Chlorophenol degradation coupled to sulfate reduction

[This corrects the article on p. 3257 in vol. 56.].     

Chlorophenol degradation coupled to sulfate reduction.

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Chlorophenol degradation by Phanerochaete chrysosporium

Degradation of chlorophenols by P. chrysosporium in static cultures has been studied. The influences of mycelium acclimation, co-substrate concentration and nitrogen source on phenol degradation were analyzed. With non-acclimated mycelium the maximal concentrations degraded were 150 ppm of o-chorophenol and 100 ppm of the isomers m- and p-chlorophenol. The substituted ortho-position on the aromatic ring was the preferred attack position. Meta- and para-positions were less reactive and resulted in a slower degradation rate than the ortho position. Nevertheless, with acclimated mycelium, an increase in the ability to degrade chlorophenol and a higher reactivity in meta- and para-positions were observed (degraded chlorophenol increased by up to 70% for the o-isomer and 50% for the m- and p-isomers with respect to non-acclimated mycelium). A decrease in glucose concentration caused a decrease in chlorophenol degradation rate. Twelve days were needed for complete degradation of o-chlorophenol with 10 g/l of glucose and 22 days when glucose concentration was decreased to 2.5 g/l. The reduction of ammonium tartrate caused a greater lag time, but not a decrease in chlorophenol degradation rate. Replacement of ammonium tartrate by ammonium chloride caused a decrease in chlorophenol degradation rate.     

Direct cathodic electron uptake coupled to sulfate reduction by Desulfovibrio ferrophilus IS5 biofilms

Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero-valent iron (Fe⁰) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe⁰-corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate-starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO₄²⁻ reduction. The insensitivity of Fe⁰ corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe⁰ corrosion.     

Anaerobic degradation of benzene in BTX mixtures dependent on sulfate reduction

Abstract Enrichment cultures from marine sediments mineralized benzene while using sulfate as the terminal electron acceptor. Parallel cultures using river marsh sediment displayed no activity. Mineralization was confirmed by release of ¹⁴CO₂ from radiolabeled benzene. The dependence on sulfate reduction was demonstrated by stoichiometric balances and the use of specific inhibitors. This work supports recent observations that anaerobic benzene degradation takes place coupled to sulfate reduction.     

Chlorophenol hydroxylases encoded by plasmid pJP4 differentially contribute to chlorophenoxyacetic acid degradation

Phenoxyalkanoic compounds are used worldwide as herbicides. Cupriavidus necator JMP134(pJP4) catabolizes 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), using tfd functions carried on plasmid pJP4. TfdA cleaves the ether bonds of these herbicides to produce 2,4-dichlorophenol (2,4-DCP) and 4-chloro-2-methylphenol (MCP), respectively. These intermediates can be degraded by two chlorophenol hydroxylases encoded by the tfdB(I) and tfdB(II) genes to produce the respective chlorocatechols. We studied the specific contribution of each of the TfdB enzymes to the 2,4-D/MCPA degradation pathway. To accomplish this, the tfdB(I) and tfdB(II) genes were independently inactivated, and growth on each chlorophenoxyacetate and total chlorophenol hydroxylase activity were measured for the mutant strains. The phenotype of these mutants shows that both TfdB enzymes are used for growth on 2,4-D or MCPA but that TfdB(I) contributes to a significantly higher extent than TfdB(II). Both enzymes showed similar specificity profiles, with 2,4-DCP, MCP, and 4-chlorophenol being the best substrates. An accumulation of chlorophenol was found to inhibit chlorophenoxyacetate degradation, and inactivation of the tfdB genes enhanced the toxic effect of 2,4-DCP on C. necator cells. Furthermore, increased chlorophenol production by overexpression of TfdA also had a negative effect on 2,4-D degradation by C. necator JMP134 and by a different host, Burkholderia xenovorans LB400, harboring plasmid pJP4. The results of this work indicate that codification and expression of the two tfdB genes in pJP4 are important to avoid toxic accumulations of chlorophenols during phenoxyacetic acid degradation and that a balance between chlorophenol-producing and chlorophenol-consuming reactions is necessary for growth on these compounds.     

