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.

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)).

Influence of hydraulic regimes on bacterial community structure and composition in an experimental drinking water distribution system.

Microbial biofilms formed on the inner-pipe surfaces of drinking water distribution systems (DWDS) can alter drinking water quality, particularly if they are mechanically detached from the pipe wall to the bulk water, such as due to changes in hydraulic conditions. Results are presented here from applying 454 pyrosequencing of the 16S ribosomal RNA (rRNA) gene to investigate the influence of different hydrological regimes on bacterial community structure and to study the potential mobilisation of material from the pipe walls to the network using a full scale, temperature-controlled experimental pipeline facility accurately representative of live DWDS. Analysis of pyrosequencing and water physico-chemical data showed that habitat type (water vs. biofilm) and hydraulic conditions influenced bacterial community structure and composition in our experimental DWDS. Bacterial community composition clearly differed between biofilms and bulk water samples. Gammaproteobacteria and Betaproteobacteria were the most abundant phyla in biofilms while Alphaproteobacteria was predominant in bulk water samples. This suggests that bacteria inhabiting biofilms, predominantly species belonging to genera Pseudomonas, Zooglea and Janthinobacterium, have an enhanced ability to express extracellular polymeric substances to adhere to surfaces and to favour co-aggregation between cells than those found in the bulk water. Highest species richness and diversity were detected in 28 days old biofilms with this being accentuated at highly varied flow conditions. Flushing altered the pipe-wall bacterial community structure but did not completely remove bacteria from the pipe walls, particularly under highly varied flow conditions, suggesting that under these conditions more compact biofilms were generated. This research brings new knowledge regarding the influence of different hydraulic regimes on the composition and structure of bacterial communities within DWDS and the implication that this might have on drinking water quality.

Zoogloea caeni sp. nov., a floc-forming bacterium isolated from activated sludge.

Two floc-forming, nitrogen-fixing bacteria, strains EMB43(T) and EMB61, obtained from activated sludge of a domestic wastewater treatment plant in Korea, were characterized. The two strains were very closely related, sharing 99.7 % 16S rRNA gene sequence similarity and showing a level of DNA-DNA relatedness of 93 %, which suggests that they represent members of a single species. Phylogenetic analysis based on 16S rRNA gene sequences showed that the two novel isolates formed a distinct phyletic lineage within the genus Zoogloea and were related most closely to Zoogloea resiniphila DhA-35(T) and Zoogloea oryzae A-7(T), with sequence similarities of 97.2 %. Levels of DNA-DNA relatedness between strain EMB43(T) and Z. resiniphila DhA-35(T) and Z. oryzae A-7(T) were 12.8 and 7.4 %, respectively. Cells of strains EMB43(T) and EMB61 were facultatively aerobic, rod-shaped, Gram-negative and motile by means of a polar flagellum. The strains grew at temperatures of 15-40 degrees C (optimum: 25-30 degrees C) and at pH 6.0-9.0 (optimum: pH 6.5-7.5). The predominant fatty acids were C(16 : 0), C(10 : 0) 3-OH and summed feature 3 (C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH), and the predominant polar lipid was phosphatidylethanolamine. The genomic DNA G+C content was 64.9-65.0 mol% and the major isoprenoid quinone was ubiquinone-8 (Q-8). On the basis of phenotypic, chemotaxonomic and molecular data, the isolates are considered to represent a novel species of the genus Zoogloea, for which the name Zoogloea caeni sp. nov. is proposed. The type strain is EMB43(T) (=KCTC 22084(T)=DSM 19389(T)).

Impact of septic compounds and operational conditions on the microbiology of an activated sludge system.

In activated sludge (AS) biotreatment, septic compounds such as volatile organic acids and reduced sulphur compounds have been frequently cited as a major cause of Thiothrix and Type 021N filamentous bulking. These filaments are common in Canadian pulp and paper biotreatment systems, where they cause settling problems in secondary clarifiers. We conducted a 14-week study of a TMP/newsprint mill effluent to characterize the septic compounds entering the biotreatment, and to determine correlations with AS biomass characteristics and biotreatment operating parameters. A significant correlation was found between the sludge volume index, the abundance of Type 021N, and the propionic acid (PA) concentration in the primary clarified effluent. PA also induced a significant change in the flocculating bacteria size distribution determined by digital imaging. Consequently, the correlation observed between PA and Type 021N bulking is an indirect effect of inhibition of floc-forming microorganisms, giving a competitive advantage to filaments.

