Effects of Soil and Water Content on Methyl Bromide Oxidation by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea

Little information exists on the potential of NH₃-oxidizing bacteria to cooxidize halogenated hydrocarbons in soil. A study was conducted to examine the cooxidation of methyl bromide (MeBr) by an NH₃-oxidizing bacterium, Nitrosomonas europaea, under soil conditions. Soil and its water content modified the availability of NH₄⁺ and MeBr and influenced the relative rates of substrate (NH₃) and cosubstrate (MeBr) oxidations. These observations highlight the complexity associated with characterizing soil cooxidative activities when soil and water interact to differentially affect substrate and cosubstrate availabilities.     

Ammonium Removal by the Oxygen-Limited Autotrophic Nitrification-Denitrification System

The present lab-scale research reveals the potential of implementation of an oxygen-limited autotrophic nitrification-denitrification (OLAND) system with normal nitrifying sludge as the biocatalyst for the removal of nitrogen from nitrogen-rich wastewater in one step. In a sequential batch reactor, synthetic wastewater containing 1 g of NH₄⁺-N liter⁻¹ and minerals was treated. Oxygen supply to the reactor was double-controlled with a pH controller and a timer. At a volumetric loading rate (B_v) of 0.13 g of NH₄⁺-N liter⁻¹ day⁻¹, about 22% of the fed NH₄⁺-N was converted to NO₂⁻-N or NO₃⁻-N, 38% remained as NH₄⁺-N, and the other 40% was removed mainly as N₂. The specific removal rate of nitrogen was on the order of 50 mg of N liter⁻¹ day⁻¹, corresponding to 16 mg of N g of volatile suspended solids⁻¹ day⁻¹. The microorganisms which catalyzed the OLAND process are assumed to be normal nitrifiers dominated by ammonium oxidizers. The loss of nitrogen in the OLAND system is presumed to occur via the oxidation of NH₄⁺ to N₂ with NO₂⁻ as the electron acceptor. Hydroxylamine stimulated the removal of NH₄⁺ and NO₂⁻. Hydroxylamine oxidoreductase (HAO) or an HAO-related enzyme might be responsible for the loss of nitrogen.     

Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by Nitrosomonas europaea

Ammonia monooxygenase (AMO) from Nitrosomonas europaea catalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of N. europaea. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with N. europaea cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by N. europaea. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH₄⁺ in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH₃ to concentrations (8 to 80 μM) similar to the K_s value of AMO for NH₃ (~29 μM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.     

Nitrite oxidation in the upper water column and oxygen minimum zone of the eastern tropical North Pacific Ocean

Nitrogen (N) is an essential nutrient in the sea and its distribution is controlled by microorganisms. Within the N cycle, nitrite (NO₂⁻) has a central role because its intermediate redox state allows both oxidation and reduction, and so it may be used by several coupled and/or competing microbial processes. In the upper water column and oxygen minimum zone (OMZ) of the eastern tropical North Pacific Ocean (ETNP), we investigated aerobic NO₂⁻ oxidation, and its relationship to ammonia (NH₃) oxidation, using rate measurements, quantification of NO₂⁻-oxidizing bacteria via quantitative PCR (QPCR), and pyrosequencing. ¹⁵NO₂⁻ oxidation rates typically exhibited two subsurface maxima at six stations sampled: one located below the euphotic zone and beneath NH₃ oxidation rate maxima, and another within the OMZ. ¹⁵NO₂⁻ oxidation rates were highest where dissolved oxygen concentrations were <5 μM, where NO₂⁻ accumulated, and when nitrate (NO₃⁻) reductase genes were expressed; they are likely sustained by NO₃⁻ reduction at these depths. QPCR and pyrosequencing data were strongly correlated (r²=0.79), and indicated that Nitrospina bacteria numbered up to 9.25% of bacterial communities. Different Nitrospina groups were distributed across different depth ranges, suggesting significant ecological diversity within Nitrospina as a whole. Across the data set, ¹⁵NO₂⁻ oxidation rates were decoupled from ¹⁵NH₄⁺ oxidation rates, but correlated with Nitrospina (r²=0.246, P<0.05) and NO₂⁻ concentrations (r²=0.276, P<0.05). Our findings suggest that Nitrospina have a quantitatively important role in NO₂⁻ oxidation and N cycling in the ETNP, and provide new insight into their ecology and interactions with other N-cycling processes in this biogeochemically important region of the ocean.     

