Lactone Molecules Research Articles

Genetic Differences in Barley Govern the Responsiveness to N-Acyl Homoserine Lactone

Enhanced resistance in barley (Hordeum vulgare) against pathogens, such as the powdery mildew-causing fungus Blumeria graminis f. sp. hordei, is of high importance. The beneficial effects of bacterial quorum sensing molecules on resistance and plant growth have been shown in different plant species. Here, we present the effects of the N-3-oxotetradecanoyl-l-homoserine lactone (oxo-C14-HSL) on the resistance of different barley genotypes. Genetically diverse accessions of barley were identified and exposed to the beneficial, oxo-C14-HSL-producing bacterium Ensifer meliloti or the pure N-acyl homoserine lactone (AHL) molecule. Metabolic profiling along with expression analysis of selected genes and physiological assays revealed that the capacity to react varies among different barley genotypes. We demonstrate that upon pretreatment with AHL molecule, AHL-primable barley genotype expresses enhanced resistance against B. graminis f. sp. hordei. We further show that pretreatment with AHL correlates with stronger activation of barley MAP kinases and regulation of defense-related PR1 and PR17b genes after a subsequent treatment with chitin. Noticeable was the stronger accumulation of lignin. Our results suggest that appropriate genetic background is required for AHL-induced priming. At the same time, they bear potential to use these genetic features for new breeding and plant protection approaches.

Relationship Between the Quorum Network (Sensing/Quenching) and Clinical Features of Pneumonia and Bacteraemia Caused by A. baumannii.

Acinetobacter baumannii (Ab) is one of the most important pathogens associated with nosocomial infections, especially pneumonia. Interest in the Quorum network, i.e., Quorum Sensing (QS)/Quorum Quenching (QQ), in this pathogen has grown in recent years. The Quorum network plays an important role in regulating diverse virulence factors such as surface motility and bacterial competition through the type VI secretion system (T6SS), which is associated with bacterial invasiveness. In the present study, we investigated 30 clinical strains of A. baumannii isolated in the “II Spanish Study of A. baumannii GEIH-REIPI 2000-2010” (Genbank Umbrella Bioproject PRJNA422585), a multicentre study describing the relationship between the Quorum network in A. baumannii and the development of pneumonia and associated bacteraemia. Expression of the aidA gene (encoding the AidA protein, QQ enzyme) was lower (P < 0.001) in strains of A. baumannii isolated from patients with bacteraemic pneumonia than in strains isolated from patients with non-bacteraemic pneumonia. Moreover, aidA expression in the first type of strain was not regulated in the presence of environmental stress factors such as the 3-oxo-C12-HSL molecule (substrate of AidA protein, QQ activation) or H2O2 (inhibitor of AidA protein, QS activation). However, in the A. baumannii strains isolated from patients with non-bacteraemic pneumonia, aidA gene expression was regulated by stressors such as 3-oxo-C12-HSL and H2O2. In an in vivo Galleria mellonella model of A. baumannii infection, the A. baumannii ATCC 17978 strain was associated with higher mortality (100% at 24 h) than the mutant, abaI-deficient, strain (carrying a synthetase enzyme of Acyl homoserine lactone molecules) (70% at 24 h). These data suggest that the QS (abaR and abaI genes)/QQ (aidA gene) network affects the development of secondary bacteraemia in pneumonia patients and also the virulence of A. baumannii.

Aeromonas hydrophila biofilm, exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.

Conformational studies of Gram-negative bacterial quorum sensing acyl homoserine lactone (AHL) molecules: The importance of the n → π* interaction

A 1H NMR study shows the presence of intermolecular hydrogen bonds for AHLs in CDCl3 solution. A detailed computational study of the structure of AHLs and the relative stability between the extended conformations (X) and those showing n → π* interactions (np) have been carried out by means of DFT calculations. Solvent effects have been shown to be very important when stabilising np conformations, particularly with polar solvents. This was shown by the shortening of C⋯O intramolecular distances and the increase in the relative energies favouring the np conformation with the dielectric constant of the solvent. The charge transfer between the O donor and the acceptor carbonyl group, assessed by second order perturbation energies, E(2), also shows an increase in the E(2) values with the dielectric constant of the solvent.

