Effects of water flow on submerged macrophyte-biofilm systems in constructed wetlands

The effects of water flow on the leaf-biofilm interface of Vallisneria natans and Hydrilla verticillata were investigated using artificial plants as the control. Water flow inhibited the growth of two species of submerged macrophytes, reduced oxygen concentrations in plant leaves and changed oxygen profiles at the leaf-biofilm interface. The results from confocal laser scanning microscopy and multifractal analysis showed that water flow reduced biofilm thickness, changed biofilm topographic characterization and increased the percentages of single colony-like biofilm patches. A cluster analysis revealed that the bacterial compositions in biofilms were determined mainly by substrate types and were different from those in sediments. However, water flow increased the bacterial diversity in biofilms in terms of operational taxonomic unit numbers and Shannon Indices. Our results indicated that water flow can be used to regulate the biomass, distribution and bacterial diversities of epiphytic biofilms in constructed wetlands dominated by submerged macrophytes.

by the experimental system. The plant density was 400 plants per square meter and 32 the plant numbers were 144 in the static tank and 48 in the flow channel, respectively. 33 The V. natans and H. verticillata were incubated in sequence. The artificial plants 34 were set among the V. natans plants as a control. 35 In the flowing channel, the velocity range at water depth 10-20cm in the plant zone 36 was reduced from 4.5-5.5 cm s -1 to 2.6-4.7 cm s -1 (2.9-4.8 cm s -1 ) by V. natans and 37 artificial plants (H. verticillata), whereas the velocity range at water depth 0-5 cm was 38 promoted from 5.6-6.3 cm s -1 to 5.9-7.4 cm s -1 (6.0-7.4 cm s -1 ) (Supplymentary Fig. 39 2B and C). 40 Values of pH, dissolved oxygen, electrical conductivity and oxidation-reduction 41 potential determined in the center of both sampling zones illustrated that no 42 significant difference (p > 0.05) was found between the water parameters in the static 43 water and those in the flowing water (Supplymentary Table 4).

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Oxygen determination. Before the tests, the oxygen microelectrode was linearly 45 calibrated from signal readings in oxygen-free and air-saturated fresh water at the 46 experimental temperature. Data under light was gathered between 2 and 4 p.m.
whereas data under dark was obtained between 8 and 10 p.m. The motor and object 48 stage of the microelectrode system were set on the vessel wall of the tank/flume 49 ( Supplementary Fig. 5). To maintain a still water condition, the pump and valve 50 between the static tank and the flowing channel were shut down during the test on the 51 leaves in the static tank. To avoid the noticeable vibration of the microelectrode 52 caused by water fluctuation during the test on the leaves in the flowing channel, the 53 surface flow velocity in the flowing channel was adjusted to approximately 1 cm s -1 .

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Yo ung leaves of V. natans and H. verticillata at 10-15 cm below the water surface 55 were used for the puncture test. The leaf was fixed onto the object stage with two 56 small clips ( Supplementary Fig. 5) at the water depth of approximately 10 mm. The 57 oxygen microelectrode was fastened on the motorized micromanipulator, and its tip 58 was set manually approximately 1500 µm above the leaf surface. The microelectrode 59 moved vertically via the motorized micromanipulator. The motorized 60 micromanipulator was set to move 300 steps down (10 µm each step) to cover 3000 61 µm.  where is the biofilm value of the box (i, j) of size ε.

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(ε) can be described as multifractal as 6 dimension ( ) can be obtained from the partition function ( ) expressed as a 94 power law of ε with an exponent ( ) applied in statistical physics as follows: where q is the moment order (−∞ < q < ∞). ( ) can be obtained from the slope of 97 the ln χq(ε)-ln ε curve, and the generalized fractal dimension is defined as  103 We took the maximum |q| as 10 since |dαmax|/Δα and |dαmin|/Δα were all less than