Microbiology

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An ultrastructural study on rusts with special reference to Puccinia sorghi on Oxalis corniculata.
(1973) Rijkenberg, Fredericus Hermanus Johannes.; Truter, Susarah J.
No abstract available.
A histopathological study on selected bacterial vascular diseases with emphasis on ultrastructure.
(1975) Wallis, Frederick Michael.; Truter, Susarah J.
No abstract available.
Purification and electron microscopy of the tomato spotted wilt virus.
(1976) Martin, Michael Menne.; Thung, T. H.; Noordam, D.
No abstract available.
Role of marine nitrifying bacteria in a closed system with Penaeus monodon.
(1984) Pillay, Balakrishna.; Roth, G.
In recent years there has been widespread interest in rearing aquatic organisms of nutritional and commercial value (Calaprice, 1976). The most hopeful prospect for marine prawn culture in the United Kingdom (Wickins, 1976), the Americas (Hanson & Goodwin, 1977) and South Africa probably lies in intensive culture under controlled conditions. A closed system approach, in which a captive body of water is circulated, provides the scope for water quality management which results ~n maximum water utilization and minimal discharge. On the other hand, direct utilization of sea-water in open systems presents problems for aquaculture since this water is subjected to diurnal and seasonal fluctuations in temperature, salinity and turbidity, as well as contamination from industrial, agricultural and maritime sources. Furthermore, large mariculture farms release enormous amounts of organic wastes which result in eutrophication and could lead to environmental deterioration of coastal waters (Gerhardt, 1978). It is well established that circulated sea-water develops an unusual ~on~c composition as a result of the metabolic activity of the prawns and of the nitrifying bacteria in the biological filter. The changes include elevated levels of ammonia, nitrite and nitrate and reduced pH. The presence of even sublethal levels of these nitrogenous compounds ~n closed systems have been found to affect growth of penaeid spec~es (Wickins, 1976). Ammonia and nitrite, which rapidly accumulate in the water, are usually maintained at nontoxic levels by nitrification in the biological filters (Spotte, 1974; Johnson & Sieburth, 1974). The chemolithotrophic bacteria responsible for nitrification are presently classified by their · cellular morphology and by the oxidation of either ammonia and nitrite (Watson, 1974). The predominant ammonia- and nitrite-oxidizing bacteria isolated from natural environments are Nitrosomonas europaea and Nitrobacter winogradskyi, respectively (Watson et aZ., 1981). Direct observation of nitrifying bacteria in natural environments, however, has been limited to studies involving light microscopy with immunofluorescent techniques (Fliermans et aZ., 1974; Fliermans & Schmidt, 1975). The electron microscopic observation of nitrifying bacteria ~sdifficult in natural microcosms with low levels of nitrification and with the presence of sunlight and anaerobic conditions conducive to the enrichment of other bacteria with a similar ultrastructure. However, in closed systems with extremely active nitrification but poor light conditions, the occurrence of morphologically similar forms in numbers that could be easily detected by electron microscopy is unlikely (Johnsort & Sieburth, 1976). Furthermore, the cyst-like colonies of the nitrifiers are unique and are not found with the methane-oxidizing bacteria with a similar ultrastructure (Davies & Whittenbury, 1970; Smith & Ribbons, 1970), whereas the thick cell wall of the cyanobacteria (Carr & Whitton, 1973) and the distinctive cell morphologies of the purple sulphur and purple nonsulphur bacteria (Pfennig, 1967) separate them from the nitrifiers. Therefore, closed systems with active nitrification provide the ideal environment to study the activities of nitrifiers in conjunction with their relative abundance, nature and diversity. In spite of the opportunity offered by closed systems, previous studies (Kawai et aZ., 1965; Wickins, 1976; Gerhardt, 1978; Mevel & Chamroux, 1981) on nitrification have been primarily indirect observations on rates of ammonia and nitrite oxidation to nitrate (Johnson & Sieburth, 1976). Studies on the enumeration and identification of nitrifiers ~n closed systems have been seriously neglected. Kawai et aZ. (1964) included the enumeration of nitrifiers in their study on nitrification while,in a qualitative study, an attempt to identify the in situ nitrifiers 1n closed systems (Johnson & Sieburth, 1976) was not very successful. This study was undertaken to investigate the three basic aspects of nitrification necessary for the understanding of such a process in closed systems, viz., the oxidation of ammonia and nitrite to nitrate, and the enumeration and identification of the nitrifying bacteria. Prior to determining the concentrations of the nitrogenous compounds in the culture water, various methods were evaluated for their accuracy and reproducibility with both sea-water and culture water samples. This approach is necessary in order to gauge the accuracy of results obtained by such methods. Enumeration of nitrifying bacteria was preceded by an investigation on the effect of incubation time on the maximum most probable number , estimate. Such an investigation was necessary because of the inconsistent approach to the enumeration of nitrifiers in previous studies (Wilson, 1927; Walker et al., 1937; Lewis & Pramer, 1958; Molina & Rovira, 1964; Meiklejohn, 1965; Smith et al., 1968). Incubation periods appear to have been chosen arbitrarily in previous investigations. Identifi~ation of nitrifying bacteria necessitates the isolation and purification of these organisms. Isolation of nitrifiers 1S a difficult and time-consuming task (Watson et al., 1981) and could be the main reason for not being included in previous studies on nitrification. Since the success of this study depended upon the isolation and purification of these chemolithotrophs, this aspect is de~lt with in detail. The changes most likely to be associated with nitrification in a closed system were also monitored 1n the culture water. These included pH, dissolved oxygen and biochemical oxygen demand. Apart from a biological sand filter, no other form of culture water treatment was effected during the investigation. The effect of growing the "sugpo" or jumbo tiger prawn, Penaeus monodon (Kinne, 1977) for 22 weeks in a captive body of sea-water was evaluated by comparing the survival and wet mass with those reported by other workers. This study differs greatly from previous reports on nitrification in closed systems because both the "causes" and "symptoms" of this important detoxifying process are investigated. It is intended that the findings of such a study would aid culturists in exploiting the nitrifying potential of closed systems to its utmost.