Anaerobic degradation of aromatic compounds coupled to Fe(III) reduction by Ferroglobus placidus

Aromatic compounds are an important component of the organic matter in some of the anaerobic environments that hyperthermophilic microorganisms inhabit, but the potential for hyperthermophilic microorganisms to metabolize aromatic compounds has not been described previously. In this study, aromatic metabolism was investigated in the hyperthermophile Ferroglobus placidus . F. placidus grew at 85°C in anaerobic medium with a variety of aromatic compounds as the sole electron donor and poorly crystalline Fe(III) oxide as the electron acceptor. Growth coincided with Fe(III) reduction. Aromatic compounds supporting growth included benzoate, phenol, 4-hydroxybenzoate, benzaldehyde, p -hydroxybenzaldehyde and t -cinnamic acid (3-phenyl-2-propenoic acid). These aromatic compounds did not support growth when nitrate was provided as the electron acceptor, even though nitrate supports the growth of this organism with Fe(II) or H₂ as the electron donor. The stoichiometry of benzoate and phenol uptake and Fe(III) reduction indicated that F. placidus completely oxidized these aromatic compounds to carbon dioxide, with Fe(III) serving as the sole electron acceptor. This is the first example of an Archaea that can anaerobically oxidize an aromatic compound. These results also demonstrate for the first time that hyperthermophilic microorganisms can anaerobically oxidize aromatic compounds and suggest that hyperthermophiles may metabolize aromatic compounds in hot environments such as the deep hot subsurface and in marine and terrestrial hydrothermal zones in which Fe(III) is available as an electron acceptor.     

Plant sulfur metabolism--the reduction of sulfate to sulfite

Until recently the pathway by which plants reduce activated sulfate to sulfite was unresolved. Recent findings on two enzymes termed 5'-adenylylsulfate (APS) sulfotransferase and APS reductase have provided new information on this topic. On the basis of their similarities it is now proposed that these proteins are the same enzyme. These discoveries confirm that the sulfate assimilation pathway in plants differs from that in other sulfate assimilating organisms.     

Chlorophenol dechlorination and subsequent degradation in denitrifying microcosms fed low concentrations of nitrate

The potential of using nitrate as a terminal electron acceptor to stimulate anaerobic degradation of mixtures of monochlorophenols (MCPs) or dichlorophenols (DCPs) was evaluated. Contaminated and non-contaminated soils were added to water saturated anaerobic microcosms supplemented with 1 mM or 5 mM nitrate. Denitrification and dechlorination activity were present in three diverse soil types and were maintained upon refeeding both nitrate and the appropriate chlorophenol. However, dechlorination activity could only be serially transferred in enrichments with an added electron donor such as acetate. Dehalogenation activity in enrichments from four of the primary microcosms showed at least five different dechlorination reactions, each mediated by different microbial communities. Three of these are distinct ortho-dechlorinating paths; two are meta-dechlorinating and one is the para-dechlorination of 3,4-DCP. Simultaneous dechlorination and denitrification was observed and both activities could be maintained in microcosms but only in the presence of low nitrate concentrations. Dechlorination and denitrification were mediated by two separate microbial communities; one that dechlorinates without use of nitrate and one that denitrifies while oxidizing the dechlorinated aromatic ring. There was no evidence that dechlorination is mediated by the denitrifying community, however the maintenance of a denitrification potential using low (< 1 mM) nitrate concentrations may be useful for completing the food chain by stimulating the mineralization of phenol and benzoate.     

Epilimnetic sulfate reduction and its relationship to lake acidification

Sulfate reduction occurred from 0–3 cm below the surface of the epilimnetic sediments of three northwestern Ontario lakes, including L.223, which has been experimentally acidified by additions of sulfuric acid. Shallow water sites were conducive to SO₄ ^2– reduction because decomposition in these predominantly sandy sediments caused oxygen concentrations to decrease rapidly within mm below the interface. The occurrence of methanogenesis just below the depth of minimum SO₄ ^2- concentration demonstrated that availability of organic carbon was not a limiting factor for sulphate reduction.Laboratory studies showed that SO₄ ^2- reduction rates in mixed sediments were lower at pH 4 than at pH 6. However, sulfate gradients in sediments indicated that there was no effect of acidification on sulfate reduction in situ. This was probably because microbial H⁺ consumption in the epilimnetic sediments maintained steep pH gradients below the sediment-water interface. The pH increased from = 5.0 to 6.5 or higher by a depth of 3.0 cm into the sediments.     

