Search

Olive mill wastewater (OMWW) is the toxic, malodorous, recalcitrant waste from the three-phase decanter system used in the processing of olives for oil. The waste has a high organic load, and is high in polyphenols which largely account for its phytotoxicity, preventing its use in irrigation or its discharge into waterways. A white-rot basidiomycete, previously found to reduce phenols and phytotoxicity of OMWW, produced substantial laccase activity during cultivation. Putatively identified as Cerrena consors, laccase production was maximal during idiophase. Copper (0.75 mM) increased laccase activity by more than 500% and addition of spent OMWW-based culture fluid to the medium was also inductive. The laccase isoform pattern changed depending on whether fresh or aged OMWW was used as the growth medium. Moreover, OMWW was found to be a source of natural laccase mediators which appear to have increased the effectiveness of phenol removal from the wastewater. Laccases can degrade and detoxify many organic pollutants, but their use is hindered by the inability to produce them cheaply. This work also suggests that OMWW may be a useful substrate for the production of laccases from white-rot fungi that are capable of utilising toxic wastes such as OMWW containing polyphenols.

Free-solution capillary electrophoresis (CE), or capillary zone electrophoresis, with direct UV detection was used for the first time for the determination of mono- and disaccharides, sugar alcohols, and ethanol in fermentation broths. Sample preparation proved to be minimal: no derivatization or specific sample purification was needed. The CE conditions can be adapted to the type of fermentation by simply altering the background electrolyte (BGE). KOH (130 mM) or NaOH (130 mM) as the BGE led to the fastest analysis time when monitoring simple fermentations. A mixture of 65 mM NaOH and 65 mM LiOH led to a 19% improvement in resolution for a complex mixture of carbohydrates. Quantification of a simple carbohydrate fermentation by CE showed values in close agreement with that of high-performance anion exchange chromatography and high-performance liquid chromatography (HPLC) on a cation exchange resin. For complex fermentations, quantification of carbohydrates by HPLC and CE led to similar results, whereas CE requires an injection volume of only 10-20 nL. Analysis of an ethanol fermentation of hydrolyzed plant fiber demonstrated the robustness of the separation and detection of carbohydrates, as well as ethanol. Ethanol determination is achieved by coupling the CE method to pressure mobilization, using the same instrument and the same sample.

Olive mill wastewater (OMWW) is a significant pollutant and its phytotoxicity is attributed mostly to the phenols present. 220 fungi were screened for their ability to produce detoxifying enzymes and/or grow in OMWW. Four isolates, species of Cerrena, Byssochlamys, Lasiodiplodia and Bionectria were selected and compared against Phanerochaete chrysosporium for their ability to bioremediate OMWW in the presence of a competing indigenous microflora. For the first time we report that a Cerrena sp. achieved a 75% reduction of phenolics in OMWW and that, unusually, the reduction occurred within 2 h of the addition to the OMWW.

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis . Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis .

Aims: To examine the causes for variations in sensitivity and intrinsic tolerance of Pseudomonas aeruginosa to plant volatile compounds. Methods and Results: Minimum inhibitory concentrations were determined for a selection of volatile phytochemicals against P. aeruginosa using a microdilution assay. Effects on growth were also assessed in 100-ml broth cultures. The two strains of P. aeruginosa included in the study exhibited intrinsic tolerance to all compounds, with the exception of carvacrol and trans-cinnamaldehyde. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased P. aeruginosa sensitivity to all compounds except trans-cinnamaldehyde, implicating an ATP-dependent efflux mechanism in the observed tolerance. Outer membrane integrity following treatment with test compounds was assessed by measuring sensitization to detergents. Only carvacrol caused damage to the outer membrane of P. aeruginosa. Conclusions: The intrinsic tolerance of P. aeruginosa strains to plant volatile compounds is associated with an active efflux mechanism and the barrier function of the outer membrane. Significance and Impact of the Study: These findings offer an explanation for the intrinsic tolerance to plant volatile compounds exhibited by P. aeruginosa. The study also confirms that the outer membrane-permeabilizing action of carvacrol, previously reported for the gram-negative bacteria Escherichia coli and Salmonella, extends to monoterpene-tolerant strains of P. aeruginosa.

