Effects of soil pH on the growth, soil nutrient composition, and rhizosphere microbiome of Ageratina adenophora.

Ageratina adenophora is an invasive weed species found in many countries. Methods to control the spread of this weed have been largely unsuccessful. Soil pH is the most important soil factor affecting the availability of nutrients for plant and impacting its growth. Understanding the mechanisms of the influence of soil pH on the growth of A. adenophora may help to develop effective control measures. In this study, we artificially changed the soil pH in pot experiments for A. adenophora. We studied the effects of acidic (pH 5.5), weakly acidic (pH 6.5), neutral (pH 7.2), and alkaline (pH 9.0) soils on the growth, availability of soil nutrients, activity of antioxidant enzymes, levels of redox markers in the leaves, and the structure and diversity of the rhizosphere microbiome. Soil with a pH 7.2 had a higher (47.8%) below-ground height versus soils of pH 5.5 at day 10; plant had a higher (11.3%) above-ground height in pH 7.2 soils than pH 9.0 soils at day 90; no differences in the fresh and dry weights of its above- and belowground parts, plant heights, and root lengths were observed in plants growing in acid, alkaline, or neutral pH soil were observed at day 180. Correspondingly, the antioxidant enzymes SOD (superoxide dismutase), POD (peroxidase), CAT (catalase) and redox markers GSH (glutathione) and MDA (malondialdehyde) were measured in the leaves. Significant differences existed in the activities of CAT and the levels of GSH between those growing in acidic and alkaline soils and those in neutral pH soil at day 90; however, only lower (36.8%) CAT activities in those grown at pH 5.5 than those grown at pH 7.2 were found at day 180. Similarly, significant differences in available P (16.89 vs 3.04 mg Kg-1) and total K (3.67 vs 0.96 mg Kg-1), total P (0.37 vs 0.25 g Kg-1) and total N (0.45 vs 1.09 g Kg-1) concentrations were found between the rhizosphere soils of A. adenophora grown at pH 9.0 and 7.2 at day 90; no such differences were seen at day 180. High throughput analyses of the 16S rRNA and ITS fragments showed that the rhizosphere microbiome diversity and composition under different soil pH conditions changed over 180 days. The rhizosphere microbiomes differed in diversity, phylum, and generic composition and population interactions under acid and alkaline conditions versus those grown in neutral soils. Soil pH had a greater impact on the diversity and composition of the prokaryotic rhizosphere communities than those of the fungal communities. A. adenophora responded successfully to pH stress by changing the diversity and composition of the rhizosphere microbiome to maintain a balanced nutrient supply to support its normal growth. The unusual pH tolerance of A. adenophora may be one crucial reason for its successful invasion. Our results suggest that attempts use soil pH to control its invasion by changing the soil pH (for example, using lime) will fail.

Screening plant growth-promoting bacteria from the rhizosphere of invasive weed Ageratina adenophora for crop growth.

