Research Articles (Eugene Cloete)

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Microbial community study of the iron ore concentrate of the Sishen Iron Ore Mine, South Africa
(Springer, 2008-11) Williams, Peter J.; Cloete, T.E. (Thomas Eugene), 1958-
As a result of the advancing global technologies and civilisation, there has been a progressive depletion of high-grade mineral deposits. Consequently, it has become increasingly important to process lower-grade ores. Phosphorous (P) and particular potassium (K) contained in the iron ore concentrates of the Sishen Iron Ore Mine have a detrimental effect on the steel making process, whereby these alkali’s cause cracks to form in the refractory lining of blast furnaces. It is initially essential to determine which microbes are indigenously present at the Sishen Iron Ore Mine before strategising how best to employ them to industrial advantage. Therefore, the objective of this study was to determine which microorganisms are indigenous to the iron ore and soil of the Sishen Iron Ore Mine. The bacterial 16S PCR and fungal ITS PCR revealed several bacterial and fungal species present in the mine environment. The bacterial isolates were found to be closely related to Herbaspirillum species, as well as Acidithiobacillus ferrooxidans, while the fungal isolates were closely related to Aureobasidium pullulans, Phaeosphaeria nodorum, Aspergillus umigatus, and Candida parapsilosis. Isolating A. fumigatus from the iron ore/soil of the mine may indicate that A. niger, the most common fungi used for the production of citric acid, can adapt to the stringent mine environment. This would allow the application of A. niger for the production of citric acid, which may be used for the chemical leaching of the P and K from the iron ore concentrate of the Sishen Iron Ore Mine.
Resistance of bacterial cultures to non-oxidising water treatment bactericides by adaptation
(Elsevier, 1995) Brözel, Volker Siegfried; Pietersen, Belinda; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
Bacterial communities in water cooling systems treated with bactericides often become resistant to these bactericides. This has been ascribed to selection for resistant cells. Certain bacteria, having a high inherent susceptibility to water treatment bactericides become dominant in systems after bactericide treatment. We investigated the idea that bacterial isolates adapt to grow in the presence of bactericides. Pure cultures of Pseudomonas aeruginosa, P stutzeri and Bacillus cereus were cultured repeatedly in the presence of sub-inhibitory concentrations of 2,2'-methylenebis(4-chlorophenol), Na dimethyldithiocarbamate, isothiazolone and alkyl dimethyl ammonium chloride. All isolates adapted to grow in the presence of increasing concentrations of the bactericides. The phenomenon of development of bacterial resistance to water treatment bactericides was ascribed to adaptation and not to selection.
Assessment of the genetic diversity of geographically unrelated Microcystis aeruginosa strains using amplified fragment length polymorphisms (AFLPs)
(Academic Journals, 2005-05) Oberholster, Paul Johan; Botha, Anna-Maria; Muller, K.; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
Molecular marker analysis is becoming increasingly capable of identifying informative genetic variation. Amplified fragment length polymorphism markers (AFLPs) are among the recent innovations in genetic marker technologies, and provide a greater capacity for genome coverage and more reproducible results than previous technologies. We have investigated the usefulness of AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate between geographical unrelated Microcystis strains. In total 23 strains were subjected to the AFLP fingerprinting. After analysis of the data on the basis of the average linkage method, known as the Unweighted Pair Group Method using Arithmetic averages (UPGMA), a dendrogram with 4 clusters was obtained. Cluster 1 consisted mainly of the NIES strains that originated from Japan, while in cluster 2 the European strains grouped together. The South African strains that originated from the northern part of the country grouped together in cluster 3, while the strains collected from the central and southern regions grouped together with the US strains in cluster 4. The study had revealed extensive evidence for the applicability of AFLP in cyanobacterial taxonomy, and furthermore clearly demonstrates the superior discriminative power of AFLP towards the differentiation of geographical unrelated Microcystis aeruginosa strains that belong to the same species, as well as highlighting the potential of this fingerprinting method in evolutionary studies.
