Fundamental Mechanisms of Phosphate Removal by Anaerobic/Aerobic Activated Sludge in Treating Municipal Wastewater

Acetate is thought to be an important substrate for phosphate removal in anaerobic/aerobic activated sludge (AS) processes. The acetate content in municipal wastewater is low, and the main organic compounds in such wastewater are particulate organic matters (POMs) that are converted to endogenous substrates in AS processes when municipal wastewater is introduced into AS reactors. The question which then arises is which substrate, acetate or POM, is important for phosphate removal in full‐scale AS plants. The rates of phosphate release and substrate uptake were determined using AS harvested from a full‐scale anaerobic/aerobic AS plant and also AS acclimated to peptone under alternate anaerobic and aerobic conditions for 26 months. The rate of phosphate release upon POM addition per AS concentration per unit of time was about 0.84 mg PO₄‐P/(g MLSS·h) irrespective of the wastewater quality. This value was about 0.05 in the case of AS acclimated to peptone for 26 months. When the AS concentration is 2.5 g/L and the mixed liquor retention time is 2 h in the anaerobic zone, about 4.2 mg/L PO₄‐P is released upon POM addition. Hence, phosphate can be removed from municipal wastewater using full‐scale AS plants running under these conditions.     

Test of Porous Ceramic Material for the Immobilisation of Predominant Nitrifying Bacteria and for the Improvement of the AAO Process

The efficiency of nitrification in a fixed bed reactor was compared to that found in an activated sludge reactor to determine their sensitivity to changing loads and lower temperatures. Two structurally identical lab‐scale systems, using the anaerobic/anoxic/aerobic (AAO) process to remove nitrogen and phosphorus simultaneously, were operated in parallel with secondary clarifiers and sludge return. The first aerobic system was operated as an activated sludge reactor, the second system as a fixed bed reactor. The aerobic fixed bed reactor was filled with porous ceramic materials for the immobilisation of predominantly nitrifying bacteria. The removal efficiencies of 99 % NH₄⁺‐N, 90 % DOC, and 98 % PO₄^3–‐P for normal loads of 0.11 g/L d DOC, 0.06 g/L d NH₄⁺‐N, and 0.0054 g/L d PO₄^3–‐P were achieved for both systems. However, the system with an aerobic fixed bed reactor was characterised by the following advantages over the system with an activated sludge reactor: a shorter time to reach almost complete nitrification, a higher nitrification rate at higher loads of NH₄⁺‐N and a lower sensitivity of nitrification at lower temperatures down to 12 °C.     

Anionic surfactants in treated sewage and sludges: risk assessment to aquatic and terrestrial environments

Compared to low concentrations of anionic surfactants (AS) in activated sludge process effluents (ASP) (<0.2 mg/L), upflow anaerobic sludge blanket-polishing pond (UASB-PP) effluents were found to contain very high concentrations of AS (>3.5 mg/L). AS (or linear alkylbenzen sulfonate, LAS) removals >99% have been found for ASP while in case of UASB-PP it was found to be < or = 30%. AS concentrations averaged 7347 and 1452 mg/kg dry wt. in wet UASB and dried sludges, respectively. Treated sewage from UASB based sewage treatment plants (STPs) when discharged to aquatic ecosystems are likely to generate substantial risk. Post-treatment using 1-1.6d detention, anaerobic, non-algal polishing ponds was found ineffective. Need of utilizing an aerobic method of post-treatment of UASB effluent in place of an anaerobic one has been emphasized. Natural drying of UASB sludges on sludge drying beds (SDBs) under aerobic conditions results in reduction of adsorbed AS by around 80%. Application of UASB sludges on SDBs was found simple, economical and effective. While disposal of treated UASB effluent may cause risk to aquatic ecosystems, use of dried UASB sludges is not likely to cause risk to terrestrial ecosystems.     

