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Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+
Accepted Manuscript Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+ Yan Zhu, Jianhong Zhang, Ningyuan Zhu, Jun Tang, Junzhuo Liu, Pengfei Sun, Yonghong Wu, Po Keung. Wong PII: DOI: Reference:
S0960-8524(17)31114-8 http://dx.doi.org/10.1016/j.biortech.2017.07.027 BITE 18447
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
12 May 2017 2 July 2017 6 July 2017
Please cite this article as: Zhu, Y., Zhang, J., Zhu, N., Tang, J., Liu, J., Sun, P., Wu, Y., Wong, P.K., Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.027
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Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+
Yan Zhua,c, Jianhong Zhangb, Ningyuan Zhua,c, Jun Tanga,c, Junzhuo Liua, Pengfei Suna,
a
Yonghong Wua*, Po Keung Wongd
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences,
Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China b
Resources & Environment Business Dept., International Engineering Consulting
Corporation, Beijing 100048, China c
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China d
School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong
Kong SAR, China
*Corresponding author: E-mail: [email protected] Tel.: (+86)-25-8688 1330 Fax: (+86)-25-8688 1000
Graphic abstract
Abstract Upconversion phosphors (UCPs) can convert visible light into luminescence, such as UV, which can regulate the growth of microbes. Based on these fundamentals, the community composition of periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ was proposed to augment the removal of phosphorus (P) and copper (Cu). Results showed that the biofilms with community composition optimized by UCPs doped with Pr3+-Li+ had high P and Cu2+ removal rates. This was partly due to overall bacterial and algal abundance and biomass increases. The synergistic actions of algae, bacterial biomass and carbon metabolic capacity in the Pr-Li stimulated biofilms facilitated the removal of P and Cu2+. The results show that the stimulation of periphytic biofilms by lanthanide-doped UCPs is a promising approach for augmenting P and Cu2+ removal.
Keywords: Periphytic biofilm; Y2SiO5 ; Lanthanide-doped upconversion phosphors; Phosphorus; Copper
1. Introduction In recent years, inorganic compounds such as phosphorous and heavy metals have been increasingly detected in waters (Falas et al., 2016). Many promising methods have been developed to remove these inorganic compounds, such as chemical precipitation (Quan et al., 2010), ultraviolet irradiation (Sousa et al., 2017), plant and microbial methods (Ahluwalia & Goyal, 2007), and electrochemical methods (Ghafari et al., 2008). Among these methods, microbial methods have relatively high environmental safety and are low-cost. However, single species microbial communities rarely dominate natural environments, while the composition of microbial aggregates is difficult to manipulate in practice. These factors lead to variable efficiency for contaminant removal using microbial methods (Asadi et al., 2012; Lee et al., 2009). Recently, new technology based on upconversion materials doped with lanthanides has attracted research attention. Upconversion refers to nonlinear optical processes characterized by the successive absorption of two or more pump photons though long-lived energy states. This is followed by sending the output radiation at a shorter wavelength than the pump wavelength by an anti-Stokes process (Han et al., 2014). Upconversion can be induced by a low power (1–103 W cm-2) continuous wave laser, as opposed to a costly high-intensity (106–109 W cm-2) pulse laser source for the generation of a simultaneous two-photon process (Wang & Liu, 2009). Upconversion materials with lanthanide-doped crystals offer an attractive optical labeling technique in biological studies (Suyver et al., 2005). Doping is also of great importance in stabilizing a particular crystalline phase (Feng et al., 2006), modifying electronic attributes (Alivisatos, 1996), modulating magnetism (Wernsdorfer et al., 1997) and tuning emission properties (Wang et al., 2010).
Upconversion materials have been used in the fields of organic compounds (e.g. Congo red) biodegradation, biological assays and medical imaging from the late 1990s (DaCosta et al., 2014; Lin et al., 2012). Given the insights already attained in material science in relation to the design of luminescence and surface coatings, there is good potential for the use of upconversion materials in bioanalytical applications (DaCosta et al., 2014). Some research has closely examined photodynamic inactivation of microorganisms or viruses using upconversion materials (Lim et al., 2012; Wang et al., 2012), but little is known about the effects of these materials on multi-species microbial aggregates. This study proposes a novel use of upconversion materials to stimulate microbial aggregates to augment contaminant removal. Periphytic biofilms, typical microbial aggregates, are capable of remediating polluted wastewater, including organic matter and metals (Wu et al., 2014). Extracellular polymer substances (EPS) are crucial components of periphytic biofilms and are a complex high-molecular-weight mixture of polymers excreted by microorganisms, products of cell lysis and adsorbed organic matter from wastewater (Sheng et al., 2010). Functional groups of periphytic biofilm EPS, such as proteins and carbohydrates, can adsorb heavy metals (Sheng et al., 2010) and as a result, the EPS of periphytic biofilms has a strong affinity for heavy metal ions (Yang et al., 2016b). Periphytic biofilms abundant in algal biomass have also been a key component for phosphorus removal from wastewater (Sukačová et al., 2015). Periphytic biofilms play several roles in removing P from water, including P uptake and deposition, and filtering particulate P from water (Wu et al., 2011). The objectives of this study are to (i) determine the removal rate of P and Cu2+ by periphytic biofilms stimulated by UCPs doped with Pr3+-Li+, and (ii) investigate
the changes in metabolic diversity, community composition and structure of the stimulated periphytic biofilm. The results are expected to offer a promising method to enhance P and Cu removal by periphytic biofilms, and provide an insight into the responses of periphytic metabolic versatility, composition and structure to UCP exposure.
2. Materials and methods
2.1 Periphytic biofilms culture Glass bottles (20 L) sterilized by 0.1 M HCl solution for 30 min were filled with nutrient-rich water from Xuanwu Lake, Nanjing, China containing microbial sources of periphytic biofilm. The properties of the water were: pH 8, total nitrogen (TN) 2.18 mg L-1, NO3--N 0.43 mg L-1, NH4+-N 0.07 mg L-1 and PO43--P 0.24 mg L-1. Industrial Soft Carriers were used as substrates. The substrates were immersed in the water with a light intensity of 2500 Lux under 12/12 h light/dark cycle. The periphytic biofilm automatically form on the surfaces of substrates after 3 days. The periphytic biofilms were incubated at 28 ± 1 °C until the formation of dense dark green periphytic biofilms. Dense periphytic biofilms (thickness ~ 0.1-0.5 cm) were collected for the following experiments.
2.2 Preparation of lanthanide doped UCPs
All chemicals were analytical pure grade and purchased from Sigma Aldrich. The containers were sterilized with ultraviolet irradiation before utilization. The syntheses of UCPs doped with Pr3+ and Li+ (Y2SiO5: Pr3+-Li+) were conducted according to the methods developed by Cates et al. (2011).
