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Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs)
Accepted Manuscript Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs) Yu Wang, Yan Zhu, Pengfei Sun, Junzhuo Liu, Ningyuan Zhu, Jun Tang, Po Keung Wong, Hua Fan, Yonghong Wu PII: DOI: Reference:
S0960-8524(18)31613-4 https://doi.org/10.1016/j.biortech.2018.11.079 BITE 20732
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2018 20 November 2018 22 November 2018
Please cite this article as: Wang, Y., Zhu, Y., Sun, P., Liu, J., Zhu, N., Tang, J., Wong, P.K., Fan, H., Wu, Y., Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs), Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.11.079
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Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs) Yu Wanga,b, Yan Zhub,d, Pengfei Sunb, Junzhuo Liub, Ningyuan Zhu b,d, Jun Tangb*, Po Keung Wongc, Hua Fana, Yonghong Wub
a
School of Resources, Environmental & Chemical Engineering and Key Laboratory
of Poyang Lake Environment and Resource Utilization, Nanchang University, Nanchang, China b
Zigui Ecological Station for Three Gorges Dam Project, State Key Laboratory of
Soil and Sustainable Agriculture, Institute of Soil Sciences, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China c
School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong
Kong SAR, China d
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China
*Corresponding author: Jun Tang Tel : (+86)-25-8688 1286 fax: (+86)-25-8688 1330 Email: [email protected]
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Abstract: The application of periphytic biofilm in removing nitrogen from water is limited by the fluctuating nitrogen concentration. Here, we delineate a novel approach to enhance periphytic biofilm performance in nitrogen removal via upconversion luminescence of upconversion phosphors (UCPs). Nitrogen removal rates (14 d) in high nitrogen wastewater (26 mg/L) were significantly improved to 58.6% and 61.4% by UCPs doped with Pr3+ and Li+ and UCPs doped with Pr3+, respectively, and to 95.1% and 95.9% in low nitrogen surface water (2 mg/L), respectively. The stimulation of UCPs optimized the microbial community structure in the periphytic biofilms, and also resulted in good acclimation to use different carbon sources. The enhanced synergic action of cyanobacterial biomass, ratio of Gram +ve to Gram -ve bacteria and carbon source metabolic capacity contributed to the improved nitrogen removal. This novel approach is promising in nitrogen removal from wastewater and surface water with fluctuating initial nitrogen concentration.
Keywords: Periphytic biofilm; Upconversion luminescence; Microbial aggregates; Nitrogen removal; Microbial community structure.
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1. Introduction High doses of nitrogen in water can not only deteriorate aquatic ecosystems by eutrophication but also pose a potential risk to public health through human consumption of drinking water with high nitrate and nitrite contents. Periphytic biofilm, naturally growing microbial aggregates attached on submerged substrates in water, have been widely applied in many types of industrial wastewater treatment such as photoautotrophic bioreactor, constructed wetland and membrane bioreactor (Kang et al., 2018; Wu, 2016). The periphytic biofilm is also capable of enhancing water quality in surface waters under different environmental conditions due to the complex community composition, strong adaptability and complete food chains comprising of producers, consumers and decomposers (Wu et al., 2012; Wu et al., 2014). However, the efficiency of periphytic biofilm application in removing nitrogen in a broad range of initial concentrations of wastewater and surface water is limited due to the periphytic biofilm community structure changing with nitrogen levels and food sources (Daija et al., 2016; Rikmann et al., 2016; Wu et al., 2011). Many physical (e.g. UV light radiation) and chemical (e.g. chlorination addition) methods have been proposed to change microbial community composition and structure via inactivating microbial activity (Cates et al., 2012; Kubacka et al., 2008; Pires et al., 2016). Among these methods, upconversion phosphors (UCPs), e.g. upconversion luminescence agents doped with lanthanide ions, have been applied to explore the interaction with microorganisms (Cates et al., 2011).
UCPs are able to
convert low-energy photons into high-energy state, directly converting visible light into germicidal ultraviolet radiation via the photoluminescence process of upconversion (Cates & Kim, 2015; Wang et al., 2010). Due to the advantages of low auto-fluorescence background, large anti-Stokes 3
shifts, sharp emission bandwidths, high resistance to photobleaching, deep penetration and temporal resolution, lanthanide-doped UCPs have become promising alternatives for inactivation of microorganisms or viruses (Lim et al., 2012). Lanthanide-doped upconversion particles are attractive alternatives to conventionally excited luminescent labels, which can be excited by multiphoton absorption at relatively low power levels by various mechanisms and give well-structured visible/UV luminescence (Vetrone et al., 2009). Pr3+ and Li+ are often employed as dopants of upconversion luminescence agents (Zhu et al., 2018c). The alteration of the crystal phase is partly done by the core–shell structure of the phase for doped samples (Kar & Patra, 2012). Pr3+ doped crystals (oxide or fluorides) have been applied extensively, in which the 4f5d lowest states are located below the 4f2 1S0 manifold and the 1D2, 3PJ and 1I6 manifolds of 4f2 configuration can be used as intermediate states for upconversion (Cefalas et al., 1993). Y2SiO5: Pr3+ is able to use a metastable 4f intermediate state to populate the 4f5d band through absorption of blue photos (Hu et al., 2006). The lithium ion is the smallest metallic ion with an ionic radius of 0.9 Å, which has been validated in the oxide host matrices of several-fold enhancements in the emission of nanophosphors (Han et al., 2014). UVC photons are easily absorbed by RNA and DNA and can be combined with nucleic acid components, forming negative photoproducts (Cates, 2015; Cates et al., 2011). These transmutations are able to prevent transcription and result in cell death (Cates, 2015; Cates et al., 2011). It is well known that microorganisms have dissimilar sensitivity and resistance to UVC (Lee et al., 2015; MacFarlane et al., 2011). Some microorganisms, such as Escherichia coli, MS-2 and T7 (Bowker et al., 2011; Guo et al., 2012), can be inactivated in the presence of ultraviolet irradiation, while some other microorganisms such as Actinobacteria (Warnecke et al., 2005) and 4
-proteobacteria (Santos et al., 2012) grow well under ultraviolet irradiation, depending on their adaptability. Most research focuses on the effects of upconversion luminescence of UCPs on the activity of pure strain microorganisms, however, the effects of upconversion luminescence on the community composition and function of multi-species microbial aggregates it is still unknown. In a recent study, the removal efficiency of inorganic phosphorus and copper for periphytic biofilms was significantly improved by stimulating it via lanthanide-doped UCPs (Zhu et al., 2018c). The removal of inorganic phosphorus and copper by periphytic biofilm is mainly controlled by physical and chemical processes. However, nitrogen removal by periphytic biofilm is more of a biochemical process, which is more susceptible to community composition and microorganism metabolism. It is necessary to investigate whether upconversion luminescence of UCPs can augment the nitrogen removal for periphytic biofilm and how it influences the community composition and metabolism of periphytic biofilm. Thus, in this study, the upconversion luminescence of UCPs coped with Pr 3+ and Li+ was proposed to convert visible light into UVC and optimize the microbial community composition of periphytic biofilm, then improve nitrogen removal. To test this hypothesis, a bench-scale simulation of a periphytic biofilm wastewater treatment system was employed to: (i) examine the efficiency of nitrogen removal by periphytic biofilm, (ii) investigate the microbial community composition and structure, and (iii) determine the carbon source utilization capacity of the periphytic biofilm stimulated by UCPs coped with Pr3+ and Li+. These findings are expected to present a novel upconversion approach to inactivate the “ineffective” component and augment the “effective” component of the periphytic biofilm in enhancing nitrogen removal.
