Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum–Alkalilimnicola group

Four new isolates were obtained from denitrifying enrichments with various electron donors using sediment samples from hypersaline soda lakes. Based on 16S rRNA gene analysis and DNA-DNA hybridization results, they were all identified as members of the Gammaproteobacteria closely associated with the Alkalispirillum–Alkalilimnicola group. Two isolates were obtained from samples enriched with nitrate as electron acceptor and H₂ or polysulfide as electron donors, and another two strains were obtained with N₂O as the electron acceptor and sulfide or acetate as electron donors. All four new isolates, together with the type strains of the genera Alkalispirillum and Alkalilimnicola originally described as obligate aerobes, were capable of anaerobic growth with acetate using either nitrate or N₂O as electron acceptors. Their denitrification pathway, however, was disrupted at the level of nitrite. RuBisCO form I gene was detected and sequenced in the new isolates and in Alkalilimnicola halodurans but not in Alkalispirillum mobile. These data, together with the evidence of Oremland et al. (Appl Environ Microbiol 68:4795–4802, 2002) on the potential of Alkalilimnicola sp. MLHE-1 for autotrophic growth with arsenite as electron donor and nitrate as electron acceptor, demonstrate much higher metabolic diversity of this specific group of haloalkaliphilic Gammaproteobacteria than was originally anticipated.     

Denitrification at pH 4 by a soil‐derived Rhodanobacter‐dominated community

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of N₂O produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO₃^‐ and N₂O reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of N₂O as denitrification end‐product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter‐like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils.     

Ammonification in Bacillus subtilis Utilizing Dissimilatory Nitrite Reductase Is Dependent on resDE

During anaerobic nitrate respiration Bacillus subtilis reduces nitrate via nitrite to ammonia. No denitrification products were observed. B. subtilis wild-type cells and a nitrate reductase mutant grew anaerobically with nitrite as an electron acceptor. Oxygen-sensitive dissimilatory nitrite reductase activity was demonstrated in cell extracts prepared from both strains with benzyl viologen as an electron donor and nitrite as an electron acceptor. The anaerobic expression of the discovered nitrite reductase activity was dependent on the regulatory system encoded by resDE. Mutation of the gene encoding the regulatory Fnr had no negative effect on dissimilatory nitrite reductase formation.     

Research of Iron Reduction and the Iron Reductase Localization of Anammox Bacteria

The iron-reducing capability of anammox bacteria was examined in this study using Percoll purified anammox bacteria. Anammox bacteria could reduce Fe(III) to Fe(II) with organic matters as the electron donor. The activity of anammox iron-reducing process was dependent on different electron donor, acceptor and pH. The highest iron-reducing activity of anammox bacteria was achieved with Fe(III)-NTA (nitrilotriacetic acid) as electron acceptor and formate as the electron donor at pH7. Similar to other iron reducers, 80 % of the iron reductase in anammox bacteria was located in the membrane fraction. Due to the chemical oxidant of NO₂ ^? and the NO₃ ^? dependent ferrous iron oxidation by anammox bacteria, the iron-reducing activity of anammox bacteria could be severely inhibited when iron-reducing pathway and the anammox process were coupled. However, the total nitrogen removal efficiency was not significantly affected in the presence of Fe(III). The iron-reducing capability of anammox bacteria could influence both N and Fe cycle on earth, and it is a potential way for wastewater treatment.     

Studies on phosphorylation coupled with denitrification and aerobic respiration inPseudomonas denitrificans

Formation of ATP during aerobic respiration and denitrification was determined inPseudomonas denitrificans. In the intact cell system, the ATP formation associated with denitrification was almost the same as that associated with aerobic respiration when lactate was used as an electron donor. The ATP formation was inhibited by KCN, NaN₃ and DNP. No phosphate uptake occurred when NH₂OH, DMPD or TMPD was used as an electron donor, although the production of N₂O, N₂ or NO from nitrite was accelerated under anaerobic conditions. In the cell-free system, the ATP formation was also demonstrated using an ATP trapping system and lactate as a substrate. The effects of inhibitors were almost the same as those observed with the intact cells. DMPD or TMPD together with ascorbate promoted the ATP formation during aerobic oxidation by the cell-free system whereas no stimulation of ATP formation was detected during denitrification.     