Plant-growth promoting rhizobacteria (PGPR) play a vital role in soil fertility and crop production. The rhizosphere of many crop plants has been well documented by screening PGPR for their plant-growth promoting (PGP) mechanisms. However, the rhizosphere of grass species that may act as potential habitats for novel PGPR remains relatively unexplored. Ageratina adenophora is a noxious weed that has invaded more than 40 tropical and subtropical countries in Asia, Oceania, Africa, and Europe. Its presence has led to changes in plant species composition, reducing their biodiversity and destroying ecosystem function. In this study, we screened 1,200 bacterial strains isolated from the rhizosphere soil of A. adenophora in three floristic regions in Yunnan Province, China. Samples were screened for their in vitro ability for N-fixation, production of the plant growth regulator indole-3-acetic acid (IAA), and the synthesis of 1-amino-cyclopropane-1-carboxylate (ACC) deaminase, which controls the levels of ethylene in developing plant roots. We found that 144 strains showed at least one of these PGP attributes. 16S rRNA gene sequencing showed that most (62.5%) of the samples were bacteria closely related to members of the genera Pseudomonas (27 strains), Providencia (20 strains), Chryseobacterium (14 strains), Ensifer (12 strains), Enterobacter (nine strains), and Hafnia (eight strains). Their abundance and biodiversity in the soil of individual floristic regions correlate positively with the invasion history of A. adenophora. From these PGP bacterial strains, KM_A34 (Pantoea agglomerans), KM_C04 (Enterobacter asburiae), and KM_A57 (Pseudomonas putida), which had the greatest in vitro ability of N-fixation, and IAA and ACC deaminase production, respectively, were selected. The strains were evaluated for their effect on the seed germination and growth of soybean, faba bean, pea, wheat, and Chinese cabbage other than A. adenophora. Chamber experiments showed these strains significantly (P < 0.05) increased (14.2-43.4% over the controls) germination rates of the soybean, faba bean, pea, and/or Chinese cabbage seeds. They also reduced relative seed germination times (20.8-48.8% over the controls) of soy bean, faba bean and/or wheat seeds. Greenhouse pot experiments showed that they significantly (P < 0.05) promoted the aboveground and belowground height of plant foliage (12.1-23.1% and 11.5-31.4% over the controls, respectively) and/or the dry weights (16.1-33.5% and 10.6-23.4% over the controls, respectively) of the soy bean, faba bean, pea, wheat and/or Chinese cabbage. These data indicate that the rhizosphere microbiota of A. adenophora contain a PGPR pool that may be used as bioinoculants to improve the growth and productivity of these crops.

Gordonia pseudamarae sp. nov., a home for novel actinobacteria isolated from stable foams on activated sludge wastewater treatment plants.

The taxonomic status of two Gordonia strains, designated BEN371 and CON9T, isolated from stable foams on activated sludge plants was the subject of a polyphasic study which also included the type strains of Gordonia species and three authenticated Gordonia amarae strains recovered from such foams. Phylogenetic analyses of 16S rRNA gene sequences showed that these isolates formed a compact cluster suggesting a well-supported lineage together with a second branch containing the G. amarae strains. A phylogenomic tree based on sequences of 92 core genes extracted from whole genome sequences of the isolates, the G. amarae strains and Gordonia type strains confirmed the assignment of the isolates and the G. amarae strains to separate but closely associated lineages. Average nucleotide index (ANI) and digital DNA-DNA hybridisation (dDDH) similarities showed that BEN371 and CON9T belonged to the same species and had chemotaxonomic and morphological features consistent with their assignment to the genus Gordonia. The isolates and the G. amarae strains were distinguished using a range of phenotypic features and by low ANI and dDDH values of 84.2 and 27.0 %, respectively. These data supplemented with associated genome characteristics show that BEN371 and CON9T represent a novel species of the genus Gordonia. The name proposed for members of this taxon is Gordonia pseudamarae sp. nov. with isolate CON9T (=DSM 43602T=JCM 35249T) as the type strain.

The phylogeny, ecology and ecophysiology of the glycogen accumulating organism (GAO) Defluviicoccus in wastewater treatment plants.

This comprehensive review looks critically what is known about members of the genus Defluviicoccus, an example of a glycogen accumulating organism (GAO), in wastewater treatment plants, but found also in other habitats. It considers the operating conditions thought to affect its performance in activated sludge plants designed to remove phosphorus microbiologically, including the still controversial view that it competes with the polyphosphate accumulating bacterium Ca. Accumulibacter for readily biodegradable substrates in the anaerobic zone receiving the influent raw sewage. It looks at its present phylogeny and what is known about it's physiology and biochemistry under the highly selective conditions of these plants, where the biomass is recycled continuously through alternative anaerobic (feed); aerobic (famine) conditions encountered there. The impact of whole genome sequence data, which have revealed considerable intra- and interclade genotypic diversity, on our understanding of its in situ behaviour is also addressed. Particular attention is paid to the problems in much of the literature data based on clone library and next generation DNA sequencing data, where Defluviicoccus identification is restricted to genus level only. Equally problematic, in many publications no attempt has been made to distinguish between Defluviicoccus and the other known GAO, especially Ca. Competibacter, which, as shown here, has a very different ecophysiology. The impact this has had and continues to have on our understanding of members of this genus is discussed, as is the present controversy over its taxonomy. It also suggests where research should be directed to answer some of the important research questions raised in this review.

