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Sustained high nutrient supply as an allelopathic trigger between periphytic biofilm and Microcystis aeruginosa Yonghong Wu, Jun Tang, Junzhuo Liu, Bruce Graham, Philip G. Kerr, and Hong Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01027 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Environmental Science & Technology

TOC

TSI M. aeruginosa

171 Allelopathy occurred

159 119 Slight allelopathy occurred

107 79 43

Allelopathy occurred

ACS Paragon Plus Environment


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Sustained high nutrient supply as an allelopathic trigger between periphytic

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biofilm and Microcystis aeruginosa

3 Yonghong Wua*, Jun Tanga, c, Junzhuo Liua, Bruce Grahamb, Philip G. Kerrb, Hong Chena,c

4 5 6

a

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Academy of Sciences No.71, East Beijing Road, Nanjing 210008, China

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b

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NSW, Australia

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese

School of Biomedical Sciences, Charles Sturt University, Boorooma St, Wagga Wagga, 2678,

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c

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100049, China

College of Resource and Environment, University of Chinese Academy of Sciences, Beijing

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*

Corresponding author: Dr. Yonghong Wu

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Institute of Soil Science, Chinese Academy of Sciences

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71 Beijing East Road, Nanjing 210008, China

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Telephone: (86)-25-86881330

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Fax: (86)-25-86881000

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E-mail: [email protected]

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1


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ABSTRACT:

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Allelopathy among aquatic organisms, especially microorganisms, has received growing

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attention in recent years for its role in shaping interactions with bloom-forming algae. Many

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studies have shown that allelopathy occurs and increases under nutrient limiting conditons.

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However, to date there is no reported direct evidence to indicate that allelopathy occurs under the

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condition of constant high nutrient supply. Here we report the allelopathic action of periphytic

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biofilm on bloom-forming cyanobacteria (Microcystis aeruginosa), which was triggered by the

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stress of high nutrient conditions, and continues while nutrients are maintained at high levels

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(trophic state index at 159 and 171). The experimental evidence indicates that the electron

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transport from photosystem II (PS II) to photosystem I (PS I) in M. aeruginosa is interrupted by

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the identified allelochemicals, (9Z)-Octadec-9-enoic acid and (9Z)-Hexadec-9-enoic acid,

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leading to the failure of photosynthesis and the subsequent death of M. aeruginosa. Our findings

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indicate that the nutrient stress of constant high nutrient supply may be a newly recognized

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trigger causing allelopathy between microbial competitors, and therefore opening a new direction

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for the better management of ecological processes in cyanobacteria-dominated and hyper-

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eutrophic waters.

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KEYWORDS: Allelopathy, Periphytic biofilm, M. aeruginosa, High trophic state, Electron

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transport.

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0 ACS Paragon Plus Environment


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Allelopathy refers to the direct or indirect beneficial or harmful effects of one organism on

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another organism through the emission of biochemicals, known as allelochemicals 1. With the

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frequent occurrence of harmful algal blooms globally, there is growing interest in the allelopathic

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interactions between aquatic microorganisms and bloom-forming cyanobacteria 2-4.

INTRODUCTION

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The driving force for allelopathy is a competitive stressor such as insufficient resources being

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available for the growth and cell division of all competitors and thus the allelopathic toxicity

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increases under nutrient limitation

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organisms may produce allelochemicals, which serve as a strategy to inhibit the opponents’

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access and thereby preferentially to obtain more nutrients

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weissflogii was exposed to the cell-free aqueous filtrate of Prymnesium parvum, under various

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nutrient conditions, the T. weissflogii exhibited a range of susceptibilities to the allelochemicals

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in the filtrate. Moreover, when P. parvum cells were nutrient (N, P) deficient, there was a

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dramatic increase in their production of allelopathic compounds, and overall toxicity. However,

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when under nutrient (N, P) sufficient conditions, allelopathy by P. parvum was virtually

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eliminated 9, 10.

5, 6

. Where nutrients are in limited supply, the competing

7, 8

. For instance, when Thalassiosira

59 60

Thus, it has been recognized that the allelopathy between competitors correlates with nutrient

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availability under certain low nutrient supply condition

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conditions of bioavailable nutrients often appear during allelopathy between competitors under

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low nutrient conditions 12-14. Thus, allelopathy between competitors such as: bacteria-bacteria 15,

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algae-bacteria

16, 17

, algae-algae

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8, 11, 12

. As a result, widely fluctuating

and biofilm-cyanobacteria 1


19

could occur when specific

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nutrient levels shifted drastically to low and middle trophic states. However, allelopathy has not

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been reported to be observed under nutrient-rich conditions which evokes the question as to

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whether allelopathy ever does occur when there is a steady and sufficient (and even excessive)

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nutrient supply.

