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.

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.

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.

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.

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.

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.

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.

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.

Production of soluble methane monooxygenase during growth of Methylosinus trichosporium on methanol.

Soluble methane monooxygenase (sMMO) can degrade many chlorinated and aromatic pollutants. It is produced by certain methanotrophs such as Methylosinus trichosporium when grown on methane under copper limitation but, due to its low aqueous solubility, methane cannot support dense biomass growth. Since it is water soluble, methanol may be a more attractive growth substrate, but it is widely believed that sMMO is not produced on methanol. In this study, when the growth-limiting substrate was switched from methane to methanol, in the presence of the particulate MMO inhibitor, allylthiourea, growth of M. trichosporium OB3b continued unabated and sMMO activity was completely retained. When allylthiourea was then removed, sMMO activity was maintained for an additional 24 generations, albeit at a slightly lower level due to the presence of 0.70 microM of Cu(2+) in the feed medium. While a biomass density of only 2 g l(-1) could be obtained on methane, 7.4 g l(-1) was achieved by feeding methanol exponentially, and 29 g l(-1) was obtained using a modified feeding strategy employing on-line carbon dioxide production measurement. It was concluded that methanol can be employed to produce large amounts of M. trichosporium biomass containing sMMO.

Use of allylthiourea to produce soluble methane monooxygenase in the presence of copper.

Methanotrophs expressing soluble methane monooxygenase (sMMO) may find use in a variety of industrial applications. However, sMMO expression is strongly inhibited by copper, and the growth rate may be limited by the aqueous solubility of methane. In this study, addition of allylthiourea decreased intracellular copper in Methylosinus trichosporium OB3b, allowing sMMO production at Cu/biomass ratios normally not permitting sMMO synthesis. The presence of about 1.5 micromoles intracellular Cu g(-1) dry biomass resulted in sMMO activity of about 250 micromoles 1-napthol formed per hour gram dry biomass whether this intracellular Cu concentration was achieved by Cu limitation or by allylthiourea addition. No loss of sMMO activity occurred when the growth substrate was switched from methane to methanol when allylthiourea had been added to growth medium containing copper. Addition of copper to medium that was almost copper-free increased the yield of dry biomass from methanol from 0.20 to 0.36 g g(-1), demonstrating that some copper was necessary for good growth. This study demonstrated a method by which sMMO can be produced by M. trichosporium OB3b while growing on methanol in copper-containing medium.

Fed-batch production of unsaturated medium-chain-length polyhydroxyalkanoates with controlled composition by Pseudomonas putida KT2440.

Unsaturated medium-chain-length polyhydroxyalkanoates (MCL-PHA) were produced at a productivity of 0.63-1.09 g PHA l(-1) h(-1) with final PHA content ranging from 42.6 to 55.8% in single-stage, carbon-limited, fed-batch fermentations of Pseudomonas putida KT2440. A mixture of nonanoic acid (NA) and 10-undecenoic acid (UDA=) was fed exponentially to control growth rate. Varying the specific growth rate (0.14 h(-1) vs. 0.23 h(-1)) at similar substrate feed ratios (NA:UDA= = 5:1) had little effect on the final PHA content and relative composition. However, decreasing the NA:UDA= ratio decreased the final amount of PHA produced from 56% with NA:UDA= = 5.07:1 to only 42% at NA:UDA= = 2.47:1. The molar fraction of all 3-hydroxyalkanoate monomers in the PHA product was relatively constant throughout each fermentation, indicating that the final product was homogeneous rather than a mixture of different copolymers. A linear relationship between unsaturation of the PHA produced and unsaturation of the carbon feed was found, which demonstrates the feasibility of producing unsaturated MCL-PHAs with controlled polymeric composition in a fed-batch process.

Fermentation process development for the production of medium-chain-length poly-3-hyroxyalkanoates.

This paper presents a review of the existing fermentation processes for the production of medium-chain-length poly-3-hydroxyalkanoates (MCL-PHAs). These biodegradable polymers are usually produced most efficiently from structurally related carbon sources such as alkanes and alkanoic acids. Unlike alkanoic acids, alkanes exhibit little toxicity but their low aqueous solubility limits their use in high density culture. Alkanoic acids pose little mass transfer difficulty, but their toxicity requires that their concentration be well controlled. Using presently available technology, large-scale production of MCL-PHA from octane has been reported to cost from US $5 to 10 per kilogram, with expenditures almost evenly divided between carbon source, fermentation process, and the separation process. However, MCL-PHAs, even some with functional groups in their subunits, can also be produced from cheaper unrelated carbon sources, such as glucose. Metabolic engineering and other approaches should also allow increased PHA cellular content to be achieved. These approaches, as well as a better understanding of fermentation kinetics, will likely result in increased productivity and lower production costs.

Increasing the yield of MCL-PHA from nonanoic acid by co-feeding glucose during the PHA accumulation stage in two-stage fed-batch fermentations of Pseudomonas putida KT2440.

A method was developed to increase the yield of MCL-PHA from nonanoic acid in the PHA accumulation phase. Pseudomonas putida KT2440 was grown on glucose until ammonium-limitation was imposed. In the second (accumulation) stage, either glucose, nonanoic acid, or a mixture of these carbon and energy sources was supplied. Since the medium-chain-length poly-3-hydroxyalkanoate (MCL-PHA) subunits produced are unique for each carbon source, their relative contribution to PHA yield could be calculated. Y(C7+C9)/NA was 0.254 mol mol(-1) during PHA synthesis from nonanoic acid. Y(C8+C10)/G was only 0.057 mol mol(-1) during PHA synthesis from glucose. When nonanoic acid and glucose were fed together, Y(C7+C8)/NA almost doubled to 0.450 mol mol(-1) while Y(C8+C10)/G decreased to 0.011 mol mol(-1). These results demonstrate that substantial savings can be obtained by feeding glucose with substrates that are good for PHA production but much more expensive than glucose.