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2. Materials and methods 2.1 Periphytic biofilm culture The water from Xuanwu Lake, Nanjing, China was used to cultivate periphytic biofilms (lake water parameters: total nitrogen (TN) = 2.18 mg/L, NO 3−-N = 0.43 mg/L, NH4+-N = 0.07 mg/L, PO43--P = 0.24 mg/L, pH = 8). The substrates of periphytic biofilm - fiber carriers (5 cm length, 2 cm width, 5 mm thickness, Jineng Environmental Protection Company of YiXing, China) were added to a cylinder with 5 L lake water, with a volume ratio of water to fiber carriers of 3:1. The cylinder was placed in an constant temperature incubator with light intensity of 32 W/m2, a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. After 35 days cultivation, the mature periphytic biofilm was collected for the following experiments. 2.2 Preparation of Pr and Pr-Li coped UCPs The syntheses of UCPs coped with Pr3+ (Y2SiO5: Pr3+), and Pr3+ and Li+ (Y2SiO5: Pr3+, Li+) were conducted according to previous studies (Cates, 2015; Cates et al., 2011). Briefly, nanocrystalline powder materials synthesized via sol gel method were used to investigate optical properties of the UCPs. Lanthanide-doped UCPs were prepared as follows: Yttrium nitrogen was made from 5.3 g of Y2O3 by boiling in 1:1 nitric acid (guaranteed reagent, weight ratio), then evaporated to dryness in an oven at 104 oC. Pr and Pr-Li dopants were added at the same time as the HNO3. The solution of yttrium nitrogen was then dissolved in 17.25 mL of 99.99% ethanol and 5.4 mL of water. Then, 10 mL Tetraethoxysilane (TEOS) was added followed by 10 min of stirring. Dopant concentrations were adjusted to optimize upconversion efficiency, which corresponded to the doping concentration 1% Pr 3+ for samples without lithium and 1% Pr3+ and Li+ for samples with both elements. 6
Aqueous stock solutions of Pr3+ and Li+ were prepared from Pr(NO3)3·6H2O and LiNO3·6H2O. All chemicals are of analytical pure grade and were purchased from Sigma Aldrich.
2.3 Experimental design 2.3.1 Activation process Before activation, all experimental materials (excluding the doped UCPs and periphytic biofilms) were disinfected on a super clean bench with UV illumination at 25 °C for 2 h to avoid the inclusion of exotic microorganisms. Then, 8 g (dry weight) periphytic biofilm was added to a flask with 30 mL Woods Hole culture medium (WC medium) (Hametner et al., 2014). After two days, 2 g of each doped UCP (Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+) was directly added into the flasks with periphytic biofilms. These two types of periphytic biofilms stimulated by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ were called Pr treated and Pr-Li treated periphytic biofilms. In the control, no Y2SiO5: Pr3+ or Y2SiO5: Pr3+, Li+ was added. All flasks were sealed with sealing film to prevent contamination. All flasks were then placed in an incubator (25 °C) for nine days under 12/12 h = light/dark conditions with the light intensity 32 W/m2. To improve the efficiency of periphytic biofilm stimulation, the flasks were shaken two times every day (at 9:00 am and 5:00 pm, about 10 mins for each time) by hand. After nine days, the activated periphytic biofilms and the control periphytic biofilm were filtered and washed using sterile water until no Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ was observed under fluorescence microscopy. The washed periphytic biofilms were then used for the N removal experiment. The treatments and controls were conducted in triplicate. 2.3.2 Experiments for nitrogen removal by periphytic biofilms To investigate the functioning of periphytic biofilm in removing nitrogen from 7
high concentration wastewater after activation by UCPs (Pr and Pr-Li), nitrogen removal experiments were conducted in a simulated periphytic biofilm wastewater treatment system designed according to our previous study (Liu et al., 2018; Zhu et al., 2018b). The activated and control periphytic biofilms (8.0 g, wet weight) were individually added into glass tanks with 200 mL of the high concentration wastewater [NaNO3-N (20 mg/L), NH4Cl-N (20 mg/L), KH2PO4-P (20 mg/L), C6H12O6 (5 mg/L), other compositions are the same as WC medium]. The volume ratio of wastewater to periphytic biofilm was 5:1. The glass tanks were placed in an incubator with light intensity of 32 W/m2 (provided by fluorescent lamps) (UV = 0 W/m2), a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. The removal experiment was conducted for 14 d. Thirty mL of water was extracted from 2 cm under the water surface using a syringe to detect nitrogen concentration every day. This was replaced with 30 mL fresh of wastewater injected into the tanks. To explore the ability of the stimulated periphytic biofilms to remove nitrogen from surface water that generally has low N concentration, the nitrogen removal experiment was repeated with surface water collected from Xuanwu Lake (Nanjing, China) in lieu of the high concentration wastewater mentioned above. The properties of the lake water were the same as those for culturing periphytic biofilm mentioned above. The experimental conditions and the water sampling technique were the same as for the high nitrogen concentration experiment. Both high and low nitrogen experiments were performed in triplicate. 2.4 Sample analysis X-ray diffraction (XRD) analysis was carried out on a Siemens D5005 X-ray diffractometer with Cu Kα radiation (λ = 1.54056 nm) at a power of 40 keV × 30 mA. 8
The XRD data were recorded with 2θ varying from 20° to 60° at counting time of 10 s and a scanning mode with a step size of 0.02°. The upconversion luminescence of UCPs was tested by a three-dimensional fluorescence spectrometer (F-7000, Japan). The parameters were selected as 1 nm of the slit, 450 nm of the excitation wavelength, 370 nm of the optical filters. Transmission electron microscope (JEM-1400PLUS, JEOL) was used to observe the cell structure of periphytic biofilm. Surface morphology, integral structure and element distribution on the surface of periphytic biofilm were observed by scanning electron microscopy (SEM, SU3500, Japan) and energy dispersive spectrometry (EDS, TM3000, Japan). Biolog E coplatesTM (Biolog, Hayward, USA) analysis was conducted to evaluate the carbon metabolic activity of periphytic biofilm. The detailed process is described in a previous study (More et al., 2012). The microbial structure and compositions were determined by phospholipid linked fatty acid (PLFA) profiling following the study of Zelles (1999). Briefly, 0.2 g of freeze-dried periphytic biofilm was extracted with a chloroform-methanol-citrate butter mixture. For each sample, the content of individual fatty acids was expressed as nmol FA g-1 dry periphytic biofilm biomass. The phospholipids were subjected to mild-alkali methanolysis and the resulting fatty acid methyl esters were separated by gas chromatography (Campbell et al., 2008). The PLFAs 18:3ω3c and 20:5ω3c, 20:2ω6 20:3ω6 and 20:4ω6, 18:2ω6 18:3ω6c and 18:3ω3c, 16:0 17:0 and 18:0, 16:1ω5c 16:1ω7c 18:1ω5c and 18:1ω7c were selected as indicators of cyanobacteria, protozoan biomass, fungal biomass, Gram +ve, Gram –ve , respectively (Boschker et al., 2005). The nitrogen in the wastewater and water samples were determined using a flow injection analyzer (SEALAA3, German).
