Biological phosphorus removal from abattoir wastewater at very short sludge ages mediated by novel PAO clade Comamonadaceae.

Recent increases in global phosphorus costs, together with the need to remove phosphorus from wastewater to comply with water discharge regulations, make phosphorus recovery from wastewater economically and environmentally attractive. Biological phosphorus (Bio-P) removal process can effectively capture the phosphorus from wastewater and concentrate it in a form that is easily amendable for recovery in contrast to traditional (chemical) phosphorus removal processes. However, Bio-P removal processes have historically been operated at medium to long solids retention times (SRTs, 10-20 days typically), which inherently increases the energy consumption while reducing the recoverable carbon fraction and hence makes it incompatible with the drive towards energy self-sufficient wastewater treatment plants. In this study, a novel high-rate Bio-P removal process has been developed as an energy efficient alternative for phosphorus removal from wastewater through operation at an SRT of less than 4 days. The process was most effective at an SRT of 2-2.5 days, achieving >90% phosphate removal. Further reducing the SRT to 1.7 days resulted in a loss of Bio-P activity. 16S pyrotag sequencing showed the community changed considerably with changes in the SRT, but that Comamonadaceae was consistently abundant when the Bio-P activity was evident. FISH analysis combined with DAPI staining confirmed that bacterial cells of Comamonadaceae arranged in tetrads contained polyphosphate, identifying them as the key polyphosphate accumulating organisms at these low SRT conditions. Overall, this paper demonstrates a novel, high-rate phosphorus removal process that can be effectively integrated with short SRT, energy-efficient carbon removal and recovery processes.

The marine sponge-derived inorganic polymers, biosilica and polyphosphate, as morphogenetically active matrices/scaffolds for the differentiation of human multipotent stromal cells: potential application in 3D printing and distraction osteogenesis.

The two marine inorganic polymers, biosilica (BS), enzymatically synthesized from ortho-silicate, and polyphosphate (polyP), a likewise enzymatically synthesized polymer consisting of 10 to >100 phosphate residues linked by high-energy phosphoanhydride bonds, have previously been shown to display a morphogenetic effect on osteoblasts. In the present study, the effect of these polymers on the differential differentiation of human multipotent stromal cells (hMSC), mesenchymal stem cells, that had been encapsulated into beads of the biocompatible plant polymer alginate, was studied. The differentiation of the hMSCs in the alginate beads was directed either to the osteogenic cell lineage by exposure to an osteogenic medium (mineralization activation cocktail; differentiation into osteoblasts) or to the chondrogenic cell lineage by incubating in chondrocyte differentiation medium (triggering chondrocyte maturation). Both biosilica and polyP, applied as Ca²âº salts, were found to induce an increased mineralization in osteogenic cells; these inorganic polymers display also morphogenetic potential. The effects were substantiated by gene expression studies, which revealed that biosilica and polyP strongly and significantly increase the expression of bone morphogenetic protein 2 (BMP-2) and alkaline phosphatase (ALP) in osteogenic cells, which was significantly more pronounced in osteogenic versus chondrogenic cells. A differential effect of the two polymers was seen on the expression of the two collagen types, I and II. While collagen Type I is highly expressed in osteogenic cells, but not in chondrogenic cells after exposure to biosilica or polyP, the upregulation of the steady-state level of collagen Type II transcripts in chondrogenic cells is comparably stronger than in osteogenic cells. It is concluded that the two polymers, biosilica and polyP, are morphogenetically active additives for the otherwise biologically inert alginate polymer. It is proposed that alginate, supplemented with polyP and/or biosilica, is a suitable biomaterial that promotes the growth and differentiation of hMSCs and might be beneficial for application in 3D tissue printing of hMSCs and for the delivery of hMSCs in fractures, surgically created during distraction osteogenesis.

Phosphate recovery as concentrated solution from treated wastewater by a PAO-enriched biofilm reactor.

Phosphorus recovery from wastewaters and its recycling are of importance for sustaining agricultural production. During the conventional enhanced biological phosphorus removal process, phosphorus is removed by withdrawing excess sludge from wastewater. However, excess sludge disposal is costly and energy intensive. A proposed novel process for phosphorus recovery from sewage treatment will result in no excess sludge if a polyphosphate accumulating organisms (PAOs) enrichment biofilm can be applied to effluents containing phosphate. This process allows the recovery of phosphate as phosphate-concentrated solutions by controlling PAOs to absorb and release phosphate. A reactor consisting of a modified trickling filter with a synthetic substrate (5 mg P L⁻¹) was operated to form a PAO-enriched biofilm. As a result of the enrichment, the concentration of phosphate of >100 mg P L⁻¹ was successfully achieved. During this experiment, no sludge withdrawal was carried out over the duration of the operation of 255 days. To highlight the new process, the principle of enriching PAOs on biofilm and concentrating phosphate from treated sewage is explained, and a discussion on phosphate recovery performance is given.