Briefly, the firing process of the upconversion materials was carried out using a sol-gel method (Brinker & Scherer, 2013). Pr3+ and Li+ ion doped upconversion materials were prepared as follows: 5.3 g Y2O3 and nitric acid (trace metal grade) were boiled in a ratio of 1:1 (weight ratio) to make the yttrium nitrate solution. The solution was dissolved in ethanol (200 proof) and water, which was evaporated to constant weight under 104 °C. 1 % (mol) Pr and Li solutions were added to nitric acid as dopants, forming aqueous stock solutions of Pr(NO3)3·6H2O and LiNO3·6H2O. Tetraethoxysilane (TEOS) was added as a silicon source and gelling agent and stirred to produce sol gel. Dopant concentrations were adjusted to optimize upconversion efficiency, which corresponded to the doping concentration of 1% Pr3+ and Li+ for samples with both elements. 2.3 Pollutant removal experiment First, a stimulation experiment to optimize periphytic biofilm community composition and metabolic activity was performed. Briefly, all containers were sterilized by ultraviolet radiation for 30 min. 40 g L-1 of UCPs doped with Pr3+-Li+ were individually added into containers containing 8.0 g periphytic biofilm (wet weight) and 150 mL Woods Hole culture medium (WC medium) (Shangguan et al., 2015). Containers without the addition of UCPs were employed as controls. The UCP treatments and the controls were placed in two separate incubators with the same incubator conditons: temperature 25 ± 1 °C under 12/12 h light/dark cycle. During the experiment, no UV and UVC were detected in the control. All treatments and controls were conducted in triplicate. On day 9, the stimulated periphytic biofilms were collected for the next experiments. The periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ are referred to as Pr-Li stimulated periphytic biofilms.
To avoid the inclusion of UCPs doped with Pr3+-Li+ into the wastewater treatment system, the stimulated 8.0 g periphytic biofilms (wet weight) were washed 3 times using 150 mL water (each time). The periphytic biofilms were then soaked in 150 mL water for 2 hours. After soaking, the stimulated periphytic biofilms were used for phosphorus and copper removal. First, 8.0 g periphytic biofilms (wet weight) were put into 200 mL of wastewater containing phosphorus and copper. The composition of the wastewater was: NaNO3-N (20 mg L-1), NH4Cl-N (20 mg L-1), CaCl2·2H2O (36.76 mg L-1), MgSO4·7H2O (36.97 mg L-1), NaHCO3 (12.6 mg L-1), Na2SiO3·9H2O (28.42 mg L-1), K2HPO4·3H2O-P (20 mg L-1), KH2PO4-P (20 mg L-1), H3BO3 (24 mg L-1), C6H12O6 (5 mg L-1), and 1 mL trace elements solution [Na2EDTA·2H2O (4.36 mg L-1), FeCl3·6H2O (3.15 mg L-1), CuSO4·5H2O (2.5 mg L-1), ZnSO4·7H2O (22 µg L-1), CoCl2·6H2O (10 µg L-1), MnCl2·4H2O (180 µg L-1 ), Na2MoO4·2H2O (6.3 µg L-1), Na3VO4 (18 µg L-1), vitamin B12 (135 µg L-1), thiamine (335 µg L-1), and biotin (25 mg L-1)]. The containers containing stimulated and non-stimulated (control) periphytic biofilms were placed in an incubator with light intensity of 2500 lux, a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. The indoor simulation experiments were carried out under flowing water circulation conditions. Specifically, when a 30 mL water sample was removed from 2 cm under the water surface, 30 mL of fresh wastewater was injected. Water samples were used for determining the concentrations of P and Cu2+. The experiment was performed in triplicate.
2.4 Sample analysis X-ray diffraction (XRD), a transmission electron microscope (TEM) and a fluorescence spectrometer were employed to examine the UCPs crystal structure and
upconversion luminescence. XRD analysis was done using a Siemens D5005 X-ray diffractometer with Cu Kα radiation (λ = 1.54056 nm) at a power of 40 keV × 30 mA. The XRD data were recorded with 2θ variation from 20° to 60° using a counting time of 10 s and a scanning mode with a step size of 0.02°. The cell structures of periphytic biofilms were observed by transmission electron microscope (JEM-1400PLUS, JEOL). The upconversion luminescence of UCPs was tested using a fluorescence spectrometer with an argon laser (FL3-TCSPC, Horiba Jobin Yvon Corporation, France). The exciting parameters were selected as 450 nm of the excitation wavelength, 370 nm of the optical filters, and 1 nm of the slit. The reflectivity of UCPs was measured using a spectrophotometer (CM-2300D, Japan). Biolog EcoplatesTM (Biolog, Hayward, USA) analysis was conducted to evaluate the carbon metabolic activity of periphytic biofilms. The microbial composition and structures were determined by phospholipid linked fatty acid (PLFA) profiling according to the studies performed by Gómez-Brandón et al. (2010). Briefly, 0.2 g of freeze-dried periphytic biofilms were 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 biofilms biomass. Hydrochloric acid was used as an acid catalyst for preparation of fatty acid methyl esters for gas chromatography (Agilent 6850, USA) (Berchmans & Hirata, 2008). The PLFA 18:3ω3c and 20:5ω3c were selected as indicators of algae biomass including cyanobacteria. The PLFA 20:2ω6, 20:3ω6 and 20:4ω6 were chosen as indicators of protozoa biomass, and 18:2ω6, 18:3ω6c and 18:3ω3c were chosen to represent fungi biomass. The PLFA 16:0, 17:0 and 18:0 were chosen to evaluate gram positive bacteria, and 16:1ω5c, 16:1ω7c, 18:1ω5c and 18:1ω7c were chosen as indicators of gram negative bacteria (Boschker et al., 2005). The P and Cu2+ in water samples were determined using a flow injection
analyzer (SEALAA3, Germany) and inductively coupled plasma mass spectrometry (XSERIES 2 ICP-MS, USA).
2.5 Data analysis
Results are presented as mean ± 1 standard deviation (SD) of three replicates. The 96 h biolog data of the carbon sources utilized and correlations of fatty acids data were analyzed using Principal Component Analysis (PCA) and Canonical Analysis (CA) by Canoco 5. The bar charts of P and Cu2+ and XRD data were drawn by OriginPro 2016 (OriginLab Company). The comparisons of P and Cu2+ removal and PLFA data between the treatment and the control were analyzed using One-way ANOVAs using SPSS 22.0. Variance partitioning analyses were calculated using the varPart function in the vegan package by R (R version 3.3.1). Statistical significance was set at p = 0.05 for all analyses.
3. Results and discussion
3.1 Y2SiO5 crystal structure characteristics
XRD was used to represent the crystal structure of yttrium silicate (Y2SiO5) doped with Pr3+-Li+. The curves presented a strong peak when 2θ = 30.7° in accordance with the standard pattern of Y2SiO5 reported in JCPDS no. 41-0004. The crystal size of the Y2SiO5 doped with Pr3+-Li+ is 337 nm at 2θ angle 30.7°. Particle crystallite size and crystal phase affect upconversion luminescence processes and upconversion efficiency (Luo et al., 2016).