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From the view of nutrient competition, algae, or microbial aggregates such as periphytic biofilm,

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may play an important role in the primary productivity, nutrient cycling and food web

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interactions in lentic ecosystems

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periphytic biofilm with high biomass, results in competition with other organisms for nutrients 11,

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possibly changing the interactions (e.g. allelopathy) between competitors. Nutrient scarcity and

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metabolite stress are two well-recognised triggers for allelopathy 22. Similarly, constant and high

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nutrient supply and/or metabolite levels might pose stress to competitors, thereby shaping their

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interactions. Thus, in this study it was hypothesized that the presence of a suitable periphytic

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biofilm would be able to inhibit the growth of bloom-forming cyanobacteria under constant high

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nutrient conditions via allelopathy.

20

. The presence of microorganisms, particularly algae or

. Hence, any nutrient level shift might shape the microbial composition and structure

21

,

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Periphytic biofilm comprised of microbial aggregates of multi-organism communities (including

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microalgae, bacteria, fungi, yeast, protozoa, metazoa and debris) is distributed in all types of

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aquatic ecosystems 20. In such a complex and highly productive microecosystem 23 consisiting of

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heterotrophs and photoautotrophs, allelochemicals are simultaneously released from different

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microorganisms of the periphytic biofilm 24. Each community might have a specific allelopathic

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action and the complex allelopathic actions among each of the microbial communities in the



25

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periphytic biofilm may interact with others

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the microbial communities may therefore have been overestimated or underestimated because of

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the neutral, dilution, or synergic effects among these allelopathic interactions 24, 26, 27.

. The potential of each allelopathic action among

91 92

Thus, the periphytic biofilm in this study was considered as a symbiotic enclave to avoid

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investigating each allelopathic interaction between microorganisms in the microbial communities.

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To test the hypothesis mentioned above, we sought to assess (1) whether the allelopathic action

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of periphytic biofilm on M. aeruginosa occurs in a high nutrient condition (with constant nutrient

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supplementation); (2) which allelochemicals are potentially inhibiting the growth of M.

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aeruginosa; and (3) the most likely mechanism of the allelopathic action involved.

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EXPERIMENTAL SECTION

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General considerations. All experiments involving the microorganisms were carried out in

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triplicate at an environmental temperature of 28 ± 1 °C with a light intensity of 45 µE m-2 s-1

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under a 12/12 h light/dark cycle. These processes were conducted under sterile conditions.

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Preparation of nutrient medium at different trophic states. Each liter of nutrient medium

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contained 20.00 mg Na2CO3, 75.00 mg MgSO4·7H2O, 36.00 mg CaCl2·2H2O, 2.86 mg H3BO4,

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1.81 mg MnCl2·4H2O, 0.22 mg ZnSO4, 0.39 mg Na2MoO4, 0.079 mg CuSO4·5H2O, 49.40 mg

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Co(NO3)2·6H2O, 6.00 mg citric acid and 6.00 mg ammonium ferric citrate. An amended Carlson

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trophic state index (TSI) using phosphorus, nitrogen and Chl-a measurements was employed to

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evaluate the nutrient level of the medium used in the experiments 28. Different doses of NaNO3

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and K2HPO4 were added to create different initial trophic states of the experimental medium.


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The initial trophic state indices (TSI) were 43 [total nitrogen (TN): 1.0 mg L-1, total phosphorus

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(TP): 0.01 mg L-1], 79 (TN: 2.0 mg L-1, TP: 0.2 mg L-1), 107 (TN: 5.4 mg L-1, TP: 2.0 mg L-1),

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119 (TN: 12.4 mg L-1, TP: 5.4 mg L-1), 159 (TN: 278.8 mg L-1, TP: 144.0 mg L-1) and 171 (TN:

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397.7 mg L-1, TP: 402.3 mg L-1), respectively and the corresponding treatments were called TSI-

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43, TSI-79, TSI-107, TSI-119, TSI-159 and TSI-171. The pH of the experimental medium was

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adjusted to ~ 7, using 1 M HCl.

117 118

Preparation of periphytic biofilm. The periphytic biofilms on the surfaces of a sponge-like

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material made of polyethylene, and which had been anchored at the interface between the

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sediments and overlying water in Lake Dianchi, Western China, were harvested using a knife

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sterilized in 0.1 M HCl. The properties of water and sediments in the biofilm sampling sites are

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presented in Table 1. The colour of the original periphytic biofilm was brown and its thickness

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ranged from ~ 2 - 8 mm. The biofilms were removed from the mats using a knife sterilized in 0.1

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M HCl and stored at -20 °C for transportation. Before using, the periphytic biofilms with no

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zooplankton (as observed under the optical microscope at 40-times magnification) were cultured

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in sterilized BG-11 medium

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periphytic biofilm (0.225 g) was added to a flask with 150 mL of the respective growth medium

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in the series of trophic states outlined above. Due to gravitation, the periphytic biofilm grew

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primarily at the bottom of the medium.

