Candidatus 'Brocadia fulgida': an autofluorescent anaerobic ammonium oxidizing bacterium

Anaerobic ammonium oxidizing (anammox) bacteria are detected in many natural ecosystems and wastewater treatment plants worldwide. This study describes the enrichment of anammox bacteria in the presence of acetate. The results obtained extend the concept that the anammox bacteria can be enriched to high densities in the presence of substrates for heterotrophic growth. Batch experiments showed that among the tested biomass, the biomass from the Candidatus 'Brocadia fulgida' enrichment culture oxidizes acetate at the highest rate. Continuous cultivation experiments showed that in the presence of acetate, ammonium, nitrite and nitrate, Candidatus 'Brocadia fulgida' out-competed other anammox bacteria. The results indicated that Candidatus 'Brocadia fulgida' did not incorporate acetate directly into their biomass. Candidatus 'Brocadia fulgida' exhibited the common characteristics of anammox bacteria: the presence of an anammoxosome and ladderane lipids and the production of hydrazine in the presence of hydroxylamine. Interestingly, the biofilm aggregates of this species showed strong autofluorescence. It is the only known anammox species exhibiting this feature. The autofluorescent extracellular polymeric substance had two excitation (352 and 442 nm) and two emission (464 and 521 nm) maxima.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Effects of inorganic carbon limitation on anaerobic ammonium oxidation (anammox) activity

Anammox bacteria are chemoautotrophic bacteria that oxidize ammonium with nitrite as the electron acceptor and with CO₂ as the main carbon source. The effects of inorganic carbon (IC) limitation on anammox bacteria were investigated using continuous feeding tests. In this study, a gel carrier with entrapped anammox sludge was used. It was clearly shown that the anammox activity deteriorated with a decrease in the influent IC concentration. The relationship between the influent IC concentration and the anammox activity was analyzed using Michaelis-Menten kinetics, and the apparent K_m was determined to be 1.2 mg-C/L. The activity could be recovered by adding IC to the influent. The consumption ratio of IC to ammonium was not constant and mainly depended on the influent ratio of the IC to ammonium concentrations (inf.IC/inf.NH₄-N). The results indicated that an inf.IC/inf.NH₄-N ratio of 0.2 in the anammox reactor was ideal for the anammox process using gel cubes.     

Ladderane lipid distribution in four genera of anammox bacteria

Intact ladderane phospholipids and core lipids were studied in four species of anaerobic ammonium oxidizing (anammox) bacteria, each representing one of the four known genera. Each species of anammox bacteria contained C(18) and C(20) ladderane fatty acids with either 3 or 5 linearly condensed cyclobutane rings and a ladderane monoether containing a C(20) alkyl moiety with 3 cyclobutane rings. The presence of ladderane lipids in all four anammox species is consistent with their putative physiological role to provide a dense membrane around the anammoxosome, the postulated site of anammox catabolism. In contrast to the core lipids, large variations were observed in the distribution of ladderane phospholipids, i.e. different combinations of hydrophobic tail (ladderane, straight chain and methyl branched fatty acid) types attached to the glycerol backbone sn-1 position, in combination with different types of polar headgroup (phosphocholine, phosphoethanolamine or phosphoglycerol) attached to the sn-3 position. Intact ladderane lipids made up a high percentage of the lipid content in the cells of "Candidatus Kuenenia stuttgartiensis", suggesting that ladderane lipids are also present in membranes other than the anammoxosome. Finally, all four investigated species contained a C(27) hopanoid ketone and bacteriohopanetetrol, which, indicates that hopanoids are anaerobically synthesised by anammox bacteria.     

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO₃⁻, NO₂⁻ or NH₄⁺) for growth. Different approaches were considered to study the effect of NH₄⁺ on nitrogen assimilation in Hfx. mediterranei cells grown in KNO₃ medium. The NH₄⁺ addition to KNO₃ medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH₄⁺ media. Both activities increased when NH₄⁺ was removed from culture, showing that the negative effect of NH₄⁺ on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO₃⁻ and NH₄⁺, NH₄⁺ was preferentially consumed, but NO₃⁻ uptake was not completely inhibited by NH₄⁺ at prolonged time scale. The addition of MSX to NH₄⁺ or NO₃⁻ cultures results in an increase of Nas and NiR activities, suggesting that NH₄⁺ assimilation, rather than NH₄⁺ per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nitrate reductase activity and growth response of forest species to ammonium and nitrate sources of nitrogen

