Doctoral Degrees (Microbiology)

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Phycoremediation of industrial and municipal domestic wastewaters with concomitant biomass propagation for bioenergy production.
(2022) Gumbi, S'fiso Thuthukani.; Olaniran, Ademola Olufolahan.
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.
Pentachlorophenol (PCP) is a persistent organic compound that bio-accumulates in the environment due to its recalcitrant nature and has been listed as a priority pollutant due to its toxicological properties. The recent incidences of xenobiotic at different sites and provinces in South Africa, other African Countries and Europe is worrisome and required a proactive measure. Biodegradation has been projected as one of the best ways to ameliorate recalcitrant impacted sites. This study thus aims to isolate and characterize PCP-degrading microorganism from the environment; degrade PCP and other compounds with the isolate in batch culture; optimize biotransformation processes for effective and efficient transformation; determine biodegradation kinetic parameters; profile metabolites produced; detect and amplify PCPdegrading genes from the selected isolate; map the degradation pathway; clone and overexpress the catabolic genes heterologous; purified and characterized the protein both biologically and structurally and sequence the whole-genome of the isolate with the view to determine the evolution and arrangements of PCP-catabolic genes in the genome of the isolate as well as exploring other potentials of the isolates. A PCP-degrading strain was isolated and characterized using a PCR amplification and analysis of the 16S rRNA. Biodegradation process parameters were optimized using response surface methodology. Degradation kinetics were determined via substrate inhibition models, while PCP transformation was evaluated by spectrophotometric and GC-MS analysis. Catabolic genes were detected and amplified via PCR. Genes were cloned via heat-shock technique using chemically competent cells. Proteins purification, digestion and sequencing were done using affinity chromatography, tryptic digestion, and Liquid Chromatography–Mass Spectrometry (LC-MS) techniques respectively. Pacific Biosciences RS II sequencer with the Single Molecule, Real-Time (SMRT) Link was used to sequence the whole genome of the isolate. Coting’s were assembled and analysed using the HGAP4-de-novo assembly application. Genes were annotated on the Rapid Annotation using Subsystem Technology tool kit (RASTtk) and ab initio prediction (PROKKA) using the prokaryotic genome annotation pipelines. Metabolic model pathways of the bacterium was reconstructed using the RAST SEED Viewer. Primarily, the isolate was identified as Bacillus cereus strain AOA-CPS1 (BcAOA) based on the 16S rDNA sequence analysis. However, a quality control test by NCBI for the submitted whole genome sequence of the strain, using an average nucleotide identity (which compares the submitted genome sequence against the whole genomes of the type strains that are already in GenBank) resulted in the renaming of BcAOA as Bacillus tropicus strain AOA-CPS1 (BtAOA). BcAOA was renamed as BtAOA (based on the whole genome data submitted at NCBI under accession number CP049019). The bacterium degraded 74% of PCP (within 9 days at initial PCP concentration of 350 mg l-1 in a batch culture) and other chlorophenolic compounds in co-metabolism. The degradation followed first and zero-order kinetics at low and high PCP concentration, respectively with biokinetic constants: maximum degradation rate (0.0996 mg l-1 h-1); substrate inhibition constant (723.75 mg l-1) and a halfsaturation constant (171.198 mg l-1) and R2 (0.98). The genes (pcpABCDE, cytochrome P450) encoding the enzymes involved in the biodegradation of PCP were amplified from the genomic DNA of BcAOA. Further, depending upon the genes amplified and identified metabolites using GC-MS, there are two different PCP transformation pathways were proposed in this study. At optimized conditions, BtAOA transformed 