Response of Pseudomonas aeruginosa PAO following exposure to hydrogen peroxide
(Water Research Council, 1996-07) Pietersen, Belinda; Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
Hydrogen peroxide is used in various applications to prevent, control or decrease bacterial activity in e.g. cooling water, hospitals, recreational waters and the food industry. The aim of the work reported here was to investigate the response of P. aeruginosa following exposure to hydrogen peroxide during both the logarithmic and stationary phases of growth. The catalase levels were determined following exposure to hydrogen peroxide and the general cellular response was investigated by pulse-labelling. Stationary phase cells did not demonstrate a stress response to hydrogen peroxide. Where de novo protein synthesis was inhibited, cells were less susceptible to growth inhibition, indicating an adverse stress response to hydrogen peroxide in P. aeruginosa. The addition of hydrogen peroxide to cultures in logarithmic growth phase resulted in the induction of a short lag phase. The growth rate following a return to logarithmic growth phase was lower than before addition of hydrogen peroxide, and was inversely related to the concentration of hydrogen peroxide added. Oxidising stress elicited de novo synthesis of four proteins within 5 min following exposure to stress. Cellular catalase levels doubled from 16 U/mg protein to over 30 U/mg within 10 min following exposure to oxidising stress but no new catalase isozymes were induced. Hydrogen peroxide was demonstrated to interrupt cell division as well as to decrease the ensuing rate of division in P. aeruginosa, and the culture did not exhibit an effective stress response to hydrogen peroxide.
Effect of storage time and temperature on the aerobic plate count and on the community structure of two water samples
(Water Research Council, 1991-10) Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
The effect of storage at various temperatures on the bacterial community of a cooling-water sample and a tap-water sample was determined. Samples were stored at 4, 10, 20 and 30 degrees C for 24, 48, 72 and 216 h and the aerobic plate count and bacterial community structure of each were determined using R2A and R3A agars. The culturable count (aerobic plate count on R2A/R3A agar) in both samples varied over time, even after 24 h storage at 4 degrees C, showing that bacterial communities in water are dynamic, even at refrigerator temperatures. At 4 degrees C the culturable count of cooling water initially decreased, followed by a tenfold increase. The tap-water count decreased at 4degrees C. At 10degrees C the pattern was similar. At 20 and 30 degrees C there was a tenfold increase in the culturable count of the cooling water, even after 24 h. In the cooling-water sample, the dominant isolates throughout were Pseudomonas stutzeri and an unidentified Gram negative pink isolate. This isolate was not detected in previous studies where Std I nutrient agar was used. Possibly this isolate plays an important role in cooling-water ecology, but does not grow on the conventional agars. The other isolates appeared randomly on the agar plates. The tap-water sample showed great variation in dominance of species over time. No direct tendencies of rate of decrease or increase could be detected in any of the samples, either in the culturable count or in community structure. Therefor results of analysis after storage cannot be adapted by a pre-determined factor. They must be interpreted with extreme caution, as they do not of necessity reflect the bacterial composition of the sample as drawn, both in terms of total numbers and in terms of community structure. Only counts performed on fresh samples yield reliable results on the total culturable count, and only community structures performed immediately, reflect the state of the community in the system from which the sample was drawn.
The Malthus system for biocide efficacy testing against Desulfovibrio desulfuricans
(Water Reseach Council, 1994) De Bruyn, E.E. (Engela Elizabeth); Croukamp, E.; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
Microbially influenced corrosion (MIC) makes an important contribution to corrosion in various industries. Considerable success has been achieved by the use of biocides. Little information for controlling MIC is, however, available on the effectivity of biocides against sulphate-reducing bacteria (SRB) due to the difficulties of culturing these organisms using conventional techniques. Conductance changes monitored using the Malthus system were evaluated as an alternative method of estimating numbers of Desulfovibrio desulfuricans for laboratory biocide evaluations. The correlation of log10 counts of Desulfovibrio cells in iron sulphite (IS)-medium using conventional techniques with detection times using the Malthus systems was highly significant (r=0,974), indicating that the Malthus system can be used as an alternative method to conventional media for the enumeration of SRB. Growth studies of Desulfovibrio using the Malthus system were useful in the evaluation of biocides. A 56% and a 100% kill was obtained when using 60 and 200mg/l quaternary ammonium compounds (QAC), respectively.
