Quorum sensing responses of activated sludge to free nitrous acid: Zoogloea deformation, AHL redistribution, and microbiota acclimatization.

Free nitrous acid (FNA) has been widely employed for improvement of wastewater management by altering sludge characteristic and function based on its polymer lysing and biocidal capacity. Sludge characteristic and function are commonly considered as the joint consequence of microbial individual behaviors and quorum sensing (QS) involved collective behaviours, but the role of the latter in FNA treatment was still as-yet-unidentified and addressed in this research. The results of sludge morphology and component characterized FNA-induced zoogloea deformation, including inner cell exposure, half of extracellular polymeric substances (EPS) reduction and adsorption site depletion. During zoogloea deformation, four acyl-homoserine lactones (AHLs), including C4-HSL, C8-HSL, C10-HSL and C12-HSL, transferred inward of microbiota, and their total contents reduced by 66% because of depressed signal production, augmented decomposer and recognition. Transcriptome analysis revealed that differentially expressed QS driven by AHL redistribution facilitated microbiota acclimatization including cellular motility and hydrolase synthesis for EPS consumption. Boosted motility may favor escaping from stress spot and moderating intercellular acidity based on cell motility test. Feasible EPS consumption provided nutrition for heterotrophic metabolisms testified by pure culture with EPS as sole nutrition. Our work thus comprehensively revealed QS behaviours responding to FNA and deepened the understanding to FNA treatment performance in wastewater management.

Production efficiency and properties of poly(3hydroxybutyrate-co-3hydroxyvalerate) generated via a robust bacterial consortium dominated by Zoogloea sp. using acidified discarded fruit juices as carbon source.

In the current study, a mixed microbial culture (MMC) of polyhydroxyalkanoates (PHAs) producers was developed under nutrient stress and was assessed as biocatalyst for the production of high-yielding PHAs from fermented (acidified) discarded fruit juices (DFJ). The structure of the MMC was analyzed periodically to determine its microbial dynamics, revealing that Zoogloae sp. dominated throughout the operation of the system. The efficiency of PHAs production from the MMC was further optimized in batch mode by altering the ratio of C to N, the ratio of carbon sources (propionate and butyrate), and the initial pH, and subsequently different fermentation mixtures of acidified DFJ were assessed as substrates at optimal conditions. Upon solvent extraction, the properties of recovered PHAs were analyzed, showing that in all cases P(3HB-co-3HV) was produced, with Tm ranging from 90.5 to 168.8 °C, and maximum obtained yields 54.61 ± 4.31 % and 43.27 ± 2.13 %, from synthetic substrates and DFJ, respectively. Overall, it was shown that the developed MMC can be efficiently applied as biocatalyst for the exploitation of sugary wastewaters, such as DFJ, towards bio-based and biodegradable plastics bearing the required properties to substitute fossil plastics, into the concept of a circular economy.

[Microbial Community Structure of Activated Sludge for Total Nitrogen Upgrading Project].

The activated sludge of a biochemical unit (WLK_OD) and an advanced denitrification unit (WLK_AD) were collected from a municipal wastewater treatment plant (WWTP), in which the TN concentration of effluent was less than 1.5 mg·L-1, and their microbial community structure and function profiles were analyzed using 16S rRNA gene high-throughput sequencing. The microorganisms in WLK_AD had lower evenness compared with that in WLK_OD, which was attributed to environmental selection. Furthermore, PCoA revealed that different incoming wastewaters had an impact on microbial community structure. At the phylum level, Proteobacteria (70.11%) was enriched in WLK_AD. At the genus level, Thauera, Flavobacterium, Hydrogenophaga, and Zoogloea served as distinct-dominant denitrifying bacteria in WLK_AD; however, Trichococcus (3.50%) and Terrimonas (1.10%) were enriched in WLK_OD. Through the comparison between groups (P<0.05), the biomarkers detected in each WWTP were different. Furthermore, the results of the co-occurrence network showed that the bacteria from module I had a higher proportion in WLK_AD; the bacteria from module II had a higher proportion in WLK_OD, and they were common microorganisms in WWTPs, implying that wastewater environments drpve the differences in the microbial community structure. Among the types of environmental parameters, the removal efficiency of COD and TN had the greatest impact on the microbial community by the RDA. The removal efficiency of COD was positively correlated with the dominant bacteria from WLK_OD, such as Saccharibacteria, Thermomarinilinea, Terrimonas, and Comamonas; the removal efficiency of TN was positively correlated with the denitrifying bacteria from WLK_AD, such as Dokdonella, Thauera, Flavobacterium, and Zoogloea. WLK_AD was enriched with Novosphingobium, Dokdonella, Thauera, and Sphingomonas, which synergistically removed TN, leading to the TN of the effluent being less than 1.5 mg·L-1. Moreover, based on the results of function prediction, WLK_AD had a higher proportion of genes that could code the denitrification enzymes.