Comparative Genomics of Members of the Genus Defluviicoccus With Insights Into Their Ecophysiological Importance.

Members of the genus Defluviicoccus occur often at high abundances in activated sludge wastewater treatment plants designed to remove phosphorus, where biomass is subjected to alternating anaerobic feed/aerobic famine conditions, believed to favor the proliferation of organisms like Ca. Accumulibacter and other phosphate-accumulating organisms (PAO), and Defluviicoccus. All have a capacity to assimilate readily metabolizable substrates and store them intracellularly during the anaerobic feed stage so that under the subsequent famine aerobic stage, these can be used to synthesize polyphosphate reserves by the PAO and glycogen by Defluviicoccus. Consequently, Defluviicoccus is described as a glycogen-accumulating organism or GAO. Because they share a similar anaerobic phenotype, it has been proposed that at high Defluviicoccus abundance, the PAO are out-competed for assimilable metabolites anaerobically, and hence aerobic P removal capacity is reduced. Several Defluviicoccus whole genome sequences have been published (Ca. Defluviicoccus tetraformis, Defluviicoccus GAO-HK, and Ca. Defluviicoccus seviourii). The available genomic data of these suggest marked metabolic differences between them, some of which have ecophysiological implications. Here, we describe the whole genome sequence of the type strain Defluviicoccus vanusT , the only cultured member of this genus, and a detailed comparative re-examination of all extant Defluviicoccus genomes. Each, with one exception, which appears not to be a member of this genus, contains the genes expected of GAO members, in possessing multiple copies of those for glycogen biosynthesis and catabolism, and anaerobic polyhydroxyalkanoate (PHA) synthesis. Both 16S rRNA and genome sequence data suggest that the current recognition of four clades is insufficient to embrace their phylogenetic biodiversity, but do not support the view that they should be re-classified into families other than their existing location in the Rhodospirillaceae. As expected, considerable variations were seen in the presence and numbers of genes encoding properties associated with key substrate assimilation and metabolic pathways. Two genomes also carried the pit gene for synthesis of the low-affinity phosphate transport protein, pit, considered by many to distinguish all PAO from GAO. The data re-emphasize the risks associated with extrapolating the data generated from a single Defluviicoccus population to embrace all members of that genus.

In vitro co-metabolism of epigallocatechin-3-gallate (EGCG) by the mucin-degrading bacterium Akkermansia muciniphila.

Akkermansia muciniphila is a Gram-negative bacterium that resides within the gut mucus layer, and plays an important role in promoting gut barrier integrity, modulating the immune response and inhibiting gut inflammation. Growth stimulation of A. muciniphila by polyphenols including epigallocatechin-3-gallate (EGCG) from difference sources is well-documented. However, no published in vitro culture data on utilization of polyphenols by A. muciniphila are available, and the mechanism of growth-stimulating prebiotic effect of polyphenols on it remains unclear. Here in vitro culture studies have been carried out on the metabolism of EGCG by A. muciniphila in the presence of either mucin or glucose. We found that A. muciniphila did not metabolize EGCG alone but could co-metabolize it together with both these substrates in the presence of mineral salts and amino acids for mucin and protein sources for glucose. Our metabolomic data show that A. muciniphila converts EGCG to gallic acid, epigallocatechin, and (-)-epicatechin through ester hydrolysis. The (-)-epicatechin formed is then further converted to hydroxyhydroquinone. Co-metabolism of A. muciniphila of EGCG together with either mucin or glucose promoted substantially its growth, which serves as a further demonstration of the growth-promoting effect of polyphenols on A. muciniphila and provides an important addition to the currently available proposed mechanisms of polyphenolic prebiotic effects on A. muciniphila.

Compositional and functional profiling of the rhizosphere microbiomes of the invasive weed Ageratina adenophora and native plants.

