Team:Aachen/HP/Gold Integrated

iGEM Team Aachen 2017

Developing an Idea

With the help of industry and research, we developed our idea to meet both scientific and societal standards. Starting with a system focused on NaCl-Uptake and a vague application in water treatment we concluded in creating the vision of microbial supercollectors for all kinds of substances, proving this vision by storing potassium in a novel yeast. Additionally, we found clear answers on biosafety and fields of application, using well-established membrane technology to remove yeast from water in an energy-efficient way.

Read the following flowchart to gather an overview of our project development and how we were influenced during our journey. By clicking the figures and pictures you get detailed information on the different cooperations.

Werra Salinization

All over Germany you can find huge white mountains consisting of 96% salt. These are products of the same industry, also responsible for the extremely high concentration of salt in the Werra, potash production. To get an insight into the seriousness of this problem we travelled to Gerstungen, a small town located directly at the Werra besides a plant producing potash.

The high burden of the river Werra in central Germany was the main motivation for our project and we got in contact with the people that struggle the most with the consequences of rigorous disposal of salinized wastewater and talked Ulf Frank, Manager of the water distributer of Gerstungen about the main inducer of the high concentration of NaCl in the Werra: The potash mining company K+S, which was sadly not willing to cooperate with us. They dispose water with high salt concentrations into the Werra, dump pure NaCl on hills up to 200m high or inject it into the ground. The consequences for the locals are devastating. Not only the tap-water coming from the ground is salinized, the sunset happens to be two hours earlier because of of the saltmountains. In Mr Franks eyes, the main problem is that K+S is allowed by law to produce and spread their wastewaters without consequences. K+S, however, is a huge company, with a profit of 130.5 million € in 2016, raising the question on the environmental responsibility of K+S Kali. Due to Mr Frank, the CEO of K+S changed in in April 2017, and showed openness for dialogue, giving the possibility for change and action with new ways to treat the sallinized wastewater.

Initial Idea

The Werra is polluted with high concentrations of NaCl due to wastewaters from potash-mining. A modified yeast, added to wastewater treatment plants as an add-on to the bacterial sludge to desalinate the wastewater would solve this issue.

We have identified NaCl as a toxin for aqueous ecosystems, destroying waters all over the world - including Germany, a country not related to water problems at all. To take action on this environmental issue we will develop a yeast which is capable of taking up and storing these ions in its vacuole. The question is, to what extent microorganisms are able to purify wastewaters. The answer is simple because we humans are doing exactly this already since decades. We treat wastewater in wastewater treatment plants with bacteria which are able to degrade N- and C-compounds within a few hours. Up until now, there is no way to handle contaminations of ions in wastewater with microorganisms.

Our yeast would close this gap, simply by being added to purification steps of a wastewater treatment plant, for example as an additional part of the activated sludge in the biological phase of water treatment. This technology could be used to desalinate all kinds of salinated waters like industrial or municipal wastewaters or even seawater and salinated rivers like the Werra in Germany. The figure below shows the potential integration of yeast into the process of a wastewater treatment plant.

Looking for Industrial Applications

To investigate whether the wastewater pollution with NaCl is a problem only related to potash-mining or rather a common problem of industrial wastewaters we got into contact with the Chemelot Industrial Site located in the Netherlands. We visited Chemelot’s biological wastewater treatment plant.

Looking for Industrial Applications

Together with deputies from Sitech and Brigthlands we discussed current hurdles of industrial water treatment in the Netherlands and possible solutions. Additionally, Sitech provided us with numbers on the concentrations of different ions and metals in Chemelot’s wastewater.

The cooperation with Sitech and Brightlands depicted that high sodium and chloride concentrations are not an issue of chemical industries these days, however Dutch thresholds on chloride are tense and will be intensified in future, making it important for Chemelot to discuss solutions for water purification from chloride. Besides that, the cooperation revealed the need for technologies purifying water from various ions like Sulphate or Phosphate or altering substances like Heavy Metals or organic molecules, concluding in the question whether these substances could be taken up by yeast acting as a microbial dustbin.

