Chemical disinfection of Legionella in hot water systems biofilm: a pilot-scale 1 study

Legionella bacteria encounter optimum growing conditions in hot water systems and cooling towers. A pilot-scale 1 unit was built in order to study the biofilm disinfection. It consisted of two identical loops, one used as a control and the other as a 'Test Loop'. A combination of a bio-detergent and a biocide (hydrogen peroxide + peracetic acid) was applied in the Test Loop three times under the same conditions at 100 and 1,000 mg/L with a contact time of 24 and 3-6 hours, respectively. Each treatment test was preceded by a three week period of biofilm re-colonization. Initial concentrations of culturable Legionella into biofilm were close to 10(3) CFU/cm2. Results showed that culturable Legionella spp. in biofilm were no longer detectable three days following each treatment. evertheless, initial Legionella spp. concentrations were recovered 7 days after the treatments (in two cases). Before the tests, Legionella spp. and L. pneumophila PCR counts were both about 10(4) GU/cm2 in biofilm and they both decreased by 1 to 2 log units 72 hours after each treatment. The three tests had a good but transient efficiency on Legionella disinfection in biofilm.     

Water reuse for urban landscape irrigation: aspersion and health related regulations.

The Mediterranean seaside resort of Le Grau du Roi includes 40 hectares of landscaped areas spray irrigated with river water supplied through a separate network. Wastewater collected from several municipalities is treated in an activated sludge wastewater treatment plant (WWTP) and polished in waste stabilization ponds (WSPs). Planned substitution of treated wastewater for river water is hindered by spray irrigation prohibition within a 100 m distance from houses and recreational areas. WWTP and WSP effluents were monitored for pathogens with a particular attention to Legionella in Spring and Summer 2006. Helminth eggs, salmonellae and enteroviruses were never detected neither in WWTP effluent nor in the ponds. Legionella spp content was slightly higher or of the order of magnitude of river water contents. Regarding Legionella pneumophila contents, WSP effluent did not significantly differ from the river water. E.coli and enterococci contents in WSP effluents complied with the "excellent quality" criteria of the European Directive for coastal bathing waters. Therefore, substituting WSP effluents to river water is unlikely to alter health risks related to spray irrigation and, in this case, the buffer zone required by the French water reuse guidelines appears being short of support.     

Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis.

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean = 35%, standard deviation = 8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.     

Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption

A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10%. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1%) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.     

Butanol and ethanol production from tapioca starch wastewater by Clostridium spp

Total (TWW) and tapioca starch wash wastewater (TSWW) from a cassava processing plant in Thailand were analyzed for their composition with a view to evaluate their potential as substrates for solvent production by ABE fermentation with Clostridium spp. Starch was detected at a 1.63-fold higher level in the TWW than that in the TSWW (24.4% and 15.0% (w/w), respectively). The chemical oxygen demand (COD) was broadly similar (20,093 and 20,433 mg/L), but the biological oxygen demand (BOD) was 1.84-fold higher (18,000 and 9,750 mg/L) in the TWW than that in the TSWW. Thus, the TSWW was selected as a substrate to evaluate its potential for butanol and ethanol production by two Clostridium spp. The combined ethanol plus butanol production in the TSWW at pH 6.5 was higher than that at pH 4.5, being around 27.8- and 3.4-fold higher in C. butyricum TISTR 1032 and C. acetobutylicum ATCC 824, respectively. In both strains, the butanol (and combined butanol plus ethanol) production level was improved at pH 5.5. The addition of yeast extract increased the bacterial cell production, but did not significantly improve solvent productivity in C. acetobutylicum, and even decreased butanol production by C. butyricum.     

Microbiological quality of reclaimed water used for golf courses' irrigation.

