Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphate removal

Activated sludge processes with alternating anaerobic and aerobic conditions (the anaerobic-aerobic process) have been successfully used for enhanced biological phosphate removal (EBPR) from wastewater. It is known that polyphosphate-accumulating bacteria (PAB) play an essential role for EBPR in the anaerobic-aerobic process. The present paper reviews limited information available on the metabolism and the microbial community structure of EBPR, highlighting the microbial ecological selection of PAB in EBPR processes. Exposure of microorganisms to alternate carbon-rich anaerobic environments and carbon-poor aerobic environments in the anaerobic-aerobic process induces the key metabolic characteristics of PAB, which include organic substrate uptake followed by its conversion to stored polyhydroxyalkanoate (PHA) and hydrolysis of intracellular polyphosphate accompanied by subsequent Pi release under anaerobic conditions. Intracellular glycogen is assumed to function as a regulator of the redox balance in the cell. Storage of glycogen is a key strategy for PAB to maintain the redox balance in the anaerobic uptake of various organic substrates, and hence to win in the microbial selection. Acinetobacter spp., Microlunatus phosphovorus, Lampropedia spp., and the Rhodocyclus group have been reported as candidates of PAB. PAB may not be composed of a few limited genospecies, but involve phylogenetically and taxonomically diverse groups of bacteria. To define microbial community structure of EBPR processes, it is needed to look more closely into the occurrence and behavior of each species of PAB in various EBPR processes mainly by molecular methods because many of PAB seem to be impossible to culture.     

High diversity of potential nitrate-reducing Fe(II)-oxidizing bacteria enriched from activated sludge

Applied Microbiology and Biotechnology - Nitrate-dependent Fe(II) oxidation (NDFO) has been discovered in various environments including activated sludge and can potentially be used to remove...     

Molecular detection and diversity of medium-chain-length polyhydroxyalkanoates-producing bacteria enriched from activated sludge

AIMS: Knowledge of the species composition of complex bacterial communities is still very limited. The main objectives of this study were to identify medium-chain-length polyhydroxyalkanoates (mcl-PHAs)-producing bacteria from activated sludge fed with methanol as well as to characterize their PHA operon. METHODS AND RESULTS: The identification was based on PCR amplification of mcl-PHA synthase gene fragments. In the analysed sample, four isolates possessing mcl-PHA synthesis systems were distinguished. The results of a 16S rDNA sequence analysis revealed that three strains belonged to Pseudomonas species and the fourth one was characterized as Comamonas testosteroni. CONCLUSIONS: The results of this study indicate that the PCR-RFLP approach is an excellent way to identify mcl-PHA-synthesizing micro-organisms. The discovery of 4 genetic variants, among the 20 analysed, demonstrates that microbial diversity of activated sludge is high and thus offers a great opportunity for the discovery of novel gene forms. SIGNIFICANCE AND IMPACT OF THE STUDY: An important discovery of this study is that C. testosteroni could harbour mcl-PHA operon. Moreover, the results obtained indicate that PHAs synthesis ability can be spread by horizontal gene transfer. The results of a comparative phylogenetic analysis revealed that mcl-PHA-synthesizing bacteria can be divided into Pseudomonas fluorescens and Pseudomonas aeruginosa groups.     

Stalked bacteria in activated sludge

Summary A laboratory-scale activated sludge unit was fed continuously with a simulated industrial wastewater, consisting of a dilute solution of inorganic salts, at a rate giving a mean retention time of about 3 days. The system produced a well-settling sludge which on examination by electron microscopy was found to contain considerable numbers of stalked bacteria. These were identified as Caulobacter, which have the ability to attach to surfaces and other organisms by means of a prosthecal holdfast and to flourish in waters with a low content of organic nutrients, and whose occurrence in activated sludge has not apparently been previously recorded. Conditions advantageous to Caulobacter generally prevail in activated sludge systems when these operate in growth phases tending to produce well-settling sludge. Since their holdfast gives Caulobacter the ability to initiate and enlarge microbial clusters by attachment, it is suggested that Caulobacter contribute to microbial floc-formation in activated sludge.     

ERIC-PCR-based strain-specific detection of phenol-degrading bacteria in activated sludge of wastewater treatment systems

Aims:  To evaluate the use of Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)-derived probes and primers to specifically detect bacterial strains in an activated sludge microbial community.
Methods and Results:  ERIC-PCR was performed on two phenol-degrading bacterial strains, Arthrobacter nicotianae P1-7 and Klebsiella sp. P8-14. Their amplicons were DIG labelled for use as probes and then hybridized with ERIC-PCR fingerprints. The results showed the distinct band patterns for both bacterial strains. Strain-specific PCR primers were designed based on the sequences of ERIC-PCR bands. The DNA of each of these strains was successfully detected from its mixture with activated sludge DNA, either by using their respective ERIC-PCR-based probes for hybridization or by using species-specific primers for amplification, with higher sensitivity by latter method.
Conclusions:  Two phenol-degrading bacterial strains were identified from a mixture of activated sludge by using ERIC-PCR-based methods.
Significance and Impact of the Study:  The study demonstrated that the bacteria, which have important functions in complex wastewater treatment microbial communities, could be specifically detected by using ERIC-PCR fingerprint-based hybridization or amplification.     

