The rate of mass transfer of lower molecular weight hydrocarbons (naphtha) from bitumen drops in mature fine tailings of oil sand tailings ponds (OSTPs) may control their bioavailability and the associated rate of GHG production. Experiments were conducted using bitumen drops spiked with o-xylene and 1-methylnaphthalene to determine the mass transfer rate of these naphtha components from bitumen drops. The results were compared to simulations using a multi-component numerical model that accounted for transport in the drop and across the oil-water interface. The results demonstrate rate-limited mass transfer, with aqueous concentrations after 60 days of dissolution that were different than those in equilibrium with the initial drop composition (less for o-xylene and greater for 1-methylnaphthalene). The simulations suggest that mole fractions were unchanged at the center of the drop, resulting in concentration gradients out to the oil-water interface. Numerical simulations conducted using different drop sizes and bitumen viscosities also suggest the potential for persistent naphtha dissolution, where the time required to deplete 80% of the o-xylene and 1-methylnaphthalene mass from an oil drop was estimated to be on the order of months to years for mm-sized drops, and years to decades for cm-sized drops assuming instantaneous biodegradation in the aqueous phase surrounding the bitumen.

Recombinant Cupriavidus necator H16/pMPJAS03, expressing a P. putida KT2440 enoyl-CoA hydratase (phaJ), was able to synthesize short-chain-length/ medium-chain-length (scl-mcl) PHA copolymers with a high content of mcl subunits using its native poly(3-hydroxyalkanoate) synthase. The cells were cultivated on fructose with canola oil or canola oil/decanoic acid (DA) mixtures in fed-batch fermentations. The recombinant C. necator H16 (without any synthase modification) produced a polymer composed of 3-hydroxybutyrate (3HB) with mclsubunits, including 3-hydroxyhexanoate (3HHx), and about 300-fold more 3-hydroxyoctanoate (3HO) than the yields reported in previous studies, as well as a significant amount of 3-hydroxydecanoate (3HD). Increasing the DA content in the feed from 0% to 15% v/v increased the molar content of the 3HD subunits from 1.2 to 2.1 mol%. The presence of larger monomers, such as 3HO and 3HD, decreased the crystallinity and melting temperature and modified the mechanical properties of the polymers. Thus, replacing either of the two gene products (phaJ or phaC1) required to produce PHA from CoA-3-hydroxy fatty acids with broader spectrum enzymes, is suitable for the production of commercially useful scl-mcl-PHA.

We examined greenhouse gas (GHG) production upon the addition of ammonium and phosphate to mature fine tailing (MFT) samples from Alberta’s Pond 2/3 (at 5 and 15 m) and Pond 7 (12.5 m) in microcosm studies. The methane production rate in unamended Pond 2/3 MFT correlated with sample age; the production rate was higher in the less dense, more recently discharged MFT samples and lower in the denser, deeper sample. Adding small amounts of naphtha increased methane production, but there was no correlation with increasing naphtha, indicating that naphtha may partition into bitumen, reducing its bioavailability. Although non-detectable phosphate and low ammonium in the pore water indicate that these nutrients were potentially limiting microbial activity, their addition did not significantly affect methanogenesis but somewhat enhanced sulphate and nitrate reduction. Neither ammonium nor phosphate were detected in the pore water when added at low concentrations, but when added at high concentrations, 25–35% phosphate and 30–45% ammonium were lost. These ions likely sorbed to MFT minerals such as kaolinite, which have microbial activity governed by phosphate/ammonium desorption. Hence, multiple limitations affected microbial activity. Sulphate was less effective than nitrate was in inhibiting methanogenesis because H2S may be a less effective inhibitor than NOx− intermediates are, and/or H2S may be more easily abiotically removed. With nitrate reduction, N2O, a potent GHG was produced but eventually metabolized.

Abstract Blends of polycaprolactone (PCL) and poly(3-hydroxyoctanoate) P(3HO) were prepared by melt compounding. These immiscible blends exhibited droplet-matrix morphology at compositions up to 30 wt% P(3HO). Even though the addition of amorphous P(3HO) decreased the crystallinity of PCL, the crystallization temperature of the blends increased by 6 to 7 8C. Blends containing up to 30 wt% P(3HO) had higher crystallization rates, and lower crystallization half-times compared to neat PCL. The viscosity of PCL decreased upon addition of P(3HO), making the blends suitable for processing using a 3D bioplotter. Compositions with 10 to 30 wt% P(3HO) were ideal for processing, because of their improved crystallization kinetics, reduced stickiness and good flow properties. Estimation of the interfacial tension by fitting the Palierne model to the linear viscoelastic properties of the blends revealed good compatibility, which gave rise to synergistic effects in the thermal and mechanical properties. The fibres prepared by 3D bioplotting maintained droplet matrix morphology, with finer particle size than the original compounded material. In addition to favourable viscosity and thermal properties, the extruded fibres containing 30 wt% P(3HO) had comparable modulus to the neat PCL, while exhibiting good ductility. These blends may be suitable alternatives to PCL for biomedical applications, because they provide a range of crystallinities, crystallization rates and viscosities.

