Browsing by Author "Schmidt, Stefan."

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Aerobic utilization of selected pharmaceutical and personal care product by estuarine heterotrophic bacteria.
(2014) Bulannga, Rendani Bridghette.; Schmidt, Stefan.
Pharmaceutical and personal care products (PPCPs) constitute a broad class of organic compounds, some of which belong to the list of the OECD high production volume (HPV) chemicals. These compounds have emerged as environmental contaminants with potentially detrimental effects. They have been detected in various environmental compartments typically in a nano- to microgram range and sewage treatment plants represent the major point source for the aquatic environment. Salicylic acid, a monohydroxybenzoic acid, is widely used in cosmetic and therapeutic products and is listed as HPV chemical. Benzyl salicylate and phenyl salicylate are diaryl ester compounds commonly used in pharmaceutical formulations, fragrances and household detergents. Benzyl salicylate is listed as HPV chemical. The fate of salicylic acid in the environment has been reported previously while those of benzyl salicylate and phenyl salicylate are unknown. Although studies are available on the microbial degradation of aromatic compounds, studies exclusive to the catabolism of PPCPs by marine heterotrophic bacterial isolates are rather limited. Therefore, the aim of this thesis was to characterize PPCPs (salicylic acid, benzyl salicylate and phenyl salicylate) utilizing bacteria from an estuarine environment (Durban Harbour, KwaZulu-Natal, South Africa). Selective enrichments were employed using artificial seawater medium typically supplemented with 2 mM of the target compounds (salicylic, benzyl salicylate or phenyl salicylate). After successive subculturing, bacteria capable of utilizing target compounds as sole carbon and energy source were characterized by morphological and physiological features, 16S rRNA gene sequence and MALDI-TOF MS analysis. Growth kinetics were assessed by monitoring the optical density, cell count and protein formation over time. The utilization of salicylic acid and phenyl salicylate was verified using UV spectroscopy and HPLC and the key reactions involved were verified by determining the specific oxygen uptake rates using resting cells and specific activities of representative enzymes. A Gram-negative coccus shaped bacterium belonging to the genus Acinetobacter degrading salicylic acid and phenyl salicylate, a Gram-negative rod shaped marine bacterium belonging to the genus Oceanimonas degrading salicylic acid and phenyl salicylate and a Gram-negative rod shaped bacterium belonging to the genus Pseudomonas utilizing benzyl salicylate in the presence and absence of synthetic surfactants (Tween 80) were isolated. The growth of Acinetobacter and Oceanimonas species was dependent on salicylic acid and phenyl salicylate as carbon source as growth was only observed when the carbon source was present and the compound was degraded almost to completion. Growth of Pseudomonas with benzyl salicylate was enhanced in the presence of surfactant. All three strains did not have an obligate requirement for NaCl. Acinetobacter and Oceanimonas strains were tolerant to high concentrations of salicylic acid and were inhibited at a concentration above 20 mM while phenyl salicylate did not show toxic effects on the strains; instead growth increased with the increase in concentration. Salicylic acid was utilized via catechol by both strains as they showed high specific oxygen uptake rates and catechol-1, 2-dioxygenase activity for this chemical. Phenyl salicylate was hydrolyzed at the ester bond to phenol and salicylic acid, as these were the metabolites that accumulated during growth with phenyl salicylate. The mono-aromatic metabolites resulting from the hydrolysis of diaryl substrate were further metabolized via catechol. Microbial catabolic activities were solely responsible for the loss of contaminant in the medium as confirmed by abiotic controls. Heterotrophic bacteria can therefore play an important role in the removal of contaminants from marine environments.
Analysis of indigenous herbivore faecal matter as a potential source of hydrolytically active microorganisms.
(2014) Ndlela, Luyanda Lindelwa.; Schmidt, Stefan.
Abstract available in PDF file.
Assessment of the diversity of bacteria and methanogenic Archaea in Zebra faeces.
(2013) Naidoo, Kewreshini Kasturi.; Schmidt, Stefan.
