Harnessing Fermentation May Enhance the Performance of Biological Sulfate-Reducing Bioreactors.

Biological sulfate reduction (BSR) represents a promising strategy for bioremediation of sulfate-rich waste streams, yet the impact of metabolic interactions on performance is largely unexplored. Here, genome-resolved metagenomics was used to characterize 17 microbial communities in reactors treating synthetic sulfate-contaminated solutions. Reactors were supplemented with lactate or acetate and a small amount of fermentable substrate. Of the 163 genomes representing all the abundant bacteria, 130 encode 321 NiFe and FeFe hydrogenases and all genomes of the 22 sulfate-reducing microorganisms (SRM) encode genes for H2 uptake. We observed lactate oxidation solely in the first packed bed reactor zone, with propionate and acetate oxidation in the middle and predominantly acetate oxidation in the effluent zone. The energetics of these reactions are very different, yet sulfate reduction kinetics were unaffected by the type of electron donor available. We hypothesize that the comparable rates, despite the typically slow growth of SRM on acetate, are a result of the consumption of H2 generated by fermentation. This is supported by the sustained performance of a predominantly acetate-supplemented stirred tank reactor dominated by diverse fermentative bacteria encoding FeFe hydrogenase genes and SRM capable of acetate and hydrogen consumption and CO2 assimilation. Thus, addition of fermentable substrates to stimulate syntrophic relationships may improve the performance of BSR reactors supplemented with inexpensive acetate.

Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts.

BACKGROUND: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. RESULTS: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. CONCLUSIONS: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.

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.

Bioreactor microbial ecosystems for thiocyanate and cyanide degradation unravelled with genome-resolved metagenomics.

Gold ore processing uses cyanide (CN(-) ), which often results in large volumes of thiocyanate- (SCN(-) ) contaminated wastewater requiring treatment. Microbial communities can degrade SCN(-) and CN(-) , but little is known about their membership and metabolic potential. Microbial-based remediation strategies will benefit from an ecological understanding of organisms involved in the breakdown of SCN(-) and CN(-) into sulfur, carbon and nitrogen compounds. We performed metagenomic analysis of samples from two laboratory-scale bioreactors used to study SCN(-) and CN(-) degradation. Community analysis revealed the dominance of Thiobacillus spp., whose genomes harbour a previously unreported operon for SCN(-) degradation. Genome-based metabolic predictions suggest that a large portion of each bioreactor community is autotrophic, relying not on molasses in reactor feed but using energy gained from oxidation of sulfur compounds produced during SCN(-) degradation. Heterotrophs, including a bacterium from a previously uncharacterized phylum, compose a smaller portion of the reactor community. Predation by phage and eukaryotes is predicted to affect community dynamics. Genes for ammonium oxidation and denitrification were detected, indicating the potential for nitrogen removal, as required for complete remediation of wastewater. These findings suggest optimization strategies for reactor design, such as improved aerobic/anaerobic partitioning and elimination of organic carbon from reactor feed.

The effect of degree and timing of nitrogen limitation on lipid productivity in Chlorella vulgaris.

Improvements in lipid productivity would enhance the economic feasibility of microalgal biodiesel. In order to optimise lipid productivity, both the growth rate and lipid content of algal cells must be maximised. The lipid content of many microalgae can be enhanced through nitrogen limitation, but at the expense of biomass productivity. This suggests that a two-stage nitrogen supply strategy might improve lipid productivity. Two different nitrogen supply strategies were investigated for their effect on lipid productivity in Chlorella vulgaris. The first was an initial nitrogen-replete stage, designed to optimise biomass productivity, followed by nitrogen limitation to enhance lipid content (two-stage batch) and the second was an initial nitrogen-limited stage, designed to maximise lipid content, followed by addition of nitrogen to enhance biomass concentration (fed-batch). Volumetric lipid yield in nitrogen-limited two-stage batch and fed-batch was compared with that achieved in nitrogen-replete and nitrogen-limited batch culture. In a previous work, maximum lipid productivity in batch culture was found at an intermediate level of nitrogen limitation (starting nitrate concentration of 170 mg L(-1)). Overall lipid productivity was not improved by using fed-batch or two-stage culture strategies, although these strategies showed higher volumetric lipid concentrations than nitrogen-replete batch culture. The dilution of cultures prior to nitrogen deprivation led to increased lipid accumulation, indicating that the availability of light influenced the rate of lipid accumulation. However, dilution did not lead to increased lipid productivity due to the resulting lower biomass concentration.