An Epidemiological study of gentamicin resistant gram negative bacteria with particular reference to pseudomonas aeruginosa at King Edward V111 Hospital, Durban
(1985) Bhana, Ratilal Hargovind.
The sources of gentamicin resistant pseudomonads and enterobacteria were studied in detail. A total of 1703 gentamicin resistant gram negative bacilli (GRGNB) isolated from patients, staff and their immediate environment were studied over a 6 month period . Of these 954 were isolated from clinical specimens obtained from patients and 540 from their immediate environment. A furthur 209 stains were isolated from the staff members who were responsible for the care of these patients. Pseudomonas aeruginosa; pyocin type 1 phage type F7 and .serotype 11 was the commonest isolate. It constituted 24,9% of all isolates in this study. This organism was distributed in all the wards investigated and was isolated throughout the 6 month study period. This strain, therefore, appears to be part of the "resident'' flora of King Edward Vlll Hospital for it was found on patients, staff and their immediate environment. Among the Enterobacteriaceae, Klebsiella pneumoniae was the commonest isolate and made up 13,6 % of all isolates. All the isolates obtained in this study were resistant to five of more antibiotics tested (gentamicin, tobramycin, kanamycin, streptomycin, carberricillin, polymyxin B amikacin and sisomicin). Of 310 staff members screened 25,2% harboured GRGNB on their hands. Among patients the commonest source of GRGNB was stool which yielded 141 (14,8 %) of the clinical isolates. Of the environmental sources studied, sinks harboured 87 (14%) GRGNB. The isolates from the environment and staff members were identical to patient strains. The significance of these findings is discussed.
Adhension of Candida albicans to host cells in culutre.
(1989) Maiter, Aziza Ismail.; Alexander, D. M.; Oellermann, Rolf Alfred.
No abstract available.
Studies on the causal agent of leaf scald disease in sugarcane.
(1989) Seetal, Ashwin Rabichand.; Oellermann, Rolf Alfred.; Roth, G.
No abstract available.
Quantification of fungal degradation of pinus patula and eucalyptus grandis.
(1992) Singh, Vahunth.; Baecker, Albin A. W.
Previous studies of fungal decay have mainly examined long term effects of wood decay. In contrast, the present work, was designed to quantify fungal degradation of wood during incipient decay. Three facultatively anaerobic, dimorphic fungi were isolated from the rumen of sheep. These fungi were identified as Mucor racemosus, Candida tropicalis and Geotrichum capitatum. Scanning electron microscopy showed that these fungi colonised Pinus patula and Eucalyptus grandis extensively but did not appear to degrade the wood. The obligate anaerobe Neocallimastix frontalis colonised wood very sparsely, whereas the white rot bas id iomycetes Cori 01 us versicolor, and Phanaerochaete chrysosporium, and the brown rotters Coniophora puteana and Lentinus lepideus, colonised wood under both aerobic and anaerobic conditions. The extents of colonisation were greater under aerobic conditions. The work then quantified the effects of the basidiomycetes C. versicolor, P. chrysosporium, C. puteana and L .lepideus, and the non-decay mould, M. racemosus in individual and coculture experiments. Wood colonisation was quantified by Kjeldahl nitrogen determinations converted to biomass assays, and degradation was quantified by weight losses, and Klason lignin determinations. Furthermore, the degraded wood samples were also analysed by HPLC analysis of hydrolysates and their sugar contents were determined to establish whether the glucose of cellulose and xylose + mannose of hemicellulose had been utilised by the respective fungi. The extent and nature of sugar utilisation by monocultures and cocultures in wood were then compared with the biomass and degradation data. statistical analyses of' these comparisons correlated the extents of colonisation, degradation, and the patterns of wood sugars predominantly utilised by each fungus. The results of the corresponding glucose, xylose and 'lignin analyses confirmed the brown rot physiological capacity of C.puteana in both'woods. The white rot fungi behaved as simultaneous rotters and,
Molecular and biochemical characterisation of ethanolic D-xylose fermenting Pichia stipitis, Candida shehatae and their fusants.