Fig 1: Average concentrations of chloride (left) and sulphate (right) in the efflux of the IAZI in mg/l in the months January to July 2017. Red line indicates the threshold for chloride in mg/l in drinking water in Germany and the Netherlands.

Consequently, we shifted our project from assimilating only NaCl to proving that the uptake of substances by yeast is not bordered to NaCl, but gives possibilities to purify water from ions, molecules or metals. To prove this concept, we integrated herbal potassium and sulphate uptake transporters into the yeast’s membrane. (Read more: Project Description)

Separation and subsequent use of the yeast cells after taking up salts emerged as central questions to be dealt with. Therefore, we focused our efforts on answering these within our further project development.

Statements of the deputies on European environmental action on water pollution raised the political question, why the protection of rivers running to various countries is not equal in the affected countries and why environmental action in general is dissimilar in different European countries (see Shaping Politics).

Fig.2: Tour around Brightlands given bei Bart van As

On the Chemelot Industrial Site around 80 chemical companies with a total of 6000 employees are producing a range of products from fertilizers to bulk polymers. The site holds a biological wastewater treatment plant (IAZI) which is operated by Sitech Services. Additionally, a research campus, Brightlands, located on the industrial site (Brightlands Chemelot Campus), initially founded by DSM and Maastricht University, was and is built since 2013. Brightlands is focusing on research on sustainable materials & manufacturing, medicine and food & nutrition.

Both Sitech and Brightlands agreed to support our work on biological wastewater management with regards to content. The cooperation included a tour around the wastewater treatment plant and Brightlands Chemelot Campus, discussions on current issues of water treatment and allocation of numbers concerning the concentrations of ions in the wastewater leaving the IAZI.

Fig 3: Entrance gate of Brightlands at Chemelot

The tour around the IAZI was given by Wiel Felder, Plant Manager of the IAZI. The IAZI has a capacity of 1.000.000 human equivalents, removing most of N- and C-compounds, and additional molecules or heavy metals. The purified wastewater is then led into the river Meuse. Due to internal regulations, we were not allowed to take pictures of the plant itself.

Secondly, we had a discussion on current issues of the IAZI and our project with employees of Sitech and Brightlands. Regulations for wastewater in the Netherlands are higher than for example in Germany, France or Belgium. One reason for this is that the Netherlands are using the Meuse as a source for drinking water, concluding in much higher restrictions due to priorities of drinking water. Therefore, an industrial site like Chemelot must put high affords into cleaning their wastewater sustainably for this manner. Additionally, the Dutch government is planning to limit the critical values on especially Temperature, Chloride, and N-compounds like Nitrate in future. Chemelot tries to prepare for this case already nowadays by talking to research teams like iGEM Aachen.

Our discussions revealed that the IAZI is working well, however, there are issues which must be tackled. The IAZI is facing problems with organic molecules, site products of chemical industries, partly not identified yet. An example for this is pyrazole, which was identified in 2015. Pyrazole is classified as a harmful substance by the Globally Harmonized System of Classification and Labelling of Chemicals. Due to strong Dutch environmental policy, Chemelot had to fear to stop production, if the source of pyrazole was not detected. Although pyrazole could be removed by Chemelot in time, this case shows the importance of research on novel water treatment methods already nowadays.

To investigate the significance of NaCl in industrial wastewater plants, Sitech provided us with averaged concentrations of several ions measured in the effluent of the IAZI between January and July 2017. Measured ions were sulphate and chloride, whereas sodium is not measured at the IAZI. The corresponding graphs below show that Chemelot is currently not facing problems with chloride, but concentrations of sulphate are higher than the threshold for sulphate in drinking water. Rene Borman, CEO of Sitech Services, confirmed that for the IAZI, chloride, sulphate and additionally phosphate are interesting and relevant to investigate feasibility with our project.

Fig 4: Average concentrations of chloride (left) and sulphate (right) in the efflux of the IAZI in mg/l in the months January to July 2017. Red line indicates the threshold for chloride in mg/l in drinking water in Germany and the Netherlands.

