In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp

Aims: The purpose of this work was to investigate microbial ecology of nitrifiers at the genus level in a typical full-scale activated sludge plant. Methods and Results: Grab samples of mixed liquor were collected from a plug-flow reactor receiving domestic wastewater. Fluorescent in situ hybridization technique (FISH) was used to characterize both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in combination with Confocal Scanning Laser Microscope (CSLM). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes were used in this study. Both Nitrosomonas and Nitrosospira genera as AOB and Nitrobacter and Nitrospira genera as NOB were sought with genus specific probes Nsm156, Nsv443 and NIT3 and NSR1156, respectively. Conclusions: It was shown that Nitrosospira genus was dominant in the activated sludge system studied, although Nitrosomonas is usually assumed to be the dominant genus. At the same time, Nitrobacter genus was detected in activated sludge samples. Significance and Impact of the Study: Previous studies based on laboratory scale pilot plants employing synthetic wastewater suggested that only Nitrospira are found in wastewater treatment plants. We have shown that Nitrobacter genus might also be present. We think that these kinds of studies may not give a valid indication of the microbial diversity of the real full-scale plants fed with domestic wastewater.     

Nitrotoga-like bacteria are previously unrecognized key nitrite oxidizers in full-scale wastewater treatment plants

Numerous past studies have shown members of the genus Nitrospira to be the predominant nitrite-oxidizing bacteria (NOB) in nitrifying wastewater treatment plants (WWTPs). Only recently, the novel NOB ‘Candidatus Nitrotoga arctica'' was identified in permafrost soil and a close relative was enriched from activated sludge. Still, little is known about diversity, distribution and functional importance of Nitrotoga in natural and engineered ecosystems. Here we developed Nitrotoga 16S rRNA-specific PCR primers and fluorescence in situ hybridization (FISH) probes, which were applied to screen activated sludge samples from 20 full-scale WWTPs. Nitrotoga-like bacteria were detected by PCR in 11 samples and reached abundances detectable by FISH in seven sludges. They coexisted with Nitrospira in most of these WWTPs, but constituted the only detectable NOB in two systems. Quantitative FISH revealed that Nitrotoga accounted for nearly 2% of the total bacterial community in one of these plants, a number comparable to Nitrospira abundances in other WWTPs. Spatial statistics revealed that Nitrotoga coaggregated with ammonia-oxidizing bacteria, strongly supporting a functional role in nitrite oxidation. This activity was confirmed by FISH in combination with microradiography, which revealed nitrite-dependent autotrophic carbon fixation by Nitrotoga in situ. Correlation of the presence or absence with WWTP operational parameters indicated low temperatures as a main factor supporting high Nitrotoga abundances, although in incubation experiments these NOB remained active over an unexpected range of temperatures, and also at different ambient nitrite concentrations. In conclusion, this study demonstrates that Nitrotoga can be functionally important nitrite oxidizers in WWTPs and can even represent the only known NOB in engineered systems.     

Influence of growth manner on nitrifying bacterial communities and nitrification kinetics in three lab-scale bioreactors

The effects of growth type, including attached growth, suspended growth, and combined growth, on the characteristics of communities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were studied in three lab-scale Anaerobic/Anoxic_m-Oxic_n (AmOn) systems. These systems amplified activated sludge, biofilms, and a mixture of activated sludge and biofilm (AS-BF). Identical inocula were adopted to analyze the selective effects of mixed growth patterns on nitrifying bacteria. Fluctuations in the concentration of nitrifying bacteria over the 120 days of system operation were analyzed, as was the composition of nitrifying bacterial community in the stabilized stage. Analysis was conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR. According to the DGGE patterns, the primary AOB lineages were Nitrosomonas europaea (six sequences), Nitrosomonas oligotropha (two sequences), and Nitrosospira (one sequence). The primary subclass of NOB community was Nitrospira, in which all identified sequences belonged to Nitrospira moscoviensis (14 sequences). Nitrobacter consisted of two lineages, namely Nitrobacter vulgaris (three sequences) and Nitrobacter alkalicus (two sequences). Under identical operating conditions, the composition of nitrifying bacterial communities in the AS-BF system demonstrated significant differences from those in the activated sludge system and those in the biofilm system. Major varieties included several new, dominant bacterial sequences in the AS-BF system, such as N. europaea and Nitrosospira and a higher concentration of AOB relative to the activated sludge system. However, no similar differences were discovered for the concentration of the NOB population. A kinetic study of nitrification demonstrated a higher maximum specific growth rate of mixed sludge and a lower half-saturation constant of mixed biofilm, indicating that the AS-BF system maintained relatively good nitrifying ability.     

Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.     

Effect of pH and nitrite concentration on nitrite oxidation rate

The effect of pH and nitrite concentration on the activity of the nitrite oxidizing bacteria (NOB) in an activated sludge reactor has been determined by means of laboratory batch experiments based on respirometric techniques. The bacterial activity was measured at different pH and at different total nitrite concentrations (TNO?). The experimental results showed that the nitrite oxidation rate (NOR) depends on the TNO? concentration independently of the free nitrous acid (FNA) concentration, so FNA cannot be considered as the real substrate for NOB. NOB were strongly affected by low pH values (no activity was detected at pH 6.5) but no inhibition was observed at high pH values (activity was nearly the same for the pH range 7.5-9.95). A kinetic expression for nitrite oxidation process including switch functions to model the effect of TNO? concentration and pH inhibition is proposed. Substrate half saturation constant and pH inhibition constants have been obtained.     

Microbial diversity differences within aerobic granular sludge and activated sludge flocs

In this study, we investigated during 400 days the microbial community variations as observed from 16S DNA gene DGGE banding patterns from an aerobic granular sludge pilot plant as well as the from a full-scale activated sludge treatment plant in Epe, the Netherlands. Both plants obtained the same wastewater and had the same relative hydraulic variations and run stable over time. For the total bacterial population, a similarity analysis was conducted showing that the community composition of both sludge types was very dissimilar. Despite this difference, general bacterial population of both systems had on average comparable species richness, entropy, and evenness, suggesting that different bacteria were sharing the same functionality. Moreover, multi-dimensional scaling analysis revealed that the microbial populations of the flocculent sludge system moved closely around the initial population, whereas the bacterial population in the aerobic granular sludge moved away from its initial population representing a permanent change. In addition, the ammonium-oxidizing community of both sludge systems was studied in detail showing more unevenness than the general bacterial community. Nitrosomonas was the dominant AOB in flocculent sludge, whereas in granular sludge, Nitrosomonas and Nitrosospira were present in equal amounts. A correlation analysis of process data and microbial data from DGGE gels showed that the microbial diversity shift in ammonium-oxidizing bacteria clearly correlated with fluctuations in temperature.     

Treatment of H2S using a horizontal biotrickling filter based on biological activated carbon: reactor setup and performance evaluation

Biological treatment is an emerging and prevalent technology for treating off-gases from wastewater treatment plants. The most commonly reported odorous compound in off-gases is hydrogen sulfide (H₂S), which has a very low odor threshold. A self-designed, bench-scale, cross-flow horizontal biotrickling filter (HBF) operated with bacteria immobilized activated carbon (termed biological activated carbon—BAC), was applied for the treatment of H₂S. A mixed culture of sulfide-oxidizing bacteria dominated by Acidithiobacillus thiooxidans acclimated from activated sludge was used as bacterial seed and the biofilm was developed by culturing the bacteria in the presence of carbon pellets in mineral medium. HBF performance was evaluated systematically over 120 days, depending on a series of changing factors including inlet H₂S concentration, gas retention time (GRT), pH of recirculation solution, upset and recovery, sulfate accumulation, pressure drop, gas-liquid ratio, and shock loading. The biotrickling filter system can operate at high efficiency from the first day of operation. At a volumetric loading of 900 m³ m^–3 h^–1 (at 92 ppmv H₂S inlet concentration), the BAC exhibited maximum elimination capacity (113 g H₂S/m^–3 h^–1) and a removal efficiency of 96% was observed. If the inlet concentration was kept at around 20 ppmv, high H₂S removal (over 98%) was achieved at a GRT of 4 s, a value comparable with those currently reported for biotrickling filters. The bacterial population in the acidic biofilter demonstrated capacity for removal of H₂S over a broad pH range (pH 1–7). A preliminary investigation into the different effects of bacterial biodegradation and carbon adsorption on system performance was also conducted. This study shows the HBF to be a feasible and economic alternative to physical and chemical treatments for the removal of H₂S.     

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.     

