In situ recovery of 2,3-butanediol from fermentation by liquid–liquid extraction

End-product conversion, low product concentration and large volumes of fermentation broth, the requirements for large bioreactors, in addition to the high cost involved in generating the steam required to distil fermentation products from the broth largely contributed to the decline in fermentative products. These considerations have motivated the study of organic extractants as a means to remove the product during fermentation and minimize downstream recovery. The aim of this study is to assess the practical applicability of liquid–liquid extraction in 2,3-butanediol fermentations. Eighteen organic solvents were screened to determine their biocompatibility, and bioavailability for their effects on Klebsiella pneumoniae growth. Candidate solvents at first were screened in shake flasks for toxicity to K. pneumoniae. Cell density and substrate consumption were used as measures of cell toxicity. The possibility of employing oleyl alcohol as an extraction solvent to enhance end product in 2,3-butanediol fermentation was evaluated. Fermentation was carried out at an initial glucose concentration of 80 g/l. Oleyl alcohol did not inhibit the growth of the fermentative organism. 2,3-Butanediol production increased from 17.9 g/l (in conventional fermentation) to 23.01 g/l (in extractive fermentation). Applying oleyl alcohol as the extraction solvent, about 68% of the total 2,3-butanediol produced was extracted. An erratum to this article can be found at     

Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

Background  

Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water.     

Resolution of 1,2-epoxyhexane by Rhodotorula glutinis using a two-phase membrane bioreactor

Large-scale resolution of epoxides by the yeast Rhodotorula glutinis was demonstrated in an aqueous/organic two-phase cascade membrane bioreactor. Due to the chemical instability and low solubility of epoxides in aqueous phases, an organic solvent was introduced into the reaction mixture in order to enhance the resolution of epoxide. A cascade hollow-fiber membrane bioreactor was used (1) to minimize the toxicity of organic solvents towards the epoxide hydrolase of R. glutinis, and (2) to remove inhibitory amounts of formed diol from the yeast cell containing aqueous phase. Dodecane was selected as a suitable solvent and 1,2-epoxyhexane as a model substrate. By use of this membrane bioreactor, highly concentrated (0.9 M in dodecane) enantiopure (> 98% ee) (S)-1,2-epoxyhexane (6.5 g, 30% yield) was obtained from the racemic mixture. Received: 28 June 1999 / Received revision: 26 August 1999 / Accepted: 3 September 1999     

Solvent selection for enhanced bioproduction of 3-methylcatechol in a two-phase partitioning bioreactor

The biotransformation of toluene to 3-methycatechol (3MC) via Pseudomonas putida MC2 was used as a model system for the development of a biphasic process offering enhanced overall volumetric productivity. Three factors were investigated for the identification of an appropriate organic solvent and they included solvent toxicity, bioavailability of the solvent as well as solvent affinity for 3MC. The critical log P (log P(crit)) of the biocatalyst was found to be 3.1 and log P values were used to predict a solvent's toxicity. The presence of various functional groups of candidate solvents were used to predict the absorption of 3MC and it was found that solvents possessing polarity showed an affinity towards 3MC. Bis (2-ethylhexyl) sebecate was selected for use in the biphasic system as it fulfilled all selection criteria. A two-phase biotransformation with BES and a 50% phase volume ratio, achieved an overall volumetric productivity of 440 mg 3MC/L-h, which was an improvement by a factor of approximately 4 over previously operated systems. Additional work focused on reducing the toluene feed in order to minimize possible toxicity and decrease loss of substrate (toluene), a result of volatilization. Toluene losses were reduced by a factor of 4, compared to previously operated systems, without suffering an appreciable loss in overall volumetric productivity.     

The effect of different solvents on the ATP/ADP content and growth properties of HeLa cells

Testing of the effects of xenobiotics in cultured cells often requires the use of organic solvents to effect suspension of the test agents in cell culture media. However, the toxic effects of the solvents themselves may introduce artifacts, which obscure interpretation of the experimental results. In this article, the toxicity of different solvents commonly used for solvation of a variety of xenobiotic agents was studied. We show that ethanol, acetone, isooctane, methanol, and hexane were considerably less toxic than the more commonly used solvent, DMSO, when ATP content and growth rates of HeLa cells exposed to these solvents was measured. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 11–15, 1999     

