Biotransformations of aromatic aldehydes by acetogenic bacteria

Vanillin was subject to O demethylation and supported growth of Clostridium formicoaceticum and Clostridium thermoaceticum. Vanillin was also stimulatory to the CO-dependent growth of Peptostreptococcus productus. The aldehyde substituent of vanillin was metabolized by routes which were dependent upon both the acetogen and a co-metabolizable substrate (e.g. carbon monoxide [CO]). C. formicoaceticum and C. thermoaceticum oxidized the aldehyde group of vanillin to the carboxyl level, while P. productus reduced the aldehyde group of vanillin to the alcohol level. In contrast, during CO-dependent growth, C. thermoaceticum reduced 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol while P. productus both reduced and oxidized 4-hydroxybenzaldehyde to 4-hydroxybenzyl alcohol and 4-hydroxybenzoate, respectively. These metabolic potentials indicate aromatic aldehydes may affect the flow of reductant during acetogenesis.     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.     

On a reversible molybdenum-containing aldehyde oxidoreductase from Clostridium formicoaceticum

Clostridium formicoaceticum grown in the presence of 1 mM molybdate and about 1.5×10^-5 mM tungsten (present in the 5 g yeast extract/l of the growth medium) forms two reversible aldehyde oxidoreductases in an activity ratio of about 45:55. The fraction of 45% does not bind to the octyl-Sepharose column, whereas the 55% aldehyde oxidoreductase binds to this column. From cells grown on a synthetic medium without the addition of tungstate only about 2% of the aldehyde oxidoreductase of the crude extract binds to octyl-Sepharose. The enzyme not binding to octyl-Sepharose has been purified as judged by electrophoresis. It is pure after about 50 fold enrichment. According to SDS gel electrophoresis the enzyme consists of identical 100 kD subunits. Based on gel chromatography it seems to be a trimer. Per subunit 0.6 molybdenum, 7 iron, 6.6 acid labile sulphur, about 0.1 pterin-6-carboxylic and <0.05 tungsten have been found. The first 13 amino acids from the amino end show no similarity with the W-containing aldehyde oxidoreductase from the same bacterium. With reduced tetramethylviologen (E₀=–550 mV) the new molybdenum containing enzyme can reduce various aliphatic and aromatic acids to aldehydes. The pH optimum is at 6.0. For the dehydrogenation of butyraldehyde a rather broad pH region from pH 6 to 10 shows almost no variation of rate. From 15 different aldehydes acetaldehyde exhibits the highest rate. The K_m value for butanal is 0.002 and for propionate 7.0 mM. Compared with the tungsten enzyme the molybdenum enzyme is only moderately oxygen-sensitive.Abbreviations AOR aldehyde oxidoreductase - BV benzylviologen - MV methylviologen - NH₂CO-MV 1,1-carbamoylmethylviologen - TMV 1,1,2,2-tetramethylviologen     

Production of an aromatic aldehyde oxidase by Streptomyces viridosporus

Streptomyces viridosporus strain T7A, when grown in liquid media containing yeast extract and aromatic aldehydes, oxidized the aromatic aldehydes to the corresponding aromatic acids. Benzaldehyde, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, and protocatechualdehyde were catabolized further via the -ketoadipate and gentisate pathways. Dehydrodivanillin, isophthalaldehyde, salicylaldehyde, syringaldehyde, terephthalaldehyde, vanillin, and veratraldehyde were oxidized only as far as the corresponding aromatic acids. Phthalaldehyde and aliphatic aldehydes were not oxidized. The aromatic aldehyde oxidase, which was produced by cultures grown in either the presence or absence of aromatic aldehydes, was partially purified by ammonium sulfate precipitation and ion-exchange chromatography. It consumed molecular oxygen, oxidized aromatic aldehydes to aromatic acids, and produced hydrogen peroxide all in equimolar amounts.Paper no. 81515 of the Idaho Agricultural Experiment Station     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

A strictly anaerobic nitrate-reducing bacterium growing with resorcinol and other aromatic compounds

