Roland H. Müller

The Alkyl tert-Butyl Ether Intermediate 2-Hydroxyisobutyrate Is Degraded via a Novel Cobalamin-Dependent Mutase Pathway

ABSTRACT Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated β-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.

Correlation between cell composition and carbon conversion efficiency in microbial growth: a theoretical study

SummaryAs the macromolecular composition of microorganisms varies during their life cycle it was asked whether, and to what extent such changes exert any influence on substrate consumption, i.e. growth yield and carbon conversion efficiency, respectively. This question was dealt with in a theoretical study by use of the YAPTmax-concept. The growth substrates considered were methanol, acetate and glucose; the latter was assumed to be assimilated via both the glycolytic and the oxidative hexosemonophosphate pathway. Five fictitious biomasses were used which were altered in their proportion of polysaccharides, proteins, lipids, RNA and DNA. As a result, only small variations in the individual “biomass formulae” were obtained. On the basis of the energy balances for the syntheses of all cell constituents it was found that variations in the macromolecular composition of microbial biomass have only a slight effect on carbon conversion efficiency, amounting to maximally 3%. From the material balances it could be calculated that the upper, solely metabolism-determined limit of carbon conversion efficiency is 85% for substrates assimilated glycolytically via phosphoglycerate; for gluconeogenetic substrates, the upper limit was 75%. These limits are essentially determined by carbon loss on the way to amino acid syntheses.

Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA

A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans. This strain displays activity for the productive degradation of the two enantiomers of dichlorprop [(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP] and mecoprop [(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP] in addition phenoxyacetate herbicides, i.e. 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized. Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass [(RS)-2,4-DP]. Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l. The optimum pH value of (RS)-2,4-DP degradation was around 8. The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.

Glucose as an energy donor in acetate growing Acinetobacter calcoaceticus

Since glucose can be oxidized but not assimilated by Acinetobacter calcoaceticus 69-V the question arose whether energy generated by glucose oxidation can help incorporate carbon from heterotrophic substrates and, if so, what the efficiency of ATP production is like. For this reason this species was grown in the chemostat on acetate. After having reached steady state conditions an increasing concentration of glucose was added. This led to an increase in the biomass level from about 0.4 g/g for growth on acetate alone to 0.6–0.65 g/g in the presence of glucose, independently of either the growth rate or the steepness of the glucose gradient used. This upper value approximates about the limit of the carbon conversion efficiency calculated for non-glycolytic substrates. Glucose was almost exclusively oxidized to gluconic acid, 2- and 5-ketogluconates, and pentose 5-phosphates were found only in traces. These results demonstrate that glucose functions as an additional energy source in Acinetobacter calcoaceticus 69-V. From the transient behaviour of biomass increase and the mixing proportion at which the maximum growth yield on acetate in the presence of glucose was obtained it followed that two mol of ATP must have been generated per mol of glucose oxidized. This property is discussed in terms of coupling glucose dehydrogenase with the respiratory chain.

Degradation of fuel oxygenates and their main intermediates by Aquincola tertiaricarbonis L108.

Growth of Aquincola tertiaricarbonis L108 on the fuel oxygenates methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME), as well as on their main metabolites tert-butyl alcohol (TBA), tert-amyl alcohol (TAA) and 2-hydroxyisobutyrate (2-HIBA) was systematically investigated to characterize the range and rates of oxygenate degradation by this strain. The effective maximum growth rates for MTBE, ETBE and TAME at pH 7 and 30 degrees C were 0.045 h(-1), 0.06 h(-1) and 0.055 h(-1), respectively, whereas TAA, TBA and 2-HIBA permitted growth at rates up to 0.08 h(-1), 0.1 h(-1) and 0.17 h(-1), respectively. The experimental growth yields with all these substrates were high. Yields of 0.55 g dry mass (dm) (g MTBE)(-1), 0.53 g dm (g ETBE)(-1), 0.81 g dm (g TAME)(-1), 0.48 g dm (g TBA)(-1), 0.76 g dm (g TAA)(-1) and 0.54 g dm (g 2-HIBA)(-1) were obtained. Maximum specific degradation rates were 0.92 mmol MTBE h(-1) (g dm)(-1), 1.11 mmol ETBE h(-1) g(-1), 0.66 mmol TAME h(-1) g(-1), 1.19 mmol TAA h(-1) g(-1), 2.82 mmol TBA h(-1) g(-1), and 3.27 mmol 2-HIBA h(-1) g(-1). The relatively high rates with TBA, TAA and 2-HIBA indicate that the transformations of these metabolites did not limit the metabolism of MTBE and the related ether compounds. Despite the fact that these metabolites still carry a tertiary carbon atom that is commonly suspected to confer recalcitrance to the ether oxygenates, the transformation rates were in the same range as those with succinate and fructose. With MTBE, strain L108 grew at pHs between 5.5 and 8.0 at near-maximal rate, whereas no growth was found below pH 5.0 and above pH 9.0. The optimum growth temperature was 30 degrees C, but at 5 degrees C still about 15 % of the maximum rate remained, whereas no growth occurred at 42 degrees C. This indicates that MTBE metabolites are valuable substrates and that A. tertiaricarbonis L108 is a good candidate for bioremediation purposes. The possible origin of its exceptional metabolic capability is discussed in terms of the evolution of enzymic activities involved in the conversion of compounds carrying tertiary butyl groups.

