Nitrospira Defluvii Research Articles

Comparative metagenomic analysis from Sundarbans ecosystems advances our understanding of microbial communities and their functional roles

The Sundarbans mangrove, located at the mouth of the Ganges and Brahmaputra Rivers, is the world’s largest tidal mangrove forest. These mangroves are also one of the most striking sources of microbial diversity, essential in productivity, conservation, nutrient cycling, and rehabilitation. Hence, the main objective of this study was to use metagenome analysis and provide detailed insight into microbial communities and their functional roles in the Sundarbans mangrove ecosystem. A comparative analysis was also done with a non-mangrove region of the Sundarbans ecosystem to assess the capability of the environmental parameters to explain the variation in microbial community composition. The study found several dominant bacteria, viz., Alphaproteobacteria, Actinomycetota, Bacilli, Clostridia, Desulfobacterota, Gammaproteobacteria, and Nitrospira, from the mangrove region. The mangrove sampling site reports several salt-tolerant bacteria like Alkalibacillus haloalkaliphilus, Halomonas anticariensis, and Salinivibrio socompensis. We found some probiotic species, viz., Bacillus clausii, Lactobacillus curvatus, Vibrio mediterranei and Vibrio fluvialis, from the Sundarbans mangrove. Nitrifying bacteria in Sundarbans soils were Nitrococcus mobilis, Nitrosococcus oceani, Nitrosomonas halophila, Nitrospirade fluvii, and others. Methanogenic archaea, viz., Methanoculleus marisnigri, Methanobrevibacter gottschalkii, and Methanolacinia petrolearia, were highly abundant in the mangroves as compared to the non-mangrove soils. The identified methanotrophic bacterial species, viz., Methylobacter tundripaludum, Methylococcus capsulatus, Methylophaga thiooxydans, and Methylosarcina lacus are expected to play a significant role in the degradation of methane in mangrove soil. Among the bioremediation bacterial species identified, Pseudomonas alcaligenes, Pseudomonas mendocina, Paracoccus denitrificans, and Shewanella putrefaciens play a significant role in the remediation of environmental pollution. Overall, our study shows for the first time that the Sundarbans, the largest mangrove ecosystem in the world, has a wide range of methanogenic archaea, methanotrophs, pathogenic, salt-tolerant, probiotic, nitrifying, and bioremediation bacteria.

Side effects of the addition of an adsorbent for the nitrification performance of a microbiome in the treatment of an antibiotic mixture

This work determined the effect of biochar (BC) as an adsorbent on the nitrifying microbiome in regulating the removal, transformation, fate, toxicity, and potential environmental consequences of an antibiotic mixture containing oxytetracycline (OTC) and sulfamethoxazole (SMX). Despite the beneficial role of BC as reported in the literature, the present study revealed side effects for the nitrifying microbiome and its functioning arising from the presence of BC. Long-term monitoring revealed severe disruption to nitratation via the inhibition of both nitrite oxidizers (e.g., Nitrospira defluvii) and potential comammox species (e.g., Ca. Nitrospira nitrificans). Byproducts (BPs) more toxic than the parent compounds were found to persist at a high relative abundance, particularly in the presence of BC. Quantitative structure–activity relationship modeling determined that the physicochemical properties of the toxic BPs significantly differed from those of OTC and SMX. The results suggested that the BPs tended to mobilize and accumulate on the surface of the solids in the system (i.e., the BC and biofilm), disrupting the nitrifiers growing at the interface. Collectively, this study provides novel insights, demonstrating that the addition of adsorbents to biological systems may not necessarily be beneficial; rather, they may generate side effects for specific bacteria that have important ecosystem functions.

Effect of antibiotic cocktail exposure on functional disturbance of nitrifying microbiome

The present study quantitatively determined the degree and type of functional disturbance in the nitrifying microbiome caused by exposure to a single oxytetracycline (OTC) and a two-antibiotic mixture containing OTC and sulfamethoxazole (SMX). While the single antibiotic had a pulsed disturbance on nitritation that was recoverable within three weeks, the antibiotic mixture caused a more significant pulsed disturbance on nitritation and a potential press disturbance on nitratation that was not recoverable for over five months. Bioinformatic analysis revealed significant perturbations for both canonical nitrite-oxidizing (Nitrospira defluvii) and potential complete ammonium-oxidizing (Ca. Nitrospira nitrificans) populations that were strongly associated with the press perturbation on nitratation. In addition to this functional disturbance, the antibiotic mixture reduced the biosorption of OTC and altered its biotransformation pathways, resulting in different transformation products compared with those produced when OTC was treated as a single antibiotic. Collectively, this work elucidated how the antibiotic mixture can affect the degree, type, and duration of the functional disturbance on nitrifying microbiome and offer new insights into the environmental consequences of antibiotic residues (e.g., their fate, transformation, and ecotoxicity) when present as an antibiotic mixture rather than single antibiotics.

