Characterization of the Microbial Community and Culturable Denitrifying Bacteria in a Solidphase Denitrification Process Using Poly(ε-caprolactone) as the Carbon and Energy Source (original) (raw)
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Microbes and Environments, 2007
Two laboratory-scale solid-phase denitrification (SPD) reactors, designated reactors A and B, for nitrogen removal were constructed by acclimating activated sludge with pellets and flakes of poly(3-hydoxybutyrate-co-3hydroxyvalerate) (PHBV) as the sole added substrate under denitrifying conditions, respectively. The average denitrification rate in both reactors was 60 mg NO3 − -N g −1 (dry wt) h −1 under steady-state conditions, whereas washed sludge taken from the reactors showed an average denitrification rate of 20 mg NO3-N g −1 (dry wt) h −1 with fresh PHBV as the sole substrate. The difference in the denitrification rate between the two might be due to the bioavailability of intermediate metabolites as the substrate for denitrification, because acetate and 3-hydroxybutyrate were detected in the reactors. Most of the predominant denitrifiers isolated quantitatively by the platecounting method using non-selective agar medium were unable to degrade PHBV and were identified as members of genera of the class Betaproteobacteria by studying 16S rRNA gene sequence information. nirS and nosZ gene clone library-based analyses of the microbial community from SPD reactor A showed that most of the nirS and nosZ clones proved to be derived from members of the family Comamonadaceae and other phylogenetic groups of the Betaproteobacteria. These results suggest that the efficiency of denitrification in the PHBV-SPD process is affected by the availability of intermediate metabolites as possible reducing-power sources as well as of the solid substrate, and that particular species of the Betaproteobacteria play the primary role in denitrification in this process.
Laboratory-scale solid-phase denitrification (SPD) reactors for nitrate removal were constructed by acclimating activated sludge with poly (L-lactic acid) (PLLA) having weight-average molecular weights (Mw) of 9,900, 12,000, and 45,100 g mol −1. A good nitrate removal rate (3.5–5.3 mg NO3 −-N g [dry wt] −1 h −1) was found in the reactor containing PLLA of 9,900 g mol −1 , whereas the other two reactors with the higher Mw PLLA showed low nitrate removal efficiency. Microbial community dynamics in the low Mw PLLA-acclimated reactor were studied by 16S rRNA gene-targeted PCR-denaturing gradient gel electrophoresis and quinone profiling. Nonmetric multidimensional scaling analyses of these data sets revealed a marked population shift during acclimation of the SPD reactor with low Mw PLLA. The 16S rRNA gene clone library and culture-dependent analyses showed that bacteria belonging to the family Comamonadaceae predominated and played the primary role in denitrification in the PLLA-using reactor; however, none of the bacterial isolates from the reactor degraded PLLA. These results suggest that the nitrate removal property of the PLLA-using SPD reactor is attained through the bioavailability of hydrolysates released abiotically from the solid substrate.
Functional characterization of heterotrophic denitrifying bacteria in activated sludge
South African Journal of Science, 2007
ASTEWATER TREATMENT FACILITIES are largely accountable for the detrimental enrichment of water bodies with nitrogenous compounds, resulting in eutrophication. Denitrification is of interest as a means of removing nitrates and nitrites from water supplies because they are also hazardous to human health. The integration of biological nutrient removal into conventional wastewater treatment processes has, however, failed to take into sufficient consideration the role of key microorganisms present, specifically denitrifying bacteria. The purpose of the study reported here was to group such heterotrophic denitrifying bacteria using a series of biochemical and molecular analyses, to achieve an improved understanding of their functions. The role of the denitrifying bacteria in reducing nitrate and nitrite was monitored using the colorimetric nitrate reduction test. The genetic diversity of the culture collection was investigated by the use of denaturing gradient gel electrophoresis (DGGE), which enabled the creation of a microbial population profile of eight predominant isolates. Batch experiments were conducted on these isolates, the results of which ultimately confirmed their classification according to their respective functions, namely, incomplete denitrifiers, true denitrifiers, sequential denitrifiers, and exclusive nitrite reducers.