Properties of the sugar carrier in baker's yeast

A total of 16 hexoses and pentoses were investigated with respect to transport intoSaccharomyces cerevisiae cells. All monosaccharides were transported across the cytoplasmic membrane but only those with an equatorial hydroxyl group in positions 1 and 4 of theC1 chair conformation and those with an equatorial hydroxyl group in position 2 and an equatorial −CH₂OH group in position 5 of the1C chair conformation reached an equilibrium distribution in the entire cell water volume. Other monosaccharides reached a distribution in only 20–66% of the intracellular water. The two groups of sugars are apparently transported by different carriers (either in parallel or in series), each of them showing countertransport and an apparent activation energy of 6,700–7,800 cal/mol. The carrier transporting the perfectly distributing sugars (Group 1) is affected by uranyl nitrate but not by 2,4-dinitrophenol, the other carrier (Group 2) is apparently not susceptible to uranyl ions but is influenced by 2,4-dinitrophenol. The space of distribution of the Group 1 sugars is reduced in hypertonic media in accordance with changes of intracellular water, that of the Group 2 sugars is altered only very slightly. The carriers differ in their kinetic parametres (mobility of the loaded carriers, maximum rate of transport). There is only a very indistinct competition for transport between representatives of the two groups. Preincubation with d-galactose induces the formation or unmasking of a transport system whereafter even the Group 2 sugars reach equilibrium in the entire cell water. Part I. Fol. microbiol. 10: 30, 1965.     

Glyphosate bioavailability in soil

Biodegradation of glyphosate in sod-podzol soil by both the indigenous micro flora and the introduced strain Ochrobactrum anthropi GPK 3 was studied with respect to its sorption and mobility. The experiments were carried out in columns simulating the vertical soil profile. Soil samples studied were taken from soil horizons 0–10, 10–20, and 20–30 cm deep. It was found out that the most of the herbicide (up to 84%) was adsorbed by soil during the first 24 h; the rest (16%) remained in the soluble fraction. The adsorbed glyphosate was completely extractable by alkali. No irreversible binding of glyphosate was observed. By the end of the experiment (21st day), glyphosate was only found in extractable fractions. The comparison of the effect of the introduced O. anthropi GPK 3 and indigenous microbial community on the total toxicant content (both soluble and absorbed) in the upper 10 cm soil layer showed its reduction by 42% (21 mg/kg soil) and 10–12% (5 mg/kg soil), respectively. Simultaneously, 14–18% glyphosate moved to a lower 10–20 cm layer. Watering (that simulated rainfall) resulted in a 20% increase of its content at this depth; 6–8% of herbicide was further washed down to the 20–30 cm layer. The glyphosate mobility down the soil profile reduced its density in the upper layer, where it was available for biodegradation, and resulted in its concentration in lower horizons characterized by the absence (or low level) of biodegradative processes. It was shown for the first time how the herbicide biodegradation in soil can be increased manifold by introduction of the selected strain O. anthropi GPK 3.     

Influence of solubility-enhancing fusion proteins and organic solvents on the in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 in a micellar reaction system

The influence of the solubility-enhancing fusion proteins glutathione-S-transferase (GST) and NusA on the heterologous expression and in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 from Arabidopsis thaliana was investigated. A micellar dispersion of the water-insoluble model substrate β-apo-8′-carotenal in combination with Triton X-100 was used for the spectrophotometric in vitro assays. Specific activity in the cellular extract was twofold increased by the use of GST as a carrier protein, whereas it was decreased by 70% when fused with NusA. Reduced molar activity of the purified fusion proteins was observed, which could not be regained by proteolytic removal of the carrier protein. The addition of organic solvents in the form of short-chain aliphatic alcohols positively influenced the enzyme activity. Optimization of the reaction medium led to an 18-fold activation, and a clear correlation could be found between the organic solvent concentration required for maximum activation and the log P of the solvent. The results provide a foundation for the development towards the application of carotenoid cleavage dioxygenases as in vitro biocatalysts for the production of norisoprenoids and apocarotenals from carotenoids.     

