Influences of Extracellular Polymeric Substances on the Dewaterability of Sewage Sludge during Bioleaching

Extracellular polymeric substances (EPS) play important roles in regulating the dewaterability of sludge. This study sought to elucidate the influence of EPS on the dewaterability of sludge during bioleaching process. Results showed that, in bioleaching system with the co-inoculation of Acidithiobacillus thiooxidans TS6 and Acidithiobacillus ferrooxidans LX5 (A. t+A. f system), the capillary suction time (CST) of sludge reduced from 255.9 s to 25.45 s within 48 h, which was obviously better than the controls. The correlation analysis between sludge CST and sludge EPS revealed that the sludge EPS significantly impacted the dewaterability of sludge. Sludge CST had correlation with protein content in slime and both protein and polysaccharide contents in TB-EPS and Slime+LB+TB layers, and the decrease of protein content in slime and decreases of both protein and polysaccharide contents in TB-EPS and Slime+LB+TB layers improved sludge dewaterability during sludge bioleaching process. Moreover, the low sludge pH (2.92) and the increasing distribution of Fe in the solid phase were another two factors responsible for the improvement of sludge dewaterability during bioleaching. This study suggested that during sludge bioleaching the growth of Acidithiobacillus species resulted in the decrease of sludge pH, the increasing distribution of Fe in the solid phase, and the decrease of EPS content (mainly including protein and/or polysaccharide) in the slime, TB-EPS, and Slime+LB+TB layers, all of which are helpful for sludge dewaterability enhancement.     

Inactivation by Ionizing Radiation of Salmonella enteritidis Serotype montevideo Grown in Composed Sewage Sludge

S. enteritidis ser. montevideo were grown in composted sewage sludge to levels of approximately 10⁹/g. These bacteria were found to be inactivated by ionizing radiation at approximately the same rate (30 krads/log) as Salmonella species in liquid digested sludge.     

Long-Term Effects of Metal-Rich Sewage Sludge Application on Soil Populations of Bradyrhizobium japonicum

The application of sewage sludge to land may increase the concentration of heavy metals in soil. Of considerable concern is the effect of heavy metals on soil microorganisms, especially those involved in the biocycling of elements important to soil productivity. Bradyrhizobium japonicum is a soil bacterium involved in symbiotic nitrogen fixation with Glycine max, the common soybean. To examine the effect of metal-rich sludge application on B. japonicum, the MICs for Pb, Cu, Al, Fe, Ni, Zn, Cd, and Hg were determined in minimal media by using laboratory reference strains representing 11 common serogroups of B. japonicum. Marked differences were found among the B. japonicum strains for sensitivity to Cu, Cd, Zn, and Ni. Strain USDA 123 was most sensitive to these metals, whereas strain USDA 122 was most resistant. In field studies, a silt loam soil amended 11 years ago with 0, 56, or 112 Mg of digested sludge per ha was examined for total numbers of B. japonicum by using the most probable number method. Nodule isolates from soybean nodules grown on this soil were serologically typed, and their metal sensitivity was determined. The number of soybean rhizobia in the sludge-amended soils was found to increase with increasing rates of sludge. Soybean rhizobia strains from 11 serogroups were identified in the soils; however, no differences in serogroup distribution or proportion of resistant strains were found between the soils. Thus, the application of heavy metal-containing sewage sludge did not have a long-term detrimental effect on soil rhizobial numbers, nor did it result in a shift in nodule serogroup distribution.     

Alteration of a Salt Marsh Bacterial Community by Fertilization with Sewage Sludge

The effects of long-term fertilization with sewage sludge on the aerobic, chemoheterotrophic portion of a salt marsh bacterial community were examined. The study site in the Great Sippewissett Marsh, Cape Cod, Mass., consisted of experimental plots that were treated with different amounts of commercial sewage sludge fertilizer or with urea and phosphate. The number of CFUs, percentage of mercury- and cadmium-resistant bacteria, and percentage of antibiotic-resistant bacteria were all increased in the sludge-fertilized plots. Preliminary taxonomic characterization showed that sludge fertilization markedly altered the taxonomic distribution and reduced diversity within both the total heterotrophic and the mercury-resistant communities. In control plots, the total heterotrophic community was fairly evenly distributed among taxa and the mercury-resistant community was dominated by Pseudomonas spp. In sludge-fertilized plots, both the total and mercury-resistant communities were dominated by a single Cytophaga sp.     

