IARA

HARDWARE

BACKGROUND

The IARA project is to reduce the concentration of mercury in water for reuse. For this, we planned to build a filter that could be easy to handle, low cost and safe. The idea is to create a bacterial biofilm with our transformed bacteria in which the metal collecting proteins will be anchored. This anchoring is due to a cellulose binding tag that was added to the metal collecting protein through our biosynthetic circuit. Thus, we chose the coconut fiber substrate for bacterial biofilm growth due to the high concentration of cellulose in the fiber constitution.

In order to reduce the cost of our filter and to try to make it safer our team designed a filter model in which we could better fix the biofilm and also to kill bacteria at the end of the purification process. Below we describe in detail each part of this planning.

GREEN COCONUT FIBER

Brazil is the world's fourth largest coconut producer, producing about 2.8 million tons per year. In this production, 15% is estimated to be green coconut production [1, 2]. Thus, there is a concern to environmentally target the subproducts of coconut water consumption, since coconut shells have a decomposition of about 8 years [3]. Generally, coconut shells are deposited in open dumps, hillside drains and landfills and can become a serious environmental problem by emitting methane gas when in anaerobic conditions [4, 5, 6].

It is possible to find ways to reuse green coconut (so that it can reduce its accumulation in the environment) associated with the need to mitigate the environmental impact caused by the release of heavy metal contaminated industrial and domestic effluents. As a result, several studies have been proposed and conducted in recent years to study the filtering potential of green coconut fiber.

An alarming current problem is the presence of heavy metals in effluents due to the disposal of these tailings in rivers. Because of this, the number of articles that study the biosorption of metals by coconut fiber has been intensifying in the last decade. It has been reported in the literature that coconut fiber has a high filtering potential for lead [7], nickel [7], cadmium [8], among other metals. Therefore, using coconut fiber as a substrate for the growth of our bacterial biofilm would be somehow an optimization of a natural filter.

In addition, due to the high percentage of cellulose in its composition, which can range from about 25 to 55% [6], it is interesting that we use coconut fiber in our project due to the presence of a cellulose binding tag in our metal pickup chimera, as fiber would serve as a second anchor substrate for our chimera.

UV LIGHT

UV light is electromagnetic radiation with wavelenghts between visible light and X-rays. It can be separated into ranges, and the “germicidal UV” is considered to correspond to wavelengths between 200 nm and 300 nm. Those wavelenghts are strongly absorbed by nucleic acids.

The method of disinfection based on Ultraviolet irradiation is able to inactivate or destroy microorganisms, causing photobiochemical alterations - destroying their nucleic acids and disrupting their DNA - which prevent the performance of vital cellular functions. It happens because of the nucleic acids absorption of energy, which can result in defects including pyrimidine dimers, that can prevent replication or expression of necessary proteins. It is a very common technique in food, air and water purification.

UVGI (Ultraviolet germicidal irradiation) devices are able to produce strong enough UV-C light in circulating water systems to make them inhospitable environments to microorganisms. Its effectiveness depends on the following parameters: the microorganism exposition time to UV, the intensity and wavelength of the UV radiation and the microorganism’s ability to endure UV during exposition time. [9,10]

COCONUT FIBER

FABRICATION

To reduce filter production costs, we decided to produce coconut fiber through a green coconut.

Coconut fiber production protocol

We drilled a hole in the green coconut and removed the water. After the coconut is empty, we break the coconut, we remove the epicarp (smooth epidermis/coconut shell), the endocarp (a hard layer that surrounds the edible part) and the edible part of the fruit.

That left only the mesocarp (fiber bundle), we separated it into some "handfuls" of fiber and put it together with distilled water in the blender. After beating for a few minutes, we remove the fiber and wash it with flowing water.

The fiber, already beaten and washed, was placed in beakers and it dried in a humid incubator at 37ºC, for about 8h.

CHARACTERIZATION

Absorption of water protocol

The coconut fiber water absorption test was performed so that we could better plan the volume of fiber and the water flow we should be using in the filter.

We weighed 1g of coconut fiber, which was placed in 50mL beakers and we added different volumes of distilled water until complete material humidification (overnight), which was determined by visual observation if there is visible water in aqueous phase.