(1994) Govinden, Roshini.; Pillay, Basil Joseph.; Pillay, D.
No abstract available.
Molecular characterisation and detection of xanthomonas albilineans, the sugarcane leaf scald pathogen.
(1994) Permaul, Kugenthiren.; Pillay, Balakrishna.; Pillay, D.
No abstract available.
The influence of soil particle surfaces and soil porosity on the biodegradation of key refuse leachate organic molecules.
(1995) Du Plessis, Chris Andre.; Senior, Eric.; Hughes, Jeffrey Colin.
Many studies have been undertaken to determine the effects of soil and soil properties on migrating metal pollutants. Organic pollutants, however, in addition to their interactions with soil components , are also susceptible to degradation (catabolism) by microorganisms. Soil-microorganism-pollutant interactions have, traditionally, been studied in soil columns (microcosms). One of the shortcomings of column and in situ studies is that the identity and specific effect(s) of the soil component(s) affecting or influencing attenuation are not known and cannot readily be determined. Attenuation effects of the soil components are, therefore, difficult to interpret. ("Attenuation" in this context is the combined effects of both soil adsorption and microbial catabolism). Attenuation studies often only consider the physical conditions such as aeration, permeability, flow rate, temperature, etc. This approach assumes the soil to be a homogeneous matrix with no specific physico-chemical properties attributable to different components within the matrix. Soil physical factors suspected of influencing pollutant attenuation could be misleading without consideration of the physico-chemical interactions between soil components, microorganisms and pollutants. Adhesion of pollutants and microorganisms seems to be most important in this regard. The initial phase of this study was undertaken to examine the effects of three different soil materials on attenuation of key landfill leachate molecules. Examination of the effects of soil surface type on attenuation focused on adsorption / desorption of the pollutant molecules and microorganisms. These experiments sought to investigate the physico-chemical effects of soil, microorganism, pollutant interactions and were done as batch slurry experiments as well as in soil columns. Two soil horizons from the Inanda soil form (humic A and red apedal B) and the topsoil (vertic A) from a Rensburg soil form were used. The Inanda topsoil had a high organic matter content and both the topsoil and subsoil had a kaolinitic clay mineralogy; the Rensburg topsoil clay mineralogy was predominantly smectitic with a relatively low organic matter content. From the batch experiments, the adsorption of a hydrophobic molecule (naphthalene) and a heavy metal (cadmium) were found to be influenced to a significant extent by soil characteristics. Adsorption of naphthalene was due to the soil organic matter (SOM) content whereas cadmium adsorption was due to the cation exchange capacity (CEC) of the soil. Soil characteristics did not seem to have a significant influence on the adsorption of a water soluble compound such as phenol at the concentrations used. Attenuation of naphthalene was found to be affected by adsorption of the pollutant molecule (related to SOM) as well as the CEC of the soil. The attenuation of hydrophobic molecules can possibly be ascribed to the influence of CEC on the microbial population responsible for attenuation. This would seem to indicate interaction between the soil surfaces and the catabolizing microbial population. Desorption of the pollutant (and possibly also of the microbial population) was achieved by the addition of acetonitrile and methanol both of which reduced the polarity of the water. These solvents were also found to be toxic to the catabolizing microbial population at high concentrations. The toxicity thresholds of both solvents for catabolizing microorganisms differed significantly between soil- (> 15 %, v/v) and soil free (< 5 %, v/v) treatments. This discrepancy cannot be accounted for by adsorption and is ascribed to physico-chemical interaction between microorganisms and the soil surfaces. This interaction probably affords protection from, otherwise, toxic concentrations of solvents or metals. The important effects of soil surfaces on attenuation processes were thought to be due to the strong adsorption of naphthalene. Surface attachment of microorganisms was, however, also inferred from results obtained with phenol. This seemed to indicate that microbial attachment to soil surfaces was an important aspect in attenuation and did not occur only because of pollutant adsorption. Soil column experiments were made with both naphthalene and phenol. The naphthalene, which was adsorbed to the soil, did not leach from the columns to any appreciable extent. This was despite the addition of acetonitrile to some columns. This was probably due to greater microbial catabolism caused by desorption and, subsequent, increased soluble concentrations of the molecule. After extraction from the soil at the end of the experiment it was clear that the sterile controls held much higher concentrations of naphthalene than the experimental columns. The soil type and treatments showed little difference in the naphthalen concentration extracted from the soil columns. This did not reflect the differences found between soil materials in the batch experiments and was probably due to the masking effect of the soil physical factors on attenuation processes. Unlike naphthalene, phenol, because of its high solubility, was detected in the column leachates at relatively high concentrations. The phenol concentrations were much higher for the Inanda subsoil (approximately 4 mM) than the Inanda topsoil (approximately 2 mM) and Rensburg topsoil (< 1 mM). The Rensburg topsoil produced the lowest phenol concentrations in the leachate and this can probably be ascribed to the larger quantity of micropores in this soil. Thus, it seems that the soil physical features had a pronounced influence on attenuation. Whether this effect was directly on the studied molecule or indirectly, because of the effects on the microbial