WO2001046459A1 - Probes and primers for the detection of polyphosphate accumulating organisms in wastewater - Google Patents
Probes and primers for the detection of polyphosphate accumulating organisms in wastewater Download PDFInfo
- Publication number
- WO2001046459A1 WO2001046459A1 PCT/AU2000/001611 AU0001611W WO0146459A1 WO 2001046459 A1 WO2001046459 A1 WO 2001046459A1 AU 0001611 W AU0001611 W AU 0001611W WO 0146459 A1 WO0146459 A1 WO 0146459A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- oligonucleotide
- probe
- sludge
- rdna
- probes
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Definitions
- This invention relates to the identification of polyphosphate accumulating organisms that are capable of biologically removing phosphorus from wastewater.
- the invention relates to a method for rapidly assessing the presence or numbers of such organisms in a wastewater sample, their numbers being indicative of the phosphorus-removing capacity of the wastewater microbial community.
- Microorganisms in the activated sludge process grow as floes — three-dimensional agglomerates about 100 ⁇ m in diameter. These floes can be separated from the treated wastewater by gravity sedimentation, but such separation processes, however, are prone to failures costing hundreds of thousands of dollars in remedial action each year in Australia alone. There are two main reasons for these failures:
- the removal of P from wastewater can be achieved by chemical precipitation or by biological mechanisms in a process called enhanced biological phosphorus removal (EBPR).
- EBPR enhanced biological phosphorus removal
- the basic configuration of an EBPR activated sludge plant has the influent wastewater going into an anaerobic zone where it is mixed with the returned microbial biomass from the secondary clarifier to form the so-called mixed liquor. This mixed liquor then flows into an aerobic zone after which the biomass is separated from the treated wastewater in the secondary clarifier.
- Polyphosphate accumulating organisms PAOs
- van Loosdrecht et al, 1997) are selectively enriched in these systems and excessive phosphate accumulation occurs in the aerobic zone. Removal of a portion of the growing biomass (waste activated sludge) results in the net removal of P from the wastewater.
- An object of the invention is to provide oligonucleotides that can be used to detect polyphosphate accumulating organisms in a sample. Further objections of the invention are to provide methods of detecting, or quantifying the level of the foregoing organisms in a sample.
- an oligonucleotide probe for detecting a polyphosphate accumulating organism in a sample, said oligonucleotide having a sequence of at least 12 nucleotides that is unique to 16S rDNA of polyphosphate accumulating organisms.
- an oligonucleotide primer for PCR amplification of DNA of a polyphosphate accumulating organism said primer having a sequence of at least 12 nucleotides that is unique to 16S rDNA of polyphosphate accumulating organisms.
- a primer pair for PCR amplification of 16S rDNA of a polyphosphate accumulating organism comprising: (a) a first oligonucleotide of at least 12 nucleotides having a sequence selected from one strand of said l ⁇ S rDNA; and (b) a second oligonucleotide of at least 12 nucleotides having a sequence selected from the other strand of said 16S rDNA downstream of said first oligonucleotide sequence; wherein at least one of said first and second oligonucleotides has a sequence that is unique to 16S rDNA of polyphosphate accumulating organisms.
- a method of detecting cells of a polyphosphate accumulating organism in a sample comprising the steps of:
- step (b) contacting said fixed cells from step (a) with a labeled oligonucleotide probe under conditions which allow said probe to hybridize with 16S rRNA within said fixed cell, wherein said probe is an oligonucleotide according to the first embodiment; (c) removing unhybridized probe from said fixed cells; and
- a method of detecting a polyphosphate accumulating organism in a sample comprising the steps of: (a) obtaining nucleic acid from cells of said organism; (b) contacting nucleic acid from step (a) with a labeled or immobilised oligonucleotide probe under conditions which allow said probe to hybridize to 16S nucleic acid molecules, wherein said probe is an oligonucleotide according to the first embodiment;
- a method of detecting a polyphosphate accumulating organism in a sample comprising the steps of:
- step (b) contacting denatured genomic DNA from step (a) with a primer pair according to the third embodiment
- the invention provides methods of quantitating the number of polyphosphate accumulating organisms in a sample.
- the invention further provides a method of identifying oligonucleotide probes suitable for the detection or quantitation of polyphosphate accumulating organisms.
- FIG. 1 is a phylogenetic tree constructed from near complete 16S rDNA sequences derived from a variety of sludges and sequences from publically accessible databases.
- Figure 2 shows fluorescence in situ hybridization micrographs of mixed liquors from sequencing batch reactors.
- Figure 3 is an alignment of 16S rDNA sequences.
- Figure 4 shows the relationship between sludge P content (% of dry mass) and % cells binding all three PAO probes of Table 4 as a percentage of the EUB338-probe positive cells.
- PAO polyphosphate accumulating organism PCR polymerase chain reaction
- Td dissociation temperature TSS total suspended solids VSS volatile suspended solids
- TSS total suspended solids VSS volatile suspended solids
- the present inventors have utilized a laboratory scale sequencing batch reactor (SBR) to generate sludges enriched in polyphosphate accumulating organisms (PAOs) and have prepared 16S rDNA clone libraries from these and other sludges.
- SBR laboratory scale sequencing batch reactor
- PAOs polyphosphate accumulating organisms
- 16S rDNA clone libraries from these and other sludges.
- the inventors have surprisingly found that there are unique sequences in the 16S rDNA of PAOs, which sequences can be used as sequences for oligonucleotide probes or primers.