The need to develop a renewable, environmentally friendly source of energy has become a primary focus in modern science, with bio gas showing considerable potential. Interest in the methanogenic Archaea has therefore grown in recent years and extensive studies have been carried out to investigate the population diversity in various habitats. Presently, there are only a few studies that have evaluated the microbial communities inhabiting the gastrointestinal tract of wildlife native to southern Africa. This study aimed to investigate the microbial diversity, in particular the bacterial and methanogen communities involved in fermentative digestion in the gastrointestinal tract of zebra. Assessment of the microbial diversity in zebra faeces included both culture-based techniques and nucleic acid targeting analysis via 16S rRNA gene sequencing. Quantitative analysis using selected solid media revealed high counts for aerobic and anaerobic Bacteria (7.51x108 and 2.45x109/gram of faecal sample respectively). The majority of aerobic colonies that were detected exhibited Bacillus-like morphology. Nucleic acid based analysis of the diversity of both Bacteria and methanogenic Archaea in zebra faecal material was performed. Both manual and kit based extractions were used for DNA isolation in order to compare the efficiency of the two methods. Results show that a vigorous mechanical treatment was best for the release of DNA from the faecal matter. Amplification of target gene regions was carried out using established primer pairs (ARCH69F/ARCH915R and EUB338F/EUB907R) for methanogen and bacterial DNA respectively. Amplified 16S rRNA gene regions were cloned into a high copy number vector and random clones were selected for evaluation. Clones containing the target gene were further analysed by ARDRA and were assigned to a specific phylotype. Two bacterial (105 clones in total) and three methanogen (178 clones in total) clone libraries were constructed, of which 24 phylotypes were established for Bacteria and 25 for methanogenic Archaea. A representative of each phylotype was analysed by sequencing and further phylogenetic analysis was conducted. Six bacterial phylotypes, which represented 56% of all bacterial clones, exhibited 99% sequence similarity to Bacillus species. Six methanogen phylotypes, which exhibited 99% sequence similarity to the hydrogenotrophic species Methanobrevibacter gottschalkii strain PG, were established to be predominant in zebra faeces. These phylotypes represented 71% of all archaeal clones selected for analysis in this study.
Bacterivory of three freshwater ciliates isolated from Blackbarough Spruit in KwaZulu-Natal, South Africa.
(2021) Bulannga, Rendani Bridghette.; Schmidt, Stefan.
Abstract available in PDF.
Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization.
(American Association for the Advancement of Science., 2012) Georgiev, Ivelin S.; Doria-Rose, Nicole A.; Zhou, Tongqing.; Do Kwon, Young.; Staupe, Ryan P.; Moquin, Stephanie.; Chuang, Gwo-Yu.; Louder, Mark K.; Schmidt, Stefan.; Altae-Tran, Han R.; Bailer, Robert T.; McKee, Krisha.; Nason, Martha.; O'Dell, Sijy.; Ofek, Gilad.; Pancera, Marie.; Srivatsan, Sanjay.; Shapiro, Lawrence.; Connors, Mark.; Migueles, Stephen A.; Morris, Lynn.; Nishimura, Yoshiaki.; Martin, Malcolm A.; Mascola, John R.; Kwong, Peter D.
Serum characterization and antibody isolation are transforming our understanding of the humoral immune response to viral infection. Here, we show that epitope specificities of HIV-1–neutralizing antibodies in serum can be elucidated from the serum pattern of neutralization against a diverse panel of HIV-1 isolates. We determined “neutralization fingerprints” for 30 neutralizing antibodies on a panel of 34 diverse HIV-1 strains and showed that similarity in neutralization fingerprint correlated with similarity in epitope. We used these fingerprints to delineate specificities of polyclonal sera from 24 HIV-1–infected donors and a chimeric siman-human immunodeficiency virus–infected macaque. Delineated specificities matched published specificities and were further confirmed by antibody isolation for two sera. Patterns of virus-isolate neutralization can thus afford a detailed epitope-specific understanding of neutralizing-antibody responses to viral infection.
Improving market access for smallholder farmers : socio-economic determinants of pre-and post-harvest practices - and their potential role for microbial contamination of fresh produce.
(2017) Beharielal, Tashiana.; Thamaga-Chitja, Joyce Magoshi.; Schmidt, Stefan.
