Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems

Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle.     

Enrichment of denitrifying glycogen-accumulating organisms in anaerobic/anoxic activated sludge system

Denitrifying glycogen-accumulating organisms (DGAO) were successfully enriched in a lab-scale sequencing batch reactor (SBR) running with anaerobic/anoxic cycles and acetate feeding during the anaerobic period. Acetate was completely taken up anaerobically, which was accompanied by the consumption of glycogen and the production of poly-beta-hydroxy-alkanoates (PHA). In the subsequent anoxic stage, nitrate or nitrite was utilized as electron acceptor for the oxidation of PHA, resulting in glycogen replenishment and cell growth. The above phenotype showed by the enrichment culture demonstrates the existence of DGAO. Further, it was found that the anaerobic behavior of DGAO could be predicted well by the anaerobic GAO model of Filipe et al. (2001) and Zeng et al. (2002a). The final product of denitrification during anoxic stage was mainly nitrous oxide (N(2)O) rather than N(2). The data strongly suggests that N(2)O production may be caused by the inhibition of nitrous oxide reductase by an elevated level of nitrite accumulated during denitrification. The existence of these organisms is a concern in biological nutrient removal systems that typically have an anaerobic/anoxic/aerobic reactor sequence since they are potential competitors to the polyphosphate-accumulating organisms.     

Comparison of laboratory-scale thermophilic biofilm and activated sludge processes integrated with a mesophilic activated sludge process

A combined thermophilic-mesophilic wastewater treatment was studied using a laboratory-scale thermophilic activated sludge process (ASP) followed by mesophilic ASP or a thermophilic suspended carrier biofilm process (SCBP) followed by mesophilic ASP, both systems treating diluted molasses (dilution factor 1:500 corresponding GF/A-filtered COD (COD(filt)) of 1900+/-190 mgl(-1)). With hydraulic retention times (HRTs) of 12-18 h the thermophilic ASP and thermophilic SCBP removed 60+/-13% and 62+/-7% of COD(filt), respectively, with HRT of 8 h the removals were 48+/-1% and 69+/-4%. The sludge volume index (SVI) was notably lower in the thermophilic SCBP (measured from suspended sludge) than in the thermophilic ASP. Under the lowest HRT the mesophilic ASP gave better performance (as SVI, COD(filt), and COD(tot) removals) after the thermophilic SCBP than after the thermophilic ASP. Measured sludge yields were low (less than 0.1 kg suspended solids (SS) kg COD(filt removed)(-1)) in all processes. Both thermophilic treatments removed 80-85% of soluble COD (COD(sol)) whereas suspended COD (COD(susp)) and colloidal COD (COD(col)) were increased. Both mesophilic post-treatments removed all COD(col) and most of the COD(susp) from the thermophilic effluents. In conclusion, combined thermophilic-mesophilic treatment appeared to be easily operable and produced high effluent quality.     

Parameters affecting protein production from brewery waste water in a multi-channel laboratory-scale activated sludge system

Summary With the aim of studying the possible utilization of brewery waste water activated sludge for animal feeding, the influence of the solids retention time (SRT) and nitrogen supplementation were investigated, especially with respect to biomass production and biomass composition. It was found that the SRT strongly influenced both parameters. At an SRT of from 4 to 6 days excellent biomass production was obtained. This biomass had the highest protein content and the daily protein production was four times higher than at a SRT of 20 days. Supplementation with urea doubled the protein production, lowered the carbohydrate and poly--hydroxybutyric acid content, but increased the nucleic acid content. The COD removal was better and phosphorus removal increased. In order to study these variables, a multi-channel laboratory system was designed. Because of its simplicity in operation and its versatility this system is described in detail.     

