Characterization of two ammonia-oxidizing bacteria isolated from reactors operated with low dissolved oxygen concentrations

AIMS: To obtain ammonia-oxidizing bacterial (AOB) strains inhabiting low dissolved oxygen (DO) environments and to characterize them to better understand their function and ecology. METHODS AND RESULTS: Using a serial dilution method, two AOB strains (ML1 and NL7) were isolated from chemostat reactors operated with low DO concentrations (0.12-0.24 mg l(-1)). Phylogenetically, strains ML1 and NL7 are affiliated to AOB within the Nitrosomonas europaea and Nitrosomonas oligotropha lineages, respectively. Kinetically, strain ML1 had high affinity for oxygen (0.24 +/- 0.13 mg l(-1)) and low affinity for ammonia (1.62 +/- 0.97 mg N l(-1)), while strain NL7 had high affinity for ammonia (0.48 +/- 0.35 mg l(-1)), but a surprisingly low affinity for oxygen (1.22 +/- 0.43 mg l(-1)). A co-culture experiment was used to iteratively estimate decay constants for both strains. CONCLUSIONS: The results indicated that AOB without high affinity for oxygen may have other mechanisms to persist in low DO environments, with high affinity for ammonia being important. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a method to determine AOB growth kinetic parameters without assuming or neglecting decay constant. And, this is the first report on oxygen affinity constant of a N. oligotropha strain.     

Inoculum effects on community composition and nitritation performance of autotrophic nitrifying biofilm reactors with counter‐diffusion geometry

The link between nitritation success in a membrane‐aerated biofilm reactor (MABR) and the composition of the initial ammonia‐ and nitrite‐oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat‐sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO₂^‐‐N/NH₄⁺‐N) were obtained in the Inoculum B‐ (55.2–56.4%) versus the Inoculum A‐ (20.2–22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100–150 µm), but the AOB profiles [based on 16S rRNA gene targeted real‐time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B‐ versus A‐initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A‐ and B‐initiated reactors were Nitrosospira spp. (48.9–61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8–63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell^?1 h^?1 versus 0.79 fmol cell^?1 h^?1). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.     

High levels of nitrifying bacteria in intermittently aerated reactors treating high ammonia wastewater

Changes in the fractions of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in two laboratory-scale reactors were investigated using 16S rRNA probe hybridizations. The reactors were operated in intermittent aeration mode and different aeration cycles to treat anaerobically digested swine wastewater with ammonia concentrations up to 175 mg NH(3)-N/L. High ammonia removals (>98.8%) were achieved even with increased nitrogen loads and lower aeration: non-aeration time ratios of 1h:3h. Nitrosomonas/Nitrosococcus mobilis were the dominant ammonia-oxidizing bacteria in the reactors. Nitrospira-like organisms were the dominant nitrite-oxidizing bacteria during most of the investigation, but were occasionally outcompeted by Nitrobacter. High levels of nitrifiers were measured in the biomass of both reactors, and ammonia-oxidizing bacteria and nitrite-oxidizing bacterial levels adjusted to changing aeration: non-aeration time ratios. Theoretical ammonia-oxidizer fractions, determined by a mathematical model, were comparable to the measured values, although the measured biomass fractions were different at each stage while the theoretical values remained approximately constant. Stable ammonia removals and no nitrite accumulation were observed even when rRNA levels of ammonia oxidizers and nitrite-oxidizers reached a minimum of 7.2% and 8.6% of total rRNA, respectively. Stable nitrogen removal performance at an aeration: non-aeration ratio of 1h:3h suggests the possibility of significant savings in operational costs.     

Impact of increasing NaCl concentrations on the performance and community composition of two anaerobic reactors

The anaerobic treatment of saline effluents using halophilic and halotolerant microbial consortia is of major interest. Inhibition of anaerobic digestion is known to occur at high salt content. However, it seems that the suitable adaptation of an anaerobic sludge makes possible the treatment of saline wastewater. In this study, a non-saline anaerobic sludge was inoculated in two anaerobic batch reactors operating with a different substrate (distillery vinasse and ethanol) and subjected to increasing NaCl concentrations. The performance of the digesters appeared to be highly dependent on the nature of the substrate, and a similar level of inhibition (i.e. around 90% of the specific loading rate and specific methanogenic activity) was stated at 10 g l⁻¹ of NaCl with distillery vinasse and 60 g l⁻¹ of NaCl with ethanol. The characterization of the microflora and its adaptation to increasing NaCl conditions were also investigated using molecular tools based on the analysis of genomic 16S rDNA. The microbial communities revealed a high diversity that could be maintained in both reactors despite the increase in NaCl concentrations.     