Fresh produce is known to carry a natural microbial community however, during agricultural production and processing, any ready-to-eat fresh produce can become contaminated with pathogenic microbial organisms if inappropriate hygiene practices are used. As such fresh produce items go through minimal, if any inactivation or preservation treatments during further processing, hygiene quality and safety of the produce may be compromised thereby limiting market access and endangering consumer health. This study was conducted to determine if the Marianhill Agri-hub smallholder farmer socio-economic characteristics influence pre- and post-harvest practices and hygiene practices adopted. Furthermore, the contributions of these practices to microbial contamination of ready-to-eat fresh produce and its implications for market access, health and household food security were evaluated. Questionnaire results indicated a literate farmer community (88%), reliant on diversified income sources and farming as a livelihood strategy. Moderate interest in gaining market access to supplement household income was a key characteristic (61%). Most farmers utilized natural water sources (Mnini pond, Mnini river, stream and rain-fed) for irrigation, however, only a few (18%) pre-treated water prior to irrigation. Statistical analysis (Pearson Chi-square tests) indicated that farmer education levels and exposure to prior training have a statistically significant (p<0.05) impact on selected pre-and post-harvest practices implemented, highlighting the importance of farmer education and training. Microbiological analysis of fresh produce samples such as lettuce, parsley, carrots and spinach collected over at least a three month period from the main Agri-hub, showed the presence of total (ranging from 130-79000 MPN/g) and faecal coliforms (ranging from 22-1400 MPN/g) as well as E. coli (ranging from 2.2-49 MPN/g). These values were not satisfactory with respect to total coliform levels and presence of E.coli in view of South African legislation. In irrigation water samples, faecal coliforms were present up to 7000 MPN/100ml thereby frequently not meeting the WHO irrigation water quality requirements. Additionally, a number of irrigation water samples did not meet the South African standards for irrigation water applied to minimally-processed fresh produce of ≤ 1 E. coli/100ml, with values between 9.3-1400 MPN/100ml. Salmonella spp. was not detected in fresh produce and irrigation water samples. Antibiotic susceptibility patterns of 155 randomly selected E. coli isolates from both fresh produce and irrigation water were determined using the EUCAST disk diffusion method. The highest percentage of antibiotic resistance in E.coli isolates was detected against the antibiotic streptomycin at >94%. However, while 6% of the tested E. coli isolates were susceptible to all tested antibiotics, 2% of the E. coli isolates were multidrug-resistant. Multidrug-resistant strains of E. coli are concerning, as resistance genes are easily transferable to other potentially pathogenic bacteria present on produce, which might render the treatment of such pathogens difficult. Scanning Electron Microscopy showed the ability of E.coli isolates to form biofilms on PVC coupons mimicking contact surfaces. Antibiotic resistant and biofilm forming E.coli isolated from fresh produce and associated production and processing surfaces highlight the need of implementation of appropriate pre-and post-harvest hygiene practices. Stringent microbiological quality standards governing entry into high-value markets need to be adhered to by smallholder farmers. Therefore, understanding of smallholder farmer socio-economics is imperative to improving pre-and post-harvest hygiene practices, as the use of proper hygienic pre-and post-harvest practices is essential to prevent microbial contamination and improve quality of ready-to-eat fresh produce which will, in turn, facilitate improved market access.
Investigation of selected hygiene parameters of uMbumbulu small-scale farmers' organic produce (leafy salad vegetables) and subsequent identification of factors affecting farmer practices and food security.
(2013) Mdluli, Fezile.; Chitja, Joyce Magoshi.; Schmidt, Stefan.