The known antimicrobially active oxygenated terpenes, carvone, terpinen-4-ol, α-terpineol, carveol and myrtenol were found to cause significant K+ leakage from E. coli cells. In contrast, the relatively inactive oxygenated terpenes, 1,8-cineole and p-menth-6-ene-2,8-diol, did not cause significant K+ leakage. Slight structural differences between active terpenes were found to affect the rate of K+ leakage from E. coli cells. The membrane permeabilizing effect of the active terpenes is considered a significant factor in their antimicrobial activity.

A total of 27 methanol extracts obtained from different plant parts of 10 species of rain forest trees belonging to four genera of the Flacourtiaceae and originating from Australia were investigated. In vitro cytotoxicity was measured by an ATP Lite-M assay method against the mouse P388 lymphocytic leukemia cell line. The total antioxidant activity has been assessed based on scavenging activity of stable ABTS free radicals. The minimum inhibition concentration (MIC) was determined by the dilution method performed in 96 well plates against four different microbes. The leaf extract of Casearia sp. (RB 3051), mature stem extract of Casearia grayi and stem extract of Scolopia braunii were found to have most antioxidant activity (IC50 = 2.9 microg/ml), cytotoxic activity (LC50 = 0.89 microg/ml) and antimicrobial activity against all four different microbes, respectively. The results obtained suggested that among the four genera studied Casearia is the most promising in respect of finding significant antioxidant, cytotoxic and also antimicrobial activity.

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

Four pairs of oxygenated terpenes, with closely related chemical structures but considerably different minimum inhibitory concentration values (1) against P. aeruginosa or E. coli, showed differences in rate of cells killed over 2 h. Addition of polymyxin B nonapeptide (PMBN) as an outer membrane permeabilising agent was found to significantly increase the initial rates and overall numbers of cells killed for all compounds. The hydrocarbon limonene and the ester geranyl acetate, normally inactive, were also investigated. Both compounds had little killing effect when added alone to the cells but did show an enhanced killing capacity upon the addition of PBMN.

Research into the mode of action of the essential oil of Melaleuca alternifolia (Tea tree oil) is briefly reviewed. Its mode of action is interpreted in terms of the membrane-toxicity of its monoterpenoid components and different approaches for determining cell membrane damage are discussed.

Methods for the measurement of the minimum inhibitory concentration (MIC) of antibacterial agents have been developed for water-soluble substances and require adaptation for use with water-insoluble essential oils. This paper reports a standardized agar dilution MIC method, using 0.5% v/v Tween 20 as a dispersant, which provides a reliable and reproducible technique. The method was tested using two Melaleuca alternifolia oil samples with two different levels of terpinen-4-ol (37% and 45% v/v). The MIC values of die tea tree oil samples against a wide selection of bacteria, moulds and yeast are reported.

The minimum inhibitory concentrations (MIC) of 60 terpenoids against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans have been determined. Hierarchical cluster analysis was used to group the compounds into five groups according to their activity patterns against the four microorganisms. K-Means cluster analysis was then used to confirm these groupings and to show the differences in the activity patterns of the groups. Ten molecular properties of the terpenoids, either calculated via molecular modelling or determined by direct measurement, were then used as variables in a forward stepwise discriminant analysis to identify which variables discriminated between groups. Low water solubility of Group IV compounds, mainly hydrocarbons and acetates, was found to be associated with their relative inactivity. The remaining groups, all containing oxygenated terpenoids, showed characteristic but distinct activity patterns towards the four test organisms. Hydrogen bonding parameters were found to be associated with antimicrobial activity in all cases. Activity against Gram-negative E. coli and P. aeruginosa was associated with a combination of a hydrogen bonding and size parameters. This was not found to be the case for the Gram-positive S. aureus or the yeast C. albicans. Copyright © 1999 John Wiley & Sons, Ltd.