Survival of Bacterial Indicator Species and Bacteriophages after Thermal Treatment of Sludge and Sewage

The inactivation of naturally occurring bacterial indicators and bacteriophages by thermal treatment of a dewatered sludge and raw sewage was studied. The sludge was heated at 80°C, and the sewage was heated at 60°C. In both cases phages were significantly more resistant to thermal inactivation than bacterial indicators, with the exception of spores of sulfite-reducing clostridia. Somatic coliphages and phages infecting Bacteroides fragilis were significantly more resistant than F-specific RNA phages. Similar trends were observed in sludge and sewage. The effects of thermal treatment on various phages belonging to the three groups mentioned above and on various enteroviruses added to sewage were also studied. The results revealed that the variability in the resistance of phages agreed with the data obtained with the naturally occurring populations and that the phages that were studied were more resistant to heat treatment than the enteroviruses that were studied. The phages survived significantly better than Salmonella choleraesuis, and the extents of inactivation indicated that naturally occurring bacteriophages can be used to monitor the inactivation of Escherichia coli and Salmonella.     

Biological Degradation of Crude Oil Refinery Sludge with Commercial Surfactant and Sewage Sludge by Co-Composting

This study determined the potential of surfactant and sewage sludge in enhancing degradation of oil sludge. A mixture of oil sludge, surfactant, and sewage sludge was co-composted for 24 weeks in the laboratory. Physical and chemical parameters in the compost were measured every four weeks. Isolated microorganisms were characterized by molecular techniques. The pH in all experiments remained between 8 and 6.4. CO₂ evolution reached 5503 µg/dwt/day by the twenty-fourth week. The dominant bacterial species were Acinectobacter, Rodococcus, mycobacterium, Pseudomonas, Bacillus, Arthrobacter, and Staphylococcus species and fungi were Pleurotus, Penicillium, and Aspergillus sp. TPH was reduced by 92% in the sewage sludge and surfactant treatment, 75 and 81% in other treatments, and 44.2% in the control. PAH concentrations were reduced by between 75 and 100%. The results indicate that a careful application of surfactant and sewage sludge could enhance oil sludge degradation in a compost system.     

Hygienisation and Nutrient Conservation of Sewage Sludge or Cattle Manure by Lactic Acid Fermentation

Manure from animal farms and sewage sludge contain pathogens and opportunistic organisms in various concentrations depending on the health of the herds and human sources. Other than for the presence of pathogens, these waste substances are excellent nutrient sources and constitute a preferred organic fertilizer. However, because of the pathogens, the risks of infection of animals or humans increase with the indiscriminate use of manure, especially liquid manure or sludge, for agriculture. This potential problem can increase with the global connectedness of animal herds fed imported feed grown on fields fertilized with local manures. This paper describes a simple, easy-to-use, low-tech hygienization method which conserves nutrients and does not require large investments in infrastructure. The proposed method uses the microbiotic shift during mesophilic fermentation of cow manure or sewage sludge during which gram-negative bacteria, enterococci and yeasts were inactivated below the detection limit of 3 log₁₀ cfu/g while lactobacilli increased up to a thousand fold. Pathogens like Salmonella, Listeria monocytogenes, Staphylococcus aureus, E. coli EHEC O:157 and vegetative Clostridium perfringens were inactivated within 3 days of fermentation. In addition, ECBO-viruses and eggs of Ascaris suum were inactivated within 7 and 56 days, respectively. Compared to the mass lost through composting (15–57%), the loss of mass during fermentation (< 2.45%) is very low and provides strong economic and ecological benefits for this process. This method might be an acceptable hygienization method for developed as well as undeveloped countries, and could play a key role in public and animal health while safely closing the nutrient cycle by reducing the necessity of using energy-inefficient inorganic fertilizer for crop production.     