Iron scraps packing rapidly enhances nitrogen removal in an aerobic sludge system and the mechanism

Simultaneous nitrification and denitrification (SND) has the advantage of energy saving and carbon demand reduction. Here, readily available low-cost iron scraps packing was added to an aerobic sludge system. This successfully enhanced the efficiency of total nitrogen removal from 37.7 ± 13.2 % to 62.7 ± 7.9 % over 2 days. While electrons from iron biocorrosion did not contribute to nitrate reduction, iron promoted heterotrophic denitrification. The iron scraps changed the spatial distribution of the microbial community, where more denitrification bacteria accumulated around the packing and higher denitrification capacity was noted. Metagenomic analysis of the sludge cultured in the presence of iron scraps for 2 days revealed that, with the exception of the enriched amoA/B/C gene expression, the abundance of other key nitrogen removal genes showed little variation. Furthermore, the structure of the microbial community was unchanged probably due to the relatively short culturing period. However, metatranscriptomic analysis indicated that iron increased the abundance of nitrifying bacteria (i.e. unclassified Nitrosomonas, Nitrosomonas sp. Is79A3 and Nitrospira defluvii) and promoted higher expression of nitrification genes. Notably, iron scraps packing decreased the abundance of the key denitrification bacteria Thauera sp. MZ1T from 52.92 to 7.58 %. The expression of napA/B also decreased, while expression of narG/H/I increased by 9 to 23 fold and a 2 to 3 fold over expression was noted for nirS, norB/C and nosZ in the presence of iron scraps. This suggested that aerobic denitrification was inhibited and anaerobic denitrification was promoted. This study has provided in-depth understanding of the influence of iron on SND to improve the application of iron-supported biological processes.

Metagenomic analysis of rhizosphere microbiome provides insights into occurrence of iron deficiency chlorosis in field of Asian pears

BackgroundFe-deficiency chlorosis (FDC) of Asian pear plants is widespread, but little is known about the association between the microbial communities in the rhizosphere soil and leaf chlorosis. The leaf mineral concentration, leaf subcellular structure, soil physiochemical properties, and bacterial species community and distribution had been analysed to gain insights into the FDC in Asian pear plant.ResultsThe total Fe in leaves with Fe-deficiency was positively correlated with total K, Mg, S, Cu, Zn, Mo and Cl contents, but no differences of available Fe (AFe) were detected between the rhizosphere soil of chlorotic and normal plants. Degraded ribosomes and degraded thylakloid stacks in chloroplast were observed in chlorotic leaves. The annotated microbiome indicated that there were 5 kingdoms, 52 phyla, 94 classes, 206 orders, 404 families, 1,161 genera, and 3,043 species in the rhizosphere soil of chlorotic plants; it was one phylum less and one order, 11 families, 59 genera, and 313 species more than in that of normal plant. Bacterial community and distribution patterns in the rhizosphere soil of chlorotic plants were distinct from those of normal plants and the relative abundance and microbiome diversity were more stable in the rhizosphere soils of normal than in chlorotic plants. Three (Nitrospira defluvii, Gemmatirosa kalamazoonesis, and Sulfuricella denitrificans) of the top five species (N. defluvii, G. kalamazoonesis, S. denitrificans, Candidatus Nitrosoarchaeum koreensis, and Candidatus Koribacter versatilis). were the identical and aerobic in both rhizosphere soils, but their relative abundance decreased by 48, 37, and 22%, respectively, and two of them (G. aurantiaca and Ca. S. usitatus) were substituted by an ammonia-oxidizing soil archaeon, Ca. N. koreensis and a nitrite and nitrate reduction related species, Ca. K. versatilis in that of chlorotic plants, which indicated the adverse soil aeration in the rhizosphere soil of chlorotic plants. A water-impermeable tables was found to reduce the soil aeration, inhibit root growth, and cause some absorption root death from infection by Fusarium solani.ConclusionsIt was waterlogging or/and poor drainage of the soil may inhibit Fe uptake not the amounts of AFe in the rhizosphere soil of chlorotic plants that caused FDC in this study.