The study aimed to investigate the hygiene quality of fresh agricultural produce, irrigation water and compost from four cooperatives (Jabulani, Nungwane, Senzakahle and Siyazenzela) supplying the uMbumbulu Agri-Hub Non-Governmental Organisation. In addition, the influence that socio-economic characteristics such as age, gender, level of education and training had on the uMbumbulu farmers’ hygienic practices was investigated. Questionnaires, key informant interviews and laboratory analysis were used to collect data. The most probable number (MPN) method, a microbiological technique, was used to quantify selected hygiene indicators (i.e. total and faecal coliforms including Escherichia coli) from compost, irrigation water and leafy vegetables (spinach and lettuce) during the months of October, November and December 2011. Microbiological analysis on lettuce and spinach produced by the four Agri-Hub cooperatives confirmed that these vegetables were safe to eat and unlikely to cause sickness. The irrigation water sources, vegetables and compost faecal coliform levels met national standards with faecal coliforms of <1 000 MPN/100ml for irrigation and<200 MPN/g for the leafy salad vegetables. Compost faecal coliform levels were <1000/g and E. coli levels of <30 MPN/g, these levels decreased over the 3 months. Descriptive statistics such as the Chi-Square test using IBM SPSS and a logistic regression was performed using the STATA 11 software. The sample consisted of 60% female and 40% males, most of which (73%) were above the age of 40. A total of 60% of respondents received income from farming activities, receiving revenues of between R150- R250 a week. The logistic regression indicated that farmers already receiving some income from farming activities and those that had received training on hygienic farming practices were likely to wash hands and equipment prior to entering the field compared to those who had not. These variables influenced the hygienic practices with a probability of 26% and 32% respectively at 5% significance level. The logistic regression also showed that respondents with primary or no formal education were less likely to wash hands and equipment prior to entering the field compared to those who had a secondary level education. This unlikelihood had a probability of 35% for primary education and 43% for farmers with no formal education at significance levels of 5% and 10% respectively. This study indicates how training, education and farming experience are important and effective tools in implementing good hygienic practices in small-scale farming. The study’s main recommendations are that policies encourage farmer awareness on their responsibility of producing vegetables that are of good hygienic quality, especially if such produce is to reach the market. Furthermore policies should advocate for small-scale farmer training. This training should not be limited to subsistence farming but should also aim at preparing farmers towards accessing produce markets. Farmer training in hygienic practices should aid farmers to meet the stringent market standards allowing for better access, the regular income from such activities support farming as a livelihood and bearer of food security. It must also be noted that farmers require support in attaining the various resources needed in order to successfully and continually supply markets.
Isolation and characterization of bacteriophages from wastewater as potential biocontrol agents for Escherichia coli.
(2022) Ntuli, Nontando Percevierance.; Schmidt, Stefan.
Host-specific lytic bacteriophages have regained momentum as an alternative treatment option to control and eliminate pathogenic bacteria. This study aimed to isolate, characterize and evaluate the potential application of Escherichia coli phages as a biocontrol agent in wastewater. In this study, four lytic Escherichia coli phages were isolated from wastewater for biocontrol purposes, using the double-layer method with E. coli (ATCC-25922) as a host. The phage morphology was characterized using transmission electron microscopy, with further parameters such as host range, phage stability at different temperatures, and pH values analyzed additionally. The genome of two selected phages (NPS and NPM) was sequenced, and the capacity of the phage isolate NPM to eliminate E. coli from artificial wastewater was evaluated and compared to conventional chlorination. All the four phage isolates showed typical T4 phage appearance with isometric capsids and contractile tails of different sizes, matching the family Myoviridae in the order Caudovirales. They exhibited a narrow host range limited to E. coli isolates, with two exceptions: phage NPS and NPM additionally lysed Salmonella Typhimurium (ATCC-14028). The four phage isolates were even able to lyse MDR (multidrug-resistant) E. coli isolates, such as the strain FP29. The four phages had burst sizes ranging from 70-115 per host cell and a latency period of 10- 20 minutes. All the four bacteriophages were stable at pH 5-9 but completely inactivated at pH 12. Exposure to 60°C for 10 minutes reduced phage titers by 1.5- log, while exposure to 80°C for 10 minutes completely inactivated all four phage isolates. The two genomes (NPS and NPM) were 99% identical and had similar sizes (169 536 bp), but phage NPS differed from phage NPM in view of its host range and plaque morphology. Another difference observed at the genome level was a shift of coding sequences between phage NPS and NPM. Phage isolate NPM achieved a 3.5- log reduction of E. coli cells present in artificial wastewater at an MOI of 0.1 in 120 minutes. A 90-minute chlorine treatment achieved a log reduction in the same range, highlighting that phages have the potential as environmentally friendly biocontrol agents in wastewater treatment.