Near-Bottom Pelagic Bacteria at a Deep-Water Sewage Sludge Disposal Site

The epibenthic bacterial community at deep-ocean sewage sludge disposal site DWD-106, located approximately 106 miles (ca. 196 km) off the coast of New Jersey, was assessed for changes associated with the introduction of large amounts of sewage sludge. Mixed cultures and bacterial isolates obtained from water overlying sediment core samples collected at the deep-water (2,500 m) municipal sewage disposal site were tested for the ability to grow under in situ conditions of temperature and pressure. The responses of cultures collected at a DWD-106 station heavily impacted by sewage sludge were compared with those of samples collected from a station at the same depth which was not contaminated by sewage sludge. Significant differences were observed in the ability of mixed bacterial cultures and isolates from the two sites to grow under deep-sea pressure and temperature conditions. The levels of sludge contamination were established by enumerating Clostridium perfringens, a sewage indicator bacterium, in sediment samples from the two sites. The results of hybridization experiments in which DNAs extracted directly from the water overlying sediment core samples were used indicate that the reference site epibenthic community, the disposal site epibenthic community, and the community in a surface sludge plume share many members. Decreased culturability of reference site mixed cultures in the presence of sewage sludge was observed. Thus, the culturable portions of both the autochthonous and allochthonous bacterial communities at the disposal site may be inhibited in situ, the former by sewage sludge and the latter by high pressure and low temperature.     

Benthic Distribution of Sewage Sludge Indicated by Clostridium perfringens at a Deep-Ocean Dump Site

Clostridium perfringens in sediment samples collected at the Deep Water Municipal Sewage Sludge Disposal Site (also called the 106-Mile Site), off the coast of New Jersey, was enumerated. The counts of C. perfringens found in sediment samples collected within and to the southwest of the 106-Mile Site were significantly elevated (P < 0.01) compared with counts of samples from reference stations of similar depth (2,400 to 2,700 m), topography, and distance from the continental shelf, indicating that the benthic environment was contaminated by sewage dumping at this site. Low counts of C. perfringens in sediment samples collected at stations between the base of the continental shelf and the 106-Mile Site indicated that coastal runoff was not a significant source of contamination. Elevated counts were observed for samples up to 92 km to the southwest, whereas low counts were obtained for samples from stations to the east of the 106-Mile Site. This distribution is consistent with previous model predictions of sludge deposition. In areas heavily impacted by sludge dumping, C. perfringens counts were generally highest in the top 1 cm of sediment and exceeded 9,000 CFU g (dry weight) of sediment^-1. The patterns of C. perfringens dispersal observed in this study have proved useful for selection of heavily impacted areas and control stations for further ecological evaluation by a multidisciplinary research team.     

Persistence of Viruses in Desert Soils Amended with Anaerobically Digested Sewage Sludge

