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Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles
Accepted Manuscript Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles Jun Tang, Ningyuan Zhu, Yan Zhu, Seyed Morteza Zamir, Yonghong Wu PII: DOI: Reference:
S0960-8524(17)31160-4 http://dx.doi.org/10.1016/j.biortech.2017.07.064 BITE 18484
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 May 2017 11 July 2017 12 July 2017
Please cite this article as: Tang, J., Zhu, N., Zhu, Y., Zamir, S.M., Wu, Y., Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.064
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Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles Jun Tang a,c, Ningyuan Zhu a,c, Yan Zhu a,c, Seyed Morteza Zamir b, Yonghong Wu a*
a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,
Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China b
Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University,
Tehran, Iran c
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China
*Corresponding author: Yonghong Wu Tel : (+86)-25-8688 1330 fax: (+86)-25-8688 1330 Email: [email protected]
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Abstract The responses of periphytic biofilm to CeO2 nanoparticle (CNP) exposure were explored by investigating community shifts and pollutant removal. Results showed that CNPs entered the sensitive microbial cells in the periphytic biofilm, leading to cytomembrane damage and intracellular reactive oxygen species (ROS) generation. The periphytic biofilm communities were, however, able to adapt to the prolonged exposure and maintain their pollutant removal (i.e. phosphorus, nitrogen and copper, organic matter) performance. Observations under synchrotron radiation scanning transmission X-ray microscopy revealed that fewer CNPs were distributed in algal cells compared to bacterial cells, wherein the transformation between Ce (IV) and Ce (III) occurred. High-throughput sequencing further showed that the proportion of algae, such as Leptolyngbya and Nostoc, significantly increased in the periphytic biofilm exposed to CNPs while the proportion of bacteria, such as Bacilli and Gemmatimonadetes, decreased. This change in community composition might be the primary reason for the sustained pollutant removal performance of the periphytic biofilm. Key words: Nanoparticle distribution; STXM; Microbial aggregates; Nanotoxicity; Pollutant removal.
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1. Introduction Nanomaterials, such as CeO2 nanoparticles (CNPs), are largely used as catalysts in automotive fuel additives, gas sensors, polishing materials and UV absorbers (Tella et al., 2014). These widely used CNPs eventually enter into waters (You et al., 2015). Microbial aggregates are the main form of aquatic microorganisms in the natural environment and play an important role in self-purification of water and pollutant removal in biological wastewater treatments (Wu et al., 2014). It is therefore important to investigate the responses of microbial aggregates to CNPs. Periphytic biofilm, a typical microbial aggregate, is an important ecological component of surface water and plays a major role in primary productivity, nutrient transformation and biological removal of pollutants (Wu et al., 2012). Some studies have examined the effect of nanoparticles (NPs) on the physiology and function of periphytic biofilm, such as photosynthetic yield and respiration potential (Gil-Allué et al., 2015), oxidative damage (Hou et al., 2015a), and the ability to remove pollutants (Xu et al., 2017). These studies, however, focused on the responses of biofilms using short-term (e.g. 2 h) or physiological investigations during NP (e.g. Ag-NP) exposure (Gil-Allué et al., 2015), with little attention paid to the distribution and transformation of NPs or the changes in microbial communities. Periphytic biofilm consists of a variety of microbial components including bacteria, algae, fungi, protozoa and metazoa (Shabbir et al., 2017). The sensitivity of different species to NP toxicity differs which implies a potential community shift in a microbial
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community exposed to NPs (Li et al., 2015; Wang et al., 2011). The microbial composition and diversity of periphytic biofilm play an important role in its resistance to external environmental stresses and maintaining its functions including pollutant removal (Wu et al., 2011). Thus, it is hypothesized that the periphytic biofilm exposed to CNPs will maintain its pollutant removal performance via self-regulation of its microbial composition and diversity. The distribution and transformation of CNPs can directly affect the physiology and function of microbial cells (Li et al., 2017a). Benthic microbial aggregates, such as periphytic biofilm, are a sink for CNPs when they agglomerate and settle at the bottom of water (Ferry et al., 2009; Schug et al., 2014). The fate of CNPs in the environment is critical when studying microbial cell responses as the transference of CNPs controls the bioaccumulation. In addition, the Ce (III) / Ce (IV) redox cycle in cells affects the generation of reactive oxygen species (ROS) (Tella et al., 2014). It is difficult, however, to determine the distribution and transformation of CNPs, at both cell and community level, under high resolution using common microscopy techniques such as transmission electron microscopy (TEM). Synchrotron radiation scanning transmission X-ray microscopy (STXM), an advanced in situ imaging technology, can quantitatively study the distribution and speciation of elements in vivo and has been used to explore the fate of CNPs in plants such as cucumber and soybean (Hernandez-Viezcas et al., 2013; Zhang et al., 2012). The STXM was employed in this study to study the distribution and transformation of CNPs in periphytic biofilm at both cell and community levels.
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In this study, we investigated the effects of CNPs on periphytic biofilm under prolonged exposure (30 d), using high-throughput sequencing (HTS) and STXM to reveal the microbial community changes and the fate of CNPs in periphytic biofilm. The objectives of this study were to (i) investigate the effects of CNPs on the physiological properties and pollutant (phosphorus, nitrogen and copper, organic matter) removal by periphytic biofilm, (ii) determine the microbial community composition and diversity responses, and (iii) determine the distribution and transformation of CNPs in periphytic biofilm at both cell and community level. 2. Materials and methods 2.1 Preparation of CNP solutions and periphytic biofilm CNPs were supplied by Aladdin (Shanghai, China) and the purity was over 99.7%. A 100 mg/L CNP stock suspension was prepared through the addition of 0.1 g of CNPs to 1 L of Milli-Q water (pH 7) followed by 30 min of ultra-sonication (100 W, 25 KHz, 25 °C). Then, the CNP stock suspension was diluted to 5 mg/L with Woods Hole culture medium (WC medium) (Hametner et al., 2014). This was referred to as CNP solution. The prepared CNP solutions were used for the exposure experiments. The original periphytic biofilm was collected from Xuanwu Lake, East China. The lake water parameters were: pH = 7.8; TP = 0.1 mg/L; TN = 1.90 mg/L; NH4+ = 0.53 mg/L; and NO3- = 0.73 mg/L. To stabilize and homogenize the periphytic biofilm, the collected material was mixed and cultured in WC medium in an incubator at 28 ± 1 °C with a light intensity of 2800 Lux and light/dark regime of 12 h/12 h. The periphytic 5