98.2% of 500 mg L-1 of PCP in 6 days which represent a significant 59.2% increase in PCP transformation compared to the unoptimized conditions. The kinetic parameters for PCP transformation at optimized conditions were: 1.064 ± 0.114 mg l-1 h-1 (maximum biodegradation rate); 229 ± 19.5 mg l-1 (half-saturation constant); 535 mg l-1 (inhibition constant); and R2 = 0.96. Each of the catabolic genes shared >99% sequences homologies with the corresponding genes in the genomes of their ancestors, however, their biological functions remain putative to date. The optimum temperature and pH of CpsB were 30oC and 7.0. CpsB showed functional stability between pH 6.0-7.5 and temperature 25oC- 30oC. CpsB activity was stimulated by Fe2+, Ca2+, EDTA (0.5-1.5 mM) and Dithiothreitol (0.5- 1.0 mM) but inhibited by sodium azide and sodium dodecyl sulphate (>0.5 mM). CpsB enzyme substrate reaction kinetics studies showed allosteric nature of the enzyme and followed presteady state using NADH as a co-substrate with apparent vmax, Km, kcat and kcat/Km values of 0.465 μmol.s-1, 140 μmol, 0.099 s-1 and 7.07 x 10-4 μmol-1.s-1, respectively, for the substrate PCP and 0.259 μmol.s-1, 224 μmol, 0.055 s-1 and 2.47 x 10-4 μmol-1.s-1, respectively, for co-substrate NADH. The Hill plots and M-W-C model reveal CpsB allosteric nature and belong to K-System. CpsB shared 100% sequence homology with aromatic amino acid hydroxylase and is classified as aromatic amino acid hydroxylase superfamily with multiple putative conserved domains and metal ion binding sites confirming its allosteric nature. Bacillus tropicus AOA-CPS1 Cytochrome P450 monooxygenase (P450CPS1) exhibited optimum activity at 40oC and pH 7.5. The P450CPS1 was stable between 25oC-30oC retaining 100% of its residual activity after 240 min of incubation. The activity of P450CPS1 was stimulated by Mn2+, Fe2+, and Fe3+ typical of an oxidoreductase but inhibited by 2.0 mM piperonyl butoxide and sodium dodecyl sulphate. The reaction kinetics studies showed allosteric nature of P450CPS1 showing apparent vmax, Km, kcat and kcat/Km values of 0.069 μmol.s-1, 200 μmol, 0.011 s-1 and 5.42 × 10-5 μmol-1.s-1, respectively, for the substrate PCP and 0.385 μmol.s-1, 56.46 μmol, 0.06 s-1 and 1.77 × 10-3 μmol- 1.s-1, respectively, for co-substrate NADH. CpsD showed optimal activity at pH 7.5 and temperature between 30oC–40oC. The enzyme was stable between pH 7.0 – 7.5 and temperature between 30oC and 35oC. CpsD activity was enhanced by Fe2+ ion and inhibited by sodium azide and SDS. CpsD followed Michaelis-Menten kinetic exhibiting an apparent vmax, Km, kcat and kcat/Km values of 0.071 μmol s-1, 94 μmol, 0.029 s-1 and 3.13x10-4s-1μmol-1, respectively, for substrate tetrachloro-1,4-benzoquinone. CpsD belongs to the pterin-4α-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF-1 (DCoH) superfamily, with specific conserved protein domains of pterin-4α dehydratase (PCD), validated Pterin-4α-carbinolamine dehydratase (DCoH), and coenzyme transport and metabolism proteins. CpsA showed optimum activity at 30oC and pH 9.0. CpsA was stable between 20oC-40oC, and also retained about 90% of its activity at 60oC. The enzyme retained about 90% activity between pH 9.0 and 11.5 and 60% activity at pH 13.0. CpsA was found to be Fe2+ dependent as about 90% increased activity was observed in the presence of FeSO4. CpsA showed apparent Vmax, Km, Kcat and Kcat/Km of 27.77±0.9 μM s-1, 0.990±0.03 mM, 4.20±0.04 s-1 and 4.24±0.03 s-1 mM-1, respectively at pH 9.0. CpsA 3D structure revealed a conserved 2-His-1-carboxylate facial triad motif (His 9, His 244 and Thr 11), with Fe3+ at the centre. The whole genome of the isolate comprises one chromosome and one plasmid. The metabolic reconstruction for Bacillus tropicus strain AOACPS1 showed that the organism has been exposed to various chlorophenolic compounds including 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane, 1,2-dichloroethane, 1,4- dichlorobenzene, 2,4-dichlorobenzene, atrazine, and other xenobiotics previously and it has recruited enzymes for their