Browsing by Author "Harrison, Susan"
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- ItemOpen AccessA Comparative Analysis of the Performance and the Microbial Ecology of Biological Sulphate Reducing Reactor Systems(2020) Hessler, Tomas; Huddy, Robert; Harrison, SusanAcid rock drainage (ARD) is defined as acidic waste-water contaminated with sulphate and heavy metals which is generated through the oxidation of sulphidic ores in the presence of water and oxygen. Mining activities accelerate this process by bringing these ores to the surface where they are further crushed and, eventually end up in waste rock dumps and tailing impoundments where they continue to generate ARD into perpetuity. Active mining operations are mandated to prevent the discharge of ARD into the environment. This ARD is commonly remediated by expensive yet highly effective active treatment strategies such as high-density sludge processes and reverse osmosis. South Africa has an extensive history of gold and coal mining which has left abandoned mine workings with associated waste rock dumps throughout northern and eastern parts of the country. As many of these mines have long been abandoned, the responsibility to mitigate the environmental impact of the generated ARD lies solely with government. Although these diffuse sites often generate smaller volumes of less aggressive ARD compared to that generated through mine water rebound, the sheer number and the continual ARD generation from these sites is a severe threat to South Africa's already poor water security. Biological sulphate reduction (BSR) has long been considered an attractive option for the longterm remediation of these low-volume sources of ARD – but its implementation has shown mixed success. BSR is a process catalysed through the innate metabolism of sulphate-reducing bacteria (SRB) which coexist within complex microbial communities. SRB themselves are a highly diverse group of anaerobic microorganisms which use sulphate as a terminal electron acceptor. The sulphide and bicarbonate produced during BSR can be used to precipitate heavy metals and aid in the neutralisation of the ARD, respectively. The implementation of BSR is, therefore, a comprehensive remediation strategy for diffuse sources of ARD. The study of BSR, using various reactor configurations and operating conditions shows much promise. However, the microbial ecology of the complex communities within BSR systems, and their links to the performance of BSR processes, has received far less attention in published literature. This is not a result of underappreciation of the role microbial communities but rather a historical lack of tools, specifically high-throughput techniques, available to assess complex microbial consortia. It is asserted that the success of a sustainable BSR process developed for the long-term remediation of ARD requires an in-depth understanding the microbial communities associated with this process. The identification of the microorganisms which are key to the process, thosewhich threaten the stability of the community and the optimal growth conditions of these microorganisms, can be used to inform how these bioreactors are designed and operated. This study investigated the performance and microbial ecology of several continuous BSR reactors using culture-independent metagenomic sequencing approaches. The performance and microbial ecology of these reactors were evaluated at a range of hydraulic residence times (HRT) over the course of approximately 1000 days of continuous operation, from five- through to one-day(s). The tested reactor configurations included a continuous stirred tank reactor (CSTR), an up-flow anaerobic packed bed reactor (UAPBR) and a linear flow channel reactor (LFCR) that were each operated in duplicate and supplemented with either lactate or acetate as an electron donor. The different reactor configurations and supplied electron donors, as well as the varied applied HRT, generated a range of microenvironments which were hypothesised to lead to the divergence of the initial microbial community of the inoculum and generate numerous distinct microbial communities throughout and across the reactor systems. 16S rRNA gene amplicon sequencing was used to assess the microbial community structure of the numerous populations across the reactor systems and monitor how these communities responded to the change in the applied HRT. Genome-resolved metagenomics was employed in parallel to recover the genomes of all predominant microorganisms identified through gene amplicon sequencing. This allowed the interrogation of the composition of the respective microbial communities as well as the genetic potential of each microorganism and encompassing the communities represented within specific reactor environments. The CSTRs were selected as these systems are characterised as well-mixed, support solely suspended biomass and kinetic equilibriums are achieved rapidly. This allows the performance of these reactors to be predictable and provides a benchmark to which the LFCRs and UAPBRs could be compared. The lactate-supplemented CSTR performed largely as anticipated based on available literature, demonstrating a maintained sulphate conversion of approximately 55% over the course of the study. The reactor achieved a maximum observed volumetric sulphate reduction rate (VSRR) of 17 mg/ℓ.h at a one-day HRT. The system supported a low SRB diversity, constituted almost entirely by a Desulfomicrobium and two Desulfovibrio operational taxonomic units (OTUs). The acetate-supplemented CSTR was able to maintain sulphate reducing performance at HRT where complete washout of SRB had been predicted based on literature. This reactor exhibited a maximum VSRR of 10.8 mg/ℓ.h at a 1.5-day HRT and was dominated by the same Desulfovibrio and Desulfomicrobium observed in the lactate-supplemented CSTR, along with several other SRB genera at lower abundance. The LFCRs demonstrated an approximately ten-fold greater biomass retention than the corresponding CSTRs. This was facilitated through the incorporation of carbon microfibres, whichfacilitated microbial colonisation and biofilm formation within the reactors. Surprisingly, the lactate-supplemented LFCR, underperformed compared to the lactate-supplemented CSTR, achieving a maximum VSRR of 14.8 mg/ℓ.h at a one-day HRT. This reduced performance, in spite of the enhanced biomass retention, was concluded to result from the out-competition of lactateoxidising SRB in the reactor by Veillonella and Enterobacter OTUs. The acetate-supplemented LFCR exhibited a period of underperformance before recovering and subsequently demonstrated a maximum VSRR of 17.1 mg/ℓ.h at a one-day HRT. Evaluations of the microbial communities of this system during the HRT study revealed a dramatic shift in the SRB communities from being dominated by Desulfatitalea and Desulfovibrio to being dominated predominantly by Desulfomicrobium and Desulfobacter. The UAPBRs are governed by plug-flow which resulted in the generation of gradients of decreasing substrates and increasing products throughout the height of the reactors. This, as hypothesised, resulted in the stratification of the microbial communities throughout the height of these reactors. This allowed many associations to be made between specific microorganisms and their ideal growth environments. Both UAPBRs demonstrated competitive sulphate reducing performance. The lactate-supplemented UAPBR proved especially successful as this system was able to maintain >95% sulphate conversion at one-day HRT, corresponding with a VSRR of 40.1 mg/ℓ.h. The performance of this reactor was attributed to the significant quantity of retained biomass and the successful harbouring of lactate-oxidising SRB towards the inlet zone of the reactor as well as propionate- and acetate-oxidising SRB towards the effluent zones of the reactor. The acetatesupplemented UAPBR exhibited a maximum VSRR of 23.2 mg/ℓ.h at a one-day HRT and a maximum sulphate conversion of 79% at a 2.3-day HRT. The stratification of the microbial communities within the acetate-supplemented UAPBR was less pronounced than the lactatesupplemented UAPBR, as a result of the fewer available volatile fatty acid species. However, the stratification which was observed in this system could be used to postulate the growth kinetics associated with the identified SRB – a Desulfobulbus was associated with rapid acetate oxidation in the inlet zone while a Desulfatitalea and a Desulfosarcina could be implicated in sulphate scavenging in the effluent zone of this reactor. This proved particularly valuable for elucidating the roles of these same SRB in the well-mixed reactor systems. Genome-resolved metagenomics was employed to recover the genomes of the microorganisms identified in these systems and determine the metabolic potential of these microorganisms. Hydrogen-evolving hydrogenase genes were found to be widespread in genomes not capable of sulphate reduction. In contrast, hydrogen-consuming hydrogenases as well as autotrophic gene pathways were common amongst SRB genomes. The ubiquity of hydrogenase genes in these environments indicated that inter-species hydrogen transfer was an important feature within thesemicrobial communities. The dual consumption of both acetate and hydrogen was concluded to have facilitated the maintained sulphate reducing performance of the acetate-supplemented reactor systems at short HRT where system failure had been predicted. Indices of replication (iRep) were used to estimate the instantaneous growth rates of the microorganisms from metagenomic shotgun sequencing datasets. This revealed that, at a four-day HRT, the microorganisms within the biofilms were comparably active to planktonic microorganisms. This, together with the dynamic changes in the composition of these biofilms during the HRT study, suggests these biofilms are even more active and competitive than previously thought. The combined use of next-generation gene amplicon sequencing and genome-resolved metagenomics has given unprecedented insights into the microbial communities of BSR reactor systems. Using this approach, it was possible to uncover a seldom discussed form of hydrogen cycling within BSR systems and has shown that there is no ‘one-size-fits-all' approach when inoculating BSR reactors. The SRB within these systems were often highly specialised to particular environments, specific electron donors and each showed differing growth kinetics. The success of long-term, semi-passive BSR reactor systems would benefit greatly from the tailoring of SRB inoculums informed by the chosen reactor configuration and operating conditions. The outcomes of the kinetic reactor experiments have led to several recommendations for the design and operation of these systems.
- ItemOpen AccessA novel semi-passive process for sulphate removal and elemental sulphur recovery centred on a hybrid linear flow channel reactor(2020) Marais, Tynan S; Harrison, Susan; van Hille, Rob; Huddy, RobertSouth Africa (SA) currently faces a major pollution problem from mining impacted water, including acid rock drainage (ARD), as a consequence of the mining activities upon which the economy has been largely built. The environmental impact of ARD has been further exacerbated by the country's water scarce status. Increasingly scarce freshwater reserves require the preservation and strategic management of the country's existing water resources to ensure sustainable water security. In SA, the primary focus on remediation of ARDcontaminated water has been based on established active technologies. However, these approaches are costly, lead to secondary challenges and are not always appropriate for the remediation of lower volume discharges. Mostly overlooked, ARD discharges from diffuse sources, associated with the SA coal mining industry, have a marked impact on the environment, similar to those originating from underground mine basins. This is due to the large number of deposits and their broad geographic distribution across largely rural areas of SA. Semi-passive ARD treatment systems present an attractive alternative treatment approach for diffuse sources, with lower capital and operational costs than active systems as well as better process control and predictability than traditional passive systems. These semi-passive systems typically target sulphate salinity through biological sulphate reduction catalysed by sulphate reducing bacteria (SRB). These anaerobic bacteria reduce sulphate, in the presence of a suitable electron donor, to sulphide and bicarbonate. However, the hydrogen sulphide product generated is highly toxic, unstable, easily re-oxidised and poses a significant threat to the environment and human health, so requires appropriate management. An attractive strategy is the reduction of sulphate to sulphide, followed by its partial oxidation to elemental sulphur, which is stable and has potential as a value-added product. A promising approach to achieve partial oxidation is the use of sulphide oxidising bacteria (SOB) in a floating sulphur biofilm (FSB). These biofilms develop naturally on the surfaces of sulphide rich wastewater streams. Its application in wastewater treatment and the feasibility of obtaining high partial oxidation rates in a linear flow channel reactor (LFCR) has been described. The use of a floating sulphur biofilm overcomes many of the drawbacks associated with conventional sulphide oxidation technologies that are costly and require precise operational control to maintain oxygen limiting conditions for partial oxidation. In the current study a hybrid LFCR, incorporating a FSB with biological sulphate reduction in a single reactor unit, was developed. The integration of the two biological processes in a single LFCR unit was successfully demonstrated as a ‘proof of concept'. The success of this system relies greatly on the development of discrete anaerobic and microaerobic zones, in the bulk liquid and at the airliquid interface, that facilitate sulphate reduction and partial sulphide oxidation, respectively. In the LFCR these environments are established as a result of the hydrodynamic properties associated with its design. Key elements of the hybrid LFCR system include the presence of a sulphate-reducing microbial community immobilised onto carbon fibres and the rapid development of a floating sulphur biofilm at the air-liquid interface. The floating sulphur biofilm consists of a complex network of bacterial cells and deposits of elemental sulphur held together by an extracellular polysaccharide matrix. During the Initial stages of FSB development, a thin transparent biofilm layer is formed by heterotrophic microorganisms. This serves as ‘scaffolding' for the subsequent attachment and colonisation of SOB. As the biofilm forms at the air-liquid interface it impedes oxygen mass transfer into the bulk volume and creates a suitable pH-redox microenvironment for partial sulphide oxidation. Under these conditions the sulphide generated in the bulk volume is oxidised at the surface. The biofilm gradually thickens as sulphur is deposited. The produced sulphur, localised within the biofilm, serves as an effective mechanism for recovering elemental sulphur while the resulting water stream is safe for discharge into the environment. The results from the initial demonstration achieved near complete reduction of the sulphate (96%) at a sulphate feed concentration of 1 g/L with effective management of the generated sulphide (95-100% removal) and recovery of a portion of the sulphur through harvesting the elemental sulphur-rich biofilm. The colonisation of the carbon microfibres by SRB ensured high biomass retention within the LFCR. This facilitated high volumetric sulphate reduction rates under the experimental conditions. Despite the lack of active mixing, at a 4-day hydraulic residence time, the system achieved volumetric sulphate reduction rates similar to that previously shown in a continuous stirred-tank reactor. The outcome of the demonstration at laboratory scale generated interest to evaluate the technology at pilot scale. This interest necessitated further development of the process with a particular focus on evaluating key challenges that would be experienced at a larger scale. A comprehensive kinetic analysis on the performance of the hybrid LFCR was conducted as a function of operational parameters, including the effect of hydraulic residence time, temperature and sulphate loading on system performance. Concurrently, the study compared the utilisation of lactate and acetate as carbon source and electron donor as well as the effect of reactor configuration on system performance. Comparative assessment of the performance between the original 2 L LFCR and an 8 L LFCR variant that reflected the pilot scale design with respect to aspect ratio was conducted. Pseudo-steady state kinetics was assessed based on carbon source utilisation, volumetric sulphate reduction, sulphide removal efficiency and elemental sulphur recovery. Additionally, the hybrid LFCR provided a unique synergistic environment for studying the co-existence of the sulphate reducing (SRB) and sulphide oxidising (SOB) microbial communities. The investigation into the microbial ecology was performed using 16S rRNA amplicon sequencing. This enabled the community structure and the relative abundance of key microbial genera to be resolved. These results were used to examine the link between process kinetics and the community dynamics as a function of hydraulic residence time. Results from this study showed that both temperature and volumetric sulphate loading rate, the latter mediated through both sulphate concentration in the feed and dilution rate, significantly influenced the kinetics of biological sulphate reduction. Partial sulphide oxidation was highly dependent on the availability and rate of sulphide production. Volumetric sulphate reduction rates (VSRR) increased linearly as hydraulic residence time (HRT) decreased. The optimal residence time was determined to be 2 days, as this supported the highest volumetric sulphate reduction rate (0.21 mmol/L.h) and conversion (98%) with effective sulphide removal (82%) in the 2 L lactate-fed LFCR. Lactate as a sole carbon source proved effective for achieving high sulphate reduction rates. Its utilisation within the process was highly dependent on the dominant metabolic pathway. The operation at high dilution rates resulted in a decrease in sulphate conversion and subsequent increase in lactate metabolism toward fermentation. This was attributed to the competitive interaction between SRB and fermentative bacteria under varying availability of lactate and concentrations of sulphate and sulphide. Acetate as a sole carbon source supported a different microbial community to lactate. The lower growth rate associated with acetate utilising SRB required longer start-up period and was highly sensitive to operational perturbations, especially the introduction of oxygen. However, biomass accumulation over long continuous operation led to an increase in performance and system stability. Microbial ecology analysis revealed that a similar community structure developed between the 2 L and 8 L lactate-fed LFCR configurations. This, in conjunction with the kinetic data analysis, confirmed that the difference in aspect ratio and scale had minimal impact on process stability and that system performance can be reproduced. The choice of carbon source selected for distinctly different, highly diverse microbial communities. This was determined using principle co-ordinate analysis (PCoA) which highlighted the variation in microbial communities as a function of diversity and relative abundance. The SRB genera Desulfarculus, Desulfovibrio and Desulfomicrobium were detected across both carbon sources. However, Desulfocurvus was found in the lactate-fed system and Desulfobacter in acetate-fed system. Other genera that predominated within the system belonged to the classes Bacteroidetes, Firmicutes and Synergistetes. The presence of Veillonella, a lactate fermenter known for competing with SRB, was detected in the lactate-fed systems. Its relative abundance corresponded well with the lactate fermentation and oxidation performance, where an apparent shift in the dominant metabolic pathway was observed at high dilution rates. Furthermore, the data also revealed preferential attachment of selective SRB onto carbon microfibers, particularly among the Desulfarculus and Desulfocurvus genera. The microbial ecology of the floating sulphur biofilm was consistent across both carbon sources. Key sulphur oxidising genera detected were Paracoccus, Halothiobacillus and Arcobacter. The most dominant genera present in the FSB were Rhizobium, well-known nitrogen fixing bacteria, and Pannonibacter. Both genera are members of the class Alphaproteobacteria, a well-known phylogenetic grouping in which the complete sulphur-oxidising, sox, enzyme system is highly conserved. An aspect often not considered in the operation of these industrial bioprocess systems is the microbial community dynamics within the system. This is particularly evident within biomass accumulating systems where the proliferation of non-SRB over time can compromise the performance and efficiency of the process. Therefore, the selection and development of robust microbial inoculums is critical for overcoming the challenges associated with scaling up, particularly with regards to start-up period, and long-term viability of sulphate reducing bioreactor systems. In the current study, long-term operation demonstrated the robustness of the hybrid LFCR process to maintain relatively stable system performance. Additionally, this study showed that process performance can be recovered through re-establishing suitable operational conditions that favor biological sulphate reduction. The ability of the system to recover after being exposed to multiple perturbations, as explored in this study, confirms the resilience and long-term viability of the hybrid process. A key feature of the hybrid process was the ability to recover the FSB intermittently without compromising biological sulphate reduction. The current research successfully demonstrated the concept of the hybrid LFCR and characterised sulphate reduction and sulphide oxidation performance across a range of operating conditions. This, in conjunction with a clearer understanding of the complex microbial ecology, illustrated that the hybrid LFCR has potential as part of a semi-passive approach for the remediation of low volume sulphate-rich waste streams, critical for treatment of diffuse ARD sources.