biofilm used in the following experiments was obtained by peeling it off the substrate using a sterilized silicone spatula when the biofilm thickness exceeded 5 mm. The dry mass to wet mass ratio of periphytic biofilm was 0.0532 ± 0.0085 (n = 10). Thus, a 5% ratio was selected as a standard for dry weight (DW) conversion of periphytic biofilm. The periphytic biofilm mass throughout the study is expressed as DW. 2.2 Experimental processes To conduct the exposure experiments, 0.05 g periphytic biofilm was added into flasks filled with 100 mL CNP solution. Then, the periphytic biofilm was cultured in an incubator (light power: 2800 lux, light/dark regime: 12 h/12 h, temperature: 28 ± 1 °C) under shaking conditions (120 rpm) for 30 d. To better investigate the response of the periphyton at the community level, the exposure experiments were performed for a long period of 30 d. The control (CK) was made by adding 0.05 g periphytic biofilm into 100 mL pure WC medium followed by periphyton culture under the same conditions as the CNP treatment. All experiments were performed in triplicate. Removal of organic matter, copper, nitrogen and phosphorus by periphytic biofilm was carried out after the exposure experiment to evaluate the influence of CNPs on the pollutant removal performance of periphytic biofilm. When the exposure experiment was completed, the CK and CNP exposed periphytic biofilm were removed from their flasks and rinsed three times with 100 mL Milli-Q water. Then, 0.05 g of CK and CNP periphytic biofilm were added into separate flasks with 100 mL wastewater and cultured in an incubator under the same experimental conditions used for the exposure 6
experiment. The main parameters of the wastewater were as follows: COD = 159.26 ± 3.12 mg/L; TN = 3.92 ± 0.19 mg/L; TP = 1.15 ± 0.11 mg/L; NO3- = 2.76 ± 0.21 mg/L; NH4+ = 0.52 ± 0.09 mg/L; PO43- = 0.76 ± 0.08 mg/L; Cu2+ = 2.95± 0.12 mg/L; Ca2+ = 2.6 ± 0.19 mg/L; and Zn2+ = 0.25 ± 0.02 mg/L. The pH was adjusted to 7.0 using 0.1M NaOH or HCl solution as required. The pollutant removal experiment was conducted in triplicate. 2.3 In situ distribution and speciation by STXM To avoid potential confusion caused by mapping CNPs inside cells due to CNP adsorption by extracellular polymeric substances (EPS) and the cell wall, EPS and NPs adsorbed by cell walls were removed before STXM analysis by the combination of ultra-sonication and EDTA treatment, followed by rinsing three times using a 0.05% (w/w) NaCl solution following (Liang et al., 2010). STXM imaging based on Ce M4 edge X-ray absorption near edge structure (XANES) was performed on the beamline BL08U1 at the Shanghai Synchrotron Radiation Facility (SSRF Shanghai, China). CeO2 and CeF3 (Aladdin, Shanghai, China) were chosen as the Ce (IV) and Ce(III) reference materials, respectively. The standard materials were ultrasonically dispersed in ethanol and deposited on tinfoil fixed on a sample holder and examined to obtain the standard spectrum. An aliquot of the periphytic biofilm sample was placed onto an X-ray transparent silicon nitride membrane (Silson Ltd, Northampton, UK) and a second silicon nitride membrane was aligned and placed on top of the periphytic biofilm; the silicon nitride sandwich was then sealed with acid-free silicone sealant and examined by 7
STXM (Dynes et al., 2006). A dual-energy method was then performed on the chosen regions of the sample and the Ce element map was calculated to obtain the distribution of the Ce component in the chosen regions (Zhang et al., 2012). 2.4 Sample and analyses The total antioxidant capacity (T-AOC) of the periphytic biofilm was determined according to a previous method (Li et al., 2017b). The minimal chlorophyll fluorescence (F0) was measured using an AquaPen-P fluorometer (Photon Systems Instruments, Brno, Czech Republic) after 25 min dark adaption. Total phosphorus (TP) and total nitrogen (TN) in the wastewater were measured by the standard methods (APHA-AWWA-WEF, 1998). Organic matter (represented by chemical oxygen demand, COD) in the wastewater was determined according to the standard potassium dichromate digestion method (GB11914-89) of the Ministry of Environmental Protection of China (Wei, 2002). The concentration of Cu2+ in the wastewater was measured by ICP-MS (7700x, Agilent, America) after filtration. The 16S rDNA high-throughput sequencing by Illumina MiSeq was employed to characterize the microbial composition and diversity of the periphytic biofilm. The processes and analyses of high-throughput sequencing were according to our previous study (Shangguan et al., 2015). The cell damage and distribution of the CNPs in microbial cells were determined by TEM (HT-7700, Hitachi, Japan) and the sample preparation was according to (Jian et al., 2016). The composition image of algae and bacteria in periphytic biofilm was detected by confocal scanning laser microscopy (CSLM) (LSM 710, Zeiss, Germany). 8
The
intracellular
ROS
of
periphytic
biofilm
was
labeled
by
10
µM
2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) for 30 min and measured by fluorescence microscopy (Nikon-80i, Nikon, Japan) at excitation/emission wavelengths of 495/525 nm (Hou et al., 2015a). 2.5 Data analyses and statistics The results were expressed as mean ± standard deviation and the significance of differences between means was evaluated at the significance level p < 0.05. Statistical analyses were performed using SPSS 19.0 and assay data were tested by One-way analysis of variance (ANOVA). The figures were drawn using Sigmaplot 12.0. 3. Results and discussion 3.1 Responses of periphytic biofilm to CNP exposure 3.1.1 Effects of CNPs on the physiological characters of periphytic biofilm Total antioxidation capacity (T-AOC), a sensitive and reliable marker to detect changes in oxidative stress in vivo, was used to determine the oxidative damage of periphytic biofilm induced by CNPs. Results showed that the T-AOC of the periphytic biofilm exposed to CNPs was decreased by 57.74% compared with CK. This result indicates that the CNPs reduced the antioxidant ability of periphytic biofilm and could inhibit the cellular activity of the microorganisms in periphytic biofilm, which further implied that some sensitive communities in the periphytic biofilm were under stress from CNPs. 9
It is well known that CNPs, and several other metal NPs, such as Ag-NPs and ZnO-NPs, have the ability to generate ROS and stimulate the peroxidation of cell membrane lipids (Gu et al., 2014; Ma et al., 2013). It has been demonstrated that NPs attacking the respiratory chain in cell to induce the incomplete reduction of oxygen and the potential redox properties between Ce(Ⅲ) and Ce(Ⅳ) states of CNPs both contribute to the generation of ROS by CNPs in cells (Zhang et al., 2011). Excessive ROS generation and oxidative injury induced by CNP exposure could significantly reduce the levels of cellular antioxidants and destroy the antioxidant system (Dogra et al., 2016; Park et al., 2008). In this study, the T-AOC of periphytic biofilm showed a significant decrease under exposure to CNPs, indicating that periphytic biofilm is sensitive to CNPs and the antioxidant system failed to maintain cellular redox equilibrium. Therefore, the main toxic effects of CNPs on periphytic biofilm were production of excess ROS and decrease of T-AOC, resulting in oxidative stress and cell damage. To estimate the effects of CNPs on photosynthetic characteristics of periphytic biofilm, the change of F0 with time was determined (Fig. 1). The F0 of periphytic biofilm exposed to CNPs decreased with time in the initial stages of exposure and reached the lowest level at day 15. It then increased and reached a stable level at day 30, with an 11.3% increase from day 15. Variation of F0 in the CK followed an opposite trend to the CNP periphytic biofilm. F0 is a basic photosynthetic character and is directly proportional to chlorophyll content (McKew et al., 2011). Therefore, the F0 increase in the recovery stage of