Physiological and genetic characteristics of two bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides.

Two strains, Rhodoferax sp. P230 and Delftia (Comamonas) acidovorans MCI, have previously been shown to carry activities for the degradation of the two enantiomers of (RS)-2-(2,4-dichlorophenoxy-)propionate (dichlorprop) and (RS)-2-(4-chloro-2-methylphenoxy-)propionate (mecoprop) and, in addition, are capable of degrading phenoxyacetate derivatives 2.4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA). Metabolism of the herbicides is initiated by alpha-ketoglutarate-dependent dioxygenases for both enantiomers of the phenoxypropionate herbicides and for 2,4-D. These activities were constitutively expressed for both enantiomers of dichlorprop in strain MC1 and for the Renantiomer in strain P230. Enzyme activities for the complete degradation of phenoxyacetate and phenoxypropionate herbicides were induced during incubation on either of these herbicides. Strain MC1 has about threefold higher activities for the degradation of dichlorprop and for growth on this substrate (mumax = 0.15 h(-1)) than strain P230; the maximum growth rate on 2,4-D amounts to 0.045 h(-1) with strain MC1. Dichlorprop is utilized faster than mecoprop and the R-enantiomers are cleaved with higher rates than the S-enantiomers. The degradation of the chlorophenolic intermediates seems to proceed via the modified ortho cleavage pathway as indicated by activities of the respective enzymes. The enzymatic results were supported by genetic investigations by which the presence of the genes tfdB (encoding a dichlorophenol hydroxylase), tfdC (encoding a chlorocatechol 1,2-dioxygenase) and tfdD (encoding a chloromuconate cycloisomerase) could be demonstrated in both strains by PCR after application of respective primers. The presence of the tfdA gene (encoding a 2,4-D/alpha-ketoglutarate dioxygenase) was only shown for strain P230 but was lacking in strain MC1. Sequence analysis of the tfd gene fragments revealed high homology to the degradative genes of other proteobacterial strains degrading chloroaromatic compounds. Strain MC1 carries a plasmid of about 120 kb which apparently harbors herbicide degradative genes as concluded from deletion mutants which have lost 2,4-D[phenoxalkanoate]/alpha-ketoglutarate dioxygenase activities for cleavage of the R- and S-enantiomer, and of 2,4-D. For strain P230, no plasmid could be demonstrated; the activity was stably conserved in this strain during growth under nonselective conditions.

Mixed Substrate Utilization in Micro-organisms: Biochemical Aspects and Energetics

SUMMARY: The energy-based classification of heterotrophic substrates requires biochemical evaluation because some substrates can be assimilated by a variety of different metabolic pathways. By using the Y ATP-concept it was shown that the classification depends on the yield of ATP and reducing equivalents already generated on the way to the precursor (phosphoglycerate). With carbon-excess substrates a part of the total substrate consumed must be oxidized to completion merely for energy production, whereas with energy-excess substrates more energy is provided on the route to the precursor than is needed for assimilation of the precursor carbon. By means of this approach it was possible to assess experimental growth yields obtained on mixed substrates and to predict the optimum mixing proportion in order to attain the maximum carbon conversion efficiency. The validity of this method was shown for some examples.

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert-butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.

Carbon conversion efficiency and limits of productive bacterial degradation of methyl tert-butyl ether and related compounds.

ABSTRACT The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the YATP concept. Experiments were conducted to derive realistic maintenance coefficients and Ks values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g−1, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient ms and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, Smin, below which growth would not further be supported. Smin strongly depended on the maximum growth rate μmax, and b and was directly correlated with the half maximum rate-associated substrate concentration Ks, meaning that any effect impacting this parameter would also change Smin. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase Ks and Smin for MTBE.

Effects of Pore-Scale Heterogeneity and Transverse Mixing on Bacterial Growth in Porous Media

Microbial degradation of contaminants in the subsurface requires the availability of nutrients; this is impacted by porous media heterogeneity and the degree of transverse mixing. Two types of microfluidic pore structures etched into silicon wafers (i.e., micromodels), (i) a homogeneous distribution of cylindrical posts and (ii) aggregates of large and small cylindrical posts, were used to evaluate the impact of heterogeneity on growth of a pure culture (Delftia acidovorans) that degrades (R)-2-(2,4-dichlorophenoxy)propionate (R-2,4-DP). Following inoculation, dissolved O2 and R-2,4-DP were introduced as two parallel streams that mixed transverse to the direction of flow. In the homogeneous micromodel, biomass growth was uniform in pore bodies along the center mixing line, while in the aggregate micromodel, preferential growth occurred between aggregates and slower less dense growth occurred throughout aggregates along the center mixing line. The homogeneous micromodel had more rapid growth overall (2 times) and more R-2,4-DP degradation (9.5%) than the aggregate pore structure (5.7%). Simulation results from a pore-scale reactive transport model indicate mass transfer limitations within aggregates along the center mixing line decreased overall reaction; hence, slower biomass growth rates relative to the homogeneous micromodel are expected. Results from this study contribute to a better understanding of the coupling between mass transfer, reaction rates, and biomass growth in complex porous media and suggest successful implementation and analysis of bioremediation systems requires knowledge of subsurface heterogeneity.