A fraction of UVC radiation with 450 nm excitation was detected during the upconversion luminescent emission of Y2SiO5: Pr3+-Li+. Stanton et al. (2012) reported
that lithium doping of Y2O3 led to enhanced X-ray emission intensities that featured slightly increased sizes and crystallinity. Pr-Li stimulated periphytic biofilm also showed incremental reflectivity with the increase in incident wavelengths. It followed that UCPs absorbed more energy in the ultraviolet wavelength. However, increasing the absorbed energy from the excitation band is needed to improve the performance of upconversion materials. 3.2 P and Cu2+ removal by stimulated periphytic biofilms The P removal rate of Pr-Li stimulated periphytic biofilms (61%) was significantly higher than that of the control (p < 0.01) (Fig. 1). Cu2+ removal efficiencies by Pr-Li stimulated periphytic biofilm were 70%, which were significantly higher than the control (61%, p < 0.01). These results suggest that UCPs doped with Pr-Li enhanced P and Cu2+ removal by periphytic biofilm. This is because UCPs doped with Li grow with an inert shell and are more competitive than non-doped UCPs, enhancing upconversion emission intensity for the improvement of the nanoparticles' crystallinity and the distortion of the local symmetry (Ding et al., 2015). While energy transfer upconversion involves two neighboring ions, UCPs can generate efficient energy transfer upconversion by using the Pr doped system itself as the sensitizer and the neighboring Li+ as the activator, producing multiphoton accumulation (Chen et al., 2014). 3.3 Microbial cellular substructure of periphytic biofilms exposed to UCPs
Observations under the optical microscope showed that cyanobacteria were abundant in the periphytic biofilms. The microbial cell structures within the control and UCPs treated periphytic biofilms were examined by transmission electron microscopy. The cell structures were intact when periphytic biofilms grew naturally in
the controls. After the periphytic biofilms were stimulated by Y2SiO5: Pr3+-Li+, microbial cell structures were maintained overall, although cytoderm and cytoplasm separation was locally observed. These observations suggest that the stimulation of periphytic biofilms by Y2SiO5: Pr3+-Li+ had a negative effect on cell structure. UCPs doped with Pr3+-Li+ could damage single cell structures (Cates & Kim, 2015). Similar to previous studies examining the impacts of UCPs on single species communities, this study demonstrated that UCPs doped with Pr 3+-Li+ could negatively affect individual microorganisms by damaging single cell structures (Cates & Kim, 2015). These impacts might lead to shifts in periphytic biofilm community composition and structure. 3.4 PLFA profiling of microbial community structures
To investigate whether the microbial community composition and structure changed during exposure to upconversion materials, periphytic biofilm was examined using PLFA analysis. PLFA can be analyzed with multivariate procedures to determine statistically significant changes in periphytic biofilm structure and relative abundances of different types of microorganisms under different doping regimes (Boschker et al., 2005).
The total PLFA content and Gram +ve bacteria of the Pr-Li stimulated periphytic biofilm (451.49 nmol g -1, 181.16 nmol g -1, respectively), were significantly higher than in the control (380.82 nmol g-1, 151.99 nmol g-1, respectively) (p < 0.01 for both comparisons). Gram –ve and algae biomass in the Pr-Li stimulated periphytic biofilm (69.12 nmol g-1, 367.72 nmol g-1, respectively) were also higher than in the control (59.07 nmol g-1, 307.38 nmol g-1, respectively) (p < 0.01 for both comparisons). These results demonstrated that exposure to Y2SiO5: Pr3+-Li+ increased the microbial
biomass of the bacteria and algae in the periphytic biofilms. Y2SiO5: Pr3+-Li+ had a slight negative effect on fungal and protozoan biomass, leading to the proportion of these microorganisms in the Pr-Li stimulated periphytic biofilms decreasing from 1.3 to 1.0% for fungi and from 1.3 to 1.0% for protozoa (Table 1). 17:0, 18:0, 15:0 iso, 20:4ω6c, 16:0 10-methyl and 17:0 iso fatty acids were detected in the control periphytic biofilm, demonstrating that the microbial community was dominated by cyanobacteria (M. aeruginosa), bacteria (e.g. Bacillus stearothermophilus and Actinomycetes sp.) and attached protozoa. The 14:0, 18:1ω9c, 16:0 and 18:2ω6c in the Pr-Li stimulated periphytic biofilm, indicate that cyanobacteria (e.g. Microcystis flos-aquae), Gram +ve bacteria and fungi dominated the periphytic biofilm. These changes show that the microbial community structures of the periphytic biofilm had been changed by UCP stimulation. This result also implies that the reduced or absent microorganisms, such as M. aeruginosa, in the periphytic biofilm stimulated by Y2SiO5: Pr3+-Li+ were an “ineffective” component in removing P and Cu2+.
Periphytic biofilms harbor a stunningly diverse collection of microbial species competing for resources and space (Hibbing et al., 2010). Microorganisms can benefit from microbial competition when competition dampens cooperative networks and increases stability, and the final commensality is beneficial for all participating organisms (Foster & Bell, 2012). Under the stimulation of UCPs, the periphytic biofilms optimized their species diversity with complex microbial interactions including cooperation and competition, leading to improved community stability (Coyte et al., 2015). These stable periphytic biofilms had high capacity for eliminating inorganic pollutants. This is because highly stable microbial communities have high
contaminant removal performance, including inorganic pollutants such as P (Miura et al., 2007), and can improve raw water quality (Luo et al., 2013). 3.5 Carbon source utilization of periphytic biofilms
The correlations between the six main carbon source groups and (Principal Component 1) PC1 or (Principal Component 2) PC2 are shown in Table 2 (only carbon sources with correlation coefficients > 0.9 were selected). PC1 accounted for most of the variance (79.45%). The variance explained by PC2 (20.55%) related to a few types of carbon sources, such as carboxylic acids. For PC1, there were six carbon sources (belonging to three groups: carbohydrates, polymers, carboxylic acids) in the control, and six carbon sources (belonging to four groups: carbohydrates, polymers, carboxylic acids, amino acids) in the Pr-Li stimulated periphytic biofilm. These results indicate that microorganisms in Pr-Li stimulated periphytic biofilm utilized different carbon sources to sustain periphytic biofilm growth and metabolism; and suggest that the Pr-Li stimulated periphytic biofilm acclimated to the UCPs.
The metabolic capacity of periphytic biofilm stimulated by UCPs was investigated by calculating the average well color development (AWCD) (Guo et al., 2010). The carbon source metabolic capacities (represented by AWCD) of the periphytic biofilms stimulated by Y2SiO5: Pr3+-Li+ were significantly improved in contrast to the control (p < 0.05, Fig. 2). 3.6 Synergic actions of algae, bacteria biomass and carbon metabolic capacity in P and Cu2+ removal Periphytic biofilm exposed to UCPs doped with Pr3+-Li+ possessed relatively higher P and Cu2+ removal capacity. To select the primary parameters explaining P
removal, variance partitioning analyses (VPA) based on algal biomass (represented by PLFAalga), bacterial biomass (PLFAbac), carbon source metabolic capacity (represented by AWCD) and P removal rate were conducted (Fig. 3). In contrast to the control (0.06), Pr-Li stimulated periphytic biofilm had higher interaction of algae, bacteria and AWCD (0.77). The contribution of algal biomass and AWCD to Pr-Li stimulated periphytic biofilm for P removal significantly increased from 0.09 to 0.11 (p < 0.05). The carbon source metabolic capacity (AWCD contribution) for Pr-Li stimulated periphytic biofilm was also related to P removal rate. These results indicated that the synergistic action of algal biomass, bacterial biomass and carbon source metabolic capacity played important roles in enhancing P removal by Pr-Li stimulated periphytic biofilms.