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at dry weight/volume = ~1:15 g L-1 for 30 days. The pre-washed

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Growth of M. aeruginosa in the absence of periphyton extract. Pure M. aeruginosa FACHB

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469 (purchased from the Freshwater Algal Culture Collection, Institute of Hydrobiology,

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Chinese Academy of Sciences, Wuhan, which had been isolated from Lake Dianchi) was pre-



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cultured to an early exponential growth phase to use as the inoculant. To determine the growth

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character of M. aeruginosa in the various trophic states as a control series, 1.0 mL of this same

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culture was added to flasks containing 150 mL experimental medium of different trophic states

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(TSI = 43, 79, 107, 119, 159 and 171). For each treatment, 1.0 mL exponentially growing culture

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of M. aeruginosa with chlorophyll-a (Chl-a) ≥ 11.6 µg L-1 was used.

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Preparation of periphyton extracts to investigate whether they inhibit cyanobacterial

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growth. To prepare the extracts, the above periphytic biofilms in different trophic states were

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filtered (paper) and 3.5 g of each of the periphytic biofilms was collected and immersed into 150

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mL deionized water for 30 min at 25 oC. The 150 mL periphytic biofilm water extract was then

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used for the preparation of BG-11 medium typically used for the culture of M. aeruginosa. To

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avoid biologically induced changes in the character of the periphytic biofilm water extract, the

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BG-11 medium was filter sterilized (0.22 µm pore-size filter, Whatman®).

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Growth of M. aeruginosa in the presence of periphyton extracts. The M. aeruginosa at an

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exponentially growing phase was added to flasks containing 150 mL sterilized BG-11 medium

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prepared using the filter sterilized periphyton biofilm extracts. Initial high concentrations of

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chlorophyll-a (Chl-a = 210 - 220 µg L-1) were achieved by adding 33 mL M. aeruginosa culture

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to each treatment. The M. aeruginosa in BG-11 formulated using deionized water (rather than

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periphytic biofilm water extract) was set as the control and was also filter-sterilized.

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Characterisation of periphytic biofilm extracts.

Periphytic biofilms co-cultured with M.

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aeruginosa were collected from the TSI-171 experiment. The periphytic biofilms were then


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extracted into water for the isolation and identification of the possible allelochemicals following

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the methods in our previous study 3. Freeze-dried biomass (5 g) was extracted with 15 mL of

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Milli-Q water for 30 min at ambient temperature (20-25 °C) and filtered (0.22 µm pore-size,

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Whatman®). The extraction steps were duplicated. The resultant solutions were combined.

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Samples were purified by passage through XAD-2 resin (Amberlite®), and eluted with

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dichloromethane (10 mL x 3). The eluted solution was dried (MgSO4), filtered and the solvent

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removed in vacuo (rotary evaporator, 10 kPa, 32 °C). The dried extract was redissolved in

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dichloromethane (0.2 mL) and injected into a GC-MS (HP-GC6890/MS5973) for analysis under

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the following conditions: HP-5 capillary column (30 m x 0.25 mm i.d.); high purity helium

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carrier gas at a constant flow speed of 1.2 mL min-1; injector temperature 280 °C; oven

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temperature programmed at 80 °C initial temperature held for 3 min, then ramped to 270 °C at

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10 °C min–1 and held at this temperature for 15 min. The injection volume was 1 µL with the

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split mode at 10:1.

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Conditions for mass spectrometry were: Ionisation voltage of 70 eV; the mass range was set

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from 50 to 800 Da; data were collected at 1.1 scans per second under entire scan mode; and

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solvent delay was 2 min. Compound identification was made by searching the NIST/EPA/NIH

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Mass Spectral Library (Data Version: NIST 05) using the Search Program in the instrument’s

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software package (Software Version 2.0d). Approximate percentages of the major components

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eluted were calculated by integrating the recorded peaks.

176 177

Toxicity of identified compounds. The following standards (chromatographic grade): (9Z)-

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octadec-9-enoic acid, (9Z)-hexadec-9-enoic acid, eicosane, tetradecanoic acid, heptadecane and

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1,3-dihydroxypropan-2-yl-(9Z)-octadec-9-enoate were purchased from Sigma-Aldrich Co., Ltd.



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Various amounts (see Results section for details) of these compounds were added to the bottom

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of flasks containing the high nutrient medium mentioned above to maximise contact with water.

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Pure cultures (1.0 mL, Chl-a = 11.6 µg L-1) of M. aeruginosa from the exponential growth phase

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were inoculated into the media in triplicate and maintained in a 250 mL flask containing 150 mL

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medium at the usual conditions.

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Concentrations of cyanobacteria were determined by counting cell numbers under a light

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microscope. The cells of M. aeruginosa were observed using transmission electron microscopy

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(TEM, TEM-H-70000FA, Hitachi). Each sample was analysed in triplicate.

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Experiment for determining the mechanism of inhibition. To elucidate the mechanism(s)

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responsible for the inhibitory growth of M. aeruginosa, (9Z)-hexadec-9-enoic acid and (9Z)-

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octadec-9-enoic acid were selected to conduct an experiment regarding photosynthesis in M.