Upgrading activated sludge systems and reduction in excess sludge

Most of 200 Activated Sludge Plant in Iran are overloaded and as a result, their efficiency is low. In this work, a pilot plant is manufactured and put into operation in one of the wastewater treatment plants in the west of Tehran. Instead of conventional activated sludge, a membrane bioreactor and an upflow anaerobic sludge blanket reactor used as a pretreatment unit in this pilot. For the sake of data accuracy and precision, an enriched municipal wastewater was opted as an influent to the pilot. Based on the attained result, the optimum retention time in this system was 4h, and the overall COD removal efficiency was 98%. As a whole, the application of this retrofit would increase the plant's capacity by a factor of 5 and reducing the excess sludge by a factor of 10. The sludge volume index in the anaerobic reactor was about 12 after granulation occurred.     

Purple nonsulfur bacteria diversity in activated sludge and its potential phosphorus-accumulating ability under different cultivation conditions

This study investigates the diversity and the potential phosphorus-accumulating ability among the purple nonsulfur (PNS) bacteria. Traditional methods and molecular biotechniques were applied. Microscopic visualization using 4′,6-diamidino-2-phenylindole staining as well as chemical analysis demonstrated that most of the isolated PNS bacteria presented different levels of phosphorus accumulation. Four of the pure cultures, denoted as Rhodopseudomonas palustris CC1, CC7, G11, and GE1, based on their differences in the PNS’s pufM gene, exhibited higher internal phosphorus content compared to other isolated strains in this study. In addition, substantial polyphosphate accumulation was observed after the bacteria entered their stationary growth phase. Among them, the isolated R. palustris G11 could accumulate internal phosphorus up to 13%–15% of its cell dry weight under anaerobic illuminated incubation conditions. When the incubation status was switched from anaerobic to aerobic, the bacterial phosphorus content had a tendency to decrease slightly or remain about the same throughout the whole aerobic stage. The growth rate and biomass were higher when the PNS bacteria grew under photoheterotrophic conditions rather than the chemoheterotrophic ones. Furthermore, the environmental pH value could affect the contents of internal bacterial phosphate. Results of this study demonstrated that PNS bacteria are a group of the polyphosphate-accumulating organisms, of which this ability had never been properly studied. The conditions that PNS bacteria accumulating polyphosphate presented from this study were unique and showed characteristics that were different from the well-known enhanced biological phosphorus removal model.     

Identity and ecophysiology of filamentous bacteria in activated sludge

Excessive growth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) can cause serious operational problems. With some filaments there may be the problem of bulking, where inadequate flocculation and settling of the biomass in the secondary clarifier results in a carryover of solids with the final treated liquid effluent. Their proliferation often encourages the development of stable foams on the surface of the reactors, and these foams may impact negatively on plant performance and operation. The availability of culture-independent molecular methods now allows us to identify many of the more common filamentous organisms encountered in WWTPs, which are phylogenetically diverse, affiliating to seven separate bacterial phyla. Furthermore, the extensive data published in the past decade on their in situ behaviour from the application of these culture-independent methods have not been summarized or reviewed critically. Hence, here, we attempt to discuss what we now know about their identity, ecophysiology and ecological niches and its practical value in better managing activated sludge processes. Some of this knowledge is already being applied to control and manage full-scale WWTPs better, and the hope is that this review will contribute towards further developments in this field of environmental microbiology.     

Competitive ability of amylolytic bacteria in activated sludge

Shifts were induced into the microbial community of activated sludge by the pulse addition of soluble starch. The subsequent changes of amylolytic and proteolytic microbial populations were recorded. Four amylolytic strains were isolated and characterized with regard to carrying capacity, specific surface and growth kinetics. The competitive ability of these strains was studied by means of two-member competition experiments. These experiments were analysed according to the Lotka-Volterra model and the de Wit method. The different results obtained suggest that the dominance of the amylolytic Pseudomonas sp. (code 01) is based on a combined occurrence of high amylolytic activity, large relative cell surface, high maximum specific growth rate and reduced sensitivity towards associated proteolytic populations.     

Ecophysiology of abundant denitrifying bacteria in activated sludge

The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20-49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.     

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.     

Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.     