Metabolic transformations and characterisation of the sludge community in an enhanced biological phosphorus removal system

Enhanced biological phosphorus removal was performed in a continuous laboratory-scale two-reactor system with sludge recirculation over a 75-day period. Influent wastewater was a synthetic medium based on acetate, and the sludge age was kept at 12 days. The adapted sludge stored poly-β-hydroxyalkanoic acids (PHA) in the anaerobic reactor with a conversion ratio of 1.45 PHA/acetic acid (based on chemical O₂ demand: COD/COD) and gave ratio of a phosphate-P release to acetic acid uptake of 0.51 P/CH₃COOH (w/w). Fractionation of anaerobic and aerobic sludges showed that the main part of phosphorus taken up, was eluted in the trichloroacetic acid fraction indicating that it was polyphosphate. A total of 60% of the phosphorus in the aerobic sludge was solubilized in the trichloroacetic acid fraction, whereas this fraction accounted for only 32% of the phosphorus in the anaerobic sludge. Only 4% of the total phosphorus in the aerobic sludge and 2% in the anaerobic sludge was found in the EDTA fraction, indicating low amounts of metal-bound phosphates. Isolation on acetate-based agar medium showed that Acinetobacter strains were present in the sludge. However, a more complete analysis of the bacterial community of the sludge was obtained by creating a clone library based on the 16S rRNA gene. A total of 51 partial clone sequences were phylogenetically evaluated. The predominating group was found in the high-(G+C) (mol%) gram-positive bacterial subphylum (31% of the sequenced clones), while the gamma proteobacteria only constituted 9.8% of the clones. Received: 12 June 1997 / Received revision: 26 September 1997 / Accepted: 28 September 1997     

Magnesium requirement for biological removal of phosphate by activated sludge

The effects of magnesium on excess uptake of phosphate in an aerobic-anaerobic activated sludge process were examined by the fill and draw procedure. The alternation of anaerobic and aerobic conditions in one cycle of fill and draw process was varied many ways.The presence of sufficient magnesium was necessary for uptake of excess phosphate. When sludge contained more phosphorus than the upper limit of phosphorus content in the usual aerobic activated sludge, 2.5% by weight, magnesium was also contained in more than an ordinary amount (0.5%). Their contents in the sludge at the end of each cycle of the process were correlated with each other by a linear equation with the correlation coefficient of 0.99. When magnesium concentration was insufficient for the uptake of excess phosphate, its concentration in the treated water was of the order of 0.1 mg/l.In the first anaerobic period both phosphate and magnesium were released, and in a successive aerobic period they were taken up again. The weight ratio of differential amounts of phosphorus and magnesium released or taken up changed with time in one cycle.Dynamic behaviors of phosphate and magnesium removal against the step change of feed magnesium concentration also showed a stoichiometric relationship supporting the correlation equation above mentioned.     

Isolation of a new type of polyphosphate accumulating bacterium and its phosphate removal characteristics

Polyphosphate accumulating bacteria were isolated from activated sludge cultured in a fill and draw system with alternating anaerobic and aerobic conditions. One of the isolates, strain NM-1 tentatively identified with the genus Micrococcus accumulated a large amount of phosphorus and its content reached 166 mg of phosphorus per g of cells. This strain accumulated phosphorus rapidly under aerobic conditions in the absence of any organic substrates, and when glucose was supplied to this strain under anaerobic conditions, it released phosphate and took up glucose in a similar manner to activated sludge that has good phosphorus eliminating capacity. Micro-aerophilic growth conditions were required for the induction of phosphorus accumulating activity.     

Anaerobic granule development for removal of pentachlorophenol in an upflow anaerobic sludge blanket (UASB) reactor

The granulation process using synthetic wastewater containing pentachlorophenol (PCP) in four 1.1 l laboratory scale upflow anaerobic sludge blanket (UASB) reactors was studied, and the anaerobic biotransformation of PCP during the granulation process investigated. After 110 days granular sludge was developed and up to 160 and 180 mg/l of PCP was added into the reactors R1 and R2, respectively, when they were inoculated with acclimated anaerobic sludge from an anaerobic digester of a citric acid plant. The inoculum was predominately composed of bacilli and filamentous bacteria. Granulation did not occur in reactors R3 and R4 which were inoculated with acclimated anaerobic sludge from aerobic sludge of the municipal sewage treatment plant which consisted mainly of cocci. Despite similar bacilli in the granule, the filamentous bacteria from reactor R1 were thicker than those of reactor R2. The granular sludge had a maximum diameter of 2.5 and 2.2 mm, and SMA of 1.44 and 1.32 gCOD/gTVS per day for reactors R1 and R2, respectively. Over 98% chemical oxygen demand (COD) removal rate and 99% of PCP removal rate were achieved when reactors R1 and R2 were operated at PCP and COD loading rates of 150 and 7.5 g/l per day, respectively. H₂-producing acetogens were the dominant anaerobes in the granular sludge.     