Acinetobacter cell biomass, growth stage and phosphorus uptake from activated sludge mixed liquor
(Elsevier, 1994) Cloete, T.E. (Thomas Eugene), 1958-; Bosch, M.; eugene.cloete@up.ac.za
Enhanced biological phosphorus removal activated sludge plants often do not remove phosphorus adequately in order to meet legal demands. Currently FeS04 is being added to almost all South African nutrient removal activated sludge systems discharging effluents to the sensitive catchments to prevent phosphorus from entering fresh water systems. In order to understand biological phosphorus removal mechanisms in order to optimise the process, the role of growth rate and phosphorus removal in Acinetobacter was investigated. Phosphorus was accumulated in the lag phase of the normal growth cycle. Little or no phosphorus was accumulated in the logarithmic growth phase, instead phosphorus was released at the beginning of logarithmic growth. Further phosphorus accumulation took place in the stationary phase, once active growth had ceased. Cells had a limit to the amount of phosphorus that could be accumulated per cell irrespective of substrate availability. It was therefore concluded, that the number of cells (biomass) in a system and their growth stage were crucial factors governing biological phosphorus removal. Maximum cell numbers should therefore be obtained and logarithmic growth should be prevented in the aerobic zone, in order to optimize biological phosphorus removal from activated sludge.
Adaptation of bacterial cultures to non-oxidising water treatment bactericides
(Water Research Council, 1993-07) Brözel, Volker Siegfried; Pietersen, Belinda; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
Bacterial communities in water-cooling systems treated with bactericides often become resistant to these bactericides. This has been ascribed to selection for resistant cells. Certain bacteria, having a high inherent susceptibility to water treatment bactericides, become dominant in systems after bactericide treatment. We investigated the idea that bacterial isolates adapt to growth in the presence of bactericides. Pure cultures of Pseudomonas stutzeri and Bacillus cereus were cultured repeatedly in the presence of sub-inhibitory concentrations of 2,2’-methylenebis(4-chlorophenol), sodium dimethyldithiocarbamate and isothiazolone. Both isolates adapted to growth in the presence of increasing concentrations of the bactericides. P. stutzeri adapted from 22 µg/ml 2,2’-methylenebis(4-chlorophenol) to 80 µ g/ml, from 12 µg/ml Na dimethyldithiocarbamate to 310 µg/ml, and from 50 µl/l isothiazolone to 250 µl/l. B. cereus adapted from 20 µg/ml 2,2’-methylenebis(4-chlorophenol) to 75 µg/ml, from 6 µg/ml Na dimethyldithiocarbamate to 132 µg/ml, and from 50 µl/l isothiazolone to 300 µl/l. The phenomenon of resistance to water treatment bactericides can be ascribed not only to selection but also to adaptation.
Fingerprinting of commercially available water treatment bactericides in South Africa
(Water Research Council, 1991-01) Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
Eighteen dominant isolates from water-cooling systems were exposed to 50mg/l of commercially available bactericides, and the kill percentage was determined after 6 h. Application costs of all bactericides giving an average kill percentage of over 90%, were compared. Low cost bactericides were re-evaluated at cost-equivalent concentrations. Dichlorophen, sulphone, a thiocarbamate and biphenol performed best, killing the full spectrum of isolates cost-effectively. Certain expensive products performed rather poorly, e.g. isothiazoline and MBT. This study highlights the selective action of many bactericides and the inherent resistance of bacteria to a number of different bactericides. This implies the importance of matching bactericides to the dominant bacteria in systems.