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2.5 Data analysis All analyses were performed in triplicate. The results are presented as mean ± 1 standard deviation (SD). The 96 h Biolog data of the carbon sources utilized and correlations of fatty acids data were conducted using Principal Component Analysis (PCA) and Canonical Analysis (CA) in Canoco 5. The bar charts of nitrogen removal and XRD data were drawn by OriginPro 2016 (OriginLab Company). The comparison of nitrogen removal after 14 d operation and PLFA data between the treatment and the control were analyzed using One-way ANOVA. Variance partitioning analyses using the varPart function in the vegan package were calculated by R software (R 3.3.1). Statistical significance was set at p = 0.05 for all analyses. 3. Results and discussion 3.1 Characteristics of UCPs doped with Pr3+ and Pr3+-Li+ As shown in the XRD patterns, the samples of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ have similar crystal structures. The two curves, a and b presented a strong peak when 2θ = 30.7°, which is consistent with the standard pattern of yttrium (JCPDS no. 41-0004). The crystal sizes of the Y2SiO5 doped with Pr3+ and Pr3+ + Li+ at 2θ angle 30.7° are 332 nm and 337 nm, respectively. The upconversion luminescence emission spectra of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ were similar. Under 450 nm excited wavelength, emissions of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ from this band produce a broad spectrum of UV radiation, including the UVC range (220-280 nm) which has a negative effect on microorganisms such as Escherischia coli (Benabbou et al., 2007). It is worth noticing that the UVC emission was enhanced by addition of lithium-ions, which shows the structural effects of improved upconversion (Liang et al., 2009; Liang et al., 2011). 10
Compared to upconversion materials synthesized in previous studies, the Pr3+ doped Y2SiO5 material (i.e. Y2SiO5: Pr3+) used in the present study has similar crystal structure and doping concentration, which achieved a high degradation performance of wastewater (Yang et al., 2013). The doping of Li+ strongly affected the crystal transition of Y2SiO5: Pr3+ resulting in high luminance intensity and upconversion efficiency (Yang et al., 2015). 3.2 UCPs distribution in the periphytic biofilms SEM images exhibit the morphological alterations of periphytic biofilm when exposed to UCPs. The Pr and Pr-Li treated periphytic biofilms were observed to be more compact than the control. Microbial communities of Pr-Li treated periphytic biofilm are more abundant than control and Pr treated periphytic biofilm. To detect the distribution of UCPs on the surface of periphytic biofilms, EDS analyses were carried out based on the SEM images. There were significant differences in trivalent praseodymium ion distributions on the surface of periphytic biofilms between UCPs treatment and control periphytic biofilms. In the control samples, there were no promethium ions distributed on the surface of periphytic biofilm, while promethium ions of 0.15% (Weight%) and 0.29% were detected in Pr treated and Pr-Li treated periphytic biofilm, respectively. These results reveal that some of the UCPs can be distributed on the surface of Pr and Pr-Li treated periphytic biofilms. 3.3 Nitrogen removal by periphytic biofilm stimulated The average nitrogen removal rates in both Pr and Pr-Li treated periphytic biofilms (58.6 % and 61.4%, respectively) in the high concentration wastewater treatment system were significantly higher than that in the control (p < 0.01, Fig. 1A). 11
In the low concentration lake water, the average nitrogen removal rates in both Pr and Pr-Li treated periphytic biofilms were 95.1% and 95.9%, respectively, which were again significantly higher than the control (p < 0.05, Fig. 1B). These results indicate that the periphytic biofilm stimulated by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ was capable of augmenting nitrogen removal from both high concentration wastewater and low concentration surface water. The enhanced nitrogen removal by Y2SiO5: Pr3+, Li+ was slightly higher than Y2SiO5: Pr3+, but the difference was not significant (p > 0.05). 3.4 Microbial community structures of periphytic biofilms According to PLFA analysis, the specific microbial community compositions were obviously different in the control and treatments, as shown in Fig. 2A. Specifically, in the control, the community was dominated by cyanobacteria (M. aeruginosa), attached protozoa and bacterium (e.g., Bacillus stearothermophilus and Actinomycetes sp.). However, the main communities in the Y2SiO5: Pr3+ treated periphytic biofilm, included bacteria (e.g., Thermocrinis rubber), blue green bacteria and cyanobacteria. Cyanobacteria (e.g., Microcystis flos-aquae), Gram +ve bacteria and fungi dominated in the Y2SiO5: Pr3+, Li+ treated periphytic biofilm. These results indicate that the community structure of the periphytic biofilms had been changed to different degrees by the UCPs. Pr-Li treated periphytic biofilm had higher microbial and cyanobacterial biomass (dominated by M. flos-aquae) than Pr treated periphytic biofilm. This is because UCP-treated periphytic biofilm with Li doped UCPs grow with an inert shell which is more competitive to UCPs (Ding et al., 2015). The UCPs with Li+ slightly enhanced the upconversion emission intensity (Bai et al., 2008), and the community showed 12
increased acclimation. Furthermore, single Pr3+ doped UCPs directly absorb light, so quenching could easily occur when the ion concentrations of the dopant and the illuminant demand are high (Balda et al., 1999). Therefore, light conversion efficiency of Pr-Li treated periphytic biofilm increased with significant effects on microorganisms, resulting in a higher proportion of cyanobacteria and bacteria. Metagenomic sequencing was used to reflect the community changes in prokaryotic (16s rRNA) and eukaryotic (18s rRNA) microorganisms in periphytic biofilm (Fig. 3). Cyanobacteria contributed 30% in the control which was lower than in Pr and Pr-Li treated periphytic biofilm (39% and 34%, respectively) (Fig. 3A). This result corresponded with the fatty acid data. The contribution of Bacteroidetes (42%) in the control was higher than in Pr and Pr-Li treated periphytic biofilm (33% and 40%, respectively). UCPs could have negative impacts on gram-negative bacteria. According to the 18S rRNA results (Fig. 3B), the contribution of SAR was 40% in the control, lower than Pr treated periphytic biofilms (41%), but higher than Pr-Li treated periphytic biofilms (31%). The contribution of Amoebozoa was 14% in the control, 18% in the Pr treated periphytic biofilms and 15% in Pr-Li treated periphytic biofilms. This result showed that Amoebozoa acclimated well to UCPs. Similar to previous studies examining the impacts of UCPs on single communities, such as bacterial and viral communities (Cates et al., 2014; Chen et al., 2014; Lim et al., 2012), this study demonstrated that UCPs doped with Pr 3+ or codoped with Pr3+ and Li+ could negatively affect sensitive individual cells, such as M. aeruginosa cells, through damaging single cell structures (Cates & Kim, 2015). These impacts successfully inactivated the “ineffective” members in the periphytic biofilm and led to the activation of some “dormant” communities such as T. rubber in Pr treated periphytic biofilm and M. flos-aquae in Pr-Li treated periphytic biofilms, 13
shifting the periphytic biofilm community composition and structure. After stimulation by UCPs, the ability of periphytic biofilm to remove N was improved significantly. The process of N removal by periphytic biofilms has been described in previous studies (Wu et al., 2018). In this study, the change in community composition of periphytic biofilms induced by upconversion fluorescence and subsequent influence on N removal capacity were investigated. When visible light is converted to ultraviolet light by UCPs, it causes stress in surrounding sensitive microorganisms, which changes the community structure in the biofilm. Meanwhile, some functional populations which could remove N might develop due to their resistance to ultraviolet light (Cates et al., 2014). Another reason for the improved N removal could be that the generation of UV light can cause EPS accumulation for periphytic biofilms which often enhances the denitrification process as an electron donor and electric medium (Zhu et al., 2018a). 3.5 Carbon source metabolic capacities of periphytic biofilms The carbon source utilization in UCP-treated periphytic biofilm was distinct from the control (Fig. 2B). The periphytic biofilm in the control favored utilization of itaconic acid, D-cellobiose, 4-hydroxybenzoic acid, D-galactonic g-lactone acid and α-ketobutric acid. The Pr treated periphytic biofilm utilized β-methyl-D-glucoside, L-serine, galacturonic acid and L-arginine. The periphytic biofilm stimulated by Y2SiO5: Pr3+, Li+ utilized N-acetyl-D-gucosamine, D-mannitol and glucose-1-phosphate.