- the probes an be used in various hybridisation techniques for detecting PAOs and the primers for PCR amplification of DNA of such organisms.
- a 16S rRNA- or rDNA-directed oligonucleotide probe or primer of the first embodiment of the invention typically has a length of about 12 to 50 nucleotides. A preferred length is 15 to 25 nucleotides.
- Particularly preferred oligonucleotides of the first embodiment are as follows: 5'-CCGTCATCTACWCAGGGTATTAAC-3' 5'-CCCTCTGCCAAACTCCAG-3' 5'-GTTAGCTACGGCACTAAAAGG-3'
- the invention also provides probes or primers for detecting organisms closely related to PAOs. With such primers mismatches at the 5' end of the primer are permissible.
- a preferred primer for detecting organisms closely related to PAOs has the following sequence:
- Specificity is the first and foremost design consideration for probes and primers. It is achieved by selecting a complementary sequence to the 16SrRNA and or 16S rDNA of a target organism with no mismatches (non-canonical base pairing). Non-target organisms must have at least one mismatch to the probe or primer sequence to ensure that hybridization will not occur.
- the optimal position of mismatches in a hybridisation probe is in the middle of the oligonucleotide and the optimal position of mismatches in a PCR primer is at the 3' (extension) end. All probes of the present invention were designed for specificity using the ARB software package (Strunk et al, unpublished.). The following parameters were subsequently assessed after the initial design in ARB.
- thermodynamic parameters of the probe or primer such as melting temperature (Tm) or dissociation temperature (Td) (Keller, 1993) determine the conditions under which the specific hybridization of the oligonucleotides will occur.
- Tm melting temperature
- Td dissociation temperature
- Accessability In the case of FISH, according to the second embodiment of the invention, accessibility of the ribosome is an important design consideration (Fuchs et al, 1998). Some regions of the 16S rRNA within the ribosome are less accessible than others, in the worst case scenario preventing the access of oligonucleotides to those sites leads to no detection. 7
- Oligonucleotides can have self complementarity resulting in either dimer formation or hairpin structures. Secondary structures of the probe are an important design parameter when used with ion-channel membrane biosensors.
- a primer according to the second embodiment of the invention like the probes of the first embodiment, typically has a length of 12 to 50 nucleotides.
- a preferred length is 12 to 22 nucleotides.
- Oligonucleotide primer pairs according to the third embodiment of the invention comprise an oligonucleotide primer that will anneal to one strand of the target sequence and a second oligonucleotide primer that will anneal to the other, complementary, strand of the target sequence.
- the second oligonucleotide primer must anneal to the complementary strand downstream of the first oligonucleotide primer sequence, which occurs in the complementary strand, to yield a double stranded amplification product in the PCR.
- the amplification product is of a size that facilitates detection.
- the first and second oligonucleotide primer sites in the target DNA are separated by 50 to 1,400 bps.
- a preferred separation is 400 to 1,000 bps.
- probes according to the first embodiment of the invention can be used as hybridisation probes to detect PAOs.
- a preferred hybridisation technique is FISH for which the specific oligonucleotides specified above are particularly suited.
- the probes can be used in any other hybridisation technique as will be discussed below.
- the probe label can be any label suitable for in situ detection of the probe-RNA hybrid.
- a preferred label is a fluorescent label such as fluorescein.
- probes of the first embodiment can be used in more general hybridisation techniques or in specialised techniques such as an ion-channel biosensor.
- nucleic acid from a PAO can be immobilised on an inert support such as a membrane. After hybridisation of the probe to the immobilised nucleic acid, the hybrid is detected by virtue of the label.
- a particularly convenient hybridization technique makes use of a slot blot manifold such as the quantitative method described by Stahl et al. (1988).
- Probes used in general hybridisation techniques can be longer than the typical length of up to 50 nucleotides
- a probe is attached to an ion-channel membrane biosensor.
- the ion-channel switch is triggered. The switching event results in a drop in electrical conductance and thereby indicates that target nucleic 8
- the label of probes according to the invention can be any of the labels known to those of skill in the art.
- the label can be a radiolabel, a reporter group or a hapten.
- the method of the fifth embodiment can also be used to quantitate the number of PAO cells in a sample. With the more general hybridisation techniques, this is done by comparing the signal obtained from the probe-nucleic acid hybrid with a reference standard or a number of standards.
- a standard is constructed comprising a known number of cells or a known amount of PAO
- An ion-channel biosensor is particularly suited to quantitative determination of cell numbers as the drop in electrical conductance on triggering of the switching event gives a quantitative measure.
- PCR is used to exponentially amplify 16SrDNA sequences using oligonucleotide primers.
- An example of its use in detecting microorganisms is given in Burrell et al. (1998).
- Detection of the amplified DNA can be by any of the methods known to those of skill in the art.
- the amplified DNA can be analysed by agarose gel electrophoresis followed by staining to identify the DNA band of the expected size.
- Other methods for the detection of amplification products include hybridisation, especially solution hybridisation, using a labeled, internal oligonucleotide probe complementary to a region of DNA lying between the ends of the amplified DNA.
- the internal oligonucleotide can be labeled using any of the labels known to those of skill in the art.
- the label can be a radiolabel or a non-radioactive label such as biotin.
- Nick-translation can also be used to label internal probes.
- Probes and primers according to the invention can be prepared by conventional methods.
- Labeling can be done, if appropriate, during synthesis of the oligonucleotide constituting the probe or primer, or can be done post-synthesis. Methods for the labeling of primers is given in standard texts such as Sambrook et al (1989).
