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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. Significant progress has been made in understanding the enzymatic degradation of hydrolyzable plastics with heteroatoms in their backbone structure, but information about the mechanisms and limiting factors for reactions of plastics containing C–C backbones is lacking.These plastics have been less well studied, and knowledge gained in related fields is invoked to propose reaction characteristics.Macromolecular architecture has been shown to govern enzymatic degradation of hydrolyzable plastics as well as abiotic reactions in polymers. We propose that this is applicable to all types of plastics as a determining factor according to the chain-flexibility hypothesis.Thermostable laccase mediator systems are promising enzyme system candidates.

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.

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.

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.

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^sub DCW^^sup -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^sub BRM^^sup -1^ was obtained within 24 h (0.34 g L^sub BRM^^sup -1^h^sup -1^) with IPTG-induced cells containing only 0.20 μmol P450 g^sub DCW^^sup -1^, when glucose (22 g L^sub BRM^^sup -1^ ) was added for cofactor regeneration.[PUBLICATION ABSTRACT]

In heap (bio)leaching processes, a substantial fraction of the valuable mineral is positioned below the ore particle surface. X-ray micro-computed tomography (μCT) was used to quantify the effect of ore type and structure and operating temperature on the leaching of this mineral, to investigate the rate-controlling factors. Mini-leaching columns containing agglomerated chalcopyrite, pyrite, and malachite ores were scanned by X-ray μCT (13.40 µm resolution) at select time intervals. The leaching of a relatively porous malachite ore was reaction-controlled, with no leaching penetration limitation into the ore particles. For two less porous ore types, the structure and higher porosity of the agglomerate rim and conditions that resulted in the degradation of the full ore matrix structure were found to be the determining variables of the leaching extent and time. In the case of a chalcopyrite ore, an enhancement of copper recovery and sulphide mineral dissolution with increasing temperature was attributable to the increased leaching penetration distance and crack development in addition to thermodynamically expected increased leaching rate. Increasing temperature did not affect the maximum penetration distance for the waste rock containing pyrite, with no crack development observed. Overall increases in iron recovery were due to accelerated leaching rates, though diffusion or mineral access limitations were evident at a higher temperature.

The presence of sulfur in coal processing wastes can lead to environmental impacts, such as acid rock drainage (ARD). However, not all sulfur species are acid-forming, and the implications of sulfur speciation when assessing acid rock drainage potential by means of static chemical tests are not well understood. This study set out to evaluate the implications of different sulfur forms on the assessment of acid rock drainage potential using static laboratory-scale tests and to investigate the reliability of methods for the analysis of such forms for the case of three South African coal processing wastes. Both the International Organization for Standardization (ISO) 157:1996 and Australian Coal Association Research Program (ACARP) C15034 protocols were found to be suitable tools for analyzing the different forms of sulfur. Acid-generating sulfur forms constituted between 53% and 64% of the total sulfur in the wastes evaluated, with the maximum potential acidity (MPA) and net acid-producing potential (NAPP) values calculated on the basis of acid-forming sulfur being significantly lower than those calculated on the basis of total sulfur content. Results also showed that the partial conversion of sulfur species under the relatively aggressive conditions of the acid-neutralizing capacity (ANC) and net acid generation (NAG) tests may overestimate the potential acid generating potential in the case of coal. These findings highlight the uncertainties associated with standard ARD static tests and the importance of taking sulfur speciation into account when calculating the MPA for coal processing wastes.

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 g DCW -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.

ARD mitigation is considered by reducing the permeability of acid-generating coal interburden waste rock dumps. Fine desulfurised benign tailings were used to conceal exposed surface active sites from air, water and more aggressive sulfur-and iron-oxidizing microorganisms in acidic environments. Test columns containing interburden waste rock co-disposed with benign tailings were configured as either alternating layered covers or as a blended mixture of tailings and discards. Demonstration of the efficacy of the procedure was assessed by analyzing effluent solution concentrations of the concurrently operated simulated ore beds following inoculation with micro-organisms implicated in bioleaching (biotic mode) or in the absence of inoculation (abiotic mode). The presence of fine tailings both minimized the infiltration of oxidants to the porous ore and void space within the packed bed and provided neutralisation. In biotic mode, tests with layered covers of desulfurised tailings were less successful at limiting microbial activity and associated accelerated oxidation reactions. Blended columns resulted in reduced leaching reactions and an increase in the mean residence time of the solution phase. By increasing the solution residence time, an aqueous cover was concurrently formed, evolving into a bifunctional mitigating measure

Characterisation of the risk of acid rock drainage is typically achieved through the quantification of acid-generating and acid-consuming components present within a sample using initial laboratory-scale, chemical static tests. Such tests, however, consider ARD generation under chemical conditions and do not account for the role of micro-organisms. Their focus is exclusively on the net potential for acid generation, with no account of metal deportment or the relative rate of acid generation and consumption. The present study investigates the ARD potential of two ultrafine coal wastes samples using the standard static tests as well as the UCT biokinetic test to account for microbial ARD generation. The deportment of metal species under each test condition was also considered. The UCT biokinetic test results supported the static test classification, adding provided preliminary kinetic data on the ARD generation. Sequential chemical extraction tests allowed for differentiation of the host minerals according to their leaching potentials, providing supporting evidence for the deportment of metal species under the characterisation tests, thereby improving the knowledge base on which to classify coal wastes as benign or otherwise.