- ItemOpen AccessAn investigation into the fundamental understanding of an activated sludge bioremediation process and optimisation of thiocyanate and cyanide destruction(2019) van Zyl, Andries Wynand; van Hille, Robert; Harrison, SusanCyanide (CN) is used in the gold mining industry to dissolve gold from free milling, complex and refractory gold containing ores. Processing sulphide containing refractory ores using biooxidation as a pre-treatment has become increasingly important due to the depletion of free milling ores. The reaction of CN with reduced sulphur species during the cyanidation process results in the formation of thiocyanate (SCN), often at relatively high concentrations (> 5 000 mg/L). The SCN and residual free CN are deported with the tailings as components of the liquid fraction. The concentration of SCN often exceeds the legislated discharge specification, necessitating on-site treatment, while water would also require treatment before on-site recycling and reuse. Biological degradation of CN and particularly SCN in these effluents provides an alternative to the more traditional processes such as SO2 treatment or UV destruction. The traditional destruction processes focus on breaking the chemical bonds, through physical or chemical means, thereby converting the toxic CN and SCN species to less toxic compounds. These processes generally suffer from high reagent cost, incomplete removal of CN and particularly SCN species and the generation of by-products which require further treatment. A number of microorganisms are capable of utilising CN and SCN as a source of sulphur, nitrogen and carbon, as well as generating energy from their oxidation. Additional removal of metal-CN complexes may be achieved by adsorption to the cell surface or extracellular polymeric substances secreted by the cells. The activated sludge tailings effluent remediation (ASTERTM) process was developed for the biological treatment of especially SCN, but also free CN and metal-cyanide complexes, such as CuCN and Zn(CN)2. The basic ASTERTM technology consists of an aerated reactor, in which SCN and CN species are oxidised and a settler to facilitate the recovery of water and potentially biomass. The desire to expand the commercial application of the technology necessitated a more complete, fundamental understanding of the ASTERTM process and required focused, in-depth research. This research aimed to define the viable operating window for SCN destruction, as well as optimising practical SCN and CN destruction process conditions. The ASTERTM process relies on a complex microbial community, so understanding the community structure and metabolic potential for SCN and CN destruction, further enhanced the fundamental and mechanistic understanding of this bioprocess. The research contributed to the fundamental understanding of this technology and enhanced the commercial application thereof. The first step in defining the operating window was to investigate the effect of feed SCN concentration on the SCN destruction ability of the mixed microbial community. Experiments were conducted at feed SCN concentrations ranging from 60- 1 800 mg/L. Complete SCN destruction was achieved across the range at ambient temperature. The maximum SCN destruction rate was 15.7 mg/L.h at an initial SCN concentration of 1 400 mg/L. Temperature was investigated in the range of 10-45°C with an initial SCN concentration range of 60-180 mg/L. A maximum SCN destruction rate of 17.4 mg/L.h was measured at 35°C, with an initial SCN concentration of 180 mg/L. A wide pH range (pH 5.0-10.0) was tolerated, with optimal performance recorded at pH 7.0. This evaluation identified not only the optimum operating pH, but also highlighted the negative impact of a sudden pH change on the efficiency of SCN destruction. Residual SCN concentrations below 1 mg/L were achieved in all cases, which would allow for discharge or recycling of treated water. Floc (sludge) formation was observed in experiments with high initial SCN concentrations and indicated a possible stress response during these batch experiments. Floc (sludge) formation were taken as microbial cells imbedded within extracellular polymeric substances and not only an aggregate of cells. Evaluating the maximum potential for SCN destruction and optimising the operating conditions and system configuration was investigated using continuous reactor experiments. A maximum SCN destruction rate of 87.4 mg/L.h (2 098 mg/L.d) was achieved at a feed SCN concentration of 1 000 mg/L and eight hour hydraulic retention time (HRT) during these experiments. The formation of substantial amounts of sludge was observed, with attachment to the reactor surfaces. The maximum feed SCN concentration, where substantial destruction was measured, was at 2 500 mg/L, achieving a practical SCN destruction rate of 972 mg/L.d. Significant inhibition of microbial inactivity was observed beyond this feed SCN concentration. The microbial community was able recover performance, within six days, after an extended period (54 days) of inactivity when the feed concentration was reduced from 3 500 mg/L SCN to 1 000 mg/L. The nature of the accumulated biofilm did not appear to change during the period of limited SCN destruction activity. Calculation of specific SCN destruction rates was not possible due to the nature of the sludge and heterogeneous dispersion of microbial members. Biomass (cells embedded in the EPS sludge) loading experiments showed SCN destruction rates increased with an increase in biomass loading, but this relationship was not proportional. A 25-fold increased biomass concentration resulted in only a 2-fold increase in destruction rate, suggesting a mass transfer limitation. The sludge most likely offers protection against unfavourable conditions, such as high residual SCN concentrations, by presenting a mass transfer barrier, resulting in an SCN concentration gradient across the sludge matrix. This enhances the robustness of the process and would facilitate rapid recovery in the case of a system upset at commercial scale. This research is the first to demonstrate the effective removal of SCN in the presence of suspended tailing solids, under conditions well suited for commercial application. The maximum SCN destruction rate achieved was 57 mg/L.h in the presence of 5.5% (m/v) solids. Sludge formation was not observed in the reactors containing solids, despite substantial sludge formation under similar operating conditions in the absence of solids, most likely due to shear-related effects. Fluctuations in pH, due to the nature of the solid material, were identified to negatively impact reactor performance and pH control was required. Moreover, the type of solid particle was found to influence the SCN destruction rate showing a need for adaptation not only to the presence of solids but also to various types of solids that are to be treated. Treatment of residual CN in solution is critical to ensure safe disposal or recycling of water. Treatment of SCN and CN was successfully demonstrated at feed concentrations up to 2 000 and 50 mg/L, respectively. The presence of residual CN (0.5 mg/L) prevented complete destruction of SCN, while complete SCN destruction was measured in the absence of CN under identical conditions. A range of reactor configurations were investigated and the optimum system required biomass retention, by means of attached biomass and complete destruction of any residual CN prior to SCN destruction. Conversion of SCN-S to SO4-S was stoichiometrically proportional in solution, while the majority of the liberated nitrogen appeared to be assimilated. Pre-colonisation of the reactor with attached biomass is beneficial and removed the need for a solid-liquid separation unit, reducing the potential footprint of the process. Additional treatment capacity could be created by operation of reactors in series. The diversity of the microbial community responsible for destruction of especially SCN were shown to be far more extensive than initially expected. Initial molecular characterisation of the microbial community selected for 185 representatives of bacterial 16S rRNA genes, of which 106 non-identical genotypes were sequenced. In contrast, for the reactor containing solids, only 48 representatives were selected and 30 genotypes were sequenced. Bacteria implicated in SCN destruction in the reactor containing suspended solids were members from the genera Bosea, Microbacterium and Thiobacillus. In the absence of solids, members capable of SCN destruction were identified from genera including Thiobacillus and Fusarium. High-throughput genome sequencing, followed by sequence assembly confirmed the dominance of Thiobacillus spp. Metabolic predictions indicated the autotrophs, gaining energy from the oxidation of reduced sulphur intermediates produced during SCN destruction were the dominant community members. The potential for ammonium oxidation and denitrification within the microbial community was identified during analysis of the metabolic potential, based on the metagenomic sequence data. These would be required for complete remediation of wastewater. The data generated during the research led to the development of a conceptual model to describe the evolution of system performance. Following inoculation with planktonic culture the SCN destruction is performed by the planktonic microbial community. An increased residual SCN concentration results in floc formation and the colonisation of reactor surfaces by attached biofilm. A concomitant decrease in planktonic cell concentration was observed, while SCN destruction rates increased. The extracellular material provided a matrix for biomass retention, resulting in high cell concentrations, and provided some protection against high SCN concentrations by providing a barrier to mass transfer. The attached biofilm developed to the point where overall SCN degradation rates may become limited by reduced oxygen penetration. The research presented in this thesis has been used to inform the design and operation of the ASTERTM process at commercial scale, specifically with respect to the benefits of attached biomass and the demonstration that the process can be used in the presence of suspended solids. The latter has been particularly important in applications where the available footprint is constrained.
- ItemOpen AccessBeyond bottlenecks: expression of complementary enzymes and permeabilization of cell membranes to improve performance of CYP153A6 in Escherichia coli whole cell biocatalysis(2022) White, Bronwyn Elizabeth; Harrison, Susan; Smit, Martha S; Fenner, CarynCytochrome P450s are a diverse and versatile class of enzymes, able to carry out oxyfunctionalisation of hydrocarbons with exceptional regio- and stereospecificity. They show great promise within the medical and fine chemical industries, and could potentially be applied for the activation of linear alkanes into platform chemicals. However, bulk chemicals require higher titres, rates and yields than fine chemicals to be economically viable. Application of these enzymes in vitro is hampered by low biocatalyst stability, inhibitory upstream processing costs, and the need for costly cofactor supplementation. In vivo operation can overcome these issues, but brings its own set of limitations. This thesis presents research on the in vivo biotransformation of n-octane to 1-octanol by CYP153A6 and its natural redox partners, ferredoxin (Fdx) and ferredoxin reductase (FdR), from Mycobacterium sp. HXN-1500. These three proteins were heterologously expressed in Escherichia coli BL21DE3. Experiments were carried out at the millilitre scale, using resting cells. Potential limitations for the whole-cell biocatalyst include insufficient expression of the CYP153A6 or its redox partners, leading to a lack of biocatalytic “active sites”; lack of oxygen for biotransformation and cell metabolism; poor transport of n-octane from the organic phase into the cytoplasm, leading to limitations in substrate; toxicity resulting from the accumulation of substrate or product; and insufficient availability of NADH cofactor. With regards to heterologous enzyme expression, the decision was made to focus on CYP153A6 expression levels (with FdR and Fdx expression levels forming part of a related project). To investigate the effects of intracellular concentration of active CYP153A6, it was necessary to find a reliable method of varying P450 expression. The haem precursors δ-aminolevulinic acid (δ-ALA) and ferric chloride were applied during the growth and expression stage, and it was found that P450 expression could be optimised by varying the concentration of these chemicals, with co-addition of the two chemicals having a synergistic effect on expression levels. However, higher intracellular levels of active CYP153A6 did not lead to increased biotransformation rates or product titres in biotransformations carried out with whole cells. This demonstrated that the biotransformation of n-octane was not limited by the availability of active CYP153A6, even at concentrations as low as 0.3 µmolP450.gDCW-1 . Furthermore, these results suggest that optimising the expression of CYP153A6 in whole-cell biocatalysts does not equate to maximising its expression. Aminolevulinic acid constitutes a substantial portion of media formulation costs in the proposed whole-cell process, and these findings allow for more efficient application of this and other precursor chemicals. Based on an analysis of the literature, it was hypothesised that oxygen was unlikely to be a limiting substrate, due to the relatively low biotransformation rates being recorded, as well as the reduced oxygen demand of resting cells. Nevertheless, two sets of experiments were carried out to assess the effects of oxygen supply to the system. In the first set, the quantity of oxygen in the headspace was increased by enlarging the vials, while keeping cross-sectional area constant. In the second set, the headspace was replenished at regular intervals to increase the average concentration of oxygen in the headspace over the course of the biotransformation. In both sets of experiments, the differences between lower-oxygen vials and higher-oxygen vials were neither substantial nor consistent. This demonstrated that biotransformations by resting cell cultures at a density of 4 – 5 gDCW.L-1 were not limited by the quantity of oxygen available. The transport of hydrocarbons across the cell envelope affects the availability of n-octane substrate in the cytoplasm, and impacts on the in situ extraction of 1-octanol product. Thus, hydrocarbon transport has the potential not only to be a rate-limiting step, but also to control levels of cytotoxicity. To explore the significance of cross-membrane transport, cell membranes were permeabilised via the application of chemicals or via mechanical breakage, and biotransformations were also carried out using cell free extract. To investigate cofactor limitations, the NADH regeneration rate of E. coli was enhanced through over-expression of glycerol dehydrogenase (GLD) and supply of glycerol as sacrificial substrate. These interventions were implemented both separately and concurrently, and the results compared to biotransformations by low-GLD, non-permeabilised cells. The interactions between substrate transport and cofactor regeneration were found to be complex In cultures of intact or chemically permeabilised cells, strains with enhanced NADH regeneration capacity produced equal, and sometimes lower, titres than control cells carrying only the empty vector. The co-expression of GLD tended to reduce product titres in high-density cultures, by exacerbating instability of the whole-cell biocatalyst, which was already substantially reduced relative to low-density cultures. In low-density cultures, co-expression of GLD was not harmful, but it did not lead to any notable improvements in product formation rates. However, across numerous cultures, enhanced cofactor regeneration capacity correlated with enhanced CYP153A6 stability. Furthermore, in many low-density cultures, the co-expression of GLD provided marginal improvements in overall stability of the whole-cell biocatalyst when biotransformations were extended beyond 48 h. Thus, under the conditions tested, n-octane biotransformation rates were not limited by the availability of NADH. At the same time, the results suggest the possibility of harnessing enhanced cofactor regeneration to extend biotransformation times, presumably by improving the overall metabolic state of the whole cell – but also show that such a strategy has a ‘tipping point', related to cell density, beyond which the presence of additional dehydrogenase becomes harmful. The application of Triton X-100 or Polymyxin B substantially increased initial product formation rates relative to untreated cells. In low-density cultures, this led to chemically permeabilised cells achieving significantly higher final titres than untreated cells. However, the use of these chemicals in high-density cultures reduced the stability of the whole-cell biocatalyst; biotransformations levelled off rapidly, limiting the maximum titres that could be obtained. The co-expression of GLD was not beneficial in these cases. Conversely, the physical breakage of cell envelopes (via passage through a high-pressure homogeniser) improved product titres and space time yields relative to unpermeabilised cells, provided strains were co-expressing GLD. This clearly demonstrated the importance of balancing the supply of alkane and cofactor, with the expression of additional GLD compensating for reductions in metabolic capacity associated with the physically broken membranes. Mechanically permeabilised cells and cell free extract remained stable over extended periods of biotransformation, even though culture densities were high in these cases. Due to their capacity to maintain initial reaction rates at high cell density, mechanically permeabilised cells produced the highest final titres of any system tested in this study. If GLD was not co-expressed (or if CYP153A6 and GLD were expressed in separate cells), the biotransformation rates of mechanically permeabilised cells were substantially reduced. These results identified cross-membrane transport of hydrocarbons as the key limitation in the biotransformation of n-octane by whole-cell biocatalysts. Supply of substrate was a rate-limiting step in the reaction (hence the increase in product formation rates in low-density cultures of permeabilised cells). The degree of cell permeabilizationalso affected how the cell responded to substrate and product toxicity (hence the stability of high-density cultures of mechanically permeabilised cells). However, cofactor regeneration rates quickly became a limiting factor if the cofactor pool was disrupted (as was the case when the cell envelope was physically broken). To improve cross-membrane transport without destroying the cell membrane, E. coli strains were engineered to express AlkL, a passive membrane transporter protein that evolved to facilitate the growth of Pseudomonas putida on alkanes. The expression strategy included manipulating the supply of haem precursors δ-ALA and FeCl3, in a drive to achieve comparable expression of CYP153A6 in strains with and without AlkL. Even though the AlkL expression strategy was not fully optimised, cells co-expressing the transport protein demonstrated 50 – 100 % improvements on their yield on biomass after 48 h. These results confirmed the importance of overcoming the cross-membrane transport limitation. Overall, the findings highlight the importance of balancing reaction components. Cross-membrane transport of hydrocarbons emerged as the key limitation in biocatalysis by CYP153A6 in whole cells. This limitation could be alleviated by permeabilising the cell. Mechanical breakage of the cell envelope was particularly effective, but this in turn impacted negatively on the cell's ability to maintain its cofactor pool, necessitating the co-expression of glycerol dehydrogenase to boost NADH regeneration rates. In this regard, the membrane channel protein AlkL showed great promise, allowing cells to increase their rates of product formation even while relying on their native cofactor pool. These performance enhancements were achieved despite low intracellular concentrations of active CYP153A6 in cells co-expressing AlkL. Furthermore, in biotransformations with intact cells, intracellular concentration of CYP153A6 could be substantially reduced (via a reduction in the concentration of haem precursor), without any negative impact on biotransformation being observed. Increasing the cell density of biotransformation cultures allowed for rapid product accumulation, but substantially reduced the stability of the whole-cell biocatalyst. This instability correlated closely with the volumetric rate of product formation, suggesting that the effect was linked to reaction or product toxicity. Isolated improvements in enzyme expression, substrate supply, cofactor regeneration, or biomass loading did not translate into improved biocatalytic performance overall. This demonstrates that the creation of a useful P450-based biocatalyst will depend on the simultaneous optimisation of multiple components. In the field of biocatalysis, the focus tends to be on the characterisation and modification of the enzymes themselves. While this enzymatic understanding is crucial, the equally important task of understanding host physiology is often not explored in the same exacting detail. Unlocking the full potential of biocatalysis within the bulk chemical space requires an understanding of the cell architectures within which the enzymes of interest must function. This study has served to shed light on the links between cells, enzymes, and reactants – and on how much there still is to understand when it comes to whole cell performance.
