Methanol-fed Denitrification Research Articles

Dynamics of a methanol-fed marine denitrifying biofilm: 1-Impact of environmental changes on the denitrification and the co-occurrence of Methylophaga nitratireducenticrescens and Hyphomicrobium nitrativorans.

BackgroundThe biofilm of a methanol-fed denitrification system that treated a marine effluent is composed of multi-species microorganisms, among which Hyphomicrobium nitrativorans strain NL23 and Methylophaga nitratireducenticrescens strain JAM1 are the principal bacteria involved in the denitrifying activities. Here, we report the capacity of the denitrifying biofilm to sustain environmental changes, and the impact of these changes on the co-occurrence of H. nitrativorans and M. nitratireducenticrescens.MethodsIn a first set of assays, the original biofilm (OB) was cultivated in an artificial seawater (ASW) medium under anoxic conditions to colonize new carriers. The new formed biofilm was then subjected to short exposures (1–5 days) of a range of NaCl, methanol, nitrate (NO3−) and nitrite (NO2−) concentrations, and to different pHs and temperatures. In a second set of assays, the OB was cultivated in ASW medium for five weeks with (i) a range of NaCl concentrations, (ii) four combinations of NO3−/methanol concentrations and temperatures, (iii) NO2−, and (iv) under oxic conditions. Finally, the OB was cultivated for five weeks in the commercial Instant Ocean (IO) seawater. The growth of the biofilm and the dynamics of NO3− and NO2− were determined. The levels of M. nitratireducenticrescens and H. nitrativorans were measured by qPCR.ResultsIn the first set of assays, the biofilm cultures had the capacity to sustain denitrifying activities in most of the tested conditions. Inhibition occurred when they were exposed to high pH (10) or to high methanol concentration (1.5%). In the second set of assays, the highest specific denitrification rates occurred with the biofilm cultures cultivated at 64.3 mM NO3− and 0.45% methanol, and at 30 °C. Poor biofilm development occurred with the biofilm cultures cultivated at 5% and 8% NaCl. In all biofilm cultures cultivated in ASW at 2.75% NaCl, H. nitrativorans strain NL23 decreased by three orders of magnitude in concentrations compared to that found in OB. This decrease coincided with the increase of the same magnitude of a subpopulation of M. nitratireducenticrescens (strain GP59 as representative). In the biofilm cultures cultivated at low NaCl concentrations (0% to 1.0%), persistence of H. nitrativorans strain NL23 was observed, with the gradual increase in concentrations of M. nitratireducenticrescens strain GP59. High levels of H. nitrativorans strain NL23 were found in the IO biofilm cultures. The concentrations of M. nitratireducenticrescens strain JAM1 were lower in most of the biofilms cultures than in OB.ConclusionsThese results demonstrate the plasticity of the marine methylotrophic denitrifying biofilm in adapting to different environmental changes. The NaCl concentration is a crucial factor in the dynamics of H. nitrativorans strain NL23, for which growth was impaired above 1% NaCl in the ASW-based biofilm cultures in favor of M. nitratireducenticrescens strain GP59.

Evaluation of the information content of long-term wastewater characteristics data in relation to activated sludge model parameters.

A parameter estimation framework was used to evaluate the ability of observed data from a full-scale nitrification-denitrification bioreactor to reduce the uncertainty associated with the bio-kinetic and stoichiometric parameters of an activated sludge model (ASM). Samples collected over a period of 150 days from the effluent as well as from the reactor tanks were used. A hybrid genetic algorithm and Bayesian inference were used to perform deterministic and parameter estimations, respectively. The main goal was to assess the ability of the data to obtain reliable parameter estimates for a modified version of the ASM. The modified ASM model includes methylotrophic processes which play the main role in methanol-fed denitrification. Sensitivity analysis was also used to explain the ability of the data to provide information about each of the parameters. The results showed that the uncertainty in the estimates of the most sensitive parameters (including growth rate, decay rate, and yield coefficients) decreased with respect to the prior information.

Methylophilaceae and Hyphomicrobium as target taxonomic groups in monitoring the function of methanol-fed denitrification biofilters in municipal wastewater treatment plants.

