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Response of biofilms-leaves of two submerged macrophytes to high ammonium
Accepted Manuscript Response of biofilms-leaves of two submerged macrophytes to high ammonium Lixue Gong, Songhe Zhang, Deqiang Chen, Kaihui Liu, Jian Lv PII:
S0045-6535(17)31569-2
DOI:
10.1016/j.chemosphere.2017.09.147
Reference:
CHEM 20020
To appear in:
ECSN
Received Date: 3 July 2017 Revised Date:
20 September 2017
Accepted Date: 30 September 2017
Please cite this article as: Gong, L., Zhang, S., Chen, D., Liu, K., Lv, J., Response of biofilmsleaves of two submerged macrophytes to high ammonium, Chemosphere (2017), doi: 10.1016/ j.chemosphere.2017.09.147. 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.
ACCEPTED MANUSCRIPT Response of biofilms-leaves of two submerged macrophytes to high
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ammonium
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Lixue Gong1, Songhe Zhang1, *, Deqiang Chen1, Kaihui Liu1, Jian Lv2
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Development on Shallow Lakes, College of Environment, Hohai University, Nanjing
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210098, China.
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Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai,
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Ministry of Education Key Laboratory of Integrated Regulation and Resource
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Shandong 264003, PR China.
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*
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Email: [email protected];
Corresponding author
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Key Laboratory of Coastal Environmental Processes and Ecological Remediation,
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ACCEPTED MANUSCRIPT Abstract: Submerged macrophytes can provide attached surface for biofilms (known
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as periphyton) growth. In the present study, the alterations in biofilms formation, and
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chemical compositions and physiological responses were investigated on leaves of
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Vallisneria asiatica and Hydrilla verticillata exposed to 0.1 mg L-1 (control) or with
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10 mg L-1 NH4+-N for 13 days. Results from physiological and biochemical indices
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(content of H2O2, malondialdehyde, total chlorophyll and activity of superoxide
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dismutase, catalase and peroxidase) showed that high ammonium caused oxidative
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damage to leaves of two species of plant. Multifractal analysis (based on scanning
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electron microscope images) showed that for the same plant, the values of width △α
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(△α=αmax-αmin) of the f(α) and ∆f (∆f = f(αmin)−f(αmax)) were smaller on leaves surface
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of two species of plant treated with 10 mg L-1 NH4+-N for 13 days than their controls,
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suggesting high ammonium treatments reduced morphological heterogeneity of leaf
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surface and enhanced area of the colony-like biofilms. X-ray photoelectron
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spectroscopy analysis showed that C, O, N and P were dominant elements on leaves
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surface of two species of plant and ammonium application increased the percentage of
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C but decreased that of O. High ammonium increased C1 (C-C or C-H) percentage
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but decreased C2 (C-O) and C3 (O-C-O or C=O) percentage on leaves surface of two
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species of plant, indicating that ammonium stress changed the surface chemical states
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and thus might reduce the capacity of leaves to adsorb nutrients from water column.
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Our results provided useful information to understand ammonium induced toxicity to
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submerged macrophytes.
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Keywords: multifractal analysis, X-ray photoelectron spectroscopy, functional
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components, ammonia nitrogen
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1. Introduction Submerged macrophytes growing under water surface are conspicuous
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components of shallow water environments (Xiao, 2014; Phillips et al., 2016), but
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they have declined extensively in eutrophic lakes due to the high nutrient (Wersal and
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Madsen, 2011), toxic substances (Nuttens et al., 2016), sediments organic enrichment
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(Raun et al., 2010), periphyton (Peterson et al., 2007) and low light (Lyche-Solheim et
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al., 2013) in recent decades. Especially, high nitrogen concentrations in water column
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have been considered as key factors causing the decline of species richness of
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submerged macrophytes in shallow lakes (James et al., 2005; Barker et al., 2008).
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Among dissolved nitrogen species, ammonium has been linked to the decrease of
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submerged macrophytes in water column (Wang et al., 2008; Yuan et al., 2013).
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Ammonium concentration above 5 mg L-1 NH4+-N that can can be considered as
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excess/high ammonium, because it can induce toxicity to submersed macrophyte
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(Wang et al., 2008 and 2010; Meng et al., 2010; Gao et al., 2015). High ammonium
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can increase free amino acids content and photorespiration, induce oxidative stress,
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affect the carbon and nitrogen metabolisms and inhibit the photosynthetic system and
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plant growth of submerged macrophytes (Wang et al., 2008; Cao et al., 2011). Though
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abundant data have been documented about the effects of ammonium on submerged
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macrophytes, the ammonium-toxicity remains far from clear.
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Aquatic plants can provide attached surface for bacteria, algae and other
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microorganisms settlement forming microbial communities (known as biofilms or
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periphyton) (Michael et al., 2008). Biofilms and submerged macrophytes compete for
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dissolved inorganic carbon in water column (Jones et al., 2002). In addition, excess
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boifilms have negative effects on their hosts through creating physical barriers to
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nutrient uptake and gas exchange, or a combination of these factors (Drake et al.,
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2003). Furthermore, the growth of epiphytic algae directly induces oxidative stress on
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V. natans and epiphytic algal biomass, which are positively correlated with nutrient
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(nitrogen and phosphorus) available in the water column (Song et al., 2015).
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Moreover, algae in biofilms can form high-oxygen, high pH, and low-carbon micro
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(Jones and Hardwick, 2000; Song et al., 2015). These data provided us useful
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information in understanding the biofilm-induced stresses on submerged macrophytes.
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However, little is known about the biofilms distribution and its potential impact on
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leaf surface properties of submerged macrophytes under high ammonium.
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Fractal analysis is a powerful tool to describe the self-similarity between the tiny
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part and the whole and to explain the complex nonlinear phenomena in nature
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(Mandelbrot, 1967; Bialowiec et al., 2010). Therefore, it has been used as a statistical
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method and mathematical model to find all possible sub-divisions of the object
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(Peitgen et al., 2004), while multifractal describes a measurement that is defined in a
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certain area or volume and can decompose the defined domain into a series of
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sub-domains in space, and according to the singularity of this measurement every
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sub-domain constitutes a single fractal (Zhang et al., 2010b). Multifractal analysis has
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been used to investigate the structural and fractal characteristics of biofilms attached
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to the surface of plants and gravels (Liang et al., 2013).
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X-ray photoelectron spectroscopy (XPS) is one of the most effective tools for
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surface analysis and is able to obtain a wealth of chemical information (Cai et al.,
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2014), because XPS survey spectra could provide the inner electron binding energy of
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almost all elements except H and He within a few surface nanometers (10 nm ) of
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materials (Matuana et al., 2001; Wepasnick et al., 2010). XPS has been used to
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describe the leaf surface properties of terrestrial plants, which are directly related with
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leaf material absorption, disease resistance and other effects (Stevens and Baker,
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1987).
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In the present study, an experiment was conducted to examine the effects of high
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ammonium on leaf-biofilms of Vallisneria asiatica (V. asiatica) and Hydrilla
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verticillata (H. verticillata). We analyzed the biofilms distribution, morphological and
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physiological alterations on plant surface and antioxidant defense system in leaves of
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two species of plants under 10 mg L-1 NH4+-N. The study was to test the following
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hypotheses: high ammonium may (1) enhance the growth of biofilms on leaves of two 4
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species of plant; (2) alter the morphological characteristics of leaves surface of two
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species of plant; and (3) change the oxidative properties of plant leaves.
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2. Materials and methods 2.1. Experimental setup
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Healthy plants of submerged macrophytes, V. asiatica and H. verticillata, were
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used in this experiment. After rinsed with tap water, these plants were acclimated in
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140 L (750*550*430 mm) plastic container with 70 mm sediments and 260 mm water
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depth for two weeks in greenhouse. V. asiatica and H. verticillata (initial optical
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density of 1:1) were treated with 0.1 mg L-1 (control) and 10 mg L-1 NH4+-N (high
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ammonium) for 13 days according to previous reports (Wang et al., 2008; Gao et al.,
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2015; Cao et al., 2011). To maintain the NH4+-N concentration, the ammonium
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concentrations were determined by continuous flow Analyzer Auto Analyzer3 (SEAL,
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Germany) every day. The water was replaced every two days to maintain the content
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of NH4+-N using static methods. In the present study, the plant samples were
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harvested at 13 days post treatments. Four replicates were used for each treatment of
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each species of plant.