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.     

Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as ΔA₇₅₀ or indirectly as O₂ evolution (in the presence or absence of ferricyanide).2. Silicomolybdate-dependent O₂ evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I).3. Silicomolybdate-dependent O₂ evolution is coupled to ATP synthesis with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis ($\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio then is about 0.3).4. Silicomolybdate-dependent O₂ evolution is also coupled to ATP-synthesis in the presence of DCMU with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 0.6–0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inbihitors.5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.     

Electron donors for biological sulfate reduction

Biological sulfate reduction is widely used for treating sulfate-containing wastewaters from industries such as mining, tannery, pulp and paper, and textiles. In biological reduction, sulfate is converted to hydrogen sulfide as the end product. The process is, therefore, ideally suited for treating metal-containing wastewater from which heavy metals are simultaneously removed through the formation of metal sulfides. Metal sulfide precipitates are more stable than metal hydroxides that are sensitive to pH change. Theoretically, conversion of 1 mol of sulfate requires 0.67 mol of chemical oxygen demand or electron donors. Sulfate rich wastewaters are usually deficient in electron donors and require external addition of electron donors in order to achieve complete sulfate reduction. This paper reviews various electron donors employed in biological sulfate reduction. Widely used electron donors include hydrogen, methanol, ethanol, acetate, lactate, propionate, butyrate, sugar, and molasses. The selection criteria for suitable electron donors are discussed.     

Revisiting the dissimilatory sulfate reduction pathway

Sulfur isotopes in the geological record integrate a combination of biological and diagenetic influences, but a key control on the ratio of sulfur isotopes in sedimentary materials is the magnitude of isotope fractionation imparted during dissimilatory sulfate reduction. This fractionation is controlled by the flux of sulfur through the network of chemical reactions involved in sulfate reduction and by the isotope effect associated with each of these chemical reactions. Despite its importance, the network of reactions constituting sulfate reduction is not fully understood, with two principle networks underpinning most isotope models. In this study, we build on biochemical data and recently solved crystal structures of enzymes to propose a revised network topology for the flow of sulfur through the sulfate reduction metabolism. This network is highly branched and under certain conditions produces results consistent with the observations that motivated previous sulfate reduction models. Our revised network suggests that there are two main paths to sulfide production: one that involves the production of thionate intermediates, and one that does not. We suggest that a key factor in determining sulfur isotope fractionation associated with sulfate reduction is the ratio of the rate at which electrons are supplied to subunits of Dsr vs. the rate of sulfite delivery to the active site of Dsr. This reaction network may help geochemists to better understand the relationship between the physiology of sulfate reduction and the isotopic record it produces.     

Studies of sulfate utilization by algae: 8. The ubiquity of sulfate reduction to thiosulfate

Cell-free extracts from several microorganisms, when prepared by methods originally devised for Chlorella pyrenoidosa (Emerson strain 3) and incubated anaerobically with ATP, Mg²⁺, and 2, 3-dimercaptopropan-1-ol, are capable of reducing sulfate-³⁵S to thiosulfate. These microorganisms include, in addition to C. pyrenoidosa (Emerson strain 3), several other strains of C. pyrenoidosa, Chlorella protothecoides, Chlorella vulgaris, Anacystis sp., Chlamydomonas reinhardi, Escherichia coli, Salmonella typhimurium, and baker's yeast. Three of these organisms, E. coli, S. typhimurium, and baker's yeast, were previously reported by others to reduce sulfate to sulfite. Moreover, three mutant strains of S. typhimurium (Ba-25, Ce-363, and Bc-482) previously reported by other workers to be unable to reduce sulfate to sulfite also cannot form thiosulfate, and one mutant strain (Cd-68) reportedly able to form sulfite can also form thiosulfate. Taken together, this suggests that thiosulfate-forming activity may be a common feature of sulfate-reducing systems, and it may be present in enzymatic systems previously thought to be forming sulfite. Reasonably conclusive identification of thiosulfate is provided by ion exchange chromatography and by paper electrophoresis; the ambiguities associated with other analytical methods are discussed.