The rhizosphere soil microbiome (RSM) plays an important role in the nutritional metabolism of the exotic weed Ageratina adenophora. However, our understanding of the composition and metabolic activity of this microbiome is limited. We used high-throughput sequencing of bacterial 16S rRNA genes and fungal internal transcribed spacer fragments in combination with transcriptome analysis to compare the composition and metabolic features of the RSMs of A. adenophora and the native plant species Artemisia indica and Imperata cylindrica. A. indica cohabitates with the weed and I. cylindrica grows in uninvaded soil areas. We found fungi belonging to the phyla Ascomycota and Basidiomycota and bacteria belonging to the phyla Proteobacteria, Acidobacteria and Bacteroidetes were highly abundant in the RSMs of A. adenophora and both native plant species. The RSM of A. adenophora differed to varying degrees in the relative abundances of bacterial and fungal phyla and genera, and in levels of expression of functional genes from those of both the native species. The RSM of A. adenophora was more metabolically active than both of these, as indicated by marked increases in the expression levels of genes associated with cell wall, membrane, and envelope biogenesis, energy production and conversion, and the transport and metabolism of carbohydrates, amino acids, coenzymes, nucleotides, and secondary metabolites. Ascomycota and Basidiomycota contributed most significantly to these differences. The composition and metabolic activities of A. adenophora RSM differed less to the RSM of A. indica than to the RSM of I. cylindrica. Fungal communities contributed most to the metabolic genes in the RSM of A. adenophora. These included the arbuscular mycorrhizal fungi Glomeromycota. The different relative abundances in the RSMs of these three plant populations may explain why A. adenophora is more successful in colonizing soils than the two native populations.

Aqueous raw and ripe Pu-erh tea extracts alleviate obesity and alter cecal microbiota composition and function in diet-induced obese rats.

Pu-erh tea is attracting increased attention worldwide because of its unique flavor and health effects, but its impact on the composition and function of the gut microbiota remains unclear. The aim of this study was to investigate the effects of aqueous extracts of fermented (ripe) and non-fermented (raw) Pu-erh teas on the composition and function of the intestinal microbiota of rats with diet-induced obesity. We conducted a comparative metagenomic and meta-proteomic investigation of the microbial communities in cecal samples taken from obese rats treated with or without extracts of raw or ripe Pu-erh teas. By analyzing the composition and diversity of 16S rRNA amplicons and expression profiles of 814 distinct proteins, we found that despite differences in the chemical compositions of raw and ripe Pu-erh teas, administration of either tea at two doses (0.15- and 0.40-g/kg body weight) significantly (P < 0.05) increased microbial diversity and changed the composition of cecal microbiota by increasing the relative abundances of Firmicutes and decreasing those of Bacteroidetes. Community metabolic processes, including sucrose metabolism, glycolysis, and syntheses of proteins, rRNAs, and antibiotics were significantly (P < 0.05) promoted or had a tendency (0.10 < P < 0.05) to be promoted due to the enrichment of relevant enzymes. Furthermore, evidence at population, molecular, and metabolic levels indicated that polyphenols of raw Pu-erh tea and their metabolites potentially promote Akkermansia muciniphila growth by stimulating a type II and III secretion system protein, the elongation factor Tu, and a glyceraldehyde-3-phosphate dehydrogenase. This study provides new evidence for the prebiotic effects of Pu-erh tea.

Effects of different carbon sources on enhanced biological phosphorus removal and "Candidatus Accumulibacter" community composition under continuous aerobic condition.

Previous studies have shown that enhanced biological phosphorus removal (EBPR) performance under continuous aerobic conditions always eventually deteriorates; however, the speed at which this happens depends on the carbon source supplied. The published data suggest that propionate is a better carbon source than acetate is for maintaining operational stability, although it is not clear why. A lab-scale sequencing batch reactor was run initially under conventional anaerobic/aerobic conditions with either acetate or propionate as the carbon source. Chemical and microbiological analyses revealed that both sources performed as expected for such systems. When continuous aerobic conditions were imposed on both these established communities, marked shifts of the "Candidatus Accumulibacter" clades were recorded for both carbon sources. Here, we discuss whether this shift could explain the prolonged EBPR stability observed with propionate.

Metabolic model for the filamentous 'Candidatus Microthrix parvicella' based on genomic and metagenomic analyses.