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aeruginosa. Aliquots of (9Z)-octadec-9-enoic acid (20 µL) and (9Z)-hexadec-9-enoic acid (10 µL)

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were added to 150 mL flasks with 100 mL of high nutrient medium mentioned above and stirred

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for 10 min to maximise contact with water.

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Pure cultures of M. aeruginosa (1.0 mL, Chl-a = 11.6 µg L-1) from the exponential growth phase

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were inoculated into the media and kept at the usual conditions for the duration of the

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experiment. Samples were collected each day for the determination of effective quantum yields

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and electron transport rates of M. aeruginosa.

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Chemical characteristic analysis. The periphytic biofilm characterisations were achieved using

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an optical microscope and a scanning electron microscope (SEM). Chl-a concentration of M.

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aeruginosa was determined by spectrophotometry using an acetone (90% v/v) extraction method


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at 663 nm and 750 nm in 1-cm path length glass cuvettes after overnight extraction 30. TN, TP,

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ammonia, nitrate, total dissolved phosphorus (TDP) and chemical oxygen demand (COD)

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concentrations were determined using Standard Methods

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water were determined with a multi-meter (Yellow Springs Instrument Company, Ohio, USA).

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The effective quantum yields and electron transport rates of the photosystem II (PS II) reaction

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centre in M. aeruginosa cells were determined with a Phyto-PAM fluorescence analyzer (Walz,

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Germany). The biomass of diatoms (represented by Chl-a) in the periphytic biofilm was also

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determined with this fluorescence analyser. The wet weight (WW) of biofilm was measured with

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wetness of 85 ± 5% at 25-30 oC after being filtered. After drying at 80 oC for 72 h, the dry

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weight (DW) of sample was determined.

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TP in sediments was measured from freeze-dried sediments (1.0 g) were burnt at 500 °C,

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followed by acid dissolution (HF + HNO3) according to the traditional method 32. The phosphate

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determination of freeze-dried sediments (1.0 g) was conducted according to the reported

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procedure

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measured total phosphorous content. Analysis of ammonia and nitrate in sediments followed

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automated (segmented flow) colorimetric procedures

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were digested using a persulfate procedure following the methods of Hosomi and Sudo (1986).

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After digestion, TN measurements were performed using Standard Methods 31. Redox and pH in

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sediments were determined by Redox and pH meters (YSI, USA).

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Statistics. Differences of M. aeruginosa growth (Chl-a concentration) between control and

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treatment were statistically analyzed (ANOVA) using SPSS 21.0. A value of p < 0.05 was

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considered statistically significant.

33

31

. Dissolved oxygen (DO) and pH in

. Organic phosphorus was calculated by subtracting inorganic phosphorus from the

31

. The sediment samples for TN analysis



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Characteristics of periphytic biofilm. Microscopic observation showed that the periphytic

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biofilm consisted of diatoms and bacteria at the beginning of the experiment (Figure 1). The

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diatoms included the following species: Melosira varians C. Agardh, Gomphonema parvulum

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Kütz., Synedra ulna Kütz., Nitzschia amphibia Grun., Fragilaria vaucheriae Kütz. Most bacteria

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were found to be bacilli and cocci. The diatom mass dominated the periphytic biofilm,

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accounting for 86.9 ± 7.6 % of the total biomass. At the end of the experiment, the dominant

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microorganisms remained diatoms and bacteria, with the same species of diatoms dominating the

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periphytic biofilm at 90.4 ± 9.3% of the total biomass. There was no evidence of “lysed or

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infected” pots in the samples at the beginning and the end of the lab experiment (Figure 1),

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indicating that algicidal microorganisms or pathogens if present were trivial.

RESULTS AND DISCUSSION

237 238

Inhibition of cyanobacterial growth by periphytic biofilm extract. The Chl-a concentrations

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of M. aeruginosa cultured in absence of periphytic biofilm rapidly increased about 100-fold

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(Figure 2a), meaning that the M. aeruginosa was able to grow in the condition of trophic state

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from TSI 43 to TSI 171. However, in the presence of periphytic biofilm there was a significantly

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different response in the Chl-a concentrations of M. aeruginosa across the various trophic states,

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with the growth for the higher and lower trophic states declining after 5 days exposure (p < 0.05)

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(Figure 2b). A similar limitation in the growth in Chl-a concentrations of M. aeruginosa after 4

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days was also seen for samples cultured in the water extracts of periphytic biofilm collected from

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different trophic states compared to the control (culture in BG-11 medium) (p < 0.05) (Figure 2c).

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These results imply that there is an optimal (mid-level) trophic state for the continued growth of


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M. aeruginosa exposed to periphytic biofilm and that higher or lower trophic states become

249

inhibitory for cyanobacterial growth over time.

