Browsing by Author "Huddy, Robert"
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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 AccessCharacterisation of three novel α-L-arabinofuranosidases from a compost metagenome(2019-04-18) Fortune, Brent; Mhlongo, Sizwe; van Zyl, Leonardo J; Huddy, Robert; Smart, Mariette; Trindade, MarlaBackground: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. Results Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0–6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s− 1 and 219 s− 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. Conclusion All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
- 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 AccessEvaluation of the ASTERTM process in the presence of suspended solids(Elsevier, 2014) van Zyl, Andries W; Huddy, Robert; Harrison, Susan T L; van Hille, Robert PThe ability to recycle and reuse process water is a major contributing factor toward increased sustainability in the mining industry. However, the presence of toxic compounds has prevented this in most bioleaching operations. The ASTERTM process has been used for the bioremediation of cyanide (CN) and thiocyanate (SCN−) containing effluents at demonstration and commercial scale, increasing the potential for recycling of the treated effluent. The process relies on a complex consortium of microorganisms and laboratory tests have shown that the biomass retention, in suspended flocs or attached biofilm, significantly improved SCN− degradation rates. The current research evaluated the process performance in the presence of suspended solids (up to 5.5% m/v) ahead of implementation at a site where complete tailings removal is not possible. Experiments were performed in four 1 l CSTRs (with three primary reactors in parallel at an 8 h residence time, feeding one secondary reactor at a 2.7 h residence time). Stable operation at the design specifications (5.5% solids, 100 mg/l SCN− feed, effluent SCN− <1 mg/l) was achieved within 50 days, including a period of adaptation. The pH had the most significant effect on performance, with significant inhibition below pH 6. The presence of gypsum and anhydrite phases in the fresh tailings was most likely responsible for the observed decrease in pH. A maximum SCN− degradation rate of >57 mg/l/h was achieved, despite no obvious floc formation. Microbial ecology studies (16S rRNA clone library) revealed reduced diversity relative to reactors operated without suspended solids.
- ItemOpen AccessInvestigating process stresses on Saccharomyces cerevisiae using isothermal microcalorimetry(2017) Myers, Matthew; Harrison, Susan TL; Tai, Siew; Huddy, Robert; Fagan-Endres, MarijkeMaximising performance of microbial processes, including yeast-based processes, in an industrial setting requires understanding of the impact of process stresses. These may be the result of process configuration, dilution, temperature changes, hydrodynamic conditions or process perturbations. Methods to determine the microbial metabolic response to such stresses have long been sought, but are typically limited, often requiring the use of a suite of methods to assess the physiological status and state. The recent technical advances in microcalorimetry suggest potential for the use of isothermal microcalorimetry (IMC) to determine yeast viability and vitality and is investigated here. IMC is a laboratory method whereby the real-time heat produced by a chemical, biological or physical process is measured in the micro to nano watt range. It is proposed that this heat production may be correlated to the physiological state of the microbial catalyst and can be used to measure the impact of different stresses. In this study, the potential of IMC as a method for exploring process stress is investigated using Saccharomyces cerevisiae and its application in the beer brewing industry as a case study. Here, it is well known that yeast viability and vitality have commercial significance. IMC is sufficiently sensitive to detect the heat given off by 1000 yeast cells. However, IMC cannot distinguish between different heat flows within a system i.e. it is non-specific. The literature demonstrates how IMC has been used in the study of numerous microbiological fields, including the growth and metabolism of yeast. Previous studies have successfully derived the specific growth rate and cell numbers of a growing yeast population from analysing power and heat curves. The specific growth activity and specific growth retardation of yeast and how these parameters relate to bactericidal and bacteriostatic effects has also been examined by a number of authors. The key objectives of this study were to determine the viability and vitality of Saccharomyces cerevisiae using IMC and to assess the impact of stresses on yeast viability and vitality. This was achieved by measuring the thermal power produced by a growing yeast suspension as a function of its overall growth and metabolism. Two industrially relevant stresses were examined: cold shock and ethanol shock. The effect of these stresses has yet to be studied using microcalorimetry. The growth of Saccharomyces cerevisiae under ethanol stress was used as an inhibition study to isolate its effects on the growth thermogram. Following the generation of thermograms under control and stress conditions using IMC, a method for their quantitative analysis was developed. Curves were fitted to the heat data using an exponential growth equation and the time for the heat flow curve to peak was determined. From the exponential curve, the specific growth rate of the yeast was determined with a high degree of repeatability. The coefficient of the exponential term in the growth equation gave highly reproducible and distinguishable results relating to the viability and vitality of the initial yeast population. The time of peak heat flow was also affected by the initial viability and vitality of the yeast and was used to estimate the initial active cell population size.
- 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.