Simultaneous removal of nitrate, tetracycline, and Pb(II) by iron oxidizing strain Zoogloea sp. FY6: Performance and mechanism.

Based on the prevalence of combined antibiotics and heavy metals contamination in the aquatic environment, this study utilized a microbial approach to achieve simultaneous removal of nitrate (NO3--N), tetracycline (TTC), and Pb(II). Zoogloea sp. FY6 could achieve an optimal NO3--N removal efficiency of 91.5% under C/N ratio of 2.0, at a pH of 6.3, and Fe(II) concentration of 20.23 mg L-1 based on response surface methodology. Additionally, strain FY6 was further found to achieve 89.9 and 81.7% removal of TTC and Pb(II) at 6 h under the optimal conditions. Finally, the results of Fluorescence excitation-emission matrix, X-ray diffraction, Fourier transform infrared spectrometer, and X-ray photoelectron spectroscopy further proved that the biologically formed nanoscale iron oxides and biological action jointly led to the removal of TTC and Pb(II). This study provided a theoretical basis for the application of microbially driven process to remove multi-pollutants in micro-polluted water bodies.

Cellulose-mediated floc formation by the activated sludge bacterium Shinella zoogloeoides ATCC 19623.

BACKGROUND: Bacterial floc formation plays a central role in the activated sludge (AS) process. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. We had demonstrated that both expolysaccharides and PEP-CTERM (a short C-terminal domain includes a near-invariant motif Pro-Glu-Pro (PEP)) proteins were required for floc-forming in Zoogloea resiniphila MMB, a dominant AS bacterium. However, the PEP-CTERM proteins are not encoded in the genome of AS bacterium Shinella zoogloeoides ATCC 19623 (formerly known as Zoogloea ramigera I-16-M) and other sequenced AS bacteria strains. The mechanism underlying floc formation of Shinella and related AS bacteria remained largely unclear. RESULTS: In this study, we have sequenced and annotated the complete genome of S. zoogloeoides ATCC 19623 (aka I-16-M), previously isolated in USA and treated as the neotype for the AS floc-forming bacterium Zoogloea ramigera I-16-M, and another AS strain XJ20 isolated in China. Mariner transposon mutagenesis had been conducted to isolate floc-forming-deficient mutants in the strain ATCC 19623 as previously performed by using Tn5 transposon three decades ago. The transposon insertional sites of multiple mutants were mapped to the gene cluster for bacterial cellulose synthesis (bcs) and secretion, and the role played by these genes in floc-formation had been further confirmed by genetic complementation. Interestingly, the restriction map of this bcs locus-flanking region was highly similar to that of the previously identified DNA fragment required for floc-formation in 1980s. Cellulase treatment abolished the floc-forming phenotype of S. zoogloeoides ATCC 19623 but not that of Z. resiniphila MMB strain. The FTIR spectral analyses revealed that the samples extracted from S. zoogloeoides ATCC 19623 were cellulose polymer. CONCLUSION: Our results indicated that we have largely reproduced and completed the unfinished pioneering work on AS floc-formation mechanism, demonstrating that the floc-formation and flocculating capability of Shinella were mediated by extracellular cellulose polymers.

Phenol and 17ß-estradiol removal by Zoogloea sp. MFQ7 and in-situ generated biogenic manganese oxides: Performance, kinetics and mechanism.