Summary Three tree species,Eucalyptus regnans (F. Muell.),E. obliqua (L'Herit.),Pinus radiata (D. Don) were grown in sand culture with different proportions of nitrate and ammonium. Nitrate Reductase Activity (NRA) was induced in root tissue of all species and in leaf tissue of the eucalypts. An increasing proportion of nitrate resulted in increasing NRA in all species and hence NRA alone is no indication of N-preference. The highest NRA was found withE. regnans, a result which has also been obtained in the mature forest. The growth ofE. regnans was least with NH₄ ⁺ alone, whereas that ofE. obliqua was least with NO₃ ^– alone. The soils of matureE. regnans forest have a high potential for nitrification while those ofE. obliqua forest show little nitrification. Thus the preference for particular N sources shown by seedlings in culture is supported by related properties of mature forests. It is postulated however, that the inducibility of a high level of RNA in seedlings is more likely a result of a preference for NO₃ ^– than a cause.     

以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除无机三态氮

【目的】揭示以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除水体中无机三态氮的特征和规律。【方法】在光照厌氧环境下,以乙酸盐为唯一有机物,在分别以氨氮、亚硝态氮、硝态氮为唯一氮源和三氮共存的模拟水体中,采用Nessler’s试剂分光光度法、N-(1-萘基)-乙二胺分光光度法和紫外分光光度法分别测定水体中氨氮、亚硝态氮和硝态氮的含量,比浊法测定菌体生物量。【结果】随着时间的延长,海洋紫色硫细菌Marichromatium gracile YL28分别在氨氮、亚硝态氮和硝态氮为唯一氮源的水体中对三氮的去除量增加,生物量增大,水体pH升高,并逐渐趋于平衡;YL28对氨氮的最大去除量和最大耐受浓度分别为9.64 mmol/L和36.64 mmol/L,当氨氮浓度低于3.21 mmol/L时,去除率可达97.61%以上;与氨氮相比,以亚硝态氮和硝态氮为唯一氮源,菌体的生长速率、生物量和水体最终pH较低,但对亚硝态氮和硝态氮的去除速率和去除量仍然很高,当亚硝态氮和硝态氮浓度分别达13.50 mmol/L和22.90 mmol/L时,YL28仍能够完全去除。在三氮共存的水体中,YL28也能良好的去除无机三态氮,对亚硝态氮和硝态氮去除能力更强。【结论】在模拟水体中,海洋紫色硫细菌YL28能够分别以氨氮、亚硝态氮和硝态氮为唯一氮源生长,具有良好的耐受和去除无机三态氮的能力,尤其对亚硝态氮具有良好的去除能力。本研究为进一步开发高效脱氮,尤其是去除亚硝态氮的不产氧光合细菌水质调节剂奠定了基础,也为微生物制剂的合理应用提供参考。     

舟山群岛海域沉积物厌氧氨氧化细菌多样性

通过构建16S rRNA基因文库和克隆测序研究了舟山海域沉积物中厌氧氨氧化细菌(AAOB)的多样性。从5个克隆文库中共获得297条16S rRNA基因序列,包含16个操作分类单元(OTUs)。离岸距离较近的3个站具有相似的群落结构,且与离岸较远的2个站具有明显差异。系统发育结果显示,Scalindua属是该海域AAOB的优势类群,95.3%的序列与Scalindua属AAOB具有较近的亲缘关系;1条序列与Kuenenia属具有较近的亲缘关系;此外还有15条序列与已知的AAOB相似性较低。相关性分析表明沉积物有机碳含量与多样性指数具有显著正相关,可能是该海域AAOB多样性变化的重要影响因子。     

Mineralisation of urea and urea derivatives in anaerobic soils

Summary The rates of mineralisation of urea and urea derivatives were studied in a laboratory anaerobic incubation experiment. Urea, urea phosphate and sulphur coated urea were hydrolysed rapidly and, even at the highest level of application, had disappeared in just over 8 days. The presence of the phosphate anion depressed pH in the early stages. Hydrolysis of the less soluble organic derivatives of urea, isobutylidine diurea, ureaform and glycoluril was very much slower and in the case of glycoluril a lag period of 8 to 16 days occurred before hydrolysis began.In the initial stages the system was anaerobic but between days 8 and 16 a change to partial aerobic conditions occurred. At this stage nitrification commenced and at day 16 nitrite was detected. Reduction of Fe(III) increased with time, reaching a maximum at day 32. More Fe(II) was produced in the presence of organic derivatives of urea than with the other fertilizers, possibly due to stabilisation by organic ligands. From day 16 nitrification, denitrification and reduction of Fe(III) proceeded together even through E_h values indicated that oxidation of Fe(II) would be expected. This did not occur until after day 32. Once nitrification began denitrification quickly followed so that for all six fertilisers, except at the highest level of application, virtually all the mineralised-N had been lost by denitrification at the end of the experiment.     