A new microdilution method has been developed for determining the minimum inhibitory concentration (MIC) of oil-based compounds. The redox dye resazurin was used to determine the MIC of a sample of the essential oil of Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of 0.15% (w/v) agar as a stabilizer overcame the problem of adequate contact between the oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid version of the assay was also developed for use as a screening method. A comparison of visual and photometric reading of the microtitre plates showed that results could be assessed without instrumentation; moreover, if the rapid assay format was used, rigorous asepsis was not necessary. Accuracy of the resazurin method was confirmed by plate counting from microwells and MIC values were compared with results obtained using an agar dilution assay. The MIC results obtained by the resazurin method were slightly lower than those obtained by agar dilution.

Essential oils, extracted from species of the genus Zieria using cold methanol extraction, were used to divide the Zieria species into eight groups based on the chemical compositions of their oils using hierarchical cluster analysis. The major components of most Zieria oils were oxygenated terpenes or other related compounds including car-3-en-2-one, chrysanthenone, eucarvone, methyl eugenol, elemicin and safrole. In several of the Zieria oils the major oxygenated monoterpene made up between 50–60% of the oil composition. Measurements of minimum inhibitory concentration, using an agar dilution method and E. coli, Staph, aureus, Ps. aeruginosa and C. albicans as test organisms, have demonstrated that essential oils from Zieria exhibit antimicrobial activity. Several of the major oxygenated compounds were tested individually and found, in most cases, to be comparable in bioactivity to the oils in which they occurred.

Cytotoxicity of Australian tea tree oil (oil of Melaleuca alternifolia) and its major oxygenated monoterpenes: terpinen-4-ol, 1,8-cineole and α-terpineol were investigated using the MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a heptaocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was α-terpineol>tea tree oil>terpinen-4-ol> 1,8-cineole and with comparison with the controls used mercuric chloride>tea tree oil>aspirin. Antimicrobial activity (MICs) displayed a similar pattern where α-terpineol>terpinen-4-ol>tea tree oil> 1,8-cin...

NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor- fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating cells displayed redistribution of free NADH with decreased relative concentration of free NADH within the nucleus whereas the majority of NADH was found in the cytoplasm. © 2012 Biophysical Society.

NADH is a naturally occurring bi-product and regulatory metabolite associated with cellular respiration. The quantification using the difference lifetime of autofluorescence of free and bound NADH has the potential to enhance the understanding of a range of cellular processes including apoptosis, cancer pathology and enzyme kinetics. Fluorescence lifetime imaging microscopy (FLIM) enables not only examination of the spatial location of the cofactor within live cells but also of its state.Here we describe the use of phasor FLIM to spatially map the fluorescence lifetimes of NADH in both free and bound form within live undifferentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. The phasor representation enables the possibility for a direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells to enable a global analysis.A comparison of myoblast cells induced to differentiate through serum starvation and undifferentiated cells show differing spatial distribution of the different forms of NADH. Cells due to undergo differentiation displayed a short lifetime representing free NADH situated around the cytoplasmic periphery and a longer lifetime attributed to the presence of bound NADH just outside of the nucleus. Differentiated cells displayed redirection of the distribution of free NADH located mainly within the nucleus while the bound form remained directly comparable to that of the other cells. Furthermore, there appears to be a spatial shift in the distribution of lifetimes at a pixel level within the phasor plot. We show that the states of differentiation of myocytes may be determined through the phasor FLIM analysis of the autofluorescent properties of NADH.

Captain Cook is reported to have used the leaves of the tea tree to brew a spicy tea and Sir Joseph Banks, the botanist with Cook’s expedition, included samples of the plant in his collection. Exactly which plants were used is not known as the name ‘tea tree’ has been used for a number of similar plants in the genera Melaleuca and Leptospermum. Whilst essential oils from several species possess antimicrobial activity, the oil which is widely used today for its antiseptic properties is the oil of Melaleuca alternifolia.

The value of gaining industry experience during undergraduate degrees is well recognised1,2 and there is much interest currently in the concept of work-integrated learning (WIL)3,4. Industry experience equips graduates with job-ready skills, but university staff have reported that time, resources, and availability of industry places are obstacles in setting up placements for students3,5. An alternative approach is to provide a ‘real-world’ learning experience within the university. In this paper we will describe an example of an ‘authentic’ learning experience that familiarises students with the procedures involved in laboratory accreditation, and enables students to develop skills that address issues identified by employers during recruitment. These issues include lack of team work, communication, understanding how industry ‘does business’, and lack of practical experience and laboratory skills2,6. Laboratory Quality Management (LQM) is a final year unit at Western Sydney University that addresses some of these issues.