The effect of nitrogen limitation on lipid productivity and cell composition in Chlorella vulgaris.

Chlorella vulgaris accumulates lipid under nitrogen limitation, but at the expense of biomass productivity. Due to this tradeoff, improved lipid productivity may be compromised, despite higher lipid content. To determine the optimal degree of nitrogen limitation for lipid productivity, batch cultures of C. vulgaris were grown at different nitrate concentrations. The growth rate, lipid content, lipid productivity and biochemical and elemental composition of the cultures were monitored for 20 days. A starting nitrate concentration of 170 mg L(-1) provided the optimal tradeoff between biomass and lipid production under the experimental conditions. Volumetric lipid yield (in milligram lipid per liter algal culture) was more than double that under nitrogen-replete conditions. Interpolation of the data indicated that the highest volumetric lipid concentration and lipid productivity would occur at nitrate concentrations of 305 and 241 mg L(-1), respectively. There was a strong correlation between the nitrogen content of the cells and the pigment, protein and lipid content, as well as biomass and lipid productivity. Knowledge of the relationships between cell nitrogen content, growth, and cell composition assists in the prediction of the nitrogen regime required for optimal productivity in batch or continuous culture. In addition to enhancing lipid productivity, nitrogen limitation improves the lipid profile for biodiesel production and reduces the requirement for nitrogen fertilizers, resulting in cost and energy savings and a reduction in the environmental burden of the process.

Sulfur oxidation kinetics of Acidithiobacillus caldus and its inhibition on exposure to thiocyanate present in cyanidation tailings wastewater.

The sulfur oxidation kinetics of an industrial strain of Acidithiobacillus caldus (At. caldus) cultured on elemental sulfur was explored in batch experiments in the absence and presence of thiocyanate (SCN-), a toxin inherent within cyanidation tailings wastewater. The Contois rate expression accurately described At. caldus sulfate generation (R2 > 0.93) and microbial growth (R2 > 0.87). For a culture maintained at 45 °C a maximum specific growth rate (µmax) of 0.105 h-1, sulfate yield from biomass (Ypx) of 4.8 × 10-9 mg SO42-.cell-1, and Contois affinity coefficient (Kx) of 1.56 × 10-8 mg S.cell-1 were established. The presence of SCN- (0 mg/L - 20 mg/L) in the bulk solution inhibited the microbial system competitively. Moreover, SCN- impeded microbial growth differentially; the rate expression was therefore partitioned with respect to SCN- concentration and inhibition constants (Ki) were determined for each region. Adaptation to discrete concentrations of SCN- (1 mg/L and 20 mg/L) improved SCN- tolerance in At. caldus; however, adapted strains exhibited reduced sulfur oxidation potential when cultured under thiocyanate-free conditions relative to the non-adapted control strain. To describe the adapted systems accurately, the Contois affinity coefficient (Kx) was revised to reflect the suspected metabolic decline. The derived Kx values increased in magnitude and affirmed an innate reduction in microbial substrate affinity or substrate adsorption capacity. Inclusion of these updated Kx constants within the rate equation suitably represented the experimental data for both adapted At. caldus strains with R2 > 0.94. Furthermore, the impact of adaptation on the inhibition kinetics and inhibition mechanism associated with SCN- exposure were reviewed. Thiocyanate inhibited sulfur oxidation non-competitively in the adapted strains, and the shift in inhibition mechanism may be attributed to the compromised metabolic state following adaptation.

Microbial immobilisation and adaptation to Cu2+ enhances microbial Fe2+ oxidation for bioleaching of printed circuit boards in the presence of mixed metal ions.