The total contribution of the three parameters of Pr-Li stimulated periphytic biofilm to Cu2+ removal slightly increased from 0.20 to 0.21 (Fig. 4). The simulated results were consistent with Cu2+ removal efficiencies with Pr-Li stimulated periphytic biofilm. These results indicated that algal and bacterial biomass and AWCD enhanced segmental Cu2+ elimination. This means that there was no significant enhancement of Cu2+ removal efficiency by the synergistic action of algae, bacteria and carbon source metabolic capacities. Ahluwalia and Goyal (2007) reported that the removal of heavy metals from aqueous solution using inactive and dead biomass (non-living biomass of algae, aquatic ferns and seaweeds, waste biomass) as potential biosorbents was an innovative and alternative technology for removing inorganic compounds and heavy metals such as Cu2+. As a chelating agent, EPS can combine with and form a complex with Cu2+, thus removing Cu2+ from wastewater (Yang et al., 2016a). The combination of Cu2+ and EPS is likely to have resulted in the changes in Cu speciation, thereby varying the toxicity of Cu to microorganisms (Wu et al., 2017).
Accordingly, the Cu2+ removal might also be attributed to the inactive and dead biomass and EPS, but further tests are needed.
In nature, the community composition of periphytic biofilm is more diverse than the samples in this study. This diversity provides sufficient microbial sources for the optimization of the microbial structure of periphytic biofilm because different microorganisms have different sensitivities to irradiation (Wu et al., 2014). For example, some microorganisms such as Escherichia coli, MS-2 and T7 (Bowker et al., 2011) are inactivated in the presence of UV and some microorganisms such as Actinobacteria (Warnecke et al., 2005) and Gammaproteobacteria (Santos et al., 2012) grow well in the presence of UV, at different rates depending on the type of microorganisms and growth conditions. This natural diversity and adaptability in conjunction with the results of this study, suggest that the stimulation of natural periphytic biofilm by upconversion phosphors doped with Pr 3+-Li+ is a potential approach to optimize microbial structure, leading to the reduction or disappearance of “ineffective” periphyton community members and the improvement of P and Cu2+ removal.
4.
Conclusions
Ultraviolet irradiation emitted by UCPs with the emission of visible light had a negative effect on a small proportion of “ineffective” periphytic biofilms components (such as M. aeruginosa) for Cu2+ and P removal. Microbial community structures doped with Y2SiO5: Pr3+-Li+ changed as demonstrated by their enhanced carbon utilization and metabolism capacities. The optimized microbial periphytic biofilm community increased P and Cu2+ removal. This was mainly due to the synergistic action of algal and bacterial biomass, and metabolic capacity. These results provide an
insight into how microbial aggregates acclimate to the luminescence conditions caused by the excitement of UCPs and improve their capacity to remove impurities from wastewater.
Acknowledgements This work was supported by the National Natural Science Foundation of China (41422111), the State Key Development Program for Basic Research of China (2015CB158200) and the Natural Science Foundation of Jiangsu Province China (BK20150066). This work was also supported by Youth Innovation Promotion Association, Chinese Academy of Sciences (2014269).
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Figure captions Fig. 1 The removal rate of P and Cu2+ by periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ and the control. * indicates significant differences in the removal rates between treatment and control (t-test, p < 0.05).
Fig. 2 The carbon metabolic capacity of periphytic biofilms, represented by the average well color development (AWCD). * refers to significant difference between the control and the treatment (t-test, p < 0.05).
Fig. 3 Variance partitioning analyses based on PLFAalga (X1), bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and P removal in the (B) Control, (C) Pr-Li stimulated periphytic biofilm (no result shown when values are approximately equal to zero or below zero). Each diagram represents the biological variation partitioned into the relative effects of each factor or a combination of factors. The corners of the triangle represent the variation explained by the factor alone. The sides of the triangles represent interactions of any two factors, and the middle of the triangles represent interactions of all three factors.
Fig. 4 Variance partitioning analyses based on PLFAalga (X1), bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and Cu2+ removal in the (B) Control, (C) Pr-Li stimulated periphytic biofilm (no result shown when values are approximately equal to zero or below zero).
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Table 1 The total PLFA (PLFAtot), protozoa (PLFApro), fungi (PLFAfun), algae (PLFAalga) (nmol g-1 dry biomass), Gram-positive (Gram +ve) bacteria (PLFApos) and Gram negative (Gram –ve) bacteria (PLFAneg) (nmol g-1) and their proportions in the control periphytic biofilm and the Pr-Li stimulated periphytic biofilm. PLFApro
PLFApos
PLFAneg
PLFAfun
PLFAalga
Gram +ve/Gram –ve
Treatments
PLFAtot
Control
380.82 ± 19.04 4.77 ± 0.24 151.99 ± 7.60 59.07 ± 2.95 4.78 ± 0.24 307.38 ± 15.37 2.57 ± 0.13
Pr-Li stimulated 451.49 ± 22.57 4.73 ± 0.24 181.16 ± 9.06 69.12 ± 3.46 4.73 ± 0.24 367.72 ± 18.38 2.62 ± 0.13
Table 2 Correlation coefficients between main carbon source groups and PC1 or PC2. Treatments Control
PC1 Carbohydrates
i-erytritol Glucose-1-phosphate α-lactose
Correlation coefficient 0.90 0.97 0.97
Stages Control
PC2 Carbohydrates Polymers
β-methyl-D-glucoside D-xylose Tween 80
Correlation coefficient 0.93 0.93 0.96
Polymers Carboxylic acids Pr-Li stimulated
Carbohydrates Polymers Carboxylic acids Amino acids
D-l-α-Glycerol-1-phosphat e Tween 40 Pyruvic acid methyl ester α-lactose α-cyclodextrine Pyruvic acid methyl ester L-arginine l-phenylalanine L-threonine
Glycogen
0.91 0.98 0.94 0.99 0.99 0.99 0.95 1.00 1.00
Phenolic acid Pr-Li stimulated
Carbohydrates Phenolic acid Carboxylic acids
2-hydroxybenzoic acid D-l-α-Glycerol-1-phosphate 4-hydroxybenzoic acid D-glucosaminic acid
0.93 1.00 0.94 0.91 0.98
Highlights · Pr-Li stimulated periphytic biofilm changed microbial community structure. · Pr-Li stimulated periphytic biofilm enhanced carbon utilization and metabolic capacity. · Synergic action of algae, bacteria biomasses and AWCD contributed to P and Cu 2+ removal.