population, is not known. Inoculation of the columns with a phenol catabolizing population had only a slight increased effect on leachate phenol concentrations from all columns. This increased effect was, however, only prolonged in the case of the Inanda subsoil. The flow rate through the columns affected leachate phenol concentration which was lower with a slower flow rate and, thus, longer retention time. From the column experiments soil physical parameters were suspected of influencing, and possibly overriding, the soil surface effects on microbial activity (capacity to catabolize a organic molecule of interest). Soil porosity, as caused by different soil materials, was suspected of being the most important soil physical parameter influencing microbial activity. To investigate the potential effect of soil porosity, relatively homogeneous porous media i.e. chromatography packing material and acid washed sand were used. These materials had more defined and distinct porosities and were considered to be suitable for investigating the fundamental influence of porosity on microbial activity. Saturated continuous flow columns were used and three types of packing configurations were tested: chromatography packing (CHROM) material (porous particles); acid washed sand (non-porous) (AWS); and a 1: 1 (w/w) mixture of chromatography packing and acid washed sand (MIX). Only a single water soluble molecule, phenol, was used in this phase of the investigation. Bacterial filtration ("filtration" as a component of "attenuation'') was found to be highest for the CHROM and lowest for the AWS materials. This difference in microbial retention affected the phenol catabolism in response to increased column dilution rates. The CHROM and MIX materials had distinctly different porosities than that of the AWS, due to the internal porosity of the chromatography packing. This greater pore size distribution in the MIX and CHROM packing materials created pores with different effective pore dilution rates within the microcosms at similar overall flow rates. The greater pore size distribution in the MIX and CHROM packing materials facilitated pore colonization since some pores did not participate, or conduct, mass flow as occurred in macropores. This led to different microcolonization effects in the macro- vs micropores. Since the MIX and CHROM packing materials had more micropore colonization sites these packing materials showed a greater range of substrate affinities (i.e. Ks values) for the phenol substrate. The extent to which micropore colonization occurred could be detected by the effect it had on phenol breakthrough curves. In the MIX and CHROM materials, microbial colonization caused blocking of micropores with a subsequent effect on the phenol breakthrough curves. The AWS material, however, which had a low inherent microporosity, showed microbially induced microporosity probably due to biofilm development. The fact that the MIX and CHROM packing materials facilitated micropore colonization was also responsible for the greater resistance to, and the recovery from , potentially inhibitory cadmium concentrations. This effect was also apparent in the presence of acetonitrile, although this effect was not identical to that observed with cadmium. Finally, column pressure build up as a function of pore clogging was determined and was found to occur in the order AWS > MIX > CHROM. This was most likely due to fewer potential liquid flow paths with a higher blocking potential in the AWS. Extrapolation of the fundamentals of the above findings led to the conclusion that soil surface- and soil porosity effects are extremely important factors in determining the behavior of soils as bioreactors.
Bioremediation of heavy metal polluted waters.
(1995) Meyer, Angela.; Wallis, Frederick Michael.
Microorganisms have the potential to remove heavy metals from polluted waters and effluents and may be used in clean-up processes. Microbial associations were enriched for and adapted to grow in nutrient solutions containing various concentrations of different metals. As immobilised cells are known to be more stable and more efficient in metal uptake than are corresponding planktonic or free-living cells the attachment of the microbial associations was investigated using a model stream and it was found that biofilm development was better on rough surfaces such as ground glass and polystyrene than on smooth surfaces such as unetched glass plates and glass beads. When comparing metal uptake by planktonic and attached microorganisms, attached populations were found to have a greater metal-uptake capacity. The uptake of individual metals from various metal combinations was tested with various proportions of pregrown metal-adapted microbial populations as inoculum and it was found that a particular metal was taken up more readily by microbial associations which had previously been exposed to that metal. Lead (Pb2+) appeared to be taken up more readily than copper (Cu2+) or cadmium (Cd2+) while Cd2+ was more actively removed than Cu2+ from solution. pH also affected metal uptake and the optimum range for Cu2+ uptake by the Cu2+ -adapted microbial association was found to be between 5.8 and 7.0. Dead microbial biomass was investigated and found to have efficient metal uptake capacity. Living mycelium from an isolated Aspergillus species showed poor uptake of Cu2+ initially, but when this fungus was pregrown and subsequently killed by moist heat treatment the non-living mycelium was efficient in removal of Pb2+ and Cu2+ ions. The optimum mycelial biomass concentration for metal uptake was also determined. The mechanism of metal uptake by this Aspergillus species was determined, using electron microscopy and EDX techniques, to be metabolism-independent biosorption onto the hyphal surface. Thus the microbial associations and fungal cultures used in this study were shown to have the potential for use in the removal of heavy metals from polluted waters.
Microbiological investigations into granular sludge from two anaerobic digesters differing in design and industrial effluent purified.
(1995) Howgrave-Graham, Alan R.; Wallis, Frederick Michael.