Polyphosphate accumulation among denitrifying bacteria in activated sludge

Bacterial polyphosphate accumulation and denitrification are important processes in biological removal of nutrients from wastewater. It has been suggested that phosphorus accumulators are able to denitrify. However, the bacteria known as the most important phosphorus accumulators, belonging to the genus Acinetobacter are generally not known to denitrify. To clarify how commonly both physiological traits are present in the same organism, we screened 165 isolates from activated sludge and wastewater for their ability to denitrify, and the ability of the denitrifying isolates to accumulate polyphosphate. Of the 165 isolates, 149 were from acetate mineral medium (87 of these identified as Acinetobacter by the API 20 NE identification system) and 16 were from nutrient broth and nitrate medium. Only 15 of 165 isolates tested showed true respiratory denitrification activity. In the presence of acetylene they converted more than 80% of 5mM NO3- to N2O in 6 days. None of the Acinetobacter isolates were among the 15 respiratory denitrifiers. The denitrifying isolates were identified as species of Pseudomonas, Agrobacterium, Pasteurella, Sphingomonas or could not be identified by the API 20 NE identification system. According to the BIOLOG identification system the denitrifiers were species of Pseudomonas, Hydrogenophaga, Citrobacter, Xanthomonas or they could not be identified. The ability of confirmed denitrifiers to accumulate phosphate was measured in experiments where cells pregrown under phosphorus limitation were exposed to phosphate (8 mg P/L) under aerobic conditions. The rates of excess phosphate uptake varied from 0.3 to more than 23 mg P/g dry matter/h. Rates for four isolates were higher than those reported for Acinetobacter strains. These results show that polyphosphate accumulation and denitrification in activated sludge can be carried out by the same organisms.     

Cost optimization of activated sludge systems

Activated sludge is a widely used aerobic biological waste-water treatment process. A rational approach to least cost design of an integrated system is described which includes the following processes: activated sludge reactor, final settling tanks, gravity thickening, and aerobic sludge digestion. Both capital and operation and maintenance costs are considered. Biological reactor design is based on microbial kinetic concepts and continuous culture of microorganisms theory. Biological solids retention time (θ_c) is utilized as the primary independent design variable to which system performance is related, e.g., effluent quality, ammonia oxidation, and excess sludge production. Liquid-biomass separation is based on the batch flux technique, a rational approach to design of gravity separators (final settling tanks). Trade-offs among reactor volume, clarifier size, recycle pumping capacity, thickener capacity, digester volume, air requirements, and sludge production are discussed. The optimum design is taken as the combination of these parameters within the acceptable design domain, determined by effluent quality criteria, that results in minimum cost. While the method described is general, design of a given treatment system depends on availability, from lab or pilot studies, of system specific numerical values for biological growth coefficients and biomass setting characteristics. A design example illustrates the approach.     

Competitive ability of amylolytic bacteria in activated sludge.

Shifts were induced into the microbial community of activated sludge by the pulse addition of soluble starch. The subsequent changes of amylolytic and proteolytic microbial populations were recorded. Four amylolytic strains were isolated and characterized with regard to carrying capacity, specific surface and growth kinetics. The competitive ability of these strains was studied by means of two-member competition experiments. These experiments were analysed according to the Lotka-Volterra model and the de Wit method. The different results obtained suggest that the dominance of the amylolytic Pseudomonas sp. (code 01) is based on a combined occurrence of high amylolytic activity, large relative cell surface, high maximum specific growth rate and reduced sensitivity towards associated proteolytic populations.     

Extraction of activated sludge bacteria exopolymers by ultrasonication

Ultrasonication for the extraction of activated sludge exopolymers was evaluated by total cell count, exopolymer extraction and transmission electron microscopy (TEM). A high deflocculation was achieved after 30 s of sonication in PBS (phosphate-buffered saline). TEM showed that cell lysis was minimal only when sludges were sonicated for 30 s. For sludges sonicated for 30, 90 and 420 s and stained with Ruthenium Red, exopolymers were not extracted on a large scale without considerable cell lysis. Sludges sonicated for 30 s in EDTA gave a larger fraction of damaged cells and also showed copious amounts of attached exopolymers.     

Ecology of bacteriophages infecting activated sludge bacteria.

Little is known about the endemic bacteriophages of activated sludge. In this investigation 49 virus-host systems were studied by isolating co-occurring bacteria and bacteriophages from the aeration basin of a sewage treatment plant during 5 successive weeks. The phage titers were high and fluctuated during the time period. The occurrence of phage-sensitive and -resistant hosts did not depend on the presence or absence of phages. Several phage-host systems expressed variable plating efficiencies. In addition, phages with broad host ranges were observed. These results show that phages are an active part of this ecosystem and that they may exert selection pressure for phage resistance on their bacterial host populations.