Additionally, Mr Borman said that they are also interested in removing traces of metals like Zink, Vanadium or Nickel. The current influx and efflux concentrations of diverse metals are shown below in Fig 3 showing that heavy metals are already eliminated efficiently by the IAZI, however, traces of metals remain in the wastewater leaving room for action on purification of water from metals

Fig 5: Average concentrations of chloride (left) and sulphate (right) in the efflux of the IAZI in mg/l in the months January to July 2017. Red line indicates the threshold for chloride in mg/l in drinking water in Germany and the Netherlands.

At the moment of the cooperation, we did not have results on efficiency and desalination period, therefore we could rather discuss on possible industrial approaches, targets of desalination and troubleshooting than on realistic feasibility. The conclusion was that our idea is interesting for industrial applications because it is a new approach to remove substances from water. However, NaCl is not a problem of all industries. Chemical industries, for example, have got altering pollutions in their wastewater. In the Netherlands, these are for example sulphate, metals or side products of polymerization. In Germany for example, research has shown that the Rhine in Germany is polluted with arrears of pharmaceutical industry.

Furthermore, we talked about the sustainability of our approach. What is happening with the yeast cells after they have taken up the ions or substances? And how are they separated from the water? Our cooperation partners have motivated us to work on a sustainable solution to use the yeast afterwards. For them, a possible way for recyclable substances like catalyzing metals is burning the cells and collecting these substances for reuse.

For us, the cooperation made clear that industries do have problems with a lot of pollutions, which are specific to their processing. In the Netherlands they are like Chemelot aware of their responsibility for their environment, meaning they are working on solutions to make wastewater even cleaner.

A microbial supercollector, specific for different kinds of ions, elements or molecules, exactly like we are doing it for sodium and chloride ions, would solve many of these issues. At the IAZI this solution could lower the amounts of sulphate or metals. This depends on the possibility to implement specific transporters into the yeast cell. To prove the concept of supercollectors for different kinds of pollutants we will create novel yeasts which are capable of taking up sulphate or potassium (see Werra) from wastewater by analogous integration of transporters from Arabidopsis thaliana into the plasma and vacuolar membrane of the yeast.(Read more: Description)

The assimilation of all kinds of substances could be possible with this technique. Nowadays for substances which transport mechanisms into the vacuole are already identified like sodium, sulphate or heavy metals. In future, specific reaction sites of known transporters could be modified with protein-engineering methods to be specific for a whole new range of substrates.

We realized that wastewater pollution is not restricited to NaCl, whereas the variety of ions and molecules causes problems. The specificity of biological transporters makes yeast suitable to be a diverse microbial supercollector for these various pollutants, being highly interesting for industrial applications. To prove that the concept derived from this cooperation works, we expanded our project and integrated transporters for the sequestration of potassium and sulphate into a novel yeast mutant.

Investigating Supporting Methods

A supercollector needs supporting methods to make it feasible. Research and discussion have identified breeding, water separation and after-usage of the yeast as questions to be answered.

To investigate possible solutions on these issues we got into contact with the WVER (Water Association Eifel-Rur), which is responsible for water treatment in the region around Aachen.

Investigating Supporting Methods

We visited the biggest municipal water treatment plant of Aachen and discussed water treatment methodologies used there with technicians of the plant. Alongside, we interviewed a chemist and a biologist researching on advances in water treatment for the WVER.

The cooperation revealed that we need to refrain from our original plan to integrate yeast into the activated sludge of municipal or industrial wastewater treatment plants. We detected the bad survival chances of yeast in competition with bacteria as the main reason. Industrial wastewaters emerged as the ideal field of application, having specific, and not strongly varying pollutants, and giving perfect adaption conditions for our yeast while municipal wastewater treatment plants are not eligible.

We were able to answer our questions on supporting methods of the yeast. Tests if yeasts cells are able to grow directly in wastewater are interesting (Link  Methods). A yeast growing directly in wastewater would produce fewer costs and be more competitive. However, a universal method of breeding is difficult to be realized due to specific wastewaters and applications in industry.

