Does Addition of Phosphate and Ammonium Nutrients Affect Microbial Activity in Froth Treatment Affected Tailings?

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.

Gold Leaching from an Oxide Ore Using Thiocyanate as a Lixiviant: Process Optimization and Kinetics.

Thiocyanate (SCN-) is a promising alternative to cyanide as a lixiviant for gold extraction and is 1000 times less toxic than cyanide. In this study, the following leaching parameters were tested to optimize the gold recovery for the first time from an oxide ore using the response surface methodology: initial thiocyanate concentration (10-500 mM), initial Fe3+ concentration (10-500 mM), and pulp density (10-50% w/v). The maximum gold recovery (96%) was achieved with 500 mM thiocyanate, 100 mM Fe3+, and 50% pulp density at 25 °C and pH = 2 for 24 h. A kinetic study on the optimum leaching condition showed that it followed the shrinking core model, in which the rate-controlling mechanism was the diffusion process. These results are discussed in the context of the published literature.

Toward Sustainable Solution for Biooxidation of Waste and Refractory Materials Using Neutrophilic and Alkaliphilic Microorganisms-A Review.

The significant increase in economic concern and environmental restrictions has resulted in increasing interest in biotechnological solutions. The application of acidophilic, sulfur-oxidizing microorganisms in biomining and in the treatment of waste matrices has been extensively explored. However, to surmount the current challenges encountered by the industrial use of acidophiles, there is an opportunity for neutrophilic and alkaliphilic microorganisms to be comprehensively considered for the biooxidation of refractory sulfide materials. This review, for the first time, provides a detailed study of neutrophiles and alkaliphiles that have potential for oxidizing sulfur-containing wastes and sulfide refractory ores to recover entrapped metals especially gold in a sustainable manner. The study illustrates the applicability of neutrophilic and alkaliphilic microorganisms to provide better and sustainable alternatives for the recovery of metals from wastes from various sources as well as refractory materials. The microorganisms summarized in this review have been successfully used in oxidizing different sulfide sources by achieving high oxidizing efficiencies (>80%) in numerous technologies. The fundamentals of biooxidation along with possible mechanisms involved in the biooxidation have been discussed in detail.

Manipulating the structure of medium-chain-length polyhydroxyalkanoate (MCL-PHA) to enhance thermal properties and crystallization kinetics.

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.

Biodegradation of CuTETA, an effluent by-product in mineral processing.

Polyamines such as triethylenetetramine (TETA) and other amine chelators are used in mineral processing applications. Formation of heavy metal complexes of these reagents as a by-product in effluent water is a recent environmental concern. In this study, Paecilomyces sp. was enriched from soil on TETA as the sole source of carbon and nitrogen and was found to degrade > 96 and 90% CuTETA complexes at initial concentrations of 0.32 and 0.79 mM respectively, following 96-h incubation. After destabilization, most of the copper (> 78%) was complexed extracellularly and the rest was associated with the cell. Mass spectroscopy results provided confirmation that copper re-complexed with small, extracellular, and organic molecules. There are no reports in the literature that Paecilomyces or any other organism can grow on TETA or CuTETA. This study is the first to show that biological destabilization of CuTETA complexes in mineral processing effluents is feasible.

Stabilization and characterization of carboxylated medium-chain-length poly(3-hydroxyalkanoate) nanosuspensions.

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 (PdI) 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±5.7nm) with narrower PdIs (0.21±0.001 to 0.25±0.003) were obtained. The combination of SDS and Triton X-100 gave the smallest particle size (97.1±1.1nm) and narrowest PdI (0.21±0.001). At higher solids content (10%), a mixture of 5mM SDS and 20mM Triton X-100 produced stable (zeta potential=-39.6± 0.9) 170.3±4.6nm nanoparticles. As carboxylation increased, particle size and hydrophobicity decreased while stability increased. When comparing nanoparticles of similar size and stability, carboxylated mcl-PHA needed ∼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.

Overproduction of MCL-PHA with high 3-hydroxydecanoate Content.

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 ß-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting ß-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 ß-oxidation knock-out mutant. Higher biomass concentration was achieved with Aa inhibition of ß-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.

Potential for mcl-PHA production from nonanoic and azelaic acids.

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 ß-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.

Biodegradation and detoxification of naphthenic acids in oil sands process affected waters.

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.

Fed-batch production of poly-3-hydroxydecanoate from decanoic acid.

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.

Toxicity and composition profiles of solid phase extracts of oil sands process-affected water.

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.

Biodegradation of naphthenic acid surrogates by axenic cultures.