Pima County, Ariz., is currently investigating the potential benefits of land application of sewage sludge. To assess risks associated with the presence of pathogenic enteric viruses present in the sludge, laboratory studies were conducted to measure the inactivation rate (k = log₁₀ reduction per day) of poliovirus type 1 and bacteriophages MS2 and PRD-1 in two sludge-amended desert agricultural soils (Brazito Sandy Loam and Pima Clay Loam). Under constant moisture (approximately -0.05 × 10⁵ Pa for both soils) and temperatures of 15, 27, and 40°C, the main factors controlling the inactivation of these viruses were soil temperature and texture. As the temperature increased from 15 to 40°C, the inactivation rate increased significantly for poliovirus and MS2, whereas, for PRD-1, a significant increase in the inactivation rate was observed only at 40°C. Clay loam soils afforded more protection to all three viruses than sandy soils. At 15°C, the inactivation rate for MS2 ranged from 0.366 to 0.394 log₁₀ reduction per day in clay loam and sandy loam soils, respectively. At 27°C, this rate increased to 0.629 log₁₀ reduction per day in clay loam soil and to 0.652 in sandy loam soil. A similar trend was observed for poliovirus at 15°C (k = 0.064 log₁₀ reduction per day, clay loam; k = 0.095 log₁₀ reduction per day, sandy loam) and 27°C (k = 0.133 log₁₀ reduction per day, clay loam; k = 0.154 log₁₀ reduction per day, sandy loam). Neither MS2 nor poliovirus was recovered after 24 h at 40°C. No reduction of PRD-1 was observed after 28 days at 15°C and after 16 days at 27°C. At 40°C, the inactivation rates were 0.208 log₁₀ reduction per day in amended clay loam soil and 0.282 log₁₀ reduction per day in sandy loam soil. Evaporation to less than 5% soil moisture completely inactivated all three viruses within 7 days at 15°C, within 3 days at 27°C, and within 2 days at 40°C regardless of soil type. This suggests that a combination of high soil temperature and rapid loss of soil moisture will significantly reduce risks caused by viruses in sludge.     

Change in Microflora of Sewage Sludge by Gamma-ray Irradiation

Total bacteria of activated dewatered sludge cake of Takasaki city which amounted to 2 × 10⁹ per gram diminished rapidly with the radiation dose, but slowly after 0.5 Mrad, and 10³ per gram survived even after 10 Mrad irradiation. However, coliforms which amounted to 8 × 10⁷ per gram were inactivated below 0.5 Mrad irradiation. The predominant bacteria in non-irradiated sludge were Pseudomonas cepacia and it mainly survived up to 2 Mrad, but Bacillus were predominant at 0.5 to 0.7 Mrad irradiation. The main residual flora from 2 to 5 Mrad was composed of Pseudomonas soranacearum, P. cepacia and P. delafieldii, and the main residual flora in more than 5 Mrad irradiated sludge was P. flava . These typical strains of Pseudomonas in phosphate buffer were radiation sensitive, and their D₁₀ values were from 0.005 to 0.021 Mrad under aerobic irradiation conditions.     

EDTA-Facilitated Phytoremediation of Soil with Heavy Metals from Sewage Sludge

The objective of this research was to determine the effect of the chelate EDTA (ethylenediaminetetraacetic acid), which is used in phytoremediation, on plant availability of heavy metals in liquid sewage sludge applied to soil. Sunflower (Helianthus annuus L.) was grown under greenhouse conditions in a commercial potting soil; the tetrasodium salt of EDTA (EDTA Na₄) was added at a rate of 1 g kg^-1 to half the pots. Immediately after seeds were planted, half of the pots with each soil (with or without EDTA) were irrigated with 60 ml sludge, and half were irrigated with 60 ml tap water. For the subsequent five irrigations, plants in soil with EDTA received either sludge or tap water containing 0.5 g EDTA Na₄ per 1000 ml, and plants in soil without EDTA received sludge or tap water without EDTA. Of the four heavy metals whose extractable concentrations in the soil were measured (Cu, Fe, Mn, and Zn), only Zn had a higher concentration in sludge-treated soil with EDTA compared to sludge-treated soil without EDTA. The concentrations of Fe, Cu, and Mn were similar in sludge-treated soil with and without EDTA. Of the three heavy metals whose total concentrations in the soil were measured (Cd, Pb, Cr), Pb (<10 mg kg^-1) and Cd (< 1 mg kg^-1) were below detection limits, and Cr was unaffected by treatment. The concentration of all measured elements in plants (Cd, Cu, Fe, Zn, Pb) was higher than the concentrations measured in the soil. With no EDTA, sludge-treated plants had a higher concentration of the five heavy elements than plants grown without sludge. Cadmium was lower in sludge-treated plants with EDTA than plants with EDTA and no sludge. After treatment with EDTA, the concentrations of Cu, Fe, and Zn were similar in plants with and without sludge. Lead was higher in plants with EDTA than plants without EDTA, showing that EDTA can facilitate phytoremediation of soil with Pb from sewage sludge.     