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periphytic biofilm exposed to CNPs might be induced by the increasing proportion of algae. This is consistent with the results of a previous study that determined NP exposure could disturb the balance between algae and bacteria and promote the growth of algae in an algal–bacterial symbiosis system (Li et al., 2015). 3.1.2 Changes in community composition and diversity of periphytic biofilm By performing OTU picks at 97% identity level, 255 and 329 OTUs based on 36743 and 42012 reads were obtained from the CNP exposed periphytic biofilm and CK, respectively (Table 1). The Chao 1 and Shannon indexes can be used to analyze the microbial richness and diversity (Ma et al., 2017). The Chao 1 richness estimators in the CNP exposed periphytic biofilm were significantly lower than the CK (p < 0.05), suggesting that the species richness of periphytic biofilm decreased in the presence of CNPs. However, the Shannon index in CNP exposed periphytic biofilm was significantly higher than the CK (p < 0.05), indicating a higher microbial diversity in biofilm exposed to CNPs. Under the prolonged exposure to CNPs, some sensitive species of periphytic biofilm may be inhibited leading to a decrease in community species richness. The proportion of the species in periphytic biofilm becomes more balanced and results in higher species evenness, which could be reflected by the increased microbial diversity (Ma et al., 2017; Wang et al., 2016). At the phylum level (Fig. 2), 12 main phyla were detected in the two periphytic biofilm communities including Firmicutes, Cyanobacteria, Gemmatimonadetes, Proteobacteria, Planctomycetes, Bacteroidetes, and Chloroflexi, which cover most of 11
the bacterial phyla found in natural aquatic environments (Liu et al., 2016). In CK, Firmicutes, Cyanobacteria, Gemmatimonadetes were the dominant species, accounting for 42.09%, 10.80%, and 21.94%, respectively. After exposure to CNPs, Cyanobacteria, Proteobacteria and Planctomycetes became the prominent phyla in the periphytic biofilm with abundances of 34.41%, 12.37%, and 15.63%, respectively. It was noted that the abundance of Firmicutes underwent a marked decline from 42.09% to 3.32% when exposed to CNPs, while the abundance of Cyanobacteria increased from 10.80% to 34.41%. To better understand the changes in microbial composition and diversity of periphytic biofilm exposed to CNPs, the microbial communities were also analyzed at the class and genus levels. At the class level, the relative abundances of Bacilli and Gemmatimonadetes sharply reduced from 41.80% and 21.94% to 3.29% and 9.80%, respectively, after the prolonged exposure to CNPs. The abundances of Cyanobacteria, Planctomycetacia and Alphaproteobacteria increased from 10.80%, 1.61% and 3.94% to 34.41%, 15.52% and 10.61%, respectively. The top 100 most abundant genera were used to evaluate the community shifts of periphytic biofilm exposed to CNPs. The abundances of algae such as Nostoc and Leptolyngbya increased from 0.08% and 2.37% to 13.28% and 13.27%, respectively, and became the dominant species in the periphytic biofilm. Reflected by the variation of F0 with time (Fig 1), the proportion of algae in periphytic biofilm exposed to CNPs decreased with time during short-term exposure (15
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d) and then increased to a stable high level after long-term exposure (30 d). Further investigation by microbial composition analysis showed the main community shifts under long-term exposure to CNPs were the increase in abundances of algae such as Nostoc and Leptolyngbya and the decrease of some sensitive bacteria such as Bacilli and Gemmatimonadetes. Algae such as Nostoc and Leptolyngbya not only have thicker cell walls, but also a mucilaginous sheath to protect the cells (Reale et al., 2015). The NPs therefore might have difficulty entering the cells of these algae in the periphytic biofilm, leading to less inhibition of algae growth. As a result, the periphytic biofilm microbial community acclimated by having more algae but less bacteria and a higher microbial diversity under the long-term exposure to CNPs. 3.2 CNPs in periphytic biofilms 3.2.1 The distribution and transformation of CNPs STXM images of Ce (IV) and Ce (III) distribution in periphyton exposed to CNPs were obtained. At the community level, CNPs were inclined to enter the cells of pelletor rod-like bacteria such as Bacilli and Gemmatimonadetes. In algae cells with filamentary (Leptolyngbya) and spherical (Nostoc) structure, few CNPs were detected. At the cellular level, more CNPs were distributed around the cytomembrane than inside the cell which might induce serious membrane damage. Meanwhile, both Ce (IV) and Ce (III) were detected in cells with the content of Ce (IV) higher than Ce (III). The element Ce exists as Ce (IV) in the CNPs, while both Ce (IV) and Ce (III) could be detected in the cells, which indicates the transformation of Ce (IV) of CNPs to Ce (III) 13
in cells (Zhang et al., 2012). Therefore, these results show that the transformation of CNPs could be detected in periphyton cells, but that Ce (IV) was still the main form under prolonged exposure. Although TEM combined with energy spectrum analysis (EDS) can reflect the distribution of NPs in cells based on cell sections, it fails to reveal the distribution of NPs in the whole microbial community and distinguish element speciation (Jian et al., 2016). According to the STXM results, more CNPs internalization and transformation of Ce (IV) to Ce (III) could be detected in the bacterial cells than algal cells, which means that the CNPs might induce more damage to bacteria than algae. Meanwhile, the HTS results showed that the periphytic biofilm community changed to consist of more algae but less bacteria, which was consistent with the distribution of CNPs in different microbial species cells. Therefore, STXM in the present study showed the CNP distribution at the community level in periphyton and revealed the transformation of Ce (IV) and Ce (III) in cells. The uneven distribution of CNPs in different microbial species confirmed that algae were not sensitive to CNP exposure, and resulted in community changes. The typical characteristics of periphytic biofilm, such as EPS and community composition, are important factors that could affect the distribution and transformation of NPs in biofilm. EPS plays an important role in the stability of NPs in the environment and could prevent the NPs from entering the cells, which could further influence the distribution and transformation of NPs (You et al., 2015). Previous studies
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have illustrated that the CNPs could both be absorbed on the EPS surrounding the biofilm and penetrate the cells (Hou et al., 2015b; You et al., 2017). There was, however, no clear understanding about the distribution of CNPs inside biofilm between cells of different species. Earlier studies have found that NPs could more easily penetrate bacterial cells (Escherichia coli) that only have simple cell walls, than the algal cells (Synechocystis) that have thick mucilaginous sheaths and complex cells walls (Zeyons et al., 2009). In the present study, we revealed that more CNPs could be detected in the bacterial cells than the algal cells in periphytic biofilm. Therefore, more EPS located outside the cells and higher algae proportion could be a greater benefit to the resistance against NPs for periphytic biofilm. Furthermore, CNPs could be reduced in organisms such as plants (cucumber plants) due to the reducing substances secreted by plants such as reducing sugar (glucose and fructose) and enzymes (reductases and ferredoxins) (Ma et al., 2015). Therefore, the reducing substances in EPS or inside cells may contribute to the transformation of CNPs in biofilm, but further study is needed to reveal where the transformation occurs and the mechanism underlying the transformation. 3.2.2 Cell damage induced by CNPs uptake To better investigate the impacts of CNPs on periphyton caused by direct NP distribution and transformation, intracellular ROS generation and cell structure damage were detected by fluorescence microscopy and TEM. Microorganisms in periphytic biofilm mainly include algae such as Leptolyngbya and Nostoc with strong spontaneous chlorophyll fluorescence, but also spherical bacterial cells without chlorophyll 15
