Isolation and characterization of 2-butoxyethanol degrading bacterial strains.

A total of 11 bacterial strains capable of completely degrading 2-butoxyethanol (2-BE) were isolated from forest soil, a biotrickling filter, a bioscrubber, and activated sludge, and identified by 16S rRNA gene sequence analysis. Eight of these strains belong to the genus Pseudomonas; the remaining three strains are Hydrogenophaga pseudoflava BOE3, Gordonia terrae BOE5, and Cupriavidus oxalaticus BOE300. In addition to 2-BE, all isolated strains were able to grow on 2-ethoxyethanol and 2-propoxyethanol, ethanol, n-hexanol, ethyl acetate, 2-butoxyacetic acid (2-BAA), glyoxylic acid, and n-butanol. Apart from the only gram-positive strain isolated, BOE5, none of the strains were able to grow on the nonpolar ethers diethyl ether, di-n-butyl ether, n-butyl vinyl ether, and dibenzyl ether, as well as on 1-butoxy-2-propanol. Strains H. pseudoflava BOE3 and two of the isolated pseudomonads, Pseudomonas putida BOE100 and P. vancouverensis BOE200, were studied in more detail. The maximum growth rates of strains BOE3, BOE100, and BOE200 at 30 °C were 0.204 h-1 at 4 mM, 0.645 h-1 at 5 mM, and 0.395 h-1 at 6 mM 2-BE, respectively. 2-BAA, n-butanol, and butanoic acid were detected as potential metabolites during the degradation of 2-BE. These findings indicate that the degradation of 2-BE by the isolated gram-negative strains proceeds via oxidation to 2-BAA with subsequent cleavage of the ether bond yielding glyoxylate and n-butanol. Since Gordonia terrae BOE5 was the only strain able to degrade nonpolar ethers like diethyl ether, the degradation pathway of 2-BE may be different for this strain.

Novel cyclohexane monooxygenase from Acidovorax sp. CHX100.

Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5-C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450 monooxygenase (CYP450chx) which catalyzed the transformation of cyclohexane to cyclohexanol. Primer walking methods categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in Escherichia coli JM109. The activity of CYP450chx was demonstrated by means of the indole co-oxidation. Biotransformation capability of CYP450chx was confirmed through the catalysis of cycloalkanes (C5-C8) to their respective cyclic alcohols.

Draft genome sequence of furan-degrading Rhodococcus erythropolis strain FUR100.

Rhodococcus erythropolis FUR100 was isolated from a mixture of soil and activated sludge. It can use furan as a sole source of carbon and energy. Its draft genome sequence may provide insight into the genetics of furan catabolism.

Degradation of 2-chlorotoluene by Rhodococcus sp. OCT 10.

A strain Rhodococcus sp. OCT 10 DSM 45596(T), exhibiting 99.9% of 16S rDNA identity with Rhodococcus wratislaviensis NCIMB 13082, was isolated from a soil sample. The strain completely mineralised 2-chlorotoluene, 2-bromotoluene, o-xylene, benzyl alcohol and benzoate. In contrast, 2-fluorotoluene was only partially mineralised. By GC-MS and (1)H-NMR analyses, 4-chloro-3-methylcatechol was identified as the central intermediate in the degradation pathway of 2-chlorotoluene. It was further degraded by enzymes of the meta cleavage pathway. Catechol 1,2-dioxygenase and chlorocatechol 1,2-dioxygenase as the initial enzymes of the ortho cleavage pathways were not detectable under these conditions. Furthermore, neither formation nor oxidation of 2-chlorobenzylic alcohol, 2-chlorobenzaldehyde, or 2-chlorobenzoate was observed, thereby excluding side chain oxidation activity.

Biodegradation of gaseous emissions of 2-chlorotoluene by strains of Rhodococcus sp. in polyurethane foam packed biotrickling filters.