'Candidatus Microthrix parvicella' is a lipid-accumulating, filamentous bacterium so far found only in activated sludge wastewater treatment plants, where it is a common causative agent of sludge separation problems. Despite attracting considerable interest, its detailed physiology is still unclear. In this study, the genome of the RN1 strain was sequenced and annotated, which facilitated the construction of a theoretical metabolic model based on available in situ and axenic experimental data. This model proposes that under anaerobic conditions, this organism accumulates preferentially long-chain fatty acids as triacylglycerols. Utilisation of trehalose and/or polyphosphate stores or partial oxidation of long-chain fatty acids may supply the energy required for anaerobic lipid uptake and storage. Comparing the genome sequence of this isolate with metagenomes from two full-scale wastewater treatment plants with enhanced biological phosphorus removal reveals high similarity, with few metabolic differences between the axenic and the dominant community 'Ca. M. parvicella' strains. Hence, the metabolic model presented in this paper could be considered generally applicable to strains in full-scale treatment systems. The genomic information obtained here will provide the basis for future research into in situ gene expression and regulation. Such information will give substantial insight into the ecophysiology of this unusual and biotechnologically important filamentous bacterium.

A metabolic model for members of the genus Tetrasphaera involved in enhanced biological phosphorus removal.

Members of the genus Tetrasphaera are considered to be putative polyphosphate accumulating organisms (PAOs) in enhanced biological phosphorus removal (EBPR) from wastewater. Although abundant in Danish full-scale wastewater EBPR plants, how similar their ecophysiology is to 'Candidatus Accumulibacter phosphatis' is unclear, although they may occupy different ecological niches in EBPR communities. The genomes of four Tetrasphaera isolates (T. australiensis, T. japonica, T. elongata and T. jenkinsii) were sequenced and annotated, and the data used to construct metabolic models. These models incorporate central aspects of carbon and phosphorus metabolism critical to understanding their behavior under the alternating anaerobic/aerobic conditions encountered in EBPR systems. Key features of these metabolic pathways were investigated in pure cultures, although poor growth limited their analyses to T. japonica and T. elongata. Based on the models, we propose that under anaerobic conditions the Tetrasphaera-related PAOs take up glucose and ferment this to succinate and other components. They also synthesize glycogen as a storage polymer, using energy generated from the degradation of stored polyphosphate and substrate fermentation. During the aerobic phase, the stored glycogen is catabolized to provide energy for growth and to replenish the intracellular polyphosphate reserves needed for subsequent anaerobic metabolism. They are also able to denitrify. This physiology is markedly different to that displayed by 'Candidatus Accumulibacter phosphatis', and reveals Tetrasphaera populations to be unusual and physiologically versatile PAOs carrying out denitrification, fermentation and polyphosphate accumulation.

Filamentous members of cluster III Defluviicoccus have the in situ phenotype expected of a glycogen-accumulating organism in activated sludge.

The in situ ecophysiology of alphaproteobacterial filamentous Cluster III Defluviicoccus present in enhanced biological phosphorus removal (EBPR)-activated sludge systems was evaluated using FISH-MAR and histochemical staining methods. These organisms, sharing the Nostocoida limicola morphotype, are known to be responsible for serious episodes of activated sludge bulking. The data presented here also demonstrate an ability to assimilate short-chain fatty acids and synthesize poly-ß-hydroxyalkanoates (PHA) anaerobically, and then utilize this stored PHA under aerobic conditions, but with no corresponding synthesis of polyphosphate. These features are consistent with an in situ phenotype of glycogen-accumulating organisms (GAO), populations thought to lower the efficiency of EBPR systems by outcompeting polyphosphate-accumulating organisms (PAO) for substrates in their anaerobic feed phase. Survey data indicate that these GAO are as commonly seen as the known PAO in full-scale EBPR-activated sludge systems, which suggest that they might play important roles there, and therefore should not be viewed just as laboratory curiosities.

Candidatus Monilibacter spp., common bulking filaments in activated sludge, are members of Cluster III Defluviicoccus.

