Kenneth F. Reardon

Biodegradation kinetics of benzene, toluene, and phenol as single and mixed substrates for Pseudomonas putida F1.

Although microbial growth on substrate mixtures is commonly encountered in bioremediation, wastewater treatment, and fermentation, mathematical modeling of mixed substrate kinetics has been limited. We report the kinetics of Pseudomonas putida F1 growing on benzene, toluene, phenol, and their mixtures, and compare mathematical models to describe these results. The three aromatics are each able to act as carbon and energy sources for this strain. Biodegradation rates were measured in batch cultivations following a protocol that eliminated mass transfer limitations for the volatile substrates and considered the culture history of the inoculum and the initial substrate to inoculum mass ratio. Toluene and benzene were better growth substrates than phenol, resulting in faster growth and higher yield coefficients. In the concentration ranges tested, toluene and benzene biodegradation kinetics were well described by the Monod model. The Monod model was also used to characterize phenol biodegradation by P. putida F1, although a small degree of substrate inhibition was noted. In mixture experiments, the rate of consumption of one substrate was found to be affected by the presence of the others, although the degree of influence varied widely. The substrates are catabolized by the same enzymatic pathway, but purely competitive enzyme kinetics did not capture the substrate interactions well. Toluene significantly inhibited the biodegradation rate of both of the other substrates, and benzene slowed the consumption of phenol (but not of toluene). Phenol had little effect on the biodegradation of either toluene or benzene. Of the models tested, a sum kinetics with interaction parameters (SKIP) model provided the best description of the paired substrate results. This model, with parameters determined from one- and two-substrate experiments, provided an excellent prediction of the biodegradation kinetics for the three-component mixture.

Flow cytometry in biotechnology

Abstract. Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

Expression of industrially relevant laccases: prokaryotic style.

Laccases are a class of multi-copper oxidases (MCOs) that catalyze the one-electron oxidation of four equivalents of a reducing substrate, with the concomitant four-electron reduction of dioxygen to water. They can catalyze a multitude of reactions, including the degradation of polymers and oxidative coupling of phenolic compounds, positioning them as significant industrial enzymes. Although fungal laccases are well known and well characterized, only recently has in silico biology led to rapid advances in the discovery, characterization and engineered expression of prokaryotic laccases. We describe the recent burgeoning of prokaryotic laccases, their catalytic properties, structural features and molecular evolution, vis-à-vis fungal laccases where possible. Special focus is given to the application of laccases to the emerging cellulosic biofuel industry.

On-line infrared spectroscopy for bioprocess monitoring

One of the major aims of bioprocess engineering is the real-time monitoring of important process variables. This is the basis of precise process control and is essential for high productivity as well as the exact documentation of the overall production process. Infrared spectroscopy is a powerful analytical technique to analyze a wide variety of organic compounds. Thus, infrared sensors are ideal instruments for bioprocess monitoring. The sensors are non-invasive, have no time delay due to sensor response times, and have no influence on the bioprocess itself. No sampling is necessary, and several components can be analyzed simultaneously. In general, the direct monitoring of substrates, products, metabolites, as well as the biomass itself is possible. In this review article, insights are provided into the different applications of infrared spectroscopy for bioprocess monitoring and the complex data interpretation. Different analytical techniques are presented as well as example applications in different areas.

Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds.

Previous work showed that random mutagenesis produced a mutant of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 containing the V106A substitution in the hydroxylase α-subunit (TomA3) that changed the color of the cell suspension from wild-type brown to green in rich medium. Here, DNA shuffling was used to isolate a random TOM mutant that turned blue due to mutation TomA3 A113V. To better understand the TOM reaction mechanism, we studied the specificity of indole hydroxylation using a spectrum of colored TOM mutants expressed in Escherichia coli TG1 and formed as a result of saturation mutagenesis at TomA3 positions A113 and V106. Colonies expressing these altered enzymes ranged in color from blue through green and purple to orange; and the enzyme products were identified using thin-layer chromatography, high performance liquid chromatography, and liquid chromatography–mass spectroscopy. Derived from the single TOM template, enzymes were identified that produced primarily isoindigo (wild-type TOM), indigo (A113V), indirubin (A113I), and isatin (A113H and V106A/A113G). The discovery that wild-type TOM formed isoindigo via C-2 hydroxylation of the indole pyrrole ring makes this the first oxygenase shown to form this compound. Variant TOM A113G was unable to form indigo, indirubin, or isoindigo (did not hydroxylate the indole pyrrole ring), but produced 4-hydroxyindole and unknown yellow compounds from C-4 hydroxylation of the indole benzene ring. Mutations at V106 in addition to A113G restored C-3 indole oxidation, so along with C-2 indole oxidation, isatin, indigo, and indirubin were formed. Other TomA3 V106/A113 mutants with hydrophobic, polar, or charged amino acids in place of the Val and/or Ala residues hydroxylated indole at the C-3 and C-2 positions, forming isatin, indigo, and indirubin in a variety of distributions. Hence, for the first time, a single enzyme was genetically modified to produce a wide range of colors from indole.

Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

ABSTRACT DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

ABSTRACT Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (Vmax of 9.3 versus 4.5 nmol · min−1 · mg of protein−1 and unchanged Km), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol · min−1 · mg of protein−1 [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (Vmax of 2.61 versus 0.95 nmol · min−1 · mg of protein−1 and unchanged Km) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol · min−1 · mg of protein−1). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol · min−1 · mg of protein−1). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.

Detection and Quantification of Functional Genes of Cellulose- Degrading, Fermentative, and Sulfate-Reducing Bacteria and Methanogenic Archaea

ABSTRACT Cellulose degradation, fermentation, sulfate reduction, and methanogenesis are microbial processes that coexist in a variety of natural and engineered anaerobic environments. Compared to the study of 16S rRNA genes, the study of the genes encoding the enzymes responsible for these phylogenetically diverse functions is advantageous because it provides direct functional information. However, no methods are available for the broad quantification of these genes from uncultured microbes characteristic of complex environments. In this study, consensus degenerate hybrid oligonucleotide primers were designed and validated to amplify both sequenced and unsequenced glycoside hydrolase genes of cellulose-degrading bacteria, hydA genes of fermentative bacteria, dsrA genes of sulfate-reducing bacteria, and mcrA genes of methanogenic archaea. Specificity was verified in silico and by cloning and sequencing of PCR products obtained from an environmental sample characterized by the target functions. The primer pairs were further adapted to quantitative PCR (Q-PCR), and the method was demonstrated on samples obtained from two sulfate-reducing bioreactors treating mine drainage, one lignocellulose based and the other ethanol fed. As expected, the Q-PCR analysis revealed that the lignocellulose-based bioreactor contained higher numbers of cellulose degraders, fermenters, and methanogens, while the ethanol-fed bioreactor was enriched in sulfate reducers. The suite of primers developed represents a significant advance over prior work, which, for the most part, has targeted only pure cultures or has suffered from low specificity. Furthermore, ensuring the suitability of the primers for Q-PCR provided broad quantitative access to genes that drive critical anaerobic catalytic processes.

Future aspects of bioprocess monitoring.

Nature has the impressive ability to efficiently and precisely control biological processes by applying highly evolved principles and using minimal space and relatively simple building blocks. The challenge is to transfer these principles into technically applicable and precise analytical systems that can be used for many applications. This article summarizes some of the new approaches in sensor technology and control strategies for different bioprocesses such as fermentations, biotransformations, and downstream processes. It focuses on bio- and chemosensors, optical sensors, DNA and protein chip technology, software sensors, and modern aspects of data evaluation for improved process monitoring and control.

Fenton's oxidation of pentachlorophenol

The combination of H(2)O(2) and Fe(II) (Fentons reaction) has been demonstrated to rapidly degrade many organics via hydroxyl radicals. However, few studies have related hydroxyl radical generation rates with measured organic chemical degradation data. The goals of this work were to investigate the kinetics, stoichiometry, and intermediates of pentachlorophenol (PCP) degradation in the Fentons reaction and to develop a mathematical model of this reaction system. Batch reaction experiments were performed to assess both initial transients and steady states, and special attention was given to the analysis of intermediates. Solutions of PCP (55 microM) and Fe(II) (200 microM) were treated with variable levels of H(2)O(2) (<850 microM), and the concentrations of these reactants and their products were measured. Partial PCP degradation and near stoichiometric dechlorination were observed at low initial H(2)O(2) concentrations. Higher H(2)O(2) doses achieved at most 70% dechlorination even though nearly all of the PCP was degraded. The reaction intermediates tetrachlorohydroquinone and dichloromaleic acid accounted for up to 5% of the PCP degraded. Organic carbon mineralization (transformation to CO(2)) was not observed. The ()OH scavenging effects of the PCP-by-products mixture were characterized as a lumped parameter in the reaction kinetics model, which provided reasonable predictions of experimental results at different oxidant concentrations and reaction time.