Immobilisation of Acinetobacter johnsonii cells within alginate beads
(Water Research Council, 1995-07) Muyima, Ndjoko Yei Osee; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
The growth and distribution of A. johnsonii cells, immobilised within alginate beads suspended in an aerated activated sludge mixed liquor medium, were assessed by viable cell counts on nutrient agar and scanning electron microscope (SEM). Both techniques indicated that A. johnsonii cells did survive and grow within alginate beads. A. johnsonii immobilised cells were metabolically active as they removed phosphate from the activated sludge mixed liquor medium. While cells were expected to occur preferably in the outer layer after a few hours of incubation, beads entrapping bacterial cells showed a random distribution of cell colonies 24h and 2 weeks after incubation. This constant random distribution might be attributed to constant aeration that would have fascilitated mass transfer added to extracellular substances which maintained daughter cells in colonies close to one another, thus preventing them from moving to the outer layer.
A bacterial population structure study of water cooling systems in South Africa
(Water Research Council, 1989-01) Cloete, Thomas Eugene; Brözel, Volker Siegfried; Pressly, J.; eugene.cloete@up.ac.za
Bacteria forming biofilms occur in all open water cooling systems where they accelerate metallic corrosion, reduce flow rate and decrease heat energy transfer rate. A population structure study of seven systems was conducted. The isolate most frequently encountered was Pseudomonas fluorescens (35,5%), the species commonly used in research regarding biofilm formation. This was followed by Chromobacter violaceum, P. pickettii, P. stutzeri and P. putida, each amounting to 6,6%. The dominant organisms occurred in two groups of over 85% relatedness between their biochemical reaction patterns. Overall four distinguishable groups occurred on the 90% similarity level.
The reaction of bacterial cultures to oxidising water treatment biocides
(Water Research Council, 1995-04) Pietersen, Belinda; Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
The possible development of resistance of bacteria found dominant in industrial water systems to the oxidising biocides hypochlorous acid and 3-bromo-1-chloro-5,5-dimethylhydantoin was investigated. Pseudomonas aeruginosa, P. stutzeri and Bacillus cereus were cultured repeatedly in the presence of sub-inhibitory concentrations of hypochlorous acid and 3-bromo-1-chloro-5,5-dimethylhydantoin. The minimum inhibitory concentrations (MIC) of hypochlorous acid and of 3-bromo-1-chloro-5,5-dimethylhydantoin decreased following initial exposure, but varied greatly during the period of the investigation. The MIC's did stabilise towards the end of the study. Whereas the isolates used did not display classical resistance, they did respond variably to successive exposures, indicating that long-term treatment of water systems with hypochlorous acid or 3-bromo-1-chloro-5,5-dimethylhydantoin would yield variable degrees of control of microbial activity.
Application of Sterikon bioindicators for the determination of bactericide concentrations
(Water Research Council, 1993-10) Cloete, Thomas Eugene; Da Silva, E.; Brözel, Volker Siegfried; eugene.cloete@up.ac.za
Biofouling in industrial water systems is normally prevented by the use of bactericides. However, bactericide programmes often fail owing to the lack of suitable techniques for determining the in situ bactericide concentration and this usually results in either inadequate or excessive bactericide concentrations. In this study, the Sterikon bioindicator was evaluated for determining the minimum inhibitory concentrations of 5 industrial bactericides (dichlorophen, sulphone, thiocarbamate, isothiazolone and a quarternary ammonium compound) for the monitoring of the concentrations of these compounds in industrial water systems. The results indicated that the Sterikon bioindicator can be used for the determination of bactericide concentrations.