Isolation and characterization of carotenoid producing microalgae from KwaZulu-Natal (South Africa).
(2017) Adedoyin, Ayodeji Emmanuel.; Schmidt, Stefan.
Abstract available in PDF file.
Isolation and characterization of diaryl ester catabolizing soil fungi.
(2019) Moonsamy, Volante.; Schmidt, Stefan.
Aromatic hydrocarbons are major organic pollutants that can persist in the environment. However, many fungi and yeasts can utilize these compounds as carbon and energy sources under aerobic conditions. Salol and benzyl salicylate are diaryl ester biocides exhibiting endocrine-disrupting properties. Using mineral salts medium containing salol and benzyl salicylate as sole carbon and energy source, aerobic enrichment cultures were established by inoculation with soil samples collected from a local animal farm and Bisley Nature Reserve Pietermaritzburg, KwaZulu-Natal. A salol utilizing fungal isolate and a benzyl salicylate utilizing yeast isolate were selected after enrichment. The fungal and yeast isolate were provisionally assigned to the genus Fusarium and Trichosporon, based on phenotypic characteristics and the sequence analysis of the ITS1-5.8S rRNA-ITS2 region. Growth kinetics of the fungus were assessed by measuring the dry weight of the biomass over time in batch cultures; the growth of the yeast was assessed via OD600 determinations and verified via microscopic cell counts. Appropriate abiotic controls showed that the concentration of tested aromatic pollutants remained stable over time while no biomass formed in biotic controls without added carbon source. Salol and benzyl salicylate utilization was verified by measuring the Chemical Oxygen Demand (COD) and UV-Vis spectra over time. COD measurements and UV spectroscopy indicated that up to 10 mM salol was catabolized completely by Fusarium sp. strain VM1 within 10 days while Trichosporon sp. strain VM2 catabolized 10 mM benzyl salicylate almost quantitatively. Specific enzyme activity determinations showed that both esterase and catechol-1,2-dioxygenase were induced by growth on salol and benzyl salicylate, indicating that the catabolism of diaryl esters is initiated by hydrolysis of the ester-linkage and the monoaromatic hydrolysis products were further metabolized via catechol and the ortho-pathway. These results indicate that members of the genus Fusarium and Trichosporon present in South African soils have the potential to eliminate diaryl esters and simple monoaromatic pollutants.
The microbiological assessment of a biofiltration system in KwaZulu-Natal (South Africa) treating borehole water containing Mn (II) and Fe (II).
(2013) Beukes, Lorika Selomi.; Schmidt, Stefan.
In the following study, the potential role that microorganisms play in the removal of Mn (II) and Fe (II) was assessed using biofilter sand and water samples collected from a biofiltration system (operated by Umgeni Water in KwaZulu-Natal, Nottingham Road, at the Nottingham combined school, South Africa) treating borehole water containing manganese and iron. Initially the presence of Mn (II) and Fe (II) oxidizing bacteria was demonstrated in the biofiltration system. Thereafter, the contribution of individual microorganisms to the overall removal of manganese and iron was assessed in the laboratory by determining the difference in metal oxidation in the presence and absence of active bacteria at neutral pH, simulating conditions in the biofilter. Controls were run to verify the elimination via physiochemical reactions occurring within the biofiltration system. Finally a diversity snapshot of the bacteria present within the biofilter matrix was established via analysis of a clone library. Viable bacterial counts for the biofiltration system were established using MSVP (minimal salts vitamins pyruvate) medium - plus added manganese sulfate or iron sulfate targeting Mn (II) and Fe (II) oxidizing bacteria - and R2A for heterotrophic bacteria. In the first experimental chapter, batch tests using MSVP were employed to determine manganese oxidation, by measuring the pH and ORP (oxidation reduction potential) in experimental flasks and controls over time. There was a clear drop in pH and a concomitant increase in ORP when an isolated manganese oxidizing strain (designated LB1) was grown in MSVP plus added manganese sulfate, indicating manganese oxidation. Based on physiological characteristics established by the VITEK-2 system as well as by 16S rRNA gene sequence analysis and MALDI-TOF (Matrix assisted laser desorption ionization-time of flight mass spectrometry) mass spectrometry of cell extracts, the isolate was identified as a member of the genus Acinetobacter. EDX (energy dispersive X-ray analysis) analysis