The differential proliferation of AOB and NOB during natural nitrifier cultivation and acclimation with raw sewage as seed sludge.

Nitrifier immigration from sewers to wastewater treatment systems is attracting increasing attention for understanding nitrifier community assembly mechanisms, and improving process modeling and operation. In this study, nitrifiers in raw sewage were cultivated and acclimated in a sequencing batch reactor (SBR) for 90 days to investigate the characteristics of the influent nitrifiers after immigration. During the experiment, specific nitrite utilization rate (SNUR) exceeded specific ammonia utilization rate (SAUR) when floc size reached 224 ± 46 μm, and nitrogen loss occurred at the same time. The ratio of nitrite oxidizing bacteria (NOB) to ammonia oxidizing bacteria (AOB) increased from 0.84 to 2.14 after cultivation. The Illumina MiSeq sequencing showed that the dominant AOB was Nitrosomonas sp. Nm84 and unidentified species, and the three most abundant Nitrospira were Nitrospira defluvii, Nitrospira calida, and unidentified Nitrospira spp. in both raw sewage and cultivated activated sludge. The shared reads of raw sewage and activated sludge were 48.76% for AOB and 89.35% for Nitrospira. These indicated that nitrifiers, especially NOB, immigrated from influent can survive and propagate in wastewater systems, which may be a significant hinder to suppress NOB in the application of advanced nitrogen remove process based on partial nitrification in the mainstream.

Comparison of the Microbial Community Structure in Nitrifying Processes Operating with Different Dissolved Oxygen Concentrations

High-throughput sequencing was applied to analyze the microbial community structure of nitrifying reactors operated with different dissolved oxygen (DO) levels. Results showed that the nitrifying reactor (RL) run with low DO (0.2-0.3 mg·L-1) exhibited greater microbial richness and diversity than the reactor run under the high DO condition[RH, DO=(2.0±0.1) mg·L-1]. In contrast, the microbial community in RH was more highly functionally organized than that in RL. Although the communities in RH and RL shared over 85% of the total sampled genetic information, the relative abundance of some individual species varied between the different DO conditions. Members of the Proteobacteria phylum, which accounted for 80.7% of the total microbes in RH, were highly enriched, and the relative abundance of Nitrosomonas reached to 65.1%. However, the microbial community in RL was dominated by Proteobacteria (43.8%), Firmicutes (20.0%), and Bacteroidetes (15.1%). In addition, a large fraction of bacteria possessing hydrolyzation and fermentation functions under anaerobic or anoxic conditions were also present in RL including Lactococcus, Anaerolineaceae, and Rhodocyclaceae. As known ammonium-oxidizing bacteria, Nitrosomonas oligotropha and Nitrosomonas europaea were enriched in the RH and RL, respectively, while Nitrospira defluvii, being a nitrite-oxidizing bacteria, dominated both reactors. Rather than DO, ammonia and nitrite availability should be key factors in the selective enrichment of these nitrifiers.

Insights into the bacterial species and communities of a full-scale anaerobic/anoxic/oxic wastewater treatment plant by using third-generation sequencing

For the first time, full-length 16S rRNA sequencing method was applied to disclose the bacterial species and communities of a full-scale wastewater treatment plant using an anaerobic/anoxic/oxic (A/A/O) process in Wuhan, China. The compositions of the bacteria at phylum and class levels in the activated sludge were similar to which revealed by Illumina Miseq sequencing. At genus and species levels, third-generation sequencing showed great merits and accuracy. Typical functional taxa classified to ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), denitrifying bacteria (DB), anaerobic ammonium oxidation bacteria (ANAMMOXB) and polyphosphate-accumulating organisms (PAOs) were presented, which were Nitrosomonas (1.11%), Nitrospira (3.56%), Pseudomonas (3.88%), Planctomycetes (13.80%), Comamonadaceae (1.83%), respectively. Pseudomonas (3.88%) and Nitrospira (3.56%) were the most predominating two genera, mainly containing Pseudomonas extremaustralis (1.69%), Nitrospira defluvii (3.13%), respectively. Bacteria regarding to nitrogen and phosphorus removal at species level were put forward. The predicted functions proved that the A/A/O process was efficient regarding nitrogen and organics removal.

Low Temperature and Neutral pH Define "Candidatus Nitrotoga sp." as a Competitive Nitrite Oxidizer in Coculture with Nitrospira defluvii.

