Quantification of Nitrifying Bacterial Populations in a Full‐Scale Nitrifying Trickling Filter Using Fluorescent In Situ Hybridization

233

119

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH 4 ؉ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ؎ 5% of the influent N at the highest surface load of approximately 8.3 g of N m؊2 day ؊1 , with N 2 as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ؎ 7.6 and 76.5 ؎ 6.4 mg of NH 4 ؉ -N g of volatile suspended solids [VSS] ؊1 day ؊1 , respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS ؊1 day ؊1 ). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH 4 ؉ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.Sustainable wastewater treatment systems are being developed that minimize energy consumption, CO 2 emission, and sludge production. However, these systems typically yield effluents rich in ammonium-nitrogen (NH 4 ϩ -N) and poor in biodegradable organic carbon, thereby making them less suitable for biological N removal through the conventional nitrification-denitrification sequence.Different N removal processes that could be successfully integrated in a sustainable wastewater treatment system are being studied. The Sharon process (single-reactor high-activity ammonium removal over nitrite) (15) uses the principle that at higher temperatures (30 to 35°C), pH 7 to 8, and a cell residence time of 1 day, aerobic ammonia-oxidizing bacteria (AAOB) are able to maintain themselves in the system while nitrite-oxidizing bacteria (NOB) are washed out. Given the reaction stoichiometry of the two groups of nitrifying bacteria (equations 1 and 2), this proces...

Section: Methodsmentioning

confidence: 99%

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؊2 day ؊1 , with N 2 as the main end product.

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mentioning

confidence: 99%

See 1 more Smart Citation

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

Pynaert

Smets

Wyffels

et al. 2003

233

119

“…Sludge samples were fixed with fresh 4% paraformaldehyde solution, washed with phosphate-buffered saline, and stored in phosphate-buffered salineethanol (1:1) at Ϫ20°C until further processing (3). FISH assays were performed with fluorescently labeled, rRNA-targeted oligonucleotide probes by the method of Biesterfeld et al (6). The following oligonucleotide probes were used (Table 1): (i) NSO190, specific for the AOB (labeled with sulfoindocyanine dye Cy3) and (ii) a mix of EUB338, EUB338-II, and EUB338-III, specific for the domain Bacteria (labeled with fluoresceine).…”

Section: Scas Reactorsmentioning

confidence: 99%

Bioaugmentation as a Tool To Protect the Structure and Function of an Activated-Sludge Microbial Community against a 3-Chloroaniline Shock Load

Boon

Top

Verstraete

et al. 2003

230

142

Bioaugmentation of bioreactors focuses on the removal of xenobiotics, with little attention typically paid to the recovery of disrupted reactor functions such as ammonium-nitrogen removal. Chloroanilines are widely used in industry as a precursor to a variety of products and are occasionally released into wastewater streams. This work evaluated the effects on activated-sludge reactor functions of a 3-chloroaniline (3-CA) pulse and bioaugmentation by inoculation with the 3-CA-degrading strain Comamonas testosteroni I2 gfp. Changes in functions such as nitrification, carbon removal, and sludge compaction were studied in relation to the sludge community structure, in particular the nitrifying populations. Denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH) were used to characterize and enumerate the ammonia-oxidizing microbial community immediately after a 3-CA shock load. Two days after the 3-CA shock, ammonium accumulated, and the nitrification activity did not recover over a 12-day period in the nonbioaugmented reactors. In contrast, nitrification in the bioaugmented reactor started to recover on day 4. The DGGE patterns and the FISH and real-time PCR data showed that the ammonia-oxidizing microbial community of the bioaugmented reactor recovered in structure, activity, and abundance, while the number of ribosomes of the ammonia oxidizers in the nonbioaugmented reactor decreased drastically and the community composition changed and did not recover. The settleability of the activated sludge was negatively influenced by the 3-CA addition, with the sludge volume index increasing by a factor of 2.3. Two days after the 3-CA shock in the nonbioaugmented reactor, chemical oxygen demand (COD) removal efficiency decreased by 36% but recovered fully by day 4. In contrast, in the bioaugmented reactor, no decrease of the COD removal efficiency was observed. This study demonstrates that bioaugmentation of wastewater reactors to accelerate the degradation of toxic chlorinated organics such as 3-CA protected the nitrifying bacterial community, thereby allowing faster recovery from toxic shocks.The main purpose of a wastewater treatment plant is to convert the organics present in an incoming wastewater stream to N 2 and CO 2 as well as to reduce the amount of suspended solids entering the environment. The biological treatment of industrial wastewaters by mixed microbial communities is, however, often disrupted by organic (e.g., chlorinated organics, phenolic compounds, surfactants, and herbicides) and inorganic (e.g., heavy metals, sulfides, and ammonia) chemicals present in the wastewater stream (7,52). This disruption of biological processes results in inhibited nitrification, decreased carbon removal, and modification of sludge compaction properties (7,29). Little is known about the composition of mixed microbial communities in reactors when biological processes are disrupted by or recovering from xenobiotic shocks. Most investigators focus on xenobiotic degradation, bu...

