Rethinking water treatment
To develop a yeast with the capability to uptake high amounts of salt, we created several mutants through genetically modifications. In addition we measured the protein expression, sodium and potassium concentration inside the cells and the relative cell amount for different mutants.
Now, we are proud to present the following results:
- Successful creation of yeast mutants through several knockouts and overexpressions and validated the gene modifications of the final 13 mutants by sequencing
- We checked protein expression of the GAL1-Promoter by qPCR
- We demonstrated higher sodium uptake by the yeast mutant “VAULTer I” compared to the wildtype and compared the growth profiles of the mutants
- We proved the adaptability of the concept of a microbial supercollector by developing a potassium uptaking mutant
Diagram 1: The ratio between the total sodium amount and the amount sequestrated of the mutants at the same cell number is shown. Cellular sodium concentrations measured by ICP-OES (inductively coupled plasma optical emission spectrometry) in the range of mg/L.
We thoughtfully planned different mutant compositions to maximize the salt uptake. We measured the growth profiles and the salt concentrations to decide which composition is the best. Due to the toxic effect of cellular sodium, we needed to carefully rise the uptake. For that reason, we used the inducible GAL1 promoter to set the overexpression to the needed rate. We worked towards the highest possible sodium uptake which the cell can survive.
The measurements demonstrated that “VAULTer I” had the most sodium accumulation of all mutants. It consists of the ENA1 Knockout, the integration and overexpression of ATNHXS1 and AVP1. As you can see in the diagram #1, VAULTer I accumulated about 39% of the total sodium amount of 0,6 M - the wildtype accumulated 8%. Furthermore, the yeast with the ENA1 knockout had a ratio of 25%. By incorporating all these mutants in one mutant (VAULTer I) we increased the uptake furthermore by approximately 14 %.
In the chart diagrams #2 and #3 below you can see the sodium uptake and the growth profiles of different mutants in comparison to the wildtype. Although VAULTer I, VAULTer II and VAULTer III have the slightest growth this can be explained by two major aspects. On the one hand, mutants with multiple gene modifications need a lot more energy for the overxpressed genes, like VAULTer I with two overexpressions, and on the other hand the sodium accumulation in the vacuole is decreasing the growth due to the toxic effect of sodium to the cell. VAULTer I can uptake high sodium amounts but in return the cell growth is limited. Quite contrary to AVP1, ATNHXS1 or NHX1.
VAULTer I as shown has the most uptake closely followed by VAULTer II and ENA. We measured an uptake of 4,7 mg/ml Na+ more than the wildtype. VAULTer II has the same genetic modifications as VAULTer I except of the knockout “NHA1” more. It seems that NHA1 as a negative impact on the sodium accumulation. We suggest that with the knockout of NHA1 the mutant gains halotolerance, leading to more growth in comparison to VAULTer I. The sodium uptake is a sensitive mechanism due the continuous ponder between death of the cells (osmotic shock / toxicity of sodium) and the interplay of the different genes we modified. NHNA shows how susceptible this mechanism is, here the interplay is overstressed. The level of cellular sodium probably exceeded the tolerable amount, since NHNA has not integrated AVP1 and AtNHXS1. The mutant could not accumulate the sodium in the vacuole. Unable to discharge the cellular sodium, the concentration gradient was greatly reduced, hence the mutant stopped the Na+ influx. All mutants, even the wildtype, accumulated more sodium. NHX1, an Na+ influx pump for prevacuolar compartments, has an uptake ratio of 12% and a total uptake of 1,86 mg/ml. VAULTer III an uptake ratio of 28% and a total of 4,1 mg/ml uptake. The integration of NHX1 in VAULTer I could furthermore enhance the up taking capabilities.
Diagram 2: The absolute sodium uptake of the mutants in mg/ml for the same cell amount is pictured. One ml of 0,6 M contains 15,3 mg of sodium. The cellular sodium concentrations are measured by ICP-OES (inductively coupled plasma optical emission spectrometry) in the range of mg/L.
Diagram 3: The relative cell mass over time is measured by a growth profiler 960 (EnzyScreen) in 0,6 M NaCl YPDGal.
Diagram 4: The absolute chloride uptake of the mutants in mg/ml for the same cell amount is pictured. One ml of 0,6 M contains 24 mg of chloride. The cellular chloride concentrations are measured by anion exchanger chromatography in the range of mg/L.
As shown in the diagram #4 VAULTer II has the highest chloride uptake with a value of 1,14 mg/ml. Closely followed by ATNHXs1 and NHX1. Due to the similarity of NHX1 and ATNHXS1 the similar behavior can be explained. To compensate the sodium (positive charged) uptake the mutants likely take up chloride (negative charged). Thus, NHNA and ENA1 with no sodium transporters have a less uptake rate. Only VAULTer I behaves different, probably caused by the negative effect AVP1 has to the chloride uptake. VAULTer I with only 0,6 mg/ml has still 50% more chloride uptake than the wildtype.
To prove the functionality of the GAL1 promotor and the transcription of the proton pump AVP1 we performed a real-time-quantitative PCR, as you can see in the diagrams below. Therefore we isolated mRNA from wildtype, AVP1 mutant grown in normal YPD-media and AVP1-mutant grown in YPD-media with 100% galactose. We followed protocol “Protocol mRNA-isolation and RT-qPCR”, using the gene ACT1 as housekeeping gen for reference. As results we identified that the concentration of AVP1-mRNA in AVP1-mutant grown on 100% galactose was about 20 to 40 times higher than in wildtype and AVP1-mutant grown in normal YPD. The results are shown in diagram #5 and #6.