Nitrification is an essential process for N removal in activated sludge to avoid toxicity of ammonium and nitrite. Besides Nitrospira, "Candidatus Nitrotoga" has been identified as a key nitrite-oxidizing bacterium (NOB) performing the second step of nitrification, nitrite oxidation to nitrate, in wastewater treatment plants (WWTPs). However, the driving forces for the dominance of Nitrotoga in certain plants have often remained unclear and could not be explained solely by temperature effects. In this study, we characterized the physiology of the ammonium-dependent Nitrotoga sp. BS with regard to temperature and pH variations and evaluated its competitiveness against Nitrospira defluvii Both NOB originated from the same WWTP and shared a comparable pH optimum of 7.3. Based on these results, coculturing experiments with these NOB were performed in batch reactors operated at either 17°C or 22°C to compare their abundances under optimal (pH 7.4) or suboptimal (pH 6.4) conditions using 1 mM nitrite. As revealed by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), and 16S amplicon sequencing, Nitrotoga sp. BS was clearly favored by its optimal growth parameters and dominated over Ns. defluvii at pH 7.4 and 17°C, whereas a pH of 6.4 was more selective for Ns. defluvii Our synthetic communities revealed that niche differentiation of NOB is influenced by a complex interaction of environmental parameters and has to be evaluated for single species.IMPORTANCE "Ca. Nitrotoga" is a NOB of high environmental relevance, but physiological data exist for only a few representatives. Initially, it was detected in specialized niches of low temperature and low nitrite concentrations, but later on, its ubiquitous distribution revealed its critical role for N removal in engineered systems like WWTPs. In this study, we analyzed the competition between Nitrotoga and Nitrospira in bioreactors and identified conditions where the K strategist Ns. defluvii was almost replaced by Nitrotoga sp. BS. We show that the pH value is an important factor that regulates the composition of the nitrite-oxidizing enrichment with a dominance of Nitrotoga sp. BS versus Ns. defluvii at a neutral pH of 7.4 in combination with a temperature of 17°C. The physiological diversity of novel Nitrotoga cultures improves our knowledge about niche differentiation of NOB with regard to functional nitrification under suboptimal conditions.

Functional Response of MBR Microbial Consortia to Substrate Stress as Revealed by Metaproteomics.

Bacterial consortia have a primary role in the biological degradations occurring in activated sludge for wastewater treatment, for their capacities to metabolize the polluting matter. Therefore, the knowledge of the main metabolic pathways for the degradation of pollutants becomes critical for a correct design and operation of wastewater treatment plants. The metabolic activity of the different bacterial groups in activated sludge is commonly investigated through respirometry. Furthermore, in the last years, the development of "omic" approaches has offered more opportunities to integrate or substitute the conventional microbiological assays and to deeply understand the taxonomy and dynamics of complex microbial consortia. In the present work, an experimental membrane bioreactor (MBR) was set up and operated for the treatment of municipal wastewater, and the effects of a sudden decrease of the organic supply on the activated sludge were investigated. Both respirometric and metaproteomic approaches revealed a resistance of autotrophic bacteria to the substrate stress, and particularly of nitrifying bacteria. Furthermore, metaproteomics allowed the identification of the taxonomy of the microbial consortium based on its protein expression, unveiling the prevalence of Sorangium and Nitrosomonas genera both before and after the organic load decrease. Moreover, it confirmed the results obtained through respirometry and revealed a general expression of proteins involved in metabolism and transport of nitrogen, or belonging to nitrifying species like Nitrosomonas europeae, Nitrosomonas sp. AL212, or Nitrospira defluvii.

Effects of Gas/Water Ratio on the Characteristics of Nitrogen Removal and the Microbial Community in Post Solid-Phase Denitrification Biofilter Process

To solve the problems of high-energy consumption, complex processes, and low nitrogen removal efficiency in the currently available low carbon source wastewater treatment processes, a novel coagulation sedimentation/post solid-phase denitrification biofilter process (CS-BAF-SPDB) was proposed. The effect of gas/water ratio on nitrogen removal efficiency of the CS-BAF-SPDB was studied. The changes in the microbial community structure along the varying gas/water ratio were investigated, and the functional bacteria were identified using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that, to realize favorable nitrifying and denitrifying performance simultaneously in the BAF-SPDB unit, the gas/water ratio of the BAF should be set at 4∶1. In addition, the influence of the macro operational parameters on the performance of the BAF and SPDB had a direct relationship with the dynamic changes in the micro microbial community. The influence of gas/water ratio on nitrification performance in the BAF was mainly embodied in the changes of composition, amount, and activity of ammonia-oxidizing bacteria Candidatus and Nitrospira defluvii and nitrite-oxidizing bacteria Nitrosomonas sp. Nm47, whereas that on denitrification performance in the SPDB was mainly embodied in the changes of composition and amount of solid carbon substrate degrading denitrifying bacteria Pseudomonas sp. and Myxobacterium AT3-03 and heterotrophic denitrifying bacteria Dechloromonas agitate, Comamonas granuli, and Rubrivivax gelatinosus.

