Juliana A. Ramsay

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.

With no inhibition of β-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 β-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.

Laccases produced by white rot fungi have been extensively evaluated for their potential to decolorize textile wastewaters which contain salts like sodium chloride and sodium sulfate. The effect of sodium chloride and sodium sulfate on Trametes versicolor laccase during the decolorization of an anthraquinone dye (Reactive Blue 19) and the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were evaluated by steady-state kinetic analysis. The results showed that, while sodium sulfate did not affect laccase activity, sodium chloride inhibited both ABTS oxidation and dye decolorization. However, the type of inhibition was substrate-dependent: it was hyperbolic, noncompetitive with ABTS and parabolic, noncompetitive with Reactive Blue 19. Furthermore, the results suggested that two chlorides may bind to laccase in the presence of the dye unlike recent inhibition models which suggest that there is only one inhibition site. This investigation is the first to provide evidence for and to propose a two-site model of laccase inhibition, providing new insight into NaCl inhibition of laccase. The proposed model is also useful to predict decolorization rates in the presence of sodium chloride and to determine operating conditions that will minimize inhibition.

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.

Although aerobic degradation of ethylene glycol is well documented, only anaerobic biodegradation via methanogenesis or fermentation has been clearly shown. Enhanced ethylene glycol degradation has been demonstrated by microorganisms in the rhizosphere of shallow-rooted plants such as alfalfa and grasses where conditions may be aerobic, but has not been demonstrated in the deeper rhizosphere of poplar or willow trees where conditions are more likely to be anaerobic. This study evaluated ethylene glycol degradation under nitrate-, and sulphate-reducing conditions by microorganisms from the rhizosphere of poplar and willow trees planted in the path of a groundwater plume containing up to 1.9 mol l⁻¹ (120 g l⁻¹) ethylene glycol and, the effect of fertilizer addition when nitrate or sulphate was provided as a terminal electron acceptor (TEA). Microorganisms in these rhizosphere soils degraded ethylene glycol using nitrate or sulphate as TEAs at close to the theoretical stoichiometric amounts required for mineralization. Although the added nitrate or sulphate was primarily used as TEA, TEAs naturally present in the soil or CO₂ produced from ethylene glycol degradation were also used, demonstrating multiple TEA usage. Anaerobic degradation produced acetaldehyde, less acetic acid, and more ethanol than under aerobic conditions. Although aerobic degradation rates were faster, close to 100% disappearance was eventually achieved anaerobically. Degradation rates under nitrate-reducing conditions were enhanced upon fertilizer addition to achieve rates similar to aerobic degradation with up to 19.3 mmol (1.20 g) of ethylene glycol degradation l⁻¹ day⁻¹ in poplar soils. This is the first study to demonstrate that microorganisms in the rhizosphere of deep rooted trees like willow and poplar can anaerobically degrade ethylene glycol. Since anaerobic biodegradation may significantly contribute to the phytoremediation of ethylene glycol in the deeper subsurface, the need for “pump and treat” or an aerobic treatment would be eliminated, hence reducing the cost of treatment.

Laccase (31.5 U of activity/g or 4.39 μg of protein/m²) from Trametes versicolor was immobilized on controlled-porosity-carrier silica beads and evaluated for the decolouration of Reactive blue 19, an anthraquinone dye. Although there was an initial, rapid adsorption of the dye to the packed bed in a recirculating reactor, about 97.5% of Reactive blue 19 removal was due to enzymatic degradation. The free enzyme lost 52% of its activity in 48 h. However, the activity of the immobilized laccase was unchanged after 4 months of storage in phosphate buffer under ambient conditions followed by three successive decolourations over 120 h. Treating the laccase immobilized beads with ethanolamine reduced dye adsorption by 40%.