Two alphaproteobacterial Neisser negative 'Nostocoida limicola' morphotypes differing slightly in their trichome diameter and filament regularity were dominant populations in the Bendigo, Victoria, Australia activated sludge community removing phosphorus (P). Neither responded to the FISH probes available for any of the other alphaproteobacterial 'N. limicola' morphotypes. Instead both fluoresced with the DF988 FISH probe designed originally to target alphaproteobacterial cluster II Defluviicoccus tetrad forming organisms. A 16S rRNA based clone library from this biomass revealed that the alphaproteobacterial clones grouped closely with Candidatus 'Monilibacter batavus' and Defluviicoccus clones in a cluster separate from the existing cluster I and II Defluviicoccus. When a FISH probe was designed against these, it only hybridized to the thinner and less abundant 'N. limicola' morphotype. Micromanipulation-RT-PCR was used to selectively recover the main 'N. limicola' morphotype and a FISH probe designed against the 16S rRNA clones generated from it showed only this filament fluoresced. From FISH based surveys, both 'N. limicola' variants occurred frequently in phosphorus removal activated sludge systems in Australia treating domestic waste. The data suggest that they represent two new strains of Candidatus 'Monilibacter', which on this evidence are filamentous members of the genus Defluviicoccus, a potential competitor for the polyphosphate accumulating organisms in these communities.

Phylogeny and in situ identification of a novel gammaproteobacterium in activated sludge.

Failure of a continuously aerated sequencing batch reactor (SBR) pilot plant-enhanced biological phosphorus removal (EBPR) process, designed to remove phosphorus from the clarified effluent from a conventional non-EBPR wastewater treatment plant, was associated with the dominance (c. 50% of the biovolume) of gammaproteobacterial coccobacilli. Flow cytometry and subsequent clone library generation from an enriched population of these Gammaproteobacteria showed that their 16S rRNA genes were most similar to partial clone sequences obtained from an actively denitrifying SBR community, and from anaerobic : aerobic EBPR communities. Under the SBR operating conditions used here, these cells stained for poly-beta-hydroxyalkanoates, but never polyphosphate. Applying FISH probes designed against them in combination with microautoradiography showed that they could also assimilate acetate 'aerobically'. FISH analyses of biomass samples from the full-scale treatment plant providing the pilot plant feed showed that they were present there in high numbers. However, they were not detected by FISH in laboratory-scale communities of the same aerated laboratory-scale EBPR process even when EBPR had failed, or from several full-scale EBPR plants or other activated sludge processes.

Biomass granulation in an aerobic:anaerobic-enhanced biological phosphorus removal process in a sequencing batch reactor with varying pH.

Long-term influences of different steady-state pH conditions on microbial community composition were determined by fluorescence in situ hybridization (FISH) in a laboratory scale reactor configured for enhanced biological phosphorus removal (EBPR). Chemical profiles were consistent with shifts in populations from polyphosphate-accumulating organisms (PAO) to glycogen-accumulating organisms (GAO) when pH fell from pH 7.5 to 7.0 and then to 6.5. While biomass was both dispersed and flocculated at pH 7.5, almost complete granulation occurred gradually after pH was dropped to 7.0, and these granules increased in size as the pH was reduced further to 6.5. Reverting back to pH 7.5 led to granule breakdown and corresponding increases in anaerobic phosphate release. Granules consisted almost entirely of Accumulibacter PAO cells, while putative GAO populations were always present in small numbers. Results suggest that low pH may contribute to granulation under these operational conditions. While chemical profiles suggested the PAO:GAO balance was changing as pH fell, FISH failed to reveal any marked corresponding increase in GAO abundances. Instead, TEM evidence suggested the Accumulibacter PAO phenotype was becoming more like that of a GAO. These data show how metabolically adaptable the Accumulibacter PAO can be under anaerobic:aerobic conditions in being able to cope with marked changes in plant conditions. They suggest that decreases in EBPR capacity may not necessarily reflect shifts in community composition, but in the existing population metabolism.

Filamentous bacterium Eikelboom type 0092 in activated sludge plants in Australia is a member of the phylum Chloroflexi.

Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-beta-hydroxyalkanoates or polyphosphate.

Elucidating further phylogenetic diversity among the Defluviicoccus-related glycogen-accumulating organisms in activated sludge.