degradation. PCP degradation by the isolate was independent of substrate concentration but highly dependent on the maximum specific growth and degradation rates. The low-affinity coefficient and high inhibition constant obtained in this study showed that the bacterium has a high affinity and tolerance to PCP, which could be explored for bulk remediation of PCP. The combination of the recombinant plasmid’s vector harbouring the PCP catabolic genes can be used for direct bioremediation of PCP in a bioreactor optimized for the growth of the hosts for overexpression of the proteins. Alternatively, concoction of the enzymes can be produced and immobilised for direct enzymatic bioremediation of PCP and other related recalcitrant xenobiotics. The study proposed that CpsD catalysed the reduction of tetrachlorobenzoquinone to tetrachloro-p-hydroquinone and released the products found in phenylalanine-hydroxylation system (PheOHS) via a Ping-Pong or atypical ternary mechanism; and regulate expression of phenylalanine 4-monooxygenase by blocking reverse flux in Bacillus tropicus AOA-CPS1 PheOHS using a probable Yin-Yang mechanism. CpsD may play a catalytic and regulatory role in Bacillus cereus PheOHS and PCP degradation pathway. Findings from this study provide new insights into the biological role of CpsA in PCP degradation and suggest alternate possible mechanism of ring-cleavage by dioxygenases. The study also provides the first experimental evidence of the involvement of a putative cytochrome p450 from Bacillus tropicus group in PCP transformation. Sequence mining and comparative analysis of the metabolic reconstruction of BtAOA with the closest strain and other closely related strains suggests that the operon encoding the first two enzymes in the PCP pathway were acquired from a pre-existing pterin-carbinolamine dehydratase subsystem. The next two enzymes were recruited (via horizontal gene transfer) from the pool of hypothetical proteins with no previous specific function while the last enzyme was recruited from pre-existing enzymes from the tricarboxylic acid cycle or serine-glyoxalase cycle via horizontal gene transfer events.
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.
The culture-independent analysis of fungal endophytes of wheat grown in KwaZulu-Natal, South Africa.
(2016) Burgdorf, Richard Jörn.; Jamal-Ally, Sumaiya Faizal.; Laing, Mark Delmege.
Fungal endophytes are of interest due to their diverse taxonomy and biological functions. A range of definitions exists based on their identity, morphology, location and relationship with their host. Fungal endophytes belong to a wide range of taxa and they are categorized by a variety of characteristics. The detection and identification of these fungal endophytes can be performed using culture-dependent and culture-independent methods. These organisms have a range of application in pharmaceutical discovery and agriculture. Agricultural applications include the exploitation of the growth promoting and protective properties of fungal endophytes in crops such as wheat. This important crop is grown in South Africa where biotic and environmental stresses pose a challenge to its cultivation. Fungal endophytes have demonstrated potential to ameliorate these challenges. Future research will reveal how they can be harnessed to fight food insecurity brought about by stress factors such as climate change. Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under Variable Pressure Scanning Electron Microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies. Denaturing gel electrophoresis (DGE) can be used in culture-independent studies of microbial community composition and the technique has several variants. This work compared two of these variants, namely denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient electrophoresis (TTGE), to establish their relative performance in terms of resolution and detection, as well as cost and preparation time. Per gel reagent and material costs and preparation times were recorded for comparison. Conversion formulae