of crystals formed in batch culture tests, containing MSVP plus either added manganese or iron sulfate, confirmed the ability of the isolate to oxidize both Mn (II) and Fe (II). The leucoberbelin blue colorimetric assay and batch tests using MSVP both demonstrated that in the presence of the isolated strain, Acinetobacter sp. LB1, the rate of Mn (II) oxidation at neutral pH was enhanced as compared to abiotic controls. In the second experimental chapter the difference in Fe (II) oxidation between biological and abiological systems at neutral pH was determined using batch tests run with Acinetobacter sp. LB1 and Fe (II) in saline. In addition, the rate of Fe (II) oxidation was also determined at acidic pH and at alkaline pH in experimental and control flasks. To determine Fe (II) removal under conditions simulating those in the biofiltration system, batch tests were set up using borehole water freshly collected from the biofiltration system. In order to verify the contribution of native microorganisms in the borehole water to Fe (II) oxidation, these flasks were spiked with bacterial strains isolated from the biofiltration system - Acinetobacter sp. LB1 and Burkholderia sp. strain LB2 - and two known iron oxidizing strains Leptothrix mobilis (DSM 10617) and Sphaerotilus natans (DSM 565) were used to determine the contribution of reference iron oxidizers to Fe (II) oxidation. A separate set of the same flasks with the addition of filter sand was used to qualitatively demonstrate iron oxidation as it would occur within the biofiltration system. The ferrozine assay was employed to quantify the amount of Fe (II) in batch tests employing saline medium and in batch tests employing borehole water. EDX analysis was employed to confirm the presence of Fe (II) in oxidation products in the batch test flask with filter sand spiked with Acinetobacter sp. LB1. In the presence of Acinetobacter sp. LB1 at neutral pH in saline medium, the rate of Fe (II) oxidation was very similar to that in the abiological controls thus demonstrating that the presence of metabolically active microorganisms does not per se enhance the oxidation of Fe (II) like in the case of Mn (II) at neutral pH. Surprisingly, in the heat inactivated control, apparently the highest amount of Fe (II) was oxidized. As expected, at acidic pH very little oxidation of Fe (II) took place and at alkaline pH almost all Fe (II) in the flasks was removed and small amounts oxidized as determined by the amount of Fe (III) produced. Batch tests using borehole water proved that native microorganisms within the biofiltration system were more efficient in the oxidative removal of Fe (II) from the system, in comparison to the reference iron oxidizing strains. In the final experimental chapter, the presence of biofilms with actively metabolizing cells was examined on a pooled sample of biofilter matrix from the manganese and iron filter using CLSM (confocal laser scanning microscopy) image analysis. DNA was extracted from the biofilm material associated with biofilter matrix to establish a diversity snapshot of the bacteria present within the biofilter matrix. ARDRA (amplified “rDNA” restriction analysis) analysis of the clone library revealed the presence of 15 unique OTU’s (operational taxonomic unit) based upon restriction patterns of amplified 16S rRNA genes of a total of 100 randomly selected clones. The majority of the clones were closely related to the genera Nitrospira and Lactococcus. Overall, 42% of the clones were assigned to the phylum Proteobacteria, 13% to the phylum Actinobacteria, 24% to the phylum Firmicutes and 21% to the phylum Nitrospirae. Overall, the results demonstrate that bacteria present within an established biofiltration system at neutral pH can contribute to the oxidative removal of Mn (II) and, apparently only to a smaller degree, to that of Fe (II) present in borehole water and that species within the proteobacterial genus Acinetobacter are potentially involved in the geochemical cycling of these two metals. Keywords: Biofiltration, iron and manganese oxidation, Acinetobacter sp. LB1, batch tests, 16S rRNA, MALDI-TOF MS analysis, Mn (II) and Fe (II) colorimetric assays, EDX analysis, biofilm formation, CLSM image analysis, 16S rRNA clone library Abbreviations: MSVP (minimal salts vitamins pyruvate), ORP (oxidation reduction potential), EDX (energy dispersive X-ray analysis), MALDI-TOF MS (Matrix assisted laser desorption ionization-time of flight mass spectrometry), rRNA (ribosomal RNA), ARDRA (amplified “rDNA” restriction analysis), CLSM (confocal laser scanning microscopy), OTU (operational taxonomic unit)
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.
