With a dense population and limited lands in Hong Kong, the aquaponics, which combined agriculture and aquaculture together with less plots utilization, would definitely enhance the economic benefits to related industries. Nevertheless, a simple artificial ecosystem with lack of proper care, maintenance of balance of various parameters, such as water heavy metal(Copper, Zinc, Lead, etc.) level, would be a significant difficulty. The heavy metals might lead to a poor growth or death of vegetables and fish. Therefore, it is planned to genetically transform an E.coli bacteria with the ability to regulate the level of heavy metals (with target metal as copper) in water, with utilization of Metallothionein of different species, which with ability to achieve metal chelation. Thus to improve the growth of the fish and the plants, and ultimately achieve profit maximisation of this industry.

Problems in aquaponics

Growth of plants

Fish food is an essential material for growing fish in aquaponics. Which contains different nutrients, but meanwhile, also contains many heavy metals. Noted that only approximately 1/3 of the ingested food will be consumed and assimilated by the fish while the rest 2/3 will be egested [1] back into the water system, and hence flows along the whole aquaponic system, which increases the concentration of the heavy metals in the water. Eventually , the growth of the plants is inhibited. For instance, excess amount of Mg inhibits the uptake of Potassium and Calcium. Also, excess amount of Copper, Zinc, Chromium and Nickel inhibits the uptake of Iron [2].

Growth of fish

The excess nutrients inside the uneaten fish food also support and promote the growth of the bacteria, fungi and algae in the water system, that If those organisms keep growing continuously, the dissolved oxygen level will decrease sharply. Thus, the oxygen competition between fish and the microorganisms decreases the growth rate of the fish. Besides, ammonia is produced upon microorganisms’ metabolism and hence ionises in water to give alkali (aqueous hydroxide ions), which also increases the pH value of the water. Then, the direct contact between alkalised water with the whole body of the fish and the root system of the plants provides a worse condition for their growth and survival. [3]

Poor growth or death of organisms consequently upsets the ecological balance in such systems, making them no longer sustainable, which leads to decrease of the economic benefits brought by aquaponics.

Biosorbents of heavy metals

A variety of methods can be applied to remove heavy metal pollutants in an aquatic environment. Biosorption is one of the feasible methods, and a list of biosorbents have been studied in the past, including algae, fungi and bacteria. Their ability to absorb our target metal, copper, was identified [4], among these model organisms, we will select the most promising target as the model in our project.

1) Fungi:

Fungi have a high percentage of cell wall material, which contributes to the excellent metal binding properties [5]. However, fungi do not survive well in aquatic environment and it is difficult to maintain the growth of fungi in a secondary laboratory setting. Therefore applying fungi in our project doesn't seem to be a good choice.

2) Algae:

Algae is relatively easy to grow and maintain in a secondary school laboratory setting. The algal cell wall also granted the metal adsorption ability to the algae species [4]. However, the speedy growth of algae (a.k.a. algal bloom) would disturb the aquaponics system, and decrease of the water oxygen level. Thus maintaining a recyclable environment in aquaponics would be a significant obstacle. Therefore, it is not a practical biosorption method in our project.

3) Plant species:

Making use of phytoremediation of plant by using specially selected and engineered metal-accumulating plants for environmental clean-up is also a feasible strategy. However, it shares all the same drawbacks mentioned above, plus, in our scenario, the aquaponics system cannot offer an extra space for holding another plant species.

4) Bacteria:

Bacteria seems to be the most promising model for our project. Firstly, studies have shown the native copper absorbing ability of bacteria species [4][8]. Second, the maintenance of bacteria is relatively easier with a lower cost comparing to other organisms. Thirdly, bacteria used as bio-filter are very common in aquarium and many commercially available equipment, such as bio-rings and ceramic tubes are easily found in the market by the users.

We then designed to further enhance the copper absorbing ability of the bacteria by synthetic approach. After extensive literature review and discussions, metallothionein 1 (a metal-chelating protein) was decided to be utilized in our investigation.