Due to a combination of selection criteria, sludges from upflow anaerobic digesters treating industrial waste waters consist primarily of well-settling, dense agglomerates called granules. Quantification of the component mixed microbial populations of these granules has been severely restricted by the inability of researchers to disrupt them without concomitantly destroying numerous cells. In situ quantification using light and electron microscopy is complicated by the high cell numbers and bacterial diversity; the small cell size; and the destructive nature of electron microscopy preparative techniques preventing the viewing of more than a small percentage of the population at a time. For these reasons, in this investigation, standardization of qualitative electron microscopic techniques was performed prior to their application to granules. Isolation and electron and light microscopic techniques were applied to granules from a fullscale clarigester treating effluent from a maize-processing factory. In addition, a method using montaged transmission electron micrographs (TEMs) taken along a granule radius, and image analysis, was developed for bacterial quantification within granules. This method, together with antibody probe quantification, was applied to granules from an upflow anaerobic sludge blanket (UASB) digester treating a brewery effluent. The clarigester granules contained a metabolically and morphologically diverse population of which many members were not isolated or identified. By contrast, the UASB digester granules consisted primarily of morphotypes resembling Methanothrix, Methanobacterium and Desulfobulbus, in order of predominance. However, only about one-third of the population reacted with antibody probes specific to strains of bacterial species expected to occur within these granules. According to the antibody probe library used, the Methanobacterium-like cells observed in TEMs were probably Methanobrevibacter arboriphilus. From this study it is apparent that different anaerobic digester designs, operational parameters, and the chemical composition of the waste water purified, are factors which influence the formation and maintenance of granules differing with respect to their microbial populations. Until the difficulties associated with quantification are overcome, the processes governing granule formation and/or population selection will remain obscure.
A laboratory scale study to investigate the effects of solids concentration on the efficiency of anaerobic digestion.
(1995) Naidoo, Valerie.; Senior, Eric.; Buckley, Christopher Andrew.
With the exceptions of mixing and heating mechanisms, and the recycling of settled solids, no radical changes or improvements have been made to conventional anaerobic digesters treating municipal sewage. These digesters usually function with a hydraulic retention time of 30 to 60 days and at a total solids concentration of 2.6 %(m/v). Volumetric loading is limited since high loadings effect the displacement of the slow growing methanogens. Thus, the hydraulic retention time is coupled to the solids retention time. A crossflow microfiltration unit has been constructed at Northern Waste Water Treatment Works, Durban, to concentrate sludge from a conventional anaerobic digester and, thus, facilitate operation with a higher solids concentration. In addition, this process should result in the retention of the active biomass which would otherwise be lost as a waste product of the treatment process. The solids retention time is, thus, decoupled from the hydraulic retention time. The net result could be higher volumetric loadings, increased microbial activity and increased volatile solids destruction and, hence, improvement in the efficiency of anaerobic digestion of sewage sludge. To test these, different experiments were conducted to specifically determine the effect of higher solids loads. Preliminary experiments were undertaken to determine the biodegradability of primary sludge from the Northern Waste Water Treatment Works. Results showed that primary sludge of 76% VS could be reduced to approximately 48 to 50% VS during an experimental period of 85 days. Reduction of the first 20% VS was rapid if conditions were optimum but subsequent reduction from 55 to 50% VS was slow. It was calculated that approximately 0.88 l gas was produced for every g volatile solids catabolised. Further experiments were conducted to investigate the effects of different solids concentrations on microbial activity. The results showed that the volume of gas produced increased as the solids concentration increased from 2 to 6%(m/v). Digesters with solids concentrations of 6 to 13%(m/v) produced similar volumes of gas. Digesters with solids concentrations of 6 to 13%(m/v) TS produced approximately 300 ml more gas than the control during the 20 days experimental period. The rate of gas production also increased as the solids concentration increased. However, digesters containing 11%(m/v) and 13%(m/v) TS produced similar rates. These results indicate that the introduction of concentrated sludge into the digester improves digestion efficiency. Finally, a semi-continuous digester was operated at a 30 days retention time and at optimum temperature to investigate the efficacy of digesters with increased solids concentrations. The results showed that the rate of gas production increased as the solids concentration increased from 2%(m/v)(control) to 3.8%(m/v). However, the digester operated with 4.7%(m/v) TS produced gas at a rate lower that the digester with 3.8%(m/v) TS. The volatile solids concentrations of all four digesters were similar, indicating neither favourable nor unfavourable effects from increased solids concentrations. The digesters operated with 3.8%(m/v) and 4.7%(m/v) TS produced higher concentrations of volatile acids than the control. The alkalinity concentrations (>_4000 mg t-1 ) were similar for all four digesters.
Bioremediation of oil-contaminated soil : a South African case study.
(1996) Lees, Zoë Marie.; Senior, Eric.; Hughes, Jeffrey Colin.