Effect of the dilution rate on microbial competition: r-strategist can win over k-strategist at low substrate concentration.

The conditions present in both in vitro and in vivo ecosystems determine the microbial population harbouring it. One commonly accepted theory is that a species with a high substrate affinity and low growth rate (k-strategist) will win the competition against a second species with a lower substrate affinity and higher growth rate (r-strategist) if both species are subjected to low substrate concentrations. In this study two nitrite oxidizing bacteria (NOB), Nitrospira defluvii (k-strategist) and Nitrobacter vulgaris (r-strategist), were cultivated in a continuous reactor systems. The minimal hydraulic retention time (HRT) required for maintaining the slower growing Nitrospira was first determined. A reactor containing Nitrobacter was set to the same HRT and Nitrospira was injected to evaluate the effect of the dilution rate on the competition between both species. By following the microbial population dynamics with qPCR analysis, it was shown that not only the substrate affinity drives the competition between k- and r-strategists but also the dilution rate. Experimental data and numerical simulations both revealed that the washout of Nitrobacter was significantly delayed at dilution rates close to the μmax of Nitrospira. The competition could be even reverted towards Nitrobacter (r-strategist) despite of low nitrite concentrations and dilution rates lower than the μmax of Nitrospira.

Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira

Nitrospira are a diverse group of nitrite-oxidizing bacteria and among the environmentally most widespread nitrifiers. However, they remain scarcely studied and mostly uncultured. Based on genomic and experimental data from Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II, we identified ecophysiological traits that contribute to the ecological success of Nitrospira. Unexpectedly, N. moscoviensis possesses genes coding for a urease and cleaves urea to ammonia and CO2. Ureolysis was not observed yet in nitrite oxidizers and enables N. moscoviensis to supply ammonia oxidizers lacking urease with ammonia from urea, which is fully nitrified by this consortium through reciprocal feeding. The presence of highly similar urease genes in Nitrospira lenta from activated sludge, in metagenomes from soils and freshwater habitats, and of other ureases in marine nitrite oxidizers, suggests a wide distribution of this extended interaction between ammonia and nitrite oxidizers, which enables nitrite-oxidizing bacteria to indirectly use urea as a source of energy. A soluble formate dehydrogenase lends additional ecophysiological flexibility and allows N. moscoviensis to use formate, with or without concomitant nitrite oxidation, using oxygen, nitrate, or both compounds as terminal electron acceptors. Compared with Nitrospira defluvii from lineage I, N. moscoviensis shares the Nitrospira core metabolism but shows substantial genomic dissimilarity including genes for adaptations to elevated oxygen concentrations. Reciprocal feeding and metabolic versatility, including the participation in different nitrogen cycling processes, likely are key factors for the niche partitioning, the ubiquity, and the high diversity of Nitrospira in natural and engineered ecosystems.

Comparison of the Bacterial Communities in Anaerobic, Anoxic, and Oxic Chambers of a Pilot A2O Process Using Pyrosequencing Analysis

A(2)O process is a sequential wastewater treatment process that uses anaerobic, anoxic, and oxic chambers for nitrogen and phosphorus removal. In this study, the bacterial communities among these chambers were compared, and the diversity of the bacteria involved in nitrogen and phosphorus removal was surveyed. A pilot-scale A(2)O process (50 m(3) day(-1)) was operated for more than 6 months, and bacterial 16S rRNA gene diversity was analyzed using pyrosequencing. A total of 7,447 bacterial sequence reads were obtained from anaerobic (1,546), anoxic (2,158), and oxic (3,743) chambers. Even though there were differences in the atmospheric condition and functionality, no prominent differences could be found in the bacterial community of the three chambers of the pilot A(2)O process. All sequence reads, which were taxonomically analyzed using the Eztaxon-e database, were assigned into 638 approved or tentative genera. Among them, about 72.2 % of the taxa were contained in the phyla Proteobacteria and Bacteroidetes. Phosphate-accumulating bacteria, Candidatus Accumulibacter phosphatis, and two other Accumulibacter were found to constitute 3.1 % of the identified genera. Ammonia-oxidizing bacteria, Nitrosomonas oligotropha, and four other phylotypes in the same family, Nitrosomonadaceae, constituted 0.2 and 0.9 %, respectively. Nitrite-oxidizing bacteria, Nitrospira defluvii, and other three phylotypes in the same family, Nitrospiraceae, constituted 2.5 and 0.1 %, respectively. In addition, Dokdonella and a phylotype of the phylum Chloroflexi, function in nitrogen and/or phosphate removal of which have not been reported in the A(2)O process, constituted the first and third composition among genera at 4.3 and 3.8 %, respectively.

