Microbial transformation of chlorinated benzoates

Chlorinated benzoates enter the environment through their use as herbicides or as metabolites of other halogenated compounds. Ample evidence is available indicating biodegradation of chlorinated benzoates to CO₂ and chloride in the environment under aerobic as well as anaerobic conditions. Under aerobic conditions, lower chlorinated benzoates can serve as sole electron and carbon sources supporting growth of a large list of taxonomically diverse bacterial strains. These bacteria utilize a variety of pathways ranging from those involving an initial degradative attack by dioxygenases to those initiated by hydrolytic dehalogenases. In addition to monochlorinated benzoates, several bacterial strains have been isolated that can grow on dichloro-, and trichloro- isomers of chlorobenzoates. Some aerobic bacteria are capable of cometabolizing chlorinated benzoates with simple primary substrates such as benzoate. Under anaerobic conditions, chlorinated benzoates are subject to reductive dechlorination when suitable electron-donating substrates are available. Several halorespiring bacteria are known which can use chlorobenzoates as electron acceptors to support growth. For example, Desulfomonile tiedjei catalyzes the reductive dechlorination of 3-chlorobenzoate to benzoate. The benzoate skeleton is mineralized by other microorganisms in the anaerobic environment. Various dichloro- and trichlorobenzoates are also known to be dechlorinated in anaerobic sediments.     

Biodegradation of xenobiotics by anaerobic bacteria

Xenobiotic biodegradation under anaerobic conditions such as in groundwater, sediment, landfill, sludge digesters and bioreactors has gained increasing attention over the last two decades. This review gives a broad overview of our current understanding of and recent advances in anaerobic biodegradation of five selected groups of xenobiotic compounds (petroleum hydrocarbons and fuel additives, nitroaromatic compounds and explosives, chlorinated aliphatic and aromatic compounds, pesticides, and surfactants). Significant advances have been made toward the isolation of bacterial cultures, elucidation of biochemical mechanisms, and laboratory and field scale applications for xenobiotic removal. For certain highly chlorinated hydrocarbons (e.g., tetrachlorethylene), anaerobic processes cannot be easily substituted with current aerobic processes. For petroleum hydrocarbons, although aerobic processes are generally used, anaerobic biodegradation is significant under certain circumstances (e.g., O₂-depleted aquifers, oil spilled in marshes). For persistent compounds including polychlorinated biphenyls, dioxins, and DDT, anaerobic processes are slow for remedial application, but can be a significant long-term avenue for natural attenuation. In some cases, a sequential anaerobic-aerobic strategy is needed for total destruction of xenobiotic compounds. Several points for future research are also presented in this review.     

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.     

Methanogenic transformation of aromatic hydrocarbons and phenols in groundwater aquifers

Fermentative and methanogenic bacteria have been found repeatedly as important members of microbial flora in anoxic zones of the subsurface—in pristine as well as in contaminated groundwater aquifers. These bacteria, which together with obligate proton reducers form complex methanogenic communities, are significant as decomposers of organic matter under conditions of exogenous electron acceptor depletion. Their metabolic activity has been demonstrated in laboratory microcosms derived from aquifer material, and also in the subsurface in situ. Methanogenic communities have been shown to transform numerous organic pollutants, or even to completely degrade these compounds with the production of carbon dioxide and methane. Depending on the chemical structure of the pollutant, such a compound can be used as an electron donor and a carbon/energy source for fermentative microorganisms (which is typically the case with highly reduced compounds); alternatively, a highly oxidized pollutant can be used as a potential electron acceptor or electron sink. This review addresses fermentative/methanogenic degradation of chlorinated and nonchlorinated aromatic hydrocarbons and phenols by subsurface microorganisms; for comparison, it briefly relates also other types of anaerobic transformations (under sulfate‐reducing, iron‐reducing, and denitrifying conditions). Furthermore, it outlines transformation pathways, those that are proposed as well as those that are already partially proved, for aromatic hydrocarbons and phenols under fermentative/methanogenic conditions; finally, it discusses the relevance of these processes to bioremediation of contaminated groundwater aquifers.     

Influence of alternative electron acceptors on the anaerobic biodegradability of chlorinated phenols and benzoic acids.