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2.2. Scanning electron microscope (SEM) and multifractal analysis
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SEM was used to visualize the distribution of bacteria and algae on the biofilms
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surface. For SEM analysis, plant leaves were cut into 0.6 × 0.6 cm squares, then,
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further fixed in 2.5% glutaraldehyde solution. After a double rinse with 0.1 M sodium
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phosphate buffer (PBS, pH 7.4), leaf samples were dehydrated with a serial of ethanol
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concentration (30, 50, 70, 80 and 90%) for 15 min and with three times of 100%
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ethanol for 15 min. Dried samples were analyzed by SEM (Hitachi, Japan, S-3400N
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Ⅱ).
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The combination of scanning electron microscopy and fractal theory is an
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effective tool to characterize the microstructure of biofilms, because it can reveal the
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discipline and essential connection which hide behind the complex phenomenon, part
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and entirety (Liang et al., 2013). In this study, box counting method was applied 5
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according to zhang et al (Zhang et al., 2010b). Briefly, the black-white SEM images
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were divided into many boxes of size ε×ε, where ε=1/L (L=256, 128, 64, 32, 4, 2 or 1).
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To calculate the multifractal spectrum, the biofilms value distribution probability in
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the box (i, j) was expressed as
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(1)
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Where is the biofilms value of the box (i, j) of size ε.
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Pij(ε) can be described as multifractal as
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(2)
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(3)
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where the exponent α depending upon the box (i, j) is the singularity of the
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subset of probabilities, the number of boxes of size with the same biofilms value
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distribution probability, and the fraction dimension of the α subset. The dependence of
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on α is the multifractal spectrum. Generally, the fractal dimension can be obtained
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from the partition function expressed as a power law of ε with an exponent applied in
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statistical physics as following:
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(4)
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where q is the moment order (−∞< q <∞), which
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can be obtained from the
slope of ln(χq(ε)) ~ ln(ε) curve. And the generalized fractal dimension Dq is defined
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as:
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(5)
f(α) can be obtained by performing Legendre transformation as followings: (6)
Dq=
and -
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(7)
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The value of q cannot be infinite in real calculation in practical calculations
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(Raoufi et al., 2008). Based on suggestions from (Wickens et al., 2014), we take the 6
ACCEPTED MANUSCRIPT maximum |q| as 10 if |dαmax|/∆α and |dαmin|/∆α were all less than 0.1%. The width of
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the multifractal spectrum is ∆α and the difference of the fractal dimensions of the
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maximum probability (α=αmin) and the minimum one (α=αmax) is ∆f (∆f =
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f(αmin)−f(αmax)).
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2.3. X-ray photoelectron spectroscopy (XPS) analysis
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XPS was employed to analyze the nutrient element compositions and oxidation
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state of leaf surface under high ammonia nitrogen. For XPS analysis, fresh healthy
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leaves were dried in the oven at 45℃ for 12 h after washing with distilled water. The
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charge
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electron-neutralizing gun. The XPS analyses were carried out with a PHI (5000
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VersaProbe, UK) spectrometer with a hemispherical energy analyser and using a
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monochromatic Al K a source (1486.6 eV). As the delay-line detector allows a high
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count rate the power applied to the X-ray anode was reduced to 25 W so that the
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possible X-ray induced degradation of the sample was minimized. High-resolution
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spectra were collected using an analysis area of ≈300 nm × 700 nm and either a 58.7
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eV pass energy. The takeoff angle was 90° in all measurements. The binding energies
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of the peaks were determined using the C1s peak at 284.6 eV (Fang and Wan, 2008).
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XPS peaks corresponding to C and O can be analyzed by deconvoluting using
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Gussian -Lorentzian mixed curve fitting (Kontturi, 2005).
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2.4. Aquatic parameters determinations and cells counting
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neutralization
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Parameters including dissolved oxygen
(DO), pH, oxidation-reduction
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potential (ORP), electrical conductivity (EC) was examined in water by Portable
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Instruments (YSI, WEISS instrument，American) every day. NH4+-N content in the
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water was determined by AtuoAnalyzer 3 system (SEAL, Germany). The values of
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DO, pH, EC and ORP were provided in Figure S1 (See supplementary materials).
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For cells counting, approximately 1 g leaf samples from healthy plants growing
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at 0–20 cm below the water surface were harvested to prevent contamination from
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sediments and was transferred into a sterile 100 mL polyethylene bottle containing 40 7
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were shook for 30 min at 225 r min-1 and exposed to ultra-sonication for 3 min. Three
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extracted suspensions from the same sample were combined and fixed with 2%
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formaldehyde. The 100 µL suspension samples was stained with 10 µg mL−1 4,
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6-diamino-2-phenyl indole (EDTA, 700 µL) and incubated in the dark for 30 min. The
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samples were filtered through a 0.22 µm incubated black filter. The numbers of
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bacteria on the black membrane were counted under a fluorescence microscope
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(ZEISS, Germany). According to our previous reports, biofilms can be formed rapidly
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and stabled within 10 dyas (Zhang et al., 2016). Therefore, the cell counting were
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conducted for biofilms samples at 1 day (initial microbe densities) and 13day (stable
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microbe densities).
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2.5. Physiological parameters determinations
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About 0.2 g sample of fresh leaf samples was ground in liquid nitrogen for
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chlorophyll. After extraction with 10 ml of 96% ethanol for 24 h in darkness at 4◦C,
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the samples were centrifuged at 10,000×g for 10 min. The absorbance of supernatant
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was determined at 649 and 665 nm. The contents of total chlorophyll (a+b) were
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calculated according to the previous described method (Wang et al., 2008).
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About 0.5 g fresh leaf samples was used for H2O2 determination (Wang et al.,
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2008). After ground in liquid nitrogen, leaf sample was homogenized in 3 mL of 50
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mM sodium PBS (pH 6.5) and then was centrifuged at 8000×g for 20 min. For the
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determination of H2O2, a mixture was prepared with suitable of supernatant and 2.5
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ml of 0.1% titanium sulfate in 20% (v/v) H2SO4 and centrifuged at 10000×g for 5 min.
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The absorbance of supernatant was measured at 410 nm, and the absorbance values
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were calibrated against a standard curve generated with known concentrations of
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H2O2.
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Malondialdehyde (MDA) was measured to indicate polyunsaturated fatty acid
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oxidation as a secondary end product and lipid peroxidation intensity and the injury
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degree of membrane system (Gunes et al., 2007; Li and Ni, 2009). About 0.5 g 8
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method described in previous report (Wang et al., 2010). Briefly, samples were mixed
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with 5ml of trichloroacetic acid (1%, TCA). The mixtures were centrifuged at
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15,000×g for 10 min, and 0.5 ml of supernatant was mixed with 2 ml of 20% TCA
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containing 0.5% 2-Thiobarbituric acid and heated at 96 ºC for 30 min. After cooled at
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0ºC, the mixtures were centrifuged at 10,000×g for 5 min at 4 ºC and the absorbance
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of supernatant was at 532 and 600 nm. The MDA concentration was calculated from
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the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155
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mM-1cm-1.
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2.6. Assay of Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD)
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activity
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For enzyme extraction, 1 g of plant leaf samples was ground in liquid nitrogen
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and mixed with 10 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1
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mM EDTA and 1% polyvinylpyrrolidone. The mixture was centrifuged at 15,000×g
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for 20 min at 4◦C, and the supernatant was used for the enzyme assays and protein
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determination. The determinations of protein content, SOD, CAT and POD activity
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were based on the previous described method (Wang et al., 2011).
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2.7. Data processing
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One-way analysis of variance was used to compare differences between
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treatments using SPSS 18.0 (IBM, USA) (p<0.05). Origin8.0 (OriginLab Corp, USA)
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was used to process XPS data analysis as well as fractal analysis.
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3. Results and discussion
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3.1.Biofilms community and growth analysis Figure 1
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Nutrient loading is currently the most documented factor influencing epiphyte
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communities (Michael et al., 2008). As revealed by SEM images, bacteria, algae and
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microbial aggregates were observed on leaves surface of V. asiatica and H.
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verticillata in all treatments (Fig. 1). As compared with control plants (Fig. 1a and 1c)
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and plants at 1d (Fig. S2a and S2b), biofilms almost covered all the leaf surface,
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especially on leaves of H. verticillata exposed to 10 mg L-1 NH4+-N for 13 days
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(Fig.1d). Microbes including algae, cocci and Bacillus were also detected on the
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surface of submerged macrophytes under high nitrate (Zhang et al., 2016).