With resorcinol as sole source of energy and organic carbon, two stains of gram-negative, nitrate-reducing bacteria were isolated under strictly anaerobic conditions. Strain LuBRes1 was facultatively anaerobic and catalase- and superoxide dismutase-positive. This strain was affiliated with Alcaligenes denitrificans on the basis of substrate utilization spectrum and peritrichous flagellation. Strain LuFRes1 could grow only under anaerobic conditions with oxidized nitrogen compounds as electron acceptor. Cells were catalase-negative but superoxide dismutase-positive. Since this strain was apparently an obligate nitrate reducer, it could not be grouped with any existing genus. Resorcinol was completely oxidized to CO₂ by both strains. Neither an enzyme activity reducing or hydrolyzing the resorcinol molecule, nor an acyl-CoA-synthetase activating resorcylic acids or benzoate was detected in cell-free extracts of cells grown with resorcinol. In dense cell suspensions, both strains produced a compound which was identified as 5-oxo-2-hexenoic acid by mass spectrometric analysis. This would indicate a direct, hydrolytic cleavage of the resorcinol nucleus without initial reduction.     

Screening for plasmids in the genus Clostridium

A plasmid screening was performed on 150 strains out of 75 clostridial species using a modification of the alkaline-lysis procedure. In 26 strains representing 21 species one or more plasmid bands were detected ranging in size from 3 to more than 100 kilobase pairs. Clostridium aceticum proved to contain a single small plasmid (pCA1) of 5.4 kbp as revealed by restriction analysis and electron microscopy. A physical map of pCA1 has been constructed. Spontaneous mutants of C. aceticum defective in autotrophic growth have been isolated. No direct correlation between plasmid content and autotrophy could be found.Abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - TAE Trisacetate-EDTA - Tris tris-(hydroxymethyl)aminomethane     

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg^–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose. Received: 14 May 1998 / Accepted: 7 August 1998     

Catabolism of the lignin substructure model compound dehydrodivanillin by a lignin-degrading Streptomyces

The lignin-degrading actinomycete Streptomyces viridosporus T7A readily degrades the lignin model compound dehydrodivanillin. Four mutants of this organism (produced by irradiation of spores with ultraviolet light) were shown to have lost the ability to catabolize dehydrodivanillin. These mutant strains retained an undiminished ability to degrade Douglas-fir lignin (¹⁴C-lignin ¹⁴CO₂) as compared to the wild-type strain. None of the strains accumulated detectable quantities of dehydrodivanillin when grown on lignocellulose. Thus it appears that the enzymes involved in dehydrodivanillin catabolism are not a part of the streptomycete's system for degrading polymeric lignin. It is concluded that dehydrodivanillin is probably not a relevant model compound for study of lignin polymer degradation by Streptomyces viridosporus. Since many stable mutants completely lacking DHDV-degrading ability were readily obtained, it is suggested that the relevant catabolic enzymes may be encoded on a plasmid.Abbreviations DHDV dehydrodivanillin     

Dioxygenase activity and relative behaviour of Pseudomonas strains from soil in the presence of different aromatic compounds

Ten different Pseudomonas strains isolated from contaminated soils were tested for expression of active dioxygenases. Of these, two different clusters, related to strain origin were observed. The first included two P. fluorescens strains and two P. aeruginosa strains isolated from soils polluted with polyaromatic hydrocarbons and the second two P. cepacia strains and four P. chlororaphis strains from soils with polyphenols. All the isolates showed catechol 1,2-dioxygenase basal activity, while other dioxygenases (catechol 2,3-dioxygenase, protocatechuate 2,3-, 3,4- and 4,5-dioxygenases) were detected only after growth in the presence of suitable inducers (benzoate, catechol, salicylate, phenol). Significant induction of catechol 1,2-dioxygenase, the major activity of the tested strains, was also observed when combining starvation with the presence of high molecular weight aromatic hydrocarbons with recalcitrant structures (fluoranthene, chrysene, benzanthracene, pyrene).     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Anaerobic degradation of methoxylated aromatic compounds by Clostridium methoxybenzovorans and a nitrate-reducing bacterium Thauera sp. strain Cin3,4