The table below shows the volumes used and whether or not it has been absorbed.

Volume of water (mL)	Absorbed?
2.5	yes
3.5	no
5.0	no
10.0	no
20.0	no

As can be seen from the table, of all tested volumes only 2.5mL was fully absorbed by the fiber after the analyzed period. Therefore, we know that the max absorption of our fiber is 2.5mL/g.

Fourier-transform infrared spectroscopy - FTIR

The FTIR equipment was used as part of the characterization of our fiber since it was possible to determine the presence of cellulose in our coconut fiber. This is important because as our mercury-collecting chimera has a cellulose-binding tag (inserted by us into the genetic circuit), the protein can bind to both the bacterial biofilm matrix and coconut fiber (if it had a significant percentage of cellulose).

We used the FTIR equipment from the laboratory of the Nanotechnology and Nanotoxicology Group (GNano) of our Institute. With the help of postdoctoral student Bianca Martins Estevão, we made KBr pellets with the green coconut fiber powder, and with these pellets, we were able to collect the FTIR spectrum of our fiber.

Comparative analysis of the spectrum with the literature [11,12,13] shows that there are bands attributed to hydroxyl groups (O-H cellulosic stretch) at 3430 cm^-1, axial deformation of C-H groups at 2910 cm^-1, angular deformation of C-H groups 1375 cm^-1, angular deformation of primary alcohol C-O bonds at 1167 cm^-1, C-O-C bond absorption band, representing pyranose ring vibration at 1050 cm^-1 and β-glycosidic bonds between glycan units in 901 cm^-1, characteristics of cellulose. The band at 1745 cm^-1 refers to acetate group residues from hemicellulose.

With this result, we could conclude that coconut fiber could possibly serve as a good substrate for the cellulose binding anchor of our metal-collecting chimera.

Coconut fiber cellulose FTIR spectrum after being ball milled.

THE FILTER

CONSTRUCTION OF THE BIOFILTER

The big protagonist of our project is found here as the central element in a big cylindrical filter, that we will be calling "biofilter".

This strange component of our whole equipment, has 2 essential parts: one is the punctured steel bracket, element that will hold the coconut fiber, with biofilm, still in the biofilter and allows it to work normally even in presence of flowing water (which is the idea), this part comes in pair to improve the efficiency of heavy metal pickup; the other part is the ‘shower’, that component is basically a shower for the biofilm, it was projected to distribute uniformly the water pressure that comes throw the entry above the perforated base, it has 13 identical holes evenly separated that minimizes the damage caused to the biofilm due water flow.

At the bottom of the biofilter there is an exit for the water, which will now go to the UV Decontaminator.

Our filter, as a whole equipment, has one big closed metal box, which one is an integrated and very important part for the success filtration at the end. This container has one entry for the water, that already passed throw the Bio-Filter, and one exit for the water after the UV decontamination process. The most important component of this closed box is, of course, the UV light, which one is linked directed with the cover, making maintenance easier and putting the electric elements as external as possible.

UV LIGHT BOX

To proceed with the UV sterilization , it is necessary to identify the microorganism of interest, so it become possible to adjust the technique parameters, which are the UV emitter power, exposure time and filter measures. Some examples are listed below:

Species	UV dose (μW.s/cm²)
Escherichia coli	6600
Pseudomonas fluorescens	6600
Staphylococcus aureus	6600
Pseudomonas aeruginosa	10500
Bacillus subtilis	11000
Saccharomyces cerevisiae	13200
Chlorella vulgaris (microalga)	22000
Clostridium tetani	22000
Cryptocaryon irritans	800000

Initially, it is necessary to determine the pump potency, knowing that the efficiency in the microorganisms elimination is achieved when 99,9% of the water to be filtered passes through the system for a maximum of 12 hours.

The relation between some of the filter parameters (exposure time, pump potency and filter volume) is given by:

$$T = 9.2\;\frac{V}{B}$$

with T indicating time of exposure, V the volume of the filter and B the pump power.