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In control groups, the microbe densities increased from 9.73 × 105 to 4.46 ×
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107cells g-1 Fresh Weight on leaves of H. verticillata and from 3.02 × 106 to 5.17 ×
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107cells g-1 Fresh weight on leaves of V. asiatica after 13 days (Fig. 1e and 1f). After
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treatments with 10 mg L-1 NH4+-N for 13 days, microbe densities increased
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significantly in biofilms from two species of plant and reached to 1.63 × 108 cells g-1
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Fresh weight and 3.79 × 108 cells g-1 Fresh weight, respectively, on leaves of V.
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asiatica and H. verticillata (Fig. 1e and 1f). Previous report has shown that high
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nutrimental (combinations of nitrogen and phosphorus) concentrations contributed to
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the overgrowth of the epiphytic algae on aquatic macrophytes in shallow lakes (Song
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et al., 2015), while our results demonstrated that ammonium application alone could
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also stimulate the biofilms growth.
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3.2. Multifractal analysis of leaf biofilms
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Table 1
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There are complex interactions between biofilms and its hosts. Surface
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topography and chemistry properties of substrates have an effect on cell dispersion, 10
ACCEPTED MANUSCRIPT density and clustering of microbes in biofilms (Wickens et al., 2014). Our previous
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report showed that the microbes in biofilms can be affected by substrates,
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environmental parameters and the distributions of biofilms on leaves of submerged
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macrophytes (Zhang et al., 2016). However, little is known the impacts of ammonium
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application on the distribution of biofilms. In this study, multifractal analysis was
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employed to investigate whether ammonium application can alter the morphological
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characterizations of leaf surface based on SEM images.
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In multifractal analysis, the linear regions in the graph of the curve cluster are
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known as scaling regions, and curve plot for all moments of q should be strict
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linearity that can tend to zero infinitely (ln ε→−∞). A random multifractal must have
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preferable linearity (Raoufi et al., 2008). With the different biofilms as examples,
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representative plots of ln(χq(ε)) ~ ln(ε) for values of q (ranged from -10 to 10) were
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generated (Fig. S2c and S2d). For images from treatments at 13th days, the favorable
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linear correlation were observed in the ln(χq(ε)) ~ ln(ε) plot (Fig. S2d), while linearity
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was not observed for images from two species of plant at first day (Fig.S2c). These
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results indicated that obvious multifractal characteristics can be observed on surface
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structures of leaf biofilms of two species of plant at 13th day.
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Figure 2
Multifractal spectra f(α) of the samples from two plants at 13th day and their
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important parameters were shown in Fig. 2 and Table 1, respectively. Hook-like
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multifractal spectrums f(α) of leaf biofilms at 13th day were obtained and showed that
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the shape and the width △α (△α=αmax-αmin) of the f(α) of plants treated with
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ammonium were different from their controls (Fig. 2). The αmin and αmax are the
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variables of singularity indices of the smallest and biggest biofilms area distribution
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rules with the changes of ε, respectively. The smaller αmin is, the bigger the maximum
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probability will be; and the bigger αmax is, the smaller the minimum probability will
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be. Thus the span of singularity indexes △α can quantitatively describe the degree of
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heterogeneity of biofilms distribution probability in biofilms. The width
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△α(△α=αmax-αmin) of the f(α) for leaf biofilms of V. asiatica and H. verticillata plants
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the width ∆α (∆α=∆αmax−∆αmin) of the multifractal spectra f(α) (Zhang et al., 2010b),
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in other words, the more the roughness will be. For the same plants, the ∆α (∆α=∆αmax
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−∆αmin) of control samples were higher than that of samples exposed to high
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ammonium (Table 1), suggesting that biofilm growth reduced the roughness of leaf
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surface.
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The ∆f of control was higher than that of treatments with ammonium for 13th day
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for the same plants (Table 1). The f(α) can be used to depict the distribution of
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microbes (from single clone to blocks) on plant leaves. The f(αmin) and f(αmax) showed
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the minimum and maximum subsets of biofilms distribution probability, respectively.
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The ∆f (∆f = f(αmin)−f(αmax) is the number difference between the maximum and
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minimum probability subsets. When big probability subsets predominate, ∆f > 0;
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when small probability subsets predominate, ∆f < 0. All ∆f
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0 (Table 1), indicating that the microbes in subset of maximum biofilms distribution
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probability is larger than that in the minimum subset. In this study, the △f value of
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control was bigger than that of samples exposed to high ammonia nitrogen, indicating
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the distribution probability of single colony were higher on control than that of
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samples exposed to 10 mg L-1 NH4+-N. Namely, high ammonium application
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enhanced the growth of single colony-like biofilms.
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3.3.Response of chemical properties on leaf surface to ammonium
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Figure 3
Leaf surface properties are closely related to their absorption of nutrients, gas
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exchange, regulation of transpiration and secretion (Zhang et al., 2010b). XPS
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technology has been widely used to characterize surface morphology and chemistry of
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plant materials (Cai et al., 2014). In the present study, XPS was employed to
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determine the chemical composition on leaves of two species of plant. Elements C, O,
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N and P were detected on leaves surface of two species of plant, while Cl and K were
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only detected in V. asiatica (Table S1 and Fig. 3). In general, the relative percentages 12
ACCEPTED MANUSCRIPT of C increased from 70.42% to 74.71% and from 78.45% to 83.66% on leaves surface
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of V. asiatica and H. verticillata, respectively, but decreased that of O, from 22.92%
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to 16.11% and from 15.53% to 11.9%, on leaf surfaces of V. asiatica and H.
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verticillata, respectively. Especially, the relative percentages of N increased from
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5.64% to 7.99% on leaf surfaces of V. asiatica, but decreased from 5.52% to 4.11% on
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leaf surfaces of H. verticillata. While no significant alterations in percentages of P
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were observed. These data demonstrated that ammonium application altered chemical
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composition of leaf surface.
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There were significant differences in the ratios of oxygen to carbon and nitrogen
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to carbon on different plant species. The ratio values of O/C and N/C were 0.33 and
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0.08, respectively in the control groups of V. asiatica and were 0.22 and 0.11 in the
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treatment groups for V. asiatica, respectively. Meanwhile, the values of the O/C and
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N/C were 0.20 and 0.07, respectively, in the control groups of V. asiatica, and the
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corresponding values were 0.14 and 0.05 in the treatment groups for H. verticillata
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(Table S1), respectively. Since ratio values of O/C can be used as an indicator of
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smooth or roughness of wood surface (Sinn et al., 2001), in this study, the decreased
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O/C ratio indicated that high ammonium reduced roughness of leaf-biofilms surface.
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That was in accord with the results from multifractal analysis. The alterations in N/C
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ratio may be ascribed to the instability of hydroxyl group in Cn(H2O)m that the amino
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group can bind to Cn(H2O)m by replacing the hydroxyl group.
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Figure 4
Fig. 4 shows the high-resolution C 1s signal following curve-fitting and three
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components of C were detected in all samples. The major component C1 (at ~284.4
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eV) is due to hydrocarbon (C-C or C-H). Peak value of C2 at ~285.8 eV consisted of
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carbon connect with a non-carbonyl oxygen (C-O), while C3 (at 287.1-287.8 eV)
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express the carbon attach to two non-carbonyl group oxygen (O-C-O) or a carbonyl
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oxygen (C=O). C1, C2 and C3 of C 1s peaks of leaves could indicate cuticle,
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cutinized layer and oxidation state, respectively (Cai et al., 2014). In the present study, 13
ACCEPTED MANUSCRIPT C1 was the dominant component, followed by C2 and C3 (Fig. 4). Percentages of C2
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and C3 were higher on leaves surface of control than that of ammonia-treated plants.
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Component Cl reflect aliphatic chains, while components C2 and C3 reflect hydroxyl,
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epoxy and ether functions (Genet et al., 2002). The decrease of C2 and C3 in this
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study can be explained that ammonia treatment decreased the contents of
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nonstructural carbohydrate, soluble sugar, sucrose, fructose, and starch in leaves of
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submerged macrophytes (Genet et al., 2002).
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Figure 5.