250 251

Considering the allelopathy shown in low TSI levels investigated in our previous study 3, in this

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study we focused on the investigation of allelopathy at high TSI levels (TSI = 159 and 171). The

253

TSIs were maintained at 159 to 171 during the co-culture of M. aeruginosa and periphytic

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biofilm. The TSI (TP) and TSI (TN) were also maintained at steady levels, without significant

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differences over time, ~ 256 – 280 for TSI (TP) and ~ 61.6 – 61.9 for TSI (TN). At the end of the

256

experiment, the concentrations of bioavailable nitrogen (the sum of ammonia and nitrate) and

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total dissolved phosphorus (TDP) were 153.3 mg L–1 and 28.5 mg L–1 in the TSI-159 treatment,

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and 218.7 mg L–1 and 80.5 mg L–1 in the TSI-171 treatment, respectively (Figure 2d). These

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nutrients are enough to support cyanobacterial growth and even to promote the occurrence of

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cyanobacterial blooms.

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Identification of allelochemicals and toxicity assay. There is little evidence published to

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demonstrate the level and type of allelochemicals present in periphytic biofilms when cultured

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under high trophic condition (TSI = 171) and therefore, these were extracted (but not

265

individually isolated) and identified by GC-MS. In aquatic ecosystems, it is a challenge to study

266

the allelopathy between microbial competitors

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these allelochemicals originate from the overall water ecosystem or from microbial competitors

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living in proximity 27. Also, these chemicals, and in particular the water-soluble allelochemicals,

269

are likely to be affected by water quality such as redox status and pH 36, 37. In this case, to obtain

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direct evidence of allelochemicals released and to prove the existence of allelopathy between

34-36

, because it is hard to distinguish whether



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periphytic biofilm and M. aeruginosa, non-polar/low polarity compounds were considered. Six

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such compounds in the periphytic biofilm extract (Figure S1 and Table 2) were annotated. In

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temporal order of elution from the column, these were 1,3-dihydroxypropan-2-yl (9Z)-octadec-9-

274

enoate (4.78 min), (9Z)-hexadec-9-enoic acid (7.48 min) (Figure S2), eicosane (10.48 min)

275

(Figure S3), tetradecanoic acid (12.50 min) (Figure S4), heptadecane (16.20 min) (Figure S5),

276

and (9Z)-octadec-9-enoic acid (20.84 min) (Figure S6).

277 278

The bioassay results showed that (9Z)-octadec-9-enoic acid [9ZOA], (9Z)-hexadec-9-enoic acid

279

[9ZHA], eicosane, tetradecanoic acid and heptadecane inhibited the growth of M. aeruginosa at

280

concentrations of 10 µg L-1 or higher. The inhibitory effects increased with both time and level

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of exposure to these compounds (Figure 3). It is therefore likely that these five compounds were

282

at least partially responsible, individually or in combination, for inhibiting the growth of M.

283

aeruginosa under constant high nutrient (TSI = 159-171) conditions.

284 285

All identified compounds of interest are hydrophobic. For example, the solubility of (9Z)-

286

octadec-9-enoic acid and (9Z)-hexadec-9-enoic acid are both less than 10 µg L-1 (7.05 µgL-1 for

287

(9Z)-octadec-9-enoic acid)

288

respectively and these ‘low density’ compounds form micelles when mixed with water 39 and can

289

even form an ‘oil layer’ which floats on the surface. Generally, M. aeruginosa inhabits the

290

middle and upper layers of high nutrient medium (water) due to their heliotropic response

291

Thus, these five compounds can easily make contact with M. aeruginosa cells via the suspended

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micelles when directly added to the bottom of flasks containing high nutrient medium which are

293

then gently stirred. When these hydrophobic compounds attach to the cyanobacterial surfaces,

38

. Furthermore, their densities are 0.89 g cm-3 and 0.85 g cm-3,


40

.

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their growth might be negatively affected, at least the photosynthesis of cyanobacteria could be

295

negatively affected. In addition, the quantities of the suspended micelles of these five compounds

296

increase with dosage (Figure 3), which would lead to greater stress on M. aeruginosa cells.

297 298

Negative effects on cyanobacterial photosynthesis. The unsaturated fatty acids 9ZOA and

299

9ZHA were selected to investigate the stress mechanism of periphytic biofilm extract on M.

300

aeruginosa growth because they were the most abundant of the identified compounds (Table 2).

301

Effective quantum yields and electron transport rates in photosystem II (PS II) reaction centres

302

were determined. The results show that both, effective quantum yields and electron transport

303

rates were significantly decreased in presence of 9ZOA and 9ZHA (p < 0.05) (Figure 4). The PS

304

II reaction centre is one of the two sites for light reception and conversion of light to chemical

305

energy in photosynthesis. These results suggested that the light reaction centre (PS II) of M.

306

aeruginosa was inhibited by 9ZOA and/or 9ZHA.

307 308

The results of M. aeruginosa cells visualised by TEM show that the cell structure, including the

309

thylakoid membrane, in the control was intact (Figure 5a). The connection between the

310

thylakoids and the cytoplasmic membrane in cells was disrupted after 24 h of exposure when the

311

dose of 9ZHA was 8.5 µg L-1 (Figure 5b). When the dose of 9ZOA was 8.9 µg L-1, the shape of

312

the thylakoid membrane had changed after 24 h of exposure, (Figure 5c).