These imply that the control and UCP-treated periphytic
biofilm grew based on different carbon sources, and further imply that the periphytic biofilm had already acclimated to the effects of activation by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+. 14
3.6 Synergic action of cyanobacteria, bacteria and metabolic capacity To select the primary factors affecting nitrogen removal, variance partitioning analyses (VPA) based on cyanobacterial biomass (represented by cyanobacterial PLFA), Gram +ve/Gram –ve bacteria, carbon source metabolic capacity (represented by AWCD) and nitrogen removal rate were conducted (Fig. 4). In contrast to the control, the contribution of cyanobacterial biomass in the Pr treated periphytic biofilm to nitrogen removal significantly decreased from 0.14 to 0.06 (p < 0.05) while the contribution in the Pr-Li treated periphytic biofilm significantly increased from 0.14 to 0.31 (p < 0.05). The contributions of Gram +ve/Gram –ve bacteria in both Pr and Pr-Li treated periphytic biofilms significantly increased from 0.13 to 0.20 and 0.13 to 0.32, respectively, compared to the control (p < 0.05 for both analyses). No contribution of AWCD was found in the control, while the contributions in the Pr and Pr-Li treated periphytic biofilms were relatively high, about 15 and 20%, respectively. These results indicate that cyanobacterial biomass, Gram +ve/Gram –ve bacteria and carbon source metabolic capacity played important roles in enhancing nitrogen removal in the two treated periphytic biofilms. It is noteworthy that the synergic contribution of cyanobacteria biomass, Gram +ve/Gram –ve bacteria and AWCD in the control was not significant (value < 0) while the values of synergic action in both Pr (0.42) and Pr-Li treated (0.84) periphytic biofilms were positive. Moreover, the contributions of these three parameters in both Pr and Pr-Li treated periphytic biofilm were significantly higher than that of the contribution of cyanobacterial biomass, Gram +ve/Gram –ve bacteria or AWCD alone. This indicates that the synergic action of PLFAcyanobacteria, Gram +ve/Gram –ve bacteria and AWCD played an important role in nitrogen removal. The cyanobacterial biomass (represented by PLFA) in the Pr-Li treated 15
periphytic biofilm increased while the biomass in the Pr treated periphytic biofilm decreased. This result was consistent with the changes in the contribution of cyanobacterial biomass to nitrogen removal. Both periphytic biofilms presented good acclimation in terms of carbon source metabolic versatility during the activation of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+. These changes in the carbon metabolic capacity of the periphytic biofilms also concurred with the AWCD changes. These results further indicate that cyanobacterial biomass and carbon metabolic activity played an important role in augmenting nitrogen removal by periphytic biofilm. Under stress conditions microbial communities, such as bacterial E. coli, present stronger plasticity and resistance for survival, as a result achieving high activity (Davies & Davies, 2010; Shade et al., 2012). In this study, the exposure of periphytic biofilms to Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ created unfavorable conditions, in turn leading to the formation of more robust periphytic biofilm with high carbon metabolism. The Gram +ve bacteria may be capable of augmenting strength and resistance to stress (Holzinger & Karsten, 2013; Wang et al., 2011). The UCPs stimulated changes in the Gram +ve/Gram –ve bacterial ratio, but also the kept the ratio at a positive level. The optimized ratio of Gram +ve/Gram –ve bacteria then promoted nitrogen removal by the treated periphytic biofilms. As mentioned, the periphytic biofilm is a type of microbial aggregate (Wu, 2016), whose functionality, such as nitrogen removal, is related to composition changes (Wu et al., 2011). Thus, the synergic action of multiple parameters, such as cyanobacterial biomass, Gram + ve/Gram –ve bactiera and AWCD to nitrogen removal was considered. The high contributions of the treated periphytic biofilms demonstrated that the synergic action of these three parameters was mainly responsible for the increased nitrogen removal. This result demonstrates that the stimulation by Y2SiO5: 16
Pr3+ and Y2SiO5: Pr3+, Li+ promoted the synergic action of the periphytic biofilms. 4. Conclusions In this study, the investigation of the multi-community optimization of periphytic biofilm by upconversion luminescence of UCPs advances the studies of microbial inactivation from one-species inactivation to multi-species community design. The strategy proposed in this study proved the feasibility of using upconversion materials to design robust microbial aggregates (i.e., periphytic biofilm) for nitrogen removal. The results obtained also invoke re-thinking of the type of microbial community (single- or multi-species microbial aggregates) that should be employed when evaluating the use of upconversion materials to inactivate microorganisms.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31772396, 41422111), the State Key Development Program for Basic Research of China (2015CB158200) and the Natural Science Foundation of Jiangsu Province (BK20150066, BK20181511). This work was also supported by Chinese Academy of Sciences Interdisciplinary Innovation Team. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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Figure captions
Fig. 1 Nitrogen removal rates by periphytic biofilms from (A) high concentration wastewater and (B) low high concentration lake water. The influent and effluent nitrate concentrations were the average means of running for 14 d.
Fig. 2 A. The PLFA canonical analysis (CA) of the microbial communities in periphytic biofilms; B. The principal component analysis (PCA) of different types of carbon source utilization in periphytic biofilms. Arrows of different colors represent different carbon sources: black, purple, red, orange, blue and green represent carbohydrates, polymers, phenolic acids, carboxylic acids, amines and amino acids, respectively. 1-31 represents β-methyl-D-glucoside, D-galactonic g-lactone acid, L-arginine, Pyruvic acid methyl ester, D-xylose, Galacturonic acid, l-asparagine, Tween 40, i-erytritol, 2-hydroxybenzoic acid, L-serine, Tween 80, D-mannitol, 4-hydroxybenzoic acid, l-phenylalanine, α-cyclodextrine, N-acetyl-d-glucosamine, γ-hydroxybutiric acid, L-threonine, Glycogen, D-glucosaminic acid, Itaconic acid, Glycyl-l-glutamic acid, D-cellobiose, Glucose-1-phosphate, α-ketobutiric acid, Phenylethylamine, α-lactose, D-l-α-Glycerol-1-phosphate, D-malic acid and Putrescine.
Fig. 3 (A) 16S rRNA and (B) 18S rRNA gene sequencing for microorganism composition of periphytic biofilms stimulated by upconversion materials. SAR includes three species, Heterokonts, Alveolates and Rhizaria.
Fig. 4 Variance partitioning analyses based on PLFAalga (X1), Gram +ve/Gram –ve bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and TN removal in the 21
(A) Control, (B) Pr treated periphytic biofilm and (C) Pr-Li treated periphytic biofilm (n = 5. Residual means the difference between the observed and predicted values (fitted value). (No result shown when values < 0).
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Fig. 1
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Fig. 2 A
B
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Fig. 3 A
B
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Fig. 4
B
A
C
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Highlights：
UCPs were first used to stimulate nitrogen removal of periphytic biofilms.
UCPs optimized the microbial community structure of periphytic biofilms.