Microbial quality of reclaimed water used for irrigation in two golf courses located in the southern Iberian Peninsula (Spain and Portugal) was evaluated. Bacterial indicators for faecal pollution (total and faecal coliforms, Escherichia coli and enterococci) were tested by membrane filtration using appropriate selective media. In addition, somatic E. coli bacteriophages, enteric viruses (entero-, hepatitis A and rota-) and Legionella pneumophila were also analysed. The results obtained showed that all wastewater treatment processes reduced adequately the number of indicator microorganisms although a significant correlation between pathogenic and indicator microorganisms tested was not found. L. pneumophila was detected by PCR but not confirmed by culture. Survival experiments of pathogenic microorganisms in aerosols and irrigated turf are conducted to determine the health hazards for the golf practice and to propose a microbial standard for wastewater used for irrigation of golf courses.     

The fate of legionellae within distribution pipe biofilms: measurement of their persistence, inactivation and detachment.

Distribution pipe biofilms present a currently unquantified public health risk to consumers receiving water for domestic potable and non-potable use. The aim of this study was to quantify the numbers of legionellae, used here as model bacterial pathogens, that may accumulate, persist within and detach from distribution pipe biofilms. L. pneumophila recovered by standard culture from an 8 week-old biofilm formed within a novel pilot-scale water distribution system represented 1% of those present in the adjacent bulk water. A combined chlorine concentration exceeding 0.2 mg x L(-1) eliminated culturable sessile legionellae altogether, though the reduction in FISH-positive cells represented just 75+/-25% of the original amount, compared to a 5-log reduction in culturable cells during the same period. Where there was < 0.1 mg x L(-1) combined chlorine, an exponential decay/loss of sessile L. pneumophila was observed (k = 0.37 - 0.41) over the course of a 38-day experimental period. The inoculation of the system with 1 microm fluorescent microspheres and legionellae demonstrated that removal of the latter was dominated by chemical disinfection, with erosion and biological grazing playing lesser roles. Under turbulent (Re approximately 5000) conditions, larger clusters of biofilm become detached from substrata, with more than 90% of sessile legionellae mobilised into the bulk water phase. Interaction with both biofilms and a thermophilic Acanthamoeba isolate reduced the susceptibility of legionellae to thermal inactivation by between one and two orders of magnitude, though it increased their sensitivity to chemical (free and combined chlorine) disinfection.     

Pretreatment and biotreatment of saline industrial wastewaters.

Wastewater from an Akzo Nobel production site contains more than 50 g/l total dissolved salts, mainly chlorides and sulphates, and is currently being treated after 10-20 x dilution. Biological treatment of undiluted or less diluted wastewater is very desirable for environmental and economic reasons. Possibilities were investigated in laboratory scale reactors to treat this highly saline and high strength wastewater aerobically, either after long adaptation or after removing part of the salts by a pretreatment. Adaptation and selection from mixed activated sludge populations took approximately 40 days to finally achieve a COD removal in aerobic treatment of 55-65% at two times dilution (11-16 g/l chloride and 5-7 g/l sulphate). Undiluted wastewater was not treatable. A higher removal percentage (> 80%) was possible at the original high salt concentration only when the sludge load was limited to approximately 0.4-0.5 kg COD/kg sludge/day. A longer adaptation time was required. Nanofiltration (NF) and crystallization could be used as a pretreatment to remove and recover up to 80% of the sulphate in the form of crystallized Glauber salt. Recovery strongly depended on the sulphate and chloride concentration in the NF concentrate and on crystallization temperature. The salt (sulphate) reduction through the NF improved the removal efficiency of a consecutive biotreatment only at a relatively low chloride level, demonstrating that the combination of nanofiltration-crystallization-aerobic biodegradation is less feasible for very saline wastewaters. Anaerobic pretreatment of saline waters turned out to be rather sensitive to high salinities. Only wastewater diluted to 10 g/l chloride could be treated well: sulphate concentration decreased by 80% and COD by 40%. Removal efficiencies of the combined anaerobic-aerobic treatment were approximately 80-85%, proving that this was a feasible route for 2-3 x diluted wastewater. The study has shown that several alternatives are available for treatment of the very saline wastewaters at a much lower degree of dilution than currently practiced.     