Effect of Iron Hydroxide on Phosphate Removal during Anaerobic Digestion of Activated Sludge

The addition of iron (III) hydroxide during methanogenic digestion of activated sludge by anaerobic sludge displaying an iron-reducing activity resulted in a microbial reduction of iron (III) with the formation of iron (II), capable of precipitating phosphates. The feasibility of eliminating 66.6 to 99.6% of the dissolved phosphate at initial concentrations of 1000 to 3500 mg PO^3- ₄/l by adding 6420 mg/l iron (III) hydroxide into a reactor for anaerobic fermentation of activated sludge was analyzed. The optimal ratio of iron (III) added to dissolved phosphate removed (mg) providing a 95% removal amounted to 2 : 1. These results may be used in new technology for anaerobic wastewater treatment with phosphate removal.     

Sulphide effects on the physiology of Candidatus Accumulibacter phosphatis type I

Sulphate-rich wastewaters can be generated due to (i) use of saline water as secondary-quality water for sanitation in urban environments (e.g. toilet flushing), (ii) discharge of industrial effluents, (iii) sea and brackish water infiltration into the sewage and (iv) use of chemicals, which contain sulphate, in drinking water production. In the presence of an electron donor and absence of oxygen or nitrate, sulphate can be reduced to sulphide. Sulphide can inhibit microbial processes in biological wastewater treatment systems. The objective of the present study was to assess the effects of sulphide concentration on the anaerobic and aerobic physiology of polyphosphate-accumulating organisms (PAOs). For this purpose, a PAO culture, dominated by Candidatus Accumulibacter phosphatis clade I (PAO I), was enriched in a sequencing batch reactor (SBR) fed with acetate and propionate. To assess the direct inhibition effects and their reversibility, a series of batch activity tests were conducted during and after the exposure of a PAO I culture to different sulphide concentrations. Sulphide affected each physiological process of PAO I in a different manner. At 189 mg TS-S/L, volatile fatty acid uptake was 55% slower and the phosphate release due to anaerobic maintenance increased from 8 to 18 mg PO₄-P/g VSS/h. Up to 8 mg H₂S-S/L, the decrease in aerobic phosphorus uptake rate was reversible (Ic₆₀). At higher concentrations of sulphide, potassium (>16 mg H₂S-S/L) and phosphate (>36 mg H₂S-S/L) were released under aerobic conditions. Ammonia uptake, an indicator of microbial growth, was not observed at any sulphide concentration. This study provides new insights into the potential failure of enhanced biological phosphorus removal sewage plants receiving sulphate- or sulphide-rich wastewaters when sulphide concentrations exceed 8 mg H₂S-S/L, as PAO I could be potentially inhibited.

     

Phosphorus release by pure cultures of Acinetobacter sp. : effect of the growth stage with cells cultivated on various carbon sources

In order to understand biological phosphorus removal mechanisms, the role of growth stage and volatile fatty acids (VFA) on phosphate release in the anaerobic stage of P removal by three Acinetobacter strains was investigated. The phosphate release in anaerobic conditions was affected by the physiological state of cells and by the carbon source used. When the experiments were made with stationary growth phase cells, the release of phosphate was higher for all three strains cultured on acetic, propionic and butyric acid. Cells showed a limit to the amount of phosphate that could be released from total phosphate accumulated. Only 5–38% of P accumulated by the log cells and 18–58% of total P accumulated by stationary cells could be released. The ratio between the amount of P released and organic substrate removed under anaerobic condition varies depending on VFA types and tested strains.     

Radiolabelled proteomics to determine differential functioning of Accumulibacter during the anaerobic and aerobic phases of a bioreactor operating for enhanced biological phosphorus removal