FEMS Microbiology Ecology, 2008
The denitrification capacity of different phylogenetic bacterial groups was investigated on addition of different substrates in activated sludge from two nutrientremoval plants. Nitrate/nitrite consumption rates (CRs) were calculated from nitrate and nitrite biosensor, in situ measurements. The nitrate/nitrite CRs depended on the substrate added, and acetate alone or combined with other substrates yielded the highest rates (3-6 mg N gVSS À1 h À1). The nitrate CRs were similar to the nitrite CRs for most substrates tested. The structure of the active denitrifying population was investigated using heterotrophic CO 2 microautoradiography (HetCO 2-MAR) and FISH. Probe-defined denitrifiers appeared as specialized substrate utilizers despite acetate being preferentially used by most of them. Azoarcus and Accumulibacter abundance in the two different sludges was related to differences in their substrate-specific nitrate/nitrite CRs. Aquaspirillumrelated bacteria were the most abundant potential denitrifiers (c. 20% of biovolume); however, Accumulibacter (3-7%) and Azoarcus (2-13%) may have primarily driven denitrification by utilizing pyruvate, ethanol, and acetate. Activated sludge denitrification was potentially conducted by a diverse, versatile population including not only Betaproteobacteria (Aquaspirillum, Thauera, Accumulibacter, and Azoarcus) but also some Alphaproteobacteria and Gammaproteobacteria, as indicated by the assimilation of 14 CO 2 by these probe-defined groups with a complex substrate mixture as an electron donor and nitrite as an electron acceptor in HetCO 2-MAR-FISH tests.
Microbiology Research Journal International, 2024
Denitrification is a crucial microbial process in the nitrogen cycle, transforming nitrate (NO₃⁻) into nitrogen gas (N₂), thereby mitigating nitrogen pollution in aquatic ecosystems. This microbial activity plays a vital role in wastewater treatment by removing excess nitrogen, which contributes to eutrophication and water contamination. The denitrification process involves various microbial communities, including bacteria such as Pseudomonas, Paracoccus, and Bacillus, which operate under anoxic conditions to achieve nitrogen reduction, an optimizing denitrification in wastewater treatment presents several challenges, such as maintaining ideal environmental conditions (e.g., carbon availability, oxygen levels, pH) and overcoming issues related to incomplete denitrification, which can lead to the production of harmful intermediates like nitrous oxide (N₂O). Despite these hurdles, recent advancements in microbial ecology, such as the use of biofilms, bioreactors, and genetic engineering, offer promising opportunities to enhance denitrification efficiency. This review explores the microbial ecology of the denitrification process, its application in wastewater treatment, and the challenges and opportunities associated with its practical implementation in reducing nitrogen pollution.
Heterotrophic denitrification by Gram-positive bacteria: Bacillus cereus and Bacillus tequilensis
Two bacteria were isolated from anoxic denitrifying reactor for treatment of domestic wastewater. The analysis of the 16S rDNA gene sequences showed that the isolated strains were affiliated with Bacillus cereus and Bacillus tequilensis. Denitrification was compared between Bacillus cereus and Bacillus tequilensis in this study. Two bacilli were able to denitrify and Bacillus cereus was more efficient than Bacillus tequilensis. Bacillus cereus reduced 80% of high amount of nitrate; however, Bacillus tequilensis could reduce 37.4% of nitrate. These heterotrophic bacteria are able to eliminate organic matter with the same trend reducing 74.5% for Bacillus tequilensis and 70.2% for Bacillus cereus.
Cultivation of denitrifying bacteria: Optimization of isolation conditions and diversity study
2006
An evolutionary algorithm was applied to study the complex interactions between medium parameters and their effects on the isolation of denitrifying bacteria, both in number and in diversity. Growth media with a pH of 7 and a nitrogen concentration of 3 mM, supplemented with 1 ml of vitamin solution but not with sodium chloride or riboflavin, were the most successful for the isolation of denitrifiers from activated sludge. The use of ethanol or succinate as a carbon source and a molar C/N ratio of 2.5, 20, or 25 were also favorable. After testing of 60 different medium parameter combinations and comparison with each other as well as with the standard medium Trypticase soy agar supplemented with nitrate, three growth media were highly suitable for the cultivation of denitrifying bacteria. All evaluated isolation conditions were used to study the cultivable denitrifier diversity of activated sludge from a municipal wastewater treatment plant. One hundred ninety-nine denitrifiers were isolated, the majority of which belonged to the Betaproteobacteria (50.4%) and the Alphaproteobacteria (36.8%). Representatives of Gammaproteobacteria (5.6%), Epsilonproteobacteria (2%), and Firmicutes (4%) and one isolate of the Bacteroidetes were also found. This study revealed a much more diverse denitrifying community than that previously described in cultivation-dependent research on activated sludge.
Fems Microbiology Ecology, 2007
The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20–49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.
Assessment of denitrifying bacterial composition in activated sludge
The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10 4 – 10 5 , nosZ with 10 4 –10 6 and 16S rRNA gene in the range 10 9 –10 10 copy number per ml of sludge. The cul-turable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudo-monas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat.