The Role of Particulate Organic Matter and Acetic Acid in the Removal of Phosphate in Anaerobic/Aerobic Activated Sludge Processes

Acetic acid is thought to be an important substrate for the removal of phosphate in anaerobic/aerobic activated sludge (AS) processes. However, the acetic acid content in municipal sewage is low, and the main organic compounds in such sewage are particulate organic matters (POM) that are converted to endogenous substrates (E(ntrapped) POM, i.e., EPOM) in AS processes. Thus, the question arises whether it is really acetate or POM, which is important for the removal of phosphate in full‐scale AS plants. AS was harvested from a full‐scale anaerobic/aerobic AS plant. The amount of phosphate released after the addition of acetic acid depends on the AS conditions, particularly the influent sewage quality. However, the amount of phosphate released by EPOM was not affected by the AS conditions, and the amount of phosphate released per AS concentration and per unit of time was calculated to be about 0.86 mg PO₄‐P/g MLSS/hour. When the AS concentration is 2.5 g/L and the mixed‐liquor retention time is 2 hours in the anaerobic zone, about 4 mg/L PO₄‐P is released from EPOM. Under these conditions, phosphate in such sewage is removed by full‐scale AS plants without using acetic acid. In the case of carbon deficiency, the introduction of primary sludge to the anaerobic zone promoted the release of phosphate.     

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. We investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. We observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. From these observations, we postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimisation of EBPR. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 507–515, 1999.     

Phosphate Release and Uptake by Pure Cultures of Acinetobacter sp.: Effect of the Volatile Fatty Acids Concentration

Phosphorus release and uptake by pure cultures of Acinetobacter strains were investigated under anaerobic and aerobic conditions respectively. Tests were performed to study the relationship between phosphorus release-storage reaction and behavior of extracellular organic substrates: acetic, propionic, and butyric acids have been used at four concentrations (50, 100, 500, and 1000 mg · L⁻¹) in the anaerobic step of biological phosphorus removal. The results obtained depend on the strain and the volatile fatty acid (VFA) used. Phosphorus released under anaerobic condition was not always related to the amount of VFA or phosphorus consumed. Phosphorus uptake (P-uptake) in the aerobic step was found to be independent of phosphorus release rates. The best phosphorus uptake rates were obtained by Acinetobacter lwoffi ATCC21130 and Acinetobacter calcoaceticus Genoespecie SUCT-5 with butyric acid as carbon source. Received: 20 May 1996 / Accepted: 8 July 1996     

The effect of an anoxic zone on biological phosphorus removal by a sequential batch reactor

Nitrate can affect phosphate release and lead to reduced efficiency of biological phosphorus removal process. The inhibition effect of remaining nitrate at the anaerobic/anoxic phases was investigated in a lab scale sequencing batch reactor. In this study the influence of denitrification process on reactor performance and phosphorus removal was examined. The experiments were carried out through simultaneous filling and decanting, mixing, mixing-aeration and settling modes. Glucose and acetate were used as carbon sources. The proposed treatment system was capable of removing approximately 80% of the influent PO4-P, 98% NH4-N and 97% COD at a SRT of 25 days. In the fill/decant phase, anoxic and anaerobic conditions prevailed and a large quantity of nitrate was removed in this stage. In the anoxic phase the remaining nitrate concentration was quickly reduced and a considerable amount of phosphate was released. This was attributed to the availability of acetate in this stage. For effective nitrogen and phosphate removal, a short anoxic phase was beneficial before an aerobic phase.     