- Probes and primers can be provided as kits for use in the methods of the invention.
- a kit can include one probe or primer and appropriate reagents for carrying out the method.
- kits for PCR amplification of target DNA include at least one primer pair according to the third embodiment.
- kits advantageously include at least one reference standard.
- kits according to the invention are particularly useful in this regard.
- a panel of PAO 16S rDNA sequences and sequences of 16S rDNA from other organisms must be constructed. From the panel, sequences unique to PAO 16S rDNA can be selected.
- the sequence alignment of Figure 3 constitutes a particularly suitable panel for the identification of sequences unique to PAOs
- the SBR was fitted with pH electrodes and a portable dissolved oxygen electrode, and a 6-h operating cycle consisting of 2-h anaerobic, 3.5-h aerobic and 0.5-h settling, was maintained.
- a hydraulic retention time (HRT) of 12 h was maintained as 900 mL or 1 liter of media was fed in the first 10 min of the anaerobic period, and 900 mL or 1 liter of treated supernatant was withdrawn in the last 5 min of the settling stage.
- Mixed liquor was wasted during the aeration period so that the solids retention time (SRT) was 8 to 10 d.
- the PO4-P concentration in the influent to the A sludge was increased to 57 mg P ⁇ 4-P/liter, while that in the GRC sludge was 28 mg/liter.
- the effluent PO4-P concentration inthe A sludge was always at or below the detection limit (0.05 mg P ⁇ 4-P/liter).
- the P% of the mixed A culture was 15.1%.
- the performance of the GRC reactor fluctuated over a 12 month period and at regular stable operating times, the sludge was analysed by FISH and the P% determined. Images presented in Figure 1 from the GRC sludge were when the reactor effluent was 6.7 mg P ⁇ 4-P/liter and the sludge contained 6.7% P. 10
- Reactor B was also operated as an SBR with a working volume of 1 liter, a temperature of 23.5°C + 2°, and the pH was controlled in the range 7.15-7.25 by the addition of either a 1%HC1 or a 40 g/liter Na2C ⁇ 3 solution.
- the 6-h cycle consisted of 1.83— h anaerobic, 3-h aerobic, 0.5-h, settle, and 0.67-h comprising draw, fill, and strip with nitrogen gas.
- An HRT of 12 h was maintained by withdrawing 500 mL of the reactor contents during each settle phase and replacing it with500 mL fresh nutrient feed.
- Timed operation of feed and effluent pumps, air and nitrogen flow, and mixing was by a programmable controller (Model CD-4, Chrontrol Corp., San Diego, CA).
- the SRT was maintained at 4 d (25% of the biomass wasted/d) by once per day manually withdrawing a portion of the mixed reactor contents immediately prior to the settle phase during the same cycle.
- the sludge in the reactor had a P% of 17.2%.
- Anaerobic conditions were maintained by continuous bubbling with N2 gas through a porous diffuser. of oxygen began approximately 30 min before the addition of feed. Aerobic conditions were maintained by bubbling ambient air through a porous diffuser. Air and N2 flow rates were approximately 500 mL/min. Anaerobic and aerobic conditions were verified by continuous measurements using an in-reactor oxygen electrode (M 1016-0770, New Brunswick Scientific, Edison, NJ), a dissolved oxygen meter (Model DO-40, New Brunswick Scientific) and a strip chart recorder (Model 288, Rustrak Corporation, Manchester, NH).
- Nutrient and carbon feeds were added separately.
- the nutrient feed consisted of (per liter) 259 mg NaH 2 P ⁇ 4 « 2H2 ⁇ (50 mg P/liter), 117 mg KCl, 119 mg NH4CI, 219 mg MgCl2'6H2 ⁇ , 14.4 mg MgS ⁇ 4 « 7H2 ⁇ , 45.9 mg CaCl2, 8.3 mg yeast extract, 0.24 mL 10% HCl, 0.20 mL trace element solution, and 0.15 mL FeS04 solution.
- the trace element solution consisted of (per liter) 300 mg H3BO3, 1 500 mg ZnS ⁇ 4 « 7H2 ⁇ , 75 mg KI, 300 mg CuS04 « 5H 2 0, 367 mg Co(N ⁇ 3)2'6H2 ⁇ , 150 mg Na2Mo ⁇ 4 « 2H2 ⁇ , and 1,679 mg MnS ⁇ 4 « H2 ⁇ .
- the FeS04 solution was 2,054 mg/liter FeS ⁇ 4 « 7H2 ⁇ .
- the carbon feed was added as a concentrated stock (10 mL per cycle).
- the carbon feed consisted of 425 mg QH ⁇ COONa ⁇ O and 30 mg casamino acids per liter of nutrient feed.
- the reactor was seeded with mixed liquor from the City of San Francisco Southeast Water Pollution Control Plant (SEP) which is a pure-oxygen activated sludge plant with six basins in series, the first of which functions as an anaerobic selector.
- High soluble P concentrations in the anaerobic selector were an indication of the presence of EBPR organisms in the SEP.
- Reactor analyses Performance of all three reactors (A, GRC, and B) was superficially assessed by determination of the supernatant P and acetate concentrations at the end of the anaerobic and aerobic periods, by the effluent P concentration, and by the sludge P%. P and acetate concentrations were also determined in each batch of feed prepared. At weekly or biweekly intervals during the operation of the reactors, cycle studies were conducted. Samples were collected from the 11
- Soluble orthophosphate was on GF B-filtered (P/N 1821025, Whatman International, Ltd., Maidstone, UK) or 0.45 ⁇ m membrane filtered (P/N 60172, Gelman Sciences, East Hills, NY) samples by the vanado-molybdate colorimetric method (Method 4500-P C; APHA et al, 1992).