Molecular monitoring of bacterial communities can explain and predict the stability of bioprocesses in varying physicochemical conditions. To study methanol-fed denitrification biofilters of municipal wastewater treatment plants, bacterial communities of two full-scale biofilters were compared through fingerprinting and sequencing of the 16S rRNA genes. Additionally, 16S rRNA gene fingerprinting was used for 10-week temporal monitoring of the bacterial community in one of the biofilters. Combining the data with previous study results, the family Methylophilaceae and genus Hyphomicrobium were determined as suitable target groups for monitoring. An increase in the relative abundance of Hyphomicrobium-related biomarkers occurred simultaneously with increases in water flow, NO x- load, and methanol addition, as well as a higher denitrification rate, although the dominating biomarkers linked to Methylophilaceae showed an opposite pattern. The results indicate that during increased loading, stability of the bioprocess is maintained by selection of more efficient denitrifier populations, and this progress can be analyzed using simple molecular fingerprinting.

Methylophaga and Hyphomicrobium can be used as target genera in monitoring saline water methanol-utilizing denitrification.

Which bacterial taxonomic groups can be used in monitoring saline water methanol-utilizing denitrification and whether nitrate is transformed into N2 in the process are unclear. Therefore, methylotrophic bacterial communities of two efficiently functioning (nitrate/nitrite reduction was 63-96%) tropical and cool seawater reactors at a public aquarium were investigated with clone library analysis and 454 pyrosequencing of the 16S rRNA genes. Transformation of nitrate into N2 was confirmed using 15N labeling in incubation of carrier material from the tropical reactor. Combining the data with previous study results, Methylophaga and Hyphomicrobium were determined to be suitable target genera for monitoring the function of saline water methanol-fed denitrification systems. However, monitoring was not possible at the single species level. Interestingly, potential nitrate-reducing methylotrophs within Filomicrobium and closely related Fil I and Fil II clusters were detected in the reactors suggesting that they also contributed to methylotrophic denitrification in the saline environment.

Complete Genome Sequence of Hyphomicrobium nitrativorans Strain NL23, a Denitrifying Bacterium Isolated from Biofilm of a Methanol-Fed Denitrification System Treating Seawater at the Montreal Biodome

Hyphomicrobium nitrativorans strain NL23 has been isolated from the biofilm of a denitrification system treating seawater. This strain has the capacity to denitrify using methanol as a carbon source. Here, we report the complete genome sequence of this strain in an effort to increase understanding of the function of this bacterium within the biofilm.

Hyphomicrobium nitrativorans sp. nov., isolated from the biofilm of a methanol-fed denitrification system treating seawater at the Montreal Biodome

A budding prosthecate bacterial strain, designated NL23(T), was isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. Phylogenetic analysis based on 16S rRNA (rRNA) gene sequences showed that the strain was affiliated with the genus Hyphomicrobium of the Alphaproteobacteria and was most closely related to Hyphomicrobium zavarzinii with 99.4 % sequence similarity. Despite this high level of 16S rRNA gene sequence similarity, DNA-DNA hybridization assays showed that strain NL23(T) was only distantly related to H. zavarzinii ZV-622(T) (12 %). Strain NL23(T) grew aerobically, but also had the capacity to grow under denitrifying conditions in the presence of nitrate without nitrite accumulation. Growth occurred at pH 7.0-9.5, with 0-1 % NaCl and at temperatures of 15-35 °C. Major fatty acids were C18 : 1ω7c or ω6c (84.6 %) and C18 : 0 (8.5 %), and major quinones were Q8 (5 %) and Q9 (95 %). The complete genome of the strain was sequenced and showed a DNA G+C content of 63.8 mol%. Genome analysis predicted open reading frames (ORF) encoding the key enzymes of the serine pathway as well as enzymes involved in methylotrophy. Also, ORF encoding a periplasmic nitrate reductase (Nap), a nitrite reductase (Nir), a nitric oxide reductase (Nor) and a nitrous oxide reductase (Nos) were identified. Our results support that strain NL23(T) represents a novel species within the genus Hyphomicrobium, for which the name Hyphomicrobium nitrativorans sp. nov. is proposed. The type strain is NL23(T) ( = ATCC BAA-2476(T) = LMG 27277(T)).