Treatment of odorous volatile fatty acids using a biotrickling filter

In this study, a novel fibrous bioreactor was developed for treating odorous compounds present in contaminated air. The first stage of this work was a preliminary study which aimed at investigating the feasibility of using the fibrous bioreactor for the removal of malodorous volatile fatty acids (VFA) that is a common odorous contaminant generated from anaerobic degradation of organic compounds. The kinetics of microbial growth and VFA degradation in the selected culture, and the performance of the submerged bioreactor at different VFA mass loadings were studied. Above 95% of VFA removal efficiencies were achieved at mass loadings up to 22.4 g/m(3)/h. In the second stage, the odour treatment process was scaled up with system design and operational considerations. A trickling biofilter with synthetic fibrous packing medium was employed. The effects of inlet VFA concentration and empty bed retention time (EBRT) on the process performance were investigated. The bioreactor was effective in removing VFA at mass loadings up to 32 g/m(3)/h, beyond which VFA started to accumulate in the recirculation liquid, indicating the biofilm was unable to degrade all of the VFA introduced. Although VFA accumulated in the liquid phase, the removal efficiency remained above 99%. This suggested that the biochemical reaction rather than gas-liquid mass transfer was the limiting step of the treatment process. In addition, the biotrickling filter was stable for long-term operation with relatively low and steady pressure drop, no clogging and degeneration of the packing material occurred during the four-month study.     

Divalent cation mobility throughout exponential growth and sporulation of Bacillus megaterium.

Each of the five elements considered was taken up by Bacillus megaterium during exponential growth. Initial Mg and Mn uptake was rapid and ended by mid-log. For Ca, Fe, and Zn, uptake continued throughout exponential growth. Elements were released from the cells immediately following initial uptake. For Mn, egression continued to t2, with release of 36% of total accumulated. Secondary uptake followed immediately and continued through stage V. Magnesium egression continued to t1 with release of 33% accumulated. Secondary uptake began by t5 (stage IV) and continued slowly through sporulation. Calcium egression ceased by t4 with release of 25% total accumulated. Secondary uptake began by t6 (stage V) and continued until depleted. Zinc egression stopped by t5 with release of 34% accumulated with some secondary uptake by stage V. Iron egression terminated at t4 with release of 59% of total accumulated. This was followed by secondary uptake after t12 (stage VI).     

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.     

Influence of temperature, salinity and nutrient limitation on yessotoxin production and release by the dinoflagellate Protoceratium reticulatum in batch-cultures

The toxic dinoflagellate Protoceratium reticulatum (Claparède & Lachmann) Buetschli is recurrently present in the Adriatic sea. It is the producing organism of yessotoxin (YTX) and some of its analogues and thus its presence in seawater often results in shellfish farm closure for long periods. However, molluscs become highly toxic also at the presence of low cell concentrations, due to the high YTX content present in most algal strains. As no data were available on the environmental conditions favouring growth and YTX production by Adriatic P. reticulatum strains, in the present work, we investigated the effect of nutrient limitation, salinity and temperature on growth and YTX content in P. reticulatum cultures. Liquid chromatography–mass spectrometry (LC–MS) analyses were carried out to determine YTX production as well as the difference between the YTX amount retained in cells and that released in growth medium, in order to relate cell content to excretion mechanisms. The toxin content was determined in cells collected at the stationary phase, since both toxin production and release were found to be higher in this growth stage than in the exponential phase. As for nutrient-effect, a severe P-limitation strongly affected cell growth and favoured toxin accumulation, as consequences of both impaired cell division and lower toxin release. N-limited cultures, on the contrary, had a toxin content similar to controls and the highest percentage of release. P. reticulatum was confirmed to be tolerant towards salinity changes as it could grow at salinity values in the range of 22–42. The highest YTX production was observed at intermediate salinity values (32) whereas toxin release, expressed as percentage of the total amount produced, decreased as salinity increased. P. reticulatum growth was impaired in cultures kept at 26 °C in respect to those grown at 16 and 20 °C. YTX release decreased as temperature increased; however, cells kept at 26 °C displayed a very high YTX content. The environmental implications of these physiological behaviours highlight that farmed molluscs can become less toxic in colder waters at lower salinity values.     

Uranyl precipitation by Pseudomonas aeruginosa via controlled polyphosphate metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation conditions.     

Influence of environmental parameters on polyphosphate accumulation inAcinetobacter sp.