Effects of Cationic Polyacrylamide Characteristics on Sewage Sludge Dewatering and Moisture Evaporation

The effects of the molecular weight (MW) and charge density (CD) of cationic polyacrylamide (CPAM) on sludge dewatering and moisture evaporation were investigated in this study. Results indicated that in sludge conditioning, the optimum dosages were 10, 6, 6, 4, and 4 mg g⁻¹ CPAM with 5 million MW and 20% CD, 5 million MW and 40% CD, 3 million MW and 40% CD, 8 million MW and 40% CD, and 5 million MW and 60% CD, respectively. The optimum dosage of CPAM was negatively correlated with its CD or MW if the CD or MW of CPAM was above 20% or 5 million. In the centrifugal dewatering of sludge, the moisture content in the conditioned sludge gradually decreased with the extension of centrifugation time, and the economical centrifugal force was 400×g. The moisture evaporation rates of the conditioned sludge were closely related to sludge dewaterability, which was in turn significantly correlated either positively with the solid content of sludge particles that were >2 mm in size or negatively with that of particles measuring 1 mm to 2 mm in diameter. During treatment, sludge moisture content was reduced from 80% to 20% by evaporation, and the moisture evaporation rates were 1.35, 1.49, 1.62, and 2.24 times faster in the sludge conditioned using 4 mg g⁻¹ CPAM with 5 million MW and 60% CD than in the sludge conditioned using 4 mg g⁻¹ CPAM with 8 million MW and 40% CD, 6 mg g⁻¹ CPAM with 5 million MW and 40% CD, 6 mg g⁻¹ CPAM with 3 million MW and 40% CD, and 10 mg g⁻¹ CPAM with 5 million MW and 20% CD, respectively. Hence, the CPAM with 5 million MW and 60% CD was ideal for sludge dewatering.     

Nitrogen Fixation (Acetylene Reduction) in a Salt Marsh Amended with Sewage Sludge and Organic Carbon and Nitrogen Compounds

Seasonal distribution of nitrogen fixation by Spartina alterniflora epiphytes and in surface and soil samples was investigated in a Georgia salt marsh which was amended with sewage sludge or with glucose and/or ammonium nitrate. There was no significant difference between the rates of fixation in the unamended and sewage sludge plots. Additional perturbation experiments suggested that nitrogen addition indirectly stimulates nitrogen fixation by enhancing Spartina production and root exudation. Glucose additions, on the other hand, suppressed nitrogen fixation on a long-term basis. It is suggested that the microbial population in the soil out-competed the plants for the available nitrogen and in turn suppressed plant production and possibly root exudation. A comparison of nitrogen fixation in clipped and unclipped Spartina plots substantiated the suggestion that root exudation probably supports nitrogen fixation. Fixation in the clipped plots was significantly lower (P < 0.05) than the rates in the unclipped plots.     

Ecophysiological Responses of Plants After Sewage Sludge Compost Applications

Composting is one of the most appropriate methods to recycle sewage sludge. Sewage sludge compost is a suitable solution for improving the quality of barren soil at landfill. Therefore, it is important to investigate the effects of sewage sludge compost on plants. Different compost application methods (mixing and scattering over reclaimed soil) on sawtooth oak (Quercus acutissima) and Japanese red pine (Pinus densiflora) have been tested. The application of sewage sludge compost markedly increased soil moisture and nitrogen content. Compost treatments resulted in significant increases in both plant height and biomass as compared to controls. Compost treatments led to a significant increase in the N content of plant leaves. Compost treatments resulted in significant increases in the chlorophyll content and photosynthetic rates of the plants. The scattering of compost over reclaimed soil (compost 2) resulted in lower total antioxidant activity and superoxide dismutase activity than mixing the compost with the reclaimed soil (compost 1), or in the control treatment. Since the growth rates, N content, and photosynthetic rates in compost 2 treatment were not markedly different from compost 1 treatment, it (compost 2) would be a better application method from both an ecological and economic perspective.     