Phosphorus release and uptake during start-up of a covered and non-aerated sequencing batch reactor with separate feeding of VFA and sulfate.

This study explored a sulfur cycle-associated biological phosphorus (P) removal process in a covered and non-aerated sequencing batch reactor (SBR) fed with volatile fatty acid (VFA) and sulfate separately. During the 60-day start-up, both phosphate release and uptake rates increased, while poly-phosphate cyclically increased and decreased accordingly. The P-release and P-uptake rates were associated with VFA uptake and sulfate reduction. The average ratio of potassium to phosphate during the P-uptake and P-release was also determined to be 0.29-0.31 mol K/mol P, which is close to a reported value (0.33) for biological phosphorus removal. All this evidence confirmed there was biological P removal in this reactor, in which metabolism could be different from conventional biological P removal.

[Biological characteristics of denitrifying polyphosphate-accumulating organisms].

A denitrifying phosphate-accumulating organisms (DNPAOs), which was called Q-hrb05, was isolated in the special medium from the anaerobic/aerobic/anoxic SBR reactor. Strain Q-hrb05 was identified by 16SrDNA gene analysis, and the accession number of 16SrDNA gene sequence of strain Q-hrb05 in GenBank was GU214826. Effects of the different pH values, temperature, carbon source of medium on nitrogen and phosphorus removal of strain Q-hrb05 were investigated. The result showed that strain Q-hrb05 belonged to Bacillus sp.. Meanwhile, extracellular exopolymers of strain Q-hrb05 was based on protein, about 120.6 mg x mL(-1), and it had 23.05 microg x mL(-1) nucleic acid, but little polysaccharide. There was no significant adsorption of phosphate. So phosphorus removal was mainly due to intracellular uptake. And when pH value was kept as 7, temperature was kept as 30 degrees C, and carbon source was kept sodium acetate, the highest nitrogen and phosphorus removal efficiency was achieved. Phosphorus uptake rate was averaged at 88%, and the denitrification rate reached 81%.

Direct quantification of inorganic polyphosphate in microbial cells using 4'-6-diamidino-2-phenylindole (DAPI).

Inorganic polyphosphate (polyP) is increasingly being recognized as an important phosphorus sink within the environment, playing a central role in phosphorus exchange and phosphogenesis. Yet despite the significant advances made in polyP research there is a lack of rapid and efficient analytical approaches for the quantification of polyP accumulation in microbial cultures and environmental samples. A major drawback is the need to extract polyP from cells prior to analysis. Due to extraction inefficiencies this can lead to an underestimation of both intracellular polyP levels and its environmental pool size: we observed 23-58% loss of polyP using standard solutions and current protocols. Here we report a direct fluorescence based DAPI assay system which removes the requirement for prior polyP extraction before quantification. This increased the efficiency of polyP detection by 28-55% in microbial cultures suggesting quantitative measurement of the intracellular polyP pool. It provides a direct polyP assay which combines quantification capability with technical simplicity. This is an important step forward in our ability to explore the role of polyP in cellular biology and biogeochemical nutrient cycling.

Biological phosphorus removal in sequencing batch reactor with single-stage oxic process.

The performance of biological phosphorus removal (BPR) in a sequencing batch reactor (SBR) with single-stage oxic process was investigated using simulated municipal wastewater. The experimental results showed that BPR could be achieved in a SBR without anaerobic phase, which was conventionally considered as a key phase for BPR. Phosphorus (P) concentration 0.22-1.79 mg L(-1) in effluent can be obtained after 4h aeration when P concentration in influent was about 15-20 mg L(-1), the dissolved oxygen (DO) was controlled at 3+/-0.2 mg L(-1) during aerobic phase and pH was maintained 7+/-0.1, which indicated the efficiencies of P removal were achieved 90% above. Experimental results also showed that P was mainly stored in the form of intracellular storage of polyphosphate (poly-P), and about 207.235 mg phosphates have been removed by the discharge of rich-phosphorus sludge for each SBR cycle. However, the energy storage poly-beta-hydroxyalkanoates (PHA) was almost kept constant at a low level (5-6 mg L(-1)) during the process. Those results showed that phosphate could be transformed to poly-P with single-stage oxic process without PHA accumulation, and BPR could be realized in net phosphate removal.

Microbial distribution of Accumulibacter spp. and Competibacter spp. in aerobic granules from a lab-scale biological nutrient removal system.