Optimization of biohydrogen production inoculum development via hybrid pre-treatment techniques : semi pilot scale production assessment on agro waste (potato peels)
(2015) Faloye, Funmilayo Dorcas.; Gueguim Kana, Evariste Bosco. ; Schmidt, Stefan.
The challenges of energy crisis and environmental pollution are vital issues hindering the global sustainable development as a result of over dependence on fossil fuels. These are driving the need to explore renewable and environmentally friendly energy sources. Biohydrogen has emerged as an eco-friendly renewable energy source and a suitable alternative to fossil fuels. However, the commercialization of biohydrogen energy is hindered by the high production cost and low yield which necessitates novel strategies for an economically feasible production. Some of these strategies include the development of stable inoculum, scale-up studies, and the utilization of renewable feedstock such as agro-food waste materials which are both abundant and sustainable. Inoculum pre-treatment is a vital aspect of hydrogen production technology as it contributes to the improvement of hydrogen yield. The inoculum pre-treatment method influences the community structure which in turn affects the microbial metabolism of hydrogen production. This study investigates novel inoculum development techniques and evaluates the feasibility of biohydrogen production from agro waste (potato peels). The linear and interactive effect of these techniques on inoculum efficiency as well as the key process parameters for hydrogen production from potato peels were modelled and optimized. Further assessment of the hydrogen production dynamics at the semi-pilot scale including the microbial community structure were investigated using the 16SrRNA gene clone library sequence analysis. A hybrid inoculum development technique of pH and Autoclave (PHA), pH and Heat shock (PHS) was modelled and optimized using the response surface methodology. The quadratic polynomial models had a coefficient of determination (R2) of 0.93 and 0.90 and the optimized pre-treatment conditions gave a 37.7% and 15.3% improvement on model predictions for PHA and PHS respectively. Maximum hydrogen yield of 1.19 mol H2/ mol glucose was obtained for PHA in a semi-pilot scale process. The interactive effect of a hybrid pH and microwave pre-treatment on mixed inoculum for biohydrogen production was investigated. The obtained model had a coefficient of determination (R2) of 0.87. Two semi pilot scale-up processes were carried out to assess the efficiency of the developed inoculum with and without pH control on biohydrogen production. A two fold increase in glucose utilization was obtained and a molar hydrogen yield of 2.07 mol H2/mol glucose under pH controlled fermentation compared to 1.78 mol H2/mol glucose without pH control. Methane production was not detected which suggests the effectiveness of the combined pre-treatment to enrich hydrogen producing bacteria. The developed inoculum was used to evaluate the feasibility of biohydrogen production from potato peels waste. The key process parameters of substrate concentration (g/L), nutrient supplementation (%), temperature (°C) and pH were modelled and optimized using the Artificial Neural Network (ANN) and Response surface methodology (RSM). The optimum conditions obtained were 50g/L of potato waste, 10% nutrients, 30°C and pH 6.5. A semi pilot production process under the optimized condition gave a hydrogen yield of 239.94mL/g TVS corresponding to a 28.5% improvement on hydrogen yield. Analysis of the microbial community structure showed the dominance of the genus Clostridium comprising of about 86% of the total microbial population including C. aminovalericum, C. intestinale, C. tertium, C. sartagofome, C. beijerinckii and C. butyricum in ascending order of predominance. Hydrogen consuming methanogens were not detected which further confirmed the efficiency of the hybrid inoculum pre-treatment. This study has highlighted the development of a novel hybrid inoculum pretreatment method to establish the requisite microbial community and to safeguard the stability of biohydrogen production. Furthermore, the potential of generating an economical feasible biohydrogen production process from potato waste was demonstrated in this work.
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.
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.
Wild and domestic animals as reservoirs of antibiotic resistant Escherichia coli in South Africa.
(2017) King, Tracy Leigh Bridget.; Schmidt, Stefan.
Abstract available in PDF file.