The regulation of and the optimum conditions for polyphosphate accumulation inAcinetobacter sp. were determined.Acinetobacter strain 210A accumulated polyphosphate in the presence of an intra- or extracellular energy source. The accumulation of polyphosphate during endogenous respiration was stimulated by streptomycin and inhibited by KCN. The highest amount of polyphosphate was found in cells in which energy supply was not limited, namely at low growth rates under sulphur limitation, and in the stationary phase of growth when either the nitrogen or the sulphur source was depleted. The phosphorus accumulation was not affected by the pH between 6.5 and 9. There was a pronounced effect of the temperature on phosphorus accumulation but is varied from strain to strain.Acinetobacter strain 210A accumulated more phosphate at low temperatures, strain B8 showed an optimum accumulation at 27.5° C, while strain P accumulated phosphorus independently of the temperature. The optimum temperature for growth ofAcinetobacter strains tested ranged from 25 to 33° C, and the optimum pH was between 6 and 9.     

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.     

Efect of the volatile fatty acids on phosphate uptake parameters by pure cultures of Acinetobacter

E.RUSTRIAN, J.P. DELGENES AND R. MOLETTA. 1996. Experiments were performed to examine the effect of volatile fatty acids(VFA) as carbon source, on the phoshate uptake parameters in four Acinetobacter strains. Acetic and butyric acids were equally good carbon sources for phosphate removal, while propionic acid was the least efficient substrate. The best ratios of assimilated phosphate vs VFA consumed were 0-178 wit acetic acid by Ac.calcoaceticus NRRL 4270, 0.21 with propionic acid by Ac.calcoaceticus NRRL 4270 AND 0.187 with butyric acid by Acinetobacter sp.SUCT 5.     

Uranyl Precipitation by Pseudomonas aeruginosa via Controlled Polyphosphate Metabolism

The polyphosphate kinase gene from Pseudomonas aeruginosa was overexpressed in its native host, resulting in the accumulation of 100 times the polyphosphate seen with control strains. Degradation of this polyphosphate was induced by carbon starvation conditions, resulting in phosphate release into the medium. The mechanism of polyphosphate degradation is not clearly understood, but it appears to be associated with glycogen degradation. Upon suspension of the cells in 1 mM uranyl nitrate, nearly all polyphosphate that had accumulated was degraded within 48 h, resulting in the removal of nearly 80% of the uranyl ion and >95% of lesser-concentrated solutions. Electron microscopy, energy-dispersive X-ray spectroscopy, and time-resolved laser-induced fluorescence spectroscopy (TRLFS) suggest that this removal was due to the precipitation of uranyl phosphate at the cell membrane. TRLFS also indicated that uranyl was initially sorbed to the cell as uranyl hydroxide and was then precipitated as uranyl phosphate as phosphate was released from the cell. Lethal doses of radiation did not halt phosphate secretion from polyphosphate-filled cells under carbon starvation 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.     

Inhibition of Anaerobic Phosphate Release by Nitric Oxide in Activated Sludge

Activated sludge not containing significant numbers of denitrifying, polyphosphate [poly(P)]-accumulating bacteria was grown in a fill-and-draw system and exposed to alternating anaerobic and aerobic periods. During the aerobic period, poly(P) accumulated up to 100 mg of P · g of (dry) weight. When portions of the sludge were incubated anaerobically in the presence of acetate, 80 to 90% of the intracellular poly(P) was degraded and released as orthophosphate. Degradation of poly(P) was mainly catalyzed by the concerted action of polyphosphate:AMP phosphotransferase and adenylate kinase, resulting in ATP formation. In the presence of 0.3 mM nitric oxide (NO) in the liquid-phase release of phosphate, uptake of acetate, formation of poly-β-hydroxybutyrate, utilization of glycogen, and formation of ATP were severely inhibited or completely abolished. In cell extracts of the sludge, adenylate kinase activity was completely inhibited by 0.15 mM NO. The nature of this inhibition was probably noncompetitive, similar to that with hog adenylate kinase. Activated sludge polyphosphate glucokinase was also completely inhibited by 0.15 mM NO. It is concluded that the inhibitory effect of NO on acetate-mediated phosphate release by the sludge used in this study is due to the inhibition of adenylate kinase in the phosphate-releasing organisms. The inhibitory effect of nitrate and nitrite on phosphate release is probably due to their conversion to NO. The lack of any inhibitory effect of NO on adenylate kinase of the poly(P)-accumulating Acinetobacter johnsonii 210A suggests that this type of organism is not involved in the enhanced biological phosphate removal by the sludges used.     

