Mechanisms Of Ammonium Assimilation In Chlorella Vulgaris Based On Proteomic And Isotope Fractionation Techniques

Ammonium is one of the most common nitrogen contaminants in the water environment, which has resulted in eutrophecation and a series of environmental problems. In addition, our country has explicitly put forward the target of ammonium emission by 10% during the period of twelfth five-year. Therefore, the exploration of a new efficient and economical technology for ammonium removal has brought a new challenge to wastewater treatment plants(WWTPs). Recently, microalgae have gained a lot of attentions due to their abilities in removal of ammonium and production of biofuels. Therefore, the application of microalgae-based technology would reduce the large burden of NH4+-N removal in WWPTs, and in turn reduce the risk of NH4+-N to the aquatic ecological environment.Organisms utilizing ammonium as the sole nitrogen source can discriminate between 15 N and 14 N ammonium. This selectivity leaves an isotopic signature in their biomass that provides a critical evidence for transportation and assimilation of extracellular ammonium. Proteomics can reveal the different expression proteins involved in ammonium assimilation. Previous studies showed that microalgae removed the ammonium mainly uptake into biomass, however, the mechanisms that control fractionation of different concentrations of ammonium by algae are poorly understood. The detailed mechanisms of ammonium assimilation by by algae remain mysterious. Based on the above background, this study exploited15 N isotope fractionation, the proteomic approaches coupled with Reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR) to reveal how the concentrations of NH4+-N regulate the related genes and enzymes to assimilate NH4+-N in Chlorella vulgaris(C. vulgaris), a dominant unicellular green alga in the water environment.(1) With the increase of initial NH4+-N concentration from 4 to 50 mg/L, the growth rate of C. vulgaris was increase and then decrease, and the maximum growth rate was 4.86×105cells/(mL?d) which achieved in 10 mg/L NH4+-N。The removal efficiency decreased from 100% to 0.4% the increase of initial NH4+-N concentration.Additionally, the uptake rate of a single-cell was was increase and then decrease,which achieved the maximum in 10 mg/L NH4+-N(2.05×10-10 mg/(cell?d)). Therefore,the optimal growth rate and uptake rate for single-cell were achieved at 10 mg/L of NH4+-N. Similarly, chlorophyll concentration presented the same tendency with growth rate and uptake rate, and the maximal content of chlorophyll a(6.49 mg/L),chlorophyll b(6.31 mg/L) were achieved at 10 mg/L of NH4+-N. From the effects of ammonium concentration and carbon concentration on growth, ammonium and carbon removal by C. vulgaris showed that the cells density acheived to maxmiun(12.21×105 cells/(mL?d)) under 40 mg/L of carbon concentration, and the removal efficency was improved to 100% when concentration of carbon resource was above20 mg/L. Meanwhile, the absorbed dose for carbon resource was increased under 40mg/L of carbon concentration(3.94 mg).(2) The nitrogen isotope fractionation in C. vulgaris was dependent on ambient NH4+-N concentration. Under the concentration of 10 mg/L NH4+-N, the15 N abundance of culture solution and algae cells was separately-3.61±0.06‰ and-1.99±0.05‰, the isotope enrichment factor were-1.63±0.06‰. While the15 N abundance of culture solution and algae cells was respectively-3.50±0.08‰ and-1.13±0.06‰, the isotope enrichment factor were-2.37±0.08‰ under the concentration of 4 mg/L NH4+-N. Therefore, the isotope fractionation effect was more significant under 10 mg/L of NH4+-N compared to 4 mg/L of NH4+-N. Furthermore,the biotransformation efficiency of NH4+-N in C. vulgaris was higher under 10 mg/L of NH4+-N(47.4%) compared to 4 mg/L of NH4+-N(7.0%), thus it could explain that the extracellular NH4+-N was mainly assimilated into intercellular N-contained organic compounds.(3) Based on proteomic approaches, 162 proteins showed differently up-regulated and down-regulated expression under 10 mg/L of NH4+-N compared to 4 mg/L of NH4+-N. Proteins of 92 was significantly expressed(n ≥ 3, p < 0.05) and 31 proteins identified by matrix assisted laser desorption ionization/time of flight MS(MALDI-TOF-MS) were involved in photosynthesis, glucose metabolism,tricarboxylic acid(TCA) cycle, amino acid metabolism, protein synthesis, in which amino acid metabolism was directly related in nitrogen metabolism. Glutamate metabolism was important intermediary metabolism for other amino acid metabolism and TCA cycle. Glutamine synthetase(GS) was regarded as the primary invertase of ammonium, which could rapidly convert the ammonium into glutamate and glutamine.The results of proteomic and RT-qPCR showed that GS was up-regulated 6.44-fold at proteome level and 18.03-fold at transcription level at 10 mg/L of NH4+-N. Thus, it can demonstrate that the efficient removal of ammonium by C. vulgaris was closely related in the expression of GS emzyme and gene.