The pollution of multifarious pollutants such as heavy metal, organic compounds, and nitrate are a hot research topic at present. In this study, the functions of Zoogloea sp. MFQ7 and its biological precipitation formed during bacterial manganese oxidation on the removal of phenol and 17ß-estradiol (E2) were investigated. Strain MFQ7, a manganese-oxidizing bacteria, can remove 98.34% of phenol under pH of 7.1, a temperature of 30 â„ƒ and Mn2+ concentration of 24.34 mg L-1, additionally, the optimum E2 removal by strain MFQ7 was 100.00% at pH of 7.1, temperature of 28 â„ƒ and Mn2+ concentration of 28.45 mg L-1 by using response surface methodology (RSM) based on Box-Behnken design (BBD) model. The maximum adsorption capacity of bio-precipitation for phenol and E2 was 201.15 mg g-1 and 65.90 mg g-1, respectively. Furthermore, adsorption kinetics and isotherms analysis, XPS, FTIR spectra, Mn(III) trapping experiments elucidated chemical adsorption and Mn(III) oxidation contribute to the removal of phenol and E2 by biogenic manganese oxides. These findings indicated that the adsorption and oxidation of manganese are expected to be one of the effective means to remove these typical organic pollutants containing phenol and E2.

Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

Simultaneous removal of nitrate, manganese, and tetracycline by Zoogloea sp. MFQ7: Adsorption mechanism of tetracycline by biological precipitation.

A Mn(II) oxidizing-denitrifying and tetracycline (TC) removal bacterium Zoogloea sp. MFQ7 was isolated in this study. Nitrogen removal was 83.49% by nitrogen balance experiment. The maximum removal efficiencies of nitrate, Mn(II), and TC by strain MFQ7 within 96 h was 100.00, 74.56, and 63.59% at C/N of 2.0, pH of 7.0, Mn(II) of 20 mg L-1, temperature of 30.0 °C, and TC of 0.2 mg L-1. SEM illustrated that biogenic manganese oxides (BMO) was petal-like, XRD and XPS analyses confirmed that MnO2 was the main component of BMO. Besides, the maximum adsorption capacity of BMO for TC was 52.21 mg g-1. FTIR detected the changes in TC adsorption by BMO. Pseudo-second-order model (R2 = 0.994) explained the adsorption kinetics of TC on BMO and Langmuir isotherm model (R2 = 0.983) suggested that it was homogeneous adsorption, thermodynamics data (ΔG < 0, ΔH = 18.31 kJ mol-1, ΔS = 72.8 J (mol*K)-1) confirmed that adsorption was endothermic and spontaneous.

Denitrification potential of sodium alginate gel beads immobilized iron-carbon, Zoogloea sp. L2, and riboflavin: Performance optimization and mechanism.

A kind of gel beads loaded with iron-carbon powder (Fe-C), Zoogloea sp. L2, and riboflavin (VB2) were prepared through cross-linking of sodium alginate (SA) to establish an immobilized bioreactor. The optimal ratio of SA beads was adjusted by orthogonal experiment. The change of oxidation-reduction potential (ORP) and the concentration of Fe2+ and Fe3+ showed that the addition of VB2 as a redox mediator can promote denitrification. Under the optimal conditions (carbon to nitrogen (C/N) ratio = 2.0, pH = 7.0, and hydraulic retention time (HRT) = 8 h), the nitrate removal efficiency (NRE) of bioreactor reached 98.48% (1.99 mg L-1h-1). Furthermore, Fourier transform infrared spectrometer (FTIR), Fluorescence excitation-emission matrix (EEM), X-ray diffraction (XRD), and gas chromatography (GC) analysis revealed that the immobilization and denitrification of the immobilized bioreactor were excellent. High throughput sequencing also showed that Zoogloea played a vital role in nitrate removal.

Stability of pure oxygen aeration-activated sludge system under non-steady food-to-microorganism ratio conditions during petrochemical wastewater treatment.

This study investigates the stability of a pure oxygen aeration-activated sludge system for petrochemical wastewater treatment under high organic concentration and non-steady food-to-microorganism (F/M) ratio conditions. Sludge settling characteristics maintained relatively stable conditions with an F/M ratio variation from 0.15 ± 0.04 to 0.33 ± 0.07 kg COD/kg MLSS⋅d, while the excess F/M ratio (0.44 ± 0.16 kg COD/kg MLSS⋅d) resulted in deterioration of the organic removal and sludge-water separation performances. Loosely bound extracellular polymeric substances (EPS) showed more significant effect on sludge settleability than the tightly bound EPS. The genus Hydrogenophaga was related to organic removal performance, while Zoogloea and Chitinophaga were related to the effluent quality of suspended solids. The excess F/M ratio also caused an increase in Zoogloea and Chitinophaga, whereas the toxicity of petrochemical wastewater resulted in decreased abundance of Hydrogenophaga. These changes caused deterioration of the organic removal and sludge-water separation performances.