Relative influence of temperature and electron donor and electron acceptor concentrations on bacterial sulfate reduction in saltmarsh sediment

The relative importance of three environmental variables known to influence the rate of bacterial sulfate reduction was examined using sediment from a saltmarsh pan. The variables investigated were temperature, electron donor concentration, and electron acceptor concentration. Their relative influence on the rate of bacterial sulfate reduction was examined with multiple replicate sediment samples in which the variables were experimentally adjusted. Sulfate reduction rates were measured with³⁵SO ₄ ^2– .The relative importance of each variable to sulfate reduction rate was assessed with multiple regression analysis by calculating the standardized partial regression coefficients, and the results were compared with the ranges of the three variables encountered in the natural sediment. Temperature proved to have the greatest influence, followed by electron donor and electron acceptor concentrations, in that order. The sulfate concentration was shown to have little influence on sulfate reduction rate at seawater concentrations of sulfate, but its effect increased if sulfate concentrations were diminished compared to those of seawater.     

Impacts of varying durations of passive oxygen exposure on freshwater denitrifier community structure and function

Fertilizer use has dramatically increased the availability of nitrate (NO₃ ^?) in aquatic systems. Microbe-mediated denitrification is one of the predominant means of NO₃ ^? removal from freshwaters, yet oxygenation (O₂)-induced disruptions—e.g., extreme precipitation events—can occur, resulting in a disproportional increase in nitrous oxide (N₂O) production and efflux as facultative anaerobic bacterial populations use of O₂ as a terminal electron acceptor increases. We examined the effects of 12- and 24-h passive O₂ exposure on previously anaerobic bacterial communities focusing on denitrification enzyme activity (DEA), N₂O production, and bacterial community 16S rRNA and nitrous oxide reductase gene (nosZ) profiles after 12, 24, and 48 h of anaerobic recovery. Treatments experiencing 24-h O₂ exposure had significantly higher DEA 12 h into anaerobic recovery than treatments undergoing 12-h O₂ exposure. Initial N₂O emissions were significantly lower in the 24-h O₂ exposure treatments although by 24 h a dramatic spike (tenfold relative to the 12-h O₂ exposure treatments) in N₂O concentrations was observed. However, within 6 h (30-h anaerobic recovery) these differences were gone. Community nosZ profiles experiencing 24-h O₂ exposure exhibited reduced diversity after 24-h recovery, which corresponded with an increase in N₂O emissions. However, after 48 h of anaerobic recovery, nosZ diversity had recovered. These observations highlight the effects of short-term aerobic disruption on denitrification, as well as the effects on the denitrifier community profile. Together, these data suggest that recovery to ambient N cycling is exacerbated by disturbance length due to increased lag time and subsequent loss of denitrifier community diversity.     

Comparison of the electron spin polarized spectrum found in plant photosystem I and in iron-depleted bacterial reaction centers with time-resolved K-band EPR; evidence that the photosystem I acceptor A1 is a quinone

The suggestion that the electron acceptor A₁ in plant photosystem I (PSI) is a quinone molecule is tested by comparisons with the bacterial photosystem. The electron spin polarized (ESP) EPR signal due to the oxidized donor and reduced quinone acceptor (P ₈₇₀ ⁺ Q^-) in iron-depleted bacterial reaction centers has similar spectral characteristics as the ESP EPR signal in PSI which is believed to be due to P ₇₀₀ ⁺ A ₁ ^- , the oxidized PSI donor and reduced A₁. This is also true for better resolved spectra obtained at K-band (24 GHz). These same spectral characteristics can be simulated using a powder spectrum based on the known g-anisotropy of reduced quinones and with the same parameter set for Q^- and A₁ ^-. The best resolution of the ESP EPR signal has been obtained for deuterated PSI particles at K-band. Simulation of the A₁ ^- contribution based on g-anisotropy yields the same parameters as for bacterial Q^- (except for an overall shift in the anisotropic g-factors, which have previously been determined for Q^-). These results provide evidence that A₁ is a quinone molecule. The electron spin polarized signal of P₇₀₀ ⁺ is part of the better resolved spectrum from the deuterated PSI particles. The nature of the P₇₀₀ ⁺ ESP is not clear; however, it appears that it does not exhibit the polarization pattern required by mechanisms which have been used so far to explain the ESP in PSI.Abbreviations hf hyperfine - A₀ A₀ acceptor of photosystem I - A₁ A₁ acceptor of photosystem I - Brij-58 polyoxyethylene 20 cetyl ether - CP1 photosystem I particles which lack ferridoxin acceptors - ESP electron spin polarized - EPR electron paramagnetic resonance - I intermediary electron acceptor, bacteriopheophytin - LDAO lauryldimethylamine - N-oxide, P₇₀₀ primary electron donor of photosystem I - PSI photosystem I - P₇₀₀ ^T triplet state of primary donor of photosystem I - P₈₇₀ primary donor in R. sphaeroides reaction center - Q quinore-acceptor in photosynthetic bacteria - RC reaction center     

