Phosphorus is one of the important factors causing eutrophication pollution (Kong et al. 2005; Nguyen et al. 2011). The high concentration of residual phosphorus in wastewater results in the eutrophication of water environments (Cheng et al. 2014). Studies revealed that eutrophication might occur when the total phosphorus amount achieves 200~500 mg/m2/year in the water (Strokal et al. 2014), and so methods to alleviate eutrophication by removing excess phosphorus from wastewater are of considerable interest (Wang et al. 2015; Winkler et al. 2011; Zhang et al. 2015). Enhanced biological phosphorus removal (EBPR) is one sewage treatment technique for removing phosphorus from wastewater that depends on the ability of phosphorus-accumulating organisms (PAOs) to convert soluble phosphate to the insoluble phase in the form of intracellular polyphosphate (Poly-P) (Tarayre et al. 2016). Microlunatus phosphovorus, which was first isolated and identified in 1995 (Crocetti et al. 2000; Kong, et al. 2005), can accumulate large amounts of Poly-P (Nakamura et al. 1995; Terashima et al. 2016), and this species accounts for 2.7% of all microbes and 9.0% of PAOs in EBPR systems (Coats et al. 2017; Mino 2000).

The genome sequence of M. phosphovorus was reported in 2010 (Kawakoshi et al. 2012). Genome analysis has identified several genes involved in the metabolic processing of Poly-P (Fig.1) (He and McMahon 2011; Kawakoshi, et al. 2012), including: ppk1 (MLP_47700) and ppk2 (MLP_05750, MLP_50300), which encode polyphosphate kinases (PPK) that catalyse the conversion of ATP into Poly-P; ppx2 (MLP_44770), which encodes the exopolyphosphatase PPX that subsequently hydrolyzes the terminal residues of Poly-P into inorganic phosphate (Pi); ppgk (MLP_05430, MLP_26610), encoding the polyphosphate glucokinase (PPGK) that phosphorylates glucose using Poly-P; pap (MLP_23310), encoding a polyphosphate: AMP phosphotransferase (PAP) that synthesizes ADP using Poly-P and AMP; and ppnk (MLP_17420), which encodes a polyphosphate/ATP-dependent NAD kinase (PPNK) that synthesizes NADP+ using Poly-P and ATP. In addition, the phosphate transport systems PstSCAB and Pit were also found in M. phosphovorus. The PstSCAB system, which includes MLP_47720, MLP_47730, MLP_47740 and MLP_477500, has a higher phosphorus affinity than the Pit system does and can transport phosphate into cells under low phosphorus concentrations. The Pit system, which includes MLP_00530, MLP_29830 and MLP_51060, can transport inorganic phosphate into or out of the cell under high phosphorus concentrations (Tu and Schuler 2013; van Veen 1997). Alivebynature

Previous studies revealed that, similar to other PAOs, M. phosphovorus could synthesize Poly-P under aerobic conditions and degrade Poly-P under anaerobic conditions, indicating that these different organisms possess similar Poly-P metabolic pathways (Coats, et al. 2017; Tsai and Liu 2002). We previously reported that strain JN459 was isolated from a sewage system and that it was identified as M. phosphovorus (Zhong et al. 2015). In this study, we compared the transcriptional profiles of strain JN459 under anaerobic and aerobic circumstances. Our goals were to characterize the expression patterns of genes associated with Poly-P metabolism in order to determine the mechanisms of phosphorus accumulation and release in M. phosphovorus.

MATERIALSAND METHODS
Bacterial strains and culture conditions

Strain JN459 was isolated from sludge and identified as Microlunatus phosphovorus by morphological observation and 16 S rDNA sequence analysis (Zhong, et al. 2015). The bacteria were incubated at 25 °C with rotary shaking at 250 rpm. The culture medium contained 0.2 g glucose, 0.2 g tryptone, 0.2 g monosodium glutamate, 0.2 g yeast powder, 0.176 g dipotassium hydrogen phosphate, 0.04 g ammonium sulfate, and 0.04 g magnesium sulfate heptahydrate per liter. The pH of the medium was adjusted to 7.0, and the medium was autoclaved before use.

Escherichia coli DH5a (Invitrogen) was used for general cloning purposes, and E. coli Rosetta (DE3) pLysS (Novagen) was used for protein expression. E. coli strains were grown in Lysogeny Broth (LB) or on solid LB plates at 37 oC according to standard procedures (Sambrook and Russell 2001).

Phosphorus uptake and release analysis

To investigate phosphorus accumulation and release, strain JN459 was cultivated in 50 mL of liquid medium in 250 mL flasks. The cells were collected during the exponential growth phase and transferred into 8 liters of synthetic wastewater in a Sequencing Batch Reactor (SBR). The synthetic wastewater contained 0.3 g glucose, 0.1 g tryptone, 0.01 g yeast powder, 0.15 g sodium acetate, 0.20 g ammonia chloride, 0.05 g sodium chloride, 0.1 g dipotassium hydrogen phosphate, and 0.12 g magnesium sulfate per liter, with a soluble phosphate concentration of 14.21 mg/L. The synthetic wastewater was autoclaved before use.

The bacteria were grown aerobically in the SBR at 28 oC for 74 h to reach the mid-exponential growth phase, and then alternating anaerobic and aerobic processes were operated for two cycles. During each cycle, the anaerobic process was operated for 6 h, and the air in the SBR was replaced by nitrogen (N2), maintaining less than 0.2 mg/L dissolved oxygen (DO) in the reactor. The aerobic process was also operated for 6 h, and the DO maintained at 4 mg/L in the SBR. In the meantime, samples were collected at 74 h, 80 h, 86 h and 92 h. Both soluble phosphate and Poly-P content were determined, and the cell pellets were harvested by centrifugation to prepare the total RNA and cell lysates. All experiments were conducted in triplicate.

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