were developed to standardize denaturing conditions for comparison of DGGE and TTGE gels. For all gel samples, band numbers, positions, peak height and base width were recorded. Samples run on DGGE gels tended to be clearer and more distinct from each other and DGGE tended to provide higher band numbers and better resolution. However, TTGE was quicker and cheaper to prepare. The TTGE and DGGE gel data were strongly correlated but DGGE provided more accurate dendrograms for comparisons of pure fungal isolates. Non-metric multidimensional scaling showed that TTGE data profiles were more heterogeneous, while DGGE produced tighter clustering of replicate samples. Although TTGE could be an acceptable technique for resolving DNA sequences in certain applications, DGGE is preferable for fungal wheat endophyte studies. Fungal endophyte community composition can be affected by various factors, such as the host genome. Research into the host genome effects on fungal endophyte composition can assist in harnessing the potential benefits of such relationships in agro-ecosystems. Several culture-based studies have investigated the presence of a cultivar effect on endophyte composition. However, a culture-based approach can only detect organisms that can be isolated and grown. Culture-independent methods can detect both culturable and non-culturable fungal endophytes for comparisons of fungal endophyte community composition (ECC) between wheat cultivars. Denaturing gradient gel electrophoresis (DGGE), high-resolution melt (HRM) analysis of community profiles, quantitative PCR, and sequence analysis were used to analyse and compare the fungal ECC of four wheat cultivars grown under field conditions. A significant organ and cultivar x organ interaction effects on fungal biomass were observed. A chytrid, namely Olpidium brassicae formed a significant component of the fungal endophyte community across all tissues in wheat. This finding highlighted the utility of the culture-independent in revealing cryptic interactions and endophytes, and raised questions about the factors that influence the organisms that reside within field-grown wheat. Systemic fungicides used in wheat production are pathogenic to many plant-inhabiting fungi such as fungal endophytes. The aim of the study was to reveal the effect of tebuconazole on the eukaryotic endophytes of wheat flag leaves using next generation sequencing (NGS). Treated and untreated leaves were surface sterilized prior to metagenomic DNA (mDNA) extraction. NGS was performed on DNA amplified using universal ITS primers. SCATA analysis was used for operational taxonomic unit (OTU) assignment of sequences, which were identified against CBS, UNITE and Genbank databases. A maximum likelihood (ML) tree was developed for taxonomic assignment of key genera. OTU mean read numbers and OTU richness were compared. The treatment effects were analysed using Principal Component Analysis (PCA), permutational multivariate ANOVA (PERMANOVA), distance-based test for homogeneity of multivariate dispersions (PERMDISP) and similarity percentage analysis (SIMPER). With one exception, non-wheat OTUs belonged to the Dikarya. Puccinia read numbers differed significantly (p = 0.01) between treatments and fungicide treatment tended to reduce total OTU read numbers and OTU richness. The variability of most key OTUs correlated positively with unsprayed samples. Treatment influenced OTU composition. Treated samples had the greatest homogeneity in endophyte composition and Puccinia made the greatest contribution to variation, with low contribution from the other OTUs. Dikarya were the dominant wheat flag leaf endophytes, and while the fungicide suppressed Puccinia and reduced fungal endophyte abundance, it did not significantly alter the community assemblage.
Clinical strains of mycobacterium tuberculosis induce strain-specific patterns of cytokine production, gene expression and pathway changes in pulmonary alveolar epithelial cells.
(2016) Mvubu, Nontobeko Eunice.; Pillay, Manormoney.; Pillay, Balakrishna.