313 314

When the dose of 9ZOA was increased to 35.6 µg L-1, the inner materials and thylakoid

315

membranes of M. aeruginosa started to deteriorate after 48 h exposure (Figure 5d). The

316

thylakoid membrane, which is the location of photosynthetic reactions 41, is mainly composed of



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317

membrane lipids, proteins and photosynthetic pigments. The results indicate that these two

318

compounds penetrated the cell membranes of M. aeruginosa and disrupted the PS II, leading to

319

the inhibition of growth, and eventually cell death, depending on the dose and duration of the

320

exposure.

321 322

Implication of the occurrence of allelopathy under constant high nutrient supply. Resource

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competition is one of the main causes of allelopathy between competitors

324

limitation may enhance the allelochemical contents of cyanobacteria 22, 43. In our previous studies,

325

the occurrence of allelopathy between periphytic biofilm and M. aeruginosa was associated with

326

wide fluctuations in: nutrients (TSI from 58 to 92) in water, bioavailable nitrogen (N, the total of

327

ammonia and nitrate), and total dissolved phosphorus (TDP) from 68.7-82.9% and 70.7-87.1%,

328

respectively 3, 19. In this study, the bioavailable nitrogen and phosphorus were maintained at high

329

levels, implying that the occurrence of allelopathic action of the periphytic biofilm was not

330

directly associated with shifts in these nutrient concentrations.

42

, and nutrient

331 332

It is well known that microbial aggregates such as coral-algal symbiosis are sensitive to nutrient

333

enrichment

334

nutrient enrichment, was investigated in our previous study 46. When the periphytic biofilm was

335

cultured at high nutrient levels (TSI from 158 to 206), there were dissimilarities in metabolic

336

versatility (Table S1), composition (Figure S7), phospholipid fatty acid (PLFA) content and

337

carbon utilization ability of the periphytic biofilm (Figure S8). This implies that the periphytic

338

biofilm biochemistry had altered under the stress of constant high nutrient levels.

44, 45

. Whether allelochemicals are released by periphytic biofilm, under the stress of

339


Page 17 of 34


340

Under constant high nutrient conditions, the increasing biomass of the M. aeruginosa in the

341

monoculture indicates that the cyanobacterium resists the stress of a high nutrient supply. In an

342

effort to survive, the composition and structure of the microbial community in periphytic biofilm

343

readjusts under high nutrient conditions (Figure S7 and Figure S8). It is likely that the

344

allelochemicals are released by the periphyta under high nutrient stress to suppress the growth of

345

M. aeruginosa. This phenomenon is in accordance with the traditional view that the

346

physiological stress is the cause for the occurrence of allelopathy 5, 6. Therefore, we conclude that

347

the constant high nutrient supply (TSI from 159 to 171) might be a newly recognised trigger,

348

driving the observed allelopathic response.

349 350

Environmental relevance of the identified allelochemicals.

351

allelopathic taxa identified to date suggests that allelopathy is a widespread competitive

352

strategy,2, 24 the ecological roles of eicosane, tetradecanoic acid and heptadecane identified from

353

this study have not been previously investigated. Both 9ZOA (oleic acid) and 9ZHA (palmitoleic

354

acid) are well known as antimicrobial compounds

355

allelochemicals in terrestrial plants and macrophytes

356

of the allelochemicals released by Cyanobacterium apponinum and Phormidium sp.

357

inhibit the growth of chlorophytes (Chlorella vulgaris Beij. and Monoraphidium contortum

358

(Thur.) Kom.-Legn.) 51. In addition, 9ZOA from Chlamydomonas reinhardtii is toxic to a variety

359

of algae

360

growth of barnyard grass (Echinochloa crus-galli) 53.

52

Although the diversity of

47

and have been widely reported as

48, 49

. For instance, 9ZOA is reportedly one 50

which

while 9ZHA is one of the fatty acids from rice that exerts allelopathic effects on the

361



Page 18 of 34

362

Periphytic biofilm in aquatic systems not only alters the biomass of the competitors, but also may

363

affect the spatial and temporal heterogeneity of other constituent biomass (e.g. plankton)

364

Extrapolated to a natural environment, both the rate of release and the equilibrium level of

365

allelochemicals are likely to be affected by the growth of the periphytic biofilm. Indeed, the

366

intensity of the allelopathic effect depends on the growth phase of allelopathic species

367

high nutrient (TSI = 159-171) waters where the nutrient content is sufficient to support the

368

growth of periphytic biofilm, a high productive capacity and rapidly growing periphytic biofilm

369

on newly established shoots may potentially suppress the development of the submerged plant

370

community

371

periphyton productivity rates in some waters have been shown to be higher than those measured

372

among plankton 23, which might stimulate allelopathic action. Certainly, in our previous similar

373

study conducted at low TSI levels, the results in field experiments conducted in Moon Lake,

374

China showed that the allelopathic action between periphytic biofilm and M. aeruginosa was

375

sustained for at least 10 months during the period of the excessive growth of M. aeruginosa

376

(Chl-a: 10.8 - 271.9µg L-1), effectively inhibiting the growth of the cyanobacterium 3.