Apoplastic pH and Fe3+ Reduction in Intact Sunflower Leaves

It has been hypothesized that under NO₃⁻ nutrition a high apoplastic pH in leaves depresses Fe³⁺ reductase activity and thus the subsequent Fe²⁺ transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO₃⁻ nutrition significantly increased apoplastic pH at distinct interveinal sites (pH ≥ 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO₃⁻/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH₄⁺ or NH₄NO₃ nutrition. Complementary to pH measurements, the formation of Fe²⁺-ferrozine from Fe³⁺-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe³⁺ reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5.0. In analogy to the monitoring of Fe³⁺ reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe³⁺ reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Nitrate-Dependent Ferrous Iron Oxidation by Anaerobic Ammonium Oxidation (Anammox) Bacteria

We examined nitrate-dependent Fe²⁺ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe²⁺ and reduced NO₃⁻ to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein⁻¹ min⁻¹, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH₄⁺ mg protein⁻¹ min⁻¹). A ¹⁵N tracer experiment demonstrated that coupling of nitrate-dependent Fe²⁺ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO₃⁻ by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe²⁺ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO₃⁻ ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe²⁺ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe²⁺ oxidation and NO₃⁻ reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein⁻¹ min⁻¹, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe²⁺ oxidation and the anammox reaction decreased the molar ratios of consumed NO₂⁻ to consumed NH₄⁺ (ΔNO₂⁻/ΔNH₄⁺) and produced NO₃⁻ to consumed NH₄⁺ (ΔNO₃⁻/ΔNH₄⁺). These reactions are preferable to the application of anammox processes for wastewater treatment.     

Nitrate-ammonium synergism in rice. A subcellular flux analysis

Many reports have shown that plant growth and yield is superior on mixtures of NO₃⁻ and NH₄⁺ compared with provision of either N source alone. Despite its clear practical importance, the nature of this N-source synergism at the cellular level is poorly understood. In the present study we have used the technique of compartmental analysis by efflux and the radiotracer ¹³N to measure cellular turnover kinetics, patterns of flux partitioning, and cytosolic pool sizes of both NO₃⁻ and NH₄⁺ in seedling roots of rice (Oryza sativa L. cv IR72), supplied simultaneously with the two N sources. We show that plasma membrane fluxes for NH₄⁺, cytosolic NH₄⁺ accumulation, and NH₄⁺ metabolism are enhanced by the presence of NO₃⁻, whereas NO₃⁻ fluxes, accumulation, and metabolism are strongly repressed by NH₄⁺. However, net N acquisition and N translocation to the shoot with dual N-source provision are substantially larger than when NO₃⁻ or NH₄⁺ is provided alone at identical N concentrations.     

The High Salt Requirement of the Moderate Halophile Chromohalobacter salexigens DSM3043 Can Be Met Not Only by NaCl but by Other Ions

The growth rate of Chromohalobacter salexigens DSM 3043 can be stimulated in media containing 0.3 M NaCl by a 0.7 M concentration of other salts of Na⁺, K⁺, Rb⁺, or NH₄⁺, Cl⁻, Br⁻, NO₃⁻, or SO₄²⁻ ions. To our knowledge, growth rate stimulation by a general high ion concentration has not been reported for any organism previously.     

Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.     

Evidence for the direct oxidation of organic nitrogen to N2 gas in the Arabian Sea

We performed a suite of ¹⁵N incubations (¹⁵NO₂⁻, ¹⁵NO₃⁻ and ¹⁵NH₄⁺) with and without the organic-nitrogen (N) compound allylthiourea (ATU), in the suboxic waters of the Arabian Sea. Production of ²⁹N₂ in control (-ATU) incubations with either ¹⁵NH₄⁺+¹⁴NO₂⁻, or their analogues, ¹⁵NO₂⁻+¹⁴NH₄⁺, though small, confirmed the presence of anammox. In contrast, when we added ATU, along with ¹⁵NO₂⁻ and ¹⁴NH₄⁺, there was a much greater production of ²⁹N₂, with 92% of the ¹⁵N-label being recovered as ²⁹N₂ on average. Such stimulated production of ²⁹N₂ could not be due to anammox, as the addition of ATU, along with ¹⁵NH₄⁺+¹⁴NO₂⁻, only produced ²⁹N₂ equivalent to that in the controls. The ratios of ²⁹N₂ to ³⁰N₂ produced also precluded stimulation of denitrification. We present this as evidence for a hitherto uncharacterised metabolism potentially capable of oxidising organic-N (e.g. NH₂ groups) directly to N₂ gas at the expense of NO₂⁻.     

Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge⁻¹ day⁻¹. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769–773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH₄-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea. However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.     

Varied Diazotrophies, Morphologies, and Toxicities of Genetically Similar Isolates of Cylindrospermopsis raciborskii (Nostocales, Cyanophyceae) from Northern Australia

The potentially toxic freshwater cyanobacterium Cylindrospermopsis raciborskii has become increasingly prevalent in tropical and temperate water bodies worldwide. This paper investigates the effects of different nitrogen sources (NO₃⁻, NH₄⁺, and omission of a fixed form of nitrogen) on the growth rates, morphologies, and cylindrospermopsin (CYL) concentrations (expressed as a percentage of the freeze-dried weight) of seven C. raciborskii isolates obtained from a range of water bodies in northern Australia and grown in batch culture. In general, growth rates were lowest in the absence of a fixed-nitrogen source and highest with NH₄⁺ as the nitrogen source. Conversely, the highest concentrations of CYL were recorded in cultures grown in the absence of a fixed-nitrogen source and the lowest were found in cultures supplied with NH₄⁺. Cultures supplied with NO₃⁻ were intermediate with respect to both CYL concentration and growth rate. Different nitrogen sources resulted in significant differences in the morphology of C. raciborskii trichomes. Most notable were the loss of heterocysts and the tapering of end cells in cultures supplied with NH₄⁺ and the statistically significant increase in vegetative cell length (nitrogen depleted < NO₃⁻ < NH₄⁺). The morphological changes induced by different nitrogen sources were consistent for all isolates, despite measurable differences in vegetative-cell and heterocyst dimensions among isolates. Such induced morphological variation has implications for Cylindrospermopsis taxonomy, given that distinctions between species are based on minor and overlapping differences in cell lengths and widths. The close phylogenetic association among all seven isolates was confirmed by the high level (>99.8%) of similarity of their 16S rRNA gene sequences. Another genetic technique, analysis of the HIP1 octameric-palindrome repeated sequence, showed greater heterogeneity among the isolates and appears to be a useful method for distinguishing among isolates of C. raciborskii.     

In Vitro Reconstitution of Electron Transport from Glucose-6-Phosphate and NADPH to Nitrite

An NADPH-dependent NO₂⁻-reducing system was reconstituted in vitro using ferredoxin (Fd) NADP⁺ oxidoreductase (FNR), Fd, and nitrite reductase (NiR) from the green alga Chlamydomonas reinhardtii. NO₂⁻ reduction was dependent on all protein components and was operated under either aerobic or anaerobic conditions. NO₂⁻ reduction by this in vitro pathway was inhibited up to 63% by 1 mm NADP⁺. NADP⁺ did not affect either methyl viologen-NiR or Fd-NiR activity, indicating that inhibition was mediated through FNR. When NADPH was replaced with a glucose-6-phosphate dehydrogenase (G6PDH)-dependent NADPH-generating system, rates of NO₂⁻ reduction reached approximately 10 times that of the NADPH-dependent system. G6PDH could be replaced by either 6-phosphogluconate dehydrogenase or isocitrate dehydrogenase, indicating that G6PDH functioned to: (a) regenerate NADPH to support NO₂⁻ reduction and (b) consume NADP⁺, releasing FNR from NADP⁺ inhibition. These results demonstrate the ability of FNR to facilitate the transfer of reducing power from NADPH to Fd in the direction opposite to that which occurs in photosynthesis. The rate of G6PDH-dependent NO₂⁻ reduction observed in vitro is capable of accounting for the observed rates of dark NO₃⁻ assimilation by C. reinhardtii.     

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC₅₀], 6 to 38 mg liter⁻¹) was affected much less by LAS than the growth rate and viability (EC₅₀, 3 to 14 mg liter⁻¹) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter⁻¹); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH₄⁺ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Influence of Starvation on Potential Ammonia-Oxidizing Activity and amoA mRNA Levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO_x biosensor. The apparent half-saturation constant [K_m(app)] value of the NH₃ oxidation of N. briensis was 3 μM NH₃ for cultures grown both in continuous and batch cultures as determined by a NO_x biosensor. Cells grown on the wall of the vessel had a lower K_m(app) value of 1.8 μM NH₃. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 μM N h⁻¹, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH₄⁺, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH₄⁺ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH₄⁺ availability.     