In 1990, an oil recycling plant situated in Hammarsdale, South Africa, was decommissioned and a decision was taken by management to rehabilitate the site in preparation for resale. The heavily impacted area covered over two hecatares and oil contamination penetrated soil to depths in excess of three metres, making excavation and removal of the soil very expensive. The options for remediation of the site were limited. No facility for incineration of contaminated soil exists in South Africa, and landfilling was not permitted. The emphasis in developing a remediation strategy, therefore, focussed upon the possibility of in situ remediation with minimal excavation of soil. This study, the first of its kind in South Africa, was subsequently initiated to assess the feasibility of this approach, the results of which would underpin a full-scale cleanup programme. The development of such a strategy involved four key stages of work : (1) a comprehensive site investigation to evaluate and fully understand the particular problems at the site; (2) treatability studies to determine the potential for biological treatment of the contaminated soil and the optimisation of such treatments, particularly in terms of time and cost; (3) the testing of some of the more effective treatments on a pilot-scale; and (4) recommendations for full-scale bioremediation of the contaminated site. various conditions unique to South Africa had to be considered at each stage viz. the lack of funds and remediation experience, which created numerous problems and emphasised the requirement for a simple, "low-tech" approach. Site investigations revealed that in situ remediation may be possible due to the high permeability of the sandy soils and low concentrations of heavy metals. Laboratory experiments also showed that a mixed association of indigenous microorganisms was present which, once stimulated by nutrient supplementation at C:N:P, ratios of between 10:1:1 and 20:1:1, was capable of degrading total petroleum hydrocarbons at an average rate of 11% week -1. Further experimentation, aimed at reducing the cost of remediation and improving the soil quality, focussed on the efficacy of oil solubilisers, a soil ameliorant (composted pine-bark), indigenous fungi and higher plants in the remedial process. Three commercial surfactants (Arkopal N-050, N-060 and E2491) and one natural solubiliser (soybean lecithin) were tested for their biotoxicity, solubilisation and biodegradability at various concentrations (0.01 - 1.0%). Formulation E2491 was able to support a microbial population and was selected as the preferred commercial surfactant if soil washing was to be recommended; however, lecithin was considered to be more useful in situ because of its localised solubilising effect, biological origin and nutritional contribution. The use of fungi was of particular interest in addressing the persistent organic compounds, such as the heavy fractions of oil, for which bacterial remediation methods have been slow or ineffective. While it was not possible, however, to demonstrate in the laboratory that the indigenous fungi contributed significantly towards the degradation of the contaminating oil, the basic trends revealed that the fungal component of the indigenous microbial population was readily stimulated by the addition of nutrient supplements. The bulking-up process was also a success and additional exploratory work was proposed in the form of a larger scale composting design. Finally, the potential for using higher plants and 20% (v / v) composted pinebark (in addition to nutrients) to increase the microbial degradation of the contamination was investigated in both greenhouse and field plot studies. Greenhouse investigations employed soybeans which were postulated to have soil quality and cost benefits. However, although the soybeans were found to significantly enhance the remedial process, the complex soil-contaminant- plant interactions gave rise to strange nutritional effects and, in some cases, severe stunting. In contrast, the field studies employed grasses that had previously established on the site and which ultimately demonstrated a better tolerance for the contaminated conditions. Scanning electron microscopy revealed that there were considerable differences between the root tips of soybean plants which had been grown in contaminated soil and those which had been grown in uncontaminated soil. It was concluded that toxicity symptoms, which are readily observed in the root, could be used as an early indicator for determining the suitability of vegetation for remediation purposes. In both instances, despite the differences, the addition of composted pine-bark and nutrients (nitrogen and phosphorus) resulted in total petroleum hydrocarbon reductions of >85%, illustrating the benefits of plant establishment and oxygen availability. The need to link results from laboratory or pilot-scale experiments to achieve reliable predictions of field-scale behaviour was an essential component of this research. The results of the field study provided evidence, similar to that found in the pot trial, of the accelerated disappearance of organic compounds in the rhizosphere. All experiments incorporated parallel measurements of hydrocarbon residues, microbial activity and pH changes in the contaminated soil, the results of which strongly supported the argument that biodegradation was the dominant component of the remediation process. Thus, after consideration of the significant interactions which dominated the study (time-contaminant-nutrient; time-contaminant-pine-bark; and time-contaminant- pine-bark-plant), it was clear that, aside from these limiting factors, little should preclude the in situ bioremediation of the impacted soil.
Development of a laboratory river model to determine the environmental impacts of key xenobiotic compounds.
(1996) Hunter, Charles Haig.; Senior, Eric.; Howard, John.; Bailey, Ian.
Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds.
An investigation of the dual co-disposal of a phenolic wastewater and activated sewage sludge with refuse and treatment of high-strength leachate obtained from a closed co-disposal landfill.
(1996) Percival, Lynda J.; Senior, Eric.; Southway, Colin.