A circular economy requires effective re-use of finite resources, such as metals from waste electrical and electronic equipment (WEEE). Bioleaching for extraction and recovery of base metals from printed circuit boards (PCBs) before recovering precious metals has potential to increase metal circularity. However, inhibition by base metals released from the PCBs and accumulated in PCB leachates on microbial Fe2+ oxidation, a critical bioleaching sub-process for Fe3+ regeneration, can limit this approach. Here, we explore the potential of microbial immobilisation on polyurethane foam (PUF) and adaptation to cupric ions to minimise inhibition by mixed metals released from PCBs, particularly zinc, nickel, and tin, and enhancing Fe2+ oxidation rates in PCB bioleaching systems. A mixed mesophilic culture dominant in Leptospirillum ferriphilum, Acidiplasma cupricumulans and Acidithiobacillus caldus was immobilised on PUF and adapted to 6 g/L Cu2+. Tolerance of Cu-adapted immobilised cells to the inhibitory metal ions Zn2+, Ni2+, and Sn2+, as individual (0-10 g/L) and mixed metal ions at concentrations typically leached from PCBs at solids loadings of 0-20% (mass/volume) was compared to that of non-adapted immobilised cells. Further, the impact of solutes from PCB leachates was evaluated. Inhibition by individual metal ions decreased in the order Sn2+ > Ni2+ > Zn2+. Inhibition of ferrous iron oxidation by mixed metal ions was synergistic with respect to individual metal ions. PCB leachates were more inhibitory than both mixed and individual metal ions even where metal concentration was low. Cu-adapted immobilised cells exhibited higher tolerance to increasing concentrations of inhibitory metal ions than non-adapted cells. These results are promising for the application of Cu-adapted cells in the bioleaching of PCBs and multi-metal concentrates.

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6.

BACKGROUND: 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 (0.20 µmol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. 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.

Attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum cultured under varying conditions to pyrite, chalcopyrite, low-grade ore and quartz in a packed column reactor.

The attachment of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum spp. grown on ferrous medium or adapted to a pyrite mineral concentrate to four mineral substrata, namely, chalcopyrite and pyrite concentrates, a low-grade chalcopyrite ore (0.5 wt%) and quartzite, was investigated. The quartzite represented a typical gangue mineral and served as a control. The attachment studies were carried out in a novel particle-coated column reactor. The saturated reactor containing glass beads, which were coated with fine mineral concentrates, provided a quantifiable surface area of mineral concentrate and maintained good fluid flow. A. ferrooxidans and Leptospirillum spp. had similar attachment characteristics. Enhanced attachment efficiency occurred with bacteria grown on sulphide minerals relative to those grown on ferrous sulphate in an ore-free environment. Selective attachment to sulphide minerals relative to gangue materials occurred, with mineral adapted cultures attaching to the minerals more efficiently than ferrous grown cultures. Mineral-adapted cultures showed highest levels of attachment to pyrite (74% and 79% attachment for A. ferrooxidans and L. ferriphilum, respectively). This was followed by attachment of mineral-adapted cultures to chalcopyrite (63% and 58% for A. ferrooxidans and L. ferriphilum, respectively). A. ferrooxidans and L. ferriphilum exhibited lower levels of attachment to low-grade ore and quartz relative to the sulphide minerals.

Vitamin interdependencies predicted by metagenomics-informed network analyses and validated in microbial community microcosms.

Metagenomic or metabarcoding data are often used to predict microbial interactions in complex communities, but these predictions are rarely explored experimentally. Here, we use an organism abundance correlation network to investigate factors that control community organization in mine tailings-derived laboratory microbial consortia grown under dozens of conditions. The network is overlaid with metagenomic information about functional capacities to generate testable hypotheses. We develop a metric to predict the importance of each node within its local network environments relative to correlated vitamin auxotrophs, and predict that a Variovorax species is a hub as an important source of thiamine. Quantification of thiamine during the growth of Variovorax in minimal media show high levels of thiamine production, up to 100 mg/L. A few of the correlated thiamine auxotrophs are predicted to produce pantothenate, which we show is required for growth of Variovorax, supporting that a subset of vitamin-dependent interactions are mutualistic. A Cryptococcus yeast produces the B-vitamin pantothenate, and co-culturing with Variovorax leads to a 90-130-fold fitness increase for both organisms. Our study demonstrates the predictive power of metagenome-informed, microbial consortia-based network analyses for identifying microbial interactions that underpin the structure and functioning of microbial communities.

Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon.