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High-resolution XPS O 1s peak was decomposed into two components fixed at
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about 531.2 eV ( O1) and 532.6 eV (O2), and attributed to oxygen making a double
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bond (O=C) and one or two single bonds (-O-) with carbon, respectively (Fig. 5).
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After ammonia treatment, there lative peak area ratio of C1/C2 and C1/C3 increased
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from 1.7 to 3.86 and from 5.06 to 7.42 for V. asiatica (Fig. 4a), respectively, and
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increased from 1.35 to 1.77 and 3.56 to 5.53 for H. verticillata (Fig. 4b), respectively;
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while that of O1/O2 decreased from 1.05 to 0.96 and from 1.05 to 0.99 for V. asiatica
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and H. verticillata after 10 mg L-1 NH4+-N, respectively. The reduction of C3 and O1
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(the same function group) suggests that the surface chemical states were more active
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in control plants than in plants exposed to 10 mg L-1 NH4+-N. A reduction in chemical
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active reflects a reduced capacity of leaves to adsorb the nutrients from water for two
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species of plant under ammonium stress.
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3.4.Alterations in intracellular oxidation/reduction status
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Figure 6
Reactive oxygen species (including H2O2) can be actively induced under various
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stresses. An increase of H2O2 concentration, respectively, 2.92 and 1.41 times of their
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control, was detected in leaves of V. asiatica and H. verticillata treated with 10 mg L-1
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NH4+-N for 13 days (Fig. 6a), suggesting that the cell tended to be an oxidation status
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under ammonium stress. Excess reactive oxygen species could induce the production
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of MDA, which can be produced from polyunsaturated fatty acid oxidation as a 14
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increased in two species of plant under ammonium stress, indicating cell membrane
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were damaged contributing to the leakage of plant cells (Wang et al., 2008; Wang et
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al., 2010). The activities of SOD, CAT and POD were visibly higher in the leaves of
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two species of plant at the 10 mg L-1 NH4+-N than those in their controls (Fig. 6d-f).
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SOD dismutase superoxide to H2O2, which can be decomposed to water and oxygen
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by the activities of CAT and POD (Wang et al., 2008). These results indicate that
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excessive energy is consumed by plants for biosynthesis of these enzymes to cope
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with ammonia-induced oxidative stress.
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Compared with the control, the total chlorophyll content of the V. asiatica and H.
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verticillata decreased significantly with 10 mg L-1 NH4+-N at the 13th day (p<0.05)
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(Fig. 6c). In the present study, dissolved oxygen concentration was not altered
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obviously in overlying water at 1-7 days post ammonium application, but decreased
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vastly after 7 days of ammonia exposure (Fig. S2a). The decrease of total chlorophyll
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content may be ascribed to the interaction effect of biofilms and ammonia toxicity and
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contribute to the decrease of dissolved oxygen concentrations in overlying water (Fig.
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S2a).
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Biofilms growth can have negative impacts on plant cells. For example, increase
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of biofilms area can directly increase light attenuation (Xie et al., 2013), and
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periphyton can attenuate most of the light reaching the adaxial leaves surface of
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Potamogeton perfoliatus (Toth, 2013). Low light and high nutrients can induce the
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activities of antioxidant enzymes including SOD and POD in submerged macrophytes
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Potamogeton crispus (Zhang et al., 2010a). Ammonium application stimulated the
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biofilms growth, which potentially changed the nutrient compositions and the
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chemical activity of leaf biofilms surface (revealed by XPS). Excess biofilms can also
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enhance oxidative stress (Song et al., 2015). These data suggested that biofilms
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contributed to ammonium-induced stress on submersed macrophytes.
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4. Conclusions
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asiatica and H. verticillata were investigated. The batch experiments illustrated that
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high ammonia nitrogen promoted the growth of biofilms on the leaf surface, while
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biofilms growth altered the surface topography and chemistry activity of leaf surfaces
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of two species of plant exposed to high ammonium. In addition, the leaf surface
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mainly consisted of C, N, O and P. The ratio of O/C on leaves surface of V. asiatica
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and H. verticillata decreased after treating with 10 mg L-1 NH4+-N for 13 days,
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suggesting that the chemical properties of the leaf surface were more active in the
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control group than that of plants exposed to 10 mg L-1 NH4+-N. Moreover, 10 mg L-1
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NH4+-N induced oxidative stress and antioxidant system stress response in two
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species of plant. This study provided useful information for understanding the
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oxidative mechanism of ammonium stress on submerged macrophytes.
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Acknowledgements
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This study was, in part, supported by Grants from the National Natural Science
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Foundation of China (Grant No. 51379063 and 51579075), the Natural Science
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Foundation of JiangSu Province for Excellsent Young Scholars (BK20160087), the
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Fundamental Research Funds for the Central Universities (2016B06714) and a Project
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Funded by the Priority Academic Program Development of Jiangsu Higher Education
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Institutions.
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Competing financial interests
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The authors declare no competing financial interests.
Supplementary materials Fig.S1. The daily alterations in dissolved oxygen concentration (a),
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oxidation-reduction potential (ORP) (b), pH (c) and electrical conductivity (d) during
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experiment.
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Fig. S2. Representative Plots of ln(χq(ε)) ~ ln(ε) for values of q for leaf-biofilms of macrophytes at initial (a) and at 13 day (b). 16
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Table S1. Elements compositions on leaf surface of submerged macrophytes (%)
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ACCEPTED MANUSCRIPT Figure captions Figure 1. SEM image (a-d) and biomass (e and f) of biofilms attached to leaves of V. asiatica (a, b and e) and H. verticillata (c, d and f) treated without (a and c) or
extracellular polymers；②, diatoms；③, crystal.
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with ammonia nitrogen (10 mg L-1, b and d). ① , organic matter or
Figure 2. Multifractal spectra f(α) of leaf-biofilm of Vallisneria asiatica (a) and Hydrilla verticillata (b) treated with/without 10 mg L-1 NH4+-N.
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Figure 3. Surface XPS spectra of Binding energy-Intensity on Vallisneria asiatica (a
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and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10
Figure 4. High-resolution XPS C1s spectrum of Binding energy-Intensity on from Vallisneria asiatica (a and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10 mg L-1 NH4+-N (b and d).
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Figure 5. High-resolution XPS O 1s spectrum of Binding energy-Intensity on from Vallisneria asiatica (a and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10 mg L-1 NH4+-N (b and d).
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Figure 6. Changes of different concentrations of NH4+-N on antioxidant system at
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Table 1. Multifractal parameters of leaf surface from two submersed macrophytes. Plants treatments αmin αmax ݂(αmin) ݂(αmax) △α △݂ Control 1.63 3.22 1.06 0.26 1.59 0.8 -1 V. asiatica 10 mg L 1.87 2.18 1.61 1.31 0.31 0.3 NH4-N Control 1.78 3.51 1.47 0.1 1.73 1.37 H. -1 10 mg L verticillata 1.63 2.91 1.18 0.49 1.28 0.69 NH4-N
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Table 2. Elements compositions on leaf surface of submersed macrophytes (%) V. asiatica H. verticillata Element -1 Control 10 mg L NH4-N Control 10 mg L-1 NH4-N C 70.42±0.11 74.71±0.29* 78.45±0.42 83.66±0.77* O 22.92±0.27 16.11±1.24* 15.53±0.65 11.9±1.21* N 5.64±0.42 7.99±1.17* 5.52±0.51 4.11±0.62* P 0.21±0.02 0.26±0.03 0.21±0.12 0.33±0.09 Cl 0.26±0.03 0.30±0.06 \ \ K 0.55±0.05 0.60±0.02 \ \ O/C 0.33 0.22 0.2 0.14 N/C 0.08 0.11 0.07 0.05 Note: "\" in the table indicates that the element content is below the detection limit * indicate that mean values are significantly different between NH4-N treatments and controls for the same plant (p<0.05).
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High ammonium enhanced biofilm growth Biofilm growth altered the topography leaf surface High ammonium altered chemical composition of leaf surface Biofilm growth might contribute to ammonium-induced toxicity
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DOI:
10.1016/j.chemosphere.2017.09.147
Reference:
CHEM 20020
To appear in:
ECSN
Received Date: 3 July 2017 Revised Date:
20 September 2017
Accepted Date: 30 September 2017
Please cite this article as: Gong, L., Zhang, S., Chen, D., Liu, K., Lv, J., Response of biofilmsleaves of two submerged macrophytes to high ammonium, Chemosphere (2017), doi: 10.1016/ j.chemosphere.2017.09.147. 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.