The production of short-chain-length polyhydroxyalkanoates (scl-PHAs) in a sugarcane biorefinery setting has been demonstrated to be an effective strategy to reduce production costs. Medium-chain-length PHA (mcl-PHA) have elastomeric properties and are more suitable for high value-added applications, but its industrial production is not yet established. Mcl-PHA synthesis occurs via different metabolic routes and thus requires distinct microorganisms and substrates compared to scl-PHA. In the present study, sucrose-derived carbohydrates were evaluated as co-substrates for the production of mcl-PHA from decanoic acid (DA). Fermentation strategies were investigated to produce mcl-PHA with enriched dominant monomer content, which is desirable for commercial applications. The mcl-PHA production was investigated in carbon-limited, fed-batch fermentations with wild-type and β-oxidation knockout mutant strains of Pseudomonas putida KT2440. The experimental results demonstrated that a mixture of glucose and fructose was a suitable co-feed with DA for mcl-PHA production, yielding equivalent results to those obtained with starch-derived glucose, a more traditional feedstock for PHA production. The use of a β-oxidation-impaired strain was essential to attain high dominant monomer content. A near-homopolymeric mcl-PHA was produced under exponential feeding, containing 99 mol% of 3-hydroxydecanoate. This work demonstrates the potential for near-homopolymeric mcl-PHA production in a sugarcane biorefinery, using hydrolyzed sucrose and DA.

EDITORIAL article Front. Bioeng. Biotechnol., 23 December 2020Sec. Industrial Biotechnology https://doi.org/10.3389/fbioe.2020.618383

The heterologous expression of Vitreoscilla hemoglobin (VHb) is known to enhance short-chain-length (scl) and short-chain-length-medium-chain-length (scl-mcl) polyhydroxyalkanoate (PHA) production in Escherichia coli, Aeromonas hydrophila and Pseudomonas putida, but has not been evaluated for the production of medium-chain-length (mcl) PHA in pseudomonads. Three recombinant strains of P. putida KT2440 were constructed, harboring pBBR1MCS-2 expression plasmids containing 0, 1 and 2 copies of the vgb gene. The vgb gene was placed under the control of the lac promoter, which conferred constitutive expression of vgb, since no lac repressor was produced. Mcl-PHA production was assessed in nonanoic acid-limited, fed-batch fermentations with exponential feeding. Although the VHb levels in the engineered strains were comparable to those observed in vgb-bearing pseudomonads, the recombinant strains had lower biomass production, PHA accumulation, volumetric productivity and product yield when compared to the wild-type strain. The performance of the plasmid-harboring strains decreased with increasing vgb copy numbers, presumably due to an increased metabolic burden arising from plasmid expression. All the fermentations ended at similar times due to insufficient oxygen supply and subsequent uncontrollable foaming, demonstrating that VHb production did not alleviate oxygen limitation. These results indicate that the effectiveness of VHb engineering must be assessed on a case-by-case basis.

The effects of carboxylation (via mercaptoundecanoic acid) on colloidal properties of medium-chain-length poly(3-hydroxyalkanoate) (mcl-PHA) latexes were studied. Non-ionic surfactants tested at 0.4% solids of 11 mol% carboxylated mcl-PHA produced similar particle sizes and particle size distribution (Pdl) with Triton X-100 giving the smallest size. When Triton X-100 was combined with an ionic surfactant, smaller nanoparticles (97.1 +/- 1.1 to 121.7 +/- 5.7 nm) with narrower Pdls (0.21 +/- 10.001 to 0.25 +/- 0.003) were obtained. The combination of SDS and Triton X-100 gave the smallest particle size (97.1 +/- 1.1 nm) and narrowest Pdl (0.21 +/- 0.001). At higher solids content (10%), a mixture of 5 mM SDS and 20 mM Triton X-100 produced stable (zeta potential= -39.6 +/- 0.9) 170.3 +/- 4.6 nm nanoparticles. As carboxylation increased, particle size and hydrophobicity decreased while stability increased. When comparing nanoparticles of similar size and stability, carboxylated ntcl-PHA needed similar to 50% less surfactant to make stable nanoparticles compared to aliphatic mcl-PHAs, with the amount of surfactant required decreasing as carboxylation increased. This is the first study to show that stable nanoparticle suspensions of a range of carboxylated mcl-PHAs above 0.4% solids can be made using a mixture of ionic and nonionic surfactants. (C) 2017 Elsevier B.V. All rights reserved.