S0960-8524(17)31114-8 http://dx.doi.org/10.1016/j.biortech.2017.07.027 BITE 18447
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
12 May 2017 2 July 2017 6 July 2017
Please cite this article as: Zhu, Y., Zhang, J., Zhu, N., Tang, J., Liu, J., Sun, P., Wu, Y., Wong, P.K., Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.027
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Phosphorus and Cu2+ removal by periphytic biofilm stimulated by upconversion phosphors doped with Pr3+-Li+
Yan Zhua,c, Jianhong Zhangb, Ningyuan Zhua,c, Jun Tanga,c, Junzhuo Liua, Pengfei Suna,
a
Yonghong Wua*, Po Keung Wongd
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Sciences,
Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China b
Resources & Environment Business Dept., International Engineering Consulting
Corporation, Beijing 100048, China c
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China d
School of Life Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong
Kong SAR, China
*Corresponding author: E-mail: [email protected] Tel.: (+86)-25-8688 1330 Fax: (+86)-25-8688 1000
Graphic abstract
Abstract Upconversion phosphors (UCPs) can convert visible light into luminescence, such as UV, which can regulate the growth of microbes. Based on these fundamentals, the community composition of periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ was proposed to augment the removal of phosphorus (P) and copper (Cu). Results showed that the biofilms with community composition optimized by UCPs doped with Pr3+-Li+ had high P and Cu2+ removal rates. This was partly due to overall bacterial and algal abundance and biomass increases. The synergistic actions of algae, bacterial biomass and carbon metabolic capacity in the Pr-Li stimulated biofilms facilitated the removal of P and Cu2+. The results show that the stimulation of periphytic biofilms by lanthanide-doped UCPs is a promising approach for augmenting P and Cu2+ removal.
Keywords: Periphytic biofilm; Y2SiO5 ; Lanthanide-doped upconversion phosphors; Phosphorus; Copper
1. Introduction In recent years, inorganic compounds such as phosphorous and heavy metals have been increasingly detected in waters (Falas et al., 2016). Many promising methods have been developed to remove these inorganic compounds, such as chemical precipitation (Quan et al., 2010), ultraviolet irradiation (Sousa et al., 2017), plant and microbial methods (Ahluwalia & Goyal, 2007), and electrochemical methods (Ghafari et al., 2008). Among these methods, microbial methods have relatively high environmental safety and are low-cost. However, single species microbial communities rarely dominate natural environments, while the composition of microbial aggregates is difficult to manipulate in practice. These factors lead to variable efficiency for contaminant removal using microbial methods (Asadi et al., 2012; Lee et al., 2009). Recently, new technology based on upconversion materials doped with lanthanides has attracted research attention. Upconversion refers to nonlinear optical processes characterized by the successive absorption of two or more pump photons though long-lived energy states. This is followed by sending the output radiation at a shorter wavelength than the pump wavelength by an anti-Stokes process (Han et al., 2014). Upconversion can be induced by a low power (1–103 W cm-2) continuous wave laser, as opposed to a costly high-intensity (106–109 W cm-2) pulse laser source for the generation of a simultaneous two-photon process (Wang & Liu, 2009). Upconversion materials with lanthanide-doped crystals offer an attractive optical labeling technique in biological studies (Suyver et al., 2005). Doping is also of great importance in stabilizing a particular crystalline phase (Feng et al., 2006), modifying electronic attributes (Alivisatos, 1996), modulating magnetism (Wernsdorfer et al., 1997) and tuning emission properties (Wang et al., 2010).
Upconversion materials have been used in the fields of organic compounds (e.g. Congo red) biodegradation, biological assays and medical imaging from the late 1990s (DaCosta et al., 2014; Lin et al., 2012). Given the insights already attained in material science in relation to the design of luminescence and surface coatings, there is good potential for the use of upconversion materials in bioanalytical applications (DaCosta et al., 2014). Some research has closely examined photodynamic inactivation of microorganisms or viruses using upconversion materials (Lim et al., 2012; Wang et al., 2012), but little is known about the effects of these materials on multi-species microbial aggregates. This study proposes a novel use of upconversion materials to stimulate microbial aggregates to augment contaminant removal. Periphytic biofilms, typical microbial aggregates, are capable of remediating polluted wastewater, including organic matter and metals (Wu et al., 2014). Extracellular polymer substances (EPS) are crucial components of periphytic biofilms and are a complex high-molecular-weight mixture of polymers excreted by microorganisms, products of cell lysis and adsorbed organic matter from wastewater (Sheng et al., 2010). Functional groups of periphytic biofilm EPS, such as proteins and carbohydrates, can adsorb heavy metals (Sheng et al., 2010) and as a result, the EPS of periphytic biofilms has a strong affinity for heavy metal ions (Yang et al., 2016b). Periphytic biofilms abundant in algal biomass have also been a key component for phosphorus removal from wastewater (Sukačová et al., 2015). Periphytic biofilms play several roles in removing P from water, including P uptake and deposition, and filtering particulate P from water (Wu et al., 2011). The objectives of this study are to (i) determine the removal rate of P and Cu2+ by periphytic biofilms stimulated by UCPs doped with Pr3+-Li+, and (ii) investigate
the changes in metabolic diversity, community composition and structure of the stimulated periphytic biofilm. The results are expected to offer a promising method to enhance P and Cu removal by periphytic biofilms, and provide an insight into the responses of periphytic metabolic versatility, composition and structure to UCP exposure.
2. Materials and methods
2.1 Periphytic biofilms culture Glass bottles (20 L) sterilized by 0.1 M HCl solution for 30 min were filled with nutrient-rich water from Xuanwu Lake, Nanjing, China containing microbial sources of periphytic biofilm. The properties of the water were: pH 8, total nitrogen (TN) 2.18 mg L-1, NO3--N 0.43 mg L-1, NH4+-N 0.07 mg L-1 and PO43--P 0.24 mg L-1. Industrial Soft Carriers were used as substrates. The substrates were immersed in the water with a light intensity of 2500 Lux under 12/12 h light/dark cycle. The periphytic biofilm automatically form on the surfaces of substrates after 3 days. The periphytic biofilms were incubated at 28 ± 1 °C until the formation of dense dark green periphytic biofilms. Dense periphytic biofilms (thickness ~ 0.1-0.5 cm) were collected for the following experiments.
2.2 Preparation of lanthanide doped UCPs
All chemicals were analytical pure grade and purchased from Sigma Aldrich. The containers were sterilized with ultraviolet irradiation before utilization. The syntheses of UCPs doped with Pr3+ and Li+ (Y2SiO5: Pr3+-Li+) were conducted according to the methods developed by Cates et al. (2011).
