F.J. Weber

Mechanisms of resistance of whole cells to toxic organic solvents

Abstract Many processes in modern biotechnology, particularly biotransformations and environmental bioremediation, are hindered by the toxic effects of organic solvents on whole cells. These compounds dissolve in the cell membrane, disturbing its integrity and effecting specific permeabilization. The hydrophobicity of a compound, expressed as its log P value, is a good indicator of toxicity. Substances with a log P value in the range 1–5 are, in general, toxic to whole cells. However, in recent years, there have been several reports of bacteria exhibiting resistance to toxic solvents. The main adaptative reactions are alterations in the composition of the membrane, particularly changes in fatty-acid composition, phospholipid headgroups, and in the protein content. One of the key processes in the adaptation of some Pseudomonas strains, enabling them to tolerate organic solvents appears to be the isomerization of cis - into trans-unsaturated fatty acids. A greater understanding of these adaptations should eventually allow biotransformation reactions to be carried out in inhospitable two-phase systems incorporating an organic phase.

Cis/trans isomerization of fatty acids as a defence mechanism of Pseudomonas putida strains to toxic concentrations of toluene.

Defence mechanisms of three Pseudomonas putida strains growing in the presence of toluene up to 50% (v/v) were investigated. The three strains reacted to toxic concentrations of toluene by accumulating trans unsaturated fatty acids in the membrane instead of the cis isomers. The membranes of the toluene-adapted cells possessed a higher trans/cis ratio and had a higher lipid-ordering since the transition temperature was about 7 centigrade degrees higher compared to the non-adapted cells.

Prevention of clogging in a biological trickle-bed reactor removing toluene from contaminated air.

Removal of organic compounds like toluene from waste gases with a trickle‐bed reactor can result in clogging of the reactor due to the formation of an excessive amount of biomass. We therefore limited the amount of nutrients available for growth, to prevent clogging of the reactor. As a consequence of this nutrient limitation a lower removal rate was observed. However, when a fungal culture was used to inoculate the reactor, the toluene removal rate under nutrient limiting conditions was higher. Over a period of 375 days, an average removal rate of 27 g C/(m3 h) was obtained with the reactor inoculated with the fungal culture. From the carbon balance over the reactor and the nitrogen availability it was concluded that, under these nutrient‐limited conditions, large amounts of carbohydrates are probably formed. We also studied the application of a NaOH wash to remove excess biomass, as a method to prevent clogging. Under these conditions an average toluene removal rate of 35 g C/(m3 h) was obtained. After about 50 days there was no net increase in the biomass content of the reactor. The amount of biomass which was formed in the reactor equaled the amount removed by the NaOH wash.

Defined media and inert supports: their potential as solid-state fermentation production systems

Solid-state fermentation (SSF) using inert supports impregnated with chemically defined liquid media has several potential applications in both scientific studies and in the industrial production of high-value products, such as metabolites, biological control agents and enzymes. As a result of its more defined system, SSF on inert supports offers numerous advantages, such as improved process control and monitoring, and enhanced process consistency, compared with cultivation on natural solid substrates.

A simplified material and energy balance approach for process development and scale-up of Coniothyrium minitans conidia production by solid-state cultivation in a packed-bed reactor.

Production of conidia of the biocontrol fungus Coniothyrium minitans by solid-state cultivation in a packed-bed reactor on an industrial scale is feasible. Spore yield and oxygen consumption rate of C. minitans during cultivation on oats and three inert solids (hemp, perlite, and bagasse) saturated with a liquid medium were determined in laboratory-scale experiments. The sensitivity of the fungus to reduced aw, and the water desorption isotherms of the four solid materials were also determined. C. minitans is very sensitive to reduced aw: 50% inhibition of respiration was found at aw 0.95, spore formation was completely inhibited at aw 0.97. A simplified mathematical model taking into account convective and evaporative cooling was used to simulate temperature and moisture gradients in the bed during cultivation. Adequate temperature control can be achieved with acceptable air flow rates for all four solid matrices. Moisture control is the limiting factor for cultivation in a packed bed. Oats cannot be used due to the shrinkage and aw reduction caused by evaporative cooling. Of the three inert supports tested, hemp provides the best spore yield and control of water activity, due to its high water uptake capacity. A spore yield of 9 x 10(14) conidia per m(3) packed bed can be achieved in 18 days, using hemp impregnated with a solution containing 100 g dm(-3) glucose and 20 g dm(-3) potato extract. Sufficient water is predicted to be available after 18 days, to allow a higher initial nutrient concentration, which may lead to higher spore yields.