Enhanced exopolysaccharide production and biofilm forming ability in methicillin resistant Staphylococcus sciuri isolated from dairy in response to acyl homoserine lactone (AHL).

Staphylococcus sciuri is an emerging human pathogen widely found in dairy industries. In this study, we have isolated methicillin resistant Staphylococcus sp. from biofilm formed on utensil used in the dairy society situated at Raia, Goa and was designated as NN14. The isolate NN14 was identified through 16S rRNA sequencing as S. sciuri (GenBank accession number MF621976). This report reveals that the S. sciuri strain NN14 responds positively to the, acyl-homoserine lactone (AHL) having 6-carbon long acyl chain i.e. N-hexanoyl-homoserine lactone molecule (C6-HSL) with gradual rise in their biofilm establishing potential as the concentration of AHL was increased from 250nM, 500nM to 1µM when compared to control (without C6-HSL) by performing crystal violet assay using 48 well microtiter plate. Also, exopolysaccharide (EPS) production was found to increase with gradual increase in C6-HSL concentration from 250nM, 500nM to 1µM proving potential role of EPS in biofilm formation. These results were further proved by scanning electron microscopy where increased in biofilm and EPS production with increase in C6-HSL concentration was observed. The biofilm forming capability of S. sciuri strain NN14 was found to decreased significantly when it was subjected to 10µg/ml of (R)-2-(2-hydroxynaphthalen-1-yl)-thiazolidine-4-carboxylic acid, however with the addition of 250 and 500nM, C6-HSL in presence of the antimicrobial compound (R)-2-(2-hydroxynaphthalen-1-yl)-thiazolidine-4-carboxylic acid, the biofilm development in bacterial strain NN14 was increased when compared with control. Our results demonstrated that the C6-HSL molecule neutralize the effect of antibacterial compound and enhances EPS production and biofilm development in S. sciuri.

Biofilm activity, ammonia removal and cell growth of the heterotrophic nitrifier, Acinetobacter sp., facilitated by exogenous N-acyl-homoserine lactones.

In the present study, the heterotrophic nitrification–aerobic denitrification strain, Acinetobacter sp. JQ1004, was treated with three typical N-acyl-homoserine lactone (AHL) molecules (C6-HSL, C8-HSL, and 3-oxo-C10-HSL) during the nitrogen removal process. The effects of AHLs on biofilm formation, flocculation, extracellular polymeric substance characteristics, and nitrogen removal were investigated. Findings revealed that low concentrations of these three AHLs could promote ammonia removal and cell growth as follows: C8-HSL > C6-HSL > 3-oxo-C10-HSL, whereas high concentrations suppressed nitrogen removal. Transcript levels of the amoA gene in the heterotrophic nitrification process were detected by real-time PCR, indicating that the addition of each AHL with 10 nmol L−1 could stimulate expression of amoA. Notably, the addition of C6-HSL at relative lowly concentrations significantly accelerated biofilm formation and self-aggregation of strain JQ1004. Many microbial-flocs were observed between cells using scanning electron microscopy when strains were dosed with 10 nmol L−1 C6-HSL. Excitation emission matrix spectra revealed that the addition of C6-HSL and C8-HSL at 10 nmol L−1 did not change the components and structures of the extracellular polymeric substance (EPS), but the fluorescence intensity of various components increased substantially. However, the addition of 3-oxo-C10-HSL reduced the fluorescence strength of EPS, which had no remarkable effect on biofilm formation, self-aggregation, and nitrogen removal of the strain.

"Hot Stuff": The Many Uses of a Radiolabel Assay in Detecting Acyl-Homoserine Lactone Quorum-Sensing Signals.