- ItemOpen AccessCharacterizing the potential environmental risks of South African coal processing wastes(2018) Moyo, Annah; Broadhurst, Jennifer; Amaral-Filho, Juarez; Harrison, SusanThe environmental impacts of coal processing wastes are a challenge in South Africa as large amounts of coal wastes are produced annually, pegged at 60 million tons per year according to Eberhard (2011). Whilst the fossil fuel-based industry is in decline globally, coal is likely to remain the dominant source of power in South Africa. The major environmental impacts reported in several studies are water pollution and soil quality degradation due to acid rock drainage (ARD) and its associated elevated levels of elements and salts. Several studies have shown the environmental performance of the wastes to be dependent on the geochemical properties of the wastes. Owing to the complex nature of coal wastes, their characterisation using tools developed for hard rock ores is associated with inconsistency and uncertainty. As a result, the South African coal processing wastes are poorly characterized and the associated risks not well understood. This study investigates the reliability of relevant characterisation techniques and interpretation of characterisation data in terms of the environmental risk potential of coal wastes. The outcomes of the study address some of the uncertainties and deficiencies arising from the current characterisation tools and evaluate potential environmental risks posed by coal processing wastes. Laboratory-scale characterisation of the physio-chemical properties and of ARD and elemental risk potential of two ultrafine coal waste and one discard waste sample were conducted. Evaluation of accuracy and repeatability of selected analyses was conducted on a certified coal standard. The selected analyses tested for accuracy and repeatability were total sulphur analysis by Leco and Eschka methods in addition to elemental analysis by wavelength dispersive x-ray fluorescence (WDXRF), inductively coupled plasma mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectroscopy (ICP-AES) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The ISO 157:1996 and ACARP C15034 protocols for assessment of sulphur forms were also compared and evaluated for precision using the coal standard and coal waste samples. Conversions of the sulphur species under static ARD tests were also studied to understand the sulphur species behaviour and implication on ARD potential. The mineralogy of the coal wastes was evaluated from a quantitative evaluation of minerals by scanning electron microscopy (QEMSCAN) and quantitative x-ray diffraction (QXRD) analysis. In addition, conventional net acid generating (NAG) and acid-base accounting (ABA) static tests were enhanced through extended boil NAG tests to assess the organic acids effect on the NAG capacity. The static tests were validated by theoretical ARD calculated from mineralogy as well as biokinetic shake flask tests which gave the timerelated acid generating behaviour of the coal waste samples. Sequential chemical extractions combined with a simple score and ranking protocol were subsequently used to evaluate the potential water and soil-related risks associated with environmentally available elements and salts in the coal wastes. The results showed both the Leco and Eschka methods to be highly precise (±0.01-0.03 % standard error) but the Leco was more accurate (±3.1 % compared to ±12.5 % relative standard error (RSE)). The total sulphur content of the coal processing waste was less than 2 %. The ISO157:1996 and ACARP C15034 protocols gave comparable and slightly different results but the latter was more precise in sulphate analysis. Furthermore, the ACARP protocol could differentiate the acid forming sulphates from the soluble sulphates giving a better theoretical maximum acid producing potential. The sulphur species from the two chemical methods and QEMSCAN mineralogy showed 52-61 %, 12-26 % and 21-43 % to be sulphide, sulphate and organic/low-risk sulphur respectively. The conversion of the sulphur species showed that partial solubilisation of sulphides in ANC and partial conversion of organic/low-risk sulphur under NAG tests can cause an over or underestimation of ARD potential. The static ARD tests has shown the Witbank coal discards sample to be potentially acid forming (PAF) (9.2-25.9 kg H2SO4/Ton), Waterberg coal slurry to be non-acid forming (NAF) (-68.6 to -46.8 kg H2SO4/Ton) and Witbank coal slurry to be uncertain (-12.1 to 9.9 kg H2SO4/Ton). The extended boil NAG tests showed organic acids effect on the Witbank coal slurry likely caused an overestimation of the NAG capacity. Validation of the static tests by biokinetic tests and ARD calculated from mineralogy classified both Witbank samples as PAF and the Waterberg sample as NAF. The results also showed the net acid producing potential of the coal wastes to depend on the mineralogy of the samples. The elemental results showed WDXRF and LA-ICP-MS analysed most of the elements accurately within ±10 % RSE and that a combination of techniques provides more reliable and accurate results. The analyses showed the coal waste to contain significant amounts of environmentally sensitive elements like Cr, As, Mo, Sb, Se. The ranking and scoring of potentially available elements under oxidising leach conditions evaluated Fe in Waterberg coal slurry and Witbank coal discards to pose high risk in drinking water while S (as sulphate), Pb, Sb, Mn, As, Al and Hg in the three samples pose moderate risk. This case study evaluated the accuracy and precision of commonly used analytical techniques and applicability of risk evaluation protocols for coal processing wastes. The research outcomes underlined some factors that cause uncertainty and inconsistency with the evaluation of ARD potential of coal wastes. The findings highlighted the need to validate and complement the characterisation data using various tools and risk evaluation protocols to overcome specific limitations. The results also indicated the coal wastes have the potential to cause environmental impacts from ARD and elevated concentration of elements and salts, thus providing a basis for designing and implementing waste management strategies which minimise these risks. The mineralogy and elemental composition of coal wastes showed enrichment of elements and presence of potentially usable and economically valuable constituencies for future studies on value recovery. Characterisation of coal processing wastes for air pollution impacts is recommended for future studies as well as a study of ARD behaviour under continuous flow systems to more closely represent the conditions in dump disposal scenario.
- ItemOpen AccessComparison of non-reactive solute transport models for the evaluation of fluid flow in leaching beds(2023) Odidi, Michael Dumisane; Harrison, Susan; Fagan MarijkeHeap leaching is a hydrometallurgical process used for the extraction of minerals within complex and typically low-grade ores. An important factor in the mineral dissolution process is the contact efficiency between the irrigation fluid (lixiviant) and the targeted mineral, which is influenced by both the solid and fluid properties of the system. One of the principal challenges related to the contact efficiency is preferential flow, cited to result in low extraction rates and in extreme cases, heap failure. Preferential flow reveals itself on two scales in drip irrigated heaps, referred to as the bed and solution scale. The bed scale takes a macro view of the heap and deals with uneven wetting profiles characterized by the presence of wet and dry sections. Linked to this is capillary suction effects which play an important role in the establishment of fluid flow profiles within the heap. The solution scale focuses on preferential flow behaviour in the wetted sections of the heap characterized by variations in the residence times of fluid elements. Such variations produce fast flowing, slow flowing and stagnant solution pools. Therefore, ideal solution flow behaviour in a heap result in uniform wetting at the bed scale and plug flow behaviour with similar fluid residence times at the solution scale. Though bed scale preferential flow can be visually observed, diagnosing symptoms at the solution scale typically requires the generation, analysis, and modelling of residence time distribution (RTD) curves. The main objectives of this study were to firstly explore the effects that important material and fluid properties have on the steady state fluid flow profiles in drip irrigated beds characteristic of those used in laboratory scale column leaching studies and quantified using step tracer tests. This is based on the underlying principle that the movement of inert tracer molecules within an irrigated bed at steady state is identical to the solution flow path within the bed. The second objective was to test the ability of nine empirical and semi-empirical solute transport models to adequately fit the generated flow profiles or RTD curves. The third was to compare the magnitudes of the quantified model parameters to ascertain the level of solution scale preferential flow in the different beds and determine the adequate level of model complexity needed to describe their flow profiles which facilitates identifying the controlling variables within the system. Properties of the loading material that were identified as potentially most impactful with respect to heap operations were: porosity, wettability, particle shape and size distribution. Therefore, four different materials with unique inherent characteristics were selected for this study: glass beads (GB - spherical and non-porous), glass shards (GS - irregularly shaped and non-porous), greywacke (GW - irregularly shaped, porous, and highly wettable) and malachite ore (MO - irregularly shaped, highly porous, low wettability and non-uniform composition). In terms of fluid properties, current models have already established a correlation between the concentration of dissolved chemical species within a fluid and its viscosity. This was relevant due to the variety of lixiviant compositions used in previous heap hydrology studies and the fact that the composition also varies with time within a reactive heap. To study the effects of this parameter on the establishment of flow profiles, glycerol was used as a viscosity modifier to formulate solutions with viscosities ranging from 0.8 to 2.2 cP, representative of the range experienced in heap leaching systems due to varying SO4 2- concentrations. The packed beds were characterized using their bulk densities, void age, total liquid holdups, total bed saturations, 24-hour drain-down moisture percentages, solution and tracer breakthrough times. Beds containing both narrow and mixed particle size fractions were tested. The nine solute transport models used for RTD modelling included three compartmental model configurations (CM-1, CM-2, CM-3) and the tanks-in-series (TIS) model, all empirical in nature. The five semi-empirical models selected were the advection-dispersion (AD), piston exchange (PE), piston exchange-diffusion variant (PE-D) and piston dispersion and exchange (PDE) models. A novel model formulation called the piston dispersion and exchange-diffusion variant (PDE-D) model was also coded and tested, which incorporated both the longitudinal dispersion coefficient as well as a diffusional flux mass transfer mechanism. The CM-2, AD and TIS were mono porosity models assuming all solution volumes within the beds were actively flowing which limited their ability to account for solution scale preferential flow. The CM-1, CM-3, PE, PE-D, PDE, PDE-D models were dual porous, accounting for the presence of either dead or stagnant solution volumes. The model parameters used to account for preferential flow in the RTD profiles included: the fraction of dead to total solution volume, dynamic to total saturation fraction, number of TIS, ratio of parallel continuously stirred tank volumes, longitudinal dispersion coefficient, overall mass transfer coefficient and maximum diffusional pore length. The cumulative RTD responses for the bed systems composed of narrow size fractions were noticeably impacted by particle size. These systems displayed symptoms of increased solution channelling behaviour at steady state, based on their relatively short tracer breakthrough times, as the average particle size was increased from ∼1 to 15 mm. The incorporation of semi-empirical models which could account for stagnant volumes. The main comparative modelling results across all systems studied showed that the PDE and PDE-D models were the top performers, based on a model fit analysis. This was due to their dual porous nature and relatively higher levels of complexity. The mono porosity models (CM-2, TIS and AD) performed the worst due to their inability to account for isolated and immobile liquid volumes. However, when 10 mm during agglomeration will aid in increasing the fraction of mobile (actively flowing) liquid within the heap due to the increased presence of macro voids. High levels of particle porosities (>2.5 m2/g) will also aid in this aspect. This is proposed to be due to greater void network connectivity with an increase in porosity facilitating better mass transfer. These insights were obtained through the analysis of experimentally generated data and model simulations. They have provided a better understanding of the movement of fluid molecules within drip irrigated beds, which is essential for improved leaching performance. Building on this, the next step is to consider the effects of scale up and reactive systems on both empirical and simulated data.
- ItemOpen AccessDesign of the aerobic hail reactor - towards improved energy efficiency(2022) Shaer, Gianluca Sasha Salvatore Ganter; Harrison, Susan; Tai, Siew; Fagan-Endres, MarijkeThis dissertation presents the results of an investigation into the design of a novel low aspect ratio reactor, dubbed the HAIL (horizontal air-injected loop) reactor. Current industrial high cell density aerobic reactors for cultivation of bacteria and yeast are typically either stirred tank reactors (STR's), bubble column reactors (BCR's) or airlift reactors (ALR's). These systems can attain high mass transfer rates and short mixing times; however, their energy efficiency remains a concern. Many studies have attempted to further optimise these reactors, but they are ultimately limited by their high aspect ratios. These lead to large pressure heads that the air compressor needs to overcome on sparging, contributing significantly to energy costs. Low aspect ratio (LAR) reactors, such as the wave bag, orbital shaker and raceway reactors offer an alternative to these systems, providing superior energy efficiency for both mixing and aeration. However, each has core issues preventing their usage in high cell density aerobic culture. Their maximum mass transfer coefficient is typically too low to support high cell density cultures. Additionally, these reactors tend to have poor scalability, making them unfeasible for large scale industrial usage. To overcome these challenges, the HAIL reactor makes use of a tubular loop design. The anticipated benefit of the loop design was that it forces the air to travel the length of the reactor before leaving the system, enabling significant surface aeration and residence time in the reactor. These both impact the mass transfer coefficient. Additionally, the loops can be stacked upon one another, overcoming the scalability issue. The reactor would also be energy efficient based on its LAR. To establish target performance ranges, a literature review on the gas-liquid mass transfer coefficient, mixing time and efficiency of current low and high aspect ratio (HAR) reactors was conducted. This was supplemented with experimental results (including mass transfer coefficients, cell density and viscosity) from the fed-batch STR cultivation of Saccharomyces cerevisiae, an easy to work with highly aerobic yeast. A fed-batch feeding profile was developed for this. To better compare reactor performance, a term was introduced called the mass transfer energy efficiency, with units m3 ∙h -1 ∙W-1 , obtained via the quotient of the kLa and the power input per unit volume. The literature mass transfer energy efficiency ranges for the STR, BCR and ALR were found to be 0.022-0.236 m3 ∙h -1 ∙W-1 , 0.084-0.317 m3 ∙h -1 ∙W-1 and 0.142-0.493 m3 ∙h -1 ∙W-1 respectively, with maximum kLa values ranging up to 1000 h-1 depending on the power input. Mixing times for these systems differ depending on scale and configuration, ranging from below a minute up to 20 minutes. Experimental fed-batch and sterile water systems had efficiency ranges of 0.044-0.245 m3 ∙h -1 ∙W-1 and 0.059-0.285 m3 ∙h -1 ∙W-1 respectively, with a maximum kLa of 240 h-1 and 226 h-1 . Based on cellular growth results, the theoretical minimum kLa required was calculated as 372 h-1 . The most notable literature efficiencies for LAR reactors were held by the travelling loop, raceway, and wave reactors with ranges of 0.286- 0.295 m3 ∙h -1 ∙W-1 , 0.034-0.867 m3 ∙h -1 ∙W-1 , and 0.112-0.742 m3 ∙h -1 ∙W-1 . For the wave and travelling loop reactors, mixing times below a minute were attainable. A 6.2 L proof-of-concept and 31.4 L laboratory-scale prototype of the HAIL reactor were developed. In the proof-of-concept prototype, preliminary studies were carried out on the impact of sparger depth and angle on circulation time. Using the laboratory-scale system a range of sparger designs, including different angled jets, outlet areas and a circular sparger design, were investigated. The circular sparger design was found to be the ideal sparger type. A mixing time of 7-19 minutes depending on the power input was found for the 31.4 L configuration. The power efficiency range determined was 0.120- 0.281 m3 ∙h -1 ∙W-1 ; however, the calculation used to determine this is an underapproximation. The maximum kLa of 13.84 h-1 is an order of magnitude (between 10 and 100) lower than the values that can be obtained in HAR reactors for industrial aerobic culture. It was found that HAIL reactor performance did not change substantially with an increase in viscosity from 1 to 1.4 cP. The HAIL reactor did not compete with existing low and high aspect ratio reactors in its current configuration in terms of mass transfer. Additional research on the design is recommended to enhance gas - liquid contacting and associated mass transfer. These ongoing studies will enable the potential relevance and application of the novel reactor to be determined.