Glycogen-accumulating organisms (GAO) are thought to out-compete the polyphosphate-accumulating organisms (PAO) in activated sludge communities removing phosphate (P). Two GAO groups are currently recognized, the gammaproteobacterial Candidatus'Competibacter phosphatis', and the alphaproteobacterial Defluviicoccus vanus-related tetrad forming organisms (TFOs). Both are phylogenetically diverse based on their 16S rRNA sequences, with the latter currently considered to contain members falling into three distinct clusters. This paper identifies members of an additional fourth Defluviicoccus cluster from 16S rRNA gene clone library data obtained from a laboratory-scale activated sludge plant community removing P, and details FISH probes designed against them. Probe DF181A was designed to target a single sequence and DF181B designed against the remaining sequences in the cluster. Cells hybridizing with these probes in the biomass samples tested always appeared as either TFOs or in large clusters of small cocci. Members of the Defluviicoccus-related organisms were commonly found in full-scale wastewater treatments plants, sometimes as a dominant population.

The microbiology of phosphorus removal in activated sludge processes-the current state of play.

This review discusses critically what we know and would like to know about the microbiology of phosphorus (P) removal in activated sludge systems. In particular, the description of the genome sequences of two strains of the polyphosphate accumulating organism found in these processes, Candidatus 'Accumulibacter phosphatis', allows us to address many of the previously unanswered questions relating to how these processes behave, and to raise new questions about the microbiology of P removal. This article attempts to be deliberately speculative, and inevitably subjective, but hopefully at the same time useful to those who have an active interest in these environmentally very important processes.

Medicinal importance of fungal beta-(1-->3), (1-->6)-glucans.

Non-cellulosic beta-glucans are now recognized as potent immunological activators, and some are used clinically in China and Japan. These beta-glucans consist of a backbone of glucose residues linked by beta-(1-->3)-glycosidic bonds, often with attached side-chain glucose residues joined by beta-(1-->6) linkages. The frequency of branching varies. The literature suggests beta-glucans are effective in treating diseases like cancer, a range of microbial infections, hypercholesterolaemia, and diabetes. Their mechanisms of action involve them being recognized as non-self molecules, so the immune system is stimulated by their presence. Several receptors have been identified, which include: dectin-1, located on macrophages, which mediates beta-glucan activation of phagocytosis and production of cytokines, a response co-ordinated by the toll-like receptor-2. Activated complement receptors on natural killer cells, neutrophils, and lymphocytes, may also be associated with tumour cytotoxicity. Two other receptors, scavenger and lactosylceramide, bind beta-glucans and mediate a series of signal pathways leading to immunological activation. Structurally different beta-glucans appear to have different affinities toward these receptors and thus generate markedly different host responses. However, the published data are not always easy to interpret as many of the earlier studies used crude beta-glucan preparations with, for the most part, unknown chemical structures. Careful choice of beta-glucan products is essential if their benefits are to be optimized, and a better understanding of how beta-glucans bind to receptors should enable more efficient use of their biological activities.

Gordonia defluvii sp. nov., an actinomycete isolated from activated sludge foam.

Three strains of non-motile, Gram-positive, filamentous actinomycetes, isolates J4(T), J5 and J59, initially recognized microscopically in activated sludge foam by their distinctive branching patterns, were isolated by micromanipulation. The taxonomic positions of the isolates were determined using a polyphasic approach. Almost-complete 16S rRNA gene sequences of the isolates were aligned with corresponding sequences of representatives of the suborder Corynebacterineae and phylogenetic trees were inferred using three tree-making algorithms. The organisms formed a distinct phyletic line in the Gordonia 16S rRNA gene tree. The three isolates showed 16S rRNA gene sequence similarities within the range 96.9-97.2 % with their nearest phylogenetic neighbours, namely Gordonia bronchialis DSM 43247(T) and Gordonia terrae DSM 43249(T). Strain J4(T) was shown to have a chemotaxonomic profile typical of the genus Gordonia and was readily distinguished from representatives of the genus on the basis of Curie-point pyrolysis mass spectrometric data. The isolates shared nearly identical phenotypic profiles that distinguished them from representatives of the most closely related Gordonia species. It is evident from the genotypic and phenotypic data that the three isolates belong to a novel Gordonia species. The name proposed for this taxon is Gordonia defluvii sp. nov.; the type strain is J4(T) (=DSM 44981(T)=NCIMB 14149(T)).