Role of nitrate in biological phosphorus removal in a sequencing batch reactor

Summary The effects of nitrate on phosphorus release and uptake in a sequencing batch reactor for biological phosphorus removal was investigated. The addition of nitrate decreased phosphorus release in the anaerobic stage. The synthesis of poly(hydroxyalkanoates) was decreased with the presence of nitrate, possibly due to the competitive utilization of the carbon source by PHA synthesis and denitrification of nitrate. Instead of oxygen, nitrate could be used as an electron acceptor for phosphorus removal. However, the simultaneous addition of nitrate and acetate greatly reduced the phosphorus removal rate. Phosphate and nitrate could be removed simultaneously with nitrate as the electron acceptor, and the continuous and steady feeding of nitrate was beneficial to phosphate removal.     

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.     

Selective enrichment and characterization of a phosphorus-removing bacterial consortium from activated sludge

Under alternating aerobic/anaerobic conditions and without additional carbon sources, a bacterial consortium consisting initially of 18 bacterial strains was obtained in a sequence batch reactor. The phosphorus removal capability could only be maintained using sterile filtrate of activated sludge as medium. The addition of calcium and magnesium salts, as well as vitamins and trace elements, to autoclaved sterile filtrate of activated sludge was not sufficient to achieve stable phosphorus removal. A further enrichment by subcultivation on solid, agar, freezing, and shortening of the aerobic and anaerobic phases led to a defined bacterial consortium consisting of four strains. On the basis of physiological and chemotaxonomic characterization, and partial 16S rRNA sequencing, one of the organisms was identified as Delftia acidovorans. A further isolate belonged to the Bacillus cereus group, and the third isolate was identified as Microbacterium sp.. The remaining strain seems to represent a new genus within the Flavobacteriaceae. Under continuous chemostat conditions, this consortium was able to remove up to 9.6 mg P/l phosphate in the aerobic phase and released up to 8.5 mg/l in the anaerobic phase. Up to 25 mg P-polyphosphate/g dry mass was stored under aerobic conditions.     

Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species

Acinetobacter has been found to be the major species responsible for mediating biological phosphate removal. The growth kinetics and phosphate uptake were investigated for an isolated Acinetobacter strain growing in a defined medium. The phosphate uptake is dependent on growth rate, temperature, and pH. Polyphosphate granules occurred in a balanced growth stage. The maximum phosphorus content in cells was 4.8% at the dilution rate of 12 day(-1). The specific phosphate uptake rate was found to be a quadratic polynomial function of the dilution rate. Increased calcium (up to 36 mg/L) and magnesium (up to 15 mg/L), and the addition of yeast extract (100 mg/L), primary effluent (20%), and fluoride (10 mg/L) did not affect phosphate uptake. Anaerobic conditioning (N(2) stripping), low pH (CO(2) stripping), and addition of sodium acetate under anaerobic conditions failed to stimulate immediate phosphate release. Nevertheless, After 21-24 h, the phosphate release was ca. 3, 5, and 15 mg P/g cell, respectively, for N(2) purging, the addition of acetate, and CO(2) purging. For two-stage completely stirred reactor operation, there was negligible phosphate overplus at the second reactor when phosphate was added, when the first reactor was subjected to phosphate limitation. When both phosphate and carbon limited the growth in the first reactor, there was slight phosphate accumulation under endogenous respiration conditions in the second reactor.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.