Granular sludge for simultaneous nitrification, denitrification and phosphorus removal (SNDPR) was generated and studied in a lab-scale sequencing batch reactor (SBR). The SBR was monitored for 450 days during which the biomass was transformed from flocs to granules, which persisted for the last 130 days of operation. Short sludge settling time was employed to successfully generate the granules, with the 10th and 90th percentiles of diameter being 0.7 and 1.6 mm respectively. Good phosphorus removal and nitrification occurred throughout the SBR operation but only when granules were generated were denitrification and full nutrient removal complete. Fluorescence in situ hybridization and oxygen microsensors were used to study the granules at a microscale. Accumulibacter spp. (a polyphosphate-accumulating organism, PAO) and Competibacter spp. (a glycogen non-polyphosphate-accumulating organism, GAO) were the most abundant microbial community members (together 74% of all Bacteria) and both are capable of denitrification. In the aerobic period of the SBR operation, the oxygen penetrated 250 microm into the granules leaving large anoxic zones in the centre part where denitrification can occur. In granules > 500 microm in diameter, Accumulibacter spp. was dominant in the outermost 200 microm region of the granule while Competibacter spp. dominated in the granule central zone. The stratification of these two populations between the outer aerobic and inner anoxic part of the granule was highly significant (P < 0.003). We concluded that the GAO Competibacter spp., and not the PAO Accumulibacter spp., was responsible for denitrification in this SBR. This is undesirable for SNDPR as savings in carbon demand cannot be fulfilled with phosphorus removal and denitrification being achieved by different groups of bacteria.

Polyphosphate dynamics in mycorrhizal roots during colonization of an arbuscular mycorrhizal fungus.

Inorganic polyphosphate (poly P) has been considered to be a translocatable form of phosphate (Pi) in arbuscular mycorrhizal fungi (AMF). Here we examined time-course changes in poly P content during the AMF colonization process. Onion (Allium cepa) plants were cultured with or without inoculation with Gigaspora margarita for 2-8 wk with periodic sampling. Poly P in the extracts, purified through gel filtration, was quantified by the reverse reaction of polyphosphate kinase. The length of poly P in mycorrhizal roots appeared to be shorter than in extraradical hyphae or in spores of the AMF, indicating that AMF depolymerize poly P before providing Pi to the host. The poly P content increased as colonization proceeded, and was highly correlated with the weight of the colonized roots. These results support the model that AMF supply Pi to the host through the poly P pool, and that the poly P content of a mycorrhizal root can be a good indicator of the Pi-supplying activity of AMF.

Stability of enhanced biological phosphorus removal and composition of polyphosphate granules.

The influence of varying Ca- and Mg-concentration of the influent wastewater on the enhanced biological phosphorus removal was investigated in an anaerobic-aerobic bench-scale plant. The artificial enhancement of the Mg-concentration in the influent from 15 to 24 mg l(-1) and 31 mg l(-1), respectively, caused a raise of the mean P-removal efficiency from 85 to 97%. The P-elimination was very stable in time. A chemical precipitation of magnesium ammonium phosphate could be excluded. The elemental composition of polyphosphate granules was investigated by electron microscopy and energy dispersive X-ray spectroscopy. The elements Ca, Mg and K were the principal metal components of polyphosphate granules. Concerning the metal composition, different types of granules could be distinguished. The quantitative ratios of Ca, Mg and K varied in dependence on the influent concentration of these metals. A relation between the Mg/Ca-ratio of the granules and the efficiency of enhanced biological phosphorus removal can be supposed.

Calibration and validation of an ASM3-based steady-state model for activated sludge systems--part II: Prediction of phosphorus removal.

An ASM3-based steady-state model which can be used for estimating the average nitrogen-removal, sludge-production and phosphorus-removal rates of different biological phosphorus-removing systems (AAO, UCT, intermittent processes) is developed. It considers the wastewater composition, the oxygen and nitrate input in the anaerobic compartment and the interaction between biological phosphorus removal and denitrification for different operating conditions. The model is calibrated and validated with data from a number of long-term pilot and full-scale experiments for Swiss municipal wastewater. The steady-state model is adequate for a comparison of different BPR process configurations or for a first estimation of the nutrient-removal efficiency. It allows the plant performance and key parameters to be determined very quickly. Excel spreadsheets of the model for different flow schemes are available from the corresponding author.

Polyphosphates in intraradical and extraradical hyphae of an arbuscular mycorrhizal fungus, Gigaspora margarita.

The amount of polyphosphate in the intraradical and extraradical hyphae of Gigaspora margarita was estimated from successive extractions with trichloroacetic acid (TCA), EDTA, and phenol-chloroform (PC). In the intraradical hyphae, most of the polyphosphate was present in TCA- and EDTA-soluble (short-chain and long-chain) fractions, whereas most of the polyphosphate in the extraradical hyphae was present in EDTA- and PC-soluble (long-chain and granular) fractions.

Polyphosphate-hydrolysis--a protective mechanism against alkaline stress?

Different microorganisms, including yeast and algae, accumulate large amounts of polyphosphates. However, the physiological role of polyphosphates is largely unknown. In vivo 31P NMR studies, carried out in the unicellular alga, Dunaliella salina, demonstrate the cytoplasmic alkalization induces massive hydrolysis of polyphosphates, which is correlated kinetically with the recovery of cytoplasmic pH. Analysis of acid extracts of the cells indicates that long-chain polyphosphates are hydrolysed mainly to tripolyphosphate. It is suggested that the hydrolysis of polyphosphates provides a pH-stat mechanism to counterbalance alkaline stress.