Microbiological survey of open recirculating cooling water systems and their raw water supplies at twelve fossil-fired power stations
(Water Research Council, 1995-07) Poulton, W.I.J.; Cloete, T.E. (Thomas Eugene), 1958-; Von Holy, A.; eugene.cloete@up.ac.za
Raw water supplies utilised at 12 fossil-fired power stations, as well as the corresponding open recirculating cooling water systems were surveyed. Visual inspections were carried out and the total aerobic and anaerobic bacteria, anaerobic acid-producing bacteria, Thiobacillus spp., Nitrobacter spp., sulphate-reducing bacteria (SRB) and algae were quantified. All raw water supplies and recirculating cooling waters contained all of the above groups of micro-organisms, with the exception of the two potable raw water supplies. In 75% of the systems, the numbers of SRB in the recirculating cooling waters were higher than in the corresponding raw water supplies and in 92% of the systems, the numbers of the total aerobic bacteria were higher in the recirculating cooling waters than in the raw supplies. However, no relationship between the sulphate levels in the recirculating cooling waters and the numbers of SRB could be distinguished, or between the percentage increase in the numbers of total aerobic bacteria and the cycles of concentration at which the system was operated. The frequency polygons of the occurences of total aerobic and anaerobic bacteria in the raw water supplies and the recirculating cooling waters did not follow normal distribution patterns. Visible biofouling deposits were observed at six of the power stations surveyed and the predominant algal group was the blue-green algae. However, in the raw water supplies, the predominant algal groups were green algae and diatoms. Microbiologically influenced corrosion was identified in all five of the condensers inspected. Each system was found to be unique and no generalisations in terms of presence or activity of micro-organisms could be made.
The effect of bactericide treatment on planktonic bacterial communities in water cooling systems
(Water Research Council, 1992-04) Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
Bactericides were applied to experimental open recirculating cooling-water systems at concentrations found to be effective under laboratory pure-culture conditions. Total aerobic plate counts and bacterial population structures were determined over a period of 48h. In all cases the total aerobic count increased one day after the bactericide addition, and decreased rapidly after ca. 36 to 40h. Population shifts occurred during the course of all four treatments. In all cases different species became dominant concurring with fluctuations in the planktonic plate count, indicating the stress reaction of the biofilm. The species diversity decreased after treatment with dichlorophen, triocarbamate and methylenebis-thiocyanate, and increased upon treatment with humic acid. Species susceptible to bactericides in pure culture were found to be the dominant planktonic survivors. An example is Pseudomonas stutzeri which was the dominant survivor after treatment with thiocarbamate and with diclhlorophen.
The response of Escherichia coli K12 upon exposure to hypochlorous acid and hydrogen peroxide
(Water Research Council, 1996-01) Pietersen, Belinda; Brözel, Volker Siegfried; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
The aim of the work reported here was to investigate the growth-inhibitory activity of HOCI and hydrogen peroxide toward Escherichia coli K12 during both logarithmic and stationary phases of the growth cycle, as well as the response of E. coli K12 to these oxidants. Stationary phase cultures were exposed to sub-inhibitory oxidising stress, and the minimum inhibitory concentrations (MIC) were determined during the ensuing 24h. The effect of oxidant on logarithmically growing cultures was also determined. Stationary phase cultures of E. coli K12 responded to hydrogen peroxide stress, both the MIC and the survival following exposure to high concentrations increasing following exposure to stress. By contrast stationary phase cells did not become more tolerant of high concentrations of HOCI following HOCI stress. Logarithmically growing E. coli K12 did not display increased tolerance to either inhibitory or lethal concentrations of hydrogen peroxide or HOCI following the relevant oxidising stress.
Antibody recognition of an 18 KDa protein possibly involved in phosphate removal by activated sludge
(Elsevier, 2000) Erasmus, A.S.; Van Wyngaard, S.; Verschoor, Jan Adrianus; Ehlers, Marthie Magdaleen; Cloete, Thomas Eugene; eugene.cloete@up.ac.za
Phosphate in wastewater effluent is implicated in eutrophication of water reserves. Enhanced biological phosphate removal by activated sludge is attributed to polyphosphate accumulating bacteria, which release phosphate during anaerobiosis and reincorporate it during aerobiosis. The aim of the study was to investigate whether the process of phosphate removal by activated sludge could be probed immunochemically. Antigen preparations from the aerobic and preceding anoxic zones of a phosphate removing system contained intact and lysed bacterial cells. Neither conventional nor subtractive immunisation strategies, the latter employing cyclophosphamide to immunofocus on unique epitopes in the zones, provided antibodies capable of distinguishing between these zones. However, a putatively protein-directed monoclonal antibody could distinguish between the aerobic zones of two activated sludge systems, differing only in phosphate removal ability: immunoblot showed five discrete bands, with molecular weights appearing to be multiples of 18 kDa, unique to the system successful at phosphate removal.