The constant 9.2 was determined by P. Ecobal, in a procedure described in the book “Aquatic Systems Engineering: devices and how they function” [14]. Knowing that, we proposed different filter volumes and fixed the exposure time as 12 hours, so we could determine different pump potencies:

Filter volume (L)	Pumping capacity (L/h)
20	15.33
50	38.33
100	76.66
500	383.33
1000	766.66

The last step is to analyze the UV emitter efficiency. It is known that the UV filter is only capable of eliminating microorganism with a maximum water passage of 15 watts per L/h. Thus, we have:

Pumping capacity (L/h)	UV emitter power (W)
15.33	1.02
38.33	2.55
76.66	5.11
383.33	25.55
766.66	51.11

REFERENCES

[1] SENHORAS, E. M. 2003. Estratégias de uma agenda para a cadeia agroindustrial de coco. Monografia, Instituto de Economia, UNICAMP, Campinas. 38 p.
[2] MARTINS, C. R., JESUS JR, L. A. 2013. Produção e comercialização de coco no Brasil frente aocomércio internacional: panorama 2014. Embrapa Tabuleiros Costeiros, 51 p. (Documentos/Embrapa Tabuleiros Costeiros, ISSN 1517-1329; 184).
[3] CARRIJO, O.A.; LIZ, R.S.; MAKISHIMA, N. 2002. Fibra da casca do coco verde como substrato agrícola. Horticultura Brasileira, Brasília, 20, 4, 533-535.
[4] ROSA, M. F., ABREU, F. A. P., FURTADO, A. A. L., BRÍGIDO, A. K. L., NORÕES, E. R. V. 2001. Processo agroindustrial: obtenção de pó de casca de coco verde. Fortaleza: EmbrapaAgroindústria Tropical (Comunicado Técnico, 61).
[5] BRITO, E. O., ROCHA, J. D. S., VIDAURRE, G. B., BATISTA, D. C., PASSOS, P. R. A., MARQUES, L. G. C. 2004. Propriedades de chapas produzidas com resíduos do fruto de coco e partículas de pinus. Floresta e Ambiente, 11, 2, 01-06.
[6] CABRAL, M. M. S.; ABUD, A. K. S.; ROCHA, M. S. R. S.; ALMEIDA, R. M. R. G.; GOMES, M. A. Composição da fibra da casca de coco verde in natura e após pré-tratamentos químicos. ENGEVISTA, V. 19, n.1 , p. 99-108, Janeiro 2017.
[7] FERREIRA, D.C; DA SILVA, N.A; LIMA, A.F; BEGNINI, M.L. BIOSORÇÃO DE CHUMBO E NÍQUEL PELAS FIBRAS DO COCOS NUCIFERA L. FAZU em Revista, Uberaba, n.9, p. 64-68, 2012.
[8] BEZERRA, R. S.; RIZZO; A. C. L.; AZEVEDO, B. S. M. Utilização da Fibra da Casca de Coco Verde como Suporte para Formação de Biofilme Visando o Tratamento de Efluentes. XVI Jornada de Iniciação Científica – CETEM.
[9] "Word of the Month: Ultraviolet Germicidal Irradiation (UVGI)" (PDF). NIOSH eNews. National Institute for Occupational Safety and Health. April 2008. Retrieved 4 May 2015.
[10] "SOLVE II Science Implementation". NASA. 2003. Archived from the original on February 16, 2013. Retrieved 4 May 2015.
[11] CIOLACU, D.; CIOLACU, F.; POPA, V. I. AMORPHOUS CELLULOSE – STRUCTURE AND CHARACTERIZATION. Cellulose Chem. Technol., 45 (1-2), 13-21 (2011).
[12] Hospodarova, V.; Singovszka, E.; Stevulova, N. Characterization of Cellulosic Fibers by FTIR Spectroscopy for Their Further Implementation to Building Materials. American Journal of Analytical Chemistry, 2018, 9, 303-310.
[13] Sang Youn Oh; Dong Il Yoo; Younsook Shin; Gon Seo. FTIR analysis of cellulose treated with sodium hydroxide and carbon dioxide. Carbohydrate Research 340 (2005) 417–428.
[14] ESCOBAL, Pedro Ramon; Aquatic Systems Engineering: Devices and how They Function. 1. ed. Universidade da Califórnia: Dimension Engineering Press, 1996.