Periphytic biofilms simulated by UCPs could adapt to nitrogen fluctuation.
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S0960-8524(18)31613-4 https://doi.org/10.1016/j.biortech.2018.11.079 BITE 20732
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2018 20 November 2018 22 November 2018
Please cite this article as: Wang, Y., Zhu, Y., Sun, P., Liu, J., Zhu, N., Tang, J., Wong, P.K., Fan, H., Wu, Y., Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs), Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.11.079
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Augmenting nitrogen removal by periphytic biofilm strengthened via upconversion phosphors (UCPs) Yu Wanga,b, Yan Zhub,d, Pengfei Sunb, Junzhuo Liub, Ningyuan Zhu b,d, Jun Tangb*, Po Keung Wongc, Hua Fana, Yonghong Wub
a
School of Resources, Environmental & Chemical Engineering and Key Laboratory
of Poyang Lake Environment and Resource Utilization, Nanchang University, Nanchang, China b
Zigui Ecological Station for Three Gorges Dam Project, State Key Laboratory of
Soil and Sustainable Agriculture, Institute of Soil Sciences, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China c
School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong
Kong SAR, China d
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China
*Corresponding author: Jun Tang Tel : (+86)-25-8688 1286 fax: (+86)-25-8688 1330 Email: [email protected]
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Abstract: The application of periphytic biofilm in removing nitrogen from water is limited by the fluctuating nitrogen concentration. Here, we delineate a novel approach to enhance periphytic biofilm performance in nitrogen removal via upconversion luminescence of upconversion phosphors (UCPs). Nitrogen removal rates (14 d) in high nitrogen wastewater (26 mg/L) were significantly improved to 58.6% and 61.4% by UCPs doped with Pr3+ and Li+ and UCPs doped with Pr3+, respectively, and to 95.1% and 95.9% in low nitrogen surface water (2 mg/L), respectively. The stimulation of UCPs optimized the microbial community structure in the periphytic biofilms, and also resulted in good acclimation to use different carbon sources. The enhanced synergic action of cyanobacterial biomass, ratio of Gram +ve to Gram -ve bacteria and carbon source metabolic capacity contributed to the improved nitrogen removal. This novel approach is promising in nitrogen removal from wastewater and surface water with fluctuating initial nitrogen concentration.
Keywords: Periphytic biofilm; Upconversion luminescence; Microbial aggregates; Nitrogen removal; Microbial community structure.
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1. Introduction High doses of nitrogen in water can not only deteriorate aquatic ecosystems by eutrophication but also pose a potential risk to public health through human consumption of drinking water with high nitrate and nitrite contents. Periphytic biofilm, naturally growing microbial aggregates attached on submerged substrates in water, have been widely applied in many types of industrial wastewater treatment such as photoautotrophic bioreactor, constructed wetland and membrane bioreactor (Kang et al., 2018; Wu, 2016). The periphytic biofilm is also capable of enhancing water quality in surface waters under different environmental conditions due to the complex community composition, strong adaptability and complete food chains comprising of producers, consumers and decomposers (Wu et al., 2012; Wu et al., 2014). However, the efficiency of periphytic biofilm application in removing nitrogen in a broad range of initial concentrations of wastewater and surface water is limited due to the periphytic biofilm community structure changing with nitrogen levels and food sources (Daija et al., 2016; Rikmann et al., 2016; Wu et al., 2011). Many physical (e.g. UV light radiation) and chemical (e.g. chlorination addition) methods have been proposed to change microbial community composition and structure via inactivating microbial activity (Cates et al., 2012; Kubacka et al., 2008; Pires et al., 2016). Among these methods, upconversion phosphors (UCPs), e.g. upconversion luminescence agents doped with lanthanide ions, have been applied to explore the interaction with microorganisms (Cates et al., 2011).
UCPs are able to
convert low-energy photons into high-energy state, directly converting visible light into germicidal ultraviolet radiation via the photoluminescence process of upconversion (Cates & Kim, 2015; Wang et al., 2010). Due to the advantages of low auto-fluorescence background, large anti-Stokes 3
shifts, sharp emission bandwidths, high resistance to photobleaching, deep penetration and temporal resolution, lanthanide-doped UCPs have become promising alternatives for inactivation of microorganisms or viruses (Lim et al., 2012). Lanthanide-doped upconversion particles are attractive alternatives to conventionally excited luminescent labels, which can be excited by multiphoton absorption at relatively low power levels by various mechanisms and give well-structured visible/UV luminescence (Vetrone et al., 2009). Pr3+ and Li+ are often employed as dopants of upconversion luminescence agents (Zhu et al., 2018c). The alteration of the crystal phase is partly done by the core–shell structure of the phase for doped samples (Kar & Patra, 2012). Pr3+ doped crystals (oxide or fluorides) have been applied extensively, in which the 4f5d lowest states are located below the 4f2 1S0 manifold and the 1D2, 3PJ and 1I6 manifolds of 4f2 configuration can be used as intermediate states for upconversion (Cefalas et al., 1993). Y2SiO5: Pr3+ is able to use a metastable 4f intermediate state to populate the 4f5d band through absorption of blue photos (Hu et al., 2006). The lithium ion is the smallest metallic ion with an ionic radius of 0.9 Å, which has been validated in the oxide host matrices of several-fold enhancements in the emission of nanophosphors (Han et al., 2014). UVC photons are easily absorbed by RNA and DNA and can be combined with nucleic acid components, forming negative photoproducts (Cates, 2015; Cates et al., 2011). These transmutations are able to prevent transcription and result in cell death (Cates, 2015; Cates et al., 2011). It is well known that microorganisms have dissimilar sensitivity and resistance to UVC (Lee et al., 2015; MacFarlane et al., 2011). Some microorganisms, such as Escherichia coli, MS-2 and T7 (Bowker et al., 2011; Guo et al., 2012), can be inactivated in the presence of ultraviolet irradiation, while some other microorganisms such as Actinobacteria (Warnecke et al., 2005) and 4
-proteobacteria (Santos et al., 2012) grow well under ultraviolet irradiation, depending on their adaptability. Most research focuses on the effects of upconversion luminescence of UCPs on the activity of pure strain microorganisms, however, the effects of upconversion luminescence on the community composition and function of multi-species microbial aggregates it is still unknown. In a recent study, the removal efficiency of inorganic phosphorus and copper for periphytic biofilms was significantly improved by stimulating it via lanthanide-doped UCPs (Zhu et al., 2018c). The removal of inorganic phosphorus and copper by periphytic biofilm is mainly controlled by physical and chemical processes. However, nitrogen removal by periphytic biofilm is more of a biochemical process, which is more susceptible to community composition and microorganism metabolism. It is necessary to investigate whether upconversion luminescence of UCPs can augment the nitrogen removal for periphytic biofilm and how it influences the community composition and metabolism of periphytic biofilm. Thus, in this study, the upconversion luminescence of UCPs coped with Pr 3+ and Li+ was proposed to convert visible light into UVC and optimize the microbial community composition of periphytic biofilm, then improve nitrogen removal. To test this hypothesis, a bench-scale simulation of a periphytic biofilm wastewater treatment system was employed to: (i) examine the efficiency of nitrogen removal by periphytic biofilm, (ii) investigate the microbial community composition and structure, and (iii) determine the carbon source utilization capacity of the periphytic biofilm stimulated by UCPs coped with Pr3+ and Li+. These findings are expected to present a novel upconversion approach to inactivate the “ineffective” component and augment the “effective” component of the periphytic biofilm in enhancing nitrogen removal.