This is the first study to report that bacteria from the genera Ochrobactrum, Brevundimonas and Bacillus can be isolated by growth on naphthenic acids (NAs) extracted from oil sands process water (OSPW). These pure cultures were screened for their ability to use a range of aliphatic, cyclic and aromatic NA surrogates in 96-well microtiter plates using water-soluble tetrazolium redox dyes (Biolog Redox Dye H) as the indicator of metabolic activity. Of the three cultures, Ochrobactrum showed most metabolic activity on the widest range of NA surrogates. Brevundomonas and especially Ochrobactrum had higher metabolic activity on polycyclic aromatic compounds than other classes of NA surrogates. Bacillus also oxidized a wide range of NA surrogates but not as well as Ochrobactrum. Using this method to characterize NA utilisation, one can identify which NAs or NA classes in OSPW are more readily degraded. Since aromatic NAs have been shown to have an estrogenic effect and polycyclic monoaromatic compounds have been suggested to pose the greatest environmental threat among the NAs, these bacterial genera may play an important role in detoxification of OSPW. Furthermore, this study demonstrates that bacteria belonging to the genera Ochrobactrum and Bacillus can also degrade surrogates of tricyclic NAs.

Identification of estrogenic compounds in oil sands process waters by effect directed analysis.

Using effect directed analysis, the presence of estrogenic components in untreated and biologically treated oil sands process water (OSPW) was detected with the yeast estrogenic screening assay after fractionation with solid phase extraction followed by reversed phase high performance liquid chromatography. Comparison of the composition, as determined by electrospray ionization combined with high-resolution linear trap quadropole (LTQ)-Orbitrap Velos Pro hybrid mass spectrometry (negative ion) of selected estrogenic and nonestrogenic fractions identified compounds that were uniquely present in the estrogenic samples, biologically treated and untreated. Of the 30 most abundant compounds, there were 14 possible nonaromatic structures and 16 possible aromatic structures. Based on the published literature, the latter are the most likely to cause estrogenicity and were O2, O3 and O4 C17 to C20 compounds with double bond equivalents between 6 and 10 and chemical formulas similar to estrone- and estradiol-like compounds. This study shows exact formulas and masses of possible estrogenic compounds in OSPW. These findings will help to focus study on the most environmentally significant components in OSPW.

Biodegradation of naphthenic acids in oils sands process waters in an immobilized soil/sediment bioreactor.

Aqueous extraction of bitumen in the Alberta oil sands industry produces large volumes of oil sands process water (OSPW) containing naphthenic acids (NAs), a complex mixture of carboxylic acids that are acutely toxic to aquatic organisms. Although aerobic biodegradation reduces NA concentrations and OSPW toxicity, treatment times are long, however, immobilized cell reactors have the potential to improve NA removal rates. In this study, two immobilized soil/sediment bioreactors (ISBRs) operating in series were evaluated for treatment of NAs in OSPW. A biofilm was established from microorganisms associated with sediment particles from an OSPW contaminated wetland on a non-woven textile. At 16 months of continuous operation with OSPW as the sole source of carbon and energy, 38±7% NA removal was consistently achieved at a residence time of 160 h at a removal rate of 2.32 mg NAs L(-1)d(-1). The change in NA profile measured by gas chromatography-mass spectrometry indicated that biodegradability decreased with increasing cyclicity. These results indicate that such treatment can significantly reduce NA removal rates compared to most studies, and the treatment of native process water in a bioreactor has been demonstrated. Amplification of bacterial 16S rRNA genes and sequencing using Ion Torrent sequencing characterized the reactors' biofilm populations and found as many as 235 and 198 distinct genera in the first and second bioreactor, respectively, with significant populations of ammonium- and nitrite-oxidizers.

A feeding strategy for incorporation of canola derived medium-chain-length monomers into the PHA produced by wild-type Cupriavidus necator.

The aim of this study was to increase the density of wild type Cupriavidus necator H16 biomass grown on fructose in order to produce sufficient copolymer of short-chain-length (scl) and medium-chain-length (mcl) polyhydroxyalkanoate (PHA) from canola oil for mechanical testing of the PHA. Initial batch cultivation on fructose was followed by exponential feeding of fructose at a predetermined µ to achieve 44.4 g biomass/l containing only 20 % w/w of polyhydroxybutyrate (PHB) with a Y(x/fructose) of 0.44 g/g. In a third stage, canola oil was added under N-limited conditions to produce 92 g/l of biomass with 48 % w/w scl-mcl PHA. Using known standards, the PHA composition was confirmed by GC-MS analysis as 99.81 % 3-hydroxybutyrate, 0.06 % 3-hydroxyvalerate, 0.09 % 3-hydroxyhexanoate and 0.04 % 3-hydroxyoctanoate. The melting temperature (179 °C), crystallinity (54 %), tensile stress (25.1 Mpa) and Young's modulus (698 Mpa) for a PHB standard decreased to 176 °C, 52 %, 19.1 and 443 Mpa respectively for C. necator PHA produced in the 3-stage process.

Fed-batch production of MCL-PHA with elevated 3-hydroxynonanoate content.

With no inhibition of ß-oxidation, Pseudomonas putida KT2440 produces medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) with approximately 65 mol% 3-hydroxynonanoate (HN) from nonanoic acid. Production of PHA with higher HN content and an adjustable monomeric composition was obtained using acrylic acid, a fatty acid ß-oxidation inhibitor, together with nonanoic acid and glucose as co-substrates in fed-batch fermentations. Different monomeric compositions were obtained by varying the feeding conditions to impose different specific growth rates and inhibitor feed concentrations. At a nonanoic acid: glucose: acrylic acid feed mass ratio of 1.25: 1: 0.05 and a specific growth rate of 0.15 h-1, 71.4 g L-1 biomass was produced containing 75.5% PHA with 89 mol% HN at a cumulative PHA productivity of 1.8 g L-1 h-1.