Greater than 65% of canola and high-oleic soy oil fatty acids is oleic acid, which is readily converted to nonanoic (NA) and azelaic (AzA) acids by ozonolysis. NA is an excellent substrate for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production but AzA has few uses. Pseudomonas citronellolis DSM 50332 and Pseudomonas fluorescens ATCC 17400, both able to produce mcl-PHA from fatty acids and to grow on AzA as the sole source of carbon and energy, were assessed for the accumulation of mcl-PHA from AzA and NA. In N-limited shake flasks using NA, P. citronellolis produced 32% of its dry biomass as mcl-PHA containing 78% 3-hydroxynonanoate with 22% 3-hydroxyheptanoate. Pseudomonas fluorescens produced only 2% PHA. N-limited P. citronellolis on AzA produced 20% dry weight PHA containing 75% 3-hydroxydecanoate and 25% 3-hydroxyoctanoate, indicative of de novo synthesis. Although selective pressure, including β-oxidation inhibition, under well-controlled (chemostat) conditions was applied to P. citronellolis, no side-chain carboxyl groups were detected. It was concluded that one or more of FabG and PhaJ or the PHA synthase cannot catalyze reactions involving ω-carboxy substrates. However, a process based on oleic acid could be established if Pseudomonas putida was engineered to grow on AzA.

We investigated the effects of the structure of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) on their thermal properties and crystallization kinetics. The predominantly homopolymeric poly(3-hydroxydecanoate), P(3HD)-98, and the poly(3-hydroxydodecanoate), P(3HDD), exhibited sharp crystallization peaks upon cooling, with the latter exhibiting faster crystallization rates. A chemical modification strategy involving reaction with dicumyl peroxide and triallyl trimesate coagent was implemented to introduce branching and enhance the crystallization kinetics of P(3HD-98). Increases in the exothermic crystallization temperature by 8 °C and in the overall crystallinity of the P(3HD)-98 were observed upon chemical modification. The Avrami crystallization kinetic parameters obtained by fitting the isothermal crystallization data revealed a significant increase in the crystallization rate of the modified P(3HD)-98.

Methods of producing medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) with high content of the dominant subunit, 3-hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a β-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting β-oxidation with addition of acrylic acid (Aa) to wild type P. putida KT2440 in carbon-limited, fed-batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA-F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl-PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone (YET) addition allowed the mutant strain to accumulate 74% mcl-PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/hr, at least twice that of published data for any β-oxidation knock-out mutant. Higher biomass concentration was achieved with Aa inhibition of β-oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity, and rate of crystallization.

This is the first study to examine the formulation (selection of ionic and nonionic surfactants and their concentrations) and processing conditions (ultrasonication time and amplitude, and selection of solvent) to make dense suspensions (10 and 30% (w/v)) of medium-chain-length poly(3-hydroxyalkanoate) (mcl-PHA) with particles less than 300 nm. A two-stage emulsification-solvent evaporation process was used. Previous studies made suspensions at much lower solids content (up to 0.4% (w/v)). The dispersed phase was mcl-PHA initially dissolved in methylene chloride, while the continuous phase was water containing one or more surfactants. Water miscible solvents, such as acetone and tetrahydrofuran, could not make dense suspensions of PHA nanoparticles, while those with low water solubility were effective. Among the ionic surfactants, the anionic, sodium dodecyl sulfate (SDS), and the cationic, dodecyltrimethylammonium bromide, produced the smallest particle sizes (both similar to 100 nm). Nanoparticles were more stable when SDS was combined with any of the non-ionic surfactants tested. The zeta potential of nanoparticles stabilized with SDS and polyoxyethylene octyl phenyl ether (Triton X-100) or polyoxyethylene (20) sorbitan monooleate (Tween 80) increased slightly over 30 days, indicating that they may be more effective than the other non-ionic surfactants stabilizers where a decrease was observed. Using the same surfactant formulation, similar size mcl-PHA stable nanoparticle suspensions were produced using either an ultrasonic probe or a more scalable high-shear microfluidic device. (C) 2016 Elsevier Ltd. All rights reserved.