CYP153A6 is a well-studied terminal alkane hydroxylase which has previously been expressed in Pseudomonas putida and Escherichia coli by using the pCom8 plasmid. In this study, CYP153A6 was successfully expressed in E. coli BL21(DE3) by cloning the complete operon from Mycobacterium sp. HXN-1500, also encoding the ferredoxin reductase and ferredoxin, into pET28b(+). LB medium with IPTG as well as auto-induction medium was used to express the proteins under the T7 promoter. A maximum concentration of 1.85 µM of active CYP153A6 was obtained when using auto-induction medium, while with IPTG induction of LB cultures, the P450 concentration peaked at 0.6-0.8 µM. Since more biomass was produced in auto-induction medium, the specific P450 content was often almost the same, 0.5-1.0 µmol P450 g (DCW)⁻¹, for both methods. Analytical scale whole-cell biotransformations of n-octane were conducted with resting cells, and it was found that high P450 content in biomass did not necessarily result in high octanol production. Whole cells from LB cultures induced with IPTG gave higher specific and volumetric octanol formation rates than biomass from auto-induction medium. A maximum of 8.7 g octanol L (BRM)⁻¹ was obtained within 24 h (0.34 g L (BRM)⁻¹ h⁻¹) with IPTG-induced cells containing only 0.20 µmol P450 g (DCW)⁻¹, when glucose (22 g L (BRM)⁻¹) was added for cofactor regeneration.

Integrated Kinetic Modelling and Microbial Profiling Provide Insights Into Biological Sulfate-Reducing Reactor Design and Operation.

Biological sulfate reduction (BSR) is an attractive approach for the bioremediation of sulfate-rich wastewater streams. Many sulfate-reducing microorganisms (SRM), which facilitate this process, have been well-studied in pure culture. However, the role of individual members of microbial communities within BSR bioreactors remains understudied. In this study we investigated the performance of two up-flow anaerobic packed bed reactors (UAPBRs) supplemented primarily with acetate and with lactate, respectively, during a hydraulic retention time (HRT) study set up to remediate sulfate-rich synthetic wastewater over the course of 1,000 + days. Plug-flow hydrodynamics led to a continuum of changing volumetric sulfate reduction rates (VSRRs), available electron donors, degrees of biomass retention and compositions of microbial communities throughout these reactors. Microbial communities throughout the successive zones of the reactors were resolved using 16S rRNA gene amplicon sequencing which allowed the association of features of performance with discrete microorganisms. The acetate UAPBR achieved a maximum VSRR of 23.2 mg.L-1. h-1 at a one-day HRT and a maximum sulfate conversion of the 1 g/L sulfate of 96% at a four-day HRT. The sulfate reduction reactions in this reactor could be described with a reaction order of 2.9, an important observation for optimisation and future scale-up. The lactate UAPBR achieved a 96% sulfate conversion at one-day HRT, corresponding with a VSRR of 40.1 mg.L-1. h-1. Lactate was supplied in this reactor at relatively low concentrations necessitating the subsequent use of propionate and acetate, by-products of lactate fermentation with acetate also a by-product of incomplete lactate oxidation, to achieve competitive performance. The consumption of these electron donors could be associated with specific SRM localised within biofilms of discrete zones. The sulfate reduction rates in the lactate UAPBR could be modelled as first-order reactions, indicating effective rates were conferred by these propionate- and acetate-oxidising SRM. Our results demonstrate how acetate, a low-cost substrate, can be used effectively despite low associated SRM growth rates, and that lactate, a more expensive substrate, can be used sparingly to achieve high VSRR and sulfate conversions. We further identified the preferred environment of additional microorganisms to inform how these microorganisms could be enriched or diminished in BSR reactors.

Sulfur and oxygen isotope constraints on sulfate sources and neutral rock drainage-related processes at a South African colliery.

