Lloyd J. Nadeau

The influence of acidity on microbial fuel cells containing Shewanella oneidensis

Microbial fuel cells (MFCs) traditionally operate at pH values between 6 and 8. However, the effect of pH on the growth and electron transfer abilities of Shewanella oneidensis MR-1 (wild-type) and DSP10 (spontaneous mutant), bacteria commonly used in MFCs, to electrodes has not been examined. Miniature MFCs using bare graphite felt electrodes and nanoporous polycarbonate membranes with MR-1 or DSP10 cultures generated >8W/m(3) and approximately 400muA between pH 6-7. The DSP10 strain significantly outperformed MR-1 at neutral pH but underperformed at pH 5. Higher concentrations of DSP10 were sustained at pH 7 relative to that of MR-1, whereas at pH 5 this trend was reversed indicating that cell count was not solely responsible for the observed differences in current. S. oneidensis MR-1 was determined to be more suitable than DSP10 for MFCs with elevated acidity levels. The concentration of riboflavin in the bacterial cultures was reduced significantly at pH 5 for DSP10, as determined by high performance liquid chromatography (HPLC) of the filter sterilized growth media. In addition, these results suggest that mediator biosynthesis and not solely bacterial concentration plays a significant role in current output from S. oneidensis containing MFCs.

Impedance spectroscopy as a tool for non-intrusive detection of extracellular mediators in microbial fuel cells

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio‐energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10‐based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling. Biotechnol. Bioeng. 2009; 104: 882–891.

Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45

ABSTRACT Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85°C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60°C. HAB mutase activity in extracts of P. pseudoalcaligenesJS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and thehabB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosisH37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. colicontaining M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream ofhabB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

The utility of Shewanella japonica for microbial fuel cells

Shewanella-containing microbial fuel cells (MFCs) typically use the fresh water wild-type strain Shewanella oneidensis MR-1 due to its metabolic diversity and facultative oxidant tolerance. However, S. oneidensis MR-1 is not capable of metabolizing polysaccharides for extracellular electron transfer. The applicability of Shewanella japonica (an agar-lytic Shewanella strain) for power applications was analyzed using a diverse array of carbon sources for current generation from MFCs, cellular physiological responses at an electrode surface, biofilm formation, and the presence of soluble extracellular mediators for electron transfer to carbon electrodes. Critically, air-exposed S. japonica utilizes biosynthesized extracellular mediators for electron transfer to carbon electrodes with sucrose as the sole carbon source.

Construction of Escherichia coli Strains for Conversion of Nitroacetophenones to ortho-Aminophenols

ABSTRACT The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-β-d-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.

Oxidation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5

Abstract Previous studies demonstrated that Alcaligenes eutrophus A5 transforms 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) to 4-chlorobenzoate via a meta-ring fission product. The initial reactions could be catalyzed by either monooxygenase or dioxygenase enzymes. In the present study, a transient intermediate that accumulated during the transformation of DDT by the biphenyl-grown cells was identified as 1,1,1-trichloro-2-(4-chlorophenyl-2,3-dihydro-4,6-cyclohexadiene)-2-(4′-chlorophenyl)ethane (DDT-2,3-dihydrodiol) on the basis of mass spectral analysis after n-butylboronic acid derivatization. The dihydrodiol undergoes a characteristic acid-catalyzed dehydration to produce phenols. 1H-NMR indicated a cis-relative stereochemistry. The results indicate that the biphenyl dioxygenase from A. eutrophus A5 catalyzes the dihydroxylation of DDT at the unsubstituted carbons on the aromatic ring to produce DDT-2,3-dihydrodiol.