Zoogloea dura sp. nov., a N2-fixing bacterium isolated from forest soil and emendation of the genus Zoogloea and the species Zoogloea oryzae and Zoogloea ramigera.

A motile, Gram-stain-negative, rod-shaped bacterium, designated G-4-1-14T, was obtained from forest soil sampled at Gwanggyo mountain, Gyeonggi-do, Republic of Korea. Cells were colourless, aerobic, grew optimally at 28-35 °C and hydrolysed DNA and casein. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain G-4-1-14T formed a lineage within the genus Zoogloea. The closest members were Zoogloea resiniphila ATCC 70068T (98.6 % sequence similarity), Zoogloea caeni EMB43T (98.2 %), Zoogloea oryzae A-7T (97.7 %), Zoogloea ramigera IAM 12136T (96.9 %) and Zoogloea oleivorans BucT (96.2 %). The major respiratory quinone was ubiquinone-8 and the principal polar lipids were phosphatidylethanolamine, phosphatidyl-N-methylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. The predominant cellular fatty acids were summed feature 3 (iso-C15 :0 2-OH/C16  : 1 ω7c) and C16 : 0. The DNA G+C content was 65.9 mol%. The average nucleotide identity and digital DNA-DNA hybridization relatedness values between strain G-4-1-14T and other type strains were ≤81.6 and ≤24.9 %, respectively, which are below the species demarcation thresholds. Based on the results of phenotypic, phylogenetic and genomic analyses, strain G-4-1-14T represents a novel species in the genus Zoogloea, for which the name Zoogloea dura sp. nov. is proposed. The type strain is G-4-1-14T (=KACC 21618T=NBRC 114358T). In addition, we propose emendation of the genus Zoogloea and the species Zoogloea oryzae and Zoogloea ramigera.

The mixotrophic denitrification characteristics of Zoogloea sp. L2 accelerated by the redox mediator of 2-hydroxy-1,4-naphthoquinone.

Denitrification in mixed culture system has been extensively researched to date, but few studies have focused on accelerating the process using redox mediators to promote electron transfer. Strain L2, an iron-reducing bacteria, can remove 75.44% of nitrate under temperature of 30.60 °C, pH of 6.75 and Fe2+ concentration of 27.86 mg·L-1. Additionally, the removal rate of nitrate reached 1.516 mg·L-1·h-1 in 8 h with the addition of 0.030 mmol·L-1 2-hydroxy-1,4-naphthoquinone (HNQ), which increased by 1.38 times than control group. Furthermore, analysis by fluorescence spectroscopy, flow cytometer and gas chromatography demonstrated that HNQ positively stimulated denitrification. This study provides a reference for enhancing denitrification in mixed culture and lays the foundation for the practical application of redox mediators in groundwater treatment.

Genome analysis provides insights into microaerobic toluene-degradation pathway of Zoogloea oleivorans BucT.

Zoogloea oleivorans, capable of using toluene as a sole source of carbon and energy, was earlier found to be an active degrader under microaerobic conditions in aquifer samples. To uncover the genetic background of the ability of microaerobic toluene degradation in Z. oleivorans, the whole-genome sequence of the type strain BucT was revealed. Metatranscriptomic sequence reads, originated from a previous SIP study on microaerobic toluene degradation, were mapped on the genome. The genome (5.68 Mb) had a mean G + C content of 62.5%, 5005 protein coding gene sequences and 80 RNA genes. Annotation predicted that 66 genes were involved in the metabolism of aromatic compounds. Genome analysis revealed the presence of a cluster with genes coding for a multicomponent phenol-hydroxylase system and a complete catechol meta-cleavage pathway. Another cluster flanked by mobile-element protein coding genes coded a partial catechol meta-cleavage pathway including a subfamily I.2.C-type extradiol dioxygenase. Analysis of metatranscriptomic data of a microaerobic toluene-degrading enrichment, containing Z . oleivorans as an active-toluene degrader revealed that a toluene dioxygenase-like enzyme was responsible for the ring-hydroxylation, while enzymes of the partial catechol meta-cleavage pathway coding cluster were responsible for further degradation of the aromatic ring under microaerobic conditions. This further advances our understanding of aromatic hydrocarbon degradation between fully oxic and strictly anoxic conditions.

