Robert J. Huddy

Genome-Resolved Meta-Omics Ties Microbial Dynamics to Process Performance in Biotechnology for Thiocyanate Degradation

Remediation of industrial wastewater is important for preventing environmental contamination and enabling water reuse. Biological treatment for one industrial contaminant, thiocyanate (SCN-), relies upon microbial hydrolysis, but this process is sensitive to high loadings. To examine the activity and stability of a microbial community over increasing SCN- loadings, we established and operated a continuous-flow bioreactor fed increasing loadings of SCN-. A second reactor was fed ammonium sulfate to mimic breakdown products of SCN-. Biomass was sampled from both reactors for metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one rotifer that constituted the abundant community in both reactors. We analyzed the metabolic potential and temporal dynamics of these organisms across the increasing loadings. In the SCN- reactor, Thiobacillus strains capable of SCN- degradation were highly abundant, whereas the ammonium sulfate reactor contained nitrifiers and heterotrophs capable of nitrate reduction. Key organisms in the SCN- reactor expressed proteins involved in SCN- degradation, sulfur oxidation, carbon fixation, and nitrogen removal. Lower performance at higher loadings was linked to changes in microbial community composition. This work provides an example of how meta-omics can increase our understanding of industrial wastewater treatment and inform iterative process design and development.

Genome-resolved metagenomics of a bioremediation system for degradation of thiocyanate in mine water containing suspended solid tailings.

Thiocyanate (SCN−) is a toxic compound that forms when cyanide (CN−), used to recover gold, reacts with sulfur species. SCN−‐degrading microbial communities have been studied, using bioreactors fed synthetic wastewater. The inclusion of suspended solids in the form of mineral tailings, during the development of the acclimatized microbial consortium, led to the selection of an active planktonic microbial community. Preliminary analysis of the community composition revealed reduced microbial diversity relative to the laboratory‐based reactors operated without suspended solids. Despite minor upsets during the acclimation period, the SCN− degradation performance was largely unchanged under stable operating conditions. Here, we characterized the microbial community in the SCN− degrading bioreactor that included solid particulate tailings and determined how it differed from the biofilm‐based communities in solids‐free reactor systems inoculated from the same source. Genome‐based analysis revealed that the presence of solids decreased microbial diversity, selected for different strains, suppressed growth of thiobacilli inferred to be primarily responsible for SCN− degradation, and promoted growth of Trupera, an organism not detected in the reactors without solids. In the solids reactor community, heterotrophy and aerobic respiration represent the dominant metabolisms. Many organisms have genes for denitrification and sulfur oxidation, but only one Thiobacillus sp. in the solids reactor has SCN− degradation genes. The presence of the solids prevented floc and biofilm formation, leading to the observed reduced microbial diversity. Collectively the presence of the solids and lack of biofilm community may result in a process with reduced resilience to process perturbations, including fluctuations in the influent composition and pH. The results from this investigation have provided novel insights into the community composition of this industrially relevant community, giving potential for improved process control and operation through ongoing process monitoring.

Detection and Localisation of the Abalone Probiotic Vibrio midae SY9 and Its Extracellular Protease, VmproA, within the Digestive Tract of the South African Abalone, Haliotis midae

Probiotics have been widely reported to increase the growth rate of commercially important fish and shellfish by enhancing the digestion of ingested feed through the production of extracellular enzymes such as proteases and alginases. In order to investigate this further, the objective of this study was to localise the bacterial probiont Vibrio midae SY9 and one of the extracellular proteases it produces in the digestive tract of the South African abalone Haliotis midae. This was accomplished by inserting a promotorless gfp gene into the chromosome of the bacterium which was incorporated in an artificial, fishmeal-based abalone feed. In situ histological comparison of abalone fed either a basal diet or the basal diet supplemented with V. midae SY9::Tn10.52 using a cocktail of DNA probes to the gfp gene localised the probiont to the crop/stomach and intestinal regions of the H. midae digestive tract. Generally, the ingested probiotic bacterium occurred in association with feed and particulate matter within the crop/stomach and intestinal regions, as well as adhered to the wall of the crop/stomach. Histological immunohistochemical examination using polyclonal anti-VmproA antibodies localised an extracellular protease produced by V. midae SY9 to the H. midae crop/stomach and intestine where it appeared to be associated with feed and/or other particulate matter in the abalone gut. Thus the data suggests that V. midae SY9 colonises and/or adheres to the mucous lining of the abalone gut. Furthermore, the close association observed between the bacterium, its extracellular protease and ingested feed particles supports the theory that V. midae SY9 elevates in situ digestive enzyme levels and thus enhances feed digestion in farmed abalone.

Investigating the Microbial Metabolic Activity on Mineral Surfaces of Pyrite-Rich Waste Rocks in an Unsaturated Heap-Simulating Column System

Microbial association with and colonisation of mineral surfaces plays a key role in enhancing the extraction of metals from ores during heap bioleaching processes. On the other hand, if uncontrolled, the same association can also lead to the generation of acid rock drainage (ARD) effluents from mine waste. This study aims to measure microbial metabolic activity of a mixed mesophilic culture on the surfaces of pyrite-bearing waste rocks of different grades over time. The waste rocks are milled, size fractionated and coated onto glass beads, to provide a defined surface area. The metabolic activity on the mineral surface is measured with isothermal microcalorimetry (IMC) complemented with scanning electron microscopy (SEM) and analysis of solution chemistry to measure leach agents and metal release into the pregnant leach solution (PLS). The waste rock samples showed a similar degree of leaching when the solution chemistry was analysed, despite having different sulphide content. However, when metabolic activity of the micro-organisms on the mineral surface was measured, greater activity was seen with higher sulphide content. This data informs an ongoing study to establish a flow-through configuration of the biokinetic test for ARD prediction accounting for both leach solution and microbial-mineral interaction as well as differing kinetics of acid-neutralising and generating reactions to enable the refinement of the current batch method.