Methylophaga nitratireducenticrescens sp. nov. and Methylophaga frappieri sp. nov., isolated from the biofilm of the methanol-fed denitrification system treating the seawater at the Montreal Biodome

Two bacterial strains, designated JAM1(T) and JAM7(T), were isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. They were affiliated within the genus Methylophaga of the Gammaproteobacteria by analysis of the 16S rRNA gene sequences. Strain JAM1(T) had the capacity to grow under denitrifying conditions by reducing nitrate into nitrite which is unique among the species of the genus Methylophaga. Major fatty acids were C16:1ω7c or ω6c, C16:0 and C18:1ω7c or ω6c. The major ubiquinone was Q8. Both strains required vitamin B12 and Na(+) ions for growth. The genomes of strains JAM1(T) and JAM7(T) have been completely sequenced and showed a DNA G+C content of 44.7 mol% and 47.8 mol%, respectively. Growth occurred at pH 6-11 and at 0.5-8% NaCl. Both genomes contained predicted ORFs encoding the key enzymes of the ribulose monophosphate pathway. Also, operons encoding two nitrate reductases (Nar), two nitric oxide reductases (Nor), one nitrous oxide reductase (Nos) and one truncated nitrite reductase (NirK) were clustered in a 67 kb chromosomal region in strain JAM1(T). No such operons were found in strain JAM7(T). These results supported the affiliation of the two strains as novel species within the genus Methylophaga. The names Methylophaga nitratireducenticrescens sp. nov. for type strain JAM1(T) (=DSM 25689(T)=ATCC BAA-2433(T)) and Methylophaga frappieri sp. nov. for type strain JAM7(T) (=DSM 25690(T)=ATCC BAA-2434(T)) are proposed.

High-rate denitrification using polyethylene glycol gel carriers entrapping heterotrophic denitrifying bacteria

This study evaluated the nitrogen removal performance of polyethylene glycol (PEG) gel carriers containing entrapped heterotrophic denitrifying bacteria. A laboratory-scale denitrification reactor was operated for treatment of synthetic nitrate wastewater. The nitrogen removal activity gradually increased in continuous feed experiments, reaching 4.4 kg N m−3 d−1 on day 16 (30 °C). A maximum nitrogen removal rate of 5.1 kg N m−3 d−1 was observed. A high nitrogen removal efficiency of 92% on average was observed at a high loading rate. In batch experiments, the denitrifying gel carriers were characterized by temperature. Nitrate and total nitrogen removal activities both increased with increasing temperature, reaching a maximum at 37 and 43 °C, respectively. Apparent activation energies for nitrate and nitrite reduction were 52.1 and 71.9 kJ mol−1, respectively. Clone library analysis performed on the basis of the 16S rRNA gene revealed that Hyphomicrobium was mainly involved in denitrification in the methanol-fed denitrification reactors.

Observations on the Performance and Modeling of Glycerin- and Methanol-fed Denitrification Filters

Observations on the Performance and Modeling of Glycerin- and Methanol-fed Denitrification FiltersThe objective of this investigation was to utilize the full-scale denitrification filter data from five treatment plants to explore the significance of the following variables on nitrate removal rate: (a) nitrate loading rate (NLR), (b) hydraulic loading rate (HLR), (c) temperature, and (d) PO4- P:NO3-N ratio. Much of this data was gathered in prior studies (Bilyk et al. 2008 and 2011; Ledwell et...Author(s)Katya BilykPeter DoldSamuel LedwellRon LatimerPaul PittMary SadlerSourceProceedings of the Water Environment FederationSubjectSession 32: Supplemental Carbon for DenitrificationDocument typeConference PaperPublisherWater Environment FederationPrint publication date Jan, 2011ISSN1938-6478SICI1938-6478(20110101)2011:15L.1598;1-DOI10.2175/193864711802713405Volume / Issue2011 / 15Content sourceWEFTECFirst / last page(s)1598 - 1614Copyright2011Word count112Subject keywordsDenitrificationfiltercarbonmethanolglyceringlycerolmodelingphosphorus limitation

Dissimilatory reduction of nitrate in seawater by a Methylophaga strain containing two highly divergent narG sequences