<Emphasis Type="Italic">Dethiobacter alkaliphilus </Emphasis> gen. nov. sp. nov., and <Emphasis Type="Italic">Desulfurivibrio alkaliphilus</Emphasis> gen. nov. sp. nov.: two novel representatives of reductive sulfur cycle from soda lakes

Anaerobic enrichments with H₂ as electron donor and thiosulfate/polysulfide as electron acceptor at pH 10 and 0.6 M total Na⁺ yielded two non sulfate-reducing representatives of reductive sulfur cycle from soda lake sediments. Strain AHT 1 was isolated with thiosulfate as the electron acceptor from north–eastern Mongolian soda lakes and strain AHT 2—with polysulfide as the electron acceptor from Wadi al Natrun lakes in Egypt. Both isolates represented new phylogenetic lineages: AHT 1—within Clostridiales and AHT 2—within the Deltaproteobacteria. Both bacteria are obligate anaerobes with respiratory metabolism. Both grew chemolithoautotrophically with H₂ as the electron donor and can use thiosulfate, elemental sulfur and polysulfide as the electron acceptors. AHT 2 also used nitrate as acceptor, reducing it to ammonia. During thiosulfate reduction, AHT 1 excreted sulfite. dsrAB gene was not found in either strain. Both strains were moderate salt-tolerant (grow up to 2 M total Na⁺) true alkaliphiles (grow between pH 8.5 and 10.3). On the basis of the phenotypic and phylogenetic data, strains AHT 1 and AHT 2 are proposed as new genera and species Dethiobacter alkaliphilus and Desulfurivibrio alkaliphilus, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence accession number: The GenBank/EMBL accession number of the 16S rRNA gene sequence of strains AHT 1T and AHT 2T are EF422412 and EF422413.     

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol g _VSS ^?1 day^?1 compared to lactate (32 µmol g _VSS ^?1 day^?1), acetate (26 µmol g _VSS ^?1 day^?1) and methane (4.7 µmol g _VSS ^?1 day^?1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane.     

Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil

The present experiment was designed to isolate bacterial strains from the brick kiln soil and to check the activity and enzyme kinetics of amylase from these isolates. The bacterial colonies were isolated from soil samples through the serial dilution method. The bacterial isolates were identified through morphological, electron microscopic and molecular analysis. The 16S ribosomal RNA sequences of the isolates IR-1, IR-2, IR-3, IR-8, and IR-9 showed high similarities with Bacillus tequilensis, Bacillus paramycoides, Proteus alimentorum, Bacillus wiedmannii, and Pseudomonas aeruginosa, respectively. All of the bacterial isolates showed a positive catalase activity except IR-9. Furthermore, the isolates showed variable antagonistic effects against different bacterial pathogens. All of the strains produced indole acetic acid (IAA), and the concentrations increased in the presence of tryptophan application. The isolates showed the amylase enzyme activity and maximum activity of isolates was achieved in 4% starch concentration. The IR-9 isolate showed the highest amylase activity of 5.9 U/ml. The V_max values of the extracellular amylase from different bacterial isolates ranged between 12.90 and 50.00 IU ml⁻¹. The lowest K_m value of 6.33 mg starch was recorded for IR-8 and the maximum K_cat value of 2.50 min⁻¹ was observed for IR-3. The amylase activity of the isolates was significantly affected by a range of different incubation time, temperature, and pH values. Further tests are required before the potential utilization of these isolates for amylase production, and in the biopesticide and biofertilizer applications.     

Electron acceptor availability alters carbon and energy metabolism in a thermoacidophile

The thermoacidophilic Acidianus strain DS80 displays versatility in its energy metabolism and can grow autotrophically and heterotrophically with elemental sulfur (S°), ferric iron (Fe³⁺) or oxygen (O₂) as electron acceptors. Here, we show that autotrophic and heterotrophic growth with S° as the electron acceptor is obligately dependent on hydrogen (H₂) as electron donor; organic substrates such as acetate can only serve as a carbon source. In contrast, organic substrates such as acetate can serve as electron donor and carbon source for Fe³⁺ or O₂ grown cells. During growth on S° or Fe³⁺ with H₂ as an electron donor, the amount of CO₂ assimilated into biomass decreased when cultures were provided with acetate. The addition of CO₂ to cultures decreased the amount of acetate mineralized and assimilated and increased cell production in H₂/Fe³⁺ grown cells but had no effect on H₂/S° grown cells. In acetate/Fe³⁺ grown cells, the presence of H₂ decreased the amount of acetate mineralized as CO₂ in cultures compared to those without H₂. These results indicate that electron acceptor availability constrains the variety of carbon sources used by this strain. Addition of H₂ to cultures overcomes this limitation and alters heterotrophic metabolism.     

A single step separation of PS 1, PS 2 and chlorophyll-antenna particles from spinach chloroplasts

After solubilization of photosynthetic membranes by digitonin, three main protein pigment complexes were isolated by electrophoresis with deoxycholate as detergent.The band with the slowest mobility, fraction 1, had PS 1 activity and was devoid of PS 2 activity. This fraction was four times enriched in P700 when compared with chloroplasts. Fraction 1 had little chl b, a long wavelength absorption maximum in the red, a maximum of low temperature emission fluorescence at 730nm, and a circular dichroism spectrum characteristic of PS 1 enriched fraction.Fraction 2 exhibited a PS 2 activity and no PS 1 activity. It was enriched five times in PS 2 reaction centre and had little chl b and carotenoids. The absorption maximum was at 674 nm and the low temperature fluorescence emission maximum was at 700 nm. Fraction 2 might be useful PS 2 enriched particle because of the great stability of this fraction with regard to photochemical activity and also rapidity and simplicity of its preparation.Fraction 3, which had the fastest migration, was devoid of photochemical activities; It was rich in chl b and had the fluorescence and the circular dichroism spectrum characteristic of an antenna complex.Abbreviations PS 1 (2) photosystem 1 (2) - chl chlorophyll - car carotenoid - Q primary plastoquinone electron acceptor - P700 primary electron donor of PS 1 - P680 primary electron donor of PS 2 - K₃Fe(CN)₆ potassium ferricyanide - DCMU dichlorophenyldimethylurea - DCPIP dichlorophenolindophenol - DPC diphenyl-carbazide     

Sulfur-driven autotrophic denitrification of nitric oxide for efficient nitrous oxide recovery

Nitrous oxide (N₂O) was previously deemed as a potent greenhouse gas but is actually an untapped energy source, which can accumulate during the microbial denitrification of nitric oxide (NO). Compared with the organic electron donor required in heterotrophic denitrification, elemental sulfur (S⁰) is a promising electron donor alternative due to its cheap cost and low biomass yield in sulfur-driven autotrophic denitrification. However, no effort has been made to test N₂O recovery from sulfur-driven denitrification of NO so far. Therefore, in this study, batch and continuous experiments were carried out to investigate the NO removal performance and N₂O recovery potential via sulfur-driven NO-based denitrification under various Fe(II)EDTA-NO concentrations. Efficient energy recovery was achieved, as up to 35.5%–40.9% of NO was converted to N₂O under various NO concentrations. N₂O recovery from Fe(II)EDTA-NO could be enhanced by the low bioavailability of sulfur and the acid environment caused by sulfur oxidation. The NO reductase (NOR) and N₂O reductase (N₂OR) were inhibited distinctively at relatively low NO levels, leading to efficient N₂O accumulation, but were suppressed irreversibly at NO level beyond 15 mM in continuous experiments. Such results indicated that the regulation of NO at a relatively low level would benefit the system stability and NO removal capacity during long-term system operation. The continuous operation of the sulfur-driven Fe(II)EDTA-NO-based denitrification reduced the overall microbial diversity but enriched several key microbial community. Thauera, Thermomonas, and Arenimonas that are able to carry out sulfur-driven autotrophic denitrification became the dominant organisms with their relative abundance increased from 25.8% to 68.3%, collectively.     

Comparison of Energy and Growth Yields for Desulfitobacterium dehalogenans during Utilization of Chlorophenol and Various Traditional Electron Acceptors

Desulfitobacterium dehalogenans grew with formate as the electron donor and 3-chloro-4-hydroxyphenylacetate (3-Cl-4-OHPA) as the electron acceptor, yielding Y_X/formate, Y_X/2e⁻, and Y_X/ATP ranging from 3.2 to 11.3 g of biomass (dry weight)/mol, thus indicating that energy was conserved through reductive dechlorination. Pyruvate was utilized as the electron donor and acceptor, yielding stoichiometric amounts of acetate and lactate, respectively, and a Y_{X/reduced acceptor} of 13.0 g of biomass (dry weight)/mol. The supplementation of pyruvate-containing medium with additional electron acceptors, such as 3-Cl-4-OHPA, nitrate, fumarate, or sulfite, caused pyruvate to be replaced as the electron acceptor and nearly doubled the Y_X/ATP (Y_{X/acetate formed}). A comparison of the yields for 3-Cl-4-OHPA with those for other traditional electron acceptors indicates that the dehalogenation reaction led to the formation of similar amounts of energy equivalents. The various electron acceptors were used concomitantly with 3-Cl-4-OHPA in nonacclimated cultures, but the utilization rates and amounts utilized differed.     