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO₂ ^? entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO₂ ^? production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct “Nitrosomonas-like” lineage dominated in activated sludge. Prior time series indicated that this “Nitrosomonas-like” lineage was dominant when NO₂ ^? levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO₂ ^? levels were high. This is consistent with the hypothesis that NO₂ ^? production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.     

Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge

In activated sludge, protozoa feed on free-swimming bacteria and suspended particles, inducing flocculation and increasing the turnover rate of nutrients. In this study, the effect of protozoan grazing on nitrification rates under various conditions in municipal activated sludge batch reactors was examined, as was the spatial distribution of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) within the activated sludge. The reactors were monitored for ammonia, nitrite, nitrate, and total nitrogen concentrations, and bacterial numbers in the presence and absence of cycloheximide (a protozoan inhibitor), allylthiourea (an inhibitor of ammonia oxidation), and EDTA (a deflocculating agent). The accumulations of nitrate, nitrite, and ammonia were lower in batches without than with protozoa grazing. Inhibition of ammonia oxidation also decreased the amount of nitrite and nitrate accumulation. Inhibiting protozoan grazing along with ammonia oxidation further decreased the amounts of nitrite and nitrate accumulated. Induction of deflocculation led to high nitrate accumulation, indicating high levels of nitrification; this effect was lessened in the absence of protozoan grazing. Using fluorescent in situ hybridization and confocal laser scanning microscopy, AOB and NOB were found clustered within the floc, and inhibiting the protozoa, inhibiting ammonia oxidation, or inducing flocculation did not appear to lower the number of AOB and NOB present or affect their position within the floc. These results suggest that the AOB and NOB are present but less active in the absence of protozoa.     

Estimation of nitrifier abundances in a partial nitrification reactor treating ammonium-rich saline wastewater using DGGE, T-RFLP and mathematical modeling

The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen (DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated based on the results of batch experiments using Double Monod model, and the results were comparable with those determined according to profiles of DGGE and T-RFLP.     

Impact of operational parameters on bacterial community in a full-scale municipal wastewater treatment plant

A bacterial community in activated sludge from a full-scale municipal wastewater treatment plant was monitored throughout the year with the use of FISH, RISA and DGGE techniques. In the investigated range of temperatures (11.9-21.6 degrees C), a rise in temperature resulted in a lower total bacteria richness, while organic load rate changes from 0.09 to 0.21 g COD x g TSS(-1) x d(-1) were positively correlated with the number of bands in RISA patterns. The most diverse pattern (29 different bands) was characteristic for the activated sludge sample collected at the end of January at wastewater temperature of 11.9 degrees C. The ammonia-oxidising bacteria community did not change during the study, and comprised of 4 different bacterial populations with one dominant species closely related to Nitrosospira sp. REGAU (GenBank accession number AY635572.1). The percentage of ammonia-oxidising bacteria in the activated sludge varied from 6.2 to 19.5% and depended on temperature (R = 0.61, p = 0:02) and organic load rate (R = -0.55, p = 0.04).     

Distribution and Rate of Microbial Processes in an Ammonia-Loaded Air Filter Biofilm