Hydraulic selection pressure-induced nitrifying granulation in sequencing batch reactors

The effect of hydraulic selection pressure on the development of nitrifying granules was investigated in four column-type sequencing batch reactors (SBR). The nature of SBR is cycle operation, thus SBR cycle time can serve as a main hydraulic selection pressure imposed on the microbial community in the system. No nitrifying granulation was observed in the SBR operated at the longest cycle time of 24 h, due to a very weak hydraulic selection pressure, while the washout of nitrifying sludge was found in the SBR run at the shortest cycle time of 3 h, and led to a failure of nitrifying granulation. Excellent nitrifying granules with a mean diameter of 0.25 mm and specific gravity of 1.014 were developed in a SBR operated at cycle times of 6 h and 12 h, respectively. The results further showed that a short cycle time would stimulate microbial activity, production of cell polysaccharides and also improve the cell hydrophobicity. These hydraulic selection pressure-induced microbial changes favour the formation of nitrifying granules. This work, probably for the first time, shows that nitrifying granules can be developed at a proper hydraulic selection pressure in terms of SBR cycle time. Nitrifying granulation is a novel biotechnology which has a great potential for wastewater nitrification.     

Linking microbial community structure to membrane biofouling associated with varying dissolved oxygen concentrations

In order to elucidate how dissolved oxygen (DO) concentration influenced the generation of extracellular polymeric substance (EPS) and soluble microbial products (SMP) in mixed liquor and biocake, 16S rDNA fingerprinting analyses were performed to investigate the variation of the microbial community in an aerobic membrane bioreactor (MBR). The function of microbial community structure was proved to be ultimately responsible for biofouling. Obvious microbial community succession from the subphylum of Betaproteobacteria to Deltaproteobacteria was observed in biocake. High concentration of EPS in biocake under the low DO concentration (0.5 mg L⁻¹) caused severe biofouling. The correlation coefficient of membrane fouling rate with EPS content in biocake (0.9941-0.9964) was much higher than that in mixed liquor (0.6689-0.8004).     

Neap-spring tidal effects on dissolved oxygen in two Malaysian estuaries

The longitudinal distributions of dissolved oxygen change dramatically during neap-spring tidal cycles in the lower Selangor and Klang Rivers. An oxygen deficit develops in both estuaries when the tidal range is high. The Selangor River inflow is nearly saturated with D.O., but during spring tides D.O. falls to as little as 15% of saturation in the middle reaches of the estuary. The Klang River has low D.O. freshwater input, an oxygen minimum develops during neap tides, and anoxic conditions are produced by spring tides. These oxygen minima are attributed to the high oxygen demand of sediment that rests on the bottom during neap tides and is resuspended during spring tides. The ecological effects of tidal cycling patterns of dissolved oxygen in these Malaysian estuaries need further study, within the context of land use patterns and other human activities.     

Bacterial growth and community composition in fractions of dissolved organic carbon of different molar mass from interstitial water

The ability of different molar-mass fractions of interstitial dissolved organic carbon (DOC) from a small stream to stimulate bacterial growth was determined. Growth was the greatest in the low-molar mass (<1 kDa) (LMM) fraction and the least in the high-molar mass (>10 kDa) (HMM) one. The LMM fraction consistently had a higher ratio of carbon to organic nitrogen than other fractions. The fluorescence in situ hybridization analysis revealed that bacterial populations in interstitial water were dominated by beta-Proteobacteria. gamma-Proteobacteria together with the Cytophaga-Flavobacteria cluster dominated in LMM DOC (66.3%) while the proportions of bacterial groups in HMM and medium-molar mass (MMM) fractions were more equal. The fact that gamma-Proteobacteria strongly dominates in LMM DOC fraction refutes the idea that LMM DOC is readily utilized by all heterotrophic bacteria.     