- Total orthophosphate (Pt) was by the persulfate digestion method (Method 4500-P B.5; APHA et al, 1992).
- Non-soluble phosphate (Pns) was calculated as (Pt - Psol) for samples taken at the end of the aerobic phase.
- Acetate was analyzed on filtered (GF/B or 0.45 ⁇ m membrane filters) acidified samples by flame ionization detector gas chromatography (FID GC), using a J&W Scientific DB- FFAP 0.53 mm capillary column. Samples were acidified with concentrated H3PO4 and stored at 4°C prior to analysis when 2 ⁇ L samples were injected.
- the carrier gas was N2 with a flow rate of 15 mL/min; H2 flow rate was 20 mL/min and the air flow rate was 250 mL/min to the FID.
- Oven temperature began at 90°C ramped to 110°C at 50° C/min, remained at 110°C for 30 s, and then ramped to 130°C at 50°C/min. Injector temperature was 250°C; the FID was unheated.
- PHAs Polyhydroxyalkanoates. PHAs were determined by a modification of the GC method of Riis and Mai (1988) as follows: 10 mL samples were collected on 25 mm Whatman GF/B filters and immediately dried at 100°C for 1 h then stored in a desiccator at 4°C prior to analysis; 1 mL of 4: 1 l-propanol:HCl and 1 mL of trichloroethene were added to each sample in 10 mL sample vials, which were then capped and heated to 95-100°C for 3-4 h. Samples were cooled and then extracted with 2 mL deionized water.
- PHAs in the lower phase were measured by injection of 2 ⁇ L into an FID GC (glass packed column, 10% AT- 1000 resin on Chromosorb W-AW 80-100 mesh, Varian model 3700 GC). Samples of 2 ⁇ L volume were analyzed using the following temperatures: oven, 250°C; injection port, 250°C; FID, 220°C. Benzoic acid was used as an internal standard.
- CH Carbohydrates
- TSS Total suspended solids
- VSS volatile suspended solids
- Microscopy of Mixed Cultures Mixed cultures (sludges) from the A, GRC and B reactors and from other reactors were collected, fixed and probed as reported by Bond et al. (1999a). Counting of the probed A sludge was done manually and occasionally, this mixed microbial culture required light sonication (Bond et al, 1999) to facilitate the process. Counts of ⁇ , ⁇ (including ⁇ -1 and ⁇ -2), and y-Proteobacteria, Actinobacteria, and Cytophaga-Flavobacterium were determined as proportions of all Bacteria (according to probe EUB338; Bond et al, 1999a — see below for details of probes) for the A sludge.
- Methylene Blue and Gram stains were also done on the A and GRC sludges and on other selected sludges.
- Neisser staining was as described in Eikelboom and van Buijsen (1981)
- Gram staining was by the Modified Hucker Method and India Ink staining were from Jenkins et al. (1993)
- PHB staining was as described in Murray (1981).
- Light micrographs of Gram and Methylene Blue stains were captured on a Nikon Microphot FXA microscope via a charged couple device connected to a PC. Images were prepared in Adobe Photoshop.
- FISH probed samples were viewed on both a Zeiss LSM510 and on a Zeiss Axiophot.
- the Zeiss LSM 510 confocal laser scanning microscope employed an Axiovert 100M SP inverted optical research microscope, and a Plan-Neofluar 63x/ 1.25 numerical aperture objective. Scan time was 31.8 s per frame and 4.48 ⁇ s pixel dwell time.
- An Argon laser 488 nm line and the HeNe 543 nm line was used for imaging.
- Frame size was 512x512 pixels. Images presented in Figure 1 were taken with the LSM510 and prepared in Adobe Photoshop. Clone libraries.
- Bacterial 16S rDNA clone libraries were prepared from genomic DNA extracted from frozen A, P (Bond et al, 1999) and B sludges and inserts from individual clones were amplified and grouped according to restriction fragment length polymorphism (RFLP) analysis using methods previously described (Burrell et al, 1998). Clones of RFLP-group representatives were partially sequenced using primer 530f and phylogenetically analysed (Bond et al, 1995; Burrell et al, 1998). A selection of clone inserts was fully sequenced with a range of primers (Blackall, 1994). Phylogenetic analysis of the 16S rDNA sequences was performed as described previously (Dojka et al, 1998).
- sequences were compiled using the software package SeqEd (Applied Biosystems, Australia). Each of the compiled sequences was compared to available databases using the basic local alignment search tool (BLAST; Altschul et al, 1990) to determine approximate phylogenetic affiliations. All clone sequences were examined with the CHECK_CHIMERA program (Maidak et al, 1999) to identify any chimeric sequences. The compiled sequences were aligned using the ARB software package (Strunk, et al, unpublished) and alignments were refined manually. Phylogenetic trees were constructed by carrying out evolutionary distance analyses on the 16S rDNA alignments, using the appropriate tool in the ARB database. The robustness of the tree topology was tested by bootstrap analysis, using neighbour-joining with the Kimura 2-parameter, and parsimony analysis (version 4.0b2a of PAUP*; Swofford, 1999). Probe Design, Synthesis and Use
- PAO-specific probes were designed using the probe design tool in the ARB software package (Strunk et al, unpublished). Based on comparative analysis of all sequences in the database, the program selected specific regions within the putative-PAO sequences which allowed their discrimination from all other reference sequences. Sequences were subsequently confirmed for specificity using BLAST (Altschul et al, 1990). The designed oligonucleotides were synthesised and labelled at the 5 '-end with the indocarbocyanine dye CY3 by Genset (France). These fluorescently- labelled probes were evaluated with paraformaldehyde fixed A sludge.