The in situ activity and distribution of heterotrophic and nitrifying bacteria and their potential interactions were investigated in a full-scale, two-section, trickling filter designed for biological degradation of volatile organics and NH₃ in ventilation air from pig farms. The filter biofilm was investigated by microsensor analysis, fluorescence in situ hybridization, quantitative PCR, and batch incubation activity measurements. In situ aerobic activity showed a significant decrease through the filter, while the distribution of ammonia-oxidizing bacteria (AOB) was highly skewed toward the filter outlet. Nitrite oxidation was not detected during most of the experimental period, and the AOB activity therefore resulted in NO₂⁻, accumulation, with concentrations often exceeding 100 mM at the filter inlet. The restriction of AOB to the outlet section of the filter was explained by both competition with heterotrophic bacteria for O₂ and inhibition by the protonated form of NO₂⁻, HNO₂. Product inhibition of AOB growth could explain why this type of filter tends to emit air with a rather constant NH₃ concentration irrespective of variations in inlet concentration and airflow.Emissions of NH₃, odorous organic gasses, and dust from pig facilities cause significant problems for neighbors and the surrounding natural environment. In Denmark, swine production accounts for 34% of the total atmospheric NH₃ emission, of which 50% originates from pig house emissions (19). Furthermore, multiple volatile and very odorous organic compounds are emitted with animal house exhaust air and constitute a severe nuisance in residential areas (16, 31). While biofilters based on wood chips, compost, and peat have proven efficient in removing complex mixtures of volatile organic compounds (VOC) from piggery exhaust air, biotrickling filters have been shown to be efficient in both odor and NH₃ removal (30). In these filters, airborne VOC and NH₃ are taken up by an irrigated biofilm and oxidized by organoheterotrophic and nitrifying bacteria, respectively, resulting in the production of CO₂, NO₂⁻ or NO₃⁻, and microbial biomass (43). The nitrification process, which comprises the two-step oxidation of NH₃ via NO₂⁻ to NO₃⁻, is catalyzed by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea and by nitrite-oxidizing bacteria (NOB), respectively (22, 23). However, in many biotrickling filters, NOB activity seems to be absent, resulting in NO₂⁻ being the end product of nitrification (29).Waste products often accumulate in biotrickling filters, as the water is recycled many times to minimize wastewater discharge. In particular, NO₂⁻ may accumulate to concentrations above 100 mM (29), resulting in high levels of free nitrous acid (FNA, or HNO₂), which is inhibitory to many microorganisms (2, 40, 50). As a result of an overall countercurrent air-water flow, FNA, VOC, and NH₃ concentrations are expected to decrease from the filter air inlet toward the outlet, potentially promoting a gradient of microbial processes and species distribution through the filter.Also, on a smaller scale, physical and chemical heterogeneity results in microgradients of substrates and O₂ within the biofilm that may affect the spatial distribution and rate of microbial processes. While considerable research effort has been given to the physicochemical aspects of filter optimization (11, 44), very little is known about the distribution, activity, and potential interactions of bacterial processes in the biofilm. Problems frequently arise, such as highly variable odor reduction, failed NH₃ removal, and filter clogging due to excess biofilm growth and slime excretion. Improved insight into the dynamics of the microbial activities may help control the biological processes in order to optimize and stabilize filter performance.In this study, the in situ activity and distribution of organoheterotrophic bacteria and AOB and the identity and NH₃ oxidation potential of the AOB were investigated in a two-section, full-scale biological air filter treating waste gas from a pig facility. Biofilms in different filter sections along a gradient of decreasing VOC and NH₃ load and NO₂⁻ accumulation were characterized. The goal was to attain a basic understanding of the microbial processes and thus filter function.     

The Bacterial Communities of Full-Scale Biologically Active,Granular Activated Carbon Filters Are Stable and Diverse and Potentially Contain Novel Ammonia-Oxidizing Microorganisms

The bacterial community composition of the full-scale biologically active, granular activated carbon (BAC) filters operated at the St. Paul Regional Water Services (SPRWS) was investigated using Illumina MiSeq analysis of PCR-amplified 16S rRNA gene fragments. These bacterial communities were consistently diverse (Shannon index, >4.4; richness estimates, >1,500 unique operational taxonomic units [OTUs]) throughout the duration of the 12-month study period. In addition, only modest shifts in the quantities of individual bacterial populations were observed; of the 15 most prominent OTUs, the most highly variable population (a Variovorax sp.) modulated less than 13-fold over time and less than 8-fold from filter to filter. The most prominent population in the profiles was a Nitrospira sp., representing 13 to 21% of the community. Interestingly, very few of the known ammonia-oxidizing bacteria (AOB; <0.07%) and no ammonia-oxidizing Archaea were detected in the profiles. Quantitative PCR of amoA genes, however, suggested that AOB were prominent in the bacterial communities (amoA/16S rRNA gene ratio, 1 to 10%). We conclude, therefore, that the BAC filters at the SPRWS potentially contained significant numbers of unidentified and novel ammonia-oxidizing microorganisms that possess amoA genes similar to those of previously described AOB.     

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_T1038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge.     