The impact of cleaner processing on nutrient availability in the bleached kraft industry.

The pulp and paper industry has invested heavily over recent years in cleaner processing, to reduce losses and minimise its impact on the environment. Over the past fifteen years, a New Zealand integrated bleached kraft mill has undergone a comprehensive programme of upgrades to increase production, reduce water consumption and streamline its biological treatment process. Whilst the overall discharge of contaminants from the site decreased, the treatment system performance did not show a concurrent improvement as may have been expected. Reduced BOD removal, low dissolved oxygen levels, and poor solids settlability were symptomatic of phosphorus limitation in the aerated lagoon treatment system. The wastewater entering the system was found to be phosphorus limited at a BOD:P ratio of 100:0.2. Mono-ammonium-phosphate was supplemented, at approximately 30 kg P/d, to raise the phosphorus levels to a BOD:P ratio of 100:0.3. Treatment efficiencies improved very quickly after phosphorus dosage, with a 50% reduction in BOD and TSS discharge, a significant increase in dissolved oxygen levels, and improved BOD removal (85% to 93%). This case study demonstrates that whilst more closed operation can result in reduced discharge of organic loads, there may be negative impacts on the availability of nutrients for balanced biological growth.

Zoogloea oryzae sp. nov., a nitrogen-fixing bacterium isolated from rice paddy soil, and reclassification of the strain ATCC 19623 as Crabtreella saccharophila gen. nov., sp. nov.

Two strains of free-living diazotrophs isolated from soil from a rice paddy field were characterized by using a polyphasic approach. The novel strains, A-7T and A-4, were found to be very closely related, with 99.9% 16S rRNA gene sequence similarity and a DNA-DNA hybridization value of 89.5%, suggesting that they represent a single species. 16S rRNA gene sequence analyses indicated that the two strains fell within the Zoogloea lineage, with less than 96.7 % sequence similarity to other Zoogloea species. Chemotaxonomic characteristics of the novel strains, including DNA G + C content (65.1 mol%), the major quinone system (Q-8), predominant fatty acids (16:1omega7c and 16:0) and major hydroxy fatty acids (3-OH 10:0 and 3-OH 12:0), are similar to those of the genus Zoogloea. The novel strains showed positive results for floc formation which is accepted as confirmatory for species of the genus Zoogloea. However, the novel strains can be distinguished from the other species of Zoogloea by physiological characteristics. The name Zoogloea oryzae sp. nov. is therefore proposed for the novel strains with strain A-7T (= IAM 15218T = CCTCC AB 2052005T) as the type strain. Phylogenetic and chemotaxonomic analyses indicate that strain ATCC 19623, designated as a reference strain of Zoogloea ramigera, does not belong to the genus Zoogloea but to a new genus of Alphaproteobacteria. The name Crabtreella saccharophila gen. nov., sp. nov. is proposed for strain ATCC 19623T (= IAM 12669T).

Shinella granuli gen. nov., sp. nov., and proposal of the reclassification of Zoogloea ramigera ATCC 19623 as Shinella zoogloeoides sp. nov.

The taxonomic position of a novel bacterial strain, Ch06T, isolated from an upflow anaerobic sludge blanket reactor was determined. Strain Ch06T was Gram-negative, aerobic, motile and oxidase- and catalase-positive. A comparative 16S rRNA gene sequence analysis showed a clear affiliation of strain Ch06T to the Alphaproteobacteria and it was most closely related to Zoogloea ramigera ATCC 19623 and Mycoplana dimorpha IAM 13154T (97.9 and 96.3% sequence similarity, respectively). The major respiratory quinone was Q-10 and the predominant fatty acids were C16:0, 3-OH C16:0, C18:0, C19:0 cyclo omega8c and summed feature 7 (C18:1omega7c/omega9t/omega12t, C18:1omega7c/omega9c/omega12t). On the basis of phenotypic, chemotaxonomic and phylogenetic characteristics, the novel isolate was assigned to a new genus, Shinella gen. nov., as Shinella granuli gen. nov., sp. nov. (type strain Ch06T=KCTC 12237T=JCM 13254T). It is proposed that Zoogloea ramigera ATCC 19623 is reclassified into the novel genus Shinella as Shinella zoogloeoides sp. nov. (type strain ATCC 19623T=IAM 12669T=I-16-MT).

Isolation and characterization of heterotrophic bacteria able to grow aerobically with quaternary ammonium alcohols as sole source of carbon and nitrogen.