About 60,000-70,000 tons of 2-chlorotoluene, which shows high toxicity in aquatic ecosystems, are produced worldwide and used in a tremendous field of applications. However, clear proofs of biodegradation were only presented for Comamonas testosteroni KT5 and Rhodococcus sp. OCT10. Hence, this study aims on the isolation of additional strains and their characterization in pilot-scale biotrickling filters. Three strains named OCT2, OCT9, and OCT14 of the genus Rhodococcus were isolated, able to mineralize gaseous 2-chlorotoluene like the previously isolated strain Rhodococcus sp. OCT10. The performance levels of these strains were tested in four biotrickling filters each containing 18.8 L of polyurethane foam package, showing elimination capacities of carbon (C) of 30.9 (OCT2), 30.1 (OCT9), 32.2 (OCT10), and 3.9 g C·m-3·h-1 (OCT14) at an average crude gas level of 397.6 mg C·m-3 and an empty bed residence time (EBRT) of 22.6 s. Since OCT10 showed the highest performance levels, this strain was characterized in a second biotrickling filter configuration at long-term conditions of 985 days, varying crude gas levels, EBRT and nutrient supply. Chloride balancing showed a recovery of 94.4% of 2-chlorotoluene eliminated out of the gas phase, pointing out mineralization of 2-chlorotoluene. German emission limit values were met at crude gas levels up to 750 mg C·m-3 at EBRTs of 120 s or higher. The maximum elimination capacity was 51.2 g C·m-3·h-1 at a specific freight of 51.9 g C·m-3·h-1 and an EBRT of 254 s. Performance levels were strongly boosted by addition of ammonia as nutrient and stabilized at efficiency levels higher than 90% at a feed rate of 4 g ammonium sulfate per week and 100 L of package volume. Repetitive monitoring of the established 2-chlorotoluene degrading community by BOX-PCR fingerprinting revealed a high long-term stability of OCT10, underlining its suitability in this kind of application.

Removal of cyclohexane gaseous emissions using a biotrickling filter system.

The removal of cyclohexane from gaseous emissions was studied using a biotrickling filter packed with polyurethane foam. Acivodorax sp. CHX100 was chosen as inoculum due to its ability to use cyclohexane as carbon source. Performance was evaluated by means of different resident times from 18 s to 37 s and concentration levels of 60, 90, 120, 160, 320, 480 and 720 mg C m-3, respectively. Removal efficiencies of 80%-99% and elimination capacities in the range of 5.4 g C m-3 h-1-38 g C m-3 h-1 were achieved for concentrations among 60 mg C m-3-480 mg C m-3. The removal efficiency decreased to 40% at concentrations of cyclohexane of 720 mg C m-3. The dynamics of the microbial population showed the strain CHX100 as predominant during the different operational process of biotrickling filter. The results of this study propose a novel approach for cleaning waste air containing cyclohexane by means of a biotrickling filter.

Degradation of toluene by ortho cleavage enzymes in Burkholderia fungorum†FLU100.

Burkholderia fungorum FLU100 simultaneously oxidized any mixture of toluene, benzene and mono-halogen benzenes to (3-substituted) catechols with a selectivity of nearly 100%. Further metabolism occurred via enzymes of ortho cleavage pathways with complete mineralization. During the transformation of 3-methylcatechol, 4-carboxymethyl-2-methylbut-2-en-4-olide (2-methyl-2-enelactone, 2-ML) accumulated transiently, being further mineralized only after a lag phase of 2 h in case of cells pre-grown on benzene or mono-halogen benzenes. No lag phase, however, occurred after growth on toluene. Cultures inhibited by chloramphenicol after growth on benzene or mono-halogen benzenes were unable to metabolize 2-ML supplied externally, even after prolonged incubation. A control culture grown with toluene did not show any lag phase and used 2-ML as a substrate. This means that 2-ML is an intermediate of toluene degradation and converted by specific enzymes. The conversion of 4-methylcatechol as a very minor by-product of toluene degradation in strain FLU100 resulted in the accumulation of 4-carboxymethyl-4-methylbut-2-en-4-olide (4-methyl-2-enelactone, 4-ML) as a dead-end product, excluding its nature as a possible intermediate. Thus, 3-methylcyclohexa-3,5-diene-1,2-diol, 3-methylcatechol, 2-methyl muconate and 2-ML were identified as central intermediates of productive ortho cleavage pathways for toluene metabolism in B. fungorum FLU100.

Comparison of biological and chemical treatment processes as cost-effective methods for elimination of benzoate in saline wastewaters.

Eight mixed cultures able to degrade benzoic acid under saline conditions were established and kinetic parameters were determined in batch processes with cultures SBM002 (0.5 g d(-1)·g oDM(-1)), SBM003 (0.7 g d(-1)·g oDM(-1)) and SBM007 (2.2 g d(-1)·g oDM(-1)) showing the highest degradation rates. Treatability of an industrial waste water (12 g L(-1) benzoic acid, 82 g L(-1) NaCl) by these cultures was proven in a fed-batch system (SBM002 & SBM003) and a continuous flow reactor (SBM007). The performance of the continuous flow reactor was 15-times higher compared to the fed-batch system due to the change of inocula, higher concentration of ammonia as nutrient and less accumulation of possibly toxic catecholic compounds. Average DOC reduction was found to be 98% at 100 g L(-1) NaCl and 1.2 g L(-1) benzoic acid under these conditions. Pre-treatment of the waste water via chemical precipitation by acidification to pH 3.5 diminished the concentration of benzoic acid to 2.1 g L(-1). In a combined chemical-biological process the volume of the bioreactor is reduced to 15% compared to a pure biological process. A comparison of operational costs for these three alternatives is presented.