Biomass control in waste air biotrickling filters by protozoan predation

Two protozoan species as well as an uncharacterized protozoan consortium were added to a toluene-degrading biotrickling filter to investigate protozoan predation as a means of biomass control. Wet biomass formation in 23.6-L reactors over a 77-day period was reduced from 13.875 kg in a control biotrickling filter to 11.795 kg in a biotrickling filter enriched with protozoa. The average toluene vapor elimination capacity at 1 g/m3 toluene and 64 m3/(m3. h) was 31.1 g/(m3. h) in the control and 32.2 g/(m3. h) in the biotrickling filter enriched with protozoa. At higher toluene inlet concentrations, toluene degradation rates increased and were slightly higher in the biotrickling filter enriched with protozoa. The lower rate of biomass accumulation after the addition of protozoa was due to an increase of carbon mineralization (68% as compared to 61% in the control). Apparent biomass yield coefficients in the control and enriched trickling filter were 0.72 and 0.59 g dry biomass/g toluene, respectively. The results show that protozoan predation may be a useful tool to control biomass in biotrickling filters, however, further stimulation of predation of the biomass immobilized in the reactor is required to ensure long-term stability of biotrickling filters.     

Activated sludge under free ammonia treatment using gel immobilization technology for long-term partial nitrification with different initial biomass

To achieve stable partial nitrification, activated sludge from a wastewater treatment plant using free ammonia (FA) inhibition was immobilized in a polyvinyl alcohol carrier. After FA treatment at 16.44 mg L⁻¹ for 1 day, due to the increased growth rate gap between ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB), AOB enrichment and NOB inhibition were achieved within 12 days, with AOB and NOB accounting for 65.61 and 0.05%, respectively. Subsequently, with dissolved oxygen concentrations of 4−5 mg L⁻¹, pH of 7.6–7.8 and temperature of 25 ± 1 °C, the immobilized carrier made of activated sludge achieved more than 90% and more than 86% of nitrite accumulation rate at the influent ammonia concentration of 90−110 mg L⁻¹ and 35−50 mg L⁻¹, respectively. After 50 days operation, the NOB content was 0.10%, indicating the immobilized carrier provided favorable conditions for maintaining the low NOB content. Furthermore, due to the low NOB content in the inoculum and the oxygen-limited environment formed by the increase in the AOB numbers in the carrier, immobilized carrier with different initial biomass (1, 2.5 and 5%) can achieve stable partial nitrification.     

Analysis of the rate-limiting step of an anaerobic biotrickling filter removing TCE vapors

A detailed analysis of a biotrickling filter treating trichloroethene (TCE) vapors anaerobically is presented and discussed. The biotrickling filter relies on mixed cultures containing bacteria from the genus Dehalococcoides that reductively dechlorinate TCE to ethene. After about 200 days of steady operation, as biomass in the packed bed increased, a partial loss in treatment performance was observed which prompted the present investigations. Analysis of TCE and of its degradation metabolites in the gas phase and in the trickling liquid combined with the calculation of global effectiveness factors revealed that significant mass transfer limitations existed. Depending on the conditions, either the gas film or the liquid film limited the removal of TCE. These findings were confirmed by the determination of gas and liquid films mass transfer coefficients. In all cases, removal of TCE was greater without trickling of liquid. The most plausible reason for the onset of mass transfer limitations was the decrease in the specific interfacial area brought by important biomass growth over time. Overall, this study illustrates how complex kinetic and transport limitations can vary with the operating conditions in biotrickling filters.     

Polyphosphate kinase genes from full-scale activated sludge plants

The performance of enhanced biological phosphorus removal (EBPR) wastewater treatment processes depends on the presence of bacteria that accumulate large quantities of polyphosphate. One such group of bacteria has been identified and named Candidatus Accumulibacter phosphatis. Accumulibacter-like bacteria are abundant in many EBPR plants, but not much is known about their community or population ecology. In this study, we used the polyphosphate kinase gene (ppk1) as a high-resolution genetic marker to study population structure in activated sludge. Ppk1 genes were amplified from samples collected from full-scale wastewater treatment plants of different configurations. Clone libraries were constructed using primers targeting highly conserved regions of ppk1, to retrieve these genes from activated sludge plants that did, and did not, perform EBPR. Comparative sequence analysis revealed that ppk1 fragments were retrieved from organisms affiliated with the Accumulibacter cluster from EBPR plants but not from a plant that did not perform EBPR. A new set of more specific primers was designed and validated to amplify a 1,100 bp ppk1 fragment from Accumulibacter-like bacteria. Our results suggest that the Accumulibacter cluster has finer-scale architecture than previously revealed by 16S ribosomal RNA-based analyses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.