Briefly, the firing process of the upconversion materials was carried out using a sol-gel method (Brinker & Scherer, 2013). Pr3+ and Li+ ion doped upconversion materials were prepared as follows: 5.3 g Y2O3 and nitric acid (trace metal grade) were boiled in a ratio of 1:1 (weight ratio) to make the yttrium nitrate solution. The solution was dissolved in ethanol (200 proof) and water, which was evaporated to constant weight under 104 °C. 1 % (mol) Pr and Li solutions were added to nitric acid as dopants, forming aqueous stock solutions of Pr(NO3)3·6H2O and LiNO3·6H2O. Tetraethoxysilane (TEOS) was added as a silicon source and gelling agent and stirred to produce sol gel. Dopant concentrations were adjusted to optimize upconversion efficiency, which corresponded to the doping concentration of 1% Pr3+ and Li+ for samples with both elements. 2.3 Pollutant removal experiment First, a stimulation experiment to optimize periphytic biofilm community composition and metabolic activity was performed. Briefly, all containers were sterilized by ultraviolet radiation for 30 min. 40 g L-1 of UCPs doped with Pr3+-Li+ were individually added into containers containing 8.0 g periphytic biofilm (wet weight) and 150 mL Woods Hole culture medium (WC medium) (Shangguan et al., 2015). Containers without the addition of UCPs were employed as controls. The UCP treatments and the controls were placed in two separate incubators with the same incubator conditons: temperature 25 ± 1 °C under 12/12 h light/dark cycle. During the experiment, no UV and UVC were detected in the control. All treatments and controls were conducted in triplicate. On day 9, the stimulated periphytic biofilms were collected for the next experiments. The periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ are referred to as Pr-Li stimulated periphytic biofilms.
To avoid the inclusion of UCPs doped with Pr3+-Li+ into the wastewater treatment system, the stimulated 8.0 g periphytic biofilms (wet weight) were washed 3 times using 150 mL water (each time). The periphytic biofilms were then soaked in 150 mL water for 2 hours. After soaking, the stimulated periphytic biofilms were used for phosphorus and copper removal. First, 8.0 g periphytic biofilms (wet weight) were put into 200 mL of wastewater containing phosphorus and copper. The composition of the wastewater was: NaNO3-N (20 mg L-1), NH4Cl-N (20 mg L-1), CaCl2·2H2O (36.76 mg L-1), MgSO4·7H2O (36.97 mg L-1), NaHCO3 (12.6 mg L-1), Na2SiO3·9H2O (28.42 mg L-1), K2HPO4·3H2O-P (20 mg L-1), KH2PO4-P (20 mg L-1), H3BO3 (24 mg L-1), C6H12O6 (5 mg L-1), and 1 mL trace elements solution [Na2EDTA·2H2O (4.36 mg L-1), FeCl3·6H2O (3.15 mg L-1), CuSO4·5H2O (2.5 mg L-1), ZnSO4·7H2O (22 µg L-1), CoCl2·6H2O (10 µg L-1), MnCl2·4H2O (180 µg L-1 ), Na2MoO4·2H2O (6.3 µg L-1), Na3VO4 (18 µg L-1), vitamin B12 (135 µg L-1), thiamine (335 µg L-1), and biotin (25 mg L-1)]. The containers containing stimulated and non-stimulated (control) periphytic biofilms were placed in an incubator with light intensity of 2500 lux, a light/dark regime of 12/12 h, and air temperature of 25 ± 1 °C. The indoor simulation experiments were carried out under flowing water circulation conditions. Specifically, when a 30 mL water sample was removed from 2 cm under the water surface, 30 mL of fresh wastewater was injected. Water samples were used for determining the concentrations of P and Cu2+. The experiment was performed in triplicate.
2.4 Sample analysis X-ray diffraction (XRD), a transmission electron microscope (TEM) and a fluorescence spectrometer were employed to examine the UCPs crystal structure and
upconversion luminescence. XRD analysis was done using a Siemens D5005 X-ray diffractometer with Cu Kα radiation (λ = 1.54056 nm) at a power of 40 keV × 30 mA. The XRD data were recorded with 2θ variation from 20° to 60° using a counting time of 10 s and a scanning mode with a step size of 0.02°. The cell structures of periphytic biofilms were observed by transmission electron microscope (JEM-1400PLUS, JEOL). The upconversion luminescence of UCPs was tested using a fluorescence spectrometer with an argon laser (FL3-TCSPC, Horiba Jobin Yvon Corporation, France). The exciting parameters were selected as 450 nm of the excitation wavelength, 370 nm of the optical filters, and 1 nm of the slit. The reflectivity of UCPs was measured using a spectrophotometer (CM-2300D, Japan). Biolog EcoplatesTM (Biolog, Hayward, USA) analysis was conducted to evaluate the carbon metabolic activity of periphytic biofilms. The microbial composition and structures were determined by phospholipid linked fatty acid (PLFA) profiling according to the studies performed by Gómez-Brandón et al. (2010). Briefly, 0.2 g of freeze-dried periphytic biofilms were 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 biofilms biomass. Hydrochloric acid was used as an acid catalyst for preparation of fatty acid methyl esters for gas chromatography (Agilent 6850, USA) (Berchmans & Hirata, 2008). The PLFA 18:3ω3c and 20:5ω3c were selected as indicators of algae biomass including cyanobacteria. The PLFA 20:2ω6, 20:3ω6 and 20:4ω6 were chosen as indicators of protozoa biomass, and 18:2ω6, 18:3ω6c and 18:3ω3c were chosen to represent fungi biomass. The PLFA 16:0, 17:0 and 18:0 were chosen to evaluate gram positive bacteria, and 16:1ω5c, 16:1ω7c, 18:1ω5c and 18:1ω7c were chosen as indicators of gram negative bacteria (Boschker et al., 2005). The P and Cu2+ in water samples were determined using a flow injection
analyzer (SEALAA3, Germany) and inductively coupled plasma mass spectrometry (XSERIES 2 ICP-MS, USA).
2.5 Data analysis
Results are presented as mean ± 1 standard deviation (SD) of three replicates. The 96 h biolog data of the carbon sources utilized and correlations of fatty acids data were analyzed using Principal Component Analysis (PCA) and Canonical Analysis (CA) by Canoco 5. The bar charts of P and Cu2+ and XRD data were drawn by OriginPro 2016 (OriginLab Company). The comparisons of P and Cu2+ removal and PLFA data between the treatment and the control were analyzed using One-way ANOVAs using SPSS 22.0. Variance partitioning analyses were calculated using the varPart function in the vegan package by R (R version 3.3.1). Statistical significance was set at p = 0.05 for all analyses.
3. Results and discussion
3.1 Y2SiO5 crystal structure characteristics
XRD was used to represent the crystal structure of yttrium silicate (Y2SiO5) doped with Pr3+-Li+. The curves presented a strong peak when 2θ = 30.7° in accordance with the standard pattern of Y2SiO5 reported in JCPDS no. 41-0004. The crystal size of the Y2SiO5 doped with Pr3+-Li+ is 337 nm at 2θ angle 30.7°. Particle crystallite size and crystal phase affect upconversion luminescence processes and upconversion efficiency (Luo et al., 2016).