ACCEPTED MANUSCRIPT Response of biofilms-leaves of two submerged macrophytes to high
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ammonium
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Lixue Gong1, Songhe Zhang1, *, Deqiang Chen1, Kaihui Liu1, Jian Lv2
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Development on Shallow Lakes, College of Environment, Hohai University, Nanjing
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210098, China.
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2
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Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai,
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Ministry of Education Key Laboratory of Integrated Regulation and Resource
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Shandong 264003, PR China.
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*
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Email: [email protected];
Corresponding author
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Key Laboratory of Coastal Environmental Processes and Ecological Remediation,
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ACCEPTED MANUSCRIPT Abstract: Submerged macrophytes can provide attached surface for biofilms (known
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as periphyton) growth. In the present study, the alterations in biofilms formation, and
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chemical compositions and physiological responses were investigated on leaves of
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Vallisneria asiatica and Hydrilla verticillata exposed to 0.1 mg L-1 (control) or with
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10 mg L-1 NH4+-N for 13 days. Results from physiological and biochemical indices
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(content of H2O2, malondialdehyde, total chlorophyll and activity of superoxide
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dismutase, catalase and peroxidase) showed that high ammonium caused oxidative
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damage to leaves of two species of plant. Multifractal analysis (based on scanning
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electron microscope images) showed that for the same plant, the values of width △α
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(△α=αmax-αmin) of the f(α) and ∆f (∆f = f(αmin)−f(αmax)) were smaller on leaves surface
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of two species of plant treated with 10 mg L-1 NH4+-N for 13 days than their controls,
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suggesting high ammonium treatments reduced morphological heterogeneity of leaf
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surface and enhanced area of the colony-like biofilms. X-ray photoelectron
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spectroscopy analysis showed that C, O, N and P were dominant elements on leaves
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surface of two species of plant and ammonium application increased the percentage of
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C but decreased that of O. High ammonium increased C1 (C-C or C-H) percentage
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but decreased C2 (C-O) and C3 (O-C-O or C=O) percentage on leaves surface of two
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species of plant, indicating that ammonium stress changed the surface chemical states
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and thus might reduce the capacity of leaves to adsorb nutrients from water column.
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Our results provided useful information to understand ammonium induced toxicity to
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submerged macrophytes.
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Keywords: multifractal analysis, X-ray photoelectron spectroscopy, functional
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components, ammonia nitrogen
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1. Introduction Submerged macrophytes growing under water surface are conspicuous
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components of shallow water environments (Xiao, 2014; Phillips et al., 2016), but
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they have declined extensively in eutrophic lakes due to the high nutrient (Wersal and
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Madsen, 2011), toxic substances (Nuttens et al., 2016), sediments organic enrichment
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(Raun et al., 2010), periphyton (Peterson et al., 2007) and low light (Lyche-Solheim et
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al., 2013) in recent decades. Especially, high nitrogen concentrations in water column
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have been considered as key factors causing the decline of species richness of
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submerged macrophytes in shallow lakes (James et al., 2005; Barker et al., 2008).
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Among dissolved nitrogen species, ammonium has been linked to the decrease of
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submerged macrophytes in water column (Wang et al., 2008; Yuan et al., 2013).
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Ammonium concentration above 5 mg L-1 NH4+-N that can can be considered as
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excess/high ammonium, because it can induce toxicity to submersed macrophyte
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(Wang et al., 2008 and 2010; Meng et al., 2010; Gao et al., 2015). High ammonium
53
can increase free amino acids content and photorespiration, induce oxidative stress,
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affect the carbon and nitrogen metabolisms and inhibit the photosynthetic system and
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plant growth of submerged macrophytes (Wang et al., 2008; Cao et al., 2011). Though
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abundant data have been documented about the effects of ammonium on submerged
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macrophytes, the ammonium-toxicity remains far from clear.
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Aquatic plants can provide attached surface for bacteria, algae and other
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microorganisms settlement forming microbial communities (known as biofilms or
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periphyton) (Michael et al., 2008). Biofilms and submerged macrophytes compete for
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dissolved inorganic carbon in water column (Jones et al., 2002). In addition, excess
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boifilms have negative effects on their hosts through creating physical barriers to
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nutrient uptake and gas exchange, or a combination of these factors (Drake et al.,
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2003). Furthermore, the growth of epiphytic algae directly induces oxidative stress on
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V. natans and epiphytic algal biomass, which are positively correlated with nutrient
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(nitrogen and phosphorus) available in the water column (Song et al., 2015).
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Moreover, algae in biofilms can form high-oxygen, high pH, and low-carbon micro
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(Jones and Hardwick, 2000; Song et al., 2015). These data provided us useful
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information in understanding the biofilm-induced stresses on submerged macrophytes.
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However, little is known about the biofilms distribution and its potential impact on
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leaf surface properties of submerged macrophytes under high ammonium.
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Fractal analysis is a powerful tool to describe the self-similarity between the tiny
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part and the whole and to explain the complex nonlinear phenomena in nature
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(Mandelbrot, 1967; Bialowiec et al., 2010). Therefore, it has been used as a statistical
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method and mathematical model to find all possible sub-divisions of the object
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(Peitgen et al., 2004), while multifractal describes a measurement that is defined in a
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certain area or volume and can decompose the defined domain into a series of
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sub-domains in space, and according to the singularity of this measurement every
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sub-domain constitutes a single fractal (Zhang et al., 2010b). Multifractal analysis has
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been used to investigate the structural and fractal characteristics of biofilms attached
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to the surface of plants and gravels (Liang et al., 2013).
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X-ray photoelectron spectroscopy (XPS) is one of the most effective tools for
84
surface analysis and is able to obtain a wealth of chemical information (Cai et al.,
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2014), because XPS survey spectra could provide the inner electron binding energy of
86
almost all elements except H and He within a few surface nanometers (10 nm ) of
87
materials (Matuana et al., 2001; Wepasnick et al., 2010). XPS has been used to
88
describe the leaf surface properties of terrestrial plants, which are directly related with
89
leaf material absorption, disease resistance and other effects (Stevens and Baker,
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1987).
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In the present study, an experiment was conducted to examine the effects of high
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ammonium on leaf-biofilms of Vallisneria asiatica (V. asiatica) and Hydrilla
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verticillata (H. verticillata). We analyzed the biofilms distribution, morphological and
94
physiological alterations on plant surface and antioxidant defense system in leaves of
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two species of plants under 10 mg L-1 NH4+-N. The study was to test the following
96
hypotheses: high ammonium may (1) enhance the growth of biofilms on leaves of two 4
ACCEPTED MANUSCRIPT 97
species of plant; (2) alter the morphological characteristics of leaves surface of two
98
species of plant; and (3) change the oxidative properties of plant leaves.
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2. Materials and methods 2.1. Experimental setup
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Healthy plants of submerged macrophytes, V. asiatica and H. verticillata, were
102
used in this experiment. After rinsed with tap water, these plants were acclimated in
103
140 L (750*550*430 mm) plastic container with 70 mm sediments and 260 mm water
104
depth for two weeks in greenhouse. V. asiatica and H. verticillata (initial optical
105
density of 1:1) were treated with 0.1 mg L-1 (control) and 10 mg L-1 NH4+-N (high
106
ammonium) for 13 days according to previous reports (Wang et al., 2008; Gao et al.,
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2015; Cao et al., 2011). To maintain the NH4+-N concentration, the ammonium
108
concentrations were determined by continuous flow Analyzer Auto Analyzer3 (SEAL,
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Germany) every day. The water was replaced every two days to maintain the content
110
of NH4+-N using static methods. In the present study, the plant samples were
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harvested at 13 days post treatments. Four replicates were used for each treatment of
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each species of plant.
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2.2. Scanning electron microscope (SEM) and multifractal analysis
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SEM was used to visualize the distribution of bacteria and algae on the biofilms
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surface. For SEM analysis, plant leaves were cut into 0.6 × 0.6 cm squares, then,
116
further fixed in 2.5% glutaraldehyde solution. After a double rinse with 0.1 M sodium
117
phosphate buffer (PBS, pH 7.4), leaf samples were dehydrated with a serial of ethanol
118
concentration (30, 50, 70, 80 and 90%) for 15 min and with three times of 100%
119
ethanol for 15 min. Dried samples were analyzed by SEM (Hitachi, Japan, S-3400N
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Ⅱ).