Behavior of Metals,Pathogen Parasites,and Indicator Bacteria in Sewage Effluents During Biological Treatment by Activated Sludge

The purpose of this study was to evaluate the behavior of metals, pathogen parasites, and indicator bacteria in sewage effluents during biological treatment by activated sludge in a wastewater treatment plant in Ribeirão Preto (WTP-RP), Sao Paulo, Brazil. The evaluation was done during a period of 1 year. Results showed that metal concentrations in treated effluents decreased, reaching concentrations according to those established by national regulations. The activated sludge process at the WTP-RP promoted a partial removal of parasites considered as possible indicators according to the WHO guidelines. Reduction factors varied between 18.2% and 100% for agents such as Endolimax nana, Entamoeba coli, Entamoeba hystolitica, Giardia sp., Ancylostoma sp., Ascaris sp., Fasciola hepatica, and Strongyloides stercoralis. A removal was also observed in total and fecal coliforms quantification. The present study represents an initial evaluation of the chemical and microbiological removal capacity of the WTP-RP. The results should be of interest for the authorities responsible for the environmental health at municipal, regional, national, and international levels.     

The Microbiota and Abundance of the Class 1 Integron-Integrase Gene in Tropical Sewage Treatment Plant Influent and Activated Sludge

Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.     

Impact of Sublethal Levels of Environmental Pollutants Found in Sewage Sludge on a Novel Caenorhabditis elegans Model Biosensor

A transgenic strain of the model nematode Caenorhabditis elegans in which bioluminescence reports on relative, whole-organism ATP levels was used to test an environmentally-relevant mixture of pollutants extracted from processed sewage sludge. Changes in bioluminescence, following exposure to sewage sludge extract, were used to assess relative ATP levels and overall metabolic health. Reproductive function and longevity were also monitored. A short (up to 8 h) sublethal exposure of L4 larval stage worms to sewage sludge extract had a concentration-dependent, detrimental effect on energy status, with bioluminescence decreasing to 50–60% of the solvent control (1% DMSO). Following longer exposure (22–24 h), the energy status of the nematodes showed recovery as assessed by bioluminescence. Continuous exposure to sewage sludge extract from the L4 stage resulted in a shorter median lifespan relative to that of solvent or medium control animals, but only in the presence of 400–600 µM 5-fluoro-2′-deoxyuridine (FUdR), which was incorporated to inhibit reproduction. This indicated that FUdR increased lifespan, and that the effect was counteracted by SSE. Exposure to sewage sludge extract from the L1 stage led to slower growth and a delayed onset of egg laying. When L1 exposed nematodes reached the reproductive stage, no effect on egg laying rate or egg number in the uterus was observed. DMSO itself (1%) had a significant inhibitory effect on growth and development of C. elegans exposed from the L1 stage and on reproduction when exposed from the L4 stage. Results demonstrate subtle adverse effects on C. elegans of a complex mixture of environmental pollutants that are present, individually, in very low concentrations and indicate that our biosensor of energy status is a novel, sensitive, rapid, quantitative, whole-organism test system which is suitable for high throughput risk assessment of complex pollutant mixtures.     

Dispersal of Aspergillus fumigatus from Sewage Sludge Compost Piles Subjected to Mechanical Agitation in Open Air

Aerosolization of the thermophilous fungal opportunist Aspergillus fumigatus from mechanically agitated compost piles was examined at a pilot-scale sewage sludge composting facility and two other selected test sites. Aerosols of A. fumigatus downwind from stationary compost piles were insignificant in comparison with those downwind from agitated piles. These aerosols were generated by a front-end loader moving and dropping compost. Aerial concentrations of the fungus at distances downwind from the point of emission were used to determine an emission rate for A. fumigatus associated with the moving operations. The maximum emission rate, 4.6 × 10⁶A. fumigatus particles per s, was used to calculate predicted concentrations in an unobstructed plume with restrictive, neutral, and dispersive atmospheric mixing conditions up to 1 km downwind from the emission source.     

Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like β-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg²⁺, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.