- ItemOpen AccessDesulphurisation flotation for the selective removal of pyrite from coal discards using microorganisms(2018) Msipa, Winfull Jaconia; Harrison, Susan; Fagan-Endres, MarijkeMineral beneficiation processes such as base metal and coal mining produce large amounts of waste rock and coal discards that contain significant quantities of sulphide minerals with Acid Rock Drainage (ARD) generating potential. ARD is caused by the exposure of sulphide minerals, primarily pyrite (FeS2), to both water and oxygen, and microorganisms. This is a naturally occurring process, but the exposure of the sulphide containing mining wastes greatly accelerates ARD formation. Thus, ARD is a major issue associated with inactive mines, waste rock dumps and tailings impoundments, which over time presents a major environmental risk. The desulphurisation of coal discards, mine tailings and finely divided waste rock prior to their disposal has been proposed as a method of preventing ARD formation. This involves the selective separation of residual values from the waste rock, followed by selective separation of sulphide minerals – especially pyrite – from the residual waste material using a two-stage froth flotation to obtain a values stream, a low volume sulphide-rich concentrate that can be easily contained, and a high volume benign tailings fraction that can be safely disposed of. The technical feasibility of this two-stage process has been demonstrated; however, the cost of the flotation reagents used in this process are particularly high in comparison to the other operating costs, contributing as much as 75% of the operating costs for desulphurisation of coal fines. Furthermore, apart from being expensive, many of the inorganic flotation reagents are relatively toxic and could be hazardous to the environment due to their slow degradation rate. Microorganisms and their metabolic products have been identified in literature as potential reagents that can be used in the selective separation of sulphide minerals using froth flotation. Just like conventional chemical flotation reagents, the microorganisms assist separation through surface chemical alterations that modify a mineral’s hydrophobic properties, thus facilitating bioflotation. The aim of this study was to investigate the prevention of ARD formation through the desulphurisation of pyrite-containing coal discards and base metal hard rock samples using microbial cultures as alternative bioflotation reagents. In this study the feasibility of using P. polymyxa, R. palustris, R. opacus, B. subtilis, and B. licheniformis as biocollectors for the removal of pyritic sulphur in the second stage of the two-stage desulphurisation froth flotation process was investigated. Microbial screening tests were performed using a pyrite concentrate to assess each microbial culture’s affinity to pyrite and their ability to float the mineral in a batch flotation cell. Attachment experiments and batch bioflotation tests were carried out to screen for a microbial culture that showed potential. Following attachment experiments at pH 4 and pH 7, all microorganisms except B. licheniformis exhibited attachment to pyrite. The level of attachment was different for each microbial culture. P. polymyxa had the highest percentage attachment of 95.6 ± 1.0 % at pH 4 and 97.1 ± 0.7 % at pH 7 after 20 minutes of interaction. Subsequent results from the pyrite-only bioflotation tests revealed that R. opacus, R. palustris and B. subtilis did not affect the floatability of pyrite. P. polymyxa, however, showed a significant effect on the floatability of pyrite, achieving a cumulative mass recovery of 7.0 ± 0.42 % at pH 4 and 81.3 ± 0.4 % at pH 7. Zeta-potential tests revealed that P. polymyxa had the most neutral net surface charge across the pH range tested, while the other microorganisms had a large net positive or negative charge. Based on this result, it was deduced that the hydrophobicity of P. polymyxa as a consequence of its near neutral surface strongly made it seek out a surface to attach to rather than remaining suspended in water. Hence, P. polymyxa was chosen as the bio-collector candidate for the bioflotation separation of pyritic sulphur from coal discard and base metal hard rock samples. Despite the positive batch pyrite bioflotation tests, P. polymyxa was not successful for the flotation of pyrite from the coal discards nor did it upgrade pyritic sulphur to the concentrate, with the bioflotation results not significantly different from the negative control without collector. P. polymyxa did affect the floatability of the base metal hard rock, achieving cumulative mass recoveries comparable with the chemical control using PAX. However, there was no significant upgrade of pyritic sulphur content, with the biofloat achieving 22.6 % total sulphur in the concentrate which was significantly less than the 66.4 % total sulphur recovered with PAX. The study thus yielded positive results from fundamental studies of P. polymyxa’s ability to enhance the flotability of pyrite. However, tests using actual samples were less successful. Although P. polymyxa enhanced the floatability of the base metal hard rock, it did not achieve the aim of obtaining a low volume sulphide-rich concentrate as the PAX did. Recommendations for the continuation of this work include contact angle measurements and FT-IR spectroscopy to better understand the effects of P. polymyxa attachment, as well as performing a kinetic study on the growth of P. polymyxa alongside adaptation of the microbial culture to a pyrite mineral concentrate in order to test if this can improve selective flotation of the desired mineral owing to modified surface properties.
- ItemOpen AccessDeveloping quantitative approaches to determine microbial colonisation and activity in mineral bioleaching and characterisation of acid rock drainage(2019) Makaula, Didi Xhanti; Harrison, Susan; Fagan-Endres, Marijke; Huddy, RobertColonisation of mineral surfaces by acidophilic microorganisms during bioleaching is important for accelerating the extraction of valuable metals from mineral sulfide ores of varying grades through biohydrometallurgy. It also influences acid formation and mineral deportment from sulfidic waste rock generated in mining processes and is key to its comprehensive waste rock characterisation for acid forming potential. This study assesses mixed mesophilic microbial interactions with, and colonisation of, pyrite concentrates and pyrite bearing waste rocks. The assessment of these interactions was carried out in this study in a synergistic qualitative as well as quantitative manner, with a particular focus on heap bioleaching for metal extraction and on disposal of waste rock, the latter through the case of characterisation of ARD generation potential. Using the tools developed, both the course of colonisation and development of metabolic activity with time of colonisation, as well as their correlation with leaching performance were studied. Furthermore, specific operating parameters such as ore grade and irrigation rates were explored. Finally, the application of this knowledge in a characterisation study was explored. To achieve the set of tools required for this study, two quantitative techniques were refined to characterise these microbial-mineral interactions. In the first, an isothermal microcalorimetric (IMC) method was developed and optimised to determine microbial colonisation of mineral surfaces quantitatively as a function of surface area (m-2 ). Three IMC configurations were considered: colonised pyrite-coated beads submerged in fresh media; beads submerged in cell free leachate; and beads in an unsaturated bed, each in the IMC vial. The highest heat output was measured in the unsaturated bed (263.3 mW m-2 ). The consistency of heat produced by the colonising microorganisms was determined through reproducibility studies. Using IMC, chemically and microbially facilitated pyrite oxidation rate studies were performed on unsaturated beds with varying surface area loadings, correlating to varying bead number. Results obtained showed similar normalised oxidation rates per surface area across the surface loadings. However, with more microbially colonised surface area loaded, the maximum heat generated was reached more quickly. This suggested that there was reagent (possibly O2) limitation in the system, which restricted microbial activity and its associated heat generation. Reagent limitation in the system was tested and validated through varying the O2 availability in the IMC vial by air displacement with CO2 and N2 gas, with the systems containing less O2 showing limited activity. Collectively the data showed that high activity, facilitated microbially, was achieved in unsaturated systems in a reproducible manner. Secondly, oxidation rates were determined and O2 limitation in the system was overcome. This then fundamentally informed the determination of activity from microbial-mineral interaction, using IMC, as a function of surface area. Secondly, a detachment protocol developed at UCT to recover microbial cells from surfaces of crushed and agglomerated ore to assess microbial growth rates and distribution in the ore bed, including cells in the interstitial phase and those weakly and strongly attached to the ore surface, was refined to assess colonisation of the finely milled pyrite-bearing concentrate or waste rock coated onto glass beads in continuous flow assays. The detachment protocol was assessed quantitatively by measuring initial and residual microbial activity, as a function of wash number, using IMC, thus providing a new level of confidence in the method. Mineral surfaces were visualised using scanning electron microscopy (SEM) following detachment for qualitative assessment. These data, together with microscopic enumeration of detached cells with increased number of washes, allow refinement of the assay and showed that six washes provided reliable estimation of mineral associated microbial cells. Extracellular polymeric substances (EPS) produced in this process were extracted using crown ether and the capsular bound components analysed. The analysed components included lipids (4.2 %), iron (16.4 %), DNA (26.8 %), and total carbohydrates (28.5 %), which are typical components of EPS. The carbohydrate fraction was further resolved to trehalose (26.2 %), fructose (36.5 %) and galactose (37.3 %) sugar monomers. The analysed EPS components confirmed presence of the EPS secreted by cells colonising the mineral ore or waste rock surface in a flow-through system, and visualised via SEM. The microcalorimetric approach developed together with the refined detachment method were applied to samples from a flow-through mini-column system, used to simulate microbe-mineral contacting in a heap. Here, the colonisation of pyrite concentrate by a mixed mesophilic culture of iron and sulfur oxidising microorganisms was assessed progressively over 30 days. The progression of mineral colonisation in the mini-column system was monitored using a combination of IMC, scanning electron microscopy, detachment method and conventional wet chemistry measurements. We observed an increase in the heat output from the colonised surfaces of pyrite mineral concentrate caused by oxidative reactions facilitated by mineral-microbial biofilm. This confirmed that the attached microorganisms were metabolically active and facilitated ongoing mineral leaching through regeneration of lixiviants. Correlation was shown between number of cells detached from the mineral surface and the heat generated, with a constant heat output per cell observed until day 15 of operation. Thereafter, the measured heat generated per cell increased, suggesting reduced efficiency of cell detachment owing to increasing firm attachment, or the lack in separation of single cells embedded within EPS matrix (clumps observed under light microscope after detachment). Using IMC to quantify the activity of the residual microorganisms on the mineral surface following detachment, it was confirmed that >95% of activity was detached through this protocol, hence the lower detached cell numbers determined following EPS formation were attributed to clumping of the detached cells. This correlated to an increased presence of EPS and was supported by SEM observation. Following the study of pyrite concentrate, colonisation of two pyrite bearing waste rock samples was assessed, with simultaneous establishment of the flow-through mini column biokinetic test configuration that resembles open flow in the waste rock dump. The flowthrough configuration was run alongside the refined UCT-developed batch biokinetic test using suspended mineral. In this study, two pyritic waste rock samples, liberated by milling, were characterised using three biokinetic test approaches: the slurry batch test (BT), the batch test using mineral-coated beads (BT-CB) and flow-through column test with mineral-coated beads (FT-CB). Our results have shown through static tests, solution redox potential and pH analysis that both waste rocks were acid forming. Furthermore, it was demonstrated in the FT-CB system that microbial proliferation on the waste rock surfaces progressed with time such that oxidative exothermic reactions facilitated by the increasing microbial presence on the surfaces were demonstrated using Isothermal microcalorimetry. This study presents and informs the on-going refinement of the biokinetic test through establishment of a flow-through test for ARD characterisation while providing insight into the role of the microbial phase in ARD generation. Microbial-mineral association was assessed under various operating conditions, including two solution flow rates (60 and 4 ml h -1 ) and minerals of varying sulfide content, including a pyrite concentrate (96 % pyrite), a high sulfide waste rock (33 % pyrite) and a low sulfide waste rock (14 % pyrite). Mineral grade impacted the activity of mineral associated microorganisms with higher activities observed on a mineral surface with high sulfide content. The activity measured from microorganisms that were associated with the pyrite concentrate was 827 mW m-2 at a 60 ml h -1 flow rate, whereas activity measured on low and high sulfide waste rock (PEL-LS and PEL-HS) were 293 mW m-2 and 157 mW m-2 respectively operated on the same flow rate. On decreasing the flow rate to 4 ml h -1 , the activity of microbial cells on PEL-LS and PEL-HS were 153 mW m-2 and 146 mW m-2 respectively. This study showed that the growth of microbial cell numbers coupled with metabolic activity is important to facilitate accelerated dissolution of sulfidic mineral surfaces. The rate of oxidation increased in the presence of EPS and thus EPS was further analysed, and its composition was confirmed. Overall, this study contributed to the understanding of microbial colonisation of mineral surfaces in a non-destructive quantitative manner. This study thus demonstrates the ability to measure and track both the growth and activity of microorganisms that are associated with mineral surfaces. This is important as it provides an approach to understanding microbe mineral surface interactions and, therefore, potential strategies to increase microbial colonisation of low-grade minerals that house valuable metals, during commercial heap bioleach processes. Furthermore, the ability to monitor progressive growth and activity of mineral associated microbial communities within a flow-through biokinetic test, as successfully demonstrated in this study, has the potential to significantly enhance current management of mine waste materials and ARD mitigation strategies. Therefore, on-going investigations of progressive microbe-mineral interactions will continue to be valuable both in terms of bioleaching for metal recovery and the mitigation of ARD through effective characterisation of mine waste material.
- ItemOpen AccessDevelopment of a novel bioreactor and systems for suspension cell culture in biopharmaceutical production(2021) Sharma, Rajesh; Tai, Siew; Harrison, SusanMammalian cells offer superior cellular machinery for the production of complex biological products. These cells provide proper post-translational processing machinery for recombinant protein expression to acquire the desired folding for optimal activity. With this advantage, mammalian cells have become the preferred choice for the production of biological products. These cells may grow either attached to a solid surface (adherent cells) or, where adapted, as suspension cultures. In order to grow these cells efficiently in suspension, a bioreactor is therefore required. Bioreactors play a key role in the production of biologicals. Due to the continuous advancement of medicine and the healthcare industry, the demand for biological drugs has increased in the last three decades. This has placed a significant pressure on the biopharmaceutical industry to meet this increasing demand and has become a key driving force behind the need to develop better, safer and more economical bioreactor designs and culture processes. Continuous stirred tank bioreactor is the norm for production of many bioproducts. However, these bioreactors exert high shear forces to cells due to the impeller speed, bubble disruption, and foam formation. In addition, at a large scale, improper mass transfer impairs the performance of cell lines and achieving high cell densities and prolonged viability with correct glycosylation of a secreted proteins is still a challenge during scale-up. Many cell lines, for example Vero cells, which are widely used to produce human vaccines are difficult to adapt into suspension culture. Fixed-bed bioreactors and the use of microcarriers provide an alternative platform for their growth to produce biologicals. However, a high surface area is required to achieve the high cell density which leading to an elevated cost of production (mainly from microcarriers) and ensuing a costly and technically challenging scaling-up of these systems. Other designs such as single-use bioreactors and novel bioreactors based on different operating principles have been explored, but their utilisation is limited from laboratory to pilot scale. Hence, a comprehensive bioreactor design which would be suitable for a large variety of cell lines to produce high-yielding products in suspension culture with the lowest cost and risk in the shortest span of time is still sought. In the current research, two approaches were investigated to address these challenges. Firstly, a horizontal tubular bioreactor (HTB) with a spiral impeller was designed and fabricated for the propagation of suspended mammalian cells with a focus to achieve middle to high cell density by improving mass transfer whilst reducing hydrodynamic shear and energy requirements through surface aeration. The second approach is to test the adaptation of adherent Vero cells into single-cell suspension culture in serum-free media by treating them with an anti-cancer drug, Puromycin amino nucleoside (PAN). The absence of a supporting surface for cell growth (e.g. microcarriers) and serum-free conditions are expected to reduce the cost of manufacturing and to achieve higher productivity of biological production per unit volume of bioreactor. In the first approach, the horizontal tubular vessel was designed to achieve the final volume of approximately 5.0 L. Design of the impeller is a key component that dictates the mixing patterns and mass transfer efficiency. Different geometric configurations were used to design the spiral impeller by considering various parameters such as impeller diameter, the pitch of the blade, pitch angle, height of the blade, the thickness of the blade, clearance efficiency and the position of the heating element. Another important aspect of the prototype design was incorporating an external magnetically-coupled motor drive which assisted in not only in aseptic handling but also reduction in mechanical stress and generation of fewer particles for cleanroom operations. The side plate was designed with the appropriate number of addition ports to allow execution of batches with minimum cross-contamination and for the ease of operation. Thereafter, the engineering characterisation of the HTB was carried out. The performance of the HTB was evaluated for (i) oxygen mass transfer (kLa) through the dynamic gassing-in method, (ii) mixing time and fluid flow by tracer and phenolphthalein method, (iii) minimum stirring speed (Njs) through alginate beads mimicking cell loading and modelling through modifying Zwietering equation, (iv) power consumption through heat calorimetry (temperature method) and (v) shear stress by determining specific death constant (kd) at different impeller speeds. The general characterisation profile of HTB has shown that at high agitation speed, homogeneity and mass transfer efficiency improved while power consumption increases with an increase in agitation speed. The bioreactor operated well at 2 L and 3 L capacity when the impeller is 40 - 90 % immersed in the liquid. The maximum mass transfer coefficient (kLa) of 16 h-1 was measured with a 3 L volume with an impeller speed of 500 rpm. These results are comparable with the other culture systems of the same scale. The HTB was also tested for suitability to grow mammalian cells. Three batches were carried out, of which one was with the Chinese hamster ovary (CHO) cells expressing the somatic angiotensinconverting enzyme (sACE) and the two with plain CHO cells without expressing any recombinant protein. The maximum cell density achieved was of 5.48 x 106 cells mL-1 with plain CHO cells and 4.14 x 106 cells mL-1 with CHO cells expressing sACE with a maximum protein productivity of 465 mg mL-1 . The specific death rate constant of 0.025 (h-1 ) was obtained when impeller speed was increase from 150 rpm (normal) to 300 rpm (induced shear) for 72 h. In this study, CHO cells have been successfully adapted to suspension in serum-free conditions using the slow weaning of serum method and propagated in the HTB whereas Vero cells have been adapted successfully to serum-free media in adherent conditions. Attempt to suspend Vero cells based on literature using the weaning method remains timeous. Therefore, an alternative approach was explored using an anti-cancer drug (PAN) which is known to suppress the expression of integrin (cell adhesion receptors). The expectations from this approach were that the suppression of integrin would allow cells to detach and grow as a suspended culture (Krishnamurti et al., 2001). The results indicated that the anti-cancerous drug may have modulated the structure and function of the integrin which resulted in dislodging of the cells from the surface and form clumps which were viable for a week in suspension culture without increase in cell density. The viability of the cell clumps and few suspended cells were tested by re-seeding of these cells back to tissue culture (TC) flasks in serum-containing media without the presence of PAN. The culture in the TC flask regained confluency in the 2-3 day which confirms the viability of the cells and the likeliness of integrin re-modulating itself in the absence of PAN. As the suspended Vero cells did not grow, they were not tested for growth in the HTB. To investigate the biological activity of these Vero cells, Isothermal microcalorimetry was used to evaluate the heat generation profile of the Vero cells quantitatively before and after drug treatment. The heat flow data (metabolic heat) from the treated and normal cells showed a distinct decrease in the heat generation profile which indicated that the treated cells were viable but not as active as the normal (non-treated) cells. It was evident from the heat flow data obtained for the PAN-treated Vero cells (-0.13 µW) from that of non-treated cells (13.12 µW) and thereafter when PAN-treated Vero cells regrown in serum-containing media, they regain their metabolic activities which were indicated by their heat flow values as positive control (9.30 µW), 100 µg mL-1 (10.12 µW), 200 µg mL-1 (10.18 µW), and 250 µg mL1 (9.15 µW). It is recommended that dielectric spectroscopy and total DNA in the culture from the lysed cells could also be used to measure the bioactivity of the pre and post treated cells and data can be compared with IMC for more insight into the behaviour of the cells It has been concluded that the horizontal tubular bioreactor (HTB) can sustain the middle to high cell density by imparting desired mixing and mass and heat transfer requirements whilst exerting minimum hydrodynamic shear. For the improvement of the design, it is recommended that more batches at different agitation speeds in combination with different airflow rate would further unravel the suitability of HTB to grow mammalian cells and stringently decode the optimum process conditions to achieve high cell densities with extended longevity. Additionally, changes in the pitch of the impeller blades could result in the improved fluid flow profile, mixing and mass transfer while drawing low power input. Subsequently, different modes of operation, e.g. fed-batch or continuous operation are suggested to investigate the suitability of the HTB for integrity, sterility, and possible higher productivities of products. In suspending Vero cells, it has been concluded that the presence of serum-containing media reversibly stimulates the re-modulation of the integrin which poses hurdles in suspending Vero cells by reattaching the cells to the TC flasks. Therefore, it is recommended that a thorough investigation of the drug-treated cell integrin profile is examined through fluorescence-activated cell sorting (FACS) which would give details of the inhibition of the different integrin subunits. This information could form the basis of adapting cell-lines into suspension in a single step, which is otherwise difficult to adapt.