fluorescence (CSLM image). After exposure to CNPs, the obvious generation of ROS in the spherical bacterial cells could be detected by fluorescent probes, while in the algae there was no obvious ROS generation. The generation of ROS in periphyton exposed to CNPs was consistent with the distribution of CNPs in the periphyton community. TEM images may better reflect the damage caused by CNPs on the cell structure. In bacterial cells, serious structural damage on the cell membrane and even some cell lysis could be observed in periphytic biofilm exposed to CNPs. Many CNPs got into the spherical and rod cells and some CNPs were also released with cell lysis. The algal cells exposed to CNPs were, however, intact because fewer CNPs entered these algal cells due to the protection of the thick mucilaginous sheath which could be clearly observed surrounding the cells. Accordingly, the CNPs tended to enter the bacterial cells with simple cell structures rather than algae cells with thick mucilaginous sheaths, which is one of the main reasons for more damage to bacterial cells. Therefore, the growth of bacteria such as Bacilli and Gemmatimonadetes was significantly inhibited, while the growth of algae such as Nostoc and Leptolyngbya was promoted, thereby leading to changes in the community composition of the periphytic biofilm with prolonged exposure to CNPs. 3.3 Performance in removing pollutants Organic matter (represented by COD), heavy metals such as copper, and nutrients such as nitrogen and phosphorus compounds, are common pollutants in surface water. The removal of COD, Cu, TP and TN of wastewater by periphytic biofilm exposed to
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CNPs and the CK were investigated (Fig. 3). There were no significant differences in the COD, Cu, TP and TN concentrations in wastewater over time between the periphytic biofilm exposed to CNPs and the control. Concentrations of these pollutants all decreased with time and reached removal rates of 66.7%, 61.7%, 82.5% and 80.9% for COD, TP, TN and Cu, respectively. This result implies that the periphytic biofilms exposed to CNPs were capable of maintaining their pollutant removal functioning to levels found in the CK periphytic biofilms. Interestingly, CNP exposure caused obvious adverse effects on the physiology (T-AOC and F0) of periphytic biofilm, but the performance of COD, TP, TN and Cu removal by the CNP periphytic biofilm was not influenced. Other studies obtained similar results showing that although exposure to NPs, such as Fe3O4, ZnO and TiO2, could damage some sensitive microbial cells by generation of ROS and inhibiting enzyme activity, the microbial aggregates could still maintain their pollutant removal performance (Ma et al., 2017; Mu et al., 2011). Compared to a single microbial species, periphytic biofilm consists of a heterogeneous hierarchy of trophic levels resulting in a relatively robust nature to resist external environmental stressors via community composition regulation (Wu et al., 2011). This is an acclimation process where some sensitive species are inhibited by external environmental stress while the adapted species survive in the community and periphytic biofilm develops into a new balanced community that could maintain the important functions such as pollutant removal (Hu et al., 2017; Wu et al., 2011). The regulation of microbial communities in periphytic
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biofilm to adapt to external environmental stress, such as exposure to heavy metals (Yang et al., 2016), organic pollutants (Shangguan et al., 2015) and azo dye (Shabbir et al., 2017), has been revealed in previous studies. In the present study, the community composition and diversity of periphytic biofilm changed significantly to contain more algae and less sensitive bacteria under prolonged exposure to CNPs. The periphytic biofilm could therefore maintain its COD, TP, TN and Cu removal performance via community shifts despite exposure to CNPs causing negative effects on physiology. According to the STXM and TEM results, the distribution of CNPs in different microbial species cells was uneven with more CNPs entering the bacterial cells than algal cells and inducing more serious damage to the bacterial cells. As a result, the growth of bacteria was inhibited while the algae gained a competitive advantage in the community, which eventually lead to the periphytic biofilm consisting of more algae and less bacteria under the prolonged exposure to CNPs. Therefore, the periphytic biofilm community shifts were mainly caused by the uneven distribution of CNPs at the community level which resulted in more serious cell damage to sensitive bacteria. Our results showed that although individual microorganisms were affected by CNPs exposure, the ability of periphytic biofilm to remove pollutants was sustained via community regulation. Therefore, periphytic biofilm is suitable for use in the treatment of mixed wastewater containing both NPs and other pollutants (COD, TP, TN and Cu). Further research is needed to (i) reveal how the characteristics of the NPs and biofilm influence the distributions and transformations of CNPs in biofilm and (ii)
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identify the interactions of CNPs with biomolecules both outside and inside cells. 4 Conclusion This study demonstrated uneven distribution of CNPs in the periphytic biofilm and the subsequent induced community shifts. The periphytic biofilm could maintain its COD, Cu, TP and TN removal performance via adaptation of community composition although some sensitive bacterial growth was negatively affected by CNP exposure. This study was an in-depth examination of the whole process of how CNPs affect periphytic biofilm and provide valuable information to better understand the fate of metal NPs in microbial aggregates.
Acknowledgments
We sincerely thank Dr Clare Morrison (Griffith University) for proof-reading this manuscript. 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). A part of the work was supported by the Shanghai Synchrotron Radiation Facility. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version. 19
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Figure Captions
Fig. 1. Changes in chlorophyll fluorescence (F0) of periphytic biofilm exposed to CNPs and CK. * p < 0.05. Fig. 2. Microbial community composition at the phylum level of periphytic biofilm exposed to CNPs and CK. Fig. 3. The abilities of periphytic biofilm exposed to CNPs and CK to remove organic matter (represented by COD), total phosphorus (TP), total nitrogen (TN) and copper (Cu) from wastewater.
23
Fig. 1
24
Fig. 2
25
Fig. 3
26
Table 1. Statistical summary for the pyrosequencing and microbial diversity analysis. Sample
Reads
OTU
Chao 1 richness
Shannon diversity
CK CNPs
42012 36743
329 209
365 (347, 401) 248 (226, 300)a
3.02 (3.00, 3.04) 3.44 (3.42, 3.45)a
a
The data reported are statistical differences (p < 0.05) from the control.
27
Highlights
CeO2 nanoparticles (NPs) distribution was detected by scanning transmission X-ray microscopy.
Uneven distribution of CeO2 NPs induced microbial community shifts.
Community composition of periphytic biofilm was investigated at phylum, class and genus level.
Pollutants removal was sustained via self-regulation of community composition.