55

6, 54

20

.

. In

, and therefore preserve its allelopathic action on M. aeruginosa. Moreover,

377 378

In a healthy aquatic ecosystem, the periphytic biofilm biomass can reach 10 g dry matter cm-2 of

379

surface 56. In this study, this is equal to ~67 g periphyton per cubic meter of water, meaning that

380

the presence of periphytic biofilm has no potential risk to natural waters

381

(85 days), the significant decrease in the biomass (represented by PLFA content) of periphytic

382

biofilm (Figure S8) further indicates that the growth of periphytic biofilm was restricted under

383

constant high nutrient (TSI = 159-171) conditions.

384


57, 58

. In the long-term

Page 19 of 34


385

Apart from the effects of periphytic biofilm itself on the allelochemicals, the effective

386

concentrations of these compounds might be influenced by abiotic or biotic factors in natural

387

waters 6. For instance, the microorganisms in sediments may take up and degrade the

388

allelochemicals

389

and M. aeruginosa still occurred in the presence of sediments

390

allelochemicals from these previous studies were different from the five compounds identified in

391

this study. Thus, it is suggested that the ecologically-effective concentrations of these five

392

allelochemicals mentioned above need to be determined under field conditions, when

393

considering the short- and/or long-term effectiveness of periphytic biofilm in inhibiting the

394

growth of M. aeruginosa.

59

. Our previous study showed that the allelopathy between periphytic biofilm 3, 19

. However, the identified

395 396

Ecological role of allelopathy under constant high nutrient condition. Eutrophic lakes are

397

usually phytoplankton-dominated and harmful algal blooms occur in consistently high nutrient

398

environments

399

frequency of harmful cyanobacterial blooms that appear to be increasing on a global scale, and

400

are linked to nutrient levels

401

relationship between nutrient fluctuation and the occurrence of allelopathy in constant high

402

nutrient conditions, which therefore limits successful control of harmful algal blooms.

3, 60, 61

. Thus, in such ecosystems, allelopathy has been proposed to reduce the

2, 24, 25, 62

. However, little information is currently available on the

403 404

Our results demonstrate that under high nutrient supply conditions without nutrient fluctuation

405

(e.g. trophic state index from 159-171), in hyper-eutrophic waters, periphytic biofilm releases a

406

suite of at least five allelochemicals that inhibit the growth of M. aeruginosa. Thus, regulating

407

periphytic biofilm (e.g., enlargement of periphytic biofilm biomass) in high nutrient (TSI = 159-



Page 20 of 34

408

171) waters might be a potential way to prevent harmful cyanobacterial blooms, although the

409

ecologically-effective concentrations of the five allelochemicals identified in this study need to

410

be determined under field conditions.

411 412

Without nutrient competition under constant nutrient supply conditions, these five

413

allelochemicals released by periphytic biofilm, might be the main factors directly influencing

414

phytoplankton competition, succession, and bloom formation or maintenance

415

term stress of allelochemicals also changes the rate and direction of community succession 65, 66.

416

In this study, the released allelochemicals simultaneously affect the donor (periphytic biofilm)

417

and acceptor (M. aeruginosa), resulting in co-dependence (or co-adaptation). For instance, the M.

418

aeruginosa stressed by the allelochemicals lost its competitive advantage, leading to a decrease

419

in its biomass. The shifted M. aeruginosa biomass affects the seasonal patterns of the dominant

420

cyanobacterial species, which was found in a previous study in Lake Dianchi 67.

63, 64

. The long-

421 422

In conclusion, our findings show that the occurrence of an allelopathic action of periphytic

423

biofilm on M. aeruginosa is not directly coupled to the wide fluctuation in nutrients but is driven

424

by a steady and high nutrient level. The stress of a high nutrient level might be an important and

425

newly recognised trigger in the origin of this allelopathic action, which should lead to a better

426

understanding of the interactions between periphytic biofilm and cyanobacteria. Our results do

427

not imply that a low nutrient state and wide fluctuations in nutrients are unimportant in

428

regulating interaction between periphyton and cyanobacteria; rather, they highlight that the stress

429

of nutrient scarcity and excess may be important regulators of the allelopathic action of


Page 21 of 34


430

periphytic biofilm on cyanobacteria, potentially creating a new perspective for the study of

431

allelopathy between microbial competitors in hyper-eutrophic waters.

432 433

434

Supporting information. Figures from S1 to S8 and table S1 are available free of charge via the

435

Internet at http://pubs.acs.org

ASSOCIATED CONTENT

436 437

438

This work was supported by the State Key Development Program for Basic Research of China

439

(2015CB158200), the National Natural Science Foundation of China (41422111), and the

440

Natural Science Foundation of Jiangsu Province China (BK20150066). This work was also

441

supported by Youth Innovation Promotion Association, Chinese Academy of Sciences

442

(2014269).