The quaternary ammonium alcohols (QAAs) 2,3-dihydroxypropyl-trimethyl-ammonium (TM), dimethyl-diethanol-ammonium (DM) and methyl-triethanol-ammonium (MM) are hydrolysis products of their parent esterquat surfactants, which are widely used as softeners in fabric care. We isolated several bacteria growing with QAAs as the sole source of carbon and nitrogen. The strains were compared with a previously isolated TM-degrading bacterium, which was identified as a representative of the species Pseudomonas putida (Syst. Appl. Microbiol. 24 (2001) 252). Two bacteria were isolated with DM, referred to as strains DM 1 and DM 2, respectively. Based on 16S-rDNA analysis, they provided 97% (DM 1) and 98% (DM 2) identities to the closest related strain Zoogloea ramigera Itzigsohn 1868AL. Both strains were long, slim, motile rods but only DM 1 showed the floc forming activity, which is typical for representatives of the genus Zoogloea. Using MM we isolated a Gram-negative, non-motile rod referred to as strain MM 1. The 16S-rDNA sequence of the isolated bacterium revealed 94% identities (best match) to Rhodobacter sphaeroides only. The strains MM 1 and DM 1 exclusively grew with the QAA which was used for their isolation. DM 2 was also utilizing TM as sole source of carbon and nitrogen. However, all of the isolated bacteria were growing with the natural and structurally related compound choline.

Physiological and phylogenetic diversity of bacteria growing on resin acids.

Resin acids are tricyclic diterpenes which are synthesized by trees and are a major cause of toxicity of pulp mill effluents. Bacterial strains isolated from three different sources and which grow on resin acids were physiologically characterized. Eleven strains, representating distinct groups, were further characterized physiologically and phylogenetically. The isolates had distinct specificities for use, as growth substrates, of the different resin acids tested. The isolates also used fatty acids but were generally limited in use of other diverse substrates tested. According to their 16S rDNA sequences, the representative isolates are related to members of the genera, Sphingomonas, Zoogloea, Ralstonia, Burkholderia, Pseudomonas and Mycobacterium. Analysis of whole-cell fatty acid profiles generally supported those phylogenetic relationships. However, most of the isolated did not have high similarities to reference strains in the Microbial Identification System database of fatty acid profiles or in the Biolog database of substrate oxidation patterns. Described species of Sphingomonas, Zoolgoea, Burkholderia Pseudomonas, most closely related to the isolates we characterized, failed to grow on, or degrade, resin acids. We propose recognition of Zoogloea resiniphila sp. nov., Pseudomonas vancouverensis sp. nov., P. abietaniphila sp. nov. and P. multiresinivorans sp. nov.

Proposal to reclassify Zoogloea ramigera IAM 12670 (P. R. Dugan 115) as Duganella zoogloeoides gen. nov., sp. nov.

The taxonomic position of a misclassified strain, Zoogloea remigera IAM 12670T (= ATCC 25925T = P. R. Dugan 115T), was reevaluated. A phylogenetic analysis based on 16S ribosomal rDNA sequences revealed that this organism was located in the beta subclass of the class Proteobacteria with members of the genus Telluria as its closest relatives. On the basis of phenotypic and phylogenetic information, we propose that this organism should be reclassified in a new taxon with the name Duganella zoogloeoides gen. nov., sp. nov.

Diversity and distribution of subterranean bacteria in groundwater at Oklo in Gabon, Africa, as determined by 16S rRNA gene sequencing.

This paper describes how ground water was sampled, DNA extracted, amplified and cloned and how information available in the ribosomal 16S rRNA gene was used for mapping diversity and distribution of subterranean bacteria in groundwater at the Bangombé site in the Oklo region. The results showed that this site was inhabited by a diversified population of bacteria. Each borehole was dominated by species that did not dominate in any of the other boreholes; a result that probably reflects documented differences in the geochemical environment. Two of the sequences obtained were identified at genus level to represent Acinetobacter and Zoogloea, but most of the 44 sequences found were only distantly related to species in the DNA database. The deepest borehole, BAX01 (105 m), had the highest number of bacteria and also total organic carbon (TOC). This borehole harboured only Proteobacteria beta group sequences while sequences related to Proteobacteria beta, gamma and delta groups and Gram-positive bacteria were found in the other four boreholes. Two of the boreholes, BAX02 (34 m) and BAX04 (10 m) had many 16S rRNA gene sequences in common and also had similar counts of bacteria, content of TOC, pH and equal conductivity, suggesting a hydraulic connection between them.

