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

ABSTRACT: 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.

SUBMITTER: Kamira B

PROVIDER: S-EPMC6113197 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The potential for oxidation of ammonia in anoxic marine sediments exists through anaerobic oxidation by Nitrosomonas-like organisms, utilizing nitrogen dioxide, coupling of nitrification, manganese reduction, and anaerobic oxidation of ammonium by planctomycetes (the Anammox process). Here we describe the presence of microbial communities with the potential to carry out these processes in a natural marine sediment system (Loch Duich, Scotland). Natural microbial communities of Planctomycetales-Verrucomicrobia and beta- and gamma-proteobacterial ammonia-oxidizing bacteria were characterized by analysis of 16S rRNA genes amplified using group-specific primers by PCR- and reverse transcription-PCR amplification of 16S rDNA and RNA, respectively. Amplification products were analyzed by sequencing of clones and by denaturant gradient gel electrophoresis (DGGE). Amplification of primers specific for Planctomycetales-Verrucomicrobia and beta-proteobacterial ammonia-oxidizing bacteria generated products at all sampling sites and depths, but no product was generated using primers specific for gamma-proteobacterial ammonia-oxidizing bacteria. 16S rDNA DGGE banding patterns indicated complex communities of beta-proteobacterial ammonia-oxidizing bacteria in anoxic marine sediments. Phylogenetic analysis of sequences from clones and those excised from DGGE gels suggests dominance of Nitrosospira cluster 1-like organisms and of strains belonging to a novel cluster represented in dominant bands in 16S rRNA DGGE banding patterns. Their presence indicates a group of organisms closely related to recognized beta-proteobacterial ammonia-oxidizing bacteria that may be selected in anoxic environments and may be capable of anoxic ammonia oxidation. Sequence analysis of planctomycete clone libraries and sequences excised from DGGE gels also demonstrated a diverse microbial community and suggested the presence of new subdivisions, but no sequence related to recognized Anammox organisms was detected.

Project description:Ammonia oxidation is the rate-limiting first step of nitrification and a key process in the nitrogen cycle that results in the formation of nitrite (NO2 -), which can be further oxidized to nitrate (NO3 -). In the Amazonian floodplains, soils are subjected to extended seasons of flooding during the rainy season, in which they can become anoxic and produce a significant amount of methane (CH4). Various microorganisms in this anoxic environment can couple the reduction of different ions, such as NO2 - and NO3 -, with the oxidation of CH4 for energy production and effectively link the carbon and nitrogen cycle. Here, we addressed the composition of ammonium (NH4 +) and NO3 --and NO2 --dependent CH4-oxidizing microbial communities in an Amazonian floodplain. In addition, we analyzed the influence of environmental and geochemical factors on these microbial communities. Soil samples were collected from different layers of forest and agroforest land-use systems during the flood and non-flood seasons in the floodplain of the Tocantins River, and next-generation sequencing of archaeal and bacterial 16S rRNA amplicons was performed, coupled with chemical characterization of the soils. We found that ammonia-oxidizing archaea (AOA) were more abundant than ammonia-oxidizing bacteria (AOB) during both flood and non-flood seasons. Nitrogen-dependent anaerobic methane oxidizers (N-DAMO) from both the archaeal and bacterial domains were also found in both seasons, with higher abundance in the flood season. The different seasons, land uses, and depths analyzed had a significant influence on the soil chemical factors and also affected the abundance and composition of AOA, AOB, and N-DAMO. During the flood season, there was a significant correlation between ammonia oxidizers and N-DAMO, indicating the possible role of these oxidizers in providing oxidized nitrogen species for methanotrophy under anaerobic conditions, which is essential for nitrogen removal in these soils.

Project description:Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r was the most suitable primer set for multiple molecular assessments of anammox bacteria in freshwater environments.

Project description:Background:Mono-specific aquaculture effluents contain high concentrations of nutrients and organic matter, which affect negatively the water quality of the recipient ecosystems. A fundamental feature of water quality is its transparency. The fraction of dissolved organic matter that absorbs light is named chromophoric dissolved organic matter (CDOM). A sustainable alternative to mono-specific aquaculture is the multitrophic aquaculture that includes species trophically complementary named "extractive" species that uptake the waste byproducts. Sea cucumbers are recognized as efficient extractive species due to the consumption of particulate organic matter (POM). However, the effects of sea cucumbers on CDOM are still unknown. Methods:During more than one year, we monitored CDOM in two big-volume tanks with different trophic structure. One of the tanks (-holothurian) only contained around 810 individuals of Anemonia sulcata, whereas the other tank (+holothurian) also included 90 individuals of Holothuria tubulosa and Holothuria forskali. We routinely analyzed CDOM absorption spectra and determined quantitative (absorption coefficients at 325 nm) and qualitative (spectral slopes) optical parameters in the inlet waters, within the tanks, and in their corresponding effluents. To confirm the time-series results, we also performed three experiments. Each experiment consisted of two treatments: +holothurians (+H) and -holothurians (-H). We set up three +H tanks with 80 individuals of A. sulcata and 10 individuals of H. tubulosa in each tank and four -H tanks that contained only 80 individuals of A. sulcata. Results:In the time-series, absorption coefficients at 325 nm (a325) and spectral slopes from 275 to 295 nm (S275-295) were significantly lower in the effluent of the +holothurian tank (average: 0.33 m-1 and 16 µm-1, respectively) than in the effluent of the -holothurian tank (average: 0.69 m-1 and 34 µm-1, respectively), the former being similar to those found in the inlet waters (average: 0.32 m-1 and 22 µm-1, respectively). This reduction in the absorption of the dissolved organic matter appears to be mediated by the POM consumption by holothurians. The experiments confirmed the results observed in the time-series. The a325 and S275-295 values were significantly lower in the treatment with holothurians than in the treatment without holothurians indicating a reduction in the concentration of chromophoric organic compounds, particularly of low molecular weight. Discussion:Consequently, sea cucumbers appear to improve water transparency in aquaculture tanks. The underlying mechanism of this improvement might be related to the POM consumption by holothurians, which reduces the concentration of CDOM derived from POM disaggregation or to the direct assimilation of dissolved compounds of low molecular weight as chromophoric amino acids.

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

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