Co-disposal with refuse in a controlled landfill is the cheapest option for the disposal of hazardous waste and, if carefully controlled, can be an effective treatment option. In this present study a high-strength phenolic wastewater and activated sewage sludge were co-disposed with refuse. The effectiveness of phenol catabolism at two organic loading rates (500mgt1 and 1000mgtl) was assessed in the presence of various co-disposal strategies. Leachate recycle at the lower phenol organic loading rate was found to facilitate the greatest rate of phenol catabolism. Despite the effective removal of phenol, however, leachate recycle promoted the production of high concentrations of ammoniacal-N and hydrogen sulphide. At the higher phenol organic loading rate, recirculation was ineffective in reducing the residual phenol concentration due to inhibition of the phenol-catabolisers. Microcosms operated with single elution and batch co-disposal strategies at both phenol organic loading rates resulted in serious detrimental effects on the refuse fermentation and subsequent leachate quality. A high-strength leachate obtained from a closed co-disposal site was characterised to determine its chemical composition and was assessed for its susceptibility to biological treatment. If carefully controlled, co-disposal sites should produce leachates which differ little in quality to those produced by municipal waste sites. The exceptionally high specific conductivity of the leachate used in this present study was, however, uncharacteristic of a leachate from a municipal waste site. The leachate required dilution to 25 % (v/v) before responding to aerobic biological treatment due to the presence of bactericidal/bacteriostatic components. Anaerobic treatment was ineffective even at a final dilution of 10% (v/v) of the original due to the inhibition of methanogenesis caused indirectly by the high concentration of sulphate in the leachate. Following phosphate addition, aerobic biological treatment effected a significant reduction in the chemical oxygen demand (COD) but did not reduce the ammoniacal-N concentration. Scaling and precipitation occurred following addition of the phosphate, and although these did not affect the biological process they can cause operational problems in full-scale leachate treatment plants. Ion exchange, with soil, and lime treatment, were, therefore, considered for their ability to reduce the inorganic content of the leachate prior to biological treatment. However, these particular pretreatments were unsuitable due to their ineffectiveness to reduce calcium, the main inorganic element involved in scaling, to an acceptable concentration.
The microbiology of ex situ bioremediation of petroleum hydrocarbon-contaminated soil.
(1996) Snyman, Heidi Gertruida.; Senior, Eric.; Oellermann, Rolf Alfred.
Bioremediation is the process whereby the degradation of organic polluting compounds occurs as a result of biochemical activity of macro- and microorganisms. Bioremediation of hydrocarbon contaminated soils can be practised in situ or ex situ by either stimulating the indigenous microorganisms (biostimulation) or introducing adapted microorganisms which specifically degrade a contaminant (bioaugmentation). This investigation focused on ex situ remediation processes with special attention to the processes and microbiology of landfarming and thermal bioventing. Landfarming was investigated at pilot-scale and full-scale, and thermal bioventing at laboratory and pilot-scale. This study indicated that pilot-scale bioremediation by landfarming was capable of effecting a total petroleum hydrocarbon concentration (TPHC) reduction of 94% (m1m) from an initial concentration of 320 gkg-I soil to 18 gkg-I soil over a period of 10 weeks. Reactors receiving biosupplements showed greater rates of bioremediation than those receiving nutrients. Promotion of TPHC catabolism by addition of a commercial or a site-specific microbial biosupplement was similar. Seedling experiments proved that bioremediation did not necessarily leave the soil in an optimal condition for plant growth. The full-scale landfarming operation reduced the TPHC concentrations from 5 260 - 23 000 mgkg- I to 820 - 2335 mgkg- I soil over a period of 169 days. At full-scale, the larger fraction of more recalcitrant and weathered petroleums. and the less intensive treatment resulted in a slower rate of TPHC reduction than was found in the pilot-scale study. Three distinct decreases in the TPHC were observed during the full-scale treatment. These presented an ideal opportunity to investigate the microbiology of the soil undergoing treatment. The dominant culturable microorganisms were isolated and identified. The bioremediation process was dominated by Bacillus and Pseudomonas species. The method used to study the population was, however, biased to culturable, fast growing microorganisms which represent a small portion of the total microbial population. For this reason, a method to study the total eubacterial population in situ with rRNA targeted oligonucleotide probes was adapted and found to be a valuable technique. Soil microorganisms respiratory activity was investigated at different times in the full-scale treatment. A clear correlation between activity and degradation was recorded. The effect of a supplement. anaerobically digested sludge, was also assessed by this method. Thermal bioventing was investigated as an ex situ in-vessel treatment technology for small volumes of highly contaminated soils. This proved to be a viable technique for the bioremediation of petroleum hydrocarbons at laboratory-scale. Volatilisation contributed to at least 40% of the reduction. Of the two supplements evaluated. dried sludge promoted degradation to a greater extent than chicken manure. The pilot-scale study proved that a chemical contaminant reduction of at least 50% could be achieved in 13 weeks by thermal bioventing. Of the supplemented reactors. the presence of dried sludge and commercial biosupplement etfected the largest contaminant decrease. As a possible supplement to increase the rate of bioremediation. dried anaerobically digested sludge was more effective than chicken manure. A parallel laboratory-scale experiment gave similar results. Gravimetric analyses were found to be conservative indications of the remediation process. The results of this study shed some light on our. still. limited understanding of bioremediation. The gap between the technology in the laboratory and field was narrowed and a better understanding of the soil microbiology was achieved. Due to the limited control of environmental parameters in the case of landfarming. thermal bioventing was investigated and proved to be an effective alternative. The latter technology is novel in Southern Africa.
Complex soil-microorganism-pollutant interactions underpinning bioremediation of hydrocarbon/heavy metal contaminated soil.