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2. Materials and methods 2.1 Periphytic biofilm culture The water from Xuanwu Lake, Nanjing, China was used to cultivate periphytic biofilms (lake water parameters: total nitrogen (TN) = 2.18 mg/L, NO 3−-N = 0.43 mg/L, NH4+-N = 0.07 mg/L, PO43--P = 0.24 mg/L, pH = 8). The substrates of periphytic biofilm - fiber carriers (5 cm length, 2 cm width, 5 mm thickness, Jineng Environmental Protection Company of YiXing, China) were added to a cylinder with 5 L lake water, with a volume ratio of water to fiber carriers of 3:1. The cylinder was placed in an constant temperature incubator with light intensity of 32 W/m2, a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. After 35 days cultivation, the mature periphytic biofilm was collected for the following experiments. 2.2 Preparation of Pr and Pr-Li coped UCPs The syntheses of UCPs coped with Pr3+ (Y2SiO5: Pr3+), and Pr3+ and Li+ (Y2SiO5: Pr3+, Li+) were conducted according to previous studies (Cates, 2015; Cates et al., 2011). Briefly, nanocrystalline powder materials synthesized via sol gel method were used to investigate optical properties of the UCPs. Lanthanide-doped UCPs were prepared as follows: Yttrium nitrogen was made from 5.3 g of Y2O3 by boiling in 1:1 nitric acid (guaranteed reagent, weight ratio), then evaporated to dryness in an oven at 104 oC. Pr and Pr-Li dopants were added at the same time as the HNO3. The solution of yttrium nitrogen was then dissolved in 17.25 mL of 99.99% ethanol and 5.4 mL of water. Then, 10 mL Tetraethoxysilane (TEOS) was added followed by 10 min of stirring. Dopant concentrations were adjusted to optimize upconversion efficiency, which corresponded to the doping concentration 1% Pr 3+ for samples without lithium and 1% Pr3+ and Li+ for samples with both elements. 6
Aqueous stock solutions of Pr3+ and Li+ were prepared from Pr(NO3)3·6H2O and LiNO3·6H2O. All chemicals are of analytical pure grade and were purchased from Sigma Aldrich.
2.3 Experimental design 2.3.1 Activation process Before activation, all experimental materials (excluding the doped UCPs and periphytic biofilms) were disinfected on a super clean bench with UV illumination at 25 °C for 2 h to avoid the inclusion of exotic microorganisms. Then, 8 g (dry weight) periphytic biofilm was added to a flask with 30 mL Woods Hole culture medium (WC medium) (Hametner et al., 2014). After two days, 2 g of each doped UCP (Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+) was directly added into the flasks with periphytic biofilms. These two types of periphytic biofilms stimulated by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ were called Pr treated and Pr-Li treated periphytic biofilms. In the control, no Y2SiO5: Pr3+ or Y2SiO5: Pr3+, Li+ was added. All flasks were sealed with sealing film to prevent contamination. All flasks were then placed in an incubator (25 °C) for nine days under 12/12 h = light/dark conditions with the light intensity 32 W/m2. To improve the efficiency of periphytic biofilm stimulation, the flasks were shaken two times every day (at 9:00 am and 5:00 pm, about 10 mins for each time) by hand. After nine days, the activated periphytic biofilms and the control periphytic biofilm were filtered and washed using sterile water until no Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ was observed under fluorescence microscopy. The washed periphytic biofilms were then used for the N removal experiment. The treatments and controls were conducted in triplicate. 2.3.2 Experiments for nitrogen removal by periphytic biofilms To investigate the functioning of periphytic biofilm in removing nitrogen from 7
high concentration wastewater after activation by UCPs (Pr and Pr-Li), nitrogen removal experiments were conducted in a simulated periphytic biofilm wastewater treatment system designed according to our previous study (Liu et al., 2018; Zhu et al., 2018b). The activated and control periphytic biofilms (8.0 g, wet weight) were individually added into glass tanks with 200 mL of the high concentration wastewater [NaNO3-N (20 mg/L), NH4Cl-N (20 mg/L), KH2PO4-P (20 mg/L), C6H12O6 (5 mg/L), other compositions are the same as WC medium]. The volume ratio of wastewater to periphytic biofilm was 5:1. The glass tanks were placed in an incubator with light intensity of 32 W/m2 (provided by fluorescent lamps) (UV = 0 W/m2), a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. The removal experiment was conducted for 14 d. Thirty mL of water was extracted from 2 cm under the water surface using a syringe to detect nitrogen concentration every day. This was replaced with 30 mL fresh of wastewater injected into the tanks. To explore the ability of the stimulated periphytic biofilms to remove nitrogen from surface water that generally has low N concentration, the nitrogen removal experiment was repeated with surface water collected from Xuanwu Lake (Nanjing, China) in lieu of the high concentration wastewater mentioned above. The properties of the lake water were the same as those for culturing periphytic biofilm mentioned above. The experimental conditions and the water sampling technique were the same as for the high nitrogen concentration experiment. Both high and low nitrogen experiments were performed in triplicate. 2.4 Sample analysis X-ray diffraction (XRD) analysis was carried out on a Siemens D5005 X-ray diffractometer with Cu Kα radiation (λ = 1.54056 nm) at a power of 40 keV × 30 mA. 8
The XRD data were recorded with 2θ varying from 20° to 60° at counting time of 10 s and a scanning mode with a step size of 0.02°. The upconversion luminescence of UCPs was tested by a three-dimensional fluorescence spectrometer (F-7000, Japan). The parameters were selected as 1 nm of the slit, 450 nm of the excitation wavelength, 370 nm of the optical filters. Transmission electron microscope (JEM-1400PLUS, JEOL) was used to observe the cell structure of periphytic biofilm. Surface morphology, integral structure and element distribution on the surface of periphytic biofilm were observed by scanning electron microscopy (SEM, SU3500, Japan) and energy dispersive spectrometry (EDS, TM3000, Japan). Biolog E coplatesTM (Biolog, Hayward, USA) analysis was conducted to evaluate the carbon metabolic activity of periphytic biofilm. The detailed process is described in a previous study (More et al., 2012). The microbial structure and compositions were determined by phospholipid linked fatty acid (PLFA) profiling following the study of Zelles (1999). Briefly, 0.2 g of freeze-dried periphytic biofilm was extracted with a chloroform-methanol-citrate butter mixture. For each sample, the content of individual fatty acids was expressed as nmol FA g-1 dry periphytic biofilm biomass. The phospholipids were subjected to mild-alkali methanolysis and the resulting fatty acid methyl esters were separated by gas chromatography (Campbell et al., 2008). The PLFAs 18:3ω3c and 20:5ω3c, 20:2ω6 20:3ω6 and 20:4ω6, 18:2ω6 18:3ω6c and 18:3ω3c, 16:0 17:0 and 18:0, 16:1ω5c 16:1ω7c 18:1ω5c and 18:1ω7c were selected as indicators of cyanobacteria, protozoan biomass, fungal biomass, Gram +ve, Gram –ve , respectively (Boschker et al., 2005). The nitrogen in the wastewater and water samples were determined using a flow injection analyzer (SEALAA3, German).