A fraction of UVC radiation with 450 nm excitation was detected during the upconversion luminescent emission of Y2SiO5: Pr3+-Li+. Stanton et al. (2012) reported
that lithium doping of Y2O3 led to enhanced X-ray emission intensities that featured slightly increased sizes and crystallinity. Pr-Li stimulated periphytic biofilm also showed incremental reflectivity with the increase in incident wavelengths. It followed that UCPs absorbed more energy in the ultraviolet wavelength. However, increasing the absorbed energy from the excitation band is needed to improve the performance of upconversion materials. 3.2 P and Cu2+ removal by stimulated periphytic biofilms The P removal rate of Pr-Li stimulated periphytic biofilms (61%) was significantly higher than that of the control (p < 0.01) (Fig. 1). Cu2+ removal efficiencies by Pr-Li stimulated periphytic biofilm were 70%, which were significantly higher than the control (61%, p < 0.01). These results suggest that UCPs doped with Pr-Li enhanced P and Cu2+ removal by periphytic biofilm. This is because UCPs doped with Li grow with an inert shell and are more competitive than non-doped UCPs, enhancing upconversion emission intensity for the improvement of the nanoparticles' crystallinity and the distortion of the local symmetry (Ding et al., 2015). While energy transfer upconversion involves two neighboring ions, UCPs can generate efficient energy transfer upconversion by using the Pr doped system itself as the sensitizer and the neighboring Li+ as the activator, producing multiphoton accumulation (Chen et al., 2014). 3.3 Microbial cellular substructure of periphytic biofilms exposed to UCPs
Observations under the optical microscope showed that cyanobacteria were abundant in the periphytic biofilms. The microbial cell structures within the control and UCPs treated periphytic biofilms were examined by transmission electron microscopy. The cell structures were intact when periphytic biofilms grew naturally in
the controls. After the periphytic biofilms were stimulated by Y2SiO5: Pr3+-Li+, microbial cell structures were maintained overall, although cytoderm and cytoplasm separation was locally observed. These observations suggest that the stimulation of periphytic biofilms by Y2SiO5: Pr3+-Li+ had a negative effect on cell structure. UCPs doped with Pr3+-Li+ could damage single cell structures (Cates & Kim, 2015). Similar to previous studies examining the impacts of UCPs on single species communities, this study demonstrated that UCPs doped with Pr 3+-Li+ could negatively affect individual microorganisms by damaging single cell structures (Cates & Kim, 2015). These impacts might lead to shifts in periphytic biofilm community composition and structure. 3.4 PLFA profiling of microbial community structures
To investigate whether the microbial community composition and structure changed during exposure to upconversion materials, periphytic biofilm was examined using PLFA analysis. PLFA can be analyzed with multivariate procedures to determine statistically significant changes in periphytic biofilm structure and relative abundances of different types of microorganisms under different doping regimes (Boschker et al., 2005).
The total PLFA content and Gram +ve bacteria of the Pr-Li stimulated periphytic biofilm (451.49 nmol g -1, 181.16 nmol g -1, respectively), were significantly higher than in the control (380.82 nmol g-1, 151.99 nmol g-1, respectively) (p < 0.01 for both comparisons). Gram –ve and algae biomass in the Pr-Li stimulated periphytic biofilm (69.12 nmol g-1, 367.72 nmol g-1, respectively) were also higher than in the control (59.07 nmol g-1, 307.38 nmol g-1, respectively) (p < 0.01 for both comparisons). These results demonstrated that exposure to Y2SiO5: Pr3+-Li+ increased the microbial
biomass of the bacteria and algae in the periphytic biofilms. Y2SiO5: Pr3+-Li+ had a slight negative effect on fungal and protozoan biomass, leading to the proportion of these microorganisms in the Pr-Li stimulated periphytic biofilms decreasing from 1.3 to 1.0% for fungi and from 1.3 to 1.0% for protozoa (Table 1). 17:0, 18:0, 15:0 iso, 20:4ω6c, 16:0 10-methyl and 17:0 iso fatty acids were detected in the control periphytic biofilm, demonstrating that the microbial community was dominated by cyanobacteria (M. aeruginosa), bacteria (e.g. Bacillus stearothermophilus and Actinomycetes sp.) and attached protozoa. The 14:0, 18:1ω9c, 16:0 and 18:2ω6c in the Pr-Li stimulated periphytic biofilm, indicate that cyanobacteria (e.g. Microcystis flos-aquae), Gram +ve bacteria and fungi dominated the periphytic biofilm. These changes show that the microbial community structures of the periphytic biofilm had been changed by UCP stimulation. This result also implies that the reduced or absent microorganisms, such as M. aeruginosa, in the periphytic biofilm stimulated by Y2SiO5: Pr3+-Li+ were an “ineffective” component in removing P and Cu2+.
Periphytic biofilms harbor a stunningly diverse collection of microbial species competing for resources and space (Hibbing et al., 2010). Microorganisms can benefit from microbial competition when competition dampens cooperative networks and increases stability, and the final commensality is beneficial for all participating organisms (Foster & Bell, 2012). Under the stimulation of UCPs, the periphytic biofilms optimized their species diversity with complex microbial interactions including cooperation and competition, leading to improved community stability (Coyte et al., 2015). These stable periphytic biofilms had high capacity for eliminating inorganic pollutants. This is because highly stable microbial communities have high
contaminant removal performance, including inorganic pollutants such as P (Miura et al., 2007), and can improve raw water quality (Luo et al., 2013). 3.5 Carbon source utilization of periphytic biofilms
The correlations between the six main carbon source groups and (Principal Component 1) PC1 or (Principal Component 2) PC2 are shown in Table 2 (only carbon sources with correlation coefficients > 0.9 were selected). PC1 accounted for most of the variance (79.45%). The variance explained by PC2 (20.55%) related to a few types of carbon sources, such as carboxylic acids. For PC1, there were six carbon sources (belonging to three groups: carbohydrates, polymers, carboxylic acids) in the control, and six carbon sources (belonging to four groups: carbohydrates, polymers, carboxylic acids, amino acids) in the Pr-Li stimulated periphytic biofilm. These results indicate that microorganisms in Pr-Li stimulated periphytic biofilm utilized different carbon sources to sustain periphytic biofilm growth and metabolism; and suggest that the Pr-Li stimulated periphytic biofilm acclimated to the UCPs.
The metabolic capacity of periphytic biofilm stimulated by UCPs was investigated by calculating the average well color development (AWCD) (Guo et al., 2010). The carbon source metabolic capacities (represented by AWCD) of the periphytic biofilms stimulated by Y2SiO5: Pr3+-Li+ were significantly improved in contrast to the control (p < 0.05, Fig. 2). 3.6 Synergic actions of algae, bacteria biomass and carbon metabolic capacity in P and Cu2+ removal Periphytic biofilm exposed to UCPs doped with Pr3+-Li+ possessed relatively higher P and Cu2+ removal capacity. To select the primary parameters explaining P
removal, variance partitioning analyses (VPA) based on algal biomass (represented by PLFAalga), bacterial biomass (PLFAbac), carbon source metabolic capacity (represented by AWCD) and P removal rate were conducted (Fig. 3). In contrast to the control (0.06), Pr-Li stimulated periphytic biofilm had higher interaction of algae, bacteria and AWCD (0.77). The contribution of algal biomass and AWCD to Pr-Li stimulated periphytic biofilm for P removal significantly increased from 0.09 to 0.11 (p < 0.05). The carbon source metabolic capacity (AWCD contribution) for Pr-Li stimulated periphytic biofilm was also related to P removal rate. These results indicated that the synergistic action of algal biomass, bacterial biomass and carbon source metabolic capacity played important roles in enhancing P removal by Pr-Li stimulated periphytic biofilms.