Methylophaga spp. are methylotrophs commonly associated with marine environments and have been defined as strict aerobic methylotrophs. They have been shown previously to represent 50-70% of the bacterial population in the biofilm of the methanol-fed denitrification reactor treating a large seawater aquarium at the Montreal Biodome. It was therefore surprising to find such a high concentration of Methylophaga spp. in anoxic conditions. In this study, we showed by cultivation-independent and -dependent approaches that one Methylophaga strain present in the anoxic biofilm is involved in the denitrification process. DNA stable-isotope probing (SIP) experiments in which the biofilm was cultured under denitrifying conditions with (13)C-methanol have revealed the enrichment of one particular taxon. By screening a 16S ribosomal RNA gene library derived from a (13)C-DNA fraction of the SIP gradients, 62% of the library was composed of one sequence affiliated with the genus Methylophaga. One strain, named JAM1, representing this Methylophaga species was isolated. It grows aerobically but also under denitrifying conditions by reducing nitrate into nitrite. The nitrate-reducing activity was correlated with the presence and the expression of two highly divergent narG genes (narG1 and narG2). narG1 showed a high percentage of identity with the corresponding part of narG found in Thiobacillus denitrificans, which suggests a recent acquisition of narG in strain JAM1 by horizontal gene transfer. This study provides the first direct evidence of the adaptation of a Methylophaga species to an oxygen-limited environment.

Long-term storage conditions for carriers with denitrifying biomass of the fluidized, methanol-fed denitrification reactor of the Montreal Biodome, and the impact on denitrifying activity and bacterial population

The Montreal Biodome has a denitrification system to stabilize the nitrate concentration in its 3 million liter seawater aquarium. However, this microbial process has failed periodically due to various technical problems. The system can take several weeks to recover its full denitrification capacity. In order to provide the denitrification system with a backup of active biomass, different freezing conditions for the denitrifying biomass were tested. The biomass was conserved for 1 week–17 months at −20 °C with and without glycerol or at −80 °C with and without glycerol, and the denitrifying activity was tested in batch culture for 140 h periods at various intervals. Our results showed that glycerol was required for fast recovery of the microbial community's denitrifying activity. The −20 °C and −80 °C conservation temperatures with glycerol gave similar results although there was a short period of nitrite accumulation in the −20 °C sample. There were no substantial changes in the microbial community of any of the frozen samples after 17 months of conservation as monitored by denaturing gradient gel electrophoresis. This is the first report on the long-term conservation of a complex denitrifying population by freezing.

Nitratireductor aquibiodomus gen. nov., sp. nov., a novel alpha-proteobacterium from the marine denitrification system of the Montreal Biodome (Canada).

The Montreal Biodome operates a methanol-fed denitrification system that treats the water in its three million litre marine mesocosm. An unknown bacterium, named strain NL21T, was isolated from this system on TSA and R2A agar. The organism is a Gram-negative, rod-shaped (1x3 microm) facultative aerobe. Optimal growth conditions on R2A agar are 30-35 degrees C, pH 7-7.5 and 1% (w/w) NaCl. Phylogenetic analysis of the 16S rDNA sequence reveals that strain NL21T forms a novel lineage in the family 'Phyllobacteriaceae' within the alpha2 subgroup of the Proteobacteria. The closest related genera are Aminobacter, Pseudaminobacter, Mesorhizobium and Defluvibacter. Major cellular fatty acids are C(18:1)omega7c (75%), C(19:0)omega8c cyclopropane (9.4%) and C(18:0) (4.2%). The DNA G+C content of strain NL21T (57 mol%) differs from those of all other described members of the 'Phyllobacteriaceae' (60-64 mol%). Strain NL21T reduces nitrate to nitrite, but does not reduce nitrite to nitrogen gas. Only a few sugars and amino acids can serve as carbon sources. Strain NL21T is able to grow without salt and tolerates up to 5% NaCl. Phylogenetic analysis, as well as physiological and biochemical tests, showed that strain NL21T was different from all other members of the 'Phyllobacteriaceae' with validly published names. Strain NL21T therefore represents a novel genus, for which the name Nitratireductor aquibiodomus gen. nov., sp. nov. is proposed, with the type strain NL21T (=DSM 15645T=ATCC BAA-762T).

Bacterial diversity in a marine methanol-fed denitrification reactor at the montreal biodome, Canada.

The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods. The microbiota extracted from the reactor carriers were cultivated on three media. Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined. The analysis showed that the isolate types were related to alpha-Proteobacteria. They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family. Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota. Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles. More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (gamma-Proteobacteria). Sequences from other profiles were related to bacterial species involved in denitrification. The number of species in the denitrification reactor was estimated at 15. Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE. The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged. PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations. As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.