SEM-EDS for determining the phosphorus content in activated sludge EPS
(Elsevier, 2001) Oosthuizen, Daniël Jacobus; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
Not all phosphorus removed in activated sludge systems can be accounted for by polyphosphate accumulating organisms (PAO). A method for the qualitative and quantitative in situ characterization of PAO cell clusters and closely associated extracellular polysaccharides (EPS) is described. X-ray microanalysis was performed on samples from four activated sludge plants situated in Pretoria, South Africa. Analyses were done by means of Scanning Electron Microscopy (SEM) combined with Energy Dispersive Spectrometry (EDS). Cell clusters with associated EPS on average contained between 57 and 59% phosphorus, while EPS alone contained on average between 23 and 30% phosphorus. Results suggest that phosphorus removal in activated sludge might be due not only to PAO, but also by EPS acting as a phosphorus reservoir. Extraction of EPS from two different activated sludge plants yielded different amounts of EPS, which, in combination with SEM-EDS, may shed light on different phosphate uptake abilities of different activated sludges.
Growth and phosphate uptake of immobilized Acinetobacter cells suspended in activated sludge mixed liquor
(Elsevier, 1995) Muyima, Ndjoko Yei Osee; Cloete, T.E. (Thomas Eugene), 1958-; eugene.cloete@up.ac.za
Immobilization of Acinetobacter cells can provide a method which could possibly be used to explain the mechanism of biological phosphorus removal from AS systems since this may allow the study of pure cultures in their natural habitat. Pure cultures of Acinetobacter johnsonii and Acinetobacter calcoaceticus cells were therefore immobilized within alginate beads (3% and 3.5% alginate) to assess their behaviour and particularly, their survival, leakage, growth and phosphate uptake abilities. Both species survived immobilization conditions, however, the effects on viability, leakage rate, growth and phosphate uptake were dependent upon the alginate concentration and the strain. Notwithstanding leakage, cell densities within pure cultures over 24 h were higher than initial densities due to growth. The growth rate of A. calcoaceticus immobilized cells was twice that of free cells, while A. johnsonii showed similar growth rates between immobilized and free cells. Non growing immobilized cells showed higher phosphate uptake ability than growing cells. After 24 h, A. calcoaceticus immobilized cells removed more phosphate per cell than A. johnsonii cells while both species showed higher phosphate uptake ability when immobilized in 3% alginate than in 3.5% alginate.
The effect of culture media on antigenic expression in sulphate reducing bacteria
(Springer, 2001-05) Cloete, T.E. (Thomas Eugene), 1958-; De Bruyn, E.E. (Engela Elizabeth); eugene.cloete@up.ac.za
The importance of sulfate-reducing bacteria (SRB) in nature has been widely recognized for many years. However, little is known about the ecology of SRB. The problem has been detecting, classifying, and quantifying these organisms. There are many shortcomings in the use of culture media for this purpose. As an alternative, fluorescent antibody (FA) techniques were considered as a method for the detection and identification of SRB. Antisera were prepared against whole cells of different species of SRB and evaluated for detection and identification of these organisms. Surface antigens of SRB were species specific. In addition, culture conditions influenced the expression of surface antigens, causing the antisera to be extremely specific. These results were confirmed by the sodium dodecyl sulfate-polyac-rylamide-gel electrophoresis (SDS-PAGE) profiles of membrane proteins. On the basis of this specificity, the application of FA produced against culture collection strains would have limited application for detecting, identifying, and enumerating these organisms in nature.

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