ACKNOWLEDGEMENTS

443 444 445

REFERENCES

446

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447 448 449 450

2. Poulson-Ellestad, K. L.; Jones, C. M.; Roy, J.; Viant, M. R.; Fernández, F. M.; Kubanek, J.; Nunn, B. L., Metabolomics and proteomics reveal impacts of chemically mediated competition on marine plankton. Proceedings of the National Academy of Sciences 2014, 111, (24), 90099014.

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626 627


Page 27 of 34


Figure Captions

628 629 630

Figure 1. Periphytic biofilm under (a, c) optical microscope (40-times magnification) and (b, d)

631

scanning electron microscope (SEM) (a and b at the beginning of the experiment and c, d at the

632

end of the experiment)

633

Figure 2. (a) Changes in the Chl-a concentrations of M. aeruginosa in the absence of periphytic

634

biofilms under different trophic states, (b) Changes in the Chl-a concentrations of M. aeruginosa

635

in the presence of periphytic biofilms under different trophic states, (c) Changes in the Chl-a

636

concentrations of M. aeruginosa cultured in the water extracts of periphytic biofilm collected

637

from different trophic states and the control (culture in BG-11 medium), (d) Fluctuations of

638

bioavailable nitrogen (N) and total dissolved phosphorus (TDP) in the high trophic treatments

639

(TSI = 159 and 171) during the co-culture of periphytic biofilms and M. aeruginosa.

640

Figure 3. Growth of M. aeruginosa (cell density) in sterilized high nutrient medium

641

supplemented with different doses of (a) (9Z)-octadec-9-enoic acid, (b) (9Z)-Hexadec-9-enoic

642

acid, (c) eicosane, (d) tetradecanoic acid and (e) heptadecane.

643

Figure 4. Changes in effective quantum yields and electronic transport rate in M. aeruginosa

644

photosystem two (PS II) supplemented with (9Z)-octadec-9-enoic acid (17.8 µg L-1) and (9Z)-

645

Hexadec-9-enoic acid (8.5 µg L-1).

646

Figure 5. Cell structure of M. aeruginosa observed under transmission electron microscopy. (a)

647

Control. Cell structure of M. aeruginosa was intact. (b) Bioassay with (9Z)-Hexadec-9-enoic

648

acid (8.5 µg L-1). The thylakoid membrane of M. aeruginosa was disorganized and dislodged

649

from cytoplasm. (c) Bioassay with (9Z)-octadec-9-enoic acid (8.9 µg L-1). The thylakoid

650

membrane was distorted. (d) Bioassay with (9Z)-octadec-9-enoic acid (35.6 µg L-1). The

651

thylakoid membranes of M. aeruginosa became more disorganized.

652 24 ACS Paragon Plus Environment


a

b

c

d

653

654 655

Figure 1.

656


Page 28 of 34

Page 29 of 34


657 658

659 660

Figure 2.

661 662



663 664

Figure 3.


Page 30 of 34

Page 31 of 34


665

666 667

Figure 4.

668



a

b

2 µm

2 µm

c

d

2 µm

2 µm

669 670

Page 32 of 34

Figure 5.

671 672 673


Page 33 of 34

674


Table 1. The parameters of the water and sediment used for growing periphyton (mean ± SD) Sample

Water

Sediments

Parameter Transparency (cm) pH DO (mg L-1) COD(mg L-1) TP (mg L-1) TN (mg L-1) NH4-N(mg L-1) pH Redox (mV) TN (mg g-1) Organic-P (mg g-1) Labile-P (mg g-1) Al-P (mg g-1) Fe-P (mg g-1) Ca-P (mg g-1) TP (mg g-1)

675 676


Value 16.0 ± 3.21 9.15 ± 1.24 3.14 ± 0.85 24.32 ± 4.36 0.64 ± 0.20 2.58 ± 0.46 0.69 ± 0.41 8.65 ± 0.53 -294.20 ± -18.34 3.18 ± 0.12 2.95 ± 0.53 0.065 ± 0.01 0.083 ± 0.01 5.64 ± 1.03 8.45 ± 2.21 21.37 ± 5.28


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Page 34 of 34

Table 2. Identified compounds from periphyton extracts and their relative amounts tr (min)

4.784

Identified compounds 1,3-dihydroxypropan-2-yl-(9Z)-octadec-9-enoate [DHP9ZO]

Relative amounts*

Reverse Match*

11.04

856

7.475

(9Z)-Hexadec-9-enoic acid [9ZHA]

15.24

922

10.478

Eicosane [C20]

5.92

896

12.504

Tetradecanoic acid [TDA]

3.73

912

16.198

Heptadecane [C17]

11.18

886

20.840

(9Z)-Octadec-9-enoic acid [9ZOA]

25.06

924

*Database is the NIST/EPA/NIH Mass Spectral Library (Data Version: NIST 05).

678


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