Ammonia-oxidizing Bacteria 16S rRNA Research Articles

Analysis of Unamplified 16S rRNA of Ammonia-Oxidizing Bacteria in Activated Sludge by Spectrophotometry Using Gold Nanoprobes

Analysis of Unamplified 16S rRNA of Ammonia-Oxidizing Bacteria in Activated Sludge by Spectrophotometry Using Gold Nanoprobes

Methane-generating ammonia oxidizing nitrifiers within bio-filters in aquaculture tanks

The discovery of aerobic and anammox bacteria capable of generating methane in bio-filters in freshwater aquaculture systems is generating interest in studies to understand the activity, diversity, distribution and roles of these environmental bacteria. In this study, we used microbial enrichment of bio-filters to assess their effect on water quality. Profiles of ammonia-oxidizing bacterial communities generated using nested PCR methods and DGGE were used to assess the expression of 16S rRNA genes using DNA sequencing. Five dominant ammonia-oxidizing bacterial strains–clones; KB.13, KB.15, KB.16, KB.17 and KB.18—were isolated and identified by phylogenetic analysis as environmental samples closely related to genera Methylobacillus, Stanieria, Nitrosomonas, and Heliorestis. The methyl ammonia-oxidizing microbes thereby found suggest a biochemical pathway involving electron donors and carbon sources, and all strains were functional in freshwater aquaculture systems. Environmental parameters including TN (2.69–20.43); COD (9.34–31.47); NH4+-N (0.44–11.78); NO2−N (0.00–3.67); NO3−N (0.05–1.82), mg/L and DO (1.47–10.31 µg/L) assessed varied in the ranges in the different tanks. Principal component analysis revealed that these water quality parameters significantly influenced the ammonia oxidizing microbial community composition. Temperature rises to about 40 °C significantly affected environmental characteristics—especially DO, TN and NH4+-N—and directly or indirectly affected the microbial communities. Although the nested PCR design was preferred due to its high sensitivity for amplifying specific DNA regions, a more concise method is recommended, as an equimolar mixture of degenerate PCR primer pairs, CTO189f-GC and CTO654r, never amplified only 16S rRNA of ammonia-oxidizing bacteria.

Nitrifier Gene Abundance and Diversity in Sediments Impacted by Acid Mine Drainage.

Extremely acidic and metal-rich acid mine drainage (AMD) waters can have severe toxicological effects on aquatic ecosystems. AMD has been shown to completely halt nitrification, which plays an important role in transferring nitrogen to higher organisms and in mitigating nitrogen pollution. We evaluated the gene abundance and diversity of nitrifying microbes in AMD-impacted sediments: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB). Samples were collected from the Iron Springs Mining District (Ophir, CO, United States) during early and late summer in 2013 and 2014. Many of the sites were characterized by low pH (<5) and high metal concentrations. Sequence analyses revealed AOA genes related to Nitrososphaera, Nitrosotalea, and Nitrosoarchaeum; AOB genes related to Nitrosomonas and Nitrosospira; and NOB genes related to Nitrospira. The overall abundance of AOA, AOB and NOB was examined using quantitative PCR (qPCR) amplification of the amoA and nxrB functional genes and 16S rRNA genes. Gene copy numbers ranged from 3.2 × 104 – 4.9 × 107 archaeal amoA copies ∗ μg DNA-1, 1.5 × 103 – 5.3 × 105 AOB 16S rRNA copies ∗ μg DNA-1, and 1.3 × 106 – 7.7 × 107 Nitrospira nxrB copies ∗ μg DNA-1. Overall, Nitrospira nxrB genes were found to be more abundant than AOB 16S rRNA and archaeal amoA genes in most of the sample sites across 2013 and 2014. AOB 16S rRNA and Nitrospira nxrB genes were quantified in sediments with pH as low as 3.2, and AOA amoA genes were quantified in sediments as low as 3.5. Though pH varied across all sites (pH 3.2–8.3), pH was not strongly correlated to the overall community structure or relative abundance of individual OTUs for any gene (based on CCA and Spearman correlations). pH was positivity correlated to the total abundance (qPCR) of AOB 16S rRNA genes, but not for any other genes. Metals were not correlated to the overall nitrifier community composition or abundance, but were correlated to the relative abundances of several individual OTUs. These findings extend our understanding of the distribution of nitrifying microbes in AMD-impacted systems and provide a platform for further research.