Both widespread PEP-CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP-CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK-PrsR two-component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc-forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post-translational modifications, could not rescue the long-rod morphology of the rpoN mutant. Consistently, PEP-CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc-forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.

[Operational Performance and Microbial Community Structure in a Completely Mixed Aeration System].

A completely mixed aeration system was introduced in domestic sewage treatment, and the simultaneous nitrification and denitrification process and microbial community structure were further investigated. The pilot-scale experimental results showed that the operation of the system was stable and the average chemical oxygen demand (COD), NH4+-N and total nitrogen (TN) removal were 93.2%, 96.9% and 75.2%, respectively. The effluent COD, NH4+-N and TN were better than the standard of level A stipulated by Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant (GB 18918-2002) in the absence of external carbon source. The sludge derived from the system showed a significant denitrification capacity and the denitrification rate was 2.86 times higher than that in the wastewater treatment plant. In addition, the existence of aerobic denitrifying bacteria in the system was confirmed by the PCR amplification of genes encoding perplasmic nitrate reductases. The high-throughput sequencing results revealed that Zoogloea, Thauera and Dechloromonas were dominant in the completely mixed aeration system.

Comparative genomics analyses on EPS biosynthesis genes required for floc formation of Zoogloea resiniphila and other activated sludge bacteria.

Activated sludge (AS) process has been widely utilized for municipal sewage and industrial wastewater treatment. Zoolgoea and its related floc-forming bacteria are required for formation of AS flocs which is the key to gravitational effluent-and-sludge separation and AS recycling. However, little is known about the genetics, biochemistry and physiology of Zoogloea and its related bacteria. This report deals with the comparative genomic analyses on two Zoogloea resiniphila draft genomes and the closely related proteobacterial species commonly found in AS. In particular, the metabolic processes involved in removal of organic matters, nitrogen and phosphorus were analyzed. Furthermore, it is revealed that a large gene cluster, encoding eight glycosyltransferases and other proteins involved in biosynthesis and export of extracellular polysaccharides (EPS), was required for floc formation. One of the two asparagine synthase paralogues, associated with this EPS biosynthesis gene cluster, was required for floc formation in Zoogloea. Similar EPS biosynthesis gene cluster(s) were identified in the genome of other AS proteobacteria including polyphosphate-accumulating Candidatus Accumulibacter phosphatis (CAP) and nitrifying Nitrosopira and Nitrosomonas bacteria, but the gene composition varies interspecifically and intraspecifically. Our results indicate that floc formation of desired AS bacteria, including CAP strains, facilitate their recruitment into AS and gradual enrichment via repeated AS settling and recycling processes.

Effects of DO levels on surface force, cell membrane properties and microbial community dynamics of activated sludge.

In this paper, we employ atomic force microscopy (AFM), fluorescence recovery after photobleaching (FRAP) technique, phospholipid fatty acids (PLFA) and MiSeq analysis to study the effects of traditional dissolved oxygen (DO) levels (0.71-1.32mg/L, 2.13-3.02mg/L and 4.31-5.16mg/L) on surface force, cell membrane properties and microbial community dynamics of activated sludge. Results showed that low DO level enhanced the surface force and roughness of activated sludge; the medium DO level decreased cell membrane fluidity by reducing the synthesis of branched fatty acids in the cell membrane; high DO level resulted in the highest protein content in the effluent by EEM scanning. Abundance of Micropruina, Zoogloea and Nakamurella increased and Paracoccus and Rudaea decreased with the increase of DO levels. RDA analysis suggested that saturated fatty acids (SFA), anteiso-fatty acids (AFA) and iso-fatty acids (IFA) were closely related to effluent quality as well as some genera.

Electrical stimulation on biodegradation of phenol and responses of microbial communities in conductive carriers supported biofilms of the bioelectrochemical reactor.