Although poly-3-hydroxyalkanoates (PHAs) and particularily medium-chain-length (mcl)-PHAs are likely to find industrial applications in a latex form, very few studies have examined their behavior in aqueous suspension and none have examined the dense suspensions required commercially. For this reason, the stability of mcl-PHA latexes containing saturated aliphatic (65 mol% 3-hydroxynonanoate, PHN), and for the first time, with vinyl (PHNU) or carboxylated side chains was examined. At 4 g L−1 with no stabilizing agent, PHNU nanoparticles (199.4 ± 3.6 nm) were significantly smaller than those of PHN (211.5 ± 6.4 nm) while carboxylated PHN nanoparticles (76.1 ± 6.4 nm) were substantially smaller than those of either PHN or PHNU with particles stable for more than 110 days. Increasing the PHN concentration to 10 g L−1 also resulted in stable latexes but with larger particles (410.8 ± 5.2 nm). Adjusting the pH of the suspending medium (water) before addition of the polymer (dissolved in acetone) resulted in much smaller PHN particles at pH = 11.3 (134 ± 2 nm) than at pH = 4.3 (312 ± 8 nm) at a 4 g L−1 final polymer concentration. Zeta potentials of PHN suspensions decreased with pH, likely due to the carboxyl end groups. Above a pH of 4.0, adjusting the pH after particle formation had little effect. NaCl addition could be used to agglomerate and ultimately precipitate the particles. Stabilizers such as surfactants will likely be required to produce denser mcl-PHA latexes with suitable particle size for certain applications such as coatings and toner production.

After oil sands process affected water (OSPW) was treated in a continuous flow biofilm reactor, about 40% of the organic compounds in the acid extractable fraction (AEF) including naphthenic acids (NAs) were degraded resulting in a reduction of 73% in the Microtox acute toxicity and of 22% in the yeast estrogenic assay. Using effect directed analysis, treated and untreated OSPW were fractionated by solid phase extraction and the fractions with the largest decrease in toxicity and estrogenicity were selected for analysis by electrospray ionization combined with linear ion trap and a high-resolution Orbitrap mass spectrometer (negative ion mode). The aim of this study was to determine whether compositional changes between the untreated and treated fractions provide insight related to biodegradation and detoxification of NAs. The O2S, O3S and O4S compounds were either not major contributors of toxicity or estrogenicity or the more toxic or estrogenic ones were biodegraded. The O3- and O4-NAs seem to be more readily metabolized than O2NAs and their degradation would contribute to detoxification. The decrease in acute toxicity may be associated with the degradation of C12 and C13 bicyclic and C12–C14 tricyclic NAs while the decrease in estrogenicity may be linked to the degradation of C16 O2-NAs with double bond equivalents (DBE) = 5 and 6, C16 and 17 O2-NAs with DBE = 7, and C19-O2-NAs with DBE = 8. The residual acute toxicity may be caused by recalcitrant components and/or degradation products such as the O2 bicyclic and tricyclic NAs, particularly the C14 and C15 bicyclic and C14–C16 tricyclic NAs as well as the polycyclic aromatic NAs (DBE ≥ 5 compounds). The decrease in estrogenicity may be linked to the degradation of the O3 and O4 oxidized NAs while much of the residual estrogenicity may be due to the recalcitrant polycyclic aromatic O2-NAs. Hence, treatment to further detoxify OSPW should target these compounds.

After fractionation using sequential solid phase extraction, the presence of toxic components in oil sands process-affected water (OSPW) was detected by the Microtox® acute toxicity assay using effect-directed analysis. The composition of each fraction was determined by high-resolution electrospray ionization-Orbitrap mass spectrometry. Partial least-squares discriminant analysis (PLS-DA) was used to determine which chemical constituents in all seven fractions co-varied most strongly with toxicity. Although O2 compounds with double bond equivalence (DBE) between 3 and 9 positively correlated with toxicity, C15–C18 O2-NAs with DBE=4 (tricyclic structure), as well as C14–C17 O2-NAs with DBE=3 (bicyclic structure), were found to be most likely associated with OSPW toxicity, consistent with published toxicity studies of surrogate NAs. O4, many O3 (i.e. possibly hydroxylated O2 c-NAs) and a few O2 compounds were found to negatively correlate with toxicity. The results demonstrate the utility of the fractionation and the PLS-DA approach for evaluating composition-response relationships in a complex mixture and also contribute to a better understanding of the toxic compounds in OSPW. These findings will help to focus study on the most environmentally significant components in OSPW.