A hot water extract of the Australian native sarsaparilla Smilax glyciphylla Sm. (Smilaceae) inhibited peroxidation of phosphatidylcholine liposomes initiated by Fe(2+)/ascorbate (IC50, 10 microg/mL) and AAPH (IC50, 33 microg/mL) in vitro. It also inhibited deoxyribose degradation and quenched chemically generated superoxide anion (IC50, 50 microg/mL). Reactivity towards ABTS (2,2'-azinobis(3-ethylbenzothiazoline 6-sulphonate) radical cation was equivalent to 48.4 mM TROLOX, the water soluble alpha-tocopherol analogue. Smilax glyciphylla is a rich source of the dihydrochalcone glycyphyllin. Given the reported level of activity it is unlikely that glycyphyllin would provide direct antioxidant protection in tissues affected by oxidative stress. However, consuming Smilax glyciphylla as a tea may be sufficient to reduce oxidative damage in the gastrointestinal tract. It is also possible that glycyphyllin is metabolised and adsorbed as phloretin, a compound with known anticancer properties. These findings indicate that further studies of the chemopreventative properties of Smilax glyciphylla is warranted.

Aims: This study compared the antimicrobial activity of Melaleuca alternifolia (tea tree) oil with that of some of its components, both individually and in two-component combinations. Methods and Results: Minimum inhibitory concentration and time-kill assays revealed that terpinen-4-ol, the principal active component of tea tree oil, was more active on its own than when present in tea tree oil. Combinations of terpinen-4-ol and either γ-terpinene or p-cymene produced similar activities to tea tree oil. Concentration-dependent reductions in terpinen-4-ol activity and solubility also occurred in the presence of γ-terpinene. Conclusions: Non-oxygenated terpenes in tea tree oil appear to reduce terpinen-4-ol efficacy by lowering its aqueous solubility. Significance and Impact of the Study: These findings explain why tea tree oil can be less active in vitro than terpinen-4-ol alone and further suggest that the presence of a non-aqueous phase in tea tree oil formulations may limit the microbial availability of its active components.

Twenty-eight extracts from 17 species of Australian native plants traditionally used as general anti-inflammatory medicines by Australian Aboriginal people were examined for inhibition of the enzyme xanthine oxidase (XO). The extracts from nine species were found to have more than 25% inhibition at a concentration of 100 microg/ml in the assay mixture. Extracts from three species Clerodendrum floribundum R. Br. (Verbenaceae), Eremophila maculata (Ker Gawler) (Myoporaceae) and Stemodia grossa Benth. (Scrophulariaceae) showed the greatest activity with inhibitions of 84, 61 and 57%, respectively, at 50 microg/ml, with four other species having more than 40% inhibitory activity at this concentration.

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism's susceptibility to tea tree oil and to its normally inert hydrocarbons, p-cymene and gamma-terpinene.

Octanol-water partition coefficients (Kow) for 57 terpenoids were measured using a RP-HPLC method. Sample detection was achieved with standard UV and refractive index detectors and required no special column treatment. Measured log Kow values for the terpenoids ranged from 1.81 to 4.48 with a standard error of between 0.03 and 0.08 over the entire range. Partition coefficients determined by the RP-HPLC method were compared against shake flask, atom/fragment contribution, fragment and atomistic methods. The HPLC values were found to give the best correlation with shake flask results. Log Kow values calculated by the atom/fragment contribution method gave the best correlation with the HPLC values when compared to fragment and atomistic methods.

Fifty-three human sera were examined for antibodies agglutinating erythrocytes sensitized with 3 different membrane teichoic acids from lactobacilli. Of the 32 sera containing detectable antibodies, 17 reacted with each of the teichoic acids. An examination of representative sera indicated that there were antibodies cross-reacting with the 3 teichoic acids and also with presumptive teichoic acids present in extracts of 3 oral streptococci. These results indicate that the reaction of a cell extract with a particular human serum does not necessarily indicate infection by that organism.