Metallothioneins

Metallothionein (MT) is a ubiquitous protein with a low molecular weight and was first identified in the kidney cortex as a cadmium-binding protein, in responsible for the natural accumulation of Cd in the tissue. Containing 61 to 68 amino acid residues, it has a role in the protection against metal toxicity and oxidative stress, and is involved in zinc and copper regulation [6][10].There are four main isoforms expressed in humans or mammals like rat, including metallothionein-1, metallothionein-2, metallothionein-3 and metallothionein-4. However, the metallothionein in yeast (Saccharomyces cerevisiae) only exists as metallothionein-1 (CUP1)

Metallothionein as the target of study

Metallothioneins demonstrate the following properties which made it a target of our study.

1) Ubiquity

The ubiquitous metallothioneins in eukaryotes plays an important and convenient role for us to target the metal-binding genes in certain organisms. Metallothionein has been identified in all animal phyla examined to date and also in certain fungi, plants and cyanobacteria as well as in humans [6][9], it showed that metallothionein must play an important role in living organisms to made it being conserved evolutionarily. Moreover, since it is expressed in most living organisms, the overexpression of metallothioneins will be less likely to affect the growth of our model organisms.

2) Wide Metal-chelating Capacity

Metallothioneins have been shown to chelate various heavy metals, including but not limited to cadmium, lead, copper, mercury, zinc, silver, nickel and cobalt [9]. With the wide variety of metals being binded at the same time, metallothionein would be an efficient choice, for regulating several water heavy metal levels at the same time.

Metallothionein-1

Metallothionein-1 will be the our first target to study due to the fact that metallothionein-1 is the best characterised among all the four analogs. Also, it has been demonstrated to have the highest affinity in binding copper and zinc in relative to other metals [10][11]. Therefore, we believe metallothionein-1 plays a particularly important role of copper chelation in living organisms.

Furthermore, we designed to conduct a cross-species metallothionein-1 comparison. Yeast metallothionein was identified as one of our targets since it showed a high affinity in copper binding and enhanced the copper tolerance in organisms [12][13]. However, only metallothionein-1 (CUP-1) is found to be expressed in yeast, which made metallothionein-1 the only applicable target for our study.

Design of Biofilter

The functions of proteins depends on their structure. The accumulation of excess heavy metal in organisms will cause irreversible structural change on proteins such as enzymes. Therefore, the proteins will lose their function such as boosting up chemical reaction and the failure of transferring materials in and out of the cells through channel proteins on their cell membrane. Fishes and plants in aquaponics therefore die because of nutrition deficiency.

A bacterial copper filtration device is designed in order to deal with the problem above. Bacteria such as E.coli we used in the experiment, itself can absorb heavy metals. Metallothionein-1 protein can combine with heavy metals. Therefore, E.coli transformed with metallothionein-1 gene has an advance ability to absorb heavy metals. When this genetically modified E.coli is put into the device, excess heavy metals can be absorbed and will not harm the fishes and plants.

Genetically modified E.coli will be put into the dialysis tubing, this is a measure of bio-safety, as the pore of dialysis tubing is big enough to allow the diffusion of copper ions but too small to allow the diffusion of E.coli. Therefore E.coli will not contaminate the ecosystem. In the device, E.coli will transferred from between the reservoir and the dialysis tubing to form a loop through the pumping of a peristaltic pump. After the absorption of excess copper ions, E.coli will be replaced.

Future studies

Regulation of Biosorption

Excessive biosorption may be resulted due to the over-expression of Metallothionein-1 or very strong affinity of it to heavy metals. Being the trace elements, certain kinds of heavy metals such as zinc and copper in low levels are important to the growth of organisms, especially plants. Thus, the strength of biosorption has to be regulated through a synthetic approach. This will be the focus of the next stage of our project.

Transcription of Metallothionein-1 genes is rapidly and dramatically up-regulated in response to zinc and cadmium, as well as in response to agents which cause oxidative stress and/or inflammation. The six zinc-finger Metal-responsive Transcription Factor 1 plays a central role in transcriptional activation of the Metallothionein-1 gene in response to metals and oxidative stress. Modifying this region could be a direction to regulation of biosorption.

Other possible drawbacks included that materials cycle in an ecosystem, instead of loss, the absorbed heavy metals are still accumulated inside the filters. A suitable disposal method of our used “bio-filter” should be considered, for instance, a metal recovery system for industrial usage by extracting accumulated heavy metals from biomass of microbial would be a feasible direction [15].

