Analysis and succession of nitrifying bacteria community structure in sequencing biofilm batch reactor

To reveal the succession procedure of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) community structure in sequencing biofilm batch reactor (SBBR), the molecular biological techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and real-time PCR were applied. DGGE showed that the structural diversity of the bacterial community increased during the biofilm formation period, and some kinds of populations had been highly preponderant consistently. The results of cloning and sequencing revealed that Nitrosomonas was the dominant species. The real-time PCR analysis indicated that the amount of the AOB increased significantly after the cultivation period, and the NOB gradually decreased. The AOB content on the 25th day was 17 times that of the 6th day. It also showed the biofilm formed successfully with accumulating nitrite and prepared to achieve the achievement of simultaneous nitrification and denitrification in SBBR. Furthermore, the ammonia-oxidizing rate was in correspondence with the NH₄ ⁺-N removal efficiency.     

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Population dynamics of nitrifying bacteria in an aquatic ecosystem.

In a space environment such as Space Shuttle or Space Station, animal experiments with aquatic species in a closed system pose a crucial problem in maintaining their water quality for a long term. In nature, ammonia as an animal wastes is converted by nitrifying bacteria to nitrite or nitrate compounds, which usually become nitrogen sources for plants. Thus an application of the biological reactor with such bacteria attached on some filters has been suggested and experimentally studied for efficient waste managements of ammonia. Although some successful results were reported (Kozu et al. 1995, Nagaoka et al. 1998, Nakamura et al. 1997, 1998) in the space applications, purely empirical approaches have so far been taken to develop a biological filter having a stable nitrifying activity. In this study, we constructed a mathematical model to deal with the dynamics of the ammonia nitrifying processes in a biological reactor. The model describes population dynamics of the ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria cultivated on the same filter. We estimated parameters involved in the model using the experimental data. The result shows that these estimated parameters could be applied to general cases and that the two bacteria are in a symbiotic relationship; they can better perform when both coexist, as has been empirically recognized. Based on the model analysis, we discuss how to prepare a high performance biological filter.     

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [(14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [(14)C]NAG-consuming bacteria but did not utilize [(14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms

The cross-feeding of microbial products derived from 14C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14C-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

Characterization of nitrifying bacteria immobilized in calcium alginate

Activated sludge has been fed with a medium containing ammonium ions as the sole nitrogen source. Biomass collected from this continuous culture was immobilized in calcium alginate. The influence of pH, temperature, and the size and cell load of the biocatalyst beads on the nitrifying activity was determined, as well as the storage and operational stability of the system. The results are compared with those obtained with Nitrosomonas europaea. It has been concluded that the mixed culture is more difficult to work with than the pure strain and that the reproducibility of the results is lower. The trends found, however, were largely similar, except for the operational stability which was poorer in the case of the immobilized mixed culture.     

The survival of nitrifying bacteria in the soil

Summary Nitrification as measured by the survival of nitrifying bacteria, was studied in 25 different soils from Argentine Patagonia, maintained in sterilized glass flasks in darkness for 5 years.Survival of nitrifying bacteria was related to environmental factors and to the level of mineral elements in the soils.The temperatures to which soils were submitted and the joint action of Ca and K in adequate quantities, were shown to be more important factors governing the survival of the nitrifying bacteria.P, Mg, and Cu did not influence in the initial number nor the survival of nitrifying bacteria nor did the content or organic matter and organic N and pH of the soil samples.     

Free nitrous acid and pH determine the predominant ammonia-oxidizing bacteria and amount of N2O in a partial nitrifying reactor

Applied Microbiology and Biotechnology - We investigated the effects of free ammonia (FA) and free nitrous acid (FNA) concentrations on the predominant ammonia-oxidizing bacteria (AOB) and the...     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

Identification of bacteria coexisting with anammox bacteria in an upflow column type reactor