The abundance of Zoogloea ramigera in sewage treatment plants.

Zoogloea ramigera has long been considered the typical activated sludge bacterium responsible for the formation of activated sludge flocs. On the basis of the results of a comparative sequence analysis, we designed three oligonucleotide probes complementary to characteristic regions of the 16S rRNAs of Z. ramigera ATCC 19544T (T = type strain) and two misclassified strains, Z. ramigera ATCC 25935 and ATCC 19623. Dissociation temperatures were determined, and probe specificities, as well as the potential of probes for whole-cell hybridization, were evaluated by using numerous reference organisms. Several activated sludge samples were examined with these probes by using both the in situ and dot blot hybridization methods. Only the type strain probe hybridized to cells that accumulated in the typical branched gelatinous matrices, the so-called Zoogloea fingers. This probe revealed cells in most of the activated sludge samples studied. We found that relatively high levels of Z. ramigera cells (up to approximately 10% of the total number of cells) and typical morphology tended to be linked to overloading of sewage plants. The probe directed to rejected type strain Z. ramigera ATCC 19623 bound to only a few cells. Cells that reacted with the probe complementary to Z. ramigera ATCC 25935, which was originally isolated from a trickling filter, were not observed in activated sludge.

Molecular systematics of the genus Zoogloea and emendation of the genus.

Phylogenetic relationships among strains of Zoogloea and related taxa were determined by 16S rDNA sequencing and genomic DNA hybridization techniques. The 16S rRNA gene was amplified by the polymerase chain reaction with a pair of eubacterial consensus primers and sequenced directly by using an automated fluorescent DNA sequencer. Sequence comparisons and distance matrix tree analysis revealed that Zoogloea ramigera IAM 12136 (= N. C. Dondero 106, type strain) and Zoogloea sp. ATCC 19324 formed a lineage with Rhodocyclus purpureus in the beta subclass of Proteobacteria. Z. ramigera IAM 12670 (= P. R. Dugan 115) was shown to belong to another cluster with Alcaligenes eutrophus and Pseudomonas cepacia in the beta subclass. In contrast, Z. ramigera IAM 12669 (= K. Crabtree I-16-M) proved to be a member of the alpha subclass of the Proteobacteria, closely related to Agrobacterium tumefaciens. Genomic DNA hybridization studies also showed that there is genetic diversity among the strains currently designated Z. ramigera, but typical Zoogloea strains, characterized by their production of rhodoquinones, are highly related to each other and can be regarded as a single species. On the basis of the molecular data, together with the early phenotypic and chemotaxonomic information, we have emended the generic description of Zoogloea.

Isoprenoid quinones and fatty acids of Zoogloea.

Nine Zoogloea strains including the type strain of Z. ramigera (IAM 12136 = ATCC 19544 = N.C. Dondero 106) and newly isolated strains were investigated for isoprenoid quinone composition and whole-cell fatty acid profiles. Seven of the tested strains, having phenotypic properties typical of Zoogloea, were characterized by their production of both ubiquinone-8 and rhodoquinone-8 as major quinones, whereas the remaining two strains, Z. ramigera IAM 12669 (= K. Crabtree I-16-M) and IAM 12670 (= P.R. Dugan 115), formed ubiquinone-10 and ubiquinone-8, respectively, as the sole quinone. All rhodoquinone-producing strains contained palmitoleic acid and 3-hydroxy-decanoic acid as the major components of nonpolar and hydroxylated fatty acids, respectively. Marked differences were noted in the fatty acid composition between the strains with and without rhodoquinones. The chemotaxonomic data suggested that the rhodoquinone-lacking strains should be excluded from the genus Zoogloea. Since there have been no reliable taxonomic tools for Zoogloea, rhodoquinone analysis may provide a new criterion of great promise for identifying Zoogloea strains.

Use of aromatic compounds for growth and isolation of Zoogloea.

Nine Zoogloea strains, were examined for their ability to utilize 35 aromatic compounds. Benzoate, m-toluate, and p-toluate, as well as phenol, o-cresol, m-cresol, and p-cresol, were utilized by eight strains. These strains exhibited meta cleavage of catechol and of methyl-substituted catechols. With the exception of L-tyrosine, none of the aromatic compounds tested supported growth of Z. ramigera ATCC 19623. A medium containing sodium m-toluate was used to isolate 37 zoogloea-forming bacteria from various polluted environments. The isolates were identified as strains of Zoogloea.