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The combination of scanning electron microscopy and fractal theory is an
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effective tool to characterize the microstructure of biofilms, because it can reveal the
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discipline and essential connection which hide behind the complex phenomenon, part
124
and entirety (Liang et al., 2013). In this study, box counting method was applied 5
ACCEPTED MANUSCRIPT 125
according to zhang et al (Zhang et al., 2010b). Briefly, the black-white SEM images
126
were divided into many boxes of size ε×ε, where ε=1/L (L=256, 128, 64, 32, 4, 2 or 1).
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To calculate the multifractal spectrum, the biofilms value distribution probability in
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the box (i, j) was expressed as
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Where is the biofilms value of the box (i, j) of size ε.
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Pij(ε) can be described as multifractal as
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(2)
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(3)
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where the exponent α depending upon the box (i, j) is the singularity of the
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subset of probabilities, the number of boxes of size with the same biofilms value
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distribution probability, and the fraction dimension of the α subset. The dependence of
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on α is the multifractal spectrum. Generally, the fractal dimension can be obtained
138
from the partition function expressed as a power law of ε with an exponent applied in
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statistical physics as following:
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(4)
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where q is the moment order (−∞< q <∞), which
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can be obtained from the
slope of ln(χq(ε)) ~ ln(ε) curve. And the generalized fractal dimension Dq is defined
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as:
144 145 146 147
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(5)
f(α) can be obtained by performing Legendre transformation as followings: (6)
Dq=
and -
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(7)
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The value of q cannot be infinite in real calculation in practical calculations
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(Raoufi et al., 2008). Based on suggestions from (Wickens et al., 2014), we take the 6
ACCEPTED MANUSCRIPT maximum |q| as 10 if |dαmax|/∆α and |dαmin|/∆α were all less than 0.1%. The width of
152
the multifractal spectrum is ∆α and the difference of the fractal dimensions of the
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maximum probability (α=αmin) and the minimum one (α=αmax) is ∆f (∆f =
154
f(αmin)−f(αmax)).
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2.3. X-ray photoelectron spectroscopy (XPS) analysis
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XPS was employed to analyze the nutrient element compositions and oxidation
157
state of leaf surface under high ammonia nitrogen. For XPS analysis, fresh healthy
158
leaves were dried in the oven at 45℃ for 12 h after washing with distilled water. The
159
charge
160
electron-neutralizing gun. The XPS analyses were carried out with a PHI (5000
161
VersaProbe, UK) spectrometer with a hemispherical energy analyser and using a
162
monochromatic Al K a source (1486.6 eV). As the delay-line detector allows a high
163
count rate the power applied to the X-ray anode was reduced to 25 W so that the
164
possible X-ray induced degradation of the sample was minimized. High-resolution
165
spectra were collected using an analysis area of ≈300 nm × 700 nm and either a 58.7
166
eV pass energy. The takeoff angle was 90° in all measurements. The binding energies
167
of the peaks were determined using the C1s peak at 284.6 eV (Fang and Wan, 2008).
168
XPS peaks corresponding to C and O can be analyzed by deconvoluting using
169
Gussian -Lorentzian mixed curve fitting (Kontturi, 2005).
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2.4. Aquatic parameters determinations and cells counting
of
dried
sample
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neutralization
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Parameters including dissolved oxygen
(DO), pH, oxidation-reduction
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potential (ORP), electrical conductivity (EC) was examined in water by Portable
173
Instruments (YSI, WEISS instrument，American) every day. NH4+-N content in the
174
water was determined by AtuoAnalyzer 3 system (SEAL, Germany). The values of
175
DO, pH, EC and ORP were provided in Figure S1 (See supplementary materials).
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For cells counting, approximately 1 g leaf samples from healthy plants growing
177
at 0–20 cm below the water surface were harvested to prevent contamination from
178
sediments and was transferred into a sterile 100 mL polyethylene bottle containing 40 7
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180
were shook for 30 min at 225 r min-1 and exposed to ultra-sonication for 3 min. Three
181
extracted suspensions from the same sample were combined and fixed with 2%
182
formaldehyde. The 100 µL suspension samples was stained with 10 µg mL−1 4,
183
6-diamino-2-phenyl indole (EDTA, 700 µL) and incubated in the dark for 30 min. The
184
samples were filtered through a 0.22 µm incubated black filter. The numbers of
185
bacteria on the black membrane were counted under a fluorescence microscope
186
(ZEISS, Germany). According to our previous reports, biofilms can be formed rapidly
187
and stabled within 10 dyas (Zhang et al., 2016). Therefore, the cell counting were
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conducted for biofilms samples at 1 day (initial microbe densities) and 13day (stable
189
microbe densities).
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2.5. Physiological parameters determinations
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About 0.2 g sample of fresh leaf samples was ground in liquid nitrogen for
192
chlorophyll. After extraction with 10 ml of 96% ethanol for 24 h in darkness at 4◦C,
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the samples were centrifuged at 10,000×g for 10 min. The absorbance of supernatant
194
was determined at 649 and 665 nm. The contents of total chlorophyll (a+b) were
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calculated according to the previous described method (Wang et al., 2008).
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About 0.5 g fresh leaf samples was used for H2O2 determination (Wang et al.,
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2008). After ground in liquid nitrogen, leaf sample was homogenized in 3 mL of 50
198
mM sodium PBS (pH 6.5) and then was centrifuged at 8000×g for 20 min. For the
199
determination of H2O2, a mixture was prepared with suitable of supernatant and 2.5
200
ml of 0.1% titanium sulfate in 20% (v/v) H2SO4 and centrifuged at 10000×g for 5 min.
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The absorbance of supernatant was measured at 410 nm, and the absorbance values
202
were calibrated against a standard curve generated with known concentrations of
203
H2O2.
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Malondialdehyde (MDA) was measured to indicate polyunsaturated fatty acid
205
oxidation as a secondary end product and lipid peroxidation intensity and the injury
206
degree of membrane system (Gunes et al., 2007; Li and Ni, 2009). About 0.5 g 8
ACCEPTED MANUSCRIPT powdered leaf samples was used for MDA content determination according to the
208
method described in previous report (Wang et al., 2010). Briefly, samples were mixed
209
with 5ml of trichloroacetic acid (1%, TCA). The mixtures were centrifuged at
210
15,000×g for 10 min, and 0.5 ml of supernatant was mixed with 2 ml of 20% TCA
211
containing 0.5% 2-Thiobarbituric acid and heated at 96 ºC for 30 min. After cooled at
212
0ºC, the mixtures were centrifuged at 10,000×g for 5 min at 4 ºC and the absorbance
213
of supernatant was at 532 and 600 nm. The MDA concentration was calculated from
214
the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155
215
mM-1cm-1.
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2.6. Assay of Superoxide dismutase (SOD), Catalase (CAT) and Peroxidase (POD)
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activity
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For enzyme extraction, 1 g of plant leaf samples was ground in liquid nitrogen
219
and mixed with 10 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1
220
mM EDTA and 1% polyvinylpyrrolidone. The mixture was centrifuged at 15,000×g
221
for 20 min at 4◦C, and the supernatant was used for the enzyme assays and protein
222
determination. The determinations of protein content, SOD, CAT and POD activity
223
were based on the previous described method (Wang et al., 2011).
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2.7. Data processing
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One-way analysis of variance was used to compare differences between
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treatments using SPSS 18.0 (IBM, USA) (p<0.05). Origin8.0 (OriginLab Corp, USA)
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was used to process XPS data analysis as well as fractal analysis.
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3. Results and discussion
230
3.1.Biofilms community and growth analysis Figure 1
232
Nutrient loading is currently the most documented factor influencing epiphyte
233
communities (Michael et al., 2008). As revealed by SEM images, bacteria, algae and
234
microbial aggregates were observed on leaves surface of V. asiatica and H.
235
verticillata in all treatments (Fig. 1). As compared with control plants (Fig. 1a and 1c)
236
and plants at 1d (Fig. S2a and S2b), biofilms almost covered all the leaf surface,
237
especially on leaves of H. verticillata exposed to 10 mg L-1 NH4+-N for 13 days
238
(Fig.1d). Microbes including algae, cocci and Bacillus were also detected on the
239
surface of submerged macrophytes under high nitrate (Zhang et al., 2016).