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2.5 Data analysis All analyses were performed in triplicate. The results are presented as mean ± 1 standard deviation (SD). The 96 h Biolog data of the carbon sources utilized and correlations of fatty acids data were conducted using Principal Component Analysis (PCA) and Canonical Analysis (CA) in Canoco 5. The bar charts of nitrogen removal and XRD data were drawn by OriginPro 2016 (OriginLab Company). The comparison of nitrogen removal after 14 d operation and PLFA data between the treatment and the control were analyzed using One-way ANOVA. Variance partitioning analyses using the varPart function in the vegan package were calculated by R software (R 3.3.1). Statistical significance was set at p = 0.05 for all analyses. 3. Results and discussion 3.1 Characteristics of UCPs doped with Pr3+ and Pr3+-Li+ As shown in the XRD patterns, the samples of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ have similar crystal structures. The two curves, a and b presented a strong peak when 2θ = 30.7°, which is consistent with the standard pattern of yttrium (JCPDS no. 41-0004). The crystal sizes of the Y2SiO5 doped with Pr3+ and Pr3+ + Li+ at 2θ angle 30.7° are 332 nm and 337 nm, respectively. The upconversion luminescence emission spectra of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ were similar. Under 450 nm excited wavelength, emissions of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ from this band produce a broad spectrum of UV radiation, including the UVC range (220-280 nm) which has a negative effect on microorganisms such as Escherischia coli (Benabbou et al., 2007). It is worth noticing that the UVC emission was enhanced by addition of lithium-ions, which shows the structural effects of improved upconversion (Liang et al., 2009; Liang et al., 2011). 10
Compared to upconversion materials synthesized in previous studies, the Pr3+ doped Y2SiO5 material (i.e. Y2SiO5: Pr3+) used in the present study has similar crystal structure and doping concentration, which achieved a high degradation performance of wastewater (Yang et al., 2013). The doping of Li+ strongly affected the crystal transition of Y2SiO5: Pr3+ resulting in high luminance intensity and upconversion efficiency (Yang et al., 2015). 3.2 UCPs distribution in the periphytic biofilms SEM images exhibit the morphological alterations of periphytic biofilm when exposed to UCPs. The Pr and Pr-Li treated periphytic biofilms were observed to be more compact than the control. Microbial communities of Pr-Li treated periphytic biofilm are more abundant than control and Pr treated periphytic biofilm. To detect the distribution of UCPs on the surface of periphytic biofilms, EDS analyses were carried out based on the SEM images. There were significant differences in trivalent praseodymium ion distributions on the surface of periphytic biofilms between UCPs treatment and control periphytic biofilms. In the control samples, there were no promethium ions distributed on the surface of periphytic biofilm, while promethium ions of 0.15% (Weight%) and 0.29% were detected in Pr treated and Pr-Li treated periphytic biofilm, respectively. These results reveal that some of the UCPs can be distributed on the surface of Pr and Pr-Li treated periphytic biofilms. 3.3 Nitrogen removal by periphytic biofilm stimulated The average nitrogen removal rates in both Pr and Pr-Li treated periphytic biofilms (58.6 % and 61.4%, respectively) in the high concentration wastewater treatment system were significantly higher than that in the control (p < 0.01, Fig. 1A). 11
In the low concentration lake water, the average nitrogen removal rates in both Pr and Pr-Li treated periphytic biofilms were 95.1% and 95.9%, respectively, which were again significantly higher than the control (p < 0.05, Fig. 1B). These results indicate that the periphytic biofilm stimulated by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ was capable of augmenting nitrogen removal from both high concentration wastewater and low concentration surface water. The enhanced nitrogen removal by Y2SiO5: Pr3+, Li+ was slightly higher than Y2SiO5: Pr3+, but the difference was not significant (p > 0.05). 3.4 Microbial community structures of periphytic biofilms According to PLFA analysis, the specific microbial community compositions were obviously different in the control and treatments, as shown in Fig. 2A. Specifically, in the control, the community was dominated by cyanobacteria (M. aeruginosa), attached protozoa and bacterium (e.g., Bacillus stearothermophilus and Actinomycetes sp.). However, the main communities in the Y2SiO5: Pr3+ treated periphytic biofilm, included bacteria (e.g., Thermocrinis rubber), blue green bacteria and cyanobacteria. Cyanobacteria (e.g., Microcystis flos-aquae), Gram +ve bacteria and fungi dominated in the Y2SiO5: Pr3+, Li+ treated periphytic biofilm. These results indicate that the community structure of the periphytic biofilms had been changed to different degrees by the UCPs. Pr-Li treated periphytic biofilm had higher microbial and cyanobacterial biomass (dominated by M. flos-aquae) than Pr treated periphytic biofilm. This is because UCP-treated periphytic biofilm with Li doped UCPs grow with an inert shell which is more competitive to UCPs (Ding et al., 2015). The UCPs with Li+ slightly enhanced the upconversion emission intensity (Bai et al., 2008), and the community showed 12
increased acclimation. Furthermore, single Pr3+ doped UCPs directly absorb light, so quenching could easily occur when the ion concentrations of the dopant and the illuminant demand are high (Balda et al., 1999). Therefore, light conversion efficiency of Pr-Li treated periphytic biofilm increased with significant effects on microorganisms, resulting in a higher proportion of cyanobacteria and bacteria. Metagenomic sequencing was used to reflect the community changes in prokaryotic (16s rRNA) and eukaryotic (18s rRNA) microorganisms in periphytic biofilm (Fig. 3). Cyanobacteria contributed 30% in the control which was lower than in Pr and Pr-Li treated periphytic biofilm (39% and 34%, respectively) (Fig. 3A). This result corresponded with the fatty acid data. The contribution of Bacteroidetes (42%) in the control was higher than in Pr and Pr-Li treated periphytic biofilm (33% and 40%, respectively). UCPs could have negative impacts on gram-negative bacteria. According to the 18S rRNA results (Fig. 3B), the contribution of SAR was 40% in the control, lower than Pr treated periphytic biofilms (41%), but higher than Pr-Li treated periphytic biofilms (31%). The contribution of Amoebozoa was 14% in the control, 18% in the Pr treated periphytic biofilms and 15% in Pr-Li treated periphytic biofilms. This result showed that Amoebozoa acclimated well to UCPs. Similar to previous studies examining the impacts of UCPs on single communities, such as bacterial and viral communities (Cates et al., 2014; Chen et al., 2014; Lim et al., 2012), this study demonstrated that UCPs doped with Pr 3+ or codoped with Pr3+ and Li+ could negatively affect sensitive individual cells, such as M. aeruginosa cells, through damaging single cell structures (Cates & Kim, 2015). These impacts successfully inactivated the “ineffective” members in the periphytic biofilm and led to the activation of some “dormant” communities such as T. rubber in Pr treated periphytic biofilm and M. flos-aquae in Pr-Li treated periphytic biofilms, 13
shifting the periphytic biofilm community composition and structure. After stimulation by UCPs, the ability of periphytic biofilm to remove N was improved significantly. The process of N removal by periphytic biofilms has been described in previous studies (Wu et al., 2018). In this study, the change in community composition of periphytic biofilms induced by upconversion fluorescence and subsequent influence on N removal capacity were investigated. When visible light is converted to ultraviolet light by UCPs, it causes stress in surrounding sensitive microorganisms, which changes the community structure in the biofilm. Meanwhile, some functional populations which could remove N might develop due to their resistance to ultraviolet light (Cates et al., 2014). Another reason for the improved N removal could be that the generation of UV light can cause EPS accumulation for periphytic biofilms which often enhances the denitrification process as an electron donor and electric medium (Zhu et al., 2018a). 3.5 Carbon source metabolic capacities of periphytic biofilms The carbon source utilization in UCP-treated periphytic biofilm was distinct from the control (Fig. 2B). The periphytic biofilm in the control favored utilization of itaconic acid, D-cellobiose, 4-hydroxybenzoic acid, D-galactonic g-lactone acid and α-ketobutric acid. The Pr treated periphytic biofilm utilized β-methyl-D-glucoside, L-serine, galacturonic acid and L-arginine. The periphytic biofilm stimulated by Y2SiO5: Pr3+, Li+ utilized N-acetyl-D-gucosamine, D-mannitol and glucose-1-phosphate.