The question how to ensure that genetically modified organisms are not released into the environment developed to be an important question. Membrane technology emerged as a highly interesting method to separate yeast from the treated water. After-usage should be a central question of our project development. As a possible application, drainaged yeast can boost energy production of wastewater treatment plants via fouling, making the after-usage of our yeast more sustainable.

As one field of responsibility, the wver is operating 44 regional municipal wastewater treatment plants, which are mainly processing municipal waters. We aimed to get insight if and how our project could be adapted for an implementation in municipal wastewater plants besides industrial applications. Therefore, we tried to find solutions on how to breed, separate and after-use the yeast in a sustainable and feasible way.

The WVER agreed on supporting our project. The cooperation included a tour around the water treatment plant Aachen-Soers, the biggest regional municipal wastewater treatment plant. Additionally, the wver assured to be available for us for interviews with several employees on our project idea.

The municipal wastewater treatment plant has a capacity of 400,000 human equivalents. Special for municipal wastewater plants in contrast to industrial plants is that the incoming water can differ over time and the plant must be prepared for altering amounts of water, as well as altering substances in the wastewater. After a three step purification with mechanical biological and in future ozone treatment the cleaned water is given back to the close-by river Wurm. Concentrations of substances in waters are regulated by the ‘EC-Water Framework Directive’ and local regulations. A municipal wastewater plant must clean water to meet these regulations, which can alter in different regions. In Aachen for example, pH, C-compounds, and nitrate are constantly monitored, whereas sodium, chloride or sulphate are not.

We conducted interviews with Dr. Joerrens, laboratory manager of the wver, and Dr. Brands, biologist of the laboratory, both working at the water treatment plant Düren, Germany. We wanted to know, which low-molecular contaminations municipal water treatment plants are facing and if a yeast could be included easily into plants.

Both interview-partners agreed that a microbial dustbin, as we are creating it would be more applicable for industrial uses instead of municipal wastewater treatment. In general, they have two main reasons for this:

1st

The bacteria composing the activated sludge are not defined. The bacteria-mix adapts to the nutrients which are present, meaning every water treatment plant has its own specific mix of bacteria with an evolutionary benefit. A genetically modified yeast would most likely not be able to be self-sustaining in competition with this bacterial mix.

Here, the organism composition that can utilize this sewage settles itself. We do not breed a special species ourselves. Genetically modified organisms on the other hand simply go down after a short period time. [...] They are not competitive enough.– Dr Brands, wver

2nd

Chloride, sodium, and sulfate are not monitored in municipal plants in the region. Therefore, Dr Joerrens and Dr Brands do not see the necessity to work on the sequestration of these substances. In conclusion, all the ions mentioned above are not treated in plants of the WVER and flow into the rivers. Sulphate, however, is indeed problematic for the canal system and the reduced H2S for fouling the sludge. In Dr Joerrens opinion research on sulfate separation is needed, although the concentrations of sulphate are normally not high enough to cause significant problems.

Sulphate is only a problem if we have got that much sulfate, so that we are facing huge amounts of H2S during fouling. We have got dischargers of sulfate, who generally induce inorganic salts, and that is what we are unsatisfied with. Definitely, there is a lot to be done there […]. - Dr Joerrens

Talking about chloride and sodium, Dr Joerrens sustained the insights we got from our research on the current situation in the Werra. Additionally, both Dr Joerrens and Dr Brands reported that de-icer strewed on streets can lead to significantly high amounts of NaCl in a short period of time, disturbing the sedimentation of bacteria.

With chlorides, we do not have problems here in our region. […] It is certainly a problem for the salt and potash mines. If you find a solution to lower the chloride-concentrations there this would be a big thing, because this is a huge issue. – Dr. Joerrens

The flake cohesion is partly dependent on divalent ions such as calcium. These can be "swept out" when de-icer is rinsed on the streets during thawing. [...] These bacteria float, in the worst case, from the secondary clarification into the river. - Dr Brands, wver

The discussions confirmed that our initial idea to integrate yeast into the sludge of wastewater treatment plants is not feasible due to clashes with bacteria and that it is necessary to find solutions on contaminations in the Werra or industrial wastewaters. For these issues, we are best suited. In winter months NaCl can cause issues for municipal water treatment plants, but generally, NaCl is not an issue to care. In conclusion, we will focus our project on industrial applications instead of municipal ones.