Microbial Diversity of Ammonia Oxidizing and Other Bacteria of Activated Sludge

Microbial diversity and large quantity of ammonia-oxidizing bacteria (AOB) in activated sludge were compared using PCR-DGGE and real-time PCR assays. Activated sludge samples were collected from different types of wastewater treatment plants (WWTPs). The composition of total bacteria determined by PCR-DGGE was highly diverse between the samples, whereas the community of AOB was similar across all the investigated activated sludge. Total bacterial numbers and AOB numbers in the aerated mixed liquor were in the range of 1.2×10<SUP>9</SUP> to 2.8×10<SUP>12</SUP> and 1.9×10<SUP>7</SUP> to 2.7×10<SUP>10</SUP> copies/l, respectively. Activated sludge from livestock, textile, and sewage treating WWTPs contained relatively high amoA gene copies (more than 10<SUP>5</SUP> copies/l), whereas activated sludge from food and paper WWTPs revealed a low number of the amoA gene (less than 10<SUP>3</SUP> copies/l). The value of the amoA gene copy effectively showed the difference in composition of bacteria in different activated sludge samples and this was better than the measurement with the AOB 16S rRNA or total 16S rRNA gene. These results suggest that the quantification of the amoA gene can help monitor AOB and ammonia oxidation in WWTPs.

Spatial and temporal diversity and abundance of ammonia oxidizers in semi-arid and arid soils: indications for a differential seasonal effect on archaeal and bacterial ammonia oxidizers

Besides water, nitrogen is the limiting factor for biomass production in arid ecosystems. Global climatic changes are exacerbating aridity levels, and the response of nitrogen-transforming microorganisms to these changes is not clear yet. Using semi-arid and arid ecosystems as surrogates for conditions of increased aridity, we investigated the activity, abundance, and diversity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in arid and semi-arid soils. Ammonia oxidation potentials were higher during the winter in both sites than in the summer, and higher nitrate concentrations were measured in the arid soil than in the semi-arid soil. Denaturing gradient gel electrophoresis (DGGE) patterns of AOB 16S rRNA gene fragments were similar for the arid and semi-arid soils with no seasonal variations. In contrast, the DGGE patterns of the AOA amoA gene fragments differed between the sites and a soil transfer experiment suggested that these differences are possibly associated with soil type. AOB numbers were higher during the winter than in the summer, while AOA numbers were higher during the summer. The results indicate the resistance of AOB and AOA community structure to arid conditions, albeit with seasonal variations in their abundance. Together, the results suggest the resilience of nitrification activity to increased aridity level.

Nitrogen Transformations and Diversity of Ammonia-Oxidizing Bacteria in a Desert Ephemeral Stream Receiving Untreated Wastewater

Levels of inorganic nitrogen species (ammonia, nitrite, and nitrate), ammonia oxidation potential (AOP), and diversity of ammonia-oxidizing bacteria (AOB) were studied in the sediments of a 50-km-long segment of an ephemeral stream in the Negev desert, receiving untreated wastewater. Water analysis in downstream sampling points showed reductions of 91.7% in biological oxygen demand, 87.7% in chemical oxygen demand, 73.9% in total nitrogen, and 72.8% in total ammonia nitrogen. Significant AOP levels in the sediment were detected mainly in the fall and spring seasons. Denaturing gradient gel electrophoresis of AOB 16S rRNA gene fragments showed that in most sampling points, the streambed was dominated by Nitrosospira cluster 3 strains similar to those dominating the stream bank's soils and sediments in nearby springs. Nitrosomonas strains introduced by discharged wastewater and others dominated some sections of the stream characterized by high organic carbon levels. The results suggest that climatic conditions in the Negev desert select for AOB belonging to Nitrosospira cluster 3, and these conditions dominate the aquatic environment effect along most of the stream sections. In addition, the nitrification-denitrification processes were not sufficient to reduce nitrogen levels in the sediment and prevent the eutrophication of some sections of the stream ecosystem. Thus, the discharge of high nitrogen wastewater into desert streams should be done carefully as it may endanger the already fragile ecosystem.

Detection and quantification of expression of amoA by competitive reverse transcription-PCR

Ammonia oxidation by chemolithoautotrophic ammonia-oxidizing bacteria is an important step in the biological nitrogen removal process. The first conversion step, the oxidation of ammonia to hydroxylamine is catalyzed by ammonia monooxygenase (AMO). To investigate the activity of ammonia oxidation, mRNA (designated as amoA) encoding a subunit of AMO was quantified by competitive reverse transcription (RT)-PCR. As a result, it was possible to detect and quantify amoA expression in cultured Nitrosomonas europaea and even complex microbial communities such as nitrifying bacterial aggregates by competitive RT-PCR. It was estimated that amoA concentration in cultured N. europaea was 2.3 x 10(8) copies x ml(-1). Additionally, it was calculated that the copy number of amoA in nitrifying bacterial aggregates was 1.0 x 10(12) copies x ml(-1) (5.1 x 10(10) copies x mg(-1)-dry weight). On the other hand, amoA expression in the natural activated sludge in a household Gappei-Johkaso was undetectable, whereas 16S rRNA of ammonia-oxidizing bacteria was detected by RT-PCR. Then, four days cultivation of this sludge in inorganic artificial wastewater resulted in increasing amoA expression to a quantifiable amount by competitive RT-PCR. In conclusion, the competitive RT-PCR was effective to investigate the expression of amoA as an indicator of ammonia oxidation activity by autotrophic ammonia-oxidizing bacteria.