Demonstrate

1. Theoretical basis

From our human practices, we knew that physicochemical methods are used in the treatment of effluent containing heavy metals. Although lime and ferric chloride decreased the concentration of heavy metal ions, they may also pollute the environment. This knowledge directly informed our project, we decided to create an engineered yeast to adsorb nickel ions.

We first use the surface display system of MFα1+hexa-His+ AGα1 to capture and bind nickel ions. We also use NixA to transfer nickel ions into the cells, and TgMTP1t2 to transfer nickel ions from the cells into the vacuoles.

The absorption abilities of engineered yeast and original yeast were compared, and the results indicated that the S.cerevisiae/BBa_k3126023 (hexa-His+nixA+TgMTP1t2) showed highest adsorption efficiency, with a nickel ion removal efficiency of 80%, with the test concentration of nickel ions had being reduced from 15 mg/L to 2.6 mg/L.

2. Device design

This year we built a mini-plant model of the effluent factory. It demonstrates our idea of using the engineered yeast in a real-world application. Because the engineered yeast must not get into the natural environment, we found a way to immobilize it in order to make it easier to separate from the effluent, and added semi-permeable membrane filters to the final outflow pipe. We also built software to automatically control the ingress and egress of effluent, and to use a water level detector in the treatment pool to add the correct amount of effluent to be treated in the main tank. The amount of yeast gel balls can be calculated from our data modeling.

3. Pilot device test

The engineering yeast S.cerevisiae/BBa_k3126023 (hexa-His+nixA+TgMTP1t2) were immobilized by PVA-boric acid method, and these gel balls (the dry weight of yeast is 0.5g/L) were used for the treatment of effluent containing nickel ions in pilot device, and the result indicated that after the immobilized yeast was used for Ni2+ adsorption, we replaced a new batch of immobilized yeast for Ni²⁺ adsorption, and the concentration of nickel ions in effluent was decreased from 15 mg/L to 0.469 mg/L.

4. Cost accounting

Cost is divided into yeast growth cost, device cost, and nickel ions recovery cost.

The car factory produces 5 m3 of effluent per hour. Supposing the factory works 300 days a year, 12 hours a day, each year will produce 18,000 m3 of effluent. Test samples of pre-treated effluent indicate a 15 mg/L concentration of nickel ions. From this we can calculate the yeast needed to be 18,000 kg.

(1) Yeast growth and immobilization cost

Table 1. Cost to culture 1 kg dry weight yeast

Ingredients	Price (RMB)
molasses (carbon source)	20
(NH₄)₂SO₄ (nitrogen source)	10
production cost	30

The total weight of yeast we need every year is 18000 kg, so we estimate the yeast grow cost at 540,000 RMB (≈USD76,000) per year.

PVA and other materials needed for yeast immobilization are about 10,000 RMB (≈USD1,400) per year.

(2) Device cost

The cost of the device is mainly derived from the collection pool, treatment tank, discharge tank, engineered yeast filter box, and air convection device.

When estimating the actual cost of the device, the construction materials of the collection pool, treatment tank and discharge pool are selected from C15 concrete. The water storage capacity of a single pool can reach 270m3. We discharge effluent into the collection pool through the control system. After the water level gauge detects that it reaches 3.5m, the pump moves the effluent into the treatment tank. After the treatment period, it is pumped into the discharge tank which is fitted with semi-permeable membrane on the outflow valve to prevent microbial escape. We use a Roots blower to connect the ABS piping system at the bottom of the treatment tank for air convection so the effluent in the pool is continually moved past the gel ball yeast filters. The whole process does not pollute environment.

Table 2. Budget table for a set of devices

Name	Quantity	Unit price
C15 total volume	360m³	300 RMB/m³
ABS pipe	25m	10.3 RMB/m
ABS engineering plastics	45m³	20 RMB each
Blower	1	8,000 RMB each
Water pump	2	5,000 RMB each
Control System	1 set	10,000 RMB per set
Water level gauge	3	200 RMB each
Greening	1 set	5000 RMB per set
Semipermeable membrane	1 m	22 RMB per m
Total	142,797.5 RMB (≈USD20,000)

(3) Nickel ion recovery cost

Enzyme decomposition method can be used to recovery nickel ions from yeast. The mixture of 0.6% of neutral protease and papain were used to disrupt yeast cells.

Table 3. Cost to recover nickel ions from 1 kg of yeast

Ingredients	Price (RMB)
Papain	10
neutral protease	10
Recovery cost	20

The total weight of yeast we need every year is 18,000 kg, so the nickel ion recovery cost is 360,000 RMB (≈USD50,700).

Conclusion

Based the analysis above, all the expenses mentioned above are about 1,050,000 RMB (≈USD148,000). Our project is feasible in theory and in practice. We use immobilized engineered yeast to perform nickel ion adsorption, which isn’t an environmental pollutant, unlike materials used in physicochemical methods, and can also complete the absorption and recycling of nickel ion at a much lower cost, and a far shorter period of time (hours versus days). Our system completely complies with all rules and policies approved by the iGEM Safety Committee.