Reference

Hijran Yavuzcan Yildiz, Fish Welfare in Aquaponic Systems: Its Relation to Water Quality with an Emphasis on Feed and Faeces—A Review, 2017
Gjesteland,lngrid, Study of Water Quality of Recirculated Water in Aquaponic Systems: Study of speciation of selected metals and characterization of the properties of natural organic matter,2013
Tidwell, J. (2012). Aquaculture production systems. Ames, IA: Wiley-Blackwell.
doi:10.1002/9781118250105.ch14 Chapter 14 Aquaponics—Integrating Fish and Plant Culture Nilanjana Das, R Vimala and P Karthika (2008). Biosorption of heavy metals–An overview. Indian Journal of Biotechnology. Vol 7, April 2008, pp 159-169.
Sağ, Y. (2001). Biosorption Of Heavy Metals By Fungal Biomass And Modeling Of Fungal Biosorption: A Review. Separation and Purification Methods,30(1), 1-48. doi:10.1081/spm-100102984
Sakulsak, N. (2012). Metallothionein: An Overview on its Metal Homeostatic Regulation in Mammals. International Journal of Morphology,30(3), 1007-1012. doi:10.4067/s0717-95022012000300039
Valls, M., González-Duarte, R., Atrian, S., & Lorenzo, V. D. (1998). Bioaccumulation of heavy metals with protein fusions of metallothionein to bacteriol OMPs. Biochimie,80(10), 855-861. doi:10.1016/s0300-9084(00)88880-x
Cooksey, D. A. (1993). Copper uptake and resistance in bacteria. Molecular Microbiology,7(1), 1-5. doi:10.1111/j.1365-2958.1993.tb01091.x
Dziegiel, P., Pula, B., Kobierzycki, C., Stasiolek, M., & Podhorska-Okolow, M. (2016). Metallothioneins: Structure and Functions. Metallothioneins in Normal and Cancer Cells Advances in Anatomy, Embryology and Cell Biology,3-20. doi:10.1007/978-3-319-27472-0_2
Kägi, J. H. (1991). [69] Overview of metallothionein. Metallobiochemistry Part B Metallothionein and Related Molecules Methods in Enzymology,613-626. doi:10.1016/0076-6879(91)05145-l
Tapia, L., González-Agüero, M., Cisternas, M. F., Suazo, M., Cambiazo, V., Uauy, R., & González, M. (2004). Metallothionein is crucial for safe intracellular copper storage and cell survival at normal and supra-physiological exposure levels. Biochemical Journal,378(2), 617-624. doi:10.1042/bj2003117412. Glen KAndrews, Regulation of metallothionein gene expression by oxidative stress and metal ions, 1999
Adamo, G. M., Lotti, M., Tamas, M. J., & Brocca, S. (2012). Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae. Microbiology,158(Pt_9), 2325-2335. doi:10.1099/mic.0.058024-0
Xie, X., Ma, Y., Chen, Z., Liao, R., Zhang, X., Wang, Q., & Pan, Y. (2014). Transgenic Mice Expressing Yeast CUP1 Exhibit Increased Copper Utilization from Feeds. PLoS ONE,9(9). doi:10.1371/journal.pone.0107810Ulrich, W. (n.d.). Copper-Thiolate Proteins (Metallothioneins). Copper Proteins and Copper Enzymes,151-174. doi:10.1201/9781351070898-511. Timothy A. Hovanec,Trace Elements, 1998
Arif Tasleem Jan., Mudsser Azam, Kehkashan Siddiqui, Arif Ali, Inho Choi, and Qazi Mohd. Rizwanul Haq, Heavy Metals and Human Health: Mechanistic Insight into Toxicity and Counter Defense System of Antioxidants, 2015 Dec 10. doi: 10.3390/ijms161226183
Ahluwalia, S. S., & Goyal, D. (2007). Microbial and plant derived biomass for removal of heavy metals from wastewater. Bioresource Technology,98(12), 2243-2257. doi:10.1016/j.biortech.2005.12.006