Sequential anaerobic/aerobic digestion of waste activated sludge: analysis of the process performance and kinetic study

Sequential anaerobic-aerobic digestion was applied to waste activated sludge (WAS) of a full scale wastewater treatment plant. The study was performed with the objective of testing the sequential digestion process on WAS, which is characterized by worse digestibility in comparison with the mixed sludge. Process performance was evaluated in terms of biogas production, volatile solids (VS) and COD reduction, and patterns of biopolymers (proteins and polysaccharides) in the subsequent digestion stages. VS removal efficiency of 40%, in the anaerobic phase, and an additional removal of 26%, in the aerobic one, were observed. For total COD removal efficiencies of 35% and 25% for anaerobic and aerobic stage respectively, were obtained. Kinetics of VS degradation process was analyzed by assuming a first order equation with respect to VS concentration. Evaluated kinetic parameters were 0.44 ± 0.20 d(-1) and 0.25 ± 0.15 d(-1) for the anaerobic stage and aerobic stage, respectively. With regard to biopolymers, in the anaerobic phase the content of proteins and polysaccharides increased to 50% and 69%, respectively, whereas in the subsequent aerobic phase, a decrease of 71% for proteins and 67% for polysaccharides was observed. The average specific biogas production 0.74 m(3)/(kg VS destroyed), was in the range of values reported in the specialized literature for conventional anaerobic mesophilic WAS digestion.     

好氧活性污泥体系中磺胺类抗生素生物降解的研究进展

磺胺类抗生素(Sulfonamides,SAs)是一类被广泛应用于医疗和畜禽养殖的抗菌药物,但其在人和动物肠道中的可降解性较差,会通过人及畜禽粪便和尿液的排放进入污水处理系统。目前,污水处理厂中的好氧活性污泥(Aerobic activated sludge,AAS)被证实能够有效转化SAs。文中基于对国内外文献的调研,总结了好氧活性污泥体系中SAs生物降解的研究进展,对生物降解机理、主要的生物降解途径和影响降解效率的环境因素进行了梳理与回顾,并针对现有研究的不足提出展望,以期为优化污水处理厂中SAs的生物降解提供科学依据。     

Using a carbon-based ASM3 EAWAG Bio-P for modelling the enhanced biological phosphorus removal in anaerobic/aerobic activated sludge systems

Modelling of activated sludge processes is a commonly used technique to design and optimize wastewater treatment processes. Since wastewater and activated sludge is characterized by chemical oxygen demand (COD) measurements, units of state variables describing organic matter are expressed as equivalent amounts of COD. However, current procedures for measuring it have several drawbacks, including the production of hazardous wastes, so the utility of other variables for characterizing the organic load in modelling, such as total organic carbon (TOC), warrant re-evaluation. Other advantages of TOC over COD are that it provides matrix-independent analytical results and it can be readily measured online. Proposals for TOC-based models were made in the 1990s, but they seem to have sunk into obscurity. To re-assess the value of TOC for this purpose, we have recalculated the EAWAG module for Bio-P removal coupled to the Activated Sludge Model No. 3 on a TOC basis, and tested it against data acquired in batch experiments with four single carbon sources (acetate, glucose, citrate and casein). The batch test-based calibrations showed a good match with experimental data, following modifications of the model to account for the anaerobic volumes and retention times applied in the tests.     

Model-based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate-accumulating and glycogen-accumulating organisms

An increasing number of studies shows that the glycogen-accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate-accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model-based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off-gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory-scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model-based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full-scale sludges.     

Understanding aerobic/anaerobic metabolism in Caldibacillus debilis through a comparison with model organisms