Many Proteobacteria synthesize acyl-homoserine lactone (AHL) molecules for use as signals in cell density-dependent gene regulation known as quorum sensing (QS) and response. AHL detection protocols are essential to QS researchers and several techniques are available, including a 14C-AHL radiolabel assay. This assay is based on the uptake of radiolabeled methionine by living cells and conversion of the radiolabel into S-adenosylmethionine (SAM). The radiolabeled SAM is then incorporated into AHL signal by an AHL synthase enzyme. Here we describe a methodology to perform the AHL radiolabel assay, which is unbiased, relatively fast, and very sensitive compared to other AHL detection protocols.

Crystal structures of hibiscus acid and hibiscus acid dimethyl ester isolated from Hibiscus sabdariffa (Malvaceae).

The biologically active title compounds have been isolated from Hibiscus sabdariffa plants, hibiscus acid as a dimethyl sulfoxide monosolvate [systematic name: (2S,3R)-3-hy-droxy-5-oxo-2,3,4,5-tetra-hydro-furan-2,3-di-carb-oxy-lic acid dimethyl sulfoxide monosolvate], C6H6O7·C2H6OS, (I), and hibiscus acid dimethyl ester [systematic name: dimethyl (2S,3R)-3-hy-droxy-5-oxo-2,3,4,5-tetra-hydro-furan-2,3-di-carboxyl-ate], C8H10O7, (II). Compound (I) forms a layered structure with alternating layers of lactone and solvent mol-ecules, that include a two-dimensional hydrogen-bonding construct. Compound (II) has two crystallographically independent and conformationally similar mol-ecules per asymmetric unit and forms a one-dimensional hydrogen-bonding construct. The known absolute configuration for both compounds has been confirmed.

The first report on Listeria monocytogenes producing siderophores and responds positively to N-acyl homoserine lactone (AHL) molecules by enhanced biofilm formation.

Listeria monocytogenes are Gram-positive well-known emerging food-borne pathogens causing listeriosis in humans. In the present study, we have isolated biofilm-forming Listeria sp. from utensils used by a local milk collection dairy society at Usgao Goa, which collects milk for Goa dairy. Through biochemical tests and 16S rRNA sequence analysis, the bacterium was confirmed to be L. monocytogenes and designated as strain BN3, having GenBank accession number MF095110. We report for the first time Gram-positive L. monocytogenes strain BN3 producing iron-chelating siderophores by chrome azurol S (CAS) agar test. Also, this is a first report which reveals that L. monocytogenes strain BN3 responds to N-hexanoyl-homoserine lactone molecule (C6-HSL) by gradual increase in their biofilm-forming potential with a gradual increase in AHL (C6-HSL) concentration (250, 500nM-1μM) as compared to control revealed by crystal violet assay (CV) in microtiter plate. These results were further confirmed by scanning electron microscopy (SEM). A significant decrease in biofilm formation was observed when L. monocytogenes strain BN3 was treated with 10µg/ml (R)-2-(2-hydroxynaphthalen-1-yl)thiazolidine-4-carboxylic acid, but when 250 and 500nM AHL molecules were added, biofilm formation in strain BN3 was found to be enhanced as compared to control even in the presence of antibacterial compound, (R)-2-(2-hydroxynaphthalen-1-yl)thiazolidine-4-carboxylic acid. These results revealed that AHL molecules nullify the effect of antimicrobial compound and promote biofilm formation in L. monocytogenes strain BN3.

Roles of quorum sensing molecules from Rhizobium etli RT1 in bacterial motility and biofilm formation.

Strain RT1 was isolated from root nodules of Lens culinaris (a lentil) and characterized as Rhizobium etli (a Gram-negative soil-borne bacterium) by 16S rDNA sequencing and phylogenetic analysis. The signaling molecules produced by R. etli (RT1) were detected and identified by high-performance liquid chromatography coupled with mass spectrometry. The most abundant and biologically active N-acyl homoserine lactone molecules (3-oxo-C8-HSL and 3-OH-C14-HSL) were detected in the ethyl acetate extract of RT1. The biological role of 3-oxo-C8-HSL was evaluated in RT1. Bacterial motility and biofilm formation were affected or modified on increasing concentrations of 3-oxo-C8-HSL. Results confirmed the existence of cell communication in RT1 mediated by 3-oxo-C8-HSL, and positive correlations were found among quorum sensing, motility and biofilm formation in RT1.