Biosynthesis and properties of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer.

When grown in a nonanoic acid-limited chemostat at a dilution rate of 0.25 h(-1), Pseudomonas putida KT2440 produced poly(3-hydroxynonanoate-co-3-hydroxyheptanoate) containing 68 mol % 3-hydroxynonanoate (C9) and 32 mol % 3-hydroxyheptanoate (C7). Under the same conditions, but in the presence of acrylic acid, a fatty acid ß-oxidation inhibitor, the C9 monomer content increased to 88 mol %. Cofeeding glucose (3.9 g L(-1)) and nonanoic acid (2.9 ± 0.1 g L(-1)) in continuous culture with 0.2 g L(-1) of acrylic acid in the feed, further increased the C9 content to 95 mol %. A yield of PHA from nonanoic acid of 0.93 mol mol(-1) was attained. PHA with a 3-hydroxyoctanoate (C8) content of 98 mol % was produced with the same cofeeding methodology from octanoic acid. As the dominant monomer content increased, the melting point of the poly(3-hydroxynonanoate) copolymers increased from 46 to 63 °C and that of the poly(3-hydroxyoctanoate) copolymers from 54 to 62 °C. All copolymer compositions resulted in elongation to break values of about 1300%, but tensile strength at break and Young's modulus both increased with increasing amounts of the dominant monomer.

The effect of continuous exposure of copper on the properties and extracellular polymeric substances (EPS) of bulking activated sludge.

INTRODUCTION: This study investigated the effect of copper on chemical oxygen demand (COD) removal efficiency and on the properties (mainly settling and dewatering) and the composition of extracellular polymeric substances (EPS) when 20 mg/L Cu(II) was continuously dosed to a sequencing batch reactor (SBR) inoculated with activated sludge. MATERIALS AND METHODS: The results showed that the continuous addition of 20 mg/L Cu(II) seriously inhibited the removal of sodium benzoate (provided as a model organic pollutant) by activated sludge in a SBR. RESULTS AND DISCUSSION: After 40 days of acclimation, the removal efficiency presented a slight but unsteady recovery and the settling and dewatering properties improved, indicating that sludge bulking had been inhibited. Additionally, the proportion of loosely bound EPS in the total EPS increased with time and the relative composition of the total organics was polysaccharide > humic substances > protein > DNA. CONCLUSION: The effects of copper on the composition of EPS and the settling and dewatering properties of bulking activated sludge were also discussed for the first time in this paper.

Microbial medium chainlength poly[(R)-3-hydroxyalkanoate] shows liquid crystal behaviour.

Medium chainlength (mcl) polyhydroxyalkanoates (PHAs) are a class of polymers receiving attention because of their potential as renewable, biodegradable and high tech properties. Unlike most short chain PHAs, mcl-PHAs are low crystallinity and elastomeric in character. In this paper we wish to point out that in their broad properties mcl-PHAs might be classified as thermotropic liquid crystals with dynamic conformational disorder and long range orientational order. As the characterization of mcl-PHAs progresses, their similarities to liquid crystalline elastomers are noteworthy. Wunderlich coined the acronym CONDIS from the words "conformational disorder" to categorize this type of liquid crystal. Thermal analysis reveals a T(g) of -40 to -45°C with several T(m) peaks. The chemistry of the elastomer from (13)C NMR confirms the poly(3-hydroxynonanoate), PHN, composition of the starting material along with two other samples containing double bonds: PHNU-18 and PHNU-31 where the numeral stands for the percent of double bonds.

Biodegradation of 1,4-dioxane by a Flavobacterium.

A dioxane-degrading consortium was enriched from soil obtained from a contaminated groundwater plume. The enriched consortium did not use dioxane as the sole source of carbon and energy but co-metabolized dioxane in the presence of tetrahydrofuran (THF). THF and dioxane concentrations up to 1000 ppm were degraded by the enriched consortium in about 2 weeks with a longer lag phase observable at 1000 ppm. Three colonies from the enriched consortium were then obtained on agar plates containing basal salts and glucose as the carbon source. Only one of the three colonies was capable of dioxane degradation. Further enrichment of this colony in liquid media led to a pure culture that grew on glucose and co-metabolically degraded dioxane after THF degradation. The rate and extent of dioxane degradation of this isolate increased with increasing THF concentration. This isolate was subsequently identified as a Flavobacterium by 16S rDNA sequencing. Using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of microbial populations, Flavobacterium was determined to be the dominant species in the enriched consortium and was distinct from the two other colonies that did not degrade dioxane. This is the first report of a dioxane-degrading Flavobacterium which is phylogenetically distinct from any previously identified dioxane degrader.