Nitrate, sulfate, and carbonate were used as electron acceptors to examine the anaerobic biodegradability of chlorinated aromatic compounds in estuarine and freshwater sediments. The respective denitrifying, sulfidogenic, and methanogenic enrichment cultures were established on each of the monochlorinated phenol and monochlorinated benzoic acid isomers, using sediment from the upper (freshwater) and lower (estuarine) Hudson River and the East River (estuarine) as source materials. Utilization of each chlorophenol and chlorobenzoate isomer was observed under at least one reducing condition; however, no single reducing condition permitted the metabolism of all six compounds tested. The anaerobic biodegradation of the chlorophenols and chlorobenzoates depended on the electron acceptor available and on the position of the chlorine substituent. In general, similar activities were observed under the different reducing conditions in both the freshwater and estuarine sediments. Under denitrifying conditions, degradation of 3- and 4-chlorobenzoate was accompanied by nitrate loss corresponding reasonably to the stoichiometric values expected for complete oxidation of the chlorobenzoate to CO2. Under sulfidogenic conditions, 3- and 4-chlorobenzoate, but not 2-chlorobenzoate, and all three monochlorophenol isomers were utilized, while under methanogenic conditions all compounds except 4-chlorobenzoate were metabolized. Given that the pattern of activity appears different for these chlorinated compounds under each reducing condition, their biodegradability appears to be more a function of the presence of competent microbial populations than one of inherent molecular structure.     

Microbial degradation of chlorinated phenols

Chlorophenols have been introduced into the environment through their use as biocides and as by-products of chlorine bleaching in the pulp and paper industry. Chlorophenols are subject to both anaerobic and aerobic metabolism. Under anaerobic conditions, chlorinated phenols can undergo reductive dechlorination when suitable electron-donating substrates are available. Halorespiring bacteria are known which can use both low and highly chlorinated congeners of chlorophenol as electron acceptors to support growth. Many strains of halorespiring bacteria have the capacity to eliminate ortho-chlorines; however only bacteria from the species Desulfitobacterium hafniense (formerly frappieri) can eliminate para- and meta-chlorines in addition to ortho-chlorines. Once dechlorinated, the phenolic carbon skeletons are completely converted to methane and carbon dioxide by other anaerobic microorganisms in the environment. Under aerobic conditions, both lower and higher chlorinated phenols can serve as sole electron and carbon sources supporting growth. The best studied strains utilizing pentachlorophenol belong to the genera Mycobacterium and Sphingomonas. Two main strategies are used by aerobic bacteria for the degradation of chlorophenols. Lower chlorinated phenols for the most part are initially attacked by monooxygenases yielding chlorocatechols as the first intermediates. On the other hand, polychlorinated phenols are converted to chlorohydroquinones as the initial intermediates. Fungi and some bacteria are additionally known that cometabolize chlorinated phenols.     

Degradation of halogenated aliphatic compounds: The role of adaptation

Abstract: A limited number of halogenated aliphatic compounds can serve as a growth substrate for aerobic microorganisms. Such cultures have (specifically) developed a variety of enzyme systems to degrade these compounds. Dehalogenations are of critical importance. Various heavily chlorinated compounds are not easily biodegraded, although there are no obvious biochemical or thermodynamic reasons why microorganisms should not be able to grow with any halogenated compound. The very diversity of catabolic enzymes present in cultures that degrade halogenated aliphatics and the occurrence of molecular mechanisms for genetic adaptation serve as good starting points for the evolution of catabolic pathways for compounds that are currently still resistant to biodegradation.     

Versatility of soil column experiments to study biodegradation of halogenated compounds under environmental conditions

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.Abbreviations 3-CB 3-chlorobenzoate - DCB dichlorobenzene - HCH hexachlorocyclohexane - IS insertion sequence - PER tetrachloroethylene - S_min minimal substrate concentration for growth - TCB trichlorobenzene - TRI trichloroethylene - filtration coefficient     

Biodegradation of chlorinated phenolic compounds

Chlorophenolic compounds are generated from a number of industrial manufacturing processes including pulp and paper manufacture. These compounds are found to be toxic and recalcitrant and hence their discharge into the environment must be regulated. Slow and partial degradation of chlorophenols under aerobic and anaerobic natural environment has been observed. Aerobic biodegradation of chlorophenols proceeds through the formation of catechols while under anaerobic conditions, reductive dehalogenation is the preferred metabolic pathway. Number and position of chlorine substituents on the phenolic ring has influence on the rate and extent of biodegradation of chlorophenols. In engineered systems, acclimatization of biomass to chlorophenols markedly enhances the biodegradation ability by reducing the initial lag phase and by countering inhibition. Partial removal of chlorophenols between 40-60% is usually observed in aerobic and anaerobic processes. Removal can be enhanced by a combination of aerobic and anaerobic operations.     