N-acyl-homoserine lactone dynamics during biofilm formation of a 1,2,4-trichlorobenzene mineralizing community on clay

In Gram-negative bacteria, quorum sensing systems are based on the N-acyl-homoserine lactone (AHL) molecule. The objective of this study was to investigate the role of quorum sensing systems during biofilm formation by a microbial community while degrading the pollutant. Our model system included 1,2,4-trichlorobenzene (1,2,4-TCB) and its mineralizing Gram-negative bacterial community to investigate the relationships between AHL dynamics, cell growth and pollutant degradation. Biomineralization of 1,2,4-TCB was monitored for both the planktonic bacterial community with and without sterile clay particles in liquid cultures. The bacterial growth and production of AHLs were quantified by fluorescent in situ hybridization and immunoassay analysis, respectively. A rapid production of AHLs which occurred coincided with the biofilm formation and the increase of mineralization rate of 1,2,4-TCB in liquid cultures. There is a positive correlation between the cell density of Bodertella on the clay particles and mineralization rate of 1,2,4-TCB. 3-oxo-C12:1-HSL appears to be the dominant AHL with the highest intensity and rapidly degraded by the bacterial community via two main consecutive reactions (lactone hydrolysis and decarboxylic reaction). These findings suggest that the integrated AHLs and their degraded products play a crucial role in biofilm formation and biomineralization of 1,2,4-TCB in culture.

Conformational studies of Gram-negative bacterial quorum sensing 3-oxo N-acyl homoserine lactone molecules

In their 1H NMR spectra in CDCl3 3-oxo-N-acyl homoserine lactones (OHLs) show significant downfield chemical shifts of the amide NH proton when compared to the parent N-acyl homoserine lactones (AHLs). NMR spectroscopic and DFT calculation studies have shown that this is most likely due to the presence of a stabilising intramolecular H-bond from the NH to the 3-oxo group. The 1H NMR spectra also show evidence for the enol tautomers and that the amount of enol present for a range of OHLs is 4.1–4.5% in CDCl3 and 6.5–7.2% in CD3CN. In contrast, DFT calculations show that the lowest energy enol tautomer and the keto tautomer are of equal energy in the gas phase, but that the keto tautomer is more stable in chloroform, acetonitrile and water solution. The calculations also show that there is no evidence for any n→π∗ or C5H-bonding interactions being present in either the lowest energy keto or enol tautomer of the OHLs in solution or the gas phase, which is in contrast to the reported solid-state structure.

A comparative study on the interactions of [bmim][NTf2] ionic liquid with selected four- to seven-membered-ring lactones

Experimental densities and viscosities of binary mixtures of 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ionic liquid, [bmim][NTf2], with selected lactones, namely: β-butyrolactone (BBL), γ-valerolactone (GVL), δ-valerolactone (DVL) and ε-caprolactone (ECL), are measured at atmospheric pressure (p=0.1MPa) in the temperature range from T=(293.15 to 323.15)K over the whole composition range. From the experimental density and viscosity results, we data calculated excess parameters and fitted using Redlich–Kister’s polynomial equation. The thermal expansion coefficients are also derived from the acquired experimental density values. Derived density and viscosity are compared with available literature data for the corresponding {[bmim][NTf2]+GBL (γ-butyrolactone)} binary mixtures. Volumetric and viscometric parameters indicate different interactions and geometrical fitting of the lactone molecule with ionic liquid depending on the number of carbon atoms of the lactone. Thermodynamic parameters of viscous flow are also calculated. The influence of the molecular solvent structure and effects of the lactones properties on the nature of interactions in the studied binary mixtures with [bmim][NTf2] are discussed from the obtained volumetric and viscosity values and also density functional theory calculations provided in this work.