Conductive carbon felts (Cf) were used as biofilm carriers in bioelectrochemical reactors to enhance the electrical stimulation on treatment of phenol-containing synthetic wastewater. In batch test, phenol biodegradation was accelerated under an optimum direct current (DC), which was 2mA for Cf biofilm carriers, lower than that for non-conductive white foam carriers. The stimulation effect was consistent with Adenosine Triphosphate contents in biofilms. The long-term operation further demonstrated that a high and stable phenol removal efficiency could be achieved with applied DC of 2mA, and intermittent DC application was better than continuous one, with phenol removal efficiency of over 97%. Although the quantities of whole microbial communities kept at a high level under all conditions, special microorganisms related with genera of Zoogloea and Desulfovibrio were distinctively enriched under intermittent applied DC pattern. This study shows that the electrical stimulation is potentially effective for biofilm reactors treating phenol-containing wastewater.

Nitrogen Removal Characteristics of a Newly Isolated Indigenous Aerobic Denitrifier from Oligotrophic Drinking Water Reservoir, Zoogloea sp. N299.

Nitrogen is considered to be one of the most widespread pollutants leading to eutrophication of freshwater ecosystems, especially in drinking water reservoirs. In this study, an oligotrophic aerobic denitrifier was isolated from drinking water reservoir sediment. Nitrogen removal performance was explored. The strain was identified by 16S rRNA gene sequence analysis as Zoogloea sp. N299. This species exhibits a periplasmic nitrate reductase gene (napA). Its specific growth rate was 0.22 h-1. Obvious denitrification and perfect nitrogen removal performances occurred when cultured in nitrate and nitrite mediums, at rates of 75.53%±1.69% and 58.65%±0.61%, respectively. The ammonia removal rate reached 44.12%±1.61% in ammonia medium. Zoogloea sp. N299 was inoculated into sterilized and unsterilized reservoir source waters with a dissolved oxygen level of 5-9 mg/L, pH 8-9, and C/N 1.14:1. The total nitrogen removal rate reached 46.41%±3.17% (sterilized) and 44.88%±4.31% (unsterilized). The cell optical density suggested the strain could survive in oligotrophic drinking water reservoir water conditions and perform nitrogen removal. Sodium acetate was the most favorable carbon source for nitrogen removal by strain N299 (p<0.05). High C/N was beneficial for nitrate reduction (p<0.05). The nitrate removal efficiencies showed no significant differences among the tested inoculums dosage (p>0.05). Furthermore, strain N299 could efficiently remove nitrate at neutral and slightly alkaline and low temperature conditions. These results, therefore, demonstrate that Zoogloea sp. N299 has high removal characteristics, and can be used as a nitrogen removal microbial inoculum with simultaneous aerobic nitrification and denitrification in a micro-polluted reservoir water ecosystem.

Zoogloea oleivorans sp. nov., a floc-forming, petroleum hydrocarbon-degrading bacterium isolated from biofilm.

A floc-forming, Gram-stain-negative, petroleum hydrocarbon-degrading bacterial strain, designated Buc(T), was isolated from a petroleum hydrocarbon-contaminated site in Hungary. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Buc(T) formed a distinct phyletic lineage within the genus Zoogloea. Its closest relative was found to be Zoogloea caeni EMB43(T) (97.2% 16S rRNA gene sequence similarity) followed by Zoogloea oryzae A-7(T) (95.9%), Zoogloea ramigera ATCC 19544(T) (95.5%) and Zoogloea resiniphila DhA-35(T) (95.4%). The level of DNA-DNA relatedness between strain Buc(T) and Z. caeni EMB43(T) was 31.6%. Cells of strain Buc(T) are facultatively aerobic, rod-shaped, and motile by means of a polar flagellum. The strain grew at temperatures of 5-35 °C (optimum 25-28 °C), and at pH 6.0-9.0 (optimum 6.5-7.5). The predominant fatty acids were C16:0, C10 : 0 3-OH, C12:0 and summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH). The major respiratory quinone was ubiquinone-8 (Q-8) and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 63.2 mol%. On the basis of the chemotaxonomic, molecular and phenotypic data, isolate Buc(T) is considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea oleivorans sp. nov. is proposed. The type strain is Buc(T) ( =DSM 28387(T) =NCAIM B 02570(T)).