Exocellular electron transfer in anaerobic microbial communities

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.     

Model based evaluation of a contaminant plume development under aerobic and anaerobic conditions in 2D bench-scale tank experiments

The influence of transverse mixing on competitive aerobic and anaerobic biodegradation of a hydrocarbon plume was investigated using a two-dimensional, bench-scale flow-through laboratory tank experiment. In the first part of the experiment aerobic degradation of increasing toluene concentrations was carried out by the aerobic strain Pseudomonas putida F1. Successively, ethylbenzene (injected as a mixture of unlabeled and fully deuterium-labeled isotopologues) substituted toluene; nitrate was added as additional electron acceptor and the anaerobic denitrifying strain Aromatoleum aromaticum EbN1 was inoculated to study competitive degradation under aerobic / anaerobic conditions. The spatial distribution of anaerobic degradation was resolved by measurements of compound-specific stable isotope fractionation induced by the anaerobic strain as well as compound concentrations. A fully transient numerical reactive transport model was employed and calibrated using measurements of electron donors, acceptors and isotope fractionation. The aerobic phases of the experiment were successfully reproduced using a double Monod kinetic growth model and assuming an initial homogeneous distribution of P. putida F1. Investigation of the competitive degradation phase shows that the observed isotopic pattern cannot be explained by transverse mixing driven biodegradation only, but also depends on the inoculation process of the anaerobic strain. Transient concentrations of electron acceptors and donors are well reproduced by the model, showing its ability to simulate transient competitive biodegradation.     

Denitrification at extremely high pH values by the alkaliphilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio denitrificans strain ALJD

Thioalkalivibrio denitrificans is the first example of an alkaliphilic, obligately autotrophic, sulfur-oxidizing bacterium able to grow anaerobically by denitrification. It was isolated from a Kenyan soda lake with thiosulfate as electron donor and N2O as electron acceptor at pH 10. The bacterium can use nitrite and N2O, but not nitrate, as electron acceptors during anaerobic growth on reduced sulfur compounds. Nitrate is only utilized as nitrogen source. In batch culture at pH 10, rapid growth was observed on N2O as electron acceptor and thiosulfate as electron donor. Growth on nitrite was only possible after prolonged adaptation of the culture to increasing nitrite concentrations. In aerobic thiosulfate-limited chemostats, Thioalkalivibrio denitrificans strain ALJD was able to grow between pH values of 7.5 and 10.5 with an optimum at pH 9.0. Growth of the organism in continuous culture on N2O was more stable and faster than in aerobic cultures. The pH limit for growth on N2O was 10.6. In nitrite-limited chemostat culture, growth was possible on thiosulfate at pH 10. Despite the observed inhibition of N2O reduction by sulfide, the bacterium was able to grow in sulfide-limited continuous culture with N2O as electron acceptor at pH 10. The highest anaerobic growth rate with N2O in continuous culture at pH 10 was observed with polysulfide (S8(2-)) as electron donor. Polysulfide was also the best substrate for oxygen-respiring cells. Washed cells at pH 10 oxidized polysulfide to sulfate via elemental sulfur in the presence of N2O or O2. In the absence of the electron acceptors, elemental sulfur was slowly reduced which resulted in regeneration of polysulfide. Cells of strain ALJD grown under anoxic conditions contained a soluble cd1-like cytochrome and a cytochrome-aa3-like component in the membranes.     

Impermeant electron acceptors and donors to the plasma membrane-bound respiratory chain of intact cyanobacterium Anacystis nidulans

Membrane-impermeant redox compounds ferricyanide and horse heart ferrocytochrome c acted as electron acceptor and donor, respectively, for intact cells or spheroplasts of Anacystis nidulans (Synechococcus ATCC 27144) in the dark. The anaerobic reduction of ferricyanide was faster than aerobic reduction. KCN significantly enhanced the reaction under aerobic conditions. Light did not influence ferricyanide reduction. The oxidation of exogenous ferrocytochrome c was oxygen-dependent and inhibited by KCN. Either type of redox reaction was accompanied by vectorial proton translocation out of the cells. Arrhenius plots for the temperature dependence of both ferricyanide reduction and cytochrome c oxidation gave one distinct break point reflecting the lipid phase transition temperature of the plasma membrane. The results are presented as evidence for a respiratory chain in the plasma membrane of A. nidulans.     