- ItemOpen AccessDevelopment of co-disposal methods for coal discards and fine waste for the prevention of acid mine drainage(2019) Mjonono, Donald; Kotsiopoulos, Athanasios; Harrison, SusanThe dependence on coal ores for energy supply has led to the considerable increase in coal discards (CD) and fine waste (FW) arising from mining and processing operations. These wastes typically contain sulphide minerals, which when oxidised may lead to the generation of acidic and toxic discharge. A deficit of naturally occurring neutralising minerals to counteract this acidic discharge results in acid rock drainage (ARD). Far reaching consequences on water systems, vegetation, people and wildlife ensue as a result. To minimise the environmental burden, the acidic water resulting from the oxidation of sulphide minerals present in wastes from both active and abandoned mines is often treated with alkaline materials and is further processed to remove metals. Indefinite maintenance and operational activities emanate from these treatment processes. Further, accumulating sludge from processing streams presents post-closure liabilities. To reduce the environmental footprint, mine waste management strategies have been developed to minimise the risk of ARD formation and proliferation. In this study, the co-disposal of CD and FW was investigated as a means to prevent the initiation of oxidation reactions at source. The CD fraction is sulphide rich with high acid producing potential but can be effectively utilised to construct structurally stable beds. In these beds, large voids are formed between the particles that facilitate the transport of oxygen and water to the sulphide mineral surfaces. Co-packing FW with sulphide-rich CD provides a sustainable approach to ARD prevention. The FW has a high-water retention capacity and can be used to encapsulate, seal or cap the sulphide bearing mineral surfaces. Apart from providing a physical barrier and decreasing voids, FW typically have low sulphide content and high specific surface area that result in increased release rates of any acid neutralising minerals present in these waste materials. Co-disposal techniques thereby provide a longterm end-of-pipe approach to ARD mitigation that may offset indefinite, resource intensive, treatment options. The co-disposal of CD with FW, however, is challenging particularly at large bed cross-sectional areas, as the incidence of high percolation rates increases. This is attributed to decreased inter-particle contact that emerge in packed beds with high void ratios, decreased packing density and increased susceptibility to deformation. This undesirable packing behaviour impacts negatively on bed stability culminating in particle displacement and increased likelihood for sulphide mineral oxidation. Fine wastes conceal these sulphide minerals by either filling voids between coarse particles or forming covers with capillary barriers and acid-neutralising effects. Consequently, the generation of ARD is inhibited. At increased scale, however, the ARD prevention efficiency of covers is enhanced by increasing the CD to FW proportion to result in a structure with high load and acid buffering capacity. The approach adopted in this study entails developing packing arrangements of co-mingled CD and FW in dry-mass ratios of 3:2 and 2:3, respectively, to improve bed stability and hence prevent ARD formation with scale up. In addition to mixture ratios, improved co-packing of CD and FW is contingent on the material geochemical properties and geotechnical parameters of the resulting packed structure. As such, geochemical analyses were performed to determine the acid producing and neutralising potential of the CD and FW through acid base accounting, net acid generating and biokinetic tests followed by geotechnical assessments. The static test results indicated that the high sulphur CD (2.16% S) was potentially acid forming and the low-sulphur FW (0.84% S) was non-acid forming with high acid neutralising capacity. The co-mingled CD and FW samples (ca. 1.5% S) were deemed uncertain as the net acid producing potential was near zero and the NAG pH was less than 4.5. Accordingly, biokinetic tests were conducted over 120 days to fully understand the acid generating and neutralising rates of the inoculated and uninoculated co-mingled samples. Near-neutral conditions were sustained for prolonged periods (> 90 days) in FW dominant samples (2CD:3FW) after which a transition to acidic conditions ensued. This highlighted the limited role of acid neutralising minerals in sustaining near neutral conditions. As ARD mitigation is contingent on preventing the rapid percolation of water and exclusion of oxygen from sulphide mineral surfaces, means to prevent the rapid depletion of neutralising minerals by either dilution or washout are essential in flow through systems. This can be achieved by decreasing voids to result in increased packing density and improved bed stability. Bed stability was shown to be dependent on several interrelated factors that included the degree of saturation of the particles (water to solid ratio, W/S), CD to FW ratio, packing configuration (layers or blends), and the extent of material compaction (assisted versus unassisted packing approach). These factors were integrated to produce 16 packing arrangements. The efficiency of these configurations was compared using packing density, slump and compressibility tests. Packing densities of ca. 0.8 m3solids. m-3mould coupled with low slump spread values (< 0.391 m) were obtained for wet, unassisted packings of CD-dominant layered and blended arrangements. Comparable packing densities and slump results were obtained for assisted packings of CD-dominant layered and blend configurations under dry conditions. Dry assisted packings of either CD or FW dominant layered systems resulted in ore beds with low compressive strain (< 7%), while in wetted structures, extensions of up to 30% over extended periods (>600 s) were noted. The large compressive extensions and the delay to achieving maximum compressive strains signalled the low particle consolidation and decreased bed stability of unassisted wet packings. As engineered co-disposal approaches are associated with long-term bed stability and hence prolonged ARD prevention, select packings were further analysed to validate their efficacy using kinetic column tests of increasing scale. An acidic feed of pH 2 was continuously introduced to the test columns at a flow rate of 3.5 L.m-2.h-1 to expedite the oxidation process and to assess the efficiency of the packing arrangements for ARD mitigation. Segregated disposal of CD in small scale columns (D = 0.19 m, H/D = 1.12) with inherent large voids allowed unrestricted access of the aqueous oxidants to the exposed sulphide minerals leading to rapid discharge of highly acidic effluent (ca. pH 2). For the wet, unassisted co-packed systems, structural instability was observed with the wash out of FW and subsequent fast effluent discharge rates. With the loss of the neutralising and reactive barrier due to migration, acidic conditions presented earlier in these wet packed beds (after 30 days) than in dry packed beds (after 90 days). The loss in geotechnical stability was more prevalent in blended systems than in layered configurations, with a rapid loss of geochemical stability following soon thereafter, despite similar neutralising characteristics in both packing configurations. In these blended arrangements, non-functional migration of the fine waste particles transpired to result in unhindered access of the oxidants with the acid generating minerals. With dilution and wash out of the neutralising components, acidic reactions dominated. In multi-layered systems, a cascading effect prevailed despite breakthrough in some layers such that a fail-safe condition resulted. Consequently, near-neutral effluent discharge at low flow rates transpired. This further emphasised the importance in preventing the displacement of particles to maintain bed stability in co-disposal prevention strategies. Assisted dry packings of blends and layers were anticipated to result in improved bed stability at large scale. As such, CD dominant blends (3CD:2FW) and FW dominant blend and layered (2CD:3FW) systems were investigated in large scale columns (D = 0.32 m, H/D = 1.12). These columns were similarly exposed to aggressive leach conditions over 120 days. As with the smaller scale columns, the packing efficiency in multi-layered arrangements were higher than for the blends. In the blended systems, evolving geochemical and geotechnical conditions were similar regardless of the CD:FW ratio demonstrating the complexity in achieving homogenously packed matrices at large scale. In multi-layered configuration, bed structural stability was sustained for extended periods as the stress imposed on the packed bed was uniformly distributed across the moisture retaining FW layers and dissipated within the matrix. Correspondingly, particle displacement was minimised, and with the cascading phenomena, ARD was successfully prevented over extended periods. A dry cover system composed of multi-layers of CD and FW is therefore recommended for pilot scale studies. Dry cover systems can be easily constructed and present a cost-effective approach to sustainable mine waste management. Further evaluation of the structural stability of multi-layers at large scale is required as changes in bed geometry, particle size and environmental conditions can alter the dump geotechnical properties and hence geochemical stability.
- ItemOpen AccessExploring the factors at play to make wastewater biorefineries a reality(2019) Verster, Bernelle; Harrison, SusanThis thesis concerns the topic of wastewater biorefineries (WWBR), in which wastewater is not seen simply as a waste stream to be cleaned but as a valuable material flow to be converted into bioproducts, while still meeting discharge limits at the end. To set the scene, similar developing approaches to valorise wastewaters globally are reviewed. Wastewaters in South Africa are reviewed and categorised with regards to their potential to serve as raw material, in terms of their volume, concentration and complexity. Bioproducts possible from wastewater is reviewed and evaluated. The wastewater biorefinery is conceptualised in the context of current wastewater treatment technologies and a set of evaluation criteria is developed. A multi-reactor setup is suggested in which wastewater is used, in series, as substrate by heterotrophic microbes like bacteria, photo-mixotrophic organisms like algae, macrophytes and fungi. Each reactor group is considered in detail and evaluated with regards to its suitability to the wastewater biorefinery, leading to selection of appropriate reactor designs. Stoichiometric mass balances of all unit operations are established, showing the material value flows, and combined to model this multi-bioreactor approach. Subsequently the model is tested against literature data. Finally, the applicability of the wastewater biorefinery concept for certain waste streams is assessed. The thesis contributes to the current body of knowledge in the following ways: 1. Introduction of the concept of the wastewater biorefinery (WWBR) 2. Provision of a potential preliminary guide for classification of wastewaters for use in the WWBR 3. Development of criteria for reactor evaluation for use in the WWBR 4. Development of an integrated model to interrogate bioproduction from wastewater and determine product yields associated with wastewater treatment 5. Creation of new knowledge through the interpretation of the model on different wastewater systems. The wastewater biorefinery is defined as a bioproduction system that integrates multiple unit operations to deliver compliant water as well as a bioproduct or bioproducts. It is approached through the concepts of industrial metabolism and the circular economy. Wastewater biorefineries are shown in this work to be a viable approach to improving resource efficiency while ensuring the better ecological functioning of humans within “greater than human” systems. The work places emphasis on the recovery of bioproducts that conserve molecular complexity but acknowledges that energy production for use on site and in the immediate surroundings is always an important factor in the WWBR. This thesis introduces the need to include a qualitative way to evaluate the complexity of wastewater, in addition to standard classification of volume and concentration of components. Complexity includes both composition of potentially problematic compounds and how unpredictably it changes over time. In this approach, it is preferable to generate three types of products: products of sufficient value to be economically viable; products of variable value with concomitant assimilation of major contaminants; and clean water as a product, typically through multiple unit operations, allowing multi-criteria optimisation. Through this approach, multiple criteria can be met. Function-based products specific to niche industries, particularly those which produced the wastewater of interest, are of substantive interest owing to their streamlined market uptake. This thesis explores the requirements of the products that can be produced from wastewater in a non-sterile context and suggests product groupings that meet these requirements. Products secreted into the bulk volume are difficult to recover, leading preference to biomass associated and intracellular products. The product needs to offer a selective advantage to the organisms producing it to facilitate enrichment through, ecological selection of the microbial consortium with simultaneous cell retention through reactor design and operation. Four groupings of unit operations were reviewed in detail and evaluated with regards to their suitability to the wastewater biorefinery, using a two-part set of evaluation criteria that was developed in this work, considering the reactor design, and its operation. The four unit operations each contribute a specific role to the functioning of the WWBR as a system. It is acknowledged that not all units are commercially important, and that the concept of diminishing returns should be kept in mind. The heterotrophic microbial bioreactor, of which the bacterial biocatalyst is used as a representative example, is helpful for removing a high proportion of the organic carbon. A wide range of commodity products with market potential is known to be produced through heterotrophic microbial systems. Existing heterotrophic microbial reactor systems like the aerobic granular sludge system (AGS) exist that suit the wastewater biorefinery approach particularly well, while activated sludge along with biological nutrient removal (BNR), the most commonly used reactor system in South Africa, is the least suitable to the WWBR. The photo-mixotrophic reactor represented by the algal bioreactor is helpful to scavenge high proportions of nutrients, particularly nitrogen and phosphorus. The algal bioreactor is also known to produce commodity products. Photo-mixotrophic bioreactor systems complement the heterotrophic systems but are unlikely to be the dominant reactor due to land and energy requirements. The macrophytic bioreactor is targeted for polishing the exiting stream in terms of nitrogen, phosphorus and particulates to ensure compliant, fit for purpose water as a product, with a macrophyte-based byproduct. Macrophyte bioreactors, particularly floating wetlands, are promising tertiary systems that should be viewed in conjunction with water sensitive design principles to overcome potential land availability limitations. The solids bioreactor is an emerging beneficiation technology for biotransformation of bio-slurries and the solid phases recovered during WWBR operation to generate products of value, including biosolids. Solids bioreactors have great potential but require more investigation, with key challenges being mass transfer and separation technologies. Operating waste treatment facilities as net income-producing bioprocesses require a mindset change about investment, risk and associated returns. WWBRs require higher capital investment due to the additional process units and downstream processing required and have higher operating costs due to the greater control required during the process and greater number of operators with advanced skillsets. An identification of the relevant product range and comparison between conventional processing routes and those possible from the wastewater is required on a case by case basis, and an overview is given in this thesis. Waste may need to be re-classified to be used as an intermediate by-product or raw material, requiring legal considerations in terms of both the solid waste as per the National Environmental Management Act (NEMA) and liquid waste as per the National Water Act (NWA). The added complexity of reclassifying waste as raw material needs an acknowledgement of institutional challenges such as speaking across department silo’s. In this thesis, a model of these integrated unit operations was developed to generate material inventories across the system. This can be used to evaluate possible scenarios in an integrated context using a generic flowsheet as well as mass balances generated through the model. Three case studies were examined: municipal, abattoir and pulp and paper wastewater. Municipal wastewater was chosenas it represents a complex, dilute, 'suboptimal’ wastewater stream. Abattoir wastewater was chosen as an example of a complex, nutrient-concentrated stream that may be well suited to biological transformation. Pulp and paper wastewater was chosen as an example where the biorefinery concept is already well established, and is a low complexity, low nutrient, high carbon content stream. In considering the above case studies, a number of key learnings resulted. The impact of solids removal was clear and in keeping with existing bioprocessing and wastewater treatment principles of decoupling the hydraulic and solids residence times. Low nitrogen and phosphorus content in the pulp and paper wastewater as compared to the other two case studies indicated the need to conduct integrative studies of the unit operations to determine the most appropriate unit operations across the system. The effect of improving the product conversion yields and product recovery yields were examined, and a surprising result is the amount of nutrients that remain in compliant effluent, due to the large volumes of liquid involved. This leads to the conclusion that while the WWBR is a valuable way to address resource recovery, separation at source and internal process efficiencies are critical to improve overall resource efficiency and environmental protection. With regards to municipal wastewater, which contributes by far the most in terms of volume and nutrients of wastewaters in South Africa from the perspective of reactor design for waste(water) beneficiation, considering the cleaner production principle of separation at source, along with the need to decouple the solid and hydraulic residence times, dry sanitation presents a clear argument for the best WWBR approach. This approach must acknowledge that the transport of the sanitation raw materials is more difficult if hydro-transportation is not available, and needs to ensure operator equity, health and safety, particularly in the handling of the sanitation raw materials. This thesis was developed in conjunction with the Water Research Commission (WRC) project “Introducing the wastewater biorefinery concept: A scoping study of polyglutamic acid production from a Bacillus-rich mixed culture using municipal waste water” (Verster, et al., 2014) and Water Research Commission (WRC) K5/2380 project titled “Towards Wastewater Biorefineries: integrated bioreactor and process design for combined water treatment and resource productivity” (Harrison, et al., 2017). While the project focused on a global and national review on research on wastewater biorefineries and wastewater as a resource, this thesis explores in greater depth the requirements of each of the reactor units and their integration.