Understanding the fundamental controls that govern the generation of mine drainage is essential for waste management strategies. Combining the isotopic composition of water (H and O) and dissolved sulfate (S and O) with hydrogeochemical measurements of surface and groundwater, microbial analysis, composition of sediments and precipitates, and geochemical modeling results in this study we discussed the processes that control mine water chemistry and identified the potential source(s) and possible mechanisms governing sulfate formation and transformation around a South African colliery. Compared to various South African water standards, water samples collected from the surroundings of a coal waste disposal facility had elevated Fe2+ (0.9 to 56.9 mg L-1), Ca (33.0 to 527.0 mg L-1), Mg (6.2 to 457.0 mg L-1), Mn (0.1 to 8.6 mg L-1) and SO4 (19.7 to 3440.8 mg L-1) and circumneutral pH. The pH conditions are mainly controlled by the release of H+ from pyrite oxidation and the subsequent dissolution of carbonates and aluminosilicate minerals. The phases predicted to precipitate by equilibrium calculation were green rusts, ferrihydrite, gypsum, ±epsomite. Low concentrations of deleterious metals in solution are due to their low abundance in the local host rocks, and their attenuation through adsorption onto secondary Fe precipitates and co-precipitation at the elevated pH values. The δ34S values of sulfate are enriched (-6.5 ‰ to +5.6 ‰) compared to that of pyrite sampled from the mine (mean -22.5 ‰) and overlap with that of the organic sulfur of coal from the region (-2.5 to +4.9 ‰). The presence of both sulfur reducing and oxidizing bacteria were detected in the collected sediment samples. Combined, the data are consistent with the dissolved sulfate in the sampled waters from the colliery being derived primarily from pyrite probably with the subordinate contribution of organic sulfur, followed by its partial removal through precipitation and microbially-induced reduction.

Non-Hydrolyzable Plastics - An Interdisciplinary Look at Plastic Bio-Oxidation.

Enzymatic plastic conversion has emerged recently as a potential adjunct and alternative to conventional plastic waste management technology. Publicity over progress in the enzymatic degradation of polyesters largely neglects that the majority of commercial plastics, including polyethylene, polypropylene, polystyrene and polyvinyl chloride, are still not biodegradable. Details about the mechanisms used by enzymes and an understanding of macromolecular factors influencing these have proved to be vital in developing biodegradation methods for polyesters. To expand the application of enzymatic degradation to other more recalcitrant plastics, extensive knowledge gaps need to be addressed. By drawing on interdisciplinary knowledge, we suggest that physicochemical influences also have a crucial impact on reactions in less well-studied types of plastic, and these need to be investigated in detail.

Thiocyanate and Organic Carbon Inputs Drive Convergent Selection for Specific Autotrophic Afipia and Thiobacillus Strains Within Complex Microbiomes.

Thiocyanate (SCN-) contamination threatens aquatic ecosystems and pollutes vital freshwater supplies. SCN--degrading microbial consortia are commercially adapted for remediation, but the impact of organic amendments on selection within SCN--degrading microbial communities has not been investigated. Here, we tested whether specific strains capable of degrading SCN- could be reproducibly selected for based on SCN- loading and the presence or absence of added organic carbon. Complex microbial communities derived from those used to treat SCN--contaminated water were exposed to systematically increased input SCN concentrations in molasses-amended and -unamended reactors and in reactors switched to unamended conditions after establishing the active SCN--degrading consortium. Five experiments were conducted over 790 days, and genome-resolved metagenomics was used to resolve community composition at the strain level. A single Thiobacillus strain proliferated in all reactors at high loadings. Despite the presence of many Rhizobiales strains, a single Afipia variant dominated the molasses-free reactor at moderately high loadings. This strain is predicted to break down SCN- using a novel thiocyanate desulfurase, oxidize resulting reduced sulfur, degrade product cyanate to ammonia and CO2 via cyanate hydratase, and fix CO2 via the Calvin-Benson-Bassham cycle. Removal of molasses from input feed solutions reproducibly led to dominance of this strain. Although sustained by autotrophy, reactors without molasses did not stably degrade SCN- at high loading rates, perhaps due to loss of biofilm-associated niche diversity. Overall, convergence in environmental conditions led to convergence in the strain composition, although reactor history also impacted the trajectory of community compositional change.

Cross-correlating analyses of mineral-associated microorganisms in an unsaturated packed bed flow-through column test; cell number, activity and EPS.

In heap bioleaching and waste-rock dumps, complex microbial communities exist in the flowing and interstitial liquid phases and mineral surface-associated biofilms, often embedded in extracellular polymeric substances (EPS). Microbial activity in the interstitial phase and mineral ore surface facilitates mineral degradation, resulting in either metal recovery or acidic, metal -bearing drainage from sulfidic waste-rock. Determining microbial presence and activity through microorganisms leaving the heap or dump has severe limitations. Hence, increasingly the ore-bed is sampled to quantify and characterise this. Here, methods for cell detachment and quantification, microbial activity measurement on the mineral surface and evaluation of EPS, quantitatively and biochemically, were refined and validated to assess microbial presence, using mineral coated beads in continuous flow-through columns. Number of wash steps required were assessed over increasing colonisation times over 30 days. Microbial cells colonising the mineral surface, pre- and post-washing were visualised by scanning electron microscopy (SEM) and their activity quantified by isothermal microcalorimetry (IMC). Using IMC, detachment and enumeration of detached cells, we demonstrated that 6-8 washes provided a reliable estimation of mineral-associated microorganisms, with less than 10% of cells or microbial activity associated with the surface following treatment. This allowed consolidated refinement of the protocol using traditional detachment method, SEM and IMC to provide correlative data. Extraction of EPS in a complete flow-through system is reported for the first time and the biochemical composition was similar to those reported under batch bioleaching conditions.