Bacterial Conversion of Hydroxylamino Aromatic Compounds by both Lyase and Mutase Enzymes Involves Intramolecular Transfer of Hydroxyl Groups

ABSTRACT Hydroxylamino aromatic compounds are converted to either the corresponding aminophenols or protocatechuate during the bacterial degradation of nitroaromatic compounds. The origin of the hydroxyl group of the products could be the substrate itself (intramolecular transfer mechanism) or the solvent water (intermolecular transfer mechanism). The conversion of hydroxylaminobenzene to 2-aminophenol catalyzed by a mutase from Pseudomonas pseudoalcaligenes JS45 proceeds by an intramolecular hydroxyl transfer. The conversions of hydroxylaminobenzene to 2- and 4-aminophenol by a mutase from Ralstonia eutropha JMP134 and to 4-hydroxylaminobenzoate to protocatechuate by a lyase from Comamonas acidovorans NBA-10 and Pseudomonas sp. strain 4NT were proposed, but not experimentally proved, to proceed by the intermolecular transfer mechanism. GC-MS analysis of the reaction products formed in H218O did not indicate any 18O-label incorporation during the conversion of hydroxylaminobenzene to 2- and 4-aminophenols catalyzed by the mutase from R. eutropha JMP134. During the conversion of 4-hydroxylaminobenzoate catalyzed by the hydroxylaminolyase from Pseudomonas sp. strain 4NT, only one of the two hydroxyl groups in the product, protocatechuate, was 18O labeled. The other hydroxyl group in the product must have come from the substrate. The mutase in strain JS45 converted 4-hydroxylaminobenzoate to 4-amino-3-hydroxybenzoate, and the lyase in Pseudomonas strain 4NT converted hydroxylaminobenzene to aniline and 2-aminophenol but not to catechol. The results indicate that all three types of enzyme-catalyzed rearrangements of hydroxylamino aromatic compounds proceed via intramolecular transfer of hydroxyl groups.

Production of 2-amino-5-phenoxyphenol from 4-nitrobiphenyl ether using nitrobenzene nitroreductase and hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45

Microbial metabolism of nitroarenes via o-aminophenols requires the participation of two key enzymes, a nitroreductase and an hydroxylaminobenzene mutase. The broad substrate ranges of the enzymes suggested that they could be used as biocatalysts for the production of substituted o-aminophenols. We have used enzymes from Pseudomonas pseudoalcaligenes JS45 for the conversion of 4-nitrobiphenyl ether to the corresponding o-aminophenol. Partially purified nitrobenzene nitroreductase reduced 4-nitrobiphenyl ether to the corresponding 4-hydroxylaminobiphenyl ether. Partially purified hydroxylaminobenzene mutase stoichiometrically converted the intermediate to 2-amino-5-phenoxyphenol. The results indicate that the enzyme system can be applied for the production of o-aminophenols useful as intermediates for synthesis of commercially important materials. Journal of Industrial Microbiology & Biotechnology (2000) 24, 301–305.

Biodegradation of medium chain hydrocarbons by Acinetobacter venetianus 2AW immobilized to hair-based adsorbent mats†

The natural attenuation of hydrocarbons can be hindered by their rapid dispersion in the environment and limited contact with bacteria capable of oxidizing hydrocarbons. A functionalized composite material is described herein, that combines in situ immobilized alkane‐degrading bacteria with an adsorbent material that collects hydrocarbon substrates, and facilitates biodegradation by the immobilized bacterial population. Acinetobacter venetianus 2AW was isolated for its ability to utilize hydrophobic n‐alkanes (C10–C18) as the sole carbon and energy source. Growth of strain 2AW also resulted in the production of a biosurfactant that aided in the dispersion of complex mixtures of hydrophobic compounds. Effective immobilization of strain 2AW to the surface of Ottimat™ adsorbent hair mats via vapor phase deposition of silica provided a stable and reproducible biocatalyst population that facilitates in situ biodegradation of n‐alkanes. Silica‐immobilized strain 2AW demonstrated ca. 85% removal of 1% (v/v) tetradecane and hexadecane within 24 h, under continuous flow conditions. The methodology for immobilizing whole bacterial cells at the surface of an adsorbent, for in situ degradation of hydrocarbons, has practical application in the bioremediation of oil in water emulsions. Published 2011 American Institute of Chemical Engineers Biotechnol Prog., 2011

Continuous synthesis of aminophenols from nitroaromatic compounds by combination of metal and biocatalyst

The combined action of immobilized hydroxylaminobenzene mutase and zinc in a flow-through system catalyzes the conversion of nitroaromatic compounds to the corresponding ortho-aminophenols, including a novel analog of chloramphenicol.