Regulation of Acyl Homoserine Lactone Synthesis in Pseudomonas putida JMQS1 Under Phenol Stress

The cell density-dependent gene expression in gram-negative bacteria is through the activity of acyl homoserine lactone signal molecules. The novel strain Pseudomonas putida JMQS1 isolated from detergent-contaminated soil exhibited quorum sensing along with its ability to degrade phenol. The response to Chromobacterium violaceum DSTS-1 mutant biosensor and luxI and luxR gene-specific amplification was used to characterize the quorum sensing property of the isolate. A regulation in the synthesis of various acyl homoserine lactone molecules, viz C6HSL in the initial stages of phenol stress, C8HSL during degradation, and 3OC12HSL on completion of degradation could be identified by liquid chromatography-quadrupole time of flight. Thin-layer chromatography, Fourier transform infrared spectroscopy, and gas chromatography mass spectrometry confirmed the complete degradation of phenol in 48–56 h. P. putida JMQS1 exhibited adaptation over phenol stress through the selective activation of the quorum sensing signal molecules depending on the changing physiological conditions. This study proposes an efficient method for enhancing the degradation of toxic organic pollutants by the supplementation of acyl homoserine lactone signal molecules.

Insights into the Genome Sequences of an N-Acyl Homoserine Lactone Molecule Producing Two Pseudomonas spp. Isolated from the Arctic.

We report for the first time the draft genome sequence of two psychrotrophic Pseudomonas species, Pseudomonas simiae RGCB 73 and Pseudomonas brenneri RGCB 108, from the Arctic that produce more than one acyl homoserine lactone molecule of varied N-acyl length. The study confirms the presence of a LuxR-LuxI (type) mediated quorum-sensing system in both the Pseudomonas species and enables us to understand the role of quorum sensing in their survival in extremely cold environments.

A New Acyl-homoserine Lactone Molecule Generated by Nitrobacter winogradskyi.

It is crucial to reveal the regulatory mechanism of nitrification to understand nitrogen conversion in agricultural systems and wastewater treatment. In this study, the nwiI gene of Nitrobacter winogradskyi was confirmed to be a homoserine lactone synthase by heterologous expression in Escherichia coli that synthesized several acyl-homoserine lactone signals with 7 to 11 carbon acyl groups. A novel signal, 7, 8-trans-N-(decanoyl) homoserine lactone (C10:1-HSL), was identified in both N. winogradskyi and the recombined E. coli. Furthermore, this novel signal also triggered variances in the nitrification rate and the level of transcripts for the genes involved in the nitrification process. These results indicate that quorum sensing may have a potential role in regulating nitrogen metabolism.

Defining the complementarities between antibodies and haptens to refine our understanding and aid the prediction of a successful binding interaction.

BackgroundLow molecular weight haptens (<1000 Da) cannot be recognized by the immune system unless conjugated to larger carrier molecules. Antibodies to these exceptionally small antigens can still be generated with exquisite sensitivity. A detailed understanding at the molecular level of this incredible ability of antibodies to recognize haptens, is still limited compared to other antigen classes.MethodsDifferent hapten targets with a broad range of structural flexibility and polarity were conjugated to carrier proteins, and utilized in sheep immunization. Three antibody libraries were constructed and used as potential pools to isolate specific antibodies to each target. The isolated antibodies were analysed in term of CDR length, canonical structure, and binding site shape and electrostatic potential.ResultsThe simple, chemically naïve structure of squalane (SQA) was recognized with micromolar sensitivity. An increase in structural rigidity of the hydrophobic and cyclic coprostane (COP) did not improve this binding sensitivity beyond the micromolar range, whilst the polar etioporphyrin (POR) was detected with nanomolar sensitivity. Homoserine lactone (HSL) molecules, which combine molecular flexibility and polarity, generated super-sensitive (picomolar) interactions. To better understand this range of antibody-hapten interactions, analyses were extended to examine the binding loop canonical structures and CDR lengths of a series of anti-hapten clones. Analyses of the pre and post- selection (panning of the phage displayed libraries) sequences revealed more conserved sites (123) within the post-selection sequences, when compared to their pre-selection counterparts (28). The strong selection pressure, generated by panning against these haptens resulted in the isolation of antibodies with significant sequence conservation in the FW regions, and suitable binding site cavities, representing only a relatively small subset of the available full repertoire sequence and structural diversity. As part of this process, the important influence of CDR H2 on antigen binding was observed through its direct interaction with individual antigens and indirect impact on the orientation and the pocket shape, when combined with CDRs H3 and L3. The binding pockets also displayed electrostatic surfaces that were complementary to the hydrophobic nature of COP, SQA, and POR, and the negatively charged HSL.ConclusionsThe best binding antibodies have shown improved capacity to recognize these haptens by establishing complementary binding pockets in terms of size, shape, and electrostatic potential.