Effects of 2,4-dichlorophenoxyacetic acid on haematological indices in West African dwarf goats

We investigated the possible toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D) in West African dwarf goats. The goats (20) were randomly divided into four equal groups; three of which were exposed to graded levels (low, medium and high doses) of 2,4-D for a period of 6 weeks. Blood samples were collected from the treatment group goats as well as the control group goats on weeks 0, 2, 4 and 6. The blood samples were used for analysis of haematologic indices such as packed cell volume (PCV), erythrocyte count (EC), total leucocyte count (TLC), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Total leucocyte counts were significantly reduced (p < 0.05) by the test herbicide (2,4-D) in all the treatment groups. Significant reduction in the levels of PCV, Hb concentration, MCV, MCH and MCHC were also recorded in all treatment groups. There was no significant change (p > 0.05) in the erythrocyte count value of all the four groups. The significant reduction in the haematologic indices of West African dwarf goats (WADG) as evidenced from the result of this study, suggest the possibility of 2,4-D toxicity in these goats.     

Screening for suitable solvents as substrate carriers for the microbial side-chain cleavage of sitosterol using microtitre plates

The microwell-scale approach is widely used for screening purposes and one-pot biotransformations, but it has seldom been applied to whole cell multistep biotransformations and to organic solvent screening/non-conventional medium bioconversion processes, which is an issue of major relevance when bioconversion processes are addressed. The present study aims to fill in this gap by using 24-well microtitre plates as platforms for the screening of suitable organic solvents as substrate carriers for effective biocatalysis. The side-chain cleavage of sitosterol with resting cells of Mycobacterium sp. NRRL B-3805 was used as model system. Series of miscible and immiscible alcohols with primary, secondary and tertiary structure were tested as carriers of the hydrophobic substrate, thus ruling out the effect of functionality on biocatalytic activity.Results suggest that microtitre plates may be used for solvent selection in complex bioconversion systems. The highest bioconversion yields were observed when methanol and ethanol were used as substrate carriers. An empirical correlation could be established between overall catalytic activity and physicochemical properties of the solvents.     

Plant protection and rhizosphere colonization of barley by seed inoculated herbicide degrading Burkholderia (Pseudomonas) cepacia DBO1(pRO101) in 2,4-D contaminated soil

The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading bacterium, Burkholderia cepacia (formerly Pseudomonas cepacia) DBO1(pRO101) was coated on non-sterile barley (Hordeum vulgare) seeds, which were planted in two non-sterile soils amended with varying amounts of 2,4-D herbicide. In the presence of 10 or 100 mg 2,4-D per kg soil B. cepacia DBO1(pRO101) readily colonized the root at densities up to 10⁷ CFU per cm root. In soil without 2,4-D the bacterium showed weak root colonization. The seeds coated with B. cepacia DBO1(pRO101) were able to germinate and grow in soils containing 10 or 100 mg kg^–1 2,4-D, while non-coated seeds either did not germinate or quickly withered after germination. The results suggest that colonization of the plant roots by the herbicide-degrading B. cepacia DBO1(pRO101) can protect the plant by degradation of the herbicide in the rhizosphere soil. The study shows that the ability to degrade certain pesticides should be considered, when searching for potential plant growth-promoting rhizobacteria. The role of root colonization by xenobiotic degrading bacteria is further discussed in relation to bioremediation of contaminated soils.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria <Emphasis Type="Italic">Cupriavidus necator</Emphasis> JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C.␣necator JMP134 could be isolated after soil enrichment. In␣native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5α (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.     

Nitrogen addition and rhizome severing modify clonal growth and reproductive modes of <Emphasis Type="Italic">Leymus</Emphasis> <Emphasis Type="Italic">chinensis</Emphasis> population

The resurrection flowering plant Ramonda serbica inhabits the shallow organo-mineral soil that develops in crevices on northern-facing carbonate rocks in the gorges in the Balkan Peninsula. This type of soil represents a complex substrate whose physical and chemical properties were found to be well suited to the most important requirements for the growth and development of R. serbica as well as for the plant’s survival in the state of anhydrobiosis in periods of drought stress. Considerable amount of organic matter (39.4%) in the soil resulted in the high field capacity (134 ml/100 g soil) as well as the slow changes in the amount of its available water. The suitable soil hydric status, based on the organic remains, supports the slow dehydration of this poikilohydric plant, which is extremely important in allowing the activation of the plant’s protective mechanisms. The pH of the soil solution was slighty alkaline (7.7) mostly due to carbonates in its crystallographic structure. The large amount of incompletely decomposed organic debris resulted in a marked difference between total and available nutrient concentration in the soil. Still, the adequate content of nutrients in the leaves points to efficient mineral consumption by the plant roots. The sufficient bioavailability of nutrients and water was also improved by vesicular–arbuscular mycorrhiza detected in R. serbica roots.     