Caldibacillus debilis GB1 is a facultative anaerobe isolated from a thermophilic aero-tolerant cellulolytic enrichment culture. There is a lack of representative proteomes of facultative anaerobic thermophilic Bacillaceae, exploring aerobic/anaerobic expression. The C. debilis GB1 genome was sequenced and annotated, and the proteome characterized under aerobic and anaerobic conditions while grown on cellobiose. The draft sequence of C. debilis GB1 contains a 3,340,752 bp chromosome and a 5,386 bp plasmid distributed over 49 contigs. Two-dimensional liquid chromatography mass spectrometry/mass spectrometry was used with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to compare protein expression profiles, focusing on energy production and conversion pathways. Under aerobic conditions, proteins in glycolysis and pyruvate fermentation pathways were down-regulated. Simultaneously, proteins within the tricarboxylic acid cycle, pyruvate dehydrogenase, the electron transport chain, and oxygen scavenging pathways showed increased amounts. Under anaerobic conditions, protein levels in fermentation pathways were consistent with the generated end-products: formate, acetate, ethanol, lactate, and CO₂. Under aerobic conditions CO₂ and acetate production was consistent with incomplete respiration. Through a direct comparison with gene expression profiles from Escherichia coli, we show that global regulation of core metabolism pathways is similar in thermophilic and mesophilic facultative anaerobes of the Phylum Proteobacteria and Firmicutes.     

Development of a test method to assess the sludge reduction potential of aquatic organisms in activated sludge.

This article shows the development of a quantitative sludge reduction test method, which uses the sludge consuming aquatic worm Lumbriculus variegatus (Oligochaeta, Lumbriculidae). Essential for the test are sufficient oxygen supply and the presence of a non-stirred layer of sludge for burrowing of the organisms. The test eliminates the unwanted effects of the organisms' movements, so-called bioturbation, on oxygen transport and (therefore) on sludge reduction. We used fresh untreated activated sludge grown on sewage, in order to stay close to the daily practice of sludge treatment. By separating sludge and worms, sludge reduction and worm growth are quantified independently and accurately. Predation by L. variegatus approximately doubles the decay rate of activated sludge. A minimum ratio of initial worm to sludge biomass (W(0)/S(0)) of about 0.4g worm/g sludge dry mass is required. Under the test conditions 20-40% of the predated sludge is converted into worm biomass. Our test is simple, reproducible and accurate and is done with equipment generally available in any laboratory, yielding results within a few days. The test can also be used to assess the application of mixtures of different aquatic organisms, but does not provide enough information for the design of a sludge treatment reactor.     

FACS enrichment and identification of floc-associated alphaproteobacterial tetrad-forming organisms in an activated sludge community

A precise phylogenetic identity of the Defluviicoccus-related glycogen-accumulating organisms (GAO) observed after FISH probing in a novel activated sludge process removing phosphorus was sought with the aim of exploring the phylogenetic diversity of this important group. These organisms, whose sequences were not revealed in previously generated community wide 16S rRNA gene clone libraries, were identified using flow cytometry cell sorting of FISH-positive cells. Sequencing of a 16S rRNA gene clone library created from this sorted population identified the Defluviicoccus-related GAO as being highly related to previous identified GAO from enhanced biological phosphorus removal systems, despite a marked environmental difference between the two systems.     

Detection of the microbial activity of aerobic heterotrophic, anoxic heterotrophic and aerobic autotrophic activated sludge organisms with an electrochemical sensor

The microbial activity of aerobic heterotrophic, anoxic heterotrophic and aerobic autotrophic microorganisms in biological wastewater treatment was determined by means of an electrochemical bioactivity sensor. The development of the sensor resulted in a system which can determine the microbial activities that are relevant for effective wastewater treatment. The signals of the sensor system are proportional to the substrate degradation and it can show inhibiting effects on the biomass. The most important advantages of the system are: it is independent of O₂ consumption, the three most important types of metabolic activities in wastewater technology can be measured with one sensor, furthermore the measurement is suitable for automation and it is on-line. The result is a potential for the optimization of processes based on microbial activity.     

