusarium mycotoxins from different Jiangsu Province, China for three years, and the influences of rainfall and temperature on accumulation of DON. In addition, Fusarium strains were isolated from rice and assessed for the potential to produce fumonisins and beauvericin. The findings of this study increase the knowledge on important rice fungal pathogens and provide relevant information on the high variability of these pathogens, as well as their implications for the development of further diseases. The ICS test developed in our study has advantages, such as rapid and efficient screening of samples. The data obtained from the ICS test shows good agreement with LC-MS/MS data. These results showed that the ICS test is suitable for on-site monitoring of ZEN.
Treatment of lipid-rich wastewater using free and immobilized bioemulsifier and hydrolytic enzymes from indigenous bacterial isolates.
(2017) Adetunji, Adegoke Isiaka.; Olaniran, Ademola Olufolahan.
The production and discharge of raw and poorly treated lipid-rich wastewater increase yearly due to rapid urbanization and industrial growth. This results in severe environmental and health hazards by affecting the normal operations of ecosystems. Biological approach involving synergistic application of low cost bioemulsifier and hydrolytic enzymes is an efficient, cost-effective, sustainable and eco-friendly technology for the treatment of high strength lipid-rich wastewater. Therefore, the main objective of this study was to investigate the potential of a mixture of free or immobilized bioemulsifier and hydrolytic enzymes (protease and lipase) in the reduction of pollutants present in dairy and poultry processing wastewater. Glycoprotein bioemulsifiers and hydrolytic enzymes were produced extracellularly by submerged fermentation from indigenous Acinetobacter sp. and Bacillus aryabhattai, respectively. Optimization of bioprocess parameters, using response surface methodology, revealed a 4.4- and 7.2-fold increase in protease and lipase production, respectively. The bioemulsifier from strain AB9-ES (XB9) and strain AB33-ES (YB33) formed stable emulsions only with edible oils with highest emulsification indices of 79.6 and 67.9%, respectively obtained against sunflower oil. The emulsifying activity of the bioemulsifiers was stable over broad range of temperature (4-121 ºC), moderate salinity (1-6%) and pH (5.0-10.0). Comparative study of biochemical profiling of both free and immobilized hydrolytic enzymes showed no change in the optimum temperature and pH of both enzyme preparations for maximum activity. However, in comparison to free enzymes, the immobilized enzymes recorded enhanced stability over the investigated pH and temperature ranges. Kinetics properties revealed enhanced enzyme-substrate affinity and increased catalytic efficiency of the immobilized enzymes when compared to soluble enzymes. In addition, the immobilized enzymes were more stable when stored at 4 and 25 °C and reusable for more than five consecutive cycles. These hyper-active and highly stable bioproducts were utilized in cocktail in both soluble and entrapped form for the batch biodegradation of pollutants present in lipid-rich wastewater. Biodegradability of the wastewater was assessed by measuring the reduction of COD and lipid content at time intervals under varying incubation conditions. In dairy wastewater treated at 37 °C without pH adjustment, maximum COD (60.51 and 65.19%) and lipid (47.98 and 63.53%) reduction efficiencies were recorded at 120 h using free and immobilized bioproducts, respectively. However, under these conditions, maximum COD (86.44 and 93.65%) and lipid (51.62 and 69.06%) removal efficiencies of poultry processing wastewater were observed at 120 h when treated with free and immobilized bioproducts, respectively. At temperature of 50 °C and pH 8.0, there was enhanced reduction of organic pollutants, with maximum COD (65.96 and 77.52%) and lipid (55.22 and 71.12%) removal efficiencies obtained in dairy wastewater at 72 h when using free and immobilized bioproducts, respectively. In the case of poultry processing wastewater, optimum COD (90.29 and 94.72%) and lipid (63 and 76.66%) removal was recorded at 72 h when treated with free and immobilized bioproducts, respectively. Reusability studies suggest that the immobilized bioproducts could be reused for up to six and seven times for the treatment of dairy and poultry processing wastewater, respectively. Findings from this study suggest the efficient, cost-effectiveness, sustainability and synergistic application of the developed immobilized bioemulsifier and hydrolytic enzymes in the removal of pollutants present in dairy and poultry processing wastewater.
Pit latrines in peri-urban South African community: a hygiene challenge and a health risk owing to current desludging practices and biofilm-forming, multi-drug resistant bacteria.
(2019) Beukes, Lorika Selomi.; Schmidt, Stefan.
Abstract available in pdf.
Isolation and evaluation of autochthonous microalgae strains for biodiesel production and wastewater treatment.
(2018) Odjadjare, Ejovwokoghene Collins.; Olaniran, Ademola Olufolahan.; Mutanda, Taurai.; Chen, Y F.