- ItemOpen AccessHydraulic Characteristics and Nutrient Degradation Kinetics of a Horizontally Orientated Subsurface Flow Biofilter(2023) Maraj, Kalpana; Harrison, Susan; Winter KevinPolluted runoff from densely populated and poorly serviced informal settlements is a growing issue in South Africa that leads to various health risks and environmental degradation. Surface waters affected by informal settlements are known to display high nutrient (NH3, NO2 - , NO3 - and PO4 3- ) concentrations. These nutrient concentrations are highly variable due to the fluctuations in the type and frequency of human activities occurring in the informal settlements. A decentralised, non-invasive and easy to maintain surface water rehabilitation system that is capable of handling variable inlet nutrient concentrations is therefore necessary in these areas. Engineered nature-based solutions such as horizontally orientated subsurface flow biofilters are a potential suitable remediation measure, as they are cost-effective, scalable and easy to maintain. However, the variable nutrient levels in surface waters affected by informal settlements pose a challenge to achieving consistent nutrient reduction in a system. The nutrient degradation potential of a microbially colonised horizontally orientated subsurface flow pilot-scale biofilter (length: 2 m; width: 0.44 m; depth: 0.7 m) that was packed with stones of 8-11 mm in diameter was assessed in this study. The purpose of performing controlled experiments on the pilot-scale biofilter was to enable data collection for the design of optimum full scale biofilters. Pulse tracer studies at varying flow rates (0.5 -3 L/min) determined that the flow behaviour in the pilot-scale biofilter approximated plug flow; with the extent of plug flow behaviour and degree of mixing in the radial direction increasing with a decrease in flow rate Surface runoff from the Stiebeuel River, contaminated by an upstream informal settlement called Langrug, was used as the polluted water source for the pilot-scale biofilter. Six nutrient degradation studies were carried out on the microbially colonised pilot biofilter with each study occurring over a 10-day period (228 hours). Three of the nutrient degradation studies were carried out at a flow rate of 0.5 L/min and three were carried out at a flow rate of 1.5 L/min. Water from the Stiebeuel River (200 L) was circulated through the system during the batch nutrient degradation studies with samples being taken every six hours. The inlet concentration varied in each study with an inlet concentration range of 8.41 - 24.2 mg/L NH3, 1.06 - 2.30 mg/L NO3 - and 1.45 - 6.82 mg/L PO4 3- being observed. An NH3 reduction of up to 91.8%, total nitrogen reduction of up to 82.4% and a PO4 3- reduction of up to 88.3% was observed. The main biological processes occurring within the pilot biofilter were nitrification and denitrification. An extent of nitrification of up to 91.7% and an extent of denitrification of up to 95.6% were observed in the nutrient degradation studies. Ammonia degradation and orthophosphate removal in the system was described using the double first-order in parallel reaction kinetic model which expresses the reaction kinetics as the sum of first two order reactions. The results of the nutrient degradation studies show that the microbial community in the pilot-scale biofilter system is able to metabolise moderate pulses of nutrients when fresh contaminated water is introduced to the system at varying inlet concentrations. The microbial community is able to survive under nutrient limited conditions. These findings indicate the effectiveness of stone biofilters at degrading nutrients in polluted runoff from informal settlements in a controlled batch experiment.
- ItemOpen AccessImproving an aqueous two-phase process for C-phycocyanin extraction from Spirulina(2022) Hockey, James Temlett; Harrison, Susan; Fagan-Endres, MarijkeBiotechnology and bioprocess engineering have made it possible to expand production of natural compounds, and markets have moved more towards this direction. An example of this is in pigments, where many synthetic pigments have been banned or pulled out of the market due to health concerns, while naturally-derived pigments are growing in popularity. The algal pigment, C-phycocyanin (C-PC) is a blue photosynthetic pigment of the phycobiliprotein family, found in cyanobacteria and red algae. Algal pigments like C-PC have been growing in demand and the market is expected to grow at 5-7 % annually. Phycocyanin has applications as a food and cosmetics dye, a health product with therapeutic uses, and as a diagnostic protein. Various processes have been studied to recover and purify C-PC from cyanobacteria such as Arthrospira platensis, commonly referred to as Spirulina, the most used organism for producing the pigment. The C-PC recovery process includes extraction, recovery and purification steps. One recovery and purification step reported in literature is aqueous twophase separation (ATPS), which is able to produce high purity C-PC with good recovery. At the University of Cape Town (UCT), the Centre for Bioprocess Engineering Research (CeBER) has patented a process for extracting and purifying C-PC from Spirulina using a polyethylene-glycol (PEG) and maltodextrin (MDX) ATPS. This is a less-studied form of ATPS, with most C-PC extraction studies using PEG – salt systems. The PEG – MDX system was studied due to challenges faced with C-PC recovery from the PEG phase. The patented CeBER process begins with cell disruption, a period of leaching into a buffered solution followed by cell debris removal. The C-PC is subsequently purified and recovered by the PEG – MDX ATPS and three ammonium sulfate precipitation stages, and finally dried to powder. The patented process requires optimisation and up-scaling before being commercially applied in industry, the ultimate aim of the greater project. As such, this project aimed to improve the understanding of the overall process for C-PC recovery from Spirulina and, in particular, the ATPS step involved, to improve the ATPS performance. The study also sought to produce cosmetic grade C-PC (purity number of > 1.5), and develop process options and simulations for this production based on a combination of literature and experimental results. The work was conducted in view of future up-scaling. This included study of the leaching step with the Spirulina used in this project as well as refinement and optimisation of the ATPS step. For the latter, phase diagrams for the PEG – MDX ATPS were produced to inform ATPS refinement and improvement before evaluating its performance. The phase diagrams give an understanding of how the phases partition in an ATPS, allowing prediction and optimisation of top and bottom phase compositions. Leaching experiments showed that a maximum C-PC concentration was found after 2 h with the Spirulina powder used without the need for cell disruption, and that the purity decreased slowly over time (using 100 g/L Spirulina powder in 5 g/L citrate buffer at pH 6). This recommended a relatively short leaching time be used compared to previous work using different starting material. The PEG – MDX ATPS phase diagrams produced corresponded well with similar ATPS data found in literature. The PEG molecular weight was tested for the best performance, finding that PEG 10000 performed slightly better than both PEG 6000 and PEG 20000, achieving a C-PC purification factor of 1.21 ± 0.01 at 9 wt% PEG 10000 and 20 wt% MDX, with a recovery of 95.1 ± 7.8 %. However, the PEG – MDX ATPS for C-PC purification gave lower purification factors compared to PEG – salt ATPS studies from literature. A two-stage ATPS was therefore considered, with a PEG – citrate ATPS used before the PEG – MDX ATPS. This aimed to take advantage of the good C-PC selective recovery reported in literature for PEG – salt ATPS systems while still using the PEG – MDX to separate the C-PC from the PEG phase. PEG – citrate phase diagrams were produced; these compared well with those found in literature studies. A screening of PEG molecular weights across both ATPS steps found PEG 4000 to be best, mainly due to the performance in the PEG – citrate stage. Using PEG 4000 in two factorial studies on the impact of PEG and citrate concentrations in the first ATPS, and PEG and MDX concentrations in the second ATPS, response curves for the purification, recovery and concentration of the C-PC in the desired phase were produced. A Statistica (version 13.5.0.17) model was used to predict a local optimum, where the combination of component concentrations produce C-PC at high purify, recovery and concentration. The PEG – citrate ATPS model predicted the best component concentrations to be 11 wt% PEG and 20 wt% citrate, which gave a C-PC purification factor of 1.63 ± 0.28, at a recovery of 95.6 ± 8.0 %. The PEG – MDX ATPS model predicted a 1.43 ± 0.09 C-PC purification factor and a recovery of 86.8 ± 4.7 %, using a composition of 11 wt% PEG and 22 wt% MDX. A combination of experimental results and literature data were used to underpin the simulation of five process configurations for C-PC production using SuperPro Designer (version 9.5), each targeting a cosmetic grade C-PC product or better. These sought to simplify and improve C-PC production using the PEG – MDX ATPS as the core unit procedure. The first simulation was of the original patented process (cell-disruption, leaching, ATPS, precipitation and finally freeze-drying), with the ATPS operation updated with the best case experimental results obtained in this work. The second and third simulations used the newly proposed two-stage ATPS. In the third option ultrafiltration replaced the precipitation steps. Spray-drying replaced freeze-drying as a faster and more cost-effective means of drying C-PC from the second simulation onward. The fourth simulation incorporated a pre-treatment step, using activated carbon and chitosan to adsorb contaminant proteins and purify the C-PC, before using a single PEG – MDX ATPS. The operation and performance of the pre-treatment step were based on literature information. This model used (NH4)2SO4 precipitation as in the original process and required two precipitation stages for final purification. The fifth simulation used the pre-treatment process as in simulation 4, with the second precipitation step replaced with filter-sterilisation, before spray-drying. The two-stage ATPS processes lead to slightly improved recoveries of 39.1 % and 39.5 % for the second and third of process recommendations, respectively. This is above the original process simulated to recover 38.7 % of the C-PC in the crude starting solution. The two-stage ATPS processes also have lower ammonium sulfate usage due to having fewer precipitation stages, in the case of the second process simulation, and due to replacing precipitation wit ultrafiltration in the third process. The fourth process showed a higher C-PC purity of > 4.00, compared to the 3.77 simulated for the original process, and a C-PC recovery of 42.7 % from the crude extract. The chemical consumption was similar to the original process, while decreased amounts of ammonium sulfate were required. This process showed the shortest path time of the five options. Of the process configurations presented, the fifth one is best recommended, since it leads to a short batch-time (24.8 h per 3 batches), fewer process units than the original and the lowest overall chemical usage of the five processes. It also produces higher C-PC purification and recovery with reduced complexity compared to the original process. A C-PC purity of up to or above 4.0 is estimated to be achievable with this process, at a recovery of 47.7 % (using the leached C-PC as the starting point). This process produces a C-PC quality well above cosmetic grade. The trade-off between recovery and purity could be explored to achieve a higher recovery of cosmetic grade C-PC. To summarise, phase diagrams were produced for the PEG – MDX ATPS, and the C-PC leaching was tested on the Spirulina used in this project. This ATPS was then tested before moving on to a two-stage ATPS, using a PEG – citrate stage, for which PEG – citrate phase diagrams were produced, before the PEG – MDX stage. This produced better results, comparable to PEG – salt ATPS studies found in literature. Results from the process simulations done on Superpro Designer then supported the use of a pre-treatment step before a single-phase PEG – MDX ATPS, followed by precipitation and spray drying, based on information from this study and other literature. This could lead to a feasible design for pilot-testing and a novel process for industrial C-PC production. It is recommended that the results of the simulations be tested experimentally in further studies. A thorough techno-economic analysis of the proposed processes is also required. The pre-treatment process based on adsorption requires experimental validation and pilot scale confirmation before being applied in industry. Due to the rapidly growing demand for C-PC, a commercial process capable of producing multiple grades of C-PC, from foodgrade, to cosmetic-and reagent-grade, could be lucrative for business interests involved.
- ItemOpen AccessThe influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6(BioMed Central Ltd, 2013) Olaofe, Oluwafemi; Fenner, Caryn; Gudiminchi, Rama Krishna; Smit, Martha; Harrison, SusanBACKGROUND: Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n-octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). RESULTS: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20mumol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. CONCLUSION: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
- ItemOpen AccessInvestigating red pigment production by Penicillium purpurogenum DSM 62866(2022) Horn, Caryn; Harrison, SusanThe production of a diverse range of pigments with variable properties and application potential highlights filamentous fungi as potential sources of pigments in the consumer-driven move toward natural colourant alternatives in food, cosmetic and nutraceutical products. The majority of natural pigments currently in use are obtained from sources such as insects and plants, including fruit and vegetables. These sources are inherently affected by natural variation and seasonal availability. Fungal pigments can, however, be produced in large-scale processes, under optimised and controlled conditions, with minimal dependence on weather and seasonal raw materials. Penicillium purpurogenum was selected for investigation in this study on the basis of reported pigment production without the co-production of mycotoxins, and the ability to adjust pigmentation through modifying cultivation conditions. Red pigments produced by this organism are of particular interest given the demand for this colour in food and cosmetic applications. Red pigment production by P. purpurogenum DSM 62866 was confirmed on a medium composed of 30 g.L- 1 malt extract and 3 g.L-1 soya peptone (MESP medium), following which the impact of varying cultivation conditions was investigated. Factors considered included cultivation temperature, pH and the application of buffers, and shaking speed during incubation of flask cultures. Conditions identified as beneficial for pigment production and, therefore, applied to 5 L cultivation in a benchtop bioreactor, were a temperature of 30 °C and a culture pH of 5.0 maintained through the application of a 50 mM citrate buffer. The pH, growth and pigment production trends were consistent upon scaling up the cultivation from 100 mL shake flask to 5 L bioreactor scale. The volumetric biomass concentration achieved under these base case conditions in the bioreactor system was approximately 8.4 g.L -1 dry weight with a volumetric pigment concentration, based on absorbance at 500 nm, of 24 OD units. This related to a yield of pigment on biomass of 2.86 OD units.gx -1 . Maximum biomass productivity observed between approximately 30 and 120 hours of cultivation was determined to be 0.089 ± 0.007 gx.L-1 .h-1 , with maximum pigment productivity of 0.72 ± 0.18 OD units.h- 1 observed over the period of approximately 78 to 102 hours. Pigment production was estimated to start between 54 and 69 hours of cultivation. Using the Luedeking-Piret model of product formation, pigment production was shown to not be growth-associated. It is however, biomass associated and can be formed when the culture is actively growing. The non-growth associated specific pigment production rate, β, defining the base case cultivation was estimated at 0.23 OD units.gx -1 .h-1 . The pigmentation generated by P. purpurogenum DSM 62866 was shown to be the result of a mixture of multiple polar pigments. Extraction of pigment products was achieved using ethyl acetate, with intense red colouration still, however, observed in the aqueous medium. The pigment products in the organic and aqueous phases were processed further, with isolated products submitted for mass spectrometry analysis. A major red product present in the ethyl acetate extract was suggested to be an alanine derivative of the Monascus pigment rubropunctamine based on absorbance maxima and mass spectrometry analysis. The complex nature of the medium could support the formation of a number of related pigment derivatives with properties, such as solubility, dependent on incorporation of various side chains. Investigation of shaking speed during flask cultivation and agitation speed during benchtop bioreactor cultivation revealed a relationship between pigment production by P. purpurogenum and the rate of oxygen transfer into the cultivation medium. Residual oxygen concentration was demonstrated to not be a major factor affecting pigment production. A direct relationship was observed between pigmentation and kLa defining the system over the kLa range of 20 to 25 h -1 , corresponding to a maximum oxygen transfer rate of approximately 150 to 188 mg.L-1 .h-1 . During bioreactor cultivation, red pigmentation increased from 0 OD units to approximately 25 OD units as agitation speed was increased. Potential antioxidant properties of the pigment products could explain this trend. The impact of medium composition was also investigated over a range of growth scales, namely agar plate, multiwell plate, shake flask and bioreactor cultivation. Changes to medium composition included altering the ratio of malt extract to soya peptone and investigating the impact of replacing malt extract with a marshmallow-based substrate as a simple representation of a confectionery waste stream. Across growth scales, soya peptone was demonstrated to be an important medium component for pigment production. Replacing soya peptone with peptone of animal origin during agar plate cultivation inhibited pigment production by P. purpurogenum. The altered malt extract, soya peptone medium taken forward into bioreactor cultivation was composed of half the amount of malt extract, in comparison to the base case medium, with the concentration of soya peptone unchanged (Half MESP medium). This supported equivalent volumetric pigment concentrations, but approximately half the amount of biomass in comparison to MESP medium. As observed under base case conditions, scale-up from shake flasks to the bioreactor system using the Half MESP medium had little effect on volumetric biomass and pigment concentrations achieved. The highest specific pigment productivity was, therefore, achieved when cultivating P. purpurogenum on a medium composed of 15 g.L - 1 malt extract and 3 g.L - 1 soya peptone, maintained at a pH of 5 through the application of a citrate buffer, with a cultivation temperature of 30 °C. Yield of pigment on biomass was calculated to be 6.13 OD units.gx -1 , representing a 2.1-fold increase over the base case cultivation. Maximum biomass productivity was shown to be similar to that obtained in MESP medium at 0.077 ± 0.006 gx.L-1 .h- 1 , but over a shorter period of approximately 30 to 54 hours of cultivation. Maximum pigment productivity was, however, observed over approximately the same period as that in the MESP medium and was defined by a value of 0.71 ± 0.11 OD units.h-1 . The β value in the Half MESP medium was significantly higher, at 0.98 OD units.gx -1 .h-1 , showing an increase over the base case value of 4.3-fold. When replacing the malt extract in the Half MESP medium weight-for-weight with marshmallow confectionery, to simulate a sugar-rich confectionery waste stream, growth and pigment production of P. purpurogenum was supported. Biomass concentration achieved was similar to that obtained in Half MESP medium, while the volumetric pigment concentration was significantly lower. The result was a yield of pigment on biomass of 3.01 OD units.gx - 1 , which is similar to that obtained using the base case medium. The ability of this organism to grow and produce pigments when cultivated on this alternative substrate demonstrates an opportunity for improved resource efficiency through utilisation of waste resources for conversion to a product, thereby improving economic feasibility of the process. Potential exists for improving product yields through further optimisation or supplementation studies. When considering the combined results of the MESP, Half MESP and marshmallow-based medium cultivations, it was observed that sugar concentration in the medium was a determining factor for maximum volumetric biomass concentration achieved, but not for pigment productivity. Residual sugar concentration was also demonstrated to not be a trigger for the onset of pigmentation. Pigment production was seen to coincide with sporulation of the culture, indicating that some endogenous or external factor, such a medium composition, could be involved in the simultaneous onset of these two cellular processes. Medium components supplied were, however, shown to affect maximum volumetric pigment concentration. This could be attributed to the presence of a growth factor, or equivalent component, in the malt extract or soya peptone. Given the high pigment productivity achieved in this study when using a malt extract and soya peptone based medium, P. purpurogenum DSM 62866 is a promising candidate for the production of natural colourant alternatives. Further work should focus on downstream processing and formulation as well as investigating pigment properties which are important considerations for commercialisation, such as stability and ease of application.