- the formamide concentration for optimum probe stringency was determined by performing a series of FISH experiments at 5% formamide increments starting at 0% formamide. Under all but the lowest stringency conditions, the morphologically distinct clusters of Methylene Blue positive coccobacilli were the only cells which bound the PAO-probes. Therefore, the optimum formamide concentrations were determined by reference to the coccobacillus clusters. This was necessary because there are no pure cultures whose 16S rRNA would bind the PAO-probes. A similar approach was employed by Erhart et al. (1997). Generally all three designed PAO-probes, PA0462, PA0651 and PA0846 (see below), were applied to any one individual sample spotted on the slide.
- Oligonucleotide probes were labeled with digoxigenin-ddUTP, using the Digoxigenin (DIG) oligonucleotide 3'-end labeling kit, according to the manufacturer's instructions (Boehringer Mannheim, Mannheim, Germany).
- DIG Digoxigenin
- a standard 20 ⁇ l labeling reaction involved the addition of 100 pmole of unlabeled oligonucleotide to 4 ⁇ l 25mM C0CI2 , 50 U terminal transferase, 1 ⁇ l of 1 mM digoxigenin- 11 -ddUTP, and sterile distilled water (to a final volume of 20 ⁇ l).
- the labeling reaction was incubated for 15 min at 37°C and then terminated by the addition of 1 ⁇ l of 20 mg/ml glycogen solution and 1 ⁇ l of 200 mM EDTA.
- the labeled oligonucleotide was precipitated by the addition of 0.1 volume 3 M sodium acetate and 3 volumes 100% ethanol followed by incubation at -70°C for 30 min. Following centrifugation at 12,000 g for 5 min, the ethanol was removed and the pellet was washed with 50 ⁇ l of cold 70% ethanol. After brief centrifugation, the 70% ethanol was removed and the pellet was dried under vacuum. Finally, the labeled probe was resuspended in 20 ⁇ l of steril milliQ water.
- RNA to Membrane All RNA samples were denatured by heating at 96°C for 10 min. The denatured RNA samples were slotted in a 50 ⁇ l volume onto a moistened positively charged nylon membrane (Boehringer Mannheim, Mannheim, Germany) using a PR648 slot blot apparatus (Hoefer Scientific Instruments, San Francisco, USA) under slight vacuum.
- RNA samples were then immobilised on the nylon membrane by ultra violet irradiation for 5 min or baking at 80°C for 1 hr.
- a 10 ng to 40 ng serial dilution of each denatured RNA target was immobilised.
- Hybridisations and Washes The membranes were prehybridised for 2 hr at 40°C in 5 ml of hybridisation buffer (DIG Easy Hyb; 5x SSC, 0.1% N-laurylsarcosine, 0.02% SDS and 1% blocking solution). Hybridisations were performed with 2-5 ⁇ l (0.1 ⁇ l of probe per slot; 10-fold excess) of probe in 5 ml of hybridisation buffer at 40°C for 12-16 hr. All hybridisation steps were carried out in a Hybaid Mini 10 hybridisation oven (Hybaid, United Kingdom).
- the membranes were then washed twice for 15 min at the same temperature (40°C) in wash buffer containing lx SSC (150 mM NaCl, 15 mM sodium citrate, adjusted to pH 7) and 1% SDS, followed by a 10 min wash at the determined T d value for each probe. Chemiluminescent Detection. Following hybridisation and stringency washing, membranes were rinsed for 5 min in a wash buffer containing 0.1 M maleic acid, 0.15 M NaCl and 0.3% Tween 20, adjusted to pH 7.5 with NaOH.
- membranes were blocked for 30 min in 25 ml of blocking solution which consisted of a maleic acid buffer (0.1 M maleic acid, 0.15M NaCl, adjusted to pH 7.5) containing 1% Diploma skim milk powder. Following blocking, the membrane was incubated at room temperature for 30-60 min with 2 ⁇ l of anti-DIG- alkaline phosphatase solution (Boehringer Mannheim, Mannheim, Germany) in 20 ml of blocking solution. Membranes were washed twice for 15 min at room temperature in 25 ml of the wash buffer as described above and then equilibrated in 25 ml of detection buffer (0.1 MTris-HCl, 0.1 M NaCl, pH 9.5) for 5 min.
- detection buffer 0.1 MTris-HCl, 0.1 M NaCl, pH 9.5
- CSPD chemiluminescent substrate
- the membrane was briefly removed from the hybridisation bag and blotted onto Whatman 3MM paper to remove excess CSPD solution and incubated for a further 15 min at 37°C (after resealing into the hybridisation bag) to enhance the luminescent reaction. Membranes were then visualized using the Lumilmager (Boehringer Mannheim, Mannheim, Germany) and the level of chemiluminescent signal from each of the slots was quantified using the LumiAnalyst software.
- %PAO percentage of PAOs
- P the slope of the hybridisation of probe to sample RNA
- P' the slope of the hybridisation of the probe to a pure, known control RNA (RNA transcript from a PAO clone)
- x the specific probe
- c the universal bacterial probe EUB338.
- Reactor operating data are presented in Table 1.
- Table 2 For the A and B sludges, some comparative data are also presented in Table 2.