Linking Microbial Community Dynamics in BIOX® Leaching Tanks to Process Conditions: Integrating Lab and Commercial Experience

In the commercial BIOX® process, an acidophilic mixed bacterial and archaeal community dominated by iron and sulphur oxidising microorganisms is used to facilitate the recovery of precious metals from refractory gold-bearing sulphidic mineral concentrates. Characterisation of the microbial communities associated with commercial BIOX® reactors from four continents revealed a significant shift in the microbial community structure compared to that of the seed culture, maintained at SGS (South Africa). This has motivated more detailed study of the microbial community dynamics in the process. Microbial speciation of a subset of the BIOX® reactors at Fairview mines (Barberton, South Africa) and two laboratory maintained reactors housed at Centre for Bioprocess Engineering Research, University of Cape Town, has been performed tri-annually for three years by quantitative real-time polymerase chain reaction. The laboratory BIOX® culture maintained on Fairview concentrate was dominated by the desired iron oxidiser, Leptospirillum ferriphilum, and sulphur oxidiser, Acidithiobacillus caldus, when operated under standard BIOX® conditions. Shifts in the microbial community as a result of altered operating conditions were transient and did not result in a loss of the microbial diversity of the BIOX® culture. The community structure of the Fairview mines BIOX® reactor tanks showed archaeal dominance of these communities by organisms such as the iron oxidiser Ferroplasma acidiphilum and a Thermoplasma sp. for the period monitored. Shifts in the microbial community were observed across the monitoring period and mapped to changes in performance of the commercial process plant. Understanding the effect of changes in the plant operating conditions on the BIOX® community structure may assist in providing conditions that support the desired microbial consortium for optimal biooxidation to maximize gold recovery.

Analysis of Microbial Communities Associated with Bioremediation Systems for Thiocyanate-Laden Mine Water Effluents

During the processing of refractory gold ores, cyanide (CN-) and residual sulphur species react to form an effluent stream containing thiocyanate (SCN-) and residual CN-. The release of SCN- and CN- containing effluent water to the environment is prohibited, necessitating effective treatment prior to discharge and/or reuse of contaminated plant water. Biologically mediated effluent remediation processes have been developed for commercial use, to remediate SCN- containing effluents, with the aim of enabling recycling of process water and improving the quality of effluent water prior to disposal. Bioremediation processes to treat these effluents rely on a complex consortium of microorganisms to metabolise the SCN- resulting in the production of ammonium that is in turn removed by conversion to nitrite and subsequent denitrification. Increasingly, genomic methods are being used to investigate processes in wastewater treatment to identify key microbial species and, thereby, inform the rationale design and operation of these bioremediation systems. The microbial ecology of laboratory-based SCN- degrading bioprocesses have been investigated, using genome resolved metagenomics, to provide detailed information on the community composition and metabolic profile of abundant microbial community members. Our on-going research is focused on developing a greater understanding of the heterotrophic and autotrophic populations of microorganisms within the SCN- degrading community as well as the role of the component members in SCN- destruction. We are interested in the formation of microbial biofilm and the spatial distribution of key microorganisms within the resulting biofilm communities. This information is being used to inform further rational development of SCN- degradation processes for treatment of contaminated wastewater effluents.

Comparative Analysis of the Sulfate-Reducing Performance and Microbial Colonisation of Three Continuous Reactor Configurations with Varying Degrees of Biomass Retention

Biological sulfate reduction represents an alternative and sustainable option to reduce the high sulfate load, precipitate heavy metals and neutralise the acidity associated with acid rock drainage (ARD). Sulfate-reducing enrichment cultures have been developed on simple and complex electron donors from several environmental samples and used to inoculate three reactor configurations, namely a continuous stirred tank bioreactor, up-flow anaerobic packed bed reactor and a linear flow channel reactor, with varying degrees of biomass retention provided by carbon microfibres and polyurethane foam. These matrices are included to enhance microbial attachment and colonisation, allowing for the decoupling of hydraulic retention time and biomass retention time. The bioreactor systems are operated under increasingly stringent conditions through the reduction in the hydraulic residence time. The biological sulfate reduction performance and the biomass concentration of planktonic, matrix-attached and matrix-associated communities are routinely monitored. This investigation makes use of biomass quantification of the planktonic community and, following detachment, the matrix-associated community to investigate the resultant microbial communities in these reactor systems. Evaluation of these mixed microbial communities, and their link to process performance, provides an opportunity to impact the design and operation of pilot- and industrial-scale bioprocesses.

Liquid Phase Multiplex High-Throughput Screening of Metagenomic Libraries Using p -Nitrophenyl-Linked Substrates for Accessory Lignocellulosic Enzymes

To access the genetic potential contained in large metagenomic libraries, suitable high-throughput functional screening methods are required. Here we describe a high-throughput screening approach which enables the rapid identification of metagenomic library clones expressing functional accessory lignocellulosic enzymes. The high-throughput nature of this method hinges on the multiplexing of both the E. coli metagenomic library clones and the colorimetric p-nitrophenyl linked substrates which allows for the simultaneous screening for β-glucosidases, β-xylosidases, and α-L-arabinofuranosidases. This method is readily automated and compatible with high-throughput robotic screening systems.