Date: Wednesday, February 15th, 2017

Submitted to: Chemosphere Journal

- Supporting Information-

Single chamber microbial fuel cell (SCMFC) with a cathodic

microalgal biofilm: A preliminary assessment of the generation

of bioelectricity and biodegradation of real dye textile

wastewater

Washington Logroñoa, b*, Mario Péreza, Gladys Urquizoa, Abudukeremu Kadierc, Magdy

Echeverríaa, Celso Recaldea, d, Gábor Rákhelyb, e

aCentro de Investigación de Energías Alternativas y Ambiente, Escuela Superior Politécnica

de Chimborazo, Chimborazo, EC060155, Ecuador

bDepartment of Biotechnology, University of Szeged, H-6726 Szeged, Hungary

cDepartment of Chemical and Process Engineering, Faculty of Engineering and Built

Environment, National University of Malaysia (UKM), 43600 UKM Bangi, Selangor,

Malaysia

dInstituto de Ciencia, Innovación, Tecnología y Saberes, Universidad Nacional de

Chimborazo, Riobamba, Ecuador

eInstitute of Biophysics, Biological Research Centre Hungarian Academy of Sciences,

Szeged, Hungary

*Corresponding author:

W. Logrono: Tel.: +36 307574843; fax: N.A; E-mail: logrono.w@gmail.com

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Text S1. Description of the process of digital image processing

In-house masks were applied to every SEM image of the microalgal biofilm in Adobe

Photoshop CC 2015. Suitable areas were selected with the “Pen Tool” and included in the

mask in order to avoid the undesirable areas (sections of carbon fibre, shadows or coloured as

the microalgal cells). Thereafter, the mask was transferred to a new layer with the function

“Layer via cut” to ensure that pixels were not counted twice, and it was saved as a TIF file.

The MATLAB image processing was applied to every SEM image and its mask.

Morphological operations were applied on the binary masks (dilation, edge detection with the

Canny method). Manual colouring of the area of interest was performed in order to obtain

reliable results from the image processing. During the analyses, the MATLAB functions

“imfuse” and “bwarea” were used. “Imfuse” creates composite visualizations of the original

and segmented images, which were saved to a file with the "imwrite" command (Fig. S1-B).

The latest file (fused image) was used for validation of the efficiency of the masks (the mask

covered only the area of the cells). After verifying that the masks only covered the microalgal

cells (validated image), the "bwarea" function was employed, which estimated the area of the

objects in the binary image. Its result is a scalar whose value corresponds roughly to the total

number of on pixels in the image by summing the area of each pixel in the image. The area of

an individual pixel was determined by looking at its 2-by-2 neighbourhood. The coverage

area was calculated based on the resolution and sizes of our images. Thus, the total number of

pixels can be calculated by Eq. 1:

Acoverage (%)=on pixelssegmented image

pixelsorginal image×100

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Text S2. Calculation of the cellular mass of C. vulgaris over the electrode

The cell mass (g) that could be expected on the air-exposed biocathode electrode was

calculated by considering the coverage area from the SEM picture (Text S1, Fig. S1). First, it

was assumed that the coverage area of the electrode was similar to that of the SEM image

(42%). Consequently, the approximate mass in grams of 42% coverage of the biocathode was

calculated based on the assumption that the mass of one C. vulgaris cell corresponds to 1.68

× 10-11 g/cell (Chia et al., 2013). The data for the calculation are presented below:

A BNote: Non-scaled scheme representing the cathode electrode with its dimensions. A subsection of 10 × 10 mm was sampled for SEM imaging. B: SEM image (768 × 768 pixels) depicting a microalgal coverage area of 42%.

Example

Calculation of the image properties

Data: le (cathode electrode dimension) = 50 mm

image scale = 20 μm

view field = 125 px

Calculations: Cf = image scale / view field = 0.16 μm/px

widthimage(μm) = widthimage(px) × Cf = 768 px × 0.16 μm/px = 122.88 μm

Ai = 122.88 μm × 122.88 μm = 15099.49 μm2 = 1.51 × 10-2 mm2

Ae = le × le = 50 mm × 50 mm = 2.50 × 103 mm2

Data: dc = 2.70 μm = 2.70 × 10-3 mm

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50 mm

50 m

m

x 165568.46

10 m

m

10 mm

768 px=0,123 mm

768

px

rc = dc /2 = 1.35 μm = 1.35 × 10-3 mm

picture coverage = 42%

mc (cell mass) = 1.68 × 10-11 g cell-1

Calculations: Ac = rc2 × π = (1.35 μm)2 × π = 5.73 μm2 cell-1

Cn_100% = Ai/Ac= 15099.49 μm2 / 5.73 μm2/cell = 2637.21 cell ≈ 2637 cell

Cn_42% = Cn_100% × 0.42 = 1107.63 cell ≈ 1107 cell

Atotal_100% = Cn_100% × Ac = 2637 cell × 5.73 μm2/cell = 15110.01 μm2

Atotal_42% = Cn_42% × Ac = 1107 cell × 5.73 μm2/cell = 6343.11 μm2

magnitude = Ae / Ai = 165568.46

Cn_e_42% = Cn_42% × magnitude = 1107 cell × 165568.46 ≈ 183284285 cell

me = Cn_e_42% × mc = 183284285 cell × 1.68 × 10-11 g/cell = 3.08 × 10-3 g

where le is the width and height of the cathode electrode; Cf is the calibration factor; Ae is the

area of the electrode used in the experiment; Ai is the area of the image taken with the SEM;

dc is the C. vulgaris cell diameter; rc is the radius of a C. vulgaris cell; Ac is the area of a C.