The current depletion of global fossil fuel reserves and increasing CO2 emission are generating global climate change concerns hence, the increasing calls for the development of renewable biofuels with low CO2 emission. Microalgae has been touted as the most suitable feedstock of renewable fuels due to a number of reasons including high biomass productivity, fast growth rates, cost-effective cultivation systems and alleviation of food security concerns associated with use of other feedstock for biofuel production. Bioprospecting could yet reveal microalgae with high lipid contents for biodiesel production and novel characteristics such as production of unique bioactive compounds. With an attractive climate and plenty of sunlight all year round, South Africa is in a unique position to take advantage of these organisms to guarantee her future energy needs. This research thus aims to bioprospect for indigenous strains of microalgae in aquatic habitats and wastewater treatment plants in KwaZulu-Natal province of South Africa and evaluate their potential for biofuel production and wastewater remediation. DGGE and T-RFLP fingerprinting techniques were adapted to give an overview of the diversity of microalgae in various aquatic habitats including wastewater treatment plants (WWTP), rivers, estuaries and marine ecosystems in order to identify areas for potential bioprospecting. Ten (10) microalgae species from these aquatic environments were isolated, identified and evaluated for lipid, total carbohydrate and protein accumulation using well defined procedures. The lipid profile, biodiesel characteristics as well as effect of starvation on lipid accumulation were also determined. The biomass production and simultaneous phycoremediation potential of two autochthonous isolates Asterarcys quadricellulare and Neochloris aquatica were also investigated. Microalgae classes; Trebouxiophyceae and Chlorophyceae were observed to be dominant in fresh water habitat, while those belonging to the class Ulvophycea (Oltmannseillopsis spp.) dominated the salt water environment. At the brackish water habitat, microalgae belonging to the classes Trebouxiophyceae (Chlorella minutissima) and Chlorophyceae (M. sturmi) were dominant. A shift in community structure was observed at the maturation ponds of WWTPs and in rivers over time. The growth rates of 10 isolated microalgae ranged from 0.219 ± 0.003 to 0.175 ± 0.023 gL-1day-1 while dry weight ranged from 0.433 ± 0.208 to 1.167 ± 0.153 gL-1. Chlorococcum LM1 showed high accumulation of lipid (11.93 ± 0.76 mg/L). The microalgae isolated in this study accumulated high carbohydrate content ranging from 25 to 61 % of their dry weight while protein content ranged from 1.06 ± 0.1 mg/L to 1.39 ± 0.1 mg/L accounting for 21 to 28% dry weight. Lipid accumulation also varied under nutrient limitation condition. Lipid accumulation was enhanced in some of the isolates such as Chlorococcum sp. LM1 (17.2%) and C. sorokiniana NWS5 (24%) while a decrease was observed in others such as C. minutissima TS9 (16%), N. aquatica Toti4 (3.5%) and Chlorococcum sp. LM2 (6.5%). Response to enhanced lipid accumulation via starvation seem to be unique to each algal strain irrespective of species. The lipid profile consisted mainly of saturated fatty acid such as oleic acid (C18:1), palmitic acid (C16:0) and stearic acid (C18:0) with low amounts of polyunsaturated fatty acids such as linoleic acid (C18:2 n-6). Characteristic of the biodiesel based on the lipid profile reveal a low viscosity and density. The biodiesel was determined to be of good quality with high oxidation stability, low viscosity and conformed to the ASTM guidelines. Low total phosphorus concentration in the wastewater resulted in an unbalanced N:P ratio of 44 at the Northern wastewater treatment works (NWWTW) and 4 at the Umbilo wastewater treatment works (UWWTW). Asterarcys quadricellulare utilized the wastewater for growth and reduced the COD of the wastewater effluent from the UWWTW by 12.4% in contrast to Neochloris aquatica which did not show any growth. Asterarcys quadricellulare was able to utilise the wastewater achieving a growth rate up to 0.18 day−1 in sterilized wastewater from the NWWTW and 0.17 day−1 in the unsterilized wastewater from UWWTW. Asterarcys quadricellulare accumulated high biomass of 460 mg/L compared to 180 mg/L in Neochloris aquatica. Total nitrogen (TN) and Phosphorus (TP) were reduced by 48% and 50% respectively by Asterarcys quadricellulare cultivated in sterile wastewater from NWWTW while, Neochloris reduced the TP by 37% and TN by 29%. At the UWWTP, TP and TN were reduced by 32% and 44% respectively by Asterarcys quadricellulare cultivated in sterile wastewater while 29% and 19% reduction were recorded in Neochloris aquatica. The study showed the diversity and community structure of microalgae in aquatic ecosystems in the study area. Autochthonous microalgae were rich in lipid, carbohydrate and protein and could be applied for biofuel production. Wastewater effluent can be used to generate biomass for biodiesel production while treating wastewater. However, optimization of the N:P ratio and carbon source are necessary to improve remediation and biomass productivity for future commercial scale production.
Selection and identification of novel Mycobacterium tuberculosis phage-displayed biomarkers by immunoscreening against patients’ serum samples.
(2018) Chiliza, Thamsanqa Emmanuel.; Pillay, Balakrishna.; Pillay, Manormoney.
Abstract available in pdf.
Alkalic salt-based pretreatment strategies for enhancing sugar recovery from corn cobs and process development for simultaneous saccharification and bioethanol production.
(2018) Sewsynker-Sukai, Yeshona.; Gueguim Kana, Evariste Bosco .
Abstract available in PDF file
Molecular characterization of two bacteriophage strains and their role in the gastrointestinal tract of mice.
(2018) Bao, Hongduo.; Schmidt, Stefan.; Olaniran, Ademola Olufolahan.; Wang, Ran.
Abstract available in PDF file.
Strategies for enhancing sugar recovery from sugarcane leaf waste and kinetic modelling for bioethanol production using saccharomyces cerevisiae by4743.
(2017) Moodley, Preshanthan.; Gueguim Kana, Evariste Bosco.
Abstract available in PDF file.
Heterologous expression and application of lipolytic enzymes for mitigation of lipophilic compounds in eucalyptus species.
(2017) Ramnath, Lucretia.; Govinden, Roshini.; Sithole, Bishop Bruce.
Abstract available in PDF file.

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