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In control groups, the microbe densities increased from 9.73 × 105 to 4.46 ×
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107cells g-1 Fresh Weight on leaves of H. verticillata and from 3.02 × 106 to 5.17 ×
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107cells g-1 Fresh weight on leaves of V. asiatica after 13 days (Fig. 1e and 1f). After
243
treatments with 10 mg L-1 NH4+-N for 13 days, microbe densities increased
244
significantly in biofilms from two species of plant and reached to 1.63 × 108 cells g-1
245
Fresh weight and 3.79 × 108 cells g-1 Fresh weight, respectively, on leaves of V.
246
asiatica and H. verticillata (Fig. 1e and 1f). Previous report has shown that high
247
nutrimental (combinations of nitrogen and phosphorus) concentrations contributed to
248
the overgrowth of the epiphytic algae on aquatic macrophytes in shallow lakes (Song
249
et al., 2015), while our results demonstrated that ammonium application alone could
250
also stimulate the biofilms growth.
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3.2. Multifractal analysis of leaf biofilms
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Table 1
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There are complex interactions between biofilms and its hosts. Surface
254
topography and chemistry properties of substrates have an effect on cell dispersion, 10
ACCEPTED MANUSCRIPT density and clustering of microbes in biofilms (Wickens et al., 2014). Our previous
256
report showed that the microbes in biofilms can be affected by substrates,
257
environmental parameters and the distributions of biofilms on leaves of submerged
258
macrophytes (Zhang et al., 2016). However, little is known the impacts of ammonium
259
application on the distribution of biofilms. In this study, multifractal analysis was
260
employed to investigate whether ammonium application can alter the morphological
261
characterizations of leaf surface based on SEM images.
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In multifractal analysis, the linear regions in the graph of the curve cluster are
263
known as scaling regions, and curve plot for all moments of q should be strict
264
linearity that can tend to zero infinitely (ln ε→−∞). A random multifractal must have
265
preferable linearity (Raoufi et al., 2008). With the different biofilms as examples,
266
representative plots of ln(χq(ε)) ~ ln(ε) for values of q (ranged from -10 to 10) were
267
generated (Fig. S2c and S2d). For images from treatments at 13th days, the favorable
268
linear correlation were observed in the ln(χq(ε)) ~ ln(ε) plot (Fig. S2d), while linearity
269
was not observed for images from two species of plant at first day (Fig.S2c). These
270
results indicated that obvious multifractal characteristics can be observed on surface
271
structures of leaf biofilms of two species of plant at 13th day.
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Figure 2
Multifractal spectra f(α) of the samples from two plants at 13th day and their
274
important parameters were shown in Fig. 2 and Table 1, respectively. Hook-like
275
multifractal spectrums f(α) of leaf biofilms at 13th day were obtained and showed that
276
the shape and the width △α (△α=αmax-αmin) of the f(α) of plants treated with
277
ammonium were different from their controls (Fig. 2). The αmin and αmax are the
278
variables of singularity indices of the smallest and biggest biofilms area distribution
279
rules with the changes of ε, respectively. The smaller αmin is, the bigger the maximum
280
probability will be; and the bigger αmax is, the smaller the minimum probability will
281
be. Thus the span of singularity indexes △α can quantitatively describe the degree of
282
heterogeneity of biofilms distribution probability in biofilms. The width
283
△α(△α=αmax-αmin) of the f(α) for leaf biofilms of V. asiatica and H. verticillata plants
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285
the width ∆α (∆α=∆αmax−∆αmin) of the multifractal spectra f(α) (Zhang et al., 2010b),
286
in other words, the more the roughness will be. For the same plants, the ∆α (∆α=∆αmax
287
−∆αmin) of control samples were higher than that of samples exposed to high
288
ammonium (Table 1), suggesting that biofilm growth reduced the roughness of leaf
289
surface.
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The ∆f of control was higher than that of treatments with ammonium for 13th day
291
for the same plants (Table 1). The f(α) can be used to depict the distribution of
292
microbes (from single clone to blocks) on plant leaves. The f(αmin) and f(αmax) showed
293
the minimum and maximum subsets of biofilms distribution probability, respectively.
294
The ∆f (∆f = f(αmin)−f(αmax) is the number difference between the maximum and
295
minimum probability subsets. When big probability subsets predominate, ∆f > 0;
296
when small probability subsets predominate, ∆f < 0. All ∆f
297
0 (Table 1), indicating that the microbes in subset of maximum biofilms distribution
298
probability is larger than that in the minimum subset. In this study, the △f value of
299
control was bigger than that of samples exposed to high ammonia nitrogen, indicating
300
the distribution probability of single colony were higher on control than that of
301
samples exposed to 10 mg L-1 NH4+-N. Namely, high ammonium application
302
enhanced the growth of single colony-like biofilms.
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3.3.Response of chemical properties on leaf surface to ammonium
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were actually larger than
305
Figure 3
Leaf surface properties are closely related to their absorption of nutrients, gas
306
exchange, regulation of transpiration and secretion (Zhang et al., 2010b). XPS
307
technology has been widely used to characterize surface morphology and chemistry of
308
plant materials (Cai et al., 2014). In the present study, XPS was employed to
309
determine the chemical composition on leaves of two species of plant. Elements C, O,
310
N and P were detected on leaves surface of two species of plant, while Cl and K were
311
only detected in V. asiatica (Table S1 and Fig. 3). In general, the relative percentages 12
ACCEPTED MANUSCRIPT of C increased from 70.42% to 74.71% and from 78.45% to 83.66% on leaves surface
313
of V. asiatica and H. verticillata, respectively, but decreased that of O, from 22.92%
314
to 16.11% and from 15.53% to 11.9%, on leaf surfaces of V. asiatica and H.
315
verticillata, respectively. Especially, the relative percentages of N increased from
316
5.64% to 7.99% on leaf surfaces of V. asiatica, but decreased from 5.52% to 4.11% on
317
leaf surfaces of H. verticillata. While no significant alterations in percentages of P
318
were observed. These data demonstrated that ammonium application altered chemical
319
composition of leaf surface.
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There were significant differences in the ratios of oxygen to carbon and nitrogen
321
to carbon on different plant species. The ratio values of O/C and N/C were 0.33 and
322
0.08, respectively in the control groups of V. asiatica and were 0.22 and 0.11 in the
323
treatment groups for V. asiatica, respectively. Meanwhile, the values of the O/C and
324
N/C were 0.20 and 0.07, respectively, in the control groups of V. asiatica, and the
325
corresponding values were 0.14 and 0.05 in the treatment groups for H. verticillata
326
(Table S1), respectively. Since ratio values of O/C can be used as an indicator of
327
smooth or roughness of wood surface (Sinn et al., 2001), in this study, the decreased
328
O/C ratio indicated that high ammonium reduced roughness of leaf-biofilms surface.
329
That was in accord with the results from multifractal analysis. The alterations in N/C
330
ratio may be ascribed to the instability of hydroxyl group in Cn(H2O)m that the amino
331
group can bind to Cn(H2O)m by replacing the hydroxyl group.
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Figure 4
Fig. 4 shows the high-resolution C 1s signal following curve-fitting and three
334
components of C were detected in all samples. The major component C1 (at ~284.4
335
eV) is due to hydrocarbon (C-C or C-H). Peak value of C2 at ~285.8 eV consisted of
336
carbon connect with a non-carbonyl oxygen (C-O), while C3 (at 287.1-287.8 eV)
337
express the carbon attach to two non-carbonyl group oxygen (O-C-O) or a carbonyl
338
oxygen (C=O). C1, C2 and C3 of C 1s peaks of leaves could indicate cuticle,
339
cutinized layer and oxidation state, respectively (Cai et al., 2014). In the present study, 13
ACCEPTED MANUSCRIPT C1 was the dominant component, followed by C2 and C3 (Fig. 4). Percentages of C2
341
and C3 were higher on leaves surface of control than that of ammonia-treated plants.
342
Component Cl reflect aliphatic chains, while components C2 and C3 reflect hydroxyl,
343
epoxy and ether functions (Genet et al., 2002). The decrease of C2 and C3 in this
344
study can be explained that ammonia treatment decreased the contents of
345
nonstructural carbohydrate, soluble sugar, sucrose, fructose, and starch in leaves of
346
submerged macrophytes (Genet et al., 2002).
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Figure 5.
348
High-resolution XPS O 1s peak was decomposed into two components fixed at
349
about 531.2 eV ( O1) and 532.6 eV (O2), and attributed to oxygen making a double
350
bond (O=C) and one or two single bonds (-O-) with carbon, respectively (Fig. 5).