- ItemOpen AccessInvestigating variables affecting heap (bio)leaching through determining access to sub-surface mineral grains by micro-scale X-ray tomographyGhadiri, Mahdi; Fagan-Endres, Marijke; Harrison, SusanHeap bioleaching is a hydrometallurgical technology, used to facilitate the extraction of valuable metals such as copper, gold, nickel and uranium from low-grade, typically sulphidic, ores. The process is highly complex as it is influenced by interactions of different sub-processes including flow of leaching solution around the ore particles, mass and heat transfer within and around the particles, chemical reactions, microbially-mediated reactions and microbial growth. Contact of leaching solution with mineral grains is necessary for oxidation of the sulphide minerals. However, a large fraction of the mineral grains is positioned below the surface of the ore particles and so contact with the liquid occurs through cracks and pores in the ore connected to the surface. Long extraction times and low metal recoveries typical of heap systems can be attributed to the slow leaching rate of these non-surface mineral grains as well as constraints on their accessibility. Most of the valuable grains that remain in the residue ores are non-surface grains. Therefore, investigation of the mechanism and behaviour of non-surface grain leaching and quantification of the factors contributing to their leaching is expected to be highly beneficial in the optimisation of leach conditions and recoveries. Non-surface grain leaching within large particles cannot be investigated via traditional experimental methods reliant on bulk measurements, 2D or destructive methodologies. However, it can be studied using high resolution, non-destructive 3D X-ray micro-Computed Tomography (μCT), an imaging technique for investigation of internal structure of opaque objects. X-ray μCT has previously been developed and used for investigation of different aspects of heap leaching. In the current study, the viability of using X-ray μCT to study heap bioleaching systems and affecting variables is assessed. This required establishment of procedures for measurement and analysis of sulphide and oxide mineral recoveries and leaching penetration distances. The feasibility of studying biotic heap leaching by X-ray μCT was explored through investigation of the relative energies required for high mineral resolution and avoidance of microbial inactivation. Specific bioleaching operating variables that were subsequently considered included: the accuracy and representivity of the X-ray μCT images, the influence of agglomeration pre-treatment, operating temperature, and type of ore on non-surface grain leaching. Addition of surfactants to the leaching solution was explored with the aim of changing surface activity to influence the penetration of the leach agent into pores and cracks in the ore. The effects of operating conditions on non-surface mineral grain leaching was studied using mini-column experiments. Three different low-grade ores, namely a chalcopyrite-rich ore, a malachite ore and a waste rock containing pyrite were prepared for the leaching experiment. The ores were crushed using a jaw crusher and comminuted down to 100% passing 16 mm. The products were sieved into six fractions (<0.25 mm, 0.25 - 1 mm, 1 - 2 mm, 2 - 5.6 mm, 5.6 - 8 mm, 8 - 16 mm) and each fraction then representatively split into smaller portions using a rotary splitter. One portion of each size fraction was taken for XRD, AAS and QEMSCAN analyses. Mini leaching columns were designed and constructed based on the target mineral grain distribution in the ores to ensure that the mineral grains were detectable using X-ray µCT, given its resolution limitations. The columns were charged with 50 g of agglomerated or non-agglomerated ore and lixiviant was provided at a flow rate of 2.55 mL h -1 for a period of 5.5 months for chalcopyrite and pyrite and 26 days for malachite in incubators at 30 °C, 37 °C and 65 °C. In order to select a surfactant suitable for use in a biological leach experiment, the effect of five different types and concentration of non-ionic surfactants on bioleaching microorganisms was studied in terms of microbial growth, ability for ferrous ion oxidation and chalcopyrite bioleaching. This was done in shake flask experiments using mineral concentrate. Based on the results of these experiments, Tween® 20 (10 mg L -1 ) was selected to study the effect of surfactant on non-surface mineral grain leaching in the mini-columns. Each column was scanned by X-ray μCT at 100 kV and 150 mA using a 0.38 mm copper filter and at a distance of 59.40 mm between X-ray gun and specimen. The advanced 3D analysis software Avizo® 9 was used to visualize and analyse image data. The Interactive Thresholding function in Avizo® 9 software was used for segmentation of ore particles from air and sulphide minerals from air and gangue minerals, to measure the target minerals' volume reduction during leaching. The Distance Map Algorithm was applied on a binary (segmented) image to calculate the distance of the sulphide mineral from the ore particle surface. Imaging of the whole mini-column was done before leaching and at the end of each experiment and imaging of certain sections was done at select time points during leaching to track temporal leaching dynamics. Good agreement was seen between the bulk mineral recovery data, determined using standard chemical assays, and the leaching curves generated using the X-ray µCT images for all the ores, confirming that the X-ray µCT images were a good quantitative measurement of the sulphide and oxide mineral leaching. Liquid microbial culture experiments were used to confirm that exposure to X-ray does not affect microbial activity for energy doses between 35 and 90 kV at 200-280 μA. However, X-ray exposure was found to have a slight negative influence at higher voltages of 120 and 150 kV, temporarily reducing the specific ferrous ion oxidation and suppressing the specific growth rate of the bioleaching microorganisms. The X-ray exposure thus negatively affected both the total microbial population available for leaching (population viability) as well as the metabolic activity of the individual microorganisms (population vitality). The effect of X-ray exposure on bioleaching cultures attached to a mineral surface was examined using pyrite-coated glass beads packed into mini-columns. The energy dosage limits identified in the liquid culture experiments were found to be compatible with the X-ray μCT imaging conditions (minimum energy dosage and sample position) required for acquisition of complete and accurate images of the columns at a resolution that allows identification of individual mineral grains. Following X-ray exposure, the performance of the exposed bioleaching mini-columns was equivalent to the unexposed control column. Similarly, the microbial activity and presence on the mineral surface appeared unchanged. Finally, the experiment was performed on the chalcopyrite ore and the microorganisms were found to still be able to convert Fe2+ to Fe3+ after 2 scanning runs. Thus, all sets of results confirm that X-ray μCT can be compatible with heap bioleaching experiments, while still permitting appropriate resolution of the mineral grains to make an X-ray μCT investigation worthwhile. However, cognisance that an upper limit of tolerable X-ray exposure exists must be taken. This may present a challenge if it is desired to image larger or denser ore samples which require a greater X-ray energy level for sufficient penetration of the sample by the X-rays and hence accurate imaging. In chalcopyrite leaching, increasing temperature from 37 °C to 65 °C resulted in clear enhancement of leaching based on both analysis methods, with the copper recovery increasing from 20% to 64% by the end of the leaching period, and the overall sulphide mineral dissolution increasing from 24% to 67%. Increasing temperature from 37 °C to 65 °C resulted in an increased leaching penetration distance and crack development in the particles, and thus an enhancement in copper recovery and sulphide mineral dissolution. This was in addition to the thermodynamically expected increased leaching rate. The maximum leaching penetration distance, beyond which no mineral volume change is observed, at 37 °C was 1.7 mm. This increased to 2.5 mm at 65 °C. As a result of addition of 10 mg L-1 Tween® 20 into the leaching solution, the final copper recovery was improved by 4% to 68% and the maximum penetration distance increased to 2.9 mm. However, when the availability of sulphide mineral was not rate limiting, the copper recovery and sulphide mineral volume reduction in the mini-column with surfactant was lower than the system without surfactant. This may have been due to depression of diffusion of ferric ion to the ore surface as a result of the formation of an adsorbed surfactant layer on the mineral surface. The performance with surfactant became superior as the amount of readily leachable mineral became limiting. In the pyrite waste rock, an increase in temperature did not have any effect on the maximum penetration distance and any increase in iron recovery was only for thermodynamic reasons. Similarly to the chalcopyrite ore, during the later period of leaching when readily exposed mineral grains have been depleted, the system performed better in the presence of surfactant. The addition of surfactant increased the maximum penetration distance from 2.7 to 2.9 mm. The cumulative copper recovery of 86% was obtained for malachite ore in 26 days of acid leaching and the maximum penetration distance was 2.2 mm. This study thus demonstrates the value of the X-ray µCT technique for quantitative investigation of non-surface mineral grain leaching and confirms that the maximum penetration distance can be affected with changing operation conditions or ore type. This study thus demonstrates the X-ray µCT technique for quantitative investigation of non-surface mineral grain bioleaching and confirms that the maximum penetration distance can be affected with changing operation conditions. Critically, the results confirm that X-ray μCT can be compatible with bioleaching microorganisms, while still permitting appropriate resolution of the mineral grains to make an X-ray μCT investigation worthwhile.
- ItemOpen AccessLinking microbial community dynamics and performance of a biological sulphate reducing system using a mixed volatile fatty acid stream as electron donor(2021) Motleleng, Liabo; Harrison, Susan; Smart, MarietteMining for the recovery of minerals and coal can result in acid mine drainage (AMD) which presents an environmental risk. Acid mine drainage, as the name suggests, is acidic run-off water from mostly mine waste dumps. It affects water quality by lowering its pH and increasing its metal and sulphate loading, thus making it unsuitable for use by many forms of life. AMD must therefore be treated before entering nearby water systems and soils. An effective treatment technology is considered as the one that can result in water neutralisation and removal of metals and sulphate. Biological sulphate reduction (BSR) technologies, mediated by sulphate reducing bacteria (SRB), have attracted attention as a sulphate remediation strategy as they offer a cheap alternative to other sulphate removal technologies such as chemical approaches. In addition, the concomitant generation of alkalinity and soluble sulphide assist in neutralisation and heavy metal removal. One of the challenges associated with BSR is the supply of a cost-effective carbon source which also acts as an electron donor for the anaerobic reduction of sulphate. Studies have reported that both the choice of carbon source and electron donor and the microbial communities present influence the sulphate reduction process, the former frequently defining technoeconomic feasibility. The feed sulphate concentration and residence time, together defining the volumetric sulphate loading rate, have also been reported to influence the efficacy of the sulphate reduction process and needs to be optimised for the microbial community present and the chosen electron donor. The identification and characterisation of the microbial communities involved and investigating how these change with changes in operating conditions is crucial in the optimisation of BSR processes. Currently, there are no commonly used molecular tools which can be used for routine analysis of SRB communities in real time and on a regular basis and cost effectively. This makes it difficult to understand the link between changes in the mixed BSR microbial community structure and process performance. The study presented in this thesis had three main objectives. Firstly, to evaluate the use of an anaerobic digestate, obtained from a partially anaerobically digested Cyanobacteria species (Arthrospira platensis, commonly known as Spirulina), as a carbon source and electron donor for BSR. Secondly, to validate, optimise and apply the molecular tools for analysis of the relative abundances of species within the mixed BSR microbial community in this study. Thirdly, to compare the microbial community dynamics and performance of BSR using the complex anaerobic digestate as carbon source and electron donor to BSR using a single electron donor source, lactate. Chemostat studies using a mixed SRB consortium were carried out using anaerobic digestate, characterised as containing a mixture of acetate, propionate and butyrate, as a carbon source and electron donor for BSR. Upon reaching steady-state, the concentrations of sulphate, bicarbonate, acetate, propionate and butyrate were measured and used to estimate the BSR kinetics and reaction stoichiometry. A 16S rRNA gene survey of the BSR inoculum used for this thesis was performed by constructing a 16S rRNA gene clone library and analysis of the diversity of clones was performed using amplified ribosomal DNA restriction analysis (ARDRA). These 16S rRNA sequences were used to provide insight into the diversity and phylogenetic relatedness of the bacterial community and key species within the mixed BSR inoculum. In silico analysis of the 16S rRNA sequences captured from the clone library was performed to design novel genus specific quantitative real-time PCR (qPCR) primers and to validate the specificity of previously published primers. Fluorescence in situ hybridisation (FISH) techniques were optimised for the visual characterisation of this microbial community. FISH and qPCR were then applied to assess how the mixed microbial community structure was affected by the changes in the volumetric sulphate loading rate (VSLR), mediated through dilution rate and feed sulphate concentration, when anaerobic digestate (mixed carbon source) and lactate (simple carbon source) were used as an electron donor for BSR. The results obtained were used to examine and compare the link between microbial community dynamics and performance of sulphate reducers between the mixed and the simple carbon source. The results obtained from this thesis suggested the simultaneous utilisation of all the three volatile fatty acids (acetate, propionate and butyrate) present in anaerobic digestate which contributed to the robustness of the chemostat reactors as indicated by higher sulphate, propionate and butyrate conversion efficiencies. The kinetic profiles of the volumetric sulphate reduction rate (VSRR) obtained with anaerobic digestate were well matched with the kinetics observed in previous studies when single carbon sources and electron donors were used for BSR. At a feed sulphate concentration of 1.0 g l-1 , the oxidation of acetate, propionate and butyrate and concomitant sulphate reduction were observed across the dilution rates of 0.0083 to 0.083 h -1 . The stoichiometry of BSR utilising propionate and butyrate as carbon and electron donor suggested that by increasing feed sulphate concentrations from 1.0 to 2.5 and 5.0 g l-1 acetogenic reactions were favoured at the higher dilution rates of 0.042 and 0.083 h-1 . However, increasing the feed sulphate concentration at the lower dilution rates of 0.0083 to 0.021 h-1 did not alter the oxidation of volatile fatty acids (VFAs) and concomitant sulphate reduction, suggesting that the sensitivity of the propionate and butyrate oxidisers was related to specific growth rate rather than the sulphate loading. A previous mathematical model developed by Moosa et al. (2002) was used to determine microbial growth constants (μmax and Ks) and energetic coefficients (Yx/s) for SRB at each feed sulphate concentration to describe the microbial growth kinetics obtained with anaerobic digestate. A 16S rRNA gene survey, performed by 16S rRNA library construction and 16S rRNA gene amplicon sequencing, revealed a more diverse microbial community in the inoculum obtained from a lactate operated BSR reactor than previously reported. qPCR was used to confirm the presence and relative abundance of these species within the reactors receiving anaerobic digestate or lactate as carbon source and electron donor. The 16S rRNA sequences captured were found to have high similarity to well- known SRB species belonging to the Desulfomicrobium, Desulfovibrio, Desulfuromonas, Desulfobulbus and Desulfocurvus genera. Other “non-traditional SRB” species belonging to the Firmicutes and Citrobacter genera containing a specific molecular target for the detection of SRBs, the dissimilatory sulphite reductase gene (dsrAB), within their genomes were also detected. DsrAB is the key enzyme catalysing the last and main energy-generating step during sulphate reduction. Non-SRB species present were identified as members of the Sphaerochaeta, Synergistetes, Chloroflexi, Mesotoga, Acholeplasma, Bacteriodetes, Petrimonas and Bacteriodes genera. A 16S rRNA gene survey by 16S rRNA variable region amplification from metagenomic DNA extracted from microbial biomass associated with continuous stirred tank reactors (CSTRs) operated on anaerobic digestate or lactate was performed to validate the qPCR results and assist with the identification of the “other SRB” and nonSRB species. The 16S rRNA gene survey suggested the presence of 13 known SRB species Desulfomicrobium groups (Desulfomicrobium hypogeium and Desulfomicrobium aestuarii), Desulfovibrio species (D. aminophilus, D. vulgaris, D. desulfuricans, D. intestinalis, D. oxamicus, and D. sulfodismutans), Desulfobulbus oligotrophicus, Desulfocurvus vexinensis, Desulfococcus biacutus, Desulfarculus baarsii, Desulfomonile tiedjei and Desulfobacca acetoxidans in CSTRs operated on anaerobic digestate. Only up to 10 SRB species, Desulfomicrobium hypogeium, Desulfomicrobium aestuarii, Desulfovibrio groups (Desulfovibrio aminophilus, Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Desulfovibrio intestinalis, Desulfovibrio sulfodismutans, Desulfovibrio mexicanus, Desulfobulbus oligotrophicus and Desulfocurvus vexinensis were observed in reactors with lactate, suggesting that the multiple VFAs present in the anaerobic digestate (acetate, propionate and butyrate) were able to support a more diverse SRB community than a single electron donor (lactate). Various non-SRB bacterial genera as well as known elemental sulphur reducing bacteria Desulfuromonas acetexigens and Dethiosulfovibrio acidaminovorans were also found to be present, with the latter being associated only with the lactate operated reactor. qPCR results indicated that despite being present in high proportions at the lowest VSLRs, the Desulfomicrobium species were washed out of the reactors at higher VSLRs regardless of carbon source and electron donor was provided. Species from the Desulfovibrio genera, which were present at lower abundances than the Desulfomicrobium species, were more resistant to changes in dilution rates and remained present within the reactors at the higher VSLRs, 0.104 and 0.208 g l-1 h -1 . In the reactors operated on anaerobic digestate, the decline in the abundance of Desulfovibrio species at VSLRs of 0.052 and 0.104 g l-1 h -1 , correlated with a noticeable decline in sulphate conversion from 60.4 to 49.4% at feed sulphate of 2.5 g l-1 , and from 66.9 to 22.6% at feed sulphate of 5.0 g l-1 . These findings suggest that Desulfovibrio species may play a critical role in sustained sulphate reduction at lower VSLRs. 