The epidemiological success of M. tuberculosis strains, dominant in different geographic regions globally, may be ascribed to a subversion of the host‟s protective immune response. The increasing prevalence of F15/LAM4/KZN, Beijing, F11 and F28 Mycobacterium tuberculosis strain families, coupled with rapidly evolving drug resistance within the KwaZulu-Natal province of South Africa population has resulted in a need to characterize host response associated with infection by these strains. Therefore, in this study, cytokine/chemokine production and host transcriptomics were investigated in A549 pulmonary epithelial cells infected with the F15/LAM4/KZN, Beijing, F28, F11, Unique and H37Rv strains. Cytokines/chemokines were quantified using the Bio-Plex Pro Human Cytokine 27-Plex assay at 0, 24, 48 and 72 hr post-infection. Changes in host gene expression were determined by whole genome RNA Sequencing (RNA-Seq) using the Illumina HiSeq 2000 platform. The 50 bp reads were mapped to the human genome (hg19) using Tophat (2.0.10). Differential expression was quantified using Cufflinks (2.1.0) with false discovery rate (FDR) of 0.05 and a log fold change cutoff of ≥2. R commands (Bioconductor), MeV and Ingenuity Pathway Analysis (IPA) were used to generate heat maps, network and pathways analysis. Twenty-three out of 27 analytes were detected. All strains, except the F28 strain induced an increased production of 18, and a decrease in 5 cytokines/chemokines at 24, 48 and 72 hr post-infection, compared to the uninfected control. Increased production of all 23 analytes by the F28 strain occurred at 48 and 72 hr. Among the 23 cytokines/chemokines that were detected, anti-inflammatory and pro-inflammatory cytokines, as well as chemokines were produced at the different time intervals. Compared to the other strains, high cytokine levels were induced by the F28 strain at 48 hr and F15/LAM4/KZN strain at 72 hr for most analytes. A lower cytokine production was induced by the Beijing and Unique strains at all time intervals. In the case of the laboratory strain H37Rv either a higher or lower cytokine/chemokine production was observed, compared to the clinical strains. RNA-Seq revealed differential gene expression that varied among the strains with respect to both up- and down-regulated genes: F15/LAM4/KZN (1187), Beijing (1252), F11 (1639), F28 (870), Unique (886) and H37Rv (1179). A total of 292 genes were commonly induced by all strains, of which 52 were down-regulated and 240 were up-regulated. Different strain combinations induced different genes that were involved in a variety of pathways, including immune response and apoptosis pathways. Furthermore, strain specific genes were activated by each strain as follows: F15/LAM4/KZN (138), Beijing (52), F11 (255), F28 (55), Unique (185) and H37Rv (125). The F15/LAM4/KZN, Unique and H37Rv were the only strains that had molecular signatures with overlapping functional Kegg and Reactome pathways for their specific genes. IPA analysis revealed canonical pathways that differed among the strains, with the interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways being among the top 5 pathways in all the strains. Cholesterol biosynthesis and immune related pathway enrichment was similar in the Beijing and Unique strains whilst the F15/LAM4/KZN strain showed closer relatedness to the F11 strain, and the F28 strain closely clustered to the H37Rv strain. The Beijing and Unique strains highly enriched cholesterol biosynthesis pathways compared to other clinical and laboratory H37Rv strain. The top scoring networks induced by these clinical strains varied among the strains with the associated functions. These gene networks were involved in antimicrobial response, developmental disorder, organismal injury, infectious disease and cellular development. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains, while other factors were strain specific. In conclusion, low cytokine/chemokine production and activation of immune associated pathways by the Beijing and Unique strains suggest a higher virulence for these strains compared to the F15/LAM4/KZN, F11 and F28 strains. These characteristics may explain the high transmissibility and prevalence of the Beijing strains. A similar pattern exhibited by the less prevalent, non-clustering Unique strain, may suggest some virulence attributes in common with the Beijing strain. Findings in this study have the potential to reveal useful biomarkers that can be used as targets for alternative TB therapeutics including immunomodulators that take into consideration network regulations and strain-specific pathways and molecular signatures.
Screening of aerobic endospore-forming bacterial isolates as candidate biocontrol agents against rhizoctonia solani.