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Graphical abstract
S0960-8524(17)31160-4 http://dx.doi.org/10.1016/j.biortech.2017.07.064 BITE 18484
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
23 May 2017 11 July 2017 12 July 2017
Please cite this article as: Tang, J., Zhu, N., Zhu, Y., Zamir, S.M., Wu, Y., Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.064
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.
Sustainable pollutant removal by periphytic biofilm via microbial composition shifts induced by uneven distribution of CeO2 nanoparticles Jun Tang a,c, Ningyuan Zhu a,c, Yan Zhu a,c, Seyed Morteza Zamir b, Yonghong Wu a*
a
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,
Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China b
Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University,
Tehran, Iran c
College of Resource and Environment, University of Chinese Academy of Sciences,
Beijing 100049, China
*Corresponding author: Yonghong Wu Tel : (+86)-25-8688 1330 fax: (+86)-25-8688 1330 Email: [email protected]
1
Abstract The responses of periphytic biofilm to CeO2 nanoparticle (CNP) exposure were explored by investigating community shifts and pollutant removal. Results showed that CNPs entered the sensitive microbial cells in the periphytic biofilm, leading to cytomembrane damage and intracellular reactive oxygen species (ROS) generation. The periphytic biofilm communities were, however, able to adapt to the prolonged exposure and maintain their pollutant removal (i.e. phosphorus, nitrogen and copper, organic matter) performance. Observations under synchrotron radiation scanning transmission X-ray microscopy revealed that fewer CNPs were distributed in algal cells compared to bacterial cells, wherein the transformation between Ce (IV) and Ce (III) occurred. High-throughput sequencing further showed that the proportion of algae, such as Leptolyngbya and Nostoc, significantly increased in the periphytic biofilm exposed to CNPs while the proportion of bacteria, such as Bacilli and Gemmatimonadetes, decreased. This change in community composition might be the primary reason for the sustained pollutant removal performance of the periphytic biofilm. Key words: Nanoparticle distribution; STXM; Microbial aggregates; Nanotoxicity; Pollutant removal.
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1. Introduction Nanomaterials, such as CeO2 nanoparticles (CNPs), are largely used as catalysts in automotive fuel additives, gas sensors, polishing materials and UV absorbers (Tella et al., 2014). These widely used CNPs eventually enter into waters (You et al., 2015). Microbial aggregates are the main form of aquatic microorganisms in the natural environment and play an important role in self-purification of water and pollutant removal in biological wastewater treatments (Wu et al., 2014). It is therefore important to investigate the responses of microbial aggregates to CNPs. Periphytic biofilm, a typical microbial aggregate, is an important ecological component of surface water and plays a major role in primary productivity, nutrient transformation and biological removal of pollutants (Wu et al., 2012). Some studies have examined the effect of nanoparticles (NPs) on the physiology and function of periphytic biofilm, such as photosynthetic yield and respiration potential (Gil-Allué et al., 2015), oxidative damage (Hou et al., 2015a), and the ability to remove pollutants (Xu et al., 2017). These studies, however, focused on the responses of biofilms using short-term (e.g. 2 h) or physiological investigations during NP (e.g. Ag-NP) exposure (Gil-Allué et al., 2015), with little attention paid to the distribution and transformation of NPs or the changes in microbial communities. Periphytic biofilm consists of a variety of microbial components including bacteria, algae, fungi, protozoa and metazoa (Shabbir et al., 2017). The sensitivity of different species to NP toxicity differs which implies a potential community shift in a microbial
3
community exposed to NPs (Li et al., 2015; Wang et al., 2011). The microbial composition and diversity of periphytic biofilm play an important role in its resistance to external environmental stresses and maintaining its functions including pollutant removal (Wu et al., 2011). Thus, it is hypothesized that the periphytic biofilm exposed to CNPs will maintain its pollutant removal performance via self-regulation of its microbial composition and diversity. The distribution and transformation of CNPs can directly affect the physiology and function of microbial cells (Li et al., 2017a). Benthic microbial aggregates, such as periphytic biofilm, are a sink for CNPs when they agglomerate and settle at the bottom of water (Ferry et al., 2009; Schug et al., 2014). The fate of CNPs in the environment is critical when studying microbial cell responses as the transference of CNPs controls the bioaccumulation. In addition, the Ce (III) / Ce (IV) redox cycle in cells affects the generation of reactive oxygen species (ROS) (Tella et al., 2014). It is difficult, however, to determine the distribution and transformation of CNPs, at both cell and community level, under high resolution using common microscopy techniques such as transmission electron microscopy (TEM). Synchrotron radiation scanning transmission X-ray microscopy (STXM), an advanced in situ imaging technology, can quantitatively study the distribution and speciation of elements in vivo and has been used to explore the fate of CNPs in plants such as cucumber and soybean (Hernandez-Viezcas et al., 2013; Zhang et al., 2012). The STXM was employed in this study to study the distribution and transformation of CNPs in periphytic biofilm at both cell and community levels.
4
In this study, we investigated the effects of CNPs on periphytic biofilm under prolonged exposure (30 d), using high-throughput sequencing (HTS) and STXM to reveal the microbial community changes and the fate of CNPs in periphytic biofilm. The objectives of this study were to (i) investigate the effects of CNPs on the physiological properties and pollutant (phosphorus, nitrogen and copper, organic matter) removal by periphytic biofilm, (ii) determine the microbial community composition and diversity responses, and (iii) determine the distribution and transformation of CNPs in periphytic biofilm at both cell and community level. 2. Materials and methods 2.1 Preparation of CNP solutions and periphytic biofilm CNPs were supplied by Aladdin (Shanghai, China) and the purity was over 99.7%. A 100 mg/L CNP stock suspension was prepared through the addition of 0.1 g of CNPs to 1 L of Milli-Q water (pH 7) followed by 30 min of ultra-sonication (100 W, 25 KHz, 25 °C). Then, the CNP stock suspension was diluted to 5 mg/L with Woods Hole culture medium (WC medium) (Hametner et al., 2014). This was referred to as CNP solution. The prepared CNP solutions were used for the exposure experiments. The original periphytic biofilm was collected from Xuanwu Lake, East China. The lake water parameters were: pH = 7.8; TP = 0.1 mg/L; TN = 1.90 mg/L; NH4+ = 0.53 mg/L; and NO3- = 0.73 mg/L. To stabilize and homogenize the periphytic biofilm, the collected material was mixed and cultured in WC medium in an incubator at 28 ± 1 °C with a light intensity of 2800 Lux and light/dark regime of 12 h/12 h. The periphytic 5