16S rRNA gene amplicon data validated the qPCR data showing that increasing the VSLR, resulted in a change in the SRB community structure and a decrease in the proportion of total SRB within the microbial community. In agreement with the FISH and qPCR findings, Desulfomicrobium hypogeium was identified as the most abundant operational taxonomic unit (OTU) belonging to SRB present at the lowest dilution rate (D) tested (0.0083 h-1 , retention time (RT = 1/D) of 5 d) when anaerobic digestate was used as an electron donor for BSR. Washout of most SRB species was also observed when the dilution rate was increased from 0.0083 to 0.042 h-1 (RT of 5 to 1 d) in these reactors. Species such as Desulfovibrio sulfodismutans, Desulfomonile tiedjei, the acetate oxidiser Desulfococcus biacutus and the elemental sulphur reducing Desulfuromonas acetexigens were found to tolerate higher VSLRs of 0.104 and 0.208 g l-1 h -1 (dilution rate of 0.042 h-1 ), suggesting fast enough growth rates to remain in these reactors at the higher dilution rate of 0.042 h-1 . A decrease in the abundance of the incomplete propionate oxidiser Desulfobulbus oligotrophicus correlated to a decrease in propionate oxidation at a VSLR of 0.104 and 0.208 g l-1 h -1 suggesting that this SRB was responsible for the oxidation of propionate and concomitant sulphate reduction observed in these reactors. Similar to the reactors receiving anaerobic digestate, increasing the dilution rate from 0.0083 to 0.042_h -1 (RT of 5 to 1 d) resulted in washout of most SRB OTUs in CSTRs operated on lactate. At a feed sulphate concentration of 10.0 g l-1 , increasing the dilution rate from 0.0083 to 0.042 h-1 resulted in an increase in the proportion of the lactate oxidiser Desulfocurvus vexinensis from 25 to 98% of the total SRB proportion. At this dilution rate (0.042 h-1 ), other SRB species observed were the lactate oxidisers Desulfovibrio sulfodismutans and Desulfobulbus oligotrophicus which can oxidise lactate and the product of its incomplete oxidation, propionate. Although the abundance of these two SRB at the dilution rate of 0.042 h-1 was much lower than that of Desulfocurvus vexinensis, studies with anaerobic digestate suggested Desulfobulbus oligotrophicus which was abundant at only 0.004% and was identified as the only propionate degrader in the CSTR resulted in propionate conversion of 21.7%. This suggested that the less abundant Desulfovibrio sulfodismutans and Desulfobulbus oligotrophicus may have also played a role in sulphate reduction at the dilution rate of 0.042 h-1 in the CSTR with lactate. In addition, Desulfocurvus vexinensis and Desulfobulbus oligotrophicus were able to function at a VSLR of 0.42 g l-1 h -1 which suggests these two SRB species could be used effectively to reduce sulphate to hydrogen sulphide in wastewaters with higher VSLRs of up to 0.42 g l-1 h -1 when lactate was provided as an electron donor for BSR. The acetate specialist, Desulfobacca acetoxidans, the butyrate oxidiser Desulfarculus baarsii and the propionate oxidiser Desulfobulbus oligotrophicus, were able to function at a VSLR of 0.208 g l-1 h -1 suggesting that a combination of these three SRB species could be used in BSR treatment processes with VSLRs of up to 0.208 g l-1 h -1 where anaerobic digestate is provided as an electron donor. The ability for anaerobic digestate to support diverse SRB communities even at higher VSLRs may add to the robustness of the reactors to maintain sulphate reduction even at high VSLRs. This thesis showed that both the presence and diversity of SRB species are subject to the carbon source and VSLR. To the author's knowledge, this is the first study to indicate the relationship between the change in SRB community structure and sulphate reduction performance when anaerobic digestate (a complex carbon source) is used as a carbon source and electron donor for BSR. Results from this thesis suggest that the use of a mixed volatile fatty acid stream generated for the partial digestion of a suitably digestible biomass may be used as electron donor and carbon source to support a robust BSR process for the treatment of AMD. Using a mixed volatile fatty acid stream also has potential to result in the development of a more economically viable AMD treatment process.
- ItemOpen AccessOptimisation of a linear flow channel reactor for semi-passive, simultaneous biological sulphate reduction and partial sulphide oxidation(2021) Fernandes, Sarah; Harrison, Susan; Huddy, Robert; van Hille, Rob PAcid rock drainage (ARD) is a growing concern, particularly in South Africa, as a country already classified as water scarce. ARD is defined as water that has been impacted by mining activities and typically has high levels of sulphate and heavy metals, at acidic pH. Similarly, high sulphate neutral rock drainage is of increasing concern. High sulphate content increases water salinity leading to adverse effects on human health as well as agriculture. Types of ARD and neutral rock drainage can be categorised into those that are produced in high volumes from groundwater rebound, and those that are generated from diffuse sources, as low-flow ARD. Low-flow ARD and neutral rock drainage are amenable to biological treatment of the sulphate component using sulphate reducing bacteria (SRB). Biological sulphate reduction (BSR) generates sulphide, which requires further treatment to remove it from the stream in gaseous form or as a solid sulphur-containing compound. Alternatively, it can also be used, in part, to precipitate metals present within ARD waters. Key challenges associated with SRBbased bioremediation include the cost of the supplemented electron donor needed for SRB to reduce sulphate, as well as the downstream management or treatment of the excess sulphide remaining. This investigation aimed to optimise a semi-passive treatment process which integrates BSR, and concomitant partial oxidation, by sulphur oxidising bacteria (SOB), of the sulphide produced to elemental sulphur. This is generated as a floating sulphur biofilm (FSB). These processes occur simultaneously within a linear flow channel reactor (LFCR), facilitating both treatment of the water stream to a fit-for-purpose water product, and recovery of the sulphur for use within fertilisers or fungicides. The work focused on the effective utilisation of electron donors and a sustainable option thereof, as well as the optimisation of partial sulphide oxidation and sulphur recovery. In addressing the cost of a supplemented electron donor, the use of a waste product is of interest. To explore this and build on earlier work within the Centre for Bioprocess Engineering Research (CeBER) labs, this study investigated the efficient use of the volatile fatty acids (VFAs) acetate, propionate and lactate. Firstly, the study investigated propionate, a common fermentation product of waste organic sources. It is found as a component of the effluent or digestate, of anaerobic digestion (AD) processes such as algal AD. Propionate was proposed as an attractive option for a sustainable source of electron donor. An LFCR fed with synthetic propionate showed sulphate reduction occurring via the utilisation of both propionate as well as acetate produced from propionate metabolism. Fermentative bacteria were seen to work syntrophically within the system availing a significant amount of acetate to the SRB community. This acetate was the preferred electron donor over propionate. Maximum volumetric sulphate reduction rates (VSRRs) of 190 mg/L/day were achieved in the reactor. However, detailed analysis showed few propionate-utilising SRB in the community. It was concluded that a more diverse inoculum was needed to investigate the potential of propionate more fully. Secondly, the study investigated lactate as a means to explore the efficiency of electron donors that are incompletely oxidised to acetate. Higher chain VFAs such as lactate are partially oxidised to acetate under biosulphidogenesis; however, acetate oxidation by SRB appears to be a rate-limiting step in most systems. Simultaneous incomplete oxidation of the more complex VFAs with complete oxidation of acetate are rarely reported. Acetate accumulates in the effluents of these processes and results in high chemical oxygen demand (COD) remaining which can lead to environmental impacts such as eutrophication if released into river systems. Further, utilisation of the electron donor is inefficient. In order to address this, the study presents a sequential LFCR system to increase utilisation efficiency of the incompletely oxidised VFA feed. The sequential system was developed by coupling a second reactor unit, specifically colonised with acetate-utilising SRB, to a primary reactor unit utilising lactate. The SRB community in the secondary reactor was able to oxidise acetate from the primary reactor resulting in further sulphate reduction and lower residual COD levels. Residual acetate decreased from 475 mg/L in the primary reactor to 275 mg/L in the secondary reactor. Similarly, residual sulphate decreased from 533 mg/L in the primary reactor to 150 mg/L in the secondary reactor, on a 1 g/L sulphate feed, achieving a much improved effluent water quality. A VSRR of 213 mg/L/day across the sequential system, an overall conversion of 85% and a two-fold increase in sulphate reduced / lactate consumed from 0.45 to 0.85 (g/g) were achieved. Lastly, this study investigated sulphide removal via the incorporation of elemental sulphur into a floating sulphur biofilm (FSB). The generation of an FSB within a sulphate reducing bioreactor is a passive and sustainable method of sulphide remediation, producing a value-added product of elemental sulphur. The sequential reactor system resulted in an increase of two to three-fold in the amount of elemental sulphur recovered, with an improved conversion of sulphide formed to elemental sulphur. Further, the effect of the feed inorganic ions, magnesium and phosphate, on sulphur yield and sulphide removal was studied using the sequential LFCR system. It was found that a decrease of magnesium in the media supplied resulted in an increase in sulphide conversion to sulphur from 21 to 39%, while a concomitant reduction in feed phosphate resulted in a further increase to 50% of the same. In both cases the sulphur concentration in the FSB was substantially increased. Overall, the thesis addresses the need to decrease the cost associated with the supply of a suitable electron donor, as well as improves the water quality of a BSR effluent both in terms of residual sulphate, sulphide and COD. The LFCR system studied was improved both by a sequential reactor system, allowing greater sulphate reduction, sulphide removal via elemental sulphur recovery, and substrate utilisation efficiency. Additionally, changes to the inorganic component of the feed led to further sulphide removal and elemental sulphur recovery. Propionate was concluded to have been only partially used as an electron donor for SRB, however fermentative bacteria present in the mixed community degraded propionate to acetate which was then used for BSR. The work presented here contributes towards the broader research into a semi-passive, environmentally sustainable and economically viable treatment solution for low-flow, circumneutral ARD.
- ItemRestrictedPre-treatment and anaerobic codigestion of waste activated sludge from a petrochemical wastewater treatment plant(2022) van der Merwe, Carla; Harrison, SusanDuring the aerobic treatment of wastewater, waste activated sludge (WAS) is generated which requires further treatment. The typical treatment method applied for treating the sludge is incineration. The National Environmental Management: Air Quality Act (NEM:AQA), promulgated in 2004, was amended in 2013 to include the list of activities which result in atmospheric emissions. One of the activities included in the list is the specification of the minimum emission standards (MES) for thermal treatment of hazardous waste; this includes the incineration of waste activated sludge. Alternative treatment methods should be considered to minimise emissions and to derive some benefit from the WAS. One such potential method is the pre-treatment of waste activated sludge combined with co-digestion in an anaerobic digester. This study focusses on selection of a feasible pre-treatment option for the anaerobic digestion of waste activated sludge produced in a petrochemical wastewater treatment plant (PWWTP) to improve its subsequent anaerobic digestion. Following pre-treatment, the study specifically focusses on digestion of the pre-treated sludge in an attached-growth down-flow anaerobic digester with co-digestion with an available wastewater stream containing soluble organic material. This stream will be referred to as the soluble wastewater stream (SWWS). The WAS stream is dewatered in the PWWTP to reduce the volume of the stream for treatment. The test work was done on both the “as-processed” waste activated sludge originating from the bottom of the settlers as well as the dewatered stream. The “as processed” WAS stream (which is referred to as 1 wt% WAS) has a solids concentration which could vary between 9 000 mg/L to 15 000 mg/L. The total chemical oxygen demand (TCOD) of the stream ranged from 18 000 mg/L to 25 000 mg/L, whereas the soluble chemical oxygen demand (SCOD) was measured to range between 160 mg/L to 200 mg/L. Most of the COD was contained in the sludge floc and would not be freely available for the anaerobic micro-organisms to digest. The dewatered WAS (which is referred to as 10 wt% WAS) solids concentration would vary between 90 000 mg/L to 120 000 mg/L. Due to the removal of water in the dewatering step, the TCOD would increase to between 160 000 mg/L to 180 000 mg/L, however the SCOD remained low at 160 mg/L to 200 mg/L. To anaerobically digest both these streams “as is” would be challenging and require very long retention time and large digesters, yielding low conversion into biogas. Pre-treatment options are available to lyse the cell walls of the WAS, increasing the SCOD, which could make anaerobic digestion of WAS technically feasible. The research identified thermal, chemical and thermo-chemical pre-treatment processes as feasible options for the production facility to consider. Thermal pre-treatment was done at 60, 70, 80, 100, 120 and 133°C for the 1 wt% WAS and 10 wt% WAS. It was observed that the % soluble COD increased from the baseline of 2% to 24% at 133°C which supported the findings of many researchers, stating that the degree of cell disruption improves with an increase in temperature. Chemical pre-treatment with sodium hydroxide was done at 0.2, 0.6 and 1.0 ml 48% NaOH/g solids. The fraction of soluble COD was increased to as high as 27%. Less sodium hydroxide per gram solids was required for the dewatered waste activated sludge than for the 1 wt% WAS. It was found that thermo-chemical pre-treatment of the 10 wt% WAS at 133°C with 0.3 ml 48% NaOH/g solids would improve the fraction of soluble COD to greater than 80%. The treated waste activated sludge was then co-digested in a pilot scale supporting-growth down-flow anaerobic digester to evaluate the conversion of the organics and the quantity and quality of the biogas formed. The stream used for the co-digestion was a soluble wastewater stream which contains mostly volatile fatty acids (VFA) with acetic acid as the major component. The SCOD of the SWWS would range between 12 000 mg/L to 20 000 mg/L. The volumetric ratio of the lysed WAS and SWWS streams tested were 1:20, 1:25 and 1:30 to determine which ratio would provide the required outlet specifications from the anaerobic digester, set at an effluent COD concentration of less than 3 000 mg/L and effluent suspended solids of less than 3 000 mg/L. These specifications are set based on downstream process requirements. The optimum ratio of the lysed waste activated sludge to soluble wastewater (SWWS) was 1:30 for this specific production facility to meet their effluent specifications. The COD conversion for co-digestion was 87.2 ± 0.3% and the percentage suspended solids converted in the anaerobic digester was estimated to be 57.7 ± 5.2%. The system was designed for a methane fraction of 50% as the theoretical methane fraction for acetic acid is 51%. When co-digesting the WAS with the SWWS, the methane fraction increased to 57.38 ± 2.95%. An increase in the methane fraction was expected when co-digesting the WAS with the SWWS. The theoretical methane yield for the process conditions are 0.438 l CH4/g COD converted. Based on the measured flow rates and methane analysis, the methane yield was 0.38 ± 0.02 l CH4/g COD converted, equivalent to 87% of theoretical yield. This difference could be due to biogas flow meter inaccuracy, and variation in methane fraction. A block flow diagram was developed for the proposed process solution and the main pieces of equipment sized and equipment costs estimated. This was done for a system with and without a dewatering step. The system with a dewatering step had lower capital expenditure as well as lower operating expenditure. The amount of biogas generated was sufficient to be used in a combined heat and power system to generate the steam required for the pre-treatment and sufficient electricity for the pumps used in the pre-treatment system. Thermo-chemical pre-treatment of dewatered WAS (concentrated to 10 wt% solids) from a petrochemical wastewater treatment plant combined with co-digestion of the lysed WAS with a VFA-rich stream is a technically feasible option for the treatment of WAS. This solution reduces the amount of WAS sent to the incinerator which, in turn, will improve the emissions footprint with regards to particulate matter, NOx, SOx, metals, dioxins and furan emissions. Co-digestion would also result in the WAS stream being used to generate a biogas which supports the sustainable development agenda, therefore moving up in the waste hierarchy.