Phenolic refinery wastewater biodegradation by an expanded granular sludge bed reactor.

Refinery spent caustics (SC) were diluted with sour waters (SW) in a ratio 1:7, neutralized with CO2 (SC/SW(CO2)) and 83% of H2S was striped during this procedure, remaining an aromatic portion that contained 2123, 2730 and 1379 mg L(-1) of phenol, p-cresol and o-cresol, respectively. The mixture was teated anaerobically in an EGSB reactor fed with 1.5 gCOD L(-1) d(-1), without mineral supplements causing loss of COD removal efficiency that dropped to 23%, methane production ceased and no phenol or cresols were biodegraded. The EGSB experiments were resumed by feeding the reactor with nutrients and phenol at 1.0 gCOD L(-1) d(-1). The mixture SC/SWco2 added to the phenol load, was step increased from 0.10 to 0.87 gCODL(-1) d(-1) maximum. When total organic load was increased to 1.6, COD removal efficiency was 90% and at the highest load attained, 1.87, efficiency dropped to 23% attributed to the toxic effect produced by cresols.     

Disinfection of sludge with high pathogenic content using silver and other compounds.

A physicochemical sludge with high microbial content (10(2)-10(4) FPU/g TS bacteriophages, 10(6)-10(7) MPN/g TS faecal coliforms, 10(4) MPN/g TS Salmonella spp., 10(4) MPN/g TS Shigella spp., 10(3) MPN/g TS Pseudomonas aeruginosa, 10(2) MPN/g TS Vibrio cholerae, 10(2)-10(3) cysts/g TS Giardia sp., 10(2)-10(4) oocyts/g TS Cryptosporidium sp., 168-215 viable helminth ova/g TS) was disinfected using silver, silver-copper, and silver-copper plus a synergistic agent (SA). Twenty milligrams Ag/g TS inactivated 4.8 log of faecal coliforms in 1 h; however, 40 mg Ag/g TS are needed to reduce helminth ova viability from 84 to 38.4% in the same period of time. Combinations of Ag-Cu (60:600 mg Ag-Cu/g TS) and Ag-SA (60:24 mg Ag-SA/g TS) inactivated 7.8 log of faecal coliforms and around 90% of helminth ova in 60 min. To produce USEPA class A biosolids, 10:100:8 and 5:50:13.3 mg Ag-Cu-SA/gTS are needed. Bacterial regrowth was not observed for all conditions producing <1000 MPN/gTS faecal coliforms, suggesting a residual disinfection effect. Recommended doses to produce class A biosolids inactivated 2-4 log of bacteriophages, 4 log of Salmonella spp., 4 log of Shigella spp., 3 log of Pseudomonas aeruginosa, 2 log of Vibrio cholerae, 87-99.9% of Giardia sp., and 75-99.9% of Cryptosporidium sp.     

Application of biofilm reactors to improve ammonia oxidation in low nitrogen loaded wastewater

An airlift reactor using zeolite particles as carrier material was used for the nitrification of effluents from the aquaculture industry. During the start-up the nitrogen concentration was kept around 100 mg NH4(+)-N/L to develop the nitrifying population. Later it was decreased down to around 3 mg NH4(+)-N/L and the dilution rate was increased up to 4.8 d(-1) in order to simulate the conditions in a an aquaculture waster treatment system. A nitrogen loading rate (NLR) of 535 mg NH(+)-N/m2 d was fully oxidized to nitrate. Higher values of NLRs caused nitrite accumulation. A second biofilm reactor was fed with a synthetic medium containing 50 mg NH4(+)-N/L which simulated the effluents from anaerobic units treating domestic wastewater. A nitrogen loading rate of 400 mg NH4(+)-N/L d was oxidized into nitrate with an efficiency of 60% at a dilution rate of 8 d(-1). Both biofilm systems allowed the development of a nitrifying population to treat the studied types of wastewaters.     