A novel approach to controlling dissolved oxygen levels in laboratory experiments

Hypoxia is a widespread environmental stressor that affects marine, estuarine and freshwater systems worldwide. Investigating the effects of hypoxia on aquatic animals in the natural environment is difficult and expensive. Laboratory experiments provide an alternative that allows manipulation of environmental variables and simulation of altered conditions that are expected in the future. However, controlling dissolved oxygen (DO) levels precisely in laboratory test situations is challenging and generally costly and time intensive. In this paper, we describe a novel chamber design that is capable of maintaining low DO levels precisely for a period of at least 4 days with minimal re-adjustment and nitrogen gas requirement. The system is simple, inexpensive and easy to use, and offers the additional benefit of being portable. The utility of this design is demonstrated with an experiment to test the effects of hypoxia and high salinity on hatch rates and yolk-sac larvae survival in the estuarine fish black bream Acanthopagrus butcheri (Munro, 1949). The results show that early life-stage black bream are sensitive to hypoxia and increased salinity, indicating that deterioration in estuarine environmental conditions may have negative effects on spawning success and recruitment to fish populations. This new laboratory technique will be useful for testing future scenarios for the estuaries of southern Australia as predicted by climate change models. Other potential applications and modifications for further development of this new technique are discussed.     

Priming responses of leek (Allium porrum L.) seeds to different dissolved oxygen levels in the osmoticum

Leek seeds (Allium porrum L.) cv. Winterreuzen were treated for 5 days in stirred bioreactors containing a -1.0 MPa polyethylene glycol (PEG 6000) solution. The level of dissolved oxygen was controlled from zero to approximately two and a half times that of saturation with respect to atmospheric air by bubbling through 0₂/N₂ gas blends from 0% O₂ to 50% O₂. The treatment with 0% O₂ (i.e. bubbling with N₂) gave no reduction in germination time compared to untreated seeds after allowing for the time for water imbibition. As the proportion of oxygen was increased, a rapid reduction was observed until a concentration equivalent to air was used. Further increases to the maximum, produced only a marginal further improvement. The reduction in germination times for seeds that had been cabinet dried was longer for all treatments due to the time required for re-imbibition of water but the general response to priming was the same as with undried seeds. Only dried seeds treated with air or higher oxygen concentrations showed enhanced synchronicity of germination times. The percentage germination of seeds from all treatments was the same, including the treatment with nitrogen gas. These tests help to explain the recently reported advantages of using enriched air for seed priming and are one of the necessary protocols for ensuring satisfactory bulk priming.     

Response of an antarctic lake heterotrophic community to high dissolved oxygen

The upper waters of Lake Hoare, Antarctica, contain dissolved oxygen at about three times the normal saturation (>/=42 mg liter). The response of the heterotrophic plankton community to this high dissolved oxygen was evaluated by the criteria of CFU and d-[U-C]glucose assimilated-respired. High dissolved oxygen was not inhibitory to d-[U-C]glucose assimilation-respiration compared with normal atmospheric dissolved oxygen in Lake Hoare water. The d-[U-C]glucose was assimilated and respired optimally at 12 degrees C in Lake Hoare. The d-[U-C]glucose assimilated-respired in the upper saturated atmospheric dissolved oxygen waters of Mountain Lake, Va., was inhibited in contrast to Lake Hoare (P < 0.05). CFU formation was inhibited in both lakes. CFU represent <1% of the fluorochrome-stained direct counts in Lake Hoare. Lake Hoare planktobacteria are smaller than the planktobacteria in Mountain Lake. ATP size fractionation revealed that 39% of the ATP biomass was <0.5 mum in Lake Hoare.     

不同溶氧对谷氨酸棒杆菌代谢的影响

【目的】以谷氨酸棒杆菌为研究对象,分别控制在0、30%、50%3种溶氧水平下进行发酵,分析不同溶氧水平下代谢的变化。【方法】通过检测发酵代谢物中有机酸、氨基酸的含量,以及测定代谢途径中关键酶活性及其编码基因的表达情况来考察不同溶氧水平下物质代谢发生的变化。通过检测胞内还原力和ATP的含量来分析不同溶氧水平对能量代谢产生的影响。【结果】谷氨酸棒杆菌代谢支路受溶氧的影响而发生改变,氨基酸、有机酸的产量也随之改变。特别是在低溶氧(0)情况下,细胞内氧化磷酸化减弱,导致维持生命活动所必需的ATP供应减少,因此细胞通过增强底物水平磷酸化来产生ATP以满足生命活动的需求。在此情况下,胞内NADH得到较多积累,TCA循环代谢流量减小,而转向糖酵解、乙醛酸循环等,并且这个过程伴随多种杂酸包括乳酸、缬氨酸、亮氨酸等的产生,必将影响目的产物的产量。【结论】研究结果对于进一步采取措施优化溶氧的控制策略,提高目的产物的产量具有指导意义。     