These imply that the control and UCP-treated periphytic
biofilm grew based on different carbon sources, and further imply that the periphytic biofilm had already acclimated to the effects of activation by Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+. 14
3.6 Synergic action of cyanobacteria, bacteria and metabolic capacity To select the primary factors affecting nitrogen removal, variance partitioning analyses (VPA) based on cyanobacterial biomass (represented by cyanobacterial PLFA), Gram +ve/Gram –ve bacteria, carbon source metabolic capacity (represented by AWCD) and nitrogen removal rate were conducted (Fig. 4). In contrast to the control, the contribution of cyanobacterial biomass in the Pr treated periphytic biofilm to nitrogen removal significantly decreased from 0.14 to 0.06 (p < 0.05) while the contribution in the Pr-Li treated periphytic biofilm significantly increased from 0.14 to 0.31 (p < 0.05). The contributions of Gram +ve/Gram –ve bacteria in both Pr and Pr-Li treated periphytic biofilms significantly increased from 0.13 to 0.20 and 0.13 to 0.32, respectively, compared to the control (p < 0.05 for both analyses). No contribution of AWCD was found in the control, while the contributions in the Pr and Pr-Li treated periphytic biofilms were relatively high, about 15 and 20%, respectively. These results indicate that cyanobacterial biomass, Gram +ve/Gram –ve bacteria and carbon source metabolic capacity played important roles in enhancing nitrogen removal in the two treated periphytic biofilms. It is noteworthy that the synergic contribution of cyanobacteria biomass, Gram +ve/Gram –ve bacteria and AWCD in the control was not significant (value < 0) while the values of synergic action in both Pr (0.42) and Pr-Li treated (0.84) periphytic biofilms were positive. Moreover, the contributions of these three parameters in both Pr and Pr-Li treated periphytic biofilm were significantly higher than that of the contribution of cyanobacterial biomass, Gram +ve/Gram –ve bacteria or AWCD alone. This indicates that the synergic action of PLFAcyanobacteria, Gram +ve/Gram –ve bacteria and AWCD played an important role in nitrogen removal. The cyanobacterial biomass (represented by PLFA) in the Pr-Li treated 15
periphytic biofilm increased while the biomass in the Pr treated periphytic biofilm decreased. This result was consistent with the changes in the contribution of cyanobacterial biomass to nitrogen removal. Both periphytic biofilms presented good acclimation in terms of carbon source metabolic versatility during the activation of Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+. These changes in the carbon metabolic capacity of the periphytic biofilms also concurred with the AWCD changes. These results further indicate that cyanobacterial biomass and carbon metabolic activity played an important role in augmenting nitrogen removal by periphytic biofilm. Under stress conditions microbial communities, such as bacterial E. coli, present stronger plasticity and resistance for survival, as a result achieving high activity (Davies & Davies, 2010; Shade et al., 2012). In this study, the exposure of periphytic biofilms to Y2SiO5: Pr3+ and Y2SiO5: Pr3+, Li+ created unfavorable conditions, in turn leading to the formation of more robust periphytic biofilm with high carbon metabolism. The Gram +ve bacteria may be capable of augmenting strength and resistance to stress (Holzinger & Karsten, 2013; Wang et al., 2011). The UCPs stimulated changes in the Gram +ve/Gram –ve bacterial ratio, but also the kept the ratio at a positive level. The optimized ratio of Gram +ve/Gram –ve bacteria then promoted nitrogen removal by the treated periphytic biofilms. As mentioned, the periphytic biofilm is a type of microbial aggregate (Wu, 2016), whose functionality, such as nitrogen removal, is related to composition changes (Wu et al., 2011). Thus, the synergic action of multiple parameters, such as cyanobacterial biomass, Gram + ve/Gram –ve bactiera and AWCD to nitrogen removal was considered. The high contributions of the treated periphytic biofilms demonstrated that the synergic action of these three parameters was mainly responsible for the increased nitrogen removal. This result demonstrates that the stimulation by Y2SiO5: 16
Pr3+ and Y2SiO5: Pr3+, Li+ promoted the synergic action of the periphytic biofilms. 4. Conclusions In this study, the investigation of the multi-community optimization of periphytic biofilm by upconversion luminescence of UCPs advances the studies of microbial inactivation from one-species inactivation to multi-species community design. The strategy proposed in this study proved the feasibility of using upconversion materials to design robust microbial aggregates (i.e., periphytic biofilm) for nitrogen removal. The results obtained also invoke re-thinking of the type of microbial community (single- or multi-species microbial aggregates) that should be employed when evaluating the use of upconversion materials to inactivate microorganisms.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31772396, 41422111), the State Key Development Program for Basic Research of China (2015CB158200) and the Natural Science Foundation of Jiangsu Province (BK20150066, BK20181511). This work was also supported by Chinese Academy of Sciences Interdisciplinary Innovation Team. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version.
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Figure captions
Fig. 1 Nitrogen removal rates by periphytic biofilms from (A) high concentration wastewater and (B) low high concentration lake water. The influent and effluent nitrate concentrations were the average means of running for 14 d.
Fig. 2 A. The PLFA canonical analysis (CA) of the microbial communities in periphytic biofilms; B. The principal component analysis (PCA) of different types of carbon source utilization in periphytic biofilms. Arrows of different colors represent different carbon sources: black, purple, red, orange, blue and green represent carbohydrates, polymers, phenolic acids, carboxylic acids, amines and amino acids, respectively. 1-31 represents β-methyl-D-glucoside, D-galactonic g-lactone acid, L-arginine, Pyruvic acid methyl ester, D-xylose, Galacturonic acid, l-asparagine, Tween 40, i-erytritol, 2-hydroxybenzoic acid, L-serine, Tween 80, D-mannitol, 4-hydroxybenzoic acid, l-phenylalanine, α-cyclodextrine, N-acetyl-d-glucosamine, γ-hydroxybutiric acid, L-threonine, Glycogen, D-glucosaminic acid, Itaconic acid, Glycyl-l-glutamic acid, D-cellobiose, Glucose-1-phosphate, α-ketobutiric acid, Phenylethylamine, α-lactose, D-l-α-Glycerol-1-phosphate, D-malic acid and Putrescine.
Fig. 3 (A) 16S rRNA and (B) 18S rRNA gene sequencing for microorganism composition of periphytic biofilms stimulated by upconversion materials. SAR includes three species, Heterokonts, Alveolates and Rhizaria.
Fig. 4 Variance partitioning analyses based on PLFAalga (X1), Gram +ve/Gram –ve bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and TN removal in the 21
(A) Control, (B) Pr treated periphytic biofilm and (C) Pr-Li treated periphytic biofilm (n = 5. Residual means the difference between the observed and predicted values (fitted value). (No result shown when values < 0).
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Fig. 1
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Fig. 2 A
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Fig. 3 A
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Fig. 4
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Highlights：
UCPs were first used to stimulate nitrogen removal of periphytic biofilms.
UCPs optimized the microbial community structure of periphytic biofilms.
Periphytic biofilms simulated by UCPs could adapt to nitrogen fluctuation.
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