(2016) Hunter, Charles Haig.; Schmidt, Stefan.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Bacterial-based biocontrol of soil-borne phytopathogens has gained prominence as a promising technology for developing sustainable agricultural pest control practices. Aerobic endospore-forming bacteria are seen as potential candidates for biocontrol applications due to various ecological and physiological traits which have been shown to influence plant health and disease control. Their ability to produce endospores also provides a major commercial advantage over non spore-forming bacteria. Appropriate screening methods are central to the discovery of successful biocontrol agents and should ideally be both ecologically relevant and able to evaluate a large number of isolates. A study was therefore undertaken with the aim of establishing screening methods that facilitate the selection of aerobic endospore-forming bacteria as candidate biocontrol agents against Rhizoctonia solani, an economically important fungal pathogen exhibiting a wide host range. Aerobic endospore-forming bacteria were isolated from rhizosphere material of five crop types grown in composted pine bark medium and screened for R. solani antagonism using traditional in vitro dual-culture bioassays. Isolates exhibiting antifungal activity were then evaluated in vivo for biocontrol activity against R. solani in cucumber seedling trials. Selected isolates were evaluated further using several screening approaches including: genomic fingerprinting; characterization of, and PCR-based screening for genes involved in the biosynthesis of bioactive lipopeptide compounds; and, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of rapidly screening bacterial isolates. Approximately 6% of the bacterial isolates (n=400) showed antagonism towards R. solani in vitro. Dual-culture bioassays against R. solani, Fusarium oxysporum, Botrytis cinerea and Pythium arrhenomanes revealed that the antagonistic activity amongst isolates varied considerably and was influenced by the duration of the assay. From these assays it was possible to rank isolates based on the extent and stability of the inhibitory response in vitro as well as by the spectrum of antifungal activity observed. Twenty-four isolates were selected for in vivo screening for biocontrol activity against R. solani, using susceptible cucumber plants grown under greenhouse conditions. In preliminary experiments the pathogen loading rates were shown to have a marked influence on disease severity. In experiments where R. solani was seeded in the form of colonized agar plugs, significant differences between treatments and controls were recorded and several potential biocontrol candidates were identified. A general observation was that isolates that achieved high rankings in vitro performed better in the in vivo trial than those with lesser rankings; although some exceptions were noted. These findings support the notion that fungal antagonism is an important determinant of biocontrol potential that can be used in preliminary biocontrol screening programmes. Internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR were evaluated as methods to differentiate isolates exhibiting antifungal activity in vitro. ITS-PCR distinguished three major groupings, but proved to be limited in its ability to detect inter- and intra-specific variation amongst closely related organisms. Based on 16S rRNA gene sequence analysis, two of the groups were identified as members of the “Bacillus subtilis” and “Bacillus cereus” clusters; while, the third group consisted of a single isolate identified as a strain of Brevibacillus laterosporus. RAPD-PCR revealed further levels of genetic diversity within each ITS grouping. The “Bacillus subtilis” cluster was distinguished further into four distinct groups, which based on gyrA gene fragment sequence analysis, were identified as strains of B. amyloliquefaciens subsp. plantarum and B. subtilis respectively. Sequence matches were consistent with the RAPD-PCR groupings, indicating that this method was appropriate for differentiating related isolates at the strain and possibly the sub-species level. Clonal similarities were evident for a number of strains isolated from different plant species suggesting that these may reflect populations of rhizosphere competent strains and/or plant adapted ecotypes. Strains of B. amyloliquefaciens subsp. plantarum and B. subtilis were amongst the best performers in the in vivo biocontrol seedling trial and generally performed better than the “Bacillus cereus” group of isolates. RAPD-PCR of the “Bacillus cereus” isolates showed that they exhibited greater levels of genetic heterogeneity and that the groupings detected were not consistent when different primer sets were evaluated. Genomic fingerprinting was found to provide an insight into the