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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.

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.

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.

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.

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.