Cellular Toxicity of Lipophilic Compounds: Mechanisms, Implications, and Adaptations

(1994). Cellular Toxicity of Lipophilic Compounds: Mechanisms, Implications, and Adaptations. Biocatalysis: Vol. 10, No. 1-4, pp. 113-122.

Alkene monooxygenase from Mycobacterium: a multicomponent enzyme.

A NADH- or NADPH-dependent alkene monooxygenase (AMO) activity has been detected in cell-free extracts of the ethene-utilizing Mycobacterium E3 and Mycobacterium aurum L1. The activity was not linear with protein concentration in the assay suggesting AMO is a multicomponent enzyme. The inhibition pattern of AMO activity was very similar to the inhibition patterns published for the three-component soluble methane monooxygenases. Fractionation of crude extracts revealed that combination of two fractions was required to restore alkene monooxygenase activity. The first fraction was inhibited by acetylene, indicating it contained an oxygenase component. The second fraction contained reductase activity which was absent from non-induced cells. This reductase activity is probably the NADH-acceptor reductase of AMO.

Quantitative recovery of fungal biomass grown on solid k-carrageenan media

The use of a dissolvable solid matrix, κ-carrageenan, to quantify biomass grown on solid media was studied. A firm gel was obtained with 2% (w/v) κ-carrageenan and 20 mM K+ which could be easily dissolved in demineralized water. Direct quantification of Coniothyrium minitans biomass grown on this medium was feasible. No effects of the dissolution on the amount of biomass recovered were detected.

Effect of the availability of magnesium ions in κ-carrageenan gels on the formation of conidia by Coniothyrium minitans

An isolate of Coniothyrium minitans did not sporulate on media to which 0‐05 mm magnesium was added when j-carrageenan was used to solidify the medium. Normal conidiation was observed when a technical grade agar or agar-agar was used as the gelling agent. The j-carrageenan and agars used in this study contained significant amounts of Mg. In the agar media all the Mg present was available to the fungus. By contrast, in the j-carrageenan gel a large portion of the Mg was bound by the gel and not available. Sporulation of C. minitans was only observed on media containing & 0‐17 mm of unbound Mg. The possible role of Mg in the initiation of conidium formation by C. minitans is discussed. Most microbial cultivations in industry are done in submerged culture. For some processes, however, cultivation on solid substrates in the absence of free-flowing water (solid-substrate cultivation) is advantageous. The production of high quantities of Coniothyrium minitans W. A. Campb. conidia, for instance, in stirred-tank reactors has heretofore not been reported. Numerous conidia, however, can easily be obtained when C. minitans is grown on various solid substrates (McQuilken & Whipps, 1995). These conidia are a promising biocontrol agent to treat Sclerotinia sclerotiorum contaminated soils and plants (Whipps & Gerlagh, 1992). The prospects of a biocontrol agent based on C. minitans are promising, provided that an ecient production process is developed. Optimization of the solid substrate with respect to spore formation is essential for the development of a cost-eective biocontrol agent. Quantification of biomass dry weight is important in such optimization studies. Direct gravimetric quantification of biomass is usually not possible when natural solid substrates are used, due to the close association of the microorganism with the substrate. During the development of an alternative model system based on j-carrageenan which facilitates the quantitative recovery of biomass dry matter (Weber, Tramper, & Rinzema, 1999), we found that magnesium is very important for sporulation. The present study evaluates the eect of several minerals in combination with various gelling agents on sporulation of C. minitans. We demonstrate that the amount of freely diusible magnesium strongly aects the spore yield. Furthermore, we demonstrate that the gelling agent used to mimic solid substrate cultivation may produce artefacts in medium optimization studies.