Nitrification rates in a saline medium at different dissolved oxygen concentrations

A kinetic study was carried out in a saline medium to assess the effect of O₂ on the two-step nitrification process: for the first nitritation step, 2–26 mg dissolved O₂ (DO) l^–1 was used and for the second nitratation step, 0.5–24 mg DO l^–1 was used. Nitritation rate was measured in the presence of sodium azide so as to inhibit nitratation. Ammonia-oxidizing (AOB) and nitrite-oxidizing (NOB) bacterial in the mixed culture were determined by 16 S rRNA hybridization. The affinity constants for oxygen O₂ of the AOB and the NOB were 1.66 mg O₂ l^–1 and 3 mg O₂ l^–1 respectively. The larger than the previously reported values of these constants might be due to the high salt content in the medium. High O₂ concentrations did inhibit the nitrification rate.     

Modulation of dissolved oxygen levels in a hypertidal estuary by sediment resuspension

Hyperconcentrated benthic layers, which form during neap tides, recruit much of the fine sediment population of the turbidity maximum of a hypertidal estuary. Measurements of tidal amplitude and suspended solids concentration reveal that resuspension of the hyperconcentrated layers occurs between three and eight tides after neap tides rather than during spring tides (12 to 15 tides after neaps). During these resuspension events, dissolved oxygen levels are reduced but recover by spring tides. Peak solids concentrations and critically depressed dissolved oxygen levels are out of phase with tidal current amplitude. Thus observations close to neap and spring tides do not necessaraly capture the extremes of the envelope of water quality conditions.     

Formation of nitrifying biofilms on small suspended particles in airlift reactors

For a stable and reliable operation of a BAS-reactor a high, active biomass concentration is required with mainly biofilm-covered carriers. The effect of reactor conditions on the formation of nitrifying biofilms in BAS-reactors was investigated in this article. A start-up strategy to obtain predominantly biofilm-covered carriers, based on the balancing of detachment and a biomass production per carrier surface area, proved tp be very successful. The amount of biomass and the fraction of covered carrier were high and development of nitrification activity was fast, leading to a volumetric conversion of 5 kg(N) . m(-3) . d(-1) at a hydraulic retention time of 1h. A 1-week, continuous inoculation with suspended purely nitrifying microorganisms resulted in a swift start-up compared with batch addition of a small number of biofilms with some nitrification activity. The development of nitrifying biofilms was very similar to the formation of heterotrophic biofilms. In contrast to heterotrophic bio-films, the diameter of nitrifying biofilms increased during start-up. The detachment rate from nitrifying biofilms decreased with lower concentrations of bare carrier, in a fashion comparable with heterotrophic biofilms, but the nitrifying biofilms were much more robust and resistant. Standard diffusion theory combined with reaction kinetics are capable of predicting the activity and conversion of biofilms on small suspended particles. (c) 1995 John Wiley & Sons Inc.     

Abundance and community composition of ammonia-oxidizing archaea and bacteria in two different zones of Lake Taihu

The abundance and community composition of ammonia-oxidizing archaea and ammonia-oxidizing bacteria in the surface sediments of 2 different zones (Meiliang Bay and Eastern Lake Taihu) of Lake Taihu were investigated using real-time quantitative polymerase chain reaction and clone libraries. The amoA gene copy numbers in the surface sediment of Meiliang Bay ranged from 4.91?× 10(5) to 8.65?× 10(6) copies/g dry sediment for the archaeal amoA gene and from 3.74?× 10(4) to 3.86?× 10(5) copies/g dry sediment for the bacterial amoA gene, which were significantly higher than those of Eastern Lake Taihu (P?< 0.05). Concentrations of ammonia (NH(4)(+)), total nitrogen, organic matter, and pH of the sediments exhibited significantly negative correlations with the abundance of ammonia-oxidizing archaea or ammonia-oxidizing bacteria (P?< 0.05 or P?< 0.01, respectively). The potential nitrification rates show remarkable correlations with the copy numbers of the archaeal amoA gene. Diversity of the archaeal amoA gene in Eastern Lake Taihu was significantly higher than that of Meiliang Bay, whereas the bacterial amoA gene diversity was comparable for the 2 lake zones. The data obtained in this study would be useful to elucidate the role of ammonia-oxidizing archaea and ammonia-oxidizing bacteria in the nitrogen cycle of freshwater ecosystems.