vulgaris cell; Cn_100% is the cell number at 100% coverage; Cn_42% is the cell number at 42%

coverage; Atotal_100% is the coverage area at 100% coverage; Atotal_42% is the coverage area at

42% coverage; Cn_e_42% is the cell number on the cathode electrode at 42% coverage; and me is

the mass in grams on the cathode electrode from Cn_e_42%.

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Figure S1

A B

C

Fig. S1. A: Photograph of C. vulgaris cultures that were maintained in test tubes containing

10 mL of sterile nitrogenized media at 0.3%. Fresh media was used for re-culturing and

maintaining the microalgae by inoculating 1% from a previous mature culture. B: Photograph

of the appearance of a mature culture of C. vulgaris (visible green), which was used to

immerse the cathode electrode and promote biofilm formation. C: Mature culture of C.

vulgaris observed under an optical microscope at x100 after ~30 days of operation on the air-

exposed MFC.

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Figure S2

A

B

Fig. S2. A: workflow of the digital image processing of a biocathode containing C. vulgaris.

B: the fused picture of the analysed SEM image (768 x 768 pixels) depicting 42% of the area

covered.

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Sample preparation

SEM imaging

In-house masks

Image segmentation

Manual coloring

Area calculation

Figure S3

Fig. S3. SEM image (768 × 768 pixels) at a 20 μm approximation depicting the differences in

the cell diameters of immobilized C. vulgaris (red arrows) on carbon fibres. The cells were

randomly selected and measured by using SEM VegaTC software. More significant

differences are clearly visible in the image, but they were not measured (blue arrow).

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Figure S4

Fig. S4. Initial stage of OCV increase during the first experimental batch (5 days). The

averaged OCV of the m-SCMFCs showed a linear increase of R2 = 0.92 (data not shown)

compared to that of the control with no linear-like curve shape.

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Table S1. Composition of the media Nitrofoska foliar for growing C. vulgaris

Compound AmountNitrogen (N) 25.0a

Phosphorus (P205) 10.0a

Potassium (K20) 17.5a

Magnesium (MgO) 1.57a

Manganese (Mn¿+¿ ¿ 300b

Iron (Fe)* 400b

Copper (Cu)* 50.0b

Boron (B) 150b

Molybdenum (Mo) 5.00b

Zinc (Zn)** EDTA chelated trace elements

400b

Note: a represents % and b is expressed in mg kg-1

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Table S2. Comparison of the price of a biocathode with commercially available Pt-coated carbon based electrodes.

Cathode materialCost (USD

)Reference

Biocathode 0.50* Local market.

Cathode Platinum 5.00

E-Tek, (Carbon Cloth Electrode, standard 0.5 mg/cm2 TM loading using 10% Pt on Vulcan XC-72, with standard ionomer application at 100 cm2)

0.5 mg/cm² 60% Platinum on a Vulcan Carbon Paper Electrode 18.75 www.fuelcellstore.com

0.5 mg/cm² 60% Platinum on a Vulcan Carbon Cloth Electrode 23.00 www.fuelcellstore.com

2 mg/cm² Platinum Black Carbon Cloth Electrode 40.00 www.fuelcellstore.com

2 mg/cm² Platinum Black Carbon Paper Electrode 49.06 www.fuelcellstore.com

Note: The commercial electrodes with Pt coating are much more expensive. According to the type of coating, the commercial cathode electrode is 9 to 97.12-fold more expensive than the biocathode. *: The costs were evaluated based on the local market. For example, the cost for this biocathode was calculated as follows: Nitrofoska, $0.01; distilled water, $0.10; energy, $0.01; and carbon fibre (5 x 5 cm), $0.38.

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Table S3. Cost of the SCMFC setup

Material Cost (USD)Carbon fibre 5 x 5 cm (biocathode and anode) 0.88 Acrylic frames 10 cm x 10 cm 13.50Wiring cables 0.25Isolating protection 0.10Alligator clips 0.80Stopper 0.30Microalgae cultivation 5.00Total ~21

Note: The costs were evaluated for a single reactor. The cost of the carbon fibre was calculated based on the cost of one square metre of carbon fibre ($150).

SI References

Chia, M. a, Lombardi, A.T., Melão, M.D.G.G., 2013. Growth and biochemical composition of Chlorella vulgaris in different growth media. An. Acad. Bras. Cienc. 85, 1427–38. doi:10.1590/0001-3765201393312

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