Anaerobic Ammonium Oxidation Measured in Sediments along the Thames Estuary, United Kingdom

Until recently, denitrification was thought to be the only significant pathway for N₂ formation and, in turn, the removal of nitrogen in aquatic sediments. The discovery of anaerobic ammonium oxidation in the laboratory suggested that alternative metabolisms might be present in the environment. By using a combination of ¹⁵N-labeled NH₄⁺, NO₃⁻, and NO₂⁻ (and ¹⁴N analogues), production of ²⁹N₂ and ³⁰N₂ was measured in anaerobic sediment slurries from six sites along the Thames estuary. The production of ²⁹N₂ in the presence of ¹⁵NH₄⁺ and either ¹⁴NO₃⁻ or ¹⁴NO₂⁻ confirmed the presence of anaerobic ammonium oxidation, with the stoichiometry of the reaction indicating that the oxidation was coupled to the reduction of NO₂⁻. Anaerobic ammonium oxidation proceeded at equal rates via either the direct reduction of NO₂⁻ or indirect reduction, following the initial reduction of NO₃⁻. Whether NO₂⁻ was directly present at 800 μM or it accumulated at 3 to 20 μM (from the reduction of NO₃⁻), the rate of ²⁹N₂ formation was not affected, which suggested that anaerobic ammonium oxidation was saturated at low concentrations of NO₂⁻. We observed a shift in the significance of anaerobic ammonium oxidation to N₂ formation relative to denitrification, from 8% near the head of the estuary to less than 1% at the coast. The relative importance of anaerobic ammonium oxidation was positively correlated (P < 0.05) with sediment organic content. This report of anaerobic ammonium oxidation in organically enriched estuarine sediments, though in contrast to a recent report on continental shelf sediments, confirms the presence of this novel metabolism in another aquatic sediment system.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH₂ more efficiently than it oxidized MBQH₂; both the affinity and maximal velocity of oxidation were higher for DBQH₂ than for MBQH₂. Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q^·− + O₂ ↔ Q + O₂^·−). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O₂^·− on quinone formation rates. Then, the production of H₂O₂ in laccase reactions, as a consequence of O₂^·− dismutation, confirmed that semiquinones autoxidized. The highest H₂O₂ levels were obtained with DBQH₂, indicating that DBQ^·− autoxidized to a greater extent than did MBQ^·−. Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O₂^·− consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O₂^·−, besides undergoing dismutation, reacts with Mn²⁺, Fe³⁺, and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 μM concentrations of MBQ^·− and DBQ^·− from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ^·− autoxidation rose to 13.8% and that of DBQ^·− increased to 39.9%.     

Rapid Consumption of Low Concentrations of Methyl Bromide by Soil Bacteria

A dynamic dilution system for producing low mixing ratios of methyl bromide (MeBr) and a sensitive analytical technique were used to study the uptake of MeBr by various soils. MeBr was removed within minutes from vials incubated with soils and ~10 parts per billion by volume of MeBr. Killed controls did not consume MeBr, and a mixture of the broad-spectrum antibiotics chloramphenicol and tetracycline inhibited MeBr uptake by 98%, indicating that all of the uptake of MeBr was biological and by bacteria. Temperature optima for MeBr uptake suggested a biological sink, yet soil moisture and temperature optima varied for different soils, implying that MeBr consumption activity by soil bacteria is diverse. The eucaryotic antibiotic cycloheximide had no effect on MeBr uptake, indicating that soil fungi were not involved in MeBr removal. MeBr consumption did not occur anaerobically. A dynamic flowthrough vial system was used to incubate soils at MeBr mixing ratios as low as those found in the remote atmosphere (5 to 15 parts per trillion by volume [pptv]). Soils consumed MeBr at all mixing ratios tested. Temperate forest and grassy lawn soils consumed MeBr most rapidly (rate constant [k] = 0.5 min⁻¹), yet sandy temperate, boreal, and tropical forest soils also readily consumed MeBr. Amendments of CH₄ up to 5% had no effect on MeBr uptake even at CH₄:MeBr ratios of 10⁷, and depth profiles of MeBr and CH₄ consumption exhibited very different vertical rate optima, suggesting that methanotrophic bacteria, like those presently in culture, do not utilize MeBr when it is at atmospheric mixing ratios. Data acquired with gas flux chambers in the field demonstrated the very rapid in situ consumption of MeBr by soils. Uptake of MeBr at mixing ratios found in the remote atmosphere occurs via aerobic bacterial activity, displays first-order kinetics at mixing ratios from 5 pptv to ~1 part per million per volume, and is rapid enough to account for 25% of the global annual loss of atmospheric MeBr.