Proteins synthesized by the mixed microbial community of two sequencing batch reactors run for enhanced biological phosphorus removal (EBPR) during aerobic and anaerobic reactor phases were compared, using mass spectrometry‐based proteomics and radiolabelling. Both sludges were dominated by polyphosphate‐accumulating organisms belonging to Candidatis Accumulibacter and the majority of proteins identified matched closest to these bacteria. Enzymes from the Embden–Meyerhof–Parnas pathway were identified, suggesting this is the major glycolytic pathway for these Accumulibacter populations. Enhanced aerobic synthesis of glyoxylate cycle enzymes suggests this cycle is important during the aerobic phase of EBPR. In one sludge, several TCA cycle enzymes showed enhanced aerobic synthesis, suggesting this cycle is unimportant anaerobically. The second sludge showed enhanced synthesis of TCA cycle enzymes under anaerobic conditions, suggesting full or partial TCA cycle operation anaerobically. A phylogenetic analysis of Accumulibacter polyphosphate kinase genes from each sludge demonstrated different Accumulibacter populations dominated the two sludges. Thus, TCA cycle activity differences may be due to Accumulibacter strain differences. The major fatty acids present in Accumulibacter‐dominated sludge include palmitic, hexadecenoic and cis‐vaccenic acid and fatty acid content increased by approximately 20% during the anaerobic phase. We hypothesize that this is associated with increased anaerobic phospholipid membrane biosynthesis, to accommodate intracellular polyhydroxyalkanoate granules.     

Biological Uptake of Phosphorus by Activated Sludge

The ability of activated sludge to remove phosphates was studied by adding carrier-free (32)P to raw sewage and measuring incorporation of the radioactivity into the cells over a period of time. Radioisotope determinations indicated that 48% of the (32)P radioactivity was removed by 12 hr. However, chemical methods indicated that only 30% of the orthophosphate apparently disappeared from the sewage during this period. Experiments with sludge prelabeled with (32)P indicated that considerable phosphate turnover occurred. The cells released large amounts of radioactivity as they were incorporating fresh phosphates. Starvation in isotonic saline for 18 hr caused the sludge to dump phosphate. When introduced into fresh sewage containing (32)P, the starved sludge removed about 60% of the radioactivity in 6 hr with little phosphate turnover. The ability of sludge to remove (32)P was inhibited approximately 83% by 10(-3)m 2,4-dinitrophenol. This inhibition was at the expense of the cell fraction that contained ribonucleic acid and deoxyribonucleic acid. The sludge cells released orthophosphate when exposed to the chemical agent. Experiments using (45)Ca indicated that calcium phosphate precipitation plays a minor role in phosphate removal under our experimental conditions.     

Phosphorus removal by pure and mixed cultures of microorganisms

The ability to remove inorganic phosphate from synthetic wastewater was tested with about 40 microbial strains, and Pseudomonas aeruginosa IAM 1007 was found to give good performance under aerobic conditions. However, the phosphate removal under batch anaerobic/aerobic (A/O) treatment was not satisfactory in pure cultures of several strains including P. aerginosa, and Aceinetobacter calcoaceticus, but the activated sludge from a plant with an A/O process almost depleted the phosphate. Mixed cultures of P. aeruginosa in the presence of the facultativelu anaerobic strains of A-1 or A-8 isolated from the activated sludge showed enhanced phosphate removal. This suggests a symbiotic effect among microbial species on biological removal of inorganic phosphate in the A/O process.     

Methanotroph and methanogen coupling in granular biofilm under O2-limited conditions

Summary The co-culture between Methylosinus sporium, a strictly aerobic methanotroph, and strictly anaerobic methanogens was studied in 5 L aerobic/anaerobic coupled granular sludge reactors under O₂-limited conditions. The methanogenic bacteria maintained very good metabolic activities and were able to produce sufficient methane which serviced as substrate for methanotrophic growth. Although other strictly aerobic population proliferated by two orders of magnitude after the granular sludge had been operated under O₂-limited conditions for one month, only a limited amount of the added methanotroph remained in the sludge. This result may indicate that M. sporium lacks sufficient O₂ affinity to compete with facultative bacteria for the dissolved O₂ for their growth.     

Removal of phosphate in a sequencing batch reactor by Staphylococcus auricularis

A sequencing batch reactor (SBR) was used to remove phosphate in biological wastewater treatment as an alternative to the activated sludge process, in order to improve the low removal efficiency of phosphate and the operational instability. After a cycle of 2 h anaerobic and 4 h aerobic conditions, phosphate removal was optimized. The removal efficiencies of 5 and 50 mg phosphate l^–1 by Staphylococcus auricularis under repeated anaerobic and aerobic conditions were above 90%. These results showed that a long adaptation time, one of the major problems in biological phosphate removal process, was overcome by SBR.     