High diversity and abundance of putative polyphosphate-accumulating Tetrasphaera-related bacteria in activated sludge systems

The diversity of the putative polyphosphate-accumulating genus Tetrasphaera in wastewater treatment systems with enhanced biological phosphorus removal (EBPR) was investigated using the full-cycle rRNA approach combined with microautoradiography and histochemical staining. 16S rRNA actinobacterial gene sequences were retrieved from different full-scale EBPR plants, and the sequences belonging to the genus Tetrasphaera (family Intrasporangiaceae) were found to form three clades. Quantitative FISH analyses of the communities in five full-scale EBPR plants using 10 new oligonucleotide probes were carried out. The results showed that the probe-defined Tetrasphaera displayed different morphologies and constituted up to 30% of the total biomass. It was shown that active uptake of orthophosphate and formation of polyphosphate took place in most of the probe-defined Tetrasphaera populations. However, aerobic uptake of orthophosphate only took place after uptake of certain carbon sources under anaerobic conditions and these were more diverse than hitherto assumed: amino acids, glucose, and for some also acetate. Tetrasphaera seemed to occupy a slightly different ecological niche compared with 'Candidatus Accumulibacter' contributing to a functional redundancy and stability of the EBPR process.     

Bacteriological studies of excess activated sludge subjected to aerobic mineralization

Quantitative and qualitative changes in the composition of bacterial microflora occur in the course of mineralization of excess activated sludge formed during the purification of crude oil refining wastewaters were studied. The excess activated sludge was dominated by Gram-negative bacteria belonging to the genera Pseudomonas III and IV. Mineralization of the excess sludge in both stationary and semicontinuous systems was accompanied by quantitative changes in the composition of the individual groups of Gram-negative bacteria. The frequency of Pseudomonas was reduced in favour of the Enterobacteriaceae, Achromobacter and Alcaligenes.     

The effects of hydraulic retention time and sludge retention time on the fate of di-(2-ethylhexyl) phthalate in a laboratory-scale anaerobic-anoxic-aerobic activated sludge system

A laboratory-scale anaerobic-anoxic-aerobic (AAA) activated sludge wastewater treatment system was employed to investigate the effects of hydraulic retention time (HRT) and sludge retention time (SRT) on the removal and fate of di-(2-ethylhexyl) phthalate (DEHP). In the range from 5 to 14h, HRT had no significant effect on DEHP removal. However, longer HRT increased DEHP accumulation in the system and DEHP retention in the waste sludge. When SRT was increased from 15 to 25d, DEHP removal efficiency stayed above 96%. Compared to the removal of only 88% at SRT of 10d, longer SRT enhanced DEHP degradation efficiency. The optimal HRT and SRT for both nutrients (nitrogen and phosphorus) and DEHP removal were 8h and 15d. At these retention times, about 71% of DEHP was degraded by the activated sludge process, 26% was accumulated in the system, 2% was released in the effluent, and 1% remained in the waste sludge. The anaerobic, anoxic and aerobic reactors were responsible for 15%, 19% and 62% of the overall DEHP removal, respectively.     

The activated sludge ecosystem contains a core community of abundant organisms

Understanding the microbial ecology of a system requires that the observed population dynamics can be linked to their metabolic functions. However, functional characterization is laborious and the choice of organisms should be prioritized to those that are frequently abundant (core) or transiently abundant, which are therefore putatively make the greatest contribution to carbon turnover in the system. We analyzed the microbial communities in 13 Danish wastewater treatment plants with nutrient removal in consecutive years and a single plant periodically over 6 years, using Illumina sequencing of 16S ribosomal RNA amplicons of the V4 region. The plants contained a core community of 63 abundant genus-level operational taxonomic units (OTUs) that made up 68% of the total reads. A core community consisting of abundant OTUs was also observed within the incoming wastewater to three plants. The net growth rate for individual OTUs was quantified using mass balance, and it was found that 10% of the total reads in the activated sludge were from slow or non-growing OTUs, and that their measured abundance was primarily because of immigration with the wastewater. Transiently abundant organisms were also identified. Among them the genus Nitrotoga (class Betaproteobacteria) was the most abundant putative nitrite oxidizer in a number of activated sludge plants, which challenges previous assumptions that Nitrospira (phylum Nitrospirae) are the primary nitrite-oxidizers in activated sludge systems with nutrient removal.     

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.     