Anammox process has attracted considerable attention in the recent years as an alternative to conventional nitrogen removal technologies. In this study, a column type reactor using a novel net type acrylic fiber (Biofix) support material was used for anammox treatment. The Biofix reactor was operated at a temperature of 25°C (peak summer temperature, 31.5°C). During more than 340 days of operation for synthetic wastewater treatment, the nitrogen loading rates of the reactor were increased to 3.6 kg-N/m³/d with TN removal efficiencies reaching 81.3%. When the reactor was used for raw anaerobic sludge digester liquor treatment, an average TN removal efficiency of 72% was obtained with highest removal efficiency of 81.6% at a nitrogen loading rate of 2.2 kg-N/m³/d. Results of extracellular polymeric substances (EPS) quantification revealed that protein was the most abundant component in the granular sludge and was found to be almost twice than that in the sludge attached to the biomass carriers. The anammox granules in the Biofix reactor illustrated a dense morphology substantiated by scanning electron microscopy and EPS results. The results of DNA analyses indicated that the anammox strain KSU-1 might prefer relatively low nutrient levels, while the anammox strain KU2 strain might be better suited at high nutrient concentration. Other types of bacteria were also identified with the potential of consuming dissolved oxygen in the influent and facilitating survival of anammox bacteria under aerobic conditions.     

Steady state and transient growth of autotrophic nitrifying bacteria

Growth of the autotrophic nitrifying bacteria Nitrosomonas europaea and Nitrobacter sp. was studied in continuous culture. Steady state growth kinetics of both organisms conformed with that predicted by chemostat theory, modified to account for maintenance energy requirement. Steady state data were used to calculate the maximum specific growth rate, the saturation constant for growth, the true growth yield and the maintenance coefficient. Transient growth was studied by imposing step changes in dilution rate. Step increases resulted in overshoots and oscillations in substrate concentration before establishment of a new steady state while step decreases in dilution rate were followed by monotonic changes in substrate concentration. The size of overshoots in substrate concentration following step increases in dilution rate was dependent on both the magnitude of the increase and of the dilution rate prior to the change.     

Formation and long-term stability of nitrifying granules in a sequencing batch reactor

The formation and long-term stability of nitrifying granules in a sequencing batch reactor was investigated in this study. The results showed that nitrifying granules with a size of 240 microm and SVI of 40 ml g(-1) were formed on day 21 at a settling time of 10 min. Maintaining settling time at 15 min from day 57 to 183 did not affect the physical characteristics of sludge and the fraction of suspended floc in the sludge. In addition, nitrifying granules could tolerate the fluctuations of nitrogen loading rate from 0.72 to 1.8 g l(-1)d(-1) during 2 months without the change of physical characteristics. However, it was observed that complete nitrification to nitrate and partial nitrification to nitrite by sludge converted each other corresponding to the change of the influent NH4+-N concentration. Thus, an appropriate method is needed to maintain a stable complete nitrification or partial nitrification under the conditions with changing influent NH4+-N concentrations and nitrogen loading rates.     

Settleability and kinetics of a nitrifying sludge in a sequencing batch reactor

A physiological study of a nitrifying sludge was carried out in a sequencing batch reactor (SBR). Pseudo steady-state nitrification conditions were obtained with an ammonium removal efficiency of 99% +/- 1% and 98% +/- 2% conversion of NH4+-N to NO3 - -N. The rate of biomass production was negligible (1.3 +/- 0.1 mg microbial protein-N.L(-1).d(-1)). The sludge presented good settling properties with sludge volume index values lower than 20 mL.g(-1) and an exopolymeric protein/carbohydrate ratio of 0.53 +/- 0.34. Kinetic results indicated that the nitrifying behavior of the sludge changed with the number of cycles. After 22 cycles, a decrease in the specific rate of NO3- -N production coupled with an increase in the NO2- -N accumulation were observed. These results showed that the activity of the nitrite oxidizing bacteria decreased at a longer operation time. Ammonia oxidizing bacteria were found to exhibit the best stability. After 4 months of operation, the specific rates of NH4+-N consumption and NO3- -N production were 1.72 NH4+-N per microbial protein-N per hour (g.g(-1).h(-1)) and 0.54 NO3- -N per microbial protein-N per hour (g.g(-1).h(-1)), respectively.     

硝化细菌群的富集及其去除城镇寡营养河道中氨氮污染的应用

[背景]随着工农业的发展,污水排放导致的氨氮超标逐渐成为水体污染的重要因素,脱氮已成为人们研究的重点.目前脱氮方法主要集中于硝化细菌的硝化作用,其将氨氮转化为硝酸盐氮,从而减少水体中氨氮的污染.由于工业废水和农业污水中的有机物含量较高,而且异养硝化细菌具有生长较快等优势,因此对异养菌的研究多于自养菌.然而现有的异养硝化...