This paper outlines evaluation of running a private Cloud-based system over two semesters at Edinburgh Napier University for two modules: Security Testing and Advanced Network Forensics (BEng (Hons) level and focused on Penetration testing and Malware Analysis); and Penetration Testing (MSc level). Overall, over the two semesters, these modules supported over 100 students within an isolated Cloud-based environment for penetration testing and malware analysis. These were taught using two different Cloud-based environment, one (DFET) allowed for a wide range of server and desktop instances to be used in a wide variety of network configuration, and the other (Linux Zoo) which focused on guiding students using a Capture The Flag methodology. The DFET (Digital Forensics Evaluation and Training) Cloud creates new training methods/techniques to support judicial authorities, law enforcement agencies and associated stakeholders in the fight against cybercrime through the development of a virtual (cloud-based) cybercrime training environment to include real life simulation and scenario analysis. Currently it is partly funded by DG Home – Prevention Of and Fight against Crime, and aims to improve crime detection rates by providing scenario-based training in line with the dynamic nature of cybercrime. Overall DFET aims to create a training infrastructure which can share cyber training across Europe, and allow access to hands-on environments, no matter the physical location of trainer. Its core partners are Edinburgh Napier University, Joseph Stefan Institute (JSI), Stockholm University, Police Scotland, and Aconite Internet Solutions. The current DFET Cloud contains four main cluster nodes, where each cluster node runs VMware vSphere 5.1 with VMware vCenter used to manage the instances. This gives a total of 119 GHz CPU, 520 GB of physical memory, and 18 TB of disk space. The paper shows the main architecture, and shows that all of the management components, such as a domain controller, are run as virtual machines. The paper outlines the structure of modules, and in the coverage of the labs within the Cloud environments, including the usage of Metasploit, Metasploitable and Web assessment systems. For the evaluation, the paper focuses on the performance analysis of a large-scale penetration testing coursework. The results highlights a key breakpoint in the Cloud which occurred when over 80 students where completing their Penetration Testing coursework and working for a 12pm deadline, and where the resources within the infrastructure became stressed where there was a considerably lag in remotely accessing the DFET Cloud. Overall the system coped by increasing memory allocation for vCenter, and in stopping running instances which had been dormant for several weeks. The paper will showcase the best practice used for the creation and maintenance of the Cloud environment, and how subjects such as Malware Analysis and Penetration Testing can be achieved within a private cloud environment, and where students can learn new methods, without the danger of ethical and moral problems. It will also highlight the key pointers towards scaling up the environment to support and increasing number of remote and distance learning students, including the methods used to script the creation of instances, and in the returning them back to a pool, along with enhanced failover protection, and in isolating instances using VLAN technology. The results are also backed-up by a student survey on the usage of the Cloud environment, and in how this enhanced their learning.

Poly(3-hydroxyoctanoate) (PHO), poly(lactic acid) (PLA) and PHO/PLA blends were reactively modified in the melt state, using dicumyl peroxide (DCP) and triallyl trimesate (TAM) trifunctional coagent. The viscosity and elasticity of PHO and PLA increased substantially following reactive modification. The processability of the partially cross-linked PHO was improved, whereas coagent-modified PLA displayed strain hardening and enhanced crystallization. Reactive compounding of PLA and PHO in the presence of DCP and TAM resulted in blends with enhanced strain hardening, indicative of high melt strength, and improved crystallinity compared to the unreacted blend. Furthermore these blends had a finer morphology, which was attributed to a compatibilizing effect possibly arising from copolymer formation at the interface. The elongation at break and impact properties of the blends were improved compared to the neat PLA, while the heat deflection temperature remained unaffected.

Decanoic acid is an ideal substrate for the synthesis of medium-chain-length poly-3-hydroxyalkanoate (MCL-PHA), but its use for this purpose has only previously been studied in shake-flasks likely due to its surfactant properties, low aqueous solubility and high melting temperature. A fed-batch fermentation process was developed for the production of MCL-PHA from decanoic acid using Pseudomonas putida KT2440. Decanoic acid was kept in liquid form by heating or by mixing with acetic acid to prevent crystallization. Different ratios of decanoic acid:acetic acid:glucose (DA:AA:G) were fed to produce a specific growth rate of 0.15 h(-1). This method produced a maximum of 39 g L(-1) dry biomass containing 67% MCL-PHA when the DA:AA:G ratio was 5:1:4. However, a declining growth rate occurred in the late stage of fermentation, resulting in decanoic acid accumulation in the bioreactor leading to foaming. The duration of MCL-PHA production was extended by shifting from exponential to linear feeding before accumulation of decanoic acid. This resulted in 75 g L(-1) biomass containing 74% PHA and an overall PHA productivity of 1.16 g L(-1)h(-1) with the production of each gram of PHA requiring only 1.16 g of decanoic acid. The final PHA composition (on a molar basis) was 78% 3-hydroxydecanoate, 11% 3-hydroxyoctanoate and 11% 3-hydroxyhexanoate.