PAHs concentration and toxicity in organic solvent extracts of atmospheric particulate matter and sea sediments

The concentration of polycyclic aromatic hydrocarbons (PAHs) and the toxicity to marine bacteria (Vibrio fischeri) were measured for the organic solvent extracts of sea sediments collected from an urban watershed area (Hiroshima Bay) of Japan and compared with the concentrations and toxicity of atmospheric particulate matter (PM). In atmospheric PM, the PAHs concentration was highest in fine particulate matter (FPM) collected during cold seasons. The concentrations of sea sediments were 0.01-0.001 times those of atmospheric PM. 1/EC50 was 1-10 L g(-1) PM for atmospheric PM and 0.1-1 L g(-1) dry solids for sea sediments. These results imply that toxic substances from atmospheric PM are diluted several tens or hundreds of times in sea sediments. The ratio of the 1/EC50 to PAHs concentration ((1/EC50)/16PAHs) was stable for all sea sediments (0.1-1 L μg(-1) 16PAHs) and was the same order of magnitude as that of FPM and coarse particulate matter (CPM). The ratio of sediments collected from the west was more similar to that of CPM while that from the east was more similar to FPM, possibly because of hydraulic differences among water bodies. The PAHs concentration pattern analyses (principal component analysis and isomer ratio analysis) were conducted and the results showed that the PAHs pattern in sea sediments was quite different to that of FPM and CPM. Comparison with previously conducted PAHs analyses suggested that biomass burning residues comprised a major portion of these other sources.     

Interaction of Cocos nucifera cream casein with humic acid suspensions

Two caseins labelled as CaSMG (casein without fats) and CaMG (casein with fats) were extracted from coconut cream. Both caseins were used as coagulants for the aggregation of humic acid (HA) particles in synthetic water at pH = 6 during the jar-test essays. The optimum dosage of CaMG or CaSMG and the residual turbidities of treated water obtained depend on the type of used casein (CaMG or CaSMG) and the concentration of particles in solution. The optimal doses of CaMG and CaSMG are respectively 280 and 180 mg/L for solution S(1) (HA aqueous solution at 15 mg/L), and then 340 and 240 mg/L for solution S(2) (HA aqueous solution at 25 mg/L). The residual turbidities of treated water are respectively 6.88 and 3.85 NTU for solution S(1) and 4.52 and 2.53 NTU for solution S(2). The collected sediment volumes are respectively 1.2 and 1.5 mL for solutions S(1) and S(2). The electrophoretic mobility measurement and transmission electron microscopy images of flocs formed during the flocculation essays suggest that both caseins operate through both mechanisms (charge neutralisation and bridging process mechanism), this last one seems to be predominant. The aggregates formed are the large clusters and result from adsorption of HA particles by the casein molecules.     

SBR treatment of olive mill wastewaters: dilution or pre-treatment?

The olive-oil extraction industry is an economically important activity for many countries of the Mediterranean Sea area, with Spain, Greece and Italy being the major producers. This activity, however, may represent a serious environmental problem due to the discharge of highly polluted effluents, usually referred to as 'olive mill wastewaters' (OMWs). They are characterized by high values of chemical oxygen demand (COD) (80-300 g/L), lipids, total polyphenols (TPP), tannins and other substances difficult to degrade. An adequate treatment before discharging is therefore required to reduce the pollutant load. The aim of the present paper was to evaluate performances of a biological process in a sequencing batch reactor (SBR) fed with pre-treated OMWs. Pre-treatment consisted of a combined acid cracking (AC) and granular activated carbon (GAC) adsorption process. The efficiency of the system was compared with that of an identical SBR fed with the raw wastewater only diluted. Combined AC and GAC adsorption was chosen to be used prior to the following biological process due to its capability of providing high removal efficiencies of COD and TPP and also appreciable improvement of biodegradability. Comparing results obtained with different influents showed that best performances of the SBR were obtained by feeding it with raw diluted OMWs (dOMWs) and at the lowest dilution ratio (1:25): in this case, the removal efficiencies were 90 and 76%, as average, for COD and TPP, respectively. Feeding the SBR with either the pre-treated or the raw dOMWs at 1:50 gave very similar values of COD reduction (74%); however, an improvement of the TPP removal was observed in the former case.     