351
After ammonia treatment, there lative peak area ratio of C1/C2 and C1/C3 increased
352
from 1.7 to 3.86 and from 5.06 to 7.42 for V. asiatica (Fig. 4a), respectively, and
353
increased from 1.35 to 1.77 and 3.56 to 5.53 for H. verticillata (Fig. 4b), respectively;
354
while that of O1/O2 decreased from 1.05 to 0.96 and from 1.05 to 0.99 for V. asiatica
355
and H. verticillata after 10 mg L-1 NH4+-N, respectively. The reduction of C3 and O1
356
(the same function group) suggests that the surface chemical states were more active
357
in control plants than in plants exposed to 10 mg L-1 NH4+-N. A reduction in chemical
358
active reflects a reduced capacity of leaves to adsorb the nutrients from water for two
359
species of plant under ammonium stress.
360
3.4.Alterations in intracellular oxidation/reduction status
362
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Figure 6
Reactive oxygen species (including H2O2) can be actively induced under various
363
stresses. An increase of H2O2 concentration, respectively, 2.92 and 1.41 times of their
364
control, was detected in leaves of V. asiatica and H. verticillata treated with 10 mg L-1
365
NH4+-N for 13 days (Fig. 6a), suggesting that the cell tended to be an oxidation status
366
under ammonium stress. Excess reactive oxygen species could induce the production
367
of MDA, which can be produced from polyunsaturated fatty acid oxidation as a 14
ACCEPTED MANUSCRIPT secondary end product (Wang et al., 2008; Wang et al., 2010). MDA content (Fig. 6b)
369
increased in two species of plant under ammonium stress, indicating cell membrane
370
were damaged contributing to the leakage of plant cells (Wang et al., 2008; Wang et
371
al., 2010). The activities of SOD, CAT and POD were visibly higher in the leaves of
372
two species of plant at the 10 mg L-1 NH4+-N than those in their controls (Fig. 6d-f).
373
SOD dismutase superoxide to H2O2, which can be decomposed to water and oxygen
374
by the activities of CAT and POD (Wang et al., 2008). These results indicate that
375
excessive energy is consumed by plants for biosynthesis of these enzymes to cope
376
with ammonia-induced oxidative stress.
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Compared with the control, the total chlorophyll content of the V. asiatica and H.
378
verticillata decreased significantly with 10 mg L-1 NH4+-N at the 13th day (p<0.05)
379
(Fig. 6c). In the present study, dissolved oxygen concentration was not altered
380
obviously in overlying water at 1-7 days post ammonium application, but decreased
381
vastly after 7 days of ammonia exposure (Fig. S2a). The decrease of total chlorophyll
382
content may be ascribed to the interaction effect of biofilms and ammonia toxicity and
383
contribute to the decrease of dissolved oxygen concentrations in overlying water (Fig.
384
S2a).
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Biofilms growth can have negative impacts on plant cells. For example, increase
386
of biofilms area can directly increase light attenuation (Xie et al., 2013), and
387
periphyton can attenuate most of the light reaching the adaxial leaves surface of
388
Potamogeton perfoliatus (Toth, 2013). Low light and high nutrients can induce the
389
activities of antioxidant enzymes including SOD and POD in submerged macrophytes
390
Potamogeton crispus (Zhang et al., 2010a). Ammonium application stimulated the
391
biofilms growth, which potentially changed the nutrient compositions and the
392
chemical activity of leaf biofilms surface (revealed by XPS). Excess biofilms can also
393
enhance oxidative stress (Song et al., 2015). These data suggested that biofilms
394
contributed to ammonium-induced stress on submersed macrophytes.
395
4. Conclusions
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ACCEPTED MANUSCRIPT Effects of ammonium on biofilms and antioxidant responses in leaves of V.
397
asiatica and H. verticillata were investigated. The batch experiments illustrated that
398
high ammonia nitrogen promoted the growth of biofilms on the leaf surface, while
399
biofilms growth altered the surface topography and chemistry activity of leaf surfaces
400
of two species of plant exposed to high ammonium. In addition, the leaf surface
401
mainly consisted of C, N, O and P. The ratio of O/C on leaves surface of V. asiatica
402
and H. verticillata decreased after treating with 10 mg L-1 NH4+-N for 13 days,
403
suggesting that the chemical properties of the leaf surface were more active in the
404
control group than that of plants exposed to 10 mg L-1 NH4+-N. Moreover, 10 mg L-1
405
NH4+-N induced oxidative stress and antioxidant system stress response in two
406
species of plant. This study provided useful information for understanding the
407
oxidative mechanism of ammonium stress on submerged macrophytes.
408
Acknowledgements
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This study was, in part, supported by Grants from the National Natural Science
410
Foundation of China (Grant No. 51379063 and 51579075), the Natural Science
411
Foundation of JiangSu Province for Excellsent Young Scholars (BK20160087), the
412
Fundamental Research Funds for the Central Universities (2016B06714) and a Project
413
Funded by the Priority Academic Program Development of Jiangsu Higher Education
414
Institutions.
415
Competing financial interests
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The authors declare no competing financial interests.
Supplementary materials Fig.S1. The daily alterations in dissolved oxygen concentration (a),
419
oxidation-reduction potential (ORP) (b), pH (c) and electrical conductivity (d) during
420
experiment.
421 422
Fig. S2. Representative Plots of ln(χq(ε)) ~ ln(ε) for values of q for leaf-biofilms of macrophytes at initial (a) and at 13 day (b). 16
ACCEPTED MANUSCRIPT 423
Table S1. Elements compositions on leaf surface of submerged macrophytes (%)
424
5. References
425 426 427
Barker, T., Hatton, K., O'Connor, M., Connor, L., Moss, B., 2008. Effects of nitrate load on submerged
428 429
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extracellular polymers；②, diatoms；③, crystal.
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with ammonia nitrogen (10 mg L-1, b and d). ① , organic matter or
Figure 2. Multifractal spectra f(α) of leaf-biofilm of Vallisneria asiatica (a) and Hydrilla verticillata (b) treated with/without 10 mg L-1 NH4+-N.
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Figure 3. Surface XPS spectra of Binding energy-Intensity on Vallisneria asiatica (a
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and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10
Figure 4. High-resolution XPS C1s spectrum of Binding energy-Intensity on from Vallisneria asiatica (a and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10 mg L-1 NH4+-N (b and d).
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Figure 5. High-resolution XPS O 1s spectrum of Binding energy-Intensity on from Vallisneria asiatica (a and b) and Hydrilla verticillata (c and d) treated without (a and c) or with 10 mg L-1 NH4+-N (b and d).
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Figure 6. Changes of different concentrations of NH4+-N on antioxidant system at
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Table 1. Multifractal parameters of leaf surface from two submersed macrophytes. Plants treatments αmin αmax ݂(αmin) ݂(αmax) △α △݂ Control 1.63 3.22 1.06 0.26 1.59 0.8 -1 V. asiatica 10 mg L 1.87 2.18 1.61 1.31 0.31 0.3 NH4-N Control 1.78 3.51 1.47 0.1 1.73 1.37 H. -1 10 mg L verticillata 1.63 2.91 1.18 0.49 1.28 0.69 NH4-N
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Table 2. Elements compositions on leaf surface of submersed macrophytes (%) V. asiatica H. verticillata Element -1 Control 10 mg L NH4-N Control 10 mg L-1 NH4-N C 70.42±0.11 74.71±0.29* 78.45±0.42 83.66±0.77* O 22.92±0.27 16.11±1.24* 15.53±0.65 11.9±1.21* N 5.64±0.42 7.99±1.17* 5.52±0.51 4.11±0.62* P 0.21±0.02 0.26±0.03 0.21±0.12 0.33±0.09 Cl 0.26±0.03 0.30±0.06 \ \ K 0.55±0.05 0.60±0.02 \ \ O/C 0.33 0.22 0.2 0.14 N/C 0.08 0.11 0.07 0.05 Note: "\" in the table indicates that the element content is below the detection limit * indicate that mean values are significantly different between NH4-N treatments and controls for the same plant (p<0.05).
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High ammonium enhanced biofilm growth Biofilm growth altered the topography leaf surface High ammonium altered chemical composition of leaf surface Biofilm growth might contribute to ammonium-induced toxicity
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