Biochemical and structural insights into the cytochrome P450 reductase from Candida tropicalis.

Cytochrome P450 reductases (CPRs) are diflavin oxidoreductases that supply electrons to type II cytochrome P450 monooxygenases (CYPs). In addition, it can also reduce other proteins and molecules, including cytochrome c, ferricyanide, and different drugs. Although various CPRs have been functionally and structurally characterized, the overall mechanism and its interaction with different redox acceptors remain elusive. One of the main problems regarding electron transfer between CPRs and CYPs is the so-called "uncoupling", whereby NAD(P)H derived electrons are lost due to the reduced intermediates' (FAD and FMN of CPR) interaction with molecular oxygen. Additionally, the decay of the iron-oxygen complex of the CYP can also contribute to loss of reducing equivalents during an unproductive reaction cycle. This phenomenon generates reactive oxygen species (ROS), leading to an inefficient reaction. Here, we present the study of the CPR from Candida tropicalis (CtCPR) lacking the hydrophobic N-terminal part (Δ2-22). The enzyme supports the reduction of cytochrome c and ferricyanide, with an estimated 30% uncoupling during the reactions with cytochrome c. The ROS produced was not influenced by different physicochemical conditions (ionic strength, pH, temperature). The X-ray structures of the enzyme were solved with and without its cofactor, NADPH. Both CtCPR structures exhibited the closed conformation. Comparison with the different solved structures revealed an intricate ionic network responsible for the regulation of the open/closed movement of CtCPR.

A chemo-enzymatic oxidation cascade to activate C-H bonds with in situ generated H2O2.

Continuous low-level supply or in situ generation of hydrogen peroxide (H2O2) is essential for the stability of unspecific peroxygenases, which are deemed ideal biocatalysts for the selective activation of C-H bonds. To envisage potential large scale applications of combined catalytic systems the reactions need to be simple, efficient and produce minimal by-products. We show that gold-palladium nanoparticles supported on TiO2 or carbon have sufficient activity at ambient temperature and pressure to generate H2O2 from H2 and O2 and supply the oxidant to the engineered unspecific heme-thiolate peroxygenase PaDa-I. This tandem catalyst combination facilitates efficient oxidation of a range of C-H bonds to hydroxylated products in one reaction vessel with only water as a by-product under conditions that could be easily scaled.

Stratification of microbial communities throughout a biological sulphate reducing up-flow anaerobic packed bed reactor, revealed through 16S metagenomics.

Biological sulphate reduction (BSR) is a promising low-cost treatment of acid rock drainage effluents. In this paper, the system performance and microbial ecology of a lactate supplemented BSR up-flow anaerobic packed bed reactor (UAPBR) are evaluated across reactor height and compared to a continuous stirred tank reactor (CSTR). The biomass concentrations of planktonic and biofilm communities were quantified and subsequently characterised by 16S rRNA gene amplicon sequencing. The defined microbial communities were shown to correlate with differing availability of lactate, volatile fatty acids produced from lactate degradation and sulphate concentration. The UAPBR was able to achieve near complete sulphate conversion at a 4-day hydraulic residence time (HRT) at a sulphate feed concentration of 10.41 mM (1 g/L). The high volumetric sulphate reduction rate of 0.184 mM/L.h achieved in the first third of the reactor was attributed to OTUs present in the planktonic and biofilm communities. While the scavenging of sulphate within the final third of the UAPBR was attributed to an acetate oxidising genus of SRB which was not detected in the lactate-fed CSTR. The detailed analyses of the microbial communities throughout the UAPBR and CSTR contribute to the growing understanding of the impact of the microbial communities of BSR reactors on system performance.