Redox Thermodynamics of High-Spin and Low-Spin Forms of Chlorite Dismutases with Diverse Subunit and Oligomeric Structures

Chlorite dismutases (Clds) are heme b-containing oxidoreductases that convert chlorite to chloride and dioxygen. In this work, the thermodynamics of the one-electron reduction of the ferric high-spin forms and of the six-coordinate low-spin cyanide adducts of the enzymes from Nitrobacter winogradskyi (NwCld) and Candidatus “Nitrospira defluvii” (NdCld) were determined through spectroelectrochemical experiments. These proteins belong to two phylogenetically separated lineages that differ in subunit (21.5 and 26 kDa, respectively) and oligomeric (dimeric and pentameric, respectively) structure but exhibit similar chlorite degradation activity. The E°′ values for free and cyanide-bound proteins were determined to be −119 and −397 mV for NwCld and −113 and −404 mV for NdCld, respectively (pH 7.0, 25 °C). Variable-temperature spectroelectrochemical experiments revealed that the oxidized state of both proteins is enthalpically stabilized. Molecular dynamics simulations suggest that changes in the protein structure are negligible, whereas solvent reorganization is mainly responsible for the increase in entropy during the redox reaction. Obtained data are discussed with respect to the known structures of the two Clds and the proposed reaction mechanism.

Impact of subunit and oligomeric structure on the thermal and conformational stability of chlorite dismutases

Chlorite dismutases (Cld) are unique heme b containing oxidoreductases that convert chlorite to chloride and dioxygen. Recent phylogenetic and structural analyses demonstrated that these metalloproteins significantly differ in oligomeric and subunit structure. Here we have analyzed two representatives of two phylogenetically separated lineages, namely pentameric Cld from Candidatus “Nitrospira defluvii” and dimeric Cld from Nitrobacter winogradskyi having a similar enzymatic activity at room temperature. By application of a broad set of techniques including differential scanning calorimetry, electronic circular dichroism, UV–vis and fluorescence spectroscopy the temperature-mediated and chemical unfolding of both recombinant proteins were analyzed. Significant differences in thermal and conformational stability are reported. The pentameric enzyme is very stable between pH 3 and 10 (Tm=92°C at pH 7.0) and active at high temperatures thus being an interesting candidate for bioremediation of chlorite. By contrast the dimeric protein starts to unfold already at 53°C. The observed unfolding pathways are discussed with respect to the known subunit structure and subunit interaction.

Microbial community structure in an integrated A/O reactor treating diluted livestock wastewater during start-up period

In order to investigate the correlation between reactor performance and the microorganisms, an integrated A/O reactor was operated for 72 days to treat diluted livestock wastewater. Chemical oxygen demand (COD) removal efficiency increased from 79% to 94%, with total nitrogen (TN) removal efficiency from 37% to 50% (HRT 7.4 hr) when the influent COD and TN were ca. 1500 mg/L and 95 mg/L, respectively, and the outlet COD concentration was less than 100 mg/L at the end. Microbial community was monitored during start-up period by denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA gene. DGGE profiles showed that microbial community had changed significantly during the start-up and these shifts were in accordance with the reactor performance. UPGMA clustering analysis showed that 14 anaerobic samples fell into five main groups and so did the aerobic ones, but the grouping patterns were different. Phylogenetic analysis indicated that microbial populations in the anaerobic compartment belonged to Firmicutes, Proteobacteria, Chloroflexi and Bacteroidetes, while Proteobacteria, Bacteroidetes, Firmicutes, Verrucomicrobiae and Nitrospira were present in the aerobic compartment. In the anaerobic compartment, more fermentative and acetogenic bacteria were detected during the start-up while denitrifying bacteria faded away. Two functional populations such as Nitrospira defluvii and Dechloromonas denitrificans were observed when nitrogen removal was high, indicating that simultaneous nitrification and denitrification occurred in the aerobic compartment.