Role of bicarbonate at the acceptor side of Photosystem II

Besides being the substrate for the carboxylation reaction of photosynthesis, CO₂ (bicarbonate) is required for the activity of Photosystem II (water plastoquinone oxido-reductase). It plays a role on the electron donor side as well as the electron acceptor side. In this contribution, attention will mostly be focused on the history of research into the effects of bicarbonate on electron flow reactions on the acceptor side. Donor side reactions are discussed in this issue by Alan Stemler. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel Processes for Anaerobic Sulfate Production from Elemental Sulfur by Sulfate-Reducing Bacteria

Sulfate reducers and related organisms which had previously been found to reduce Fe(III) with H₂ or organic electron donors oxidized S⁰ to sulfate when Mn(IV) was provided as an electron acceptor. Organisms catalyzing this reaction in washed cell suspensions included Desulfovibrio desulfuricans, Desulfomicrobium baculatum, Desulfobacterium autotrophicum, Desulfuromonas acetoxidans, and Geobacter metallireducens. These organisms produced little or no sulfate from S⁰ with Fe(III) as a potential electron acceptor or in the absence of an electron acceptor. In detailed studies with Desulfovibrio desulfuricans, the stoichiometry of sulfate and Mn(II) production was consistent with the reaction S⁰ + 3 MnO₂ + 4H⁺→SO₄^2- + 3Mn(II) + 2H₂O. None of the organisms evaluated could be grown with S⁰ as the sole electron donor and Mn(IV) as the electron acceptor. In contrast to the other sulfate reducers evaluated, Desulfobulbus propionicus produced sulfate from S⁰ in the absence of an electron acceptor and Fe(III) oxide stimulated sulfate production. Sulfide also accumulated in the absence of Mn(IV) or Fe(III). The stoichiometry of sulfate and sulfide production indicated that Desulfobulbus propionicus disproportionates S⁰ as follows: 4S⁰ + 4H₂O→SO₄^2- + 3HS^- + 5 H⁺. Growth of Desulfobulbus propionicus with S⁰ as the electron donor and Fe(III) as a sulfide sink and/or electron acceptor was very slow. The S⁰ oxidation coupled to Mn(IV) reduction described here provides a potential explanation for the Mn(IV)-dependent sulfate production that previous studies have observed in anoxic marine sediments. Desulfobulbus propionicus is the first example of a pure culture known to disproportionate S⁰.     

Survival of Denitrifiers in Nitrate-Free, Anaerobic Environments

Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO₃^--free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 × 10³ and 3.1 × 10⁵ cells cm^-3 in deep sediments from a river and lake, respectively, and 5.3 × 10⁶ cells cm^-3 in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predominant isolates that would grow on anaerobic medium with NO₃^-. All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of Pseudomonas and Alcaligenes. No detectable growth was observed when these strains were incubated with electron acceptors other than NO₃^- or O₂. When representative isolates were added to sterile, O₂- and NO₃^--free porewater from their original locations at their natural densities (10⁵ cells cm^-3), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [¹⁴C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O₂ or NO₃^-), ¹⁴C-labeled products, including cell biomass, ¹⁴CO₂, and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O₂ or NO₃^- and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.     

Metabolism of the Plasticizer di-n-Butylphthalate by Pseudomonas pseudoalcaligenes Under Anaerobic Conditions, with Nitrate as the Only Electron Acceptor

The metabolism of di-n-butylphthalate by a denitrifying strain of Pseudomonas pseudoalcaligenes B20b1 was studied under anaerobic conditions, with nitrate as the only electron acceptor. Thin-layer chromatography and mass spectral analysis of culture extracts (20 days at 30°C) showed mono-n-butylphthalate and phthalic acid as the only products, suggesting that one butanol moiety served essentially as the carbon source for growth and denitrification. N₂ and N₂O were detected by gas chromatography. In contrast to aerobic metabolism, phthalate was not degraded further if nitrate was the only electron acceptor.