(1996) Phaal, Clinton B.; Senior, Eric.; Du Plessis, Chris Andre.
This study evaluated the efficacy of bioremediation as a treatment option for a hydrocarbon and heavy metal contaminated soil. Microbial degradation of hydrocarbons under aerobic, nitrate-reducing and sulphate-reducing conditions was examined. Nutrient supplementation with nitrogen and phosphate as well as aeration seemed to be the most important factors for enhancing biodegradation. From initial batch studies, a carbon: nitrogen ratio of 50: 1 was found to be optimal for biodegradation. However, very low carbon to nitrogen ratios were undesirable since these inhibited microbial activity. Manipulation of the pH did not seem to be beneficial with regard to hydrocarbon biodegradation. However, low pH values induced elevated concentrations of leachate heavy metals. Aerobic conditions provided optimal conditions for hydrocarbon catabolism with up to 54% of the original contaminant degraded after 2 months of treatment. Further treatment for up to 20 months did not significantly increase hydrocarbon biodegradation. Under nitrate- and sulphatereducing conditions, 6% and 31 % respectively of the initial contaminant was degraded after 2 months while after a further 20 months, 50% and 42%, respectively were degraded. The addition of soil bulking agents and the use of sparging did not significantly increase biodegradation. Similarly, the addition of inoculum did not influence biodegradation rates to any great degree. The presence of heavy metals up to concentrations of 400 mgt1 Mn, 176 mgt1 Zn and 94 mgt1 Ni did not reduce microbial activity within the soil. During the treatment phase, heavy metal and hydrocarbon migration were limited even under water saturation and low pH conditions. A Biodegradation Index was developed and evaluated and may, potentially, find use as an in situ assessment technique for microbial hydrocarbon catabolism. The iodonitrophenyltetrazolium salt assay was also found to be an effective and rapid alternative assay for monitoring bioremediation progress.
Development of a code of practice for co-disposal to obviate inimical environmental impacts of generated gases and leachates.
(1996) Daneel, Richard A.; Senior, Eric.
Despite its phasing out in numerous countries, such as Germany and the U.S.A., co-disposal of hazardous waste with municipal solid waste continues to be widely practised in South Africa. Co-disposal utilises properties and microbial activities in the refuse to attenuate the hazardous waste and thus obviate its environmental impact potential. All landfill operations require careful planning in not only site selection criteria but also the type and amount of various wastes accepted for disposal. It is clear, however, that the practice of co-disposal requires special precautions and management as the methods employed in the landfill operation determine to a large extent the environmental effects and, thus, the public acceptability of the operations. Although co-disposal is not suitable for all industrial wastes the results of recent research efforts, conducted mainly in the U.K., have indicated that, when properly managed, co-disposal can be regarded as a safe and efficient disposal option for many hazardous wastes. Environmental awareness in many European countries ensures that numerous hazardous compounds are either recycled or recovered. Unfortunately, in South Africa the lack of similar concern has resulted in increased concentrations of toxic compounds being co-disposed on a regular basis. Since fundamental studies of this technology, pertaining to South African conditions, have been lacking laboratory models/microcosms were built to address this paucity. Model. To effect the separation of species habitat domains of component species of growth rate-dependent interacting microbial associations responsible for terminal catabolic processes of the refuse fermentation, with retention of overlapping activity domains, and so facilitate examination of species in isolation without violating the integrity of each association, multi-stage models were constructed. The accidental overgassing of the culture with liquid petroleum gas (LPG) effected interesting fermentation balance changes which also emphasised the need for an Anaerobic Bioassay Test to assess the impacts of specific perturbants. Evidence of differential susceptibility of the component species to phenol was demonstrated in this study. Microcosm. A total of 42 refuse packed single-stage glass column bioreactors were commissioned and subjected to phenol and/or anaerobically digested sewage sludge codisposal. The effects of four different operational modes: leachate discard (single elution); leachate recycle; batch; and simulated rain on the co-disposals as well as refuse catabolism per se were examined. The results of these studies indicated that protracted periods of adaption to phenol (1000 and 2000 mg l -1) could have resulted from nutrient (elemental) limitation. Circumstantial evidence was also gained which indicated that the nitrate- and sulphate-reducing bacteria (SRB) were particularly sensitive to the added xenobiotic. Further, without the effective participation of the nitrate- and SRB the active and total fermentation of both the phenol and refuse components were depressed. It was also determined that the operating regime employed was a key factor in refuse degradation although with time, and especially following the phenol resupplementations, the operating conditions played a less significant role. In general, the single elution operated columns demonstrated increased phenol removal rates which were, unfortunately, coincident with low pH values and increased leachate residual phenol concentrations. Leachate recycle, on the other hand, unlike the batch operated columns, facilitated increased pH values and methane evolutions. The simulated rain columns were characterised by rapid washout of the added phenol as well as methanogenic precursors. The sewage sludge co-disposal experiments, likewise, demonstrated that, depending on the sludge:refuse ratio, the operating regime was extremely important in optimising the refuse degradation processes although, in general, leachate recycle appeared to be the most favoured method of operation.

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