Amino acid transport in membrane vesicles of obligately anaerobic Veillonella alcalescens.

Membrane vesicles of Veillonella alcalescens, grown in the presence of L-lactate and KNO-3, actively transport amino acids under anaerobic conditions in the presence of several electron donors and the electron acceptor nitrate. The highest initial rates of uptake are obtained with L-lactate, followed by reduced nicotinamide adenine dinucleotide, glycerol-1-phosphate, formate, and L-malate.. The membrane vesicles contain the dehydrogenases for these electron donors, and these enzymes are coupled with nitrate reductase. In membrane vesicles from cells, grown in the presence of nitrate, the dehydrogenases are not coupled with fumarate reducatase, and anaerobic transport of amino acids does not occur with fumarate as electron acceptor. Under aerobic conditions none of the physiological electron donors can energize transport. However, a high rate of uptake is observed with the electron donor system ascorbate-phenazine metho-sulfate. This electron donor system also effectively energizes transport under anaerobicconditions in the presence of the electron acceptor nitrate.     

Isotope analysis as a natural reaction probe to determine mechanisms of biodegradation of 1,2-dichloroethane

1,2-Dichloroethane (1,2-DCA), a chlorinated aliphatic hydrocarbon, is a well-known groundwater contaminant. In this study, fractionation of stable carbon isotope values of 1,2-DCA during biodegradation was used as a novel reaction probe to provide information about the mechanism of 1,2-DCA biodegradation under both aerobic (O2-reducing) and anaerobic (NO3-reducing) conditions. Under O2-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.1 per thousand (+/-95% confidence intervals) was measured for the enrichment culture. Under NO3-reducing conditions, an epsilon-value of -25.8 +/- 3.5 per thousand was measured. The microbial culture produced isotopic enrichment values (epsilon) that are not only large and reproducible, but also are the same whether O2 or NO3 was used as an electron acceptor. Combining data measured under both O2- and NO3-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.6 per thousand is measured for the microbial culture during 1,2-DCA degradation. The epsilon-value can be converted into a kinetic isotope effect (KIE) value to relate the observed isotopic fractionation to the mechanism of degradation. This KIE value (1.05) is consistent with degradation via hydrolytic dehalogenation under both electron-accepting conditions. This study demonstrates the added value of compound-specific isotope analysis not only as a technique to verify the occurrence and extent of biodegradation in the field, but also as a natural reaction probe to provide insight into the enzymatic mechanism of contaminant degradation.     

Microbial degradation of chlorinated benzenes

Chlorinated benzenes are important industrial intermediates and solvents. Their widespread use has resulted in broad distribution of these compounds in the environment. Chlorobenzenes (CBs) are subject to both aerobic and anaerobic metabolism. Under aerobic conditions, CBs with four or less chlorine groups are susceptible to oxidation by aerobic bacteria, including bacteria (Burkholderia, Pseudomonas, etc.) that grow on such compounds as the sole source of carbon and energy. Sound evidence for the mineralization of CBs has been provided based on stoichiometric release of chloride or mineralization of (14)C-labeled CBs to (14)CO(2). The degradative attack of CBs by these strains is initiated with dioxygenases eventually yielding chlorocatechols as intermediates in a pathway leading to CO(2) and chloride. Higher CBs are readily reductively dehalogenated to lower chlorinated benzenes in anaerobic environments. Halorespiring bacteria from the genus Dehalococcoides are implicated in this conversion. Lower chlorinated benzenes are less readily converted, and mono-chlorinated benzene is recalcitrant to biotransformation under anaerobic conditions.     

Stimulation of anaerobic biodegradation of DDT and its metabolites in a muck soil: laboratory microcosm and mesocosm studies

The aim of this study was to evaluate the impact of selected electron donors and electron acceptors on the anaerobic biodegradation of DDT and its major metabolites in a muck soil with a long history of exposure to the pesticide. Loss of DDT was measured in anaerobic microcosms supplemented with H₂, lactate, and acetate. The greatest loss of DDT (approximately 87 %) was observed in microcosms amended with lactate and no additional electron acceptor compared to the no additional electron donor or acceptor sets. An increase in measureable concentrations of DDx was observed in un-amended microcosms. In larger scale mesocosms, significant increases in dissolved organic carbon (DOC) corresponded with low redox potentials. Increases in DOC corresponded with sharp increases in measured concentrations of DDx, followed by a decrease in measured DDT concentrations in lactate-amended mesocosms. Our studies indicate that sorbed DDx is released upon anaerobic incubation, and that indigenous microorganisms capable of DDx degradation respond to lactate additions. Both the potential for release of sorbed DDx and the potential for biodegradation of DDx should be considered during remediation of DDx-contaminated organic soils at low redox potentials.     