prevalence, distribution and possible rhizosphere competency of related strains. Liquid chromatography was used in conjunction with electrospray-ionization time-of-flight (ESI-TOF) mass spectrometry (MS) to characterize bioactive lipopeptides purified from culture supernatants of selected strains that ranked highly in the in vitro/in vivo assays. Phylogenetically related strains produced very similar lipopeptide profiles. Bacillus subtilis strains were found to produce isoforms of surfactin and fengycin. In addition to these lipopeptides, B. amyloliquefaciens subsp. plantarum strains were also found to produce isoforms of bacillomycin D or iturin A. Bacillomycin/iturin and fengycin fractions exhibited antifungal activity in vitro, whereas surfactin fractions did not. Isolates that ranked the highest in the R. solani dual-culture bioassays all produced either isoforms of bacillomycin D or iturin A. Bacillomycin D producing isolates were amongst the best performers in the in vivo biocontrol trials. Gene markers targeting the biosynthetic apparatus of the detected lipopeptide classes were then assessed for screening purposes using PCR. BACC1F/1R primers targeting the bacillomycin D synthetase C (bmyC) gene correlated well with the ESI-TOF MS findings, whereas ITUD1F/1R primers targeting the malonyl-CoA-transacylase (ituD) gene linked to iturin A biosynthesis were unable to distinguish between isolates that produced iturin or bacillomycin in culture. Disparities between some of the PCR and ESI-TOF MS results suggested that primers targeting srfA (surfactin) and fenD (fengycin) biosynthetic genes showed limited specificity amongst the strains screened. Phylogenetic comparisons of srfD and fenD gene sequences from selected strains of B. amyloliquefaciens subsp. plantarum and B. subtilis revealed that these genes clustered according to species with marked heterogeneity between clusters being evident. Using fenD gene sequence data from B. amyloliquefaciens subsp. plantarum FZB42, primers (FENG1F/1R) targeting fengycin synthetase genes of strains of B. amyloliquefaciens subsp. plantarum isolated in this study were successfully established. MALDI-TOF MS was assessed as a means of identifying isolates antagonistic to R. solani in vitro and determining their associated lipopeptide profiles. Mass spectra were obtained in the m/z range 2000 to 20000 for identification and grouping purposes and in the m/z 750 to 2500 range in order to profile lipopeptide production. The available Bruker BDal spectral library allowed for the identification of isolates to the genus level but proved to be limited for identifying environmental isolates to the species level. Extension of the library using “inhouse” mass spectra generated from isolates identified in this study significantly improved the level of isolate identification in subsequent identification runs. Cluster analysis of mass spectra allowed for the relationships between isolates to be established and provided a means of grouping closely related isolates. Strains of B. subtilis and B. amyloliquefaciens were clearly distinguished from one another and the potential for differentiating strains at the subspecies level was also shown. MALDI-TOF MS also provided a convenient means of detecting bioactive lipopeptides directly from whole cell preparations, cell extracts and crude culture filtrates. Lipopeptide profiles varied depending on taxonomic groupings. Results for isolates within the “Bacillus subtilis” group supported the earlier ESI-TOF MS findings and were found to be more reliable than PCR screening for lipopeptide synthesis genes. “Bacillus cereus” group isolates produced distinct spectral profiles with peaks that were consistent with biomarkers previously described in the literature as isoforms of the kurstakin class of lipoheptapeptides. Brevibacillus laterosporus CC-R4 yielded a unique spectral profile in the m/z 750-2000 range with mass fragments which were similar to antimicrobial compounds recently reported in the literature. Overall, MALDI-TOF MS was found to fulfil the requirement for a practical yet robust technique suitable for processing large numbers of aerobic endospore-forming bacteria for biocontrol screening. This study has shown that genomic fingerprinting and MALDI-TOF MS characterization of bacterial isolates are worthwhile additions to preliminary in vitro screening practices. They provide a level of isolate differentiation and characterization that is beneficial for selecting candidate biocontrol agents, which is not possible with traditional screening practices. Effectively, they allow traditional biocontrol screening to move away from empirically based approaches to ones which are “knowledge” based, allowing for representative groups of bacteria with specific traits to be selected for further evaluation.

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