biofilm used in the following experiments was obtained by peeling it off the substrate using a sterilized silicone spatula when the biofilm thickness exceeded 5 mm. The dry mass to wet mass ratio of periphytic biofilm was 0.0532 ± 0.0085 (n = 10). Thus, a 5% ratio was selected as a standard for dry weight (DW) conversion of periphytic biofilm. The periphytic biofilm mass throughout the study is expressed as DW. 2.2 Experimental processes To conduct the exposure experiments, 0.05 g periphytic biofilm was added into flasks filled with 100 mL CNP solution. Then, the periphytic biofilm was cultured in an incubator (light power: 2800 lux, light/dark regime: 12 h/12 h, temperature: 28 ± 1 °C) under shaking conditions (120 rpm) for 30 d. To better investigate the response of the periphyton at the community level, the exposure experiments were performed for a long period of 30 d. The control (CK) was made by adding 0.05 g periphytic biofilm into 100 mL pure WC medium followed by periphyton culture under the same conditions as the CNP treatment. All experiments were performed in triplicate. Removal of organic matter, copper, nitrogen and phosphorus by periphytic biofilm was carried out after the exposure experiment to evaluate the influence of CNPs on the pollutant removal performance of periphytic biofilm. When the exposure experiment was completed, the CK and CNP exposed periphytic biofilm were removed from their flasks and rinsed three times with 100 mL Milli-Q water. Then, 0.05 g of CK and CNP periphytic biofilm were added into separate flasks with 100 mL wastewater and cultured in an incubator under the same experimental conditions used for the exposure 6
experiment. The main parameters of the wastewater were as follows: COD = 159.26 ± 3.12 mg/L; TN = 3.92 ± 0.19 mg/L; TP = 1.15 ± 0.11 mg/L; NO3- = 2.76 ± 0.21 mg/L; NH4+ = 0.52 ± 0.09 mg/L; PO43- = 0.76 ± 0.08 mg/L; Cu2+ = 2.95± 0.12 mg/L; Ca2+ = 2.6 ± 0.19 mg/L; and Zn2+ = 0.25 ± 0.02 mg/L. The pH was adjusted to 7.0 using 0.1M NaOH or HCl solution as required. The pollutant removal experiment was conducted in triplicate. 2.3 In situ distribution and speciation by STXM To avoid potential confusion caused by mapping CNPs inside cells due to CNP adsorption by extracellular polymeric substances (EPS) and the cell wall, EPS and NPs adsorbed by cell walls were removed before STXM analysis by the combination of ultra-sonication and EDTA treatment, followed by rinsing three times using a 0.05% (w/w) NaCl solution following (Liang et al., 2010). STXM imaging based on Ce M4 edge X-ray absorption near edge structure (XANES) was performed on the beamline BL08U1 at the Shanghai Synchrotron Radiation Facility (SSRF Shanghai, China). CeO2 and CeF3 (Aladdin, Shanghai, China) were chosen as the Ce (IV) and Ce(III) reference materials, respectively. The standard materials were ultrasonically dispersed in ethanol and deposited on tinfoil fixed on a sample holder and examined to obtain the standard spectrum. An aliquot of the periphytic biofilm sample was placed onto an X-ray transparent silicon nitride membrane (Silson Ltd, Northampton, UK) and a second silicon nitride membrane was aligned and placed on top of the periphytic biofilm; the silicon nitride sandwich was then sealed with acid-free silicone sealant and examined by 7
STXM (Dynes et al., 2006). A dual-energy method was then performed on the chosen regions of the sample and the Ce element map was calculated to obtain the distribution of the Ce component in the chosen regions (Zhang et al., 2012). 2.4 Sample and analyses The total antioxidant capacity (T-AOC) of the periphytic biofilm was determined according to a previous method (Li et al., 2017b). The minimal chlorophyll fluorescence (F0) was measured using an AquaPen-P fluorometer (Photon Systems Instruments, Brno, Czech Republic) after 25 min dark adaption. Total phosphorus (TP) and total nitrogen (TN) in the wastewater were measured by the standard methods (APHA-AWWA-WEF, 1998). Organic matter (represented by chemical oxygen demand, COD) in the wastewater was determined according to the standard potassium dichromate digestion method (GB11914-89) of the Ministry of Environmental Protection of China (Wei, 2002). The concentration of Cu2+ in the wastewater was measured by ICP-MS (7700x, Agilent, America) after filtration. The 16S rDNA high-throughput sequencing by Illumina MiSeq was employed to characterize the microbial composition and diversity of the periphytic biofilm. The processes and analyses of high-throughput sequencing were according to our previous study (Shangguan et al., 2015). The cell damage and distribution of the CNPs in microbial cells were determined by TEM (HT-7700, Hitachi, Japan) and the sample preparation was according to (Jian et al., 2016). The composition image of algae and bacteria in periphytic biofilm was detected by confocal scanning laser microscopy (CSLM) (LSM 710, Zeiss, Germany). 8
The
intracellular
ROS
of
periphytic
biofilm
was
labeled
by
10
µM
2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA) for 30 min and measured by fluorescence microscopy (Nikon-80i, Nikon, Japan) at excitation/emission wavelengths of 495/525 nm (Hou et al., 2015a). 2.5 Data analyses and statistics The results were expressed as mean ± standard deviation and the significance of differences between means was evaluated at the significance level p < 0.05. Statistical analyses were performed using SPSS 19.0 and assay data were tested by One-way analysis of variance (ANOVA). The figures were drawn using Sigmaplot 12.0. 3. Results and discussion 3.1 Responses of periphytic biofilm to CNP exposure 3.1.1 Effects of CNPs on the physiological characters of periphytic biofilm Total antioxidation capacity (T-AOC), a sensitive and reliable marker to detect changes in oxidative stress in vivo, was used to determine the oxidative damage of periphytic biofilm induced by CNPs. Results showed that the T-AOC of the periphytic biofilm exposed to CNPs was decreased by 57.74% compared with CK. This result indicates that the CNPs reduced the antioxidant ability of periphytic biofilm and could inhibit the cellular activity of the microorganisms in periphytic biofilm, which further implied that some sensitive communities in the periphytic biofilm were under stress from CNPs. 9
It is well known that CNPs, and several other metal NPs, such as Ag-NPs and ZnO-NPs, have the ability to generate ROS and stimulate the peroxidation of cell membrane lipids (Gu et al., 2014; Ma et al., 2013). It has been demonstrated that NPs attacking the respiratory chain in cell to induce the incomplete reduction of oxygen and the potential redox properties between Ce(Ⅲ) and Ce(Ⅳ) states of CNPs both contribute to the generation of ROS by CNPs in cells (Zhang et al., 2011). Excessive ROS generation and oxidative injury induced by CNP exposure could significantly reduce the levels of cellular antioxidants and destroy the antioxidant system (Dogra et al., 2016; Park et al., 2008). In this study, the T-AOC of periphytic biofilm showed a significant decrease under exposure to CNPs, indicating that periphytic biofilm is sensitive to CNPs and the antioxidant system failed to maintain cellular redox equilibrium. Therefore, the main toxic effects of CNPs on periphytic biofilm were production of excess ROS and decrease of T-AOC, resulting in oxidative stress and cell damage. To estimate the effects of CNPs on photosynthetic characteristics of periphytic biofilm, the change of F0 with time was determined (Fig. 1). The F0 of periphytic biofilm exposed to CNPs decreased with time in the initial stages of exposure and reached the lowest level at day 15. It then increased and reached a stable level at day 30, with an 11.3% increase from day 15. Variation of F0 in the CK followed an opposite trend to the CNP periphytic biofilm. F0 is a basic photosynthetic character and is directly proportional to chlorophyll content (McKew et al., 2011). Therefore, the F0 increase in the recovery stage of
10