The investigation of effect of organic carbon sources addition in anaerobic–aerobic (low dissolved oxygen) sequencing batch reactor for nutrients removal from wastewaters

The effect of addition of organic carbon sources (acetic acid and waste activated sludge alkaline fermentation liquid) on anaerobic–aerobic (low dissolved oxygen, 0.15–0.45 mg/L) biological municipal wastewater treatment was investigated. The results showed that carbon source addition affected not only the transformations of polyhydroxyalkanoates (PHA), glycogen, nitrogen and phosphorus, but the net removal of nitrogen and phosphorus. The removal efficiencies of TN and TP were, respectively, 61% and 61% without organic carbon source addition, 81% and 95% with acetic acid addition, and 83% and 97% with waste activated sludge alkaline fermentation liquid addition. It seems that the alkaline fermentation liquid of waste biosolids generated in biological wastewater treatment plant can be used to replace acetic acid as an additional carbon source to improve the anaerobic–aerobic (low dissolved oxygen) municipal wastewater nutrients removal although its use was observed to cause a slight increase of effluent BOD and COD concentrations.     

Phosphate Removal from Wastewaters: A Novel Approach

The conditions necessary for the establishment and maintenance of Enhanced Biological Phosphate Removal (EBPR) from wastewaters are discussed in the light of our inability to achieve levels of EBPR from artificial sewage in a laboratory‐scale system. Adequate levels of P removal and polyP accumulation by sludge biomass could only be restored by the imposition of stringent anaerobiosis (Eh < –120 mV) and by increasing the short chain fatty acid composition of the influent. Subsequent laboratory‐scale investigations into several possible alternative strategies to achieve enhanced levels of P removal and polyP accumulation from artificial sewage medium indicated that a reduction in the operational pH of the system to approximately 5.5 could achieve comparable levels of P removal under fully‐aerobic conditions. Acid stimulated P uptake and polyP formation might serve as the basis of novel alternative technologies for eutrophication control at wastewater treatment facilities.     

The demonstration of a novel sulfur cycle-based wastewater treatment process: Sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI®) biological nitrogen removal process

Saline water supply has been successfully practiced for toilet flushing in Hong Kong since 1950s, which saves 22% of freshwater in Hong Kong. In order to extend the benefits of saline water supply into saline sewage management, we have recently developed a novel biological organics and nitrogen removal process: the Sulfate reduction, Autotrophic denitrification, and Nitrification Integrated (SANI®) process. The key features of this novel process include elimination of oxygen demand in organic matter removal and production of minimal sludge. Following the success of a 500‐day lab‐scale trial, this study reports a pilot scale evaluation of this novel process treating 10 m³/day of 6‐mm screened saline sewage in Hong Kong. The SANI® pilot plant consisted of a sulfate reduction up‐flow sludge bed (SRUSB) reactor, an anoxic bioreactor for autotrophic denitrification and an aerobic bioreactor for nitrification. The plant was operated at a steady state for 225 days, during which the average removal efficiencies of both chemical oxygen demand (COD) and total suspended solids (TSS) at 87% and no excess sludge was purposefully withdrawn. Furthermore, a tracer test revealed 5% short circuit flow and a 34.6% dead zone in the SRUSB, indicating a good possibility to further optimize the treatment capacity of the process for full‐scale application. Compared with conventional biological nitrogen removal processes, the SANI® process reduces 90% of waste sludge, which saves 35% of the energy and reduces 36% of fossil CO₂ emission. The SANI® process not only eliminates the major odor sources originating from primary treatment and subsequent sludge treatment and disposal during secondary saline sewage treatment, but also promotes saline water supply as an economic and sustainable solution for water scarcity and sewage treatment in water‐scarce coastal areas. Biotechnol. Bioeng. 2012; 109: 2778–2789. © 2012 Wiley Periodicals, Inc.     

Verification of enhanced phosphate removal capability in pure cultures of<Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> under anaerobic/aerobic conditions in an SBR

Laboratory experiments were conducted using pure cultures ofAcinetobacter under anaerobic/aerobic cyclic conditions to explain the release and uptake of soluble phosphate in an activated sludge process showing enhanced biological phosphate removal (EBPR). Under anaerobic/aerobic cyclic conditions in a Sequencing Batch Reactor (SBR), COD uptake concurrent with soluble phosphate release byAcinetobacter was not significant during the anaerobic periods, indicating that EBPR would not be established in pure cultures. However,Acinetobacter cells accumulated higher phosphate content (5.2%) in SBR than that obtained (4.3%) from batch experiments. These results suggest thatAcinetobacter sp. may not follow the proposed pattern of behavior of poly-P bacteria in EBPR activated sludge plants.