“…Fluorescence in situ hybridization (FISH) represents the "gold standard" for quantification of specific bacterial cells in the environment, against which other methods should be compared. Classical (27) and immunological (20) methods are subject to methodological biases, while nonmicroscopic 16S rRNA-based methods (8,34) or PCR-based methods (13,14,18,19) deliver a proportion of total cell counts, copy number, or relative signal intensities rather than an absolute number of cells or biomass.…”

mentioning

confidence: 99%

Agreement between Theory and Measurement in Quantification of Ammonia-Oxidizing Bacteria

Coşkuner

Ballinger

Davenport

et al. 2005

Autotrophic ammonia-oxidizing bacteria (AOB) are of vital importance to wastewater treatment plants (WWTP), as well as being an intriguing group of microorganisms in their own right. To date, corroboration of quantitative measurements of AOB by fluorescence in situ hybridization (FISH) has relied on assessment of the ammonia oxidation rate per cell, relative to published values for cultured AOB. Validation of cell counts on the basis of substrate transformation rates is problematic, however, because published cell-specific ammonia oxidation rates vary by over two orders of magnitude. We present a method that uses FISH in conjunction with confocal scanning laser microscopy to quantify AOB in WWTP, where AOB are typically observed as microcolonies. The method is comparatively simple, requiring neither detailed cell counts or image analysis, and yet it can give estimates of either cell numbers or biomass. Microcolony volume and diameter were found to have a log-normal distribution. We were able to show that virtually all (>96%) of the AOB biomass occurred as microcolonies. Counts of microcolony abundance and measurement of their diameter coupled with a calibration of microcolony dimensions against cell numbers or AOB biomass were used to determine AOB cell numbers and biomass in WWTP. Cell-specific ammonia oxidation rates varied between plants by over three orders of magnitude, suggesting that cell-specific ammonia oxidation is an important process variable. Moreover, when measured AOB biomass was compared with process-based estimates of AOB biomass, the two values were in agreement.The quantification of microbial communities and populations is an invaluable aspect of microbial ecology. In principle, the autotrophic ammonia-oxidizing bacteria (AOB) are ideal candidates for the development of quantitative tools. AOB have a coherent phylogeny and defined nutritional requirements and are of profound practical importance in natural and engineered environments.The number of individuals should be the ideal benchmark for quantitative studies. Individual counts can be converted to biomass, biovolume, or proportion of biomass, and results obtained by more indirect methods are typically compared to the number of cells per unit volume (15). Fluorescence in situ hybridization (FISH) represents the "gold standard" for quantification of specific bacterial cells in the environment, against which other methods should be compared. Classical (27) and immunological (20) methods are subject to methodological biases, while nonmicroscopic 16S rRNA-based methods (8, 34) or PCR-based methods (13,14,18,19) deliver a proportion of total cell counts, copy number, or relative signal intensities rather than an absolute number of cells or biomass.A quantitative method may be evaluated with respect to its precision and its accuracy. Wagner et al. (43) originally evaluated the accuracy of FISH counts of AOB by using cell specific oxidation rates, an approach previously used to show that most-probable-number-based methods underestimate AOB numbers...