- the comparative data are from a number of literature sources as indicated in the first column of the table.
- a Molar units are calculated as moles of glucose monomers.
- hpHA poly- ⁇ -hydroxyalkanoates.
- cna not available d ⁇ olded data are from theoretical ratios for EBPR models from Arun et al. (1988) and Smolders et al. (1994), and from Satoh et al.
- Table 1 shows that all three SBRs were performing EBPR since there was P release and acetate uptake by the biomass during the initial anaerobic stage. This can be appreciated by comparing PO4-P and acetate data in the feed and at the end of the anaerobic stage (Table 1). During the subsequent aerobic period, all sludges took up excessive amounts of P, as seen by comparing the PO4-P values at the end of the anaerobic stage with those from the effluent. A and B sludges were hyper-P removing with the sludges containing >15% PO4-P which equates to ca. 50% inorganic polyphosphate.
- the GRC sludge was a good P removing sludge being able to remove >20 mg/L of PO4-P from the wastewater (compare 28 mg PO4-P/L in the influent with 6.7 mg/L in the effluent) and contained 6.7%P (Table 1).
- Table 5 includes the group probing results from the A sludge and a number of other sludges of various P-removal capacities, ⁇ Proteobacteria, specifically ⁇ -2 proteobacteria, dominated the A sludge community strongly suggesting the PAOs are members of this bacterial subdivision. In all cases, quantification of group probings of the GRC sludge was not performed but Figure 2C (see below) shows the result from its methylene blue staining.
- EBPR sludges microscopically examined included those from the Loganholme Sewage Treatment Plant (full scale) and many laboratory scale reactors operated by researchers at the Advanced Wastewater Management Centre (A, P, GRC, Saline EBPR and denitrifying EBPR sludges; see Table 2 for the sources of these sludges).
- A, P, GRC, Saline EBPR and denitrifying EBPR sludges see Table 2 for the sources of these sludges.
- the clusters of PAO-probe binding cells were distinct and uniform and resembled cells discussed below in connection with Figures 2 A and 2C.
- HGC69a TATAGTTACCACCGCCGT 23S, 1901-1918 Actinobacteria 25 (Roller et al., 1994) °
- PA0462 CCGTCATCTAC(A/T)CAGGGTATTAAC 1166SS,, 462-485 PAO cluster (see Fig. 1) 35 This study
- PA0651 CCCTCTGCCAAACTCCAG 16S, 651-668 PAO cluster (see Fig. 1) 35 This study
- the group probing broadly highlighted the PAOs as ⁇ -2 Proteobacteria (see Table 5).
- the ⁇ -2 Proteobacteria probe (BTW023a) was originally designed only as a competitor for the ⁇ -1 Proteobacteria probe (BONE23a; Amann et al, 1996). Its specificity is broad since it targets (with no mismatches) members of the ⁇ -3 Proteobacteria, some ⁇ Proteobacteria and a Green-non-sulfur division clone, OPB9, in addition to ⁇ -2 Proteobacteria. Therefore, additional more-specific probes were required to target the ⁇ -2 Proteobacteria group.
- Figure 1 shows a phylogenetic tree of the near complete sequenced ⁇ -2 Proteobacteria clones from which the PAO probes were designed, and the specificity of the probes.
- the 16S rDNA sequences were determined from sludges A, B, P, SBRH, SBR1, SBR2 and GC (Gold Coast, Queensland, Australia).
- the probe BTW023a also targets (with no mismatches) members of the ⁇ -3 Proteobacteria, ⁇ Proteobacteria; lodobacter spp., Chromo bacterium spp., Chromatium spp. and a Green-non-sulfur division clone, OPB9.
- the scale indicates 0.02 nucleotide changes per nucleotide position.
- Figures 2A and 2B show confocal laser scanning micrographs of sludges dual probed with EUB338 (25 ng, fluorescein-labelled) and a mixture of all three PAO probes (Table 4, each 25 ng, CY3 labeled). Images were collected for fluorescein and CY3 channels, artificially coloured and superimposed. Arrowed cells are the PAOs since they are dual labelled with EUB338 (grey-coloured cells) and PAO probes (bright coloured cells that appear white in the image).
- Figure 2 A shows a mixed liquor from SBR A with operating data as given in Table 1.
- Figure 2B shows lightly sonicated mixed liquor from an EBPR SBR (ca. 10%P in the sludge) operating at 3.5% NaCl in a study of seafood processing wastewater.
- Figure 2C is a bright field micrograph of GRC sludge as operated according to data in
- FIG. 2C cells were methylene blue stained which stains for polyphosphate.
- the arrowed cells in Figure 2C are those with polyphosphate and their cellular size, morphology and arrangement match the bright cells in Figure 2A.
- the cells indicated in Figure 2C with an arrow having a diamond shaped tail do not contain polyphosphate.
- the length of the bar in Figure 2C is 6 ⁇ m.
- FIGS. 2D and 2E are of a single cluster of cells from the SBR A sludge that was first probed with the labelled PAO probes ( Figures 2D) and then stained with methylene blue ( Figure 2E).
- the arrowed cells in Figure 2D are the "bright cells" and were found to correspond to the cells stained with methylene blue in Figure 2E which are again arrowed.
- cells that did not bind the PAO probes were considerably darker and did not stain with the methylene blue. These cells are again indicated with an arrow having a diamond shaped tail.
- the bar in Figure 2D represents 4 ⁇ m.
- FIG. 2 shows that in all of the sludges, characteristic clusters of coccobacilli bound the PAO-probes and depending upon EBPR performance, greater or fewer clusters were present.