Isolation,identification of sludge-lysing strain and its utilization in thermophilic aerobic digestion for waste activated sludge

A strain of sludge-lysing bacteria was isolated from waste activated sludge (WAS) in this study. The result of 16S rRNA gene analysis demonstrated that it was a species of new genus Brevibacillus (named Brevibacillus sp. KH3). The strain could release the protease with molecule weight of about 40 kDa which could enhance the efficiency of sludge thermophilic aerobic digestion. During the sterilized sludge digestion experiment inoculated with Brevibacillus sp. KH3, the maximum protease activity was 0.41 U/ml at pH 8 and 50 °C, and maximum TSS removal ratio achieved 32.8% after 120 h digestion at pH 8 and 50 °C. In the case of un-sterilized sludge digestion inoculated with Brevibacillus sp. KH3, TSS removal ratio in inoculated-group was 54.8%, increasing at 11.86% compared with un-inoculation (46.2%). The result demonstrated that inoculation of Brevibacillus sp. KH3 could help to degrade the EPS and promote the collapse of cells and inhibit the growth of certain kinds of microorganisms. It indicated that Brevibacillus sp. KH3 strain had a high potential to enhance WAS-degradation efficiency in thermophilic aerobic digestion.     

Simulation of an aerobic composting of activated sludge using a statistical procedure

Summary The time courses for the important factors in the composting process for activated sludge, that is the amount of CO₂ generated, the temperature, weight change and water content of the composting materials, were simulated by means of regression analysis. Furthermore, we could accurately estimate the temperature change of the composting materials in the early phase of composting from the heat balance including microbial heat generation estimated by regression analysis. Combining this simulation method with the Simplex method, we determined the optimum aeration regimen that minimized the time required for the temperature to increase to 65°C, at which most pathogens in compost materials are killed, to be 16.7 h.

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Résumé L'évolution chronologique au cours du compostage des facteurs importants (quantité de CO₂ produite, température, poids, et teneur en eau) a été simulée par une analyse de regression linéaire. D'autre part, la variation de température pendant la première phase du compostage a pu être éstimée de façon précise d'après le bilan thermique de la production microbienne de chaleur, déterminé par analyse de regression linéaire. En combinant cette simulation avec la méthode Simplex, on a déterminé un taux optimum d'aération réduisant à 16,7 h le temps nécessaire pour que la température s'élève à 65°C, c'est à dire au niveau où la plupart des bactéries pathogènes du compost sont tuées.

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Nomenclature A Area of heat transfer (m²) - C _pg Specific heat capacity of humid inlet air (kcal/°C kg dry air) - C _ps Specific heat capacity of solid material in the compost (kcal/kg °C) - E Euclid distance - F Aeration rate (kg dry air/h) - H Humidity of inlet air (kg H₂O/kg dry air) - H ^* Humidity at saturation at the inside temperature of the rotating drum (kg H₂O/kg dry air) - CO₂ evolution rate (l/h) - M Total weight of composting material (kg) - M ₀ Initial total weight of composting materials (kg) - P Number of independent variables - Q _W Latent heat of water vaporization (kcal/kg) - q _a Rate of heat loss carried away in the aerating gas (kcal/h) - q _l Rate of heat loss by conduction through the wall of the rotating drum (kcal/h) - q _r Rate of heat generation by microorganisms in the composting process (kcal/h) - q _w Rate of heat loss through evaporation of water (kcal/h) - R Evaporation rate of water (kg/h) - t Composting time (h) - T _c Temperature of composting material (°C) - T _d Temperature in condenser (°C) - t _f Final time of composting (h) - t _g,in Temperature of inlet gas (°C) - t _g,out Temperature of outlet gas (°C) - t _out Temperature outside of the rotating drum (°C) - U Overall heat transfer coefficient for the heat loss through the wall of the rotating drum (kcal/m² °C h) - w _c Water content (%) - W _c Amount of water in the composting materials (kg) - x _i Independent variable - a ₀,a ₁, ...,e ₃,e ₄ Regression coefficient - a ₀,a ₁...,e ₃,e ₄ Standard deviation