The total contribution of the three parameters of Pr-Li stimulated periphytic biofilm to Cu2+ removal slightly increased from 0.20 to 0.21 (Fig. 4). The simulated results were consistent with Cu2+ removal efficiencies with Pr-Li stimulated periphytic biofilm. These results indicated that algal and bacterial biomass and AWCD enhanced segmental Cu2+ elimination. This means that there was no significant enhancement of Cu2+ removal efficiency by the synergistic action of algae, bacteria and carbon source metabolic capacities. Ahluwalia and Goyal (2007) reported that the removal of heavy metals from aqueous solution using inactive and dead biomass (non-living biomass of algae, aquatic ferns and seaweeds, waste biomass) as potential biosorbents was an innovative and alternative technology for removing inorganic compounds and heavy metals such as Cu2+. As a chelating agent, EPS can combine with and form a complex with Cu2+, thus removing Cu2+ from wastewater (Yang et al., 2016a). The combination of Cu2+ and EPS is likely to have resulted in the changes in Cu speciation, thereby varying the toxicity of Cu to microorganisms (Wu et al., 2017).
Accordingly, the Cu2+ removal might also be attributed to the inactive and dead biomass and EPS, but further tests are needed.
In nature, the community composition of periphytic biofilm is more diverse than the samples in this study. This diversity provides sufficient microbial sources for the optimization of the microbial structure of periphytic biofilm because different microorganisms have different sensitivities to irradiation (Wu et al., 2014). For example, some microorganisms such as Escherichia coli, MS-2 and T7 (Bowker et al., 2011) are inactivated in the presence of UV and some microorganisms such as Actinobacteria (Warnecke et al., 2005) and Gammaproteobacteria (Santos et al., 2012) grow well in the presence of UV, at different rates depending on the type of microorganisms and growth conditions. This natural diversity and adaptability in conjunction with the results of this study, suggest that the stimulation of natural periphytic biofilm by upconversion phosphors doped with Pr 3+-Li+ is a potential approach to optimize microbial structure, leading to the reduction or disappearance of “ineffective” periphyton community members and the improvement of P and Cu2+ removal.
4.
Conclusions
Ultraviolet irradiation emitted by UCPs with the emission of visible light had a negative effect on a small proportion of “ineffective” periphytic biofilms components (such as M. aeruginosa) for Cu2+ and P removal. Microbial community structures doped with Y2SiO5: Pr3+-Li+ changed as demonstrated by their enhanced carbon utilization and metabolism capacities. The optimized microbial periphytic biofilm community increased P and Cu2+ removal. This was mainly due to the synergistic action of algal and bacterial biomass, and metabolic capacity. These results provide an
insight into how microbial aggregates acclimate to the luminescence conditions caused by the excitement of UCPs and improve their capacity to remove impurities from wastewater.
Acknowledgements This work was supported by the National Natural Science Foundation of China (41422111), the State Key Development Program for Basic Research of China (2015CB158200) and the Natural Science Foundation of Jiangsu Province China (BK20150066). This work was also supported by Youth Innovation Promotion Association, Chinese Academy of Sciences (2014269).
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Figure captions Fig. 1 The removal rate of P and Cu2+ by periphytic biofilms stimulated by UCPs doped with Pr3+-Li+ and the control. * indicates significant differences in the removal rates between treatment and control (t-test, p < 0.05).
Fig. 2 The carbon metabolic capacity of periphytic biofilms, represented by the average well color development (AWCD). * refers to significant difference between the control and the treatment (t-test, p < 0.05).
Fig. 3 Variance partitioning analyses based on PLFAalga (X1), bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and P removal in the (B) Control, (C) Pr-Li stimulated periphytic biofilm (no result shown when values are approximately equal to zero or below zero). Each diagram represents the biological variation partitioned into the relative effects of each factor or a combination of factors. The corners of the triangle represent the variation explained by the factor alone. The sides of the triangles represent interactions of any two factors, and the middle of the triangles represent interactions of all three factors.
Fig. 4 Variance partitioning analyses based on PLFAalga (X1), bacteria (X2), carbon source metabolic capacity (AWCD) (X3) and Cu2+ removal in the (B) Control, (C) Pr-Li stimulated periphytic biofilm (no result shown when values are approximately equal to zero or below zero).
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Table 1 The total PLFA (PLFAtot), protozoa (PLFApro), fungi (PLFAfun), algae (PLFAalga) (nmol g-1 dry biomass), Gram-positive (Gram +ve) bacteria (PLFApos) and Gram negative (Gram –ve) bacteria (PLFAneg) (nmol g-1) and their proportions in the control periphytic biofilm and the Pr-Li stimulated periphytic biofilm. PLFApro
PLFApos
PLFAneg
PLFAfun
PLFAalga
Gram +ve/Gram –ve
Treatments
PLFAtot
Control
380.82 ± 19.04 4.77 ± 0.24 151.99 ± 7.60 59.07 ± 2.95 4.78 ± 0.24 307.38 ± 15.37 2.57 ± 0.13
Pr-Li stimulated 451.49 ± 22.57 4.73 ± 0.24 181.16 ± 9.06 69.12 ± 3.46 4.73 ± 0.24 367.72 ± 18.38 2.62 ± 0.13
Table 2 Correlation coefficients between main carbon source groups and PC1 or PC2. Treatments Control
PC1 Carbohydrates
i-erytritol Glucose-1-phosphate α-lactose
Correlation coefficient 0.90 0.97 0.97
Stages Control
PC2 Carbohydrates Polymers
β-methyl-D-glucoside D-xylose Tween 80
Correlation coefficient 0.93 0.93 0.96
Polymers Carboxylic acids Pr-Li stimulated
Carbohydrates Polymers Carboxylic acids Amino acids
D-l-α-Glycerol-1-phosphat e Tween 40 Pyruvic acid methyl ester α-lactose α-cyclodextrine Pyruvic acid methyl ester L-arginine l-phenylalanine L-threonine
Glycogen
0.91 0.98 0.94 0.99 0.99 0.99 0.95 1.00 1.00
Phenolic acid Pr-Li stimulated
Carbohydrates Phenolic acid Carboxylic acids
2-hydroxybenzoic acid D-l-α-Glycerol-1-phosphate 4-hydroxybenzoic acid D-glucosaminic acid
0.93 1.00 0.94 0.91 0.98
Highlights · Pr-Li stimulated periphytic biofilm changed microbial community structure. · Pr-Li stimulated periphytic biofilm enhanced carbon utilization and metabolic capacity. · Synergic action of algae, bacteria biomasses and AWCD contributed to P and Cu 2+ removal.