- Figure 2 shows that in the Loganholme sludge, a full-scale activated sludge plant treating domestic wastewater with an influent containing ca. 10 mg P0 4 -P/liter, moderate numbers of clusters were observed. Large numbers of the clusters were observed in the hyper-P-removing systems like the A sludge ( Figure 2A).
- RNA transcripts were generated from several 16S rDNA clones one of which was SBRA220 (see above), for use as standards.
- the 16S rDNA inserts in an M13 vector were PCR-amplified using vector primers flanking the insert or the universal bacterial primer, 1492R.
- Purified PCR products were used as templates for in vitro transciption using either T7 or SP6 RNA polymerase as appropriate.
- Purified RNA transcripts were estimated to have a size of approximately 1,500 bp, equivalent to 16S rRNA extracted from E. coli.
- the concentration of RNA in transcript preparations was 320-660 ng/ ⁇ l.
- RNA was extracted from activated sludge samples by lysis of cells, homogenation in the presence of highly denaturing guanidinuim isothiocyanate-containing buffer, and application of ethanolic homogenate to an RNeasy mini spin column.
- concentration of RNA extracted from each of the samples was determined using the GeneQuant RNA DNA calculator and was found to range from 50 ng/ ⁇ l to 400 ng/ ⁇ l.
- RNA extracts were obtained from experimental reactor sludges (see above) and full-scale activated sludge samples collected from seven wastewater treatment facilities within south-east Queensland, Australia.
- the FISH analysis was conducted on the GRC sludge at varying but stable P-removal efficiencies, the A sludge (see above), and the Q, P and S sludges of Bond et al. (Bond et al, 1999a; Bond et al, 1999b; Bond et al, 1998).
- the results of the FISH and slot blot analyses are presented in Figure 4 A and Figure 4B, respectively. Large black triangles indicate results obtained using both methods while the grey triangles represent slot blot hybridisation results for the full-scale sludges.
- the small triangles in Figure 4A are results for which there are no corresponding slot blot data.
- Figure 4 shows that there is a definite positive correlation between the proportion of PAO probe-binding cells and the sludge P%.
- the FISH analysis gave a regression value of 0.937 while an even high value of 0.979 was obtained with slot blot hybridisation.
- the FISH and slot blot evaluations were nevertheless comparable.
- the slot blot analysis also demonstrated that such a hybridisation technique can be used to accurately determine the proportion of PAOs in environmental RNA samples.
- the PAO-probes designed from a group of highly related clone sequences (greater than or equal to 98% identical) affiliated with the ⁇ -2 Proteobacteria, bound the same cell clusters in the A sludge, as bound the probe for ⁇ -2 Proteobacteria.
- the closest pure-cultured bacterial relatives to the ⁇ -2 Proteobacteria clone sequences ( Figure 1) are from Rhodocyclus (R. tenuis and R. purpureus) and Propionibacter pelophilus.
- PAO-probe positive cells matched the morphology, size and arrangement of those staining positive for polyphosphate by the methylene blue stain ( Figure 2).
- Figure 2 One demonstration of the simultaneous use of the three PAO-probes with another sludge is given in Figure 2.
- Bond P. L., R. Erhart, M. Wagner, J. Keller, and L. L. Blackall. 1999a.
- Microthrix parvicella in activated sludge. Syst. Appl. Microbiol. 20:310-318. Fuchs, B. M., G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann. 1998. Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes. Appl. Environ. Microbiol. 64:4973-4982. Fuhs, G. W., and M. Chen. 1975. Microbiological basis of phosphate removal in the activated sludge process for the treatment of wastewater. Microb. Ecol. 2: 119-138.
- ARB a software environment for sequence data.
- URL http://www.mikro.biologie.tu- muenchen.de/. Swofford, D. L. 1999.
- PAUP* Phylogenetic Analysis Using Parsimony (version 4.0b2a ). .