Signal interference effect of human paraoxonase 1 using as substrates N-hexanoyl-L-homoserine lactone and N-3-oxo-octanoyl-L-homoserine lactone on growth of pathogenic bacteria

Paraoxonase 1 (PON1) is human lactonase orginally described as enzyme that is capable of hydrolyzing organophosphates. The hypothesis suggested that this enzyme may also participate in attenuation of bacterial virulence through interfering with quorum sensing (QS). Recently, PON1 was shown to hydrolyze over 30 lactones. In the present study, human PON1 (hPON1) was purified using ammonium sulphate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.89 U/mg protein and catalyzed the hydrolysis of N-hexanoyl-L-homoserine lactone (C10HSL) and N-3-oxo-octanoyl-L-homoserine lactone (3OC8HSL). The hydrolysis reaction was analyzed with HPLC. The KM values for hPON1 using 3OC8HSL or C10HSL as subtrate were calculated as 2.71 and 0.80 mM and Vmax values were detected as 1428.57 and 45.24 µmoles mg–1 min–1, respectively. Also, effect of hPON1 on growth of pathogenic bacterial strains using the signal lactone molecules was investigated by microtiter plate assay. Our results demonstrated that hPON1 was responsible for inhibition of QS system by hydrolyzing of signal molecules and effecting bacterial growth.

Purification and antibiofilm activity of AHL-lactonase from endophytic Enterobacter aerogenes VT66

The opportunistic pathogen Pseudomonas aeruginosa uses biofilm lifestyle to resist antibiotic treatment. In our study, endophytic bacterium Enterobacter aerogenes VT66 quenched the N-acyl homoserine lactone (AHL) molecules produced by P. aeruginosa PAO1. The quorum quenching activity was attributed to the presence of AHL-lactonase. The AHL-lactonase was purified using column chromatography and purified AHL-lactonase was applied for the control of biofilm formation in P. aeruginosa PAO1. The results showed that purified AHL-lactonase obtained with a molecular weight about 30kDa was able to inhibit more than 70% of biofilm in P. aeruginosa PAO1 (P<0.001). Antibiofilm activity of AHL-lactonase was correlated well with results from staining technique used to determine inhibition of biomass and viable cell activity. Therefore, results unambiguously confirm that the AHL-lactonase from E. aerogenes VT66 could be used as antibiofilm therapeutics in P. aeruginosa associated biomedical applications.

Cell adhesion, ammonia removal and granulation of autotrophic nitrifying sludge facilitated by N-acyl-homoserine lactones

In this study, six N-acyl-homoserine lactone (AHL) molecules (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were each dosed into a bioreactor and seeded using autotrophic nitrifying sludge (ANS). The effects of the AHLs on cell adhesion, nitrification and sludge granulation were investigated. The results indicated that the efficiencies of cell adhesion and ammonia removal both had a close correlation with the side chain length and β position substituent group of the AHLs. The best-performing AHL in terms of accelerating bacterial attached-growth was 3-oxo-C6-HSL, whereas C6-HSL outperformed the others in terms of the ammonia degradation rate. The addition of 3-oxo-C6-HSL or C6-HSL increased the biomass growth rate, microbial activity, extracellular proteins and nitrifying bacteria, which can accelerate the formation of nitrifying granules. Consequently, selecting AHL molecules that could improve bacteria in attached-growth mode and nitrification efficiency simultaneously will most likely facilitate the rapid granulation of nitrifying sludge.