改良CASS工艺在小区生活污水处理中的应用

改良CASS工艺在小区生活污水处理及回用中应用的运行结果表明,进水CODcr为100-300mg/L,BOD5为80-150mg/L时,SS为100-200mg/L时,出水CODcr≤50mg/L,BOD5≤10mg/L时,SS≤10mg/L,完全符合回用要求。     

Evaluation of nutrient loads from a mountain forest including storm runoff loads.

Water quality and flow rates at a weir installed on the end of Aburahi-S Experimental Watershed (3.34 ha) were measured once a week from 2001 to 2003 and in appropriate intervals from 30 min to 6 h during five storm runoff events caused by each rainfall from 8 mm to 417 mm. The average annual loads of total nitrogen (TN) and total phosphorus (TP) were calculated to be 19.0 and 0.339 kg ha(-1) y(-1) from the periodical data by using the integration interval-loads method (ILM), which did not properly account for storm runoff loads. Three types of L(Q) equations (L = aQ(b)) were derived from correlations between loading rates L and flow rates Q obtained from the periodic observation and from storm runoff observation. L(Q) equation method (LQM), which was derived from the storm runoff observation and allowed for the hysteresis of discharge of materials, gave 9.68 and 0.159 kg ha(-1) y(-1), respectively, by substitution of the sequential hourly data of flow rates. L(R) equation (L = c(R - r)d) was derived from the correlations between the loads and the effective rainfall depth (R - r) measured during the storm runoff events, and L(R) equation method (LRM) calculated 9.83 +/- 1.68 and 0.175 +/- 0.0761 kg ha(-1) y(-1), respectively, by using the rainfall data for the past 16 years. The atmospheric input-fluxes of TN and TP were 16.5 and 0.791 kg ha(-1) y(-1).     

Prevalence and ecological features of deep chlorophyll layers in Lake of the Woods,a complex hydrological system with strong trophic,physical, and chemical gradients

We characterized deep chlorophyll layers (DCLs), previously unknown, in Lake of the Woods (Canada), a complex hydrological system with strong trophic, physical, and chemical gradients. Of 42 sites, five (12 %) contained at least one dense, thin metalimnetic peak – often more, overlapping or vertically stratified. In spring, highest biomass (>4 mg/L) was found at 3 m in Bigstone Bay, composed of a mixed phytoplankton community. Other, deeper, spring DCLs were dominated by the diatom Cyclotella (～1 mg/L, 21 m), dinoflagellate Gymnodinium (>1 mg/L, 17 m), cyanobacterium Dolichospermum (>3 mg/L, 10 m), or cyanobacterium Woronichinia (>1 mg/L, ～8 m). In summer, the highest biomass of a DCL was in Yellow Girl Bay (>21 mg/L), over five times higher than spring and higher than large surface blooms from the shallow, eutrophic south (Sabaskong Bay ～19 mg/L). Summer DCLs (～7 m) were not as deep as spring, owing to reduced light penetration (2.5-fold lower Zeu:Zmix). Most summer DCLs were dominated by Dolichospermum (max. >16 mg/L) but other species were also high (～1 mg/L biomass), including cyanobacterium Aphanizomenon, diatom Aulacoseira, and cryptophyte Cryptomonas. Euphotic zone in these embayments intersected with a nutrient-enriched (PO₄, Fe, Mn, Si) hypoxic hypolimnion, conditions conducive to DCLs. Other drivers of composition and activity of DCLs remain to be elucidated. These high levels of DCL biomass, up to fivefold higher than near-surface, have routinely gone unreported. Their inclusion in establishing baselines and tracking change would inform lake management decisions and qualify expectations for ecosystem response.     