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Electrostatic Separation (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020027008163A KR20020089314A (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
JP2001546955A JP2003521898A (en) | 1999-12-23 | 2000-12-28 | Probes and primers for detecting polyphosphate accumulating organisms in wastewater |
AU24934/01A AU779080B2 (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
CA002394652A CA2394652A1 (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
US10/168,337 US20030170654A1 (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
EP00988522A EP1242625A4 (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AUPQ4867 | 1999-12-23 | ||
AUPQ4867A AUPQ486799A0 (en) | 1999-12-23 | 1999-12-23 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001046459A1 true WO2001046459A1 (en) | 2001-06-28 |
Family
ID=3819006
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU2000/001611 WO2001046459A1 (en) | 1999-12-23 | 2000-12-28 | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1242625A4 (en) |
JP (1) | JP2003521898A (en) |
KR (1) | KR20020089314A (en) |
AU (1) | AUPQ486799A0 (en) |
CA (1) | CA2394652A1 (en) |
WO (1) | WO2001046459A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010133969A (en) * | 2002-08-28 | 2010-06-17 | Carl Zeiss Surgical Gmbh | Microscopy system and method for operating microscopy system |
CN114230024A (en) * | 2021-12-29 | 2022-03-25 | 华南理工大学 | Method for selectively inhibiting glycan bacteria to improve biological phosphorus removal efficiency |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100691435B1 (en) * | 2005-10-31 | 2007-03-09 | 삼성전기주식회사 | A wireless headset of mic position variable type |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE127530T1 (en) * | 1989-05-31 | 1995-09-15 | Amoco Corp | UNIVERSAL NUCLEIC ACID PROBE FOR EUBACTERIA AND METHODS. |
-
1999
- 1999-12-23 AU AUPQ4867A patent/AUPQ486799A0/en not_active Abandoned
-
2000
- 2000-12-28 JP JP2001546955A patent/JP2003521898A/en not_active Withdrawn
- 2000-12-28 CA CA002394652A patent/CA2394652A1/en not_active Abandoned
- 2000-12-28 WO PCT/AU2000/001611 patent/WO2001046459A1/en not_active Application Discontinuation
- 2000-12-28 KR KR1020027008163A patent/KR20020089314A/en not_active Application Discontinuation
- 2000-12-28 EP EP00988522A patent/EP1242625A4/en not_active Withdrawn
Non-Patent Citations (6)
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010133969A (en) * | 2002-08-28 | 2010-06-17 | Carl Zeiss Surgical Gmbh | Microscopy system and method for operating microscopy system |
US8189201B2 (en) | 2002-08-28 | 2012-05-29 | Carl Zeiss Meditec Ag | Microscopy system, microscopy method, and a method of treating an aneurysm |
US8300309B2 (en) | 2002-08-28 | 2012-10-30 | Carl Zeiss Meditec Ag | OCT measuring method and system |
US8705042B2 (en) | 2002-08-28 | 2014-04-22 | Carl Zeiss Meditec Ag | Microscopy system, microscopy method and method of treating an aneurysm |
US8810907B2 (en) | 2002-08-28 | 2014-08-19 | Carl Zeiss Meditec Ag | Surgical microscope system and method |
CN114230024A (en) * | 2021-12-29 | 2022-03-25 | 华南理工大学 | Method for selectively inhibiting glycan bacteria to improve biological phosphorus removal efficiency |
Also Published As
Publication number | Publication date |
---|---|
CA2394652A1 (en) | 2001-06-28 |
EP1242625A4 (en) | 2005-10-19 |
AUPQ486799A0 (en) | 2000-02-03 |
JP2003521898A (en) | 2003-07-22 |
KR20020089314A (en) | 2002-11-29 |
EP1242625A1 (en) | 2002-09-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wang et al. | Detailed comparison of bacterial communities during seasonal sludge bulking in a municipal wastewater treatment plant | |
Crocetti et al. | Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation | |
You et al. | Nitrification efficiency and nitrifying bacteria abundance in combined AS-RBC and A2O systems | |
Ahn et al. | Characterization of denitrifying phosphate-accumulating organisms cultivated under different electron acceptor conditions using polymerase chain reaction-denaturing gradient gel electrophoresis assay | |
Sathasivan et al. | Application of new bacterial regrowth potential method for water distribution system–a clear evidence of phosphorus limitation | |
Bjornsson et al. | Filamentous Chloroflexi (green non-sulfur bacteria) are abundant in wastewater treatment processes with biological nutrient removal | |
Guo et al. | Filamentous and non-filamentous bulking of activated sludge encountered under nutrients limitation or deficiency conditions | |
Kim et al. | Effect of nitrite concentration on the distribution and competition of nitrite-oxidizing bacteria in nitratation reactor systems and their kinetic characteristics | |
Kawaharasaki et al. | In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 4′, 6-diamidino-2-phenylindol (DAPI) at a polyphosphate-probing concentration | |
Chen et al. | Carbon uptake bioenergetics of PAOs and GAOs in full-scale enhanced biological phosphorus removal systems | |
Liu et al. | Dynamics of microbial activities and community structures in activated sludge under aerobic starvation | |
Tsai et al. | Phylogenetic and physiological diversity of tetrad-forming organisms in deteriorated biological phosphorus removal systems | |
Bouchez et al. | Successful and unsuccessful bioaugmentation experiments monitored by fluorescent in situ hybridization | |
He et al. | Fine-scale differences between Accumulibacter-like bacteria in enhanced biological phosphorus removal activated sludge | |
Schneegurt et al. | The application of molecular techniques in environmental biotechnology for monitoring | |
Zilles et al. | Presence of Rhodocyclus in a full-scale wastewater treatment plant and their participation in enhanced biological phosphorus removal | |
Liu et al. | Accumulation and isolation of simultaneous denitrifying polyphosphate-accumulating organisms in an improved sequencing batch reactor system at low temperature | |
Nielsen et al. | Use of microautoradiography and fluorescent in situ hybridization for characterization of microbial activity in activated sludge | |
Cydzik-Kwiatkowska et al. | Impact of operational parameters on bacterial community in a full-scale municipal wastewater treatment plant | |
Bond et al. | Bio-P and non-bio-P bacteria identification by a novel microbial approach | |
US20030170654A1 (en) | Probes and primers for the detection of polyphosphate accumulating organisms in wastewater | |
Guo et al. | Changes in the microbial community structure of filaments and floc formers in response to various carbon sources and feeding patterns | |
Oshiki et al. | PHA-accumulating microorganisms in full-scale wastewater treatment plants | |
Mudaly et al. | 16S rRNA in situ probing for the determination of the family level community structure implicated in enhanced biological nutrient removal | |
Forster et al. | Rains, drains and active strains: towards online assessment of wastewater bacterial communities |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2001 546955 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2394652 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1020027008163 Country of ref document: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 24934/01 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000988522 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 2000988522 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1020027008163 Country of ref document: KR |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10168337 Country of ref document: US |
|
WWG | Wipo information: grant in national office |
Ref document number: 24934/01 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000988522 Country of ref document: EP |