Enhanced Mineralization of [U-14C]2,4-Dichlorophenoxyacetic Acid in Soil from the Rhizosphere of Trifolium pratense

Enhanced biodegradation in the rhizosphere has been reported for many organic xenobiotic compounds, although the mechanisms are not fully understood. The purpose of this study was to discover whether rhizosphere-enhanced biodegradation is due to selective enrichment of degraders through growth on compounds produced by rhizodeposition. We monitored the mineralization of [U-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in rhizosphere soil with no history of herbicide application collected over a period of 0 to 116 days after sowing of Lolium perenne and Trifolium pratense. The relationships between the mineralization kinetics, the number of 2,4-D degraders, and the diversity of genes encoding 2,4-D/α-ketoglutarate dioxygenase (tfdA) were investigated. The rhizosphere effect on [¹⁴C]2,4-D mineralization (50 μg g⁻¹) was shown to be plant species and plant age specific. In comparison with nonplanted soil, there were significant (P < 0.05) reductions in the lag phase and enhancements of the maximum mineralization rate for 25- and 60-day T. pratense soil but not for 116-day T. pratense rhizosphere soil or for L. perenne rhizosphere soil of any age. Numbers of 2,4-D degraders in planted and nonplanted soil were low (most probable number, <100 g⁻¹) and were not related to plant species or age. Single-strand conformational polymorphism analysis showed that plant species had no impact on the diversity of α-Proteobacteria tfdA-like genes, although an impact of 2,4-D application was recorded. Our results indicate that enhanced mineralization in T. pratense rhizosphere soil is not due to enrichment of 2,4-D-degrading microorganisms by rhizodeposits. We suggest an alternative mechanism in which one or more components of the rhizodeposits induce the 2,4-D pathway.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

The localization of 2,4-D in leaf tissue

Summary Disruption of chloroplast structure is known to occur following applications of 2,4-dichlorophenoxyacetic acid (2,4-D) to the leaves of Phaseolus vulgaris. Although chloroplasts which had been extracted from treated tissue in an aqueous medium were free from 2,4-D and its metabolites, those extracted from freeze-dried tissue in organic solvents are the principal cell components to contain the herbicide.     

Modeling structure and flexibility of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B in organic solvents

Background  

The structure and flexibility of Candida antarctica lipase B in water and five different organic solvent models was investigated using multiple molecular dynamics simulations to describe the effect of solvents on structure and dynamics. Interactions of the solvents with the protein and the distribution of water molecules at the protein surface were examined.     

Toxicity of homologous series of organic solvents for the gram-positive bacteria Arthrobacter and Nocardia Sp. and the gram-negative bacteria Acinetobacter and Pseudomonas Sp

The toxicity of homologous series of organic solvents has been investigated for the gram-positive bacteria, Arthrobacter sp. and Nocardia sp., and the gram-negative bacteria, Acinetobacter sp. and Pseudomonas sp. The hydrophobicity of the solvent, expressed by its logP(octanol), proves to be a good measure for the toxicity of solvents in a two-phase system. The transition from toxic to nontoxic solvents occurs between logP(octanol) 3 and 5 and depends on the homologous series. No correlation has been found between the hydrophobicity of the substituent on the alkyl backbone of the solvent and the location of the transition point in toxicity. The logP(octanol), above which all solvents are nontoxic, is used to express the solvent tolerance of the bacteria. In general, the solvent tolerance of gram-negative bacteria is found to be slightly higher than that of gram-positive bacteria, but this does not hold for all homologous series of organic solvents investigated.Because the toxicity effects of organic solvents in a two-phase system can be ascribed to molecular as well as phase toxicity effects, molecular toxicity effects were investigated separately in a one-phase system with subsaturating amounts of organic solvent. The solvent concentration in the aqueous phase, at which 50% of the metabolic activity of the bacteria is lost, is used to express solvent toxicity. This concentration is found to be similar for the gram-positive Arthrobacter and the gram-negative Acinetobacter. Assuming the critical membrane concentration theory (G. J. Osborne et al. Enzyme Microb. Technol. 1990, 12: 281-291) to be valid, it can be concluded that differences in solvent tolerance between these two bacteria, cannot be ascribed to differences in response to molecular toxicity. Prediction of the toxicity of any solvent, using the critical membrane theory, appears to be possible in the case of alkanols or alkyl acetates. However, prediction of the toxicity of ethers appears to be impossible. (c) 1993 John Wiley & Sons, Inc.