Determination of ammonia-oxidizing bacteria based on time-resolved fluorescence and tandem probe

To study the ecology of ammonia-oxidizing bacteria (AOB), quantitative techniques are essential. In this report, the authors introduced an innovative method based on time-resolved fluorescence to quantify AOB as a representative of the major functional microorganisms in sewage water treatment. A bifunctional europium complex with the characteristics of long lifetime and intense luminescence was used as labels in the experiments. In the detection, the capture probe and dye-labeled reporter probe could form a two-probe tandem with the target sequence, and the determination of target DNA was done by monitoring the time-resolved fluorescence signals of europium complex-labeled reporter probe left on the glass slide surface. The experiment conditions consisting of concentration of capture probe, hybridization temperature, hybridization time and washing time were optimized. This method presents satisfactory specificity to common bacteria such as Nitrobacter winogradskyi, Escherichia coli and Paenibacillus polymyxa. The detection limit was 3.65 × 10(-11)mol L(-1). This detection system enables us to rapidly and sensitively analyze the microbial population variety in sewage water treatment.     

Nucleic acids: indicators for dynamic processes of clogging in soil filter systems.

A high-performance liquid chromatography (HPLC)-method was established to quantify the development and degradation of DNA, rRNA and tRNA in the Langenalb soil filter fed with combined sewer overflow. The DNA served as a parameter for the microbial biomass and the RNA/DNA ratio for the growth rate. For nucleic acids, quantification samples were taken at different depths and sections of the clogged and the unclogged soil filter. Highest DNA concentrations were found at a depth of 0-2 cm in the clogged soil filter. During a starvation period of three months the DNA- and RNA-concentrations decreased twofold at depths between 0-8 cm. The higher nucleic acid concentrations at depths of 10-40 cm in the unclogged soil filter were attributed to a shift of the starved microorganisms. Similar RNA/DNA ratios in the clogged and the unclogged soil filter indicate similar growth rates and metabolic activities. It could be proven that the clogging of the soil filter was due to the microbial biomass and the DNA concentration, respectively.     

Autotrophic nitrogen removal from anaerobic supernatant of Florence's WWTP digesters.

In municipal WWTP with anaerobic sludge digestion, 10-20% of total nitrogen load comes from the return supernatant produced by the final sludge dewatering. In recent years a completely autotrophic nitrogen removal process based on Anammox biomass has been tested in a few European countries, in order to treat anaerobic supernatant and to increase the COD/N ratio in municipal wastewater. This work reports the experimental results of the SHARON-ANAMMOX process application to anaerobic supernatant taken from the urban Florentine area wastewater treatment plant (S. Colombano WWTP). A nitritation labscale chemostat (7.4 L) has been started-up seeded with the S. Colombano WWTP nitrifying activated sludge. During the experimental period, nitrite oxidising bacteria wash-out was steadily achieved with a retention time ranging from 1 to 1.5 d at 35 degrees C. The Anammox inoculum sludge was taken from a pilot plant at EAWAG (Zurich). Anammox biomass has been enriched at 33 degrees C with anaerobic supernatant diluted with sodium nitrite solution until reaching a maximum specific nitrogen removal rate of 0.065 kgN kg(-1) VSS d(-1), which was 11 times higher than the one found in inoculum sludge (0.005 kgN kg(-1) VSS d(-1). In a lab-scale SBR reactor (4 L), coupled with nitritation bioreactor, specific nitrogen removal rate (doubling time equal to 26 d at 35 degrees C and at nitrite-limiting condition) reached the value of 0.22 kgN kg(-1) VSS d(-1), which was approximately 44 times larger than the rate measured in the inoculum Anammox sludge.