1,2,3-三氯丙烷降解机制与污染场地修复技术研究进展

1,2,3-三氯丙烷(1,2,3-trichloropropane,1,2,3-TCP)是一种人工合成的脂肪族氯代烃,在工、农业生产中得到广泛应用。1,2,3-TCP作为环氧氯丙烷工业生产的中间产物,可作为前体物质用于生产土壤熏蒸剂、有机溶剂等。因其环境持久性、迁移性和生态毒性,国内外机构逐渐开始关注该有机氯污染物的环境归趋、生态健康风险和环境管控。当前,1,2,3-TCP污染物的降解与场地修复仍然是研究热点,但是对于1,2,3-TCP降解转化机制尚缺乏深入研究与总结。鉴于此,文中在讨论1,2,3-TCP的来源、环境污染、生态效应及物理化学降解方法与技术等的基础上,进一步综述了1,2,3-TCP的微生物降解与修复机制(如好氧共代谢降解、厌氧降解等);重点讨论了地下水等厌氧环境中1,2,3-TCP的厌氧微生物降解转化途径与机制,并从热力学角度论证了厌氧条件下1,2,3-TCP作为电子受体被有机卤呼吸微生物利用并降解的可行性;最后,对1,2,3-TCP污染场地原位生物修复进行了总结并对未来研究发展方向进行了展望。     

The expression of redox proteins of denitrification inThiosphaera pantotropha grown with oxygen,nitrate, and nitrous oxide as electron acceptors

The redox proteins and enzymes involved in denitrification inThiosphaera pantotropha exhibited a differential expression in response to oxygen. Pseudoazurin was completely repressed during batch or continuous culture under oxic conditions. Cytochromecd ₁ nitrite reductase was also heavily repressed after aerobic growth. Nitrite, nitric oxide, and nitrous oxide reductase activities were detected in intact cells under some conditions of aerobic growth, indicating that aerobic denitrification might occur in some circumstances. However, the rates of denitrification were much lower after aerobic growth than after anaerobic growth. Growth with nitrous oxide as sole electron acceptor mimicked aerobic growth in some respects, implying that expression of parts of the denitrification apparatus might be controlled by the redox state of a component of the electron transport chain rather than by oxygen itself. Nevertheless, the regulation of expression of nitrous oxide reductase was linked to the oxygen concentration.     

Sequential application of electron donors and humic acids for the anaerobic bioremediation of chlorinated aliphatic hydrocarbons

In situ anaerobic bioremediation of chlorinated solvents such as perchloroethene (PCE) frequently faces the problem of accumulating toxic, lower chlorinated compounds such as dichloroethene (cis-DCE) and vinyl chloride (VC). In the present study, the efficacy of the sequential application of electron donors, supporting reductive dechlorination, and of humic acids, acting as extracellular electron shuttles facilitating the anaerobic oxidation of recalcitrant intermediates, was explored in microcosm studies. Upon one initial dose of lactose, supplied in a 1000-fold superstoichiometric electron equivalent ratio, PCE was completely converted into cis-DCE within 35 days. Repeated electron donor additions did not entail exhaustive cis-DCE degradation over incubation time (120 days). Although the electron donor was quickly converted into fatty acids, about 30% of added reducing equivalents were recovered as acetate after four months of operation, indicating the inhibition of acetoclastic methanogenesis. In the next step, the substoichiometric addition of anthraquinone-2,6-disulfonate, a humic acid model compound, effected the complete removal of the accumulated cis-DCE within 15 days, probably as a result of the participation of the quinone in the biotic or abiotic anaerobic oxidation of cis-DCE. Cis-DCE degradation was not connected to the accumulation of VC, rendering the proposed two-step treatment an efficient and environmentally compliant remedy for anaerobic groundwater bodies contaminated with chlorinated solvents.