The coupling of growth of the o-demethylating bacterium, Clostridium methoxybenzovorans SR3, with a nitrate-reducing bacterium able to degrade aromatic compounds, Thauera sp. Cin3,4, allowed complete mineralization of poorly oxidizable methoxylated aromatic compounds such as vanillate, isovanillate, vanilline, anisate, ferulate and veratrate. C. methoxybenzovorans o-demethylated these aromatic compounds to their corresponding hydroxylated derivatives and fermented the side chains to acetate and butyrate. The hydroxylated compounds and the fermentation end-products in the C. methoxybenzovorans spent growth medium were then completely metabolized to CO₂ on inoculation with the Thauera strain. Kinetic studies with veratrate indicated that C. methoxybenzovorans initially o-demethylated the substrate to vanillate and then further to protocatechuate together with the production of acetate and butyrate from the demethylated side chains. Protocatechuate, acetate and butyrate were then utilized as a carbon source by the Thauera strain aerobically or anaerobically in the presence of nitrate. The results therefore suggest that mono- or dimethoxylated aromatic compounds can be completely mineralized by coupling the growth of a fermentative bacterium with a nitrate-reducing bacterium, and a metabolic pathway for this is proposed.     

Complete anaerobic oxidation of hydroquinone by Desulfococcus sp. strain Hy5: indications of hydroquinone carboxylation to gentisate

The sulfate-reducing strain Hy5 was able to grow with hydroquinone as sole source of carbon and energy. In experiments with dense cell suspensions, several indications were found that gentisate was the first intermediate in anaerobic degradation of hydroquinone: (1) degradation of hydroquinone was accelerated by addition of bicarbonate; (2) cell suspensions grown with hydroquinone oxidized gentisate at a rate similar to that of suspensions grown with gentisate, whereas the latter were not able to degrade hydroquinone in the presence of chloramphenicol; (3) in SDS-PAGE analysis of cell-free extracts of strain Hy5, two additional protein bands were found after growth with hydroquinone that were not detected in cells grown with gentisate, probably representing a hydroquinone carboxylating enzyme. A corresponding enzyme activity could not be detected. In cell-free extracts of hydroquinone-grown strain Hy5, the specific acyl-CoA ligase activity with gentisate as substrate was detected at 70 nmol x mg^-1 x min^-1. Gentisyl-CoA was enzymatically reduced to several unidentified nonaromatic products in the presence of dithionite-reduced methyl viologen.     

Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or l-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2–3.5 U per mg of protein. Reduced benzyl viologen, FMNH₂ and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates.The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. These results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - O.D optical density - DTE dithioerythritol     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate

The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH₃) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO₂. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultanous adaptation: cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on tolucne; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.     

Utilization of aromatic amino acids as nitrogen sources in Brevibacterium linens: an inducible aromatic amino acid aminotransferase

The first step of the utilization of the aromatic amino acids as sole nitrogen sources by Brevibacterium linens strain 47 was found to be a transamination. The deaminated metabolites of the amino acids were detected in culture supernatants, and the enzyme activity was identified in cell free extracts. The cells contained increased aromatic amino acid aminotransferase activities on growth on the aromatic amino acids as sole nitrogen sources. Two aromatic aminotransferases (AT-I and AT-II) were separated upon diethylaminoethyl-Trisacryl M column chromatography of cell free extracts. Only AT-I was responsible for the increased level of aromatic amino acid aminotransferase activity of induced cells. The results suggested a catabolic role of AT-I in vivo.Abbreviations DNP dinitrophenyl - HPLC high performance liquid chromatography - PLP pyridoxal-5-phosphate     

Anaerobic degradation of phenol by pure cultures of newly isolated denitrifying pseudomonads

From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO₂ with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gramnegative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.     

Evidence for two uptake systems in Rhizobium leguminosarum for hydroxy-aromatic compounds metabolized by the 3-oxoadipate pathway

Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii have separate uptake systems for 4-hydroxybenzoate and protocatechuate. The 4-hydroxybenzoate uptake system (pobP) is inhibited by a range of compounds with substitution at the 4-position on the aromatic ring whereas the uptake system for protocatechuate (pcaP) is markedly inhibited only by other dihydroxybenzoic acids. The rate of 4-hydroxybenzoate uptake is very low in Rhizobium leguminosarum and Rhizobium trifolii grown on protocatechuate but mutants defective in 4-hydroxybenzoate uptake transport protocatechuate at rates similar to the wild-type grown under similar conditions.     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1