Kinetics of ammonium and nitrate uptake among wild and cultivated tomatoes

Summary Concentration dependence of net ammonium and nitrate uptake was monitored for a cultivar of tomato, Lycopersicon esculentum, and two accessions of a neotropical wild relative, L. hirsutum. The kinetics of net NH ₄ ⁺ uptake differed among these taxa and were not dependent on the ionic composition of the nutrient solution. The kinetics of net NO ₃ ^- uptake were dependent on the composition of the nutrient solution; the presence of NH ₄ ⁺ or Cl^- enhanced net NO ₃ ^- uptake for the cultivated species and for a highland accession of the wild species. The capacity for net NO ₃ ^- uptake was greater than the capacity for net NH ₄ ⁺ uptake in all three taxa; the proportion of NO ₃ ^- to NH ₄ ⁺ absorbed was much greater for the wild taxa. Our data suggest that NO ₃ ^- may be a more important source of mineral nitrogen than NH ₄ ⁺ for these tropical taxa.     

Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage

The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at −20°C with and without glycerol as cryoprotectant and at 4 and 20°C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4°C with and without nitrate and at 20°C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4°C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.     

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O₂ ha⁻¹ day⁻¹. As the oxygen transfer rate of an abiotic control column was only 17 kg O₂ ha⁻¹ day⁻¹, biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.     

Studies on dissimilatory sulfate-reducing bacteria that decompose fatty acids II. Incomplete oxidation of propionate by Desulfobulbus propionicus gen. nov., sp. nov.

A new type of sulfate-reducing bacteria with ellipsoidal to lemon-shaped cells was regularly enriched from anaerobic freshwater and marine mud samples when mineral media with propionate and sulfate were used. Three strains (1pr3, 2pr4, 3pr10) were isolated in pure culture. Propionate, lactate and alcohols were used as electron donors and carbon sources. Growth on H₂ required acetate as a carbon source in the presence of CO₂. Stoichiometric measurements revealed that oxidation of propionate was incomplete and led to acetate as an endproduct. Instead of sulfate, strain 1pr3 was shown to reduce sulfite and thiosulfate to H₂S; nitrate also served as electron acceptor and was reduced to ammonia. With lactate or pyruvate, all three strains were able to grow without external electron acceptor and formed propionate and acetate as fermentation products. None of the strains contained desulfoviridin. In strain 1pr3 cytochromes of the b- and c-type were identified. Strain 1pr3 is described as type strain of the new species and genus, Desulfobulbus propionicus.     

Metabolic pathway to propionate of Pectinatus frisingensis,a strictly anaerobic beer-spoilage bacterium

      Pectinatus frisingensis, a recently described species of anaerobic mesophilic beer-spoilage bacteria, grows by fermenting various organic compounds, and produces mainly propionate, acetate, and succinate. Although acrylate and succinate were both dismutated by dense resting-cell suspensions, propionate production proceeded through the succinate pathway: [3-¹³C]pyruvate consumption led to equal ¹³C-labeling of propionate on methyl and methylene groups. Growth on glucose or glycerol led to a similar propionate to acetate ratio, suggesting dihydroxyacetone phosphate as being a common metabolic intermediate. Diacetyl, 1,3-propanediol, and 2,3-butanediol were not growth substrates or fermentation products, but they were all dismutated by dense resting-cell suspensions to acetate and propionate. Acetoin was a minor fermentation product. The consumption of [2-¹³C] or [3-¹³C]pyruvate by dense resting-cell suspensions demonstrated the involvement of two equivalent pyruvate molecules during acetoin production. Key enzymes involved in this metabolism were measured in anoxic cell-free extracts. A tentative metabolic pathway to the main fermentation products was proposed from the above results. Received: 17 February 1994 / Accepted: 30 August 1994     

Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation

The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.     

Isolation, characterization, and nitrification potential of a methylotroph and two heterotrophic bacteria from a consortium showing methane-dependent nitrification

Abstract A methanotrophic nitrifying consortium was previously obtained from a humisol which showed CH₄-dependent nitrification. Although the methanotroph could not be obtained in pure culture, three other members of the consortium have been isolated: An obligately methylotrophic Methylobacillus (Is-1) which grows only on CH₃OH and does not nitrify; a Pseudomonas (Is-2) which grows on Is-1 culture filtrate and produces NO₂⁻, NO₃⁻ and N₂O from NH₂OH, and NO₃⁻ from NO₂⁻; and a second Pseudomonas (Is-3) which produces NO₃⁻ from NH₄⁺ or NO₂⁻, and N₂O from NH₂OH. A model is proposed for the trophic relations and nitrogen transformations in the consortium which may apply to some natural systems.