Diagram 5: The amounts of mRNA of the AVP1-gene are pictured, comparing wildtype S. cerevisiae (WT), a mutant strain containing AVP1 growing in YPD (GLU) an a mutant AVP1-strain growing in YPD with 100% galactose (GAL). The relative comparison of AVP1-mRNA amount shows that AVP1 mutant growing in YPD media with galactose exhibit a 20-40 times higher transcription of the AVP1-gene than AVP1 mutant growing in normal YPD-media or wildtype growing in normal YPD media. The showed results were measured with RT-PCR Detection system developed by BIORAD. The measurements were performed in duplicates (#1 AVP1 and #2 AVP1)
Diagram 6: Pictured is a melting curve of the amplified cDNA of AVP1 (see above). The diagram was recorded measuring the negative alteration of fluorescence of the given samples with increasing temperature. Samples measured are: cDNA of AVP1-mRNA extracted from wildtype (6x), cDNA of AVP1-mRNA extracted from not induced AVP1-mutant (6x), cDNA of AVP1-mRNA extracted from induced AVP1-mutant (6x) and negative control without template DNA (4x). The showed results were measured with RT-PCR Detection system developed by BIORAD immediately after RT-qPCR.
Our aim of the project SALT VAULT is to develop a microbial supercollector for pollutive ions. To show the adaptability of this idea we additionally created a mutant called “NAKT”, uptaking potassium instead of sodium. We created NAKT through the knockout of ENA1 and NHA1 and the overexpression of the foreign gene AtAKT1 from A. Thaliana. In the chart on the right you can see the uptaking efficiency between VAULTer I and NAKT. In 0,6 M KCl media NAKT had the efficiency to take up potassium of appr. 54% of VAULTer I taking up sodium in 0,6M NaCl- media.
Potassium uptake of NAKT ratio to sodium uptake of VAULTer by different concentrations Shown is the relative efficiency between the potassium uptake of NAKT and the sodium uptake of VAUlter with adjusted cell number.
The relative cell mass of NAKT and AtAVENA over time is measured by a growth profiler 960 (EnzyScreen) in 0,6 M NaCl YPDGal.
In the End, we successfully developed mutants capable of accumulating high amounts of sodium and potassium. Our idea of a microbial supercollector worked out so far for sodium and potassium.
Osmotolerance and Membrane Experiment
To further demonstrate the feasibility of the application we carried out two more experiments, generating valuable information of osmotolerance and separation of yeast- cells from water.
Osmotolerance and Membrane Experiment
Intracellular accumulation of NaCl significantly intensifies the osmotic pressure on yeast. Bursting of the cells would resolve ions, making Salt Vaults useless. In previous years, the high osmotolerance of yeast and its mechansims have been identified.
Read the following text to gain information on the mechanisms of yeast to handle high osmotic pressusres.
We have conducted an experiment to prove the capability of yeast to resists a hypo osmotic shock. We inoculated the mutants AVPA (AVP1 Integration + ENA1 Knock-Out) and VAULTer II in YPD-media. The grown cultures were centrifuged and resuspended in double-distilled water to create a strong hypo-osmotic shock. Additionally, wildtype was inoculated and resuspended in both double-distilled water and normal YPD-media as a control. After 15 minutes the cultures were microscoped on cellular damages.
Fig.1: Microscopical pictures of yeast wildtype without a hypo-osmotic shock (left) and VAULTer II after 15 minutes of incubation in double-distilled water (right)
The microscopical pictures show intact cells for every examined mutant and the wildtype without a hypo-osmotic shock, proving the osmotic tolerance of both the yeast wildtype and our mutants to withstand a high hypo-osmotic shock. To create the same osmotic pressure we created with the experiment, a high uptake rate of NaCl has to be developed, making osmotic pressure not a limiting step in the development of Salt Vaults.
Ultrafiltraion to separate yeast from water
In cooperation with General Electric within our Integrated Human Practices (Link: IHP, GE), we developed a continuous experiment to prove and test the capability of ultrafiltration membranes to separate water from yeast. This is important to guarantee the complete isolation of the genetically modified yeast from the environment in an industrial water treatment application.
The figure below shows the setup of the experiment. Click below to read detailed information on the complete setup and flux numbers.
GE provided us with a small-scale ultrafiltration membrane with a surface area of 0.025m2. Membranes, used in water treatment plants, are run with a flux over the membrane of less than 15.0 L/(h*m2 membrane surface area). Together with GE, we decided to use a flux of 25.0 L/(h*m2), resulting in a flux of 0.625 L/h over the membrane.
Assuming a membrane surface of 300m2, a realistic number in water treatment plants, the set-up would be able to purify 7500 L/h of water per hour. However, the small-scale experiment does not permit predictions on the efficiency of the method in industrial scale.
As a reaction medium, we used a murky water-yeast mixture. We developed a continuous process by pumping 0.625 L/h of the mixture into the bioreactor. On the other side 0.625 L/h were pumped out of the bioreactor using a piston pump. The pump created a low-pressure of around 0.5 bar, to squeeze water through the membrane, while yeasts are hold back. As a filtration method, we used dead-end filtration.
Fig1: Experimental set-up of the experiment to separate yeast from water using an ultra-filtration membrane
After filtering, the efflux of the bioreactor was microscoped on cell amounts. Additionally, the cultures were centrifuged and the pellet investigated.
Fig2: Microscopical picture of culture after membrane treatment
The experiment proved complete separation of yeast from the mixture. Centrifugation and microscoping did not show any cells. Additionally, the opacity of the media shows significant differences.