periphytic biofilm exposed to CNPs might be induced by the increasing proportion of algae. This is consistent with the results of a previous study that determined NP exposure could disturb the balance between algae and bacteria and promote the growth of algae in an algal–bacterial symbiosis system (Li et al., 2015). 3.1.2 Changes in community composition and diversity of periphytic biofilm By performing OTU picks at 97% identity level, 255 and 329 OTUs based on 36743 and 42012 reads were obtained from the CNP exposed periphytic biofilm and CK, respectively (Table 1). The Chao 1 and Shannon indexes can be used to analyze the microbial richness and diversity (Ma et al., 2017). The Chao 1 richness estimators in the CNP exposed periphytic biofilm were significantly lower than the CK (p < 0.05), suggesting that the species richness of periphytic biofilm decreased in the presence of CNPs. However, the Shannon index in CNP exposed periphytic biofilm was significantly higher than the CK (p < 0.05), indicating a higher microbial diversity in biofilm exposed to CNPs. Under the prolonged exposure to CNPs, some sensitive species of periphytic biofilm may be inhibited leading to a decrease in community species richness. The proportion of the species in periphytic biofilm becomes more balanced and results in higher species evenness, which could be reflected by the increased microbial diversity (Ma et al., 2017; Wang et al., 2016). At the phylum level (Fig. 2), 12 main phyla were detected in the two periphytic biofilm communities including Firmicutes, Cyanobacteria, Gemmatimonadetes, Proteobacteria, Planctomycetes, Bacteroidetes, and Chloroflexi, which cover most of 11
the bacterial phyla found in natural aquatic environments (Liu et al., 2016). In CK, Firmicutes, Cyanobacteria, Gemmatimonadetes were the dominant species, accounting for 42.09%, 10.80%, and 21.94%, respectively. After exposure to CNPs, Cyanobacteria, Proteobacteria and Planctomycetes became the prominent phyla in the periphytic biofilm with abundances of 34.41%, 12.37%, and 15.63%, respectively. It was noted that the abundance of Firmicutes underwent a marked decline from 42.09% to 3.32% when exposed to CNPs, while the abundance of Cyanobacteria increased from 10.80% to 34.41%. To better understand the changes in microbial composition and diversity of periphytic biofilm exposed to CNPs, the microbial communities were also analyzed at the class and genus levels. At the class level, the relative abundances of Bacilli and Gemmatimonadetes sharply reduced from 41.80% and 21.94% to 3.29% and 9.80%, respectively, after the prolonged exposure to CNPs. The abundances of Cyanobacteria, Planctomycetacia and Alphaproteobacteria increased from 10.80%, 1.61% and 3.94% to 34.41%, 15.52% and 10.61%, respectively. The top 100 most abundant genera were used to evaluate the community shifts of periphytic biofilm exposed to CNPs. The abundances of algae such as Nostoc and Leptolyngbya increased from 0.08% and 2.37% to 13.28% and 13.27%, respectively, and became the dominant species in the periphytic biofilm. Reflected by the variation of F0 with time (Fig 1), the proportion of algae in periphytic biofilm exposed to CNPs decreased with time during short-term exposure (15
12
d) and then increased to a stable high level after long-term exposure (30 d). Further investigation by microbial composition analysis showed the main community shifts under long-term exposure to CNPs were the increase in abundances of algae such as Nostoc and Leptolyngbya and the decrease of some sensitive bacteria such as Bacilli and Gemmatimonadetes. Algae such as Nostoc and Leptolyngbya not only have thicker cell walls, but also a mucilaginous sheath to protect the cells (Reale et al., 2015). The NPs therefore might have difficulty entering the cells of these algae in the periphytic biofilm, leading to less inhibition of algae growth. As a result, the periphytic biofilm microbial community acclimated by having more algae but less bacteria and a higher microbial diversity under the long-term exposure to CNPs. 3.2 CNPs in periphytic biofilms 3.2.1 The distribution and transformation of CNPs STXM images of Ce (IV) and Ce (III) distribution in periphyton exposed to CNPs were obtained. At the community level, CNPs were inclined to enter the cells of pelletor rod-like bacteria such as Bacilli and Gemmatimonadetes. In algae cells with filamentary (Leptolyngbya) and spherical (Nostoc) structure, few CNPs were detected. At the cellular level, more CNPs were distributed around the cytomembrane than inside the cell which might induce serious membrane damage. Meanwhile, both Ce (IV) and Ce (III) were detected in cells with the content of Ce (IV) higher than Ce (III). The element Ce exists as Ce (IV) in the CNPs, while both Ce (IV) and Ce (III) could be detected in the cells, which indicates the transformation of Ce (IV) of CNPs to Ce (III) 13
in cells (Zhang et al., 2012). Therefore, these results show that the transformation of CNPs could be detected in periphyton cells, but that Ce (IV) was still the main form under prolonged exposure. Although TEM combined with energy spectrum analysis (EDS) can reflect the distribution of NPs in cells based on cell sections, it fails to reveal the distribution of NPs in the whole microbial community and distinguish element speciation (Jian et al., 2016). According to the STXM results, more CNPs internalization and transformation of Ce (IV) to Ce (III) could be detected in the bacterial cells than algal cells, which means that the CNPs might induce more damage to bacteria than algae. Meanwhile, the HTS results showed that the periphytic biofilm community changed to consist of more algae but less bacteria, which was consistent with the distribution of CNPs in different microbial species cells. Therefore, STXM in the present study showed the CNP distribution at the community level in periphyton and revealed the transformation of Ce (IV) and Ce (III) in cells. The uneven distribution of CNPs in different microbial species confirmed that algae were not sensitive to CNP exposure, and resulted in community changes. The typical characteristics of periphytic biofilm, such as EPS and community composition, are important factors that could affect the distribution and transformation of NPs in biofilm. EPS plays an important role in the stability of NPs in the environment and could prevent the NPs from entering the cells, which could further influence the distribution and transformation of NPs (You et al., 2015). Previous studies
14
have illustrated that the CNPs could both be absorbed on the EPS surrounding the biofilm and penetrate the cells (Hou et al., 2015b; You et al., 2017). There was, however, no clear understanding about the distribution of CNPs inside biofilm between cells of different species. Earlier studies have found that NPs could more easily penetrate bacterial cells (Escherichia coli) that only have simple cell walls, than the algal cells (Synechocystis) that have thick mucilaginous sheaths and complex cells walls (Zeyons et al., 2009). In the present study, we revealed that more CNPs could be detected in the bacterial cells than the algal cells in periphytic biofilm. Therefore, more EPS located outside the cells and higher algae proportion could be a greater benefit to the resistance against NPs for periphytic biofilm. Furthermore, CNPs could be reduced in organisms such as plants (cucumber plants) due to the reducing substances secreted by plants such as reducing sugar (glucose and fructose) and enzymes (reductases and ferredoxins) (Ma et al., 2015). Therefore, the reducing substances in EPS or inside cells may contribute to the transformation of CNPs in biofilm, but further study is needed to reveal where the transformation occurs and the mechanism underlying the transformation. 3.2.2 Cell damage induced by CNPs uptake To better investigate the impacts of CNPs on periphyton caused by direct NP distribution and transformation, intracellular ROS generation and cell structure damage were detected by fluorescence microscopy and TEM. Microorganisms in periphytic biofilm mainly include algae such as Leptolyngbya and Nostoc with strong spontaneous chlorophyll fluorescence, but also spherical bacterial cells without chlorophyll 15
fluorescence (CSLM image). After exposure to CNPs, the obvious generation of ROS in the spherical bacterial cells could be detected by fluorescent probes, while in the algae there was no obvious ROS generation. The generation of ROS in periphyton exposed to CNPs was consistent with the distribution of CNPs in the periphyton community. TEM images may better reflect the damage caused by CNPs on the cell structure. In bacterial cells, serious structural damage on the cell membrane and even some cell lysis could be observed in periphytic biofilm exposed to CNPs. Many CNPs got into the spherical and rod cells and some CNPs were also released with cell lysis. The algal cells exposed to CNPs were, however, intact because fewer CNPs entered these algal cells due to the protection of the thick mucilaginous sheath which could be clearly observed surrounding the cells. Accordingly, the CNPs tended to enter the bacterial cells with simple cell structures rather than algae cells with thick mucilaginous sheaths, which is one of the main reasons for more damage to bacterial cells. Therefore, the growth of bacteria such as Bacilli and Gemmatimonadetes was significantly inhibited, while the growth of algae such as Nostoc and Leptolyngbya was promoted, thereby leading to changes in the community composition of the periphytic biofilm with prolonged exposure to CNPs. 3.3 Performance in removing pollutants Organic matter (represented by COD), heavy metals such as copper, and nutrients such as nitrogen and phosphorus compounds, are common pollutants in surface water. The removal of COD, Cu, TP and TN of wastewater by periphytic biofilm exposed to
16
CNPs and the CK were investigated (Fig. 3). There were no significant differences in the COD, Cu, TP and TN concentrations in wastewater over time between the periphytic biofilm exposed to CNPs and the control. Concentrations of these pollutants all decreased with time and reached removal rates of 66.7%, 61.7%, 82.5% and 80.9% for COD, TP, TN and Cu, respectively. This result implies that the periphytic biofilms exposed to CNPs were capable of maintaining their pollutant removal functioning to levels found in the CK periphytic biofilms. Interestingly, CNP exposure caused obvious adverse effects on the physiology (T-AOC and F0) of periphytic biofilm, but the performance of COD, TP, TN and Cu removal by the CNP periphytic biofilm was not influenced. Other studies obtained similar results showing that although exposure to NPs, such as Fe3O4, ZnO and TiO2, could damage some sensitive microbial cells by generation of ROS and inhibiting enzyme activity, the microbial aggregates could still maintain their pollutant removal performance (Ma et al., 2017; Mu et al., 2011). Compared to a single microbial species, periphytic biofilm consists of a heterogeneous hierarchy of trophic levels resulting in a relatively robust nature to resist external environmental stressors via community composition regulation (Wu et al., 2011). This is an acclimation process where some sensitive species are inhibited by external environmental stress while the adapted species survive in the community and periphytic biofilm develops into a new balanced community that could maintain the important functions such as pollutant removal (Hu et al., 2017; Wu et al., 2011). The regulation of microbial communities in periphytic
17
biofilm to adapt to external environmental stress, such as exposure to heavy metals (Yang et al., 2016), organic pollutants (Shangguan et al., 2015) and azo dye (Shabbir et al., 2017), has been revealed in previous studies. In the present study, the community composition and diversity of periphytic biofilm changed significantly to contain more algae and less sensitive bacteria under prolonged exposure to CNPs. The periphytic biofilm could therefore maintain its COD, TP, TN and Cu removal performance via community shifts despite exposure to CNPs causing negative effects on physiology. According to the STXM and TEM results, the distribution of CNPs in different microbial species cells was uneven with more CNPs entering the bacterial cells than algal cells and inducing more serious damage to the bacterial cells. As a result, the growth of bacteria was inhibited while the algae gained a competitive advantage in the community, which eventually lead to the periphytic biofilm consisting of more algae and less bacteria under the prolonged exposure to CNPs. Therefore, the periphytic biofilm community shifts were mainly caused by the uneven distribution of CNPs at the community level which resulted in more serious cell damage to sensitive bacteria. Our results showed that although individual microorganisms were affected by CNPs exposure, the ability of periphytic biofilm to remove pollutants was sustained via community regulation. Therefore, periphytic biofilm is suitable for use in the treatment of mixed wastewater containing both NPs and other pollutants (COD, TP, TN and Cu). Further research is needed to (i) reveal how the characteristics of the NPs and biofilm influence the distributions and transformations of CNPs in biofilm and (ii)
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identify the interactions of CNPs with biomolecules both outside and inside cells. 4 Conclusion This study demonstrated uneven distribution of CNPs in the periphytic biofilm and the subsequent induced community shifts. The periphytic biofilm could maintain its COD, Cu, TP and TN removal performance via adaptation of community composition although some sensitive bacterial growth was negatively affected by CNP exposure. This study was an in-depth examination of the whole process of how CNPs affect periphytic biofilm and provide valuable information to better understand the fate of metal NPs in microbial aggregates.
Acknowledgments
We sincerely thank Dr Clare Morrison (Griffith University) for proof-reading this manuscript. 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). A part of the work was supported by the Shanghai Synchrotron Radiation Facility. Appendix A. Supplementary data Supplementary data associated with this article can be found in the online version. 19
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Figure Captions
Fig. 1. Changes in chlorophyll fluorescence (F0) of periphytic biofilm exposed to CNPs and CK. * p < 0.05. Fig. 2. Microbial community composition at the phylum level of periphytic biofilm exposed to CNPs and CK. Fig. 3. The abilities of periphytic biofilm exposed to CNPs and CK to remove organic matter (represented by COD), total phosphorus (TP), total nitrogen (TN) and copper (Cu) from wastewater.
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Fig. 1
24
Fig. 2
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Fig. 3
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Table 1. Statistical summary for the pyrosequencing and microbial diversity analysis. Sample
Reads
OTU
Chao 1 richness
Shannon diversity
CK CNPs
42012 36743
329 209
365 (347, 401) 248 (226, 300)a
3.02 (3.00, 3.04) 3.44 (3.42, 3.45)a
a
The data reported are statistical differences (p < 0.05) from the control.
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Highlights
CeO2 nanoparticles (NPs) distribution was detected by scanning transmission X-ray microscopy.
Uneven distribution of CeO2 NPs induced microbial community shifts.
Community composition of periphytic biofilm was investigated at phylum, class and genus level.
Pollutants removal was sustained via self-regulation of community composition.
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Graphical abstract