The second part of the interviews focused on possible methods to implement our yeast into wastewater treatment. 3 main subject areas were discussed.

1st Breeding conditions of the yeast

As described above we refrained from adding the yeast to the bacterial sludge. Therefore, a separate basin only containing yeast would be needed. In this basin, the yeast would need nutrients, especially a carbon-source. For Dr Brands this is a problem for municipal wastewaters, but not for industrial applications. Wastewaters which contain the nutrients already would additionally lower costs, meaning the easiest way would be to breed the yeast directly in the wastewater.

You need to look for specific branches of industry that have wastewater with the necessary nutrients and sugars for yeasts. As on the sewage treatment plant here with such a sewage mixture, this will certainly not be realizable. – Dr Brands

2nd Separation of yeast from cleaned water

We need to make sure that a genetically modified yeast is not flushed out with the effluent of the water treatment plant. We asked if Dr Brands and Dr Joerrens could think of possibilities to solve this issue. Therefore, we discussed if sedimentation of the yeast, being the most conventional method for bacteria, due to flocculation is realistic. Yeast do not sediment, instead they appeared to float on the water when entering a wver wastewater treatment plant accidentally.

We once had a high induce of baking yeast in one of our smaller sewage treatment plants. […] The floating sludge was then skimmed and fouled. - Dr Joerrens

Obviously, this means that an alternative separation method is needed. Dr. Brands pointed out that there are wastewater treatment plants using membrane technology. In those plants, the bacterial sludge is not separated via sedimentations but via a close-meshed membrane.

Principle of a wastewater treatment plant based on membrane technology for separation compared to a classic treatment plant with secondary clarification based on sedimentation.

For Dr. Brands, membrane separation is more energy-costly but way more efficient then sedimentation, that’s why it is used already nowadays in wastewater treatment plants to hold back, for example, antibiotic resistant strains. In her eyes, this makes it an ideal system for us to investigate.

We have two sewage treatment plants, which are operated with membrane filters. […] In this bacterial sewage mixture, membrane units are placed in the aeration tank, and the permeate, for example, the purified wastewater, is sucked out under vacuum, keeping the biomass back. […] The membrane system can definitely keep the yeasts back. – Dr Brands

In conclusion, we worked on a membrane system to guarantee complete separation of yeast and investigated the balance of energy of this method. Therefore, we cooperated with the RWTH Institute on sanitary environmental engineering as a research group for membranes and General Electric as a producer of membranes.

3rd After-usage of yeast after taking up substances.

Similar to conventional desalination methods for seawater like reverse osmosis or Multi-Stage Flash Evaporation (MSFE) the ions or substances taken up by yeast only assimilate and are not spoilt. This raises the question what to do with the yeast after purifying water.

It seems nearby to re-use the yeast as a nutrient for agriculture. There are two essential problems opposing this solution. First, it is not allowed to spread genetically modified organisms in Germany, meaning the yeast would have to be killed and denaturated, concluding in a yeast extract. Secondly, the yeast extract would contain the substances taken up. It seems feasible to create a NaCl containing yeast extract for feeding since most organisms are dependent on NaCl as well. But what is with yeast containing sulfate or other toxic substances? There is no possibility to spread those substances within feeding stuff. Another possibility for that yeast is burning the cells. A method could collect remaining substances after burning for further use, for example recycling of metals used as catalyzers (Input from Bert van As, Chemelot), or diminish them in a controlled way. However, just burning the cells does not seem to be sustainable.

We utilized the knowledge of the WVER to discuss possible solutions for this issue. Dr Joerrens came up with a thought we have not been thinking about before. On a wastewater treatment plant he has experienced that yeast added to fouling towers had a highly positive effect. Many wastewater treatment plants in Germany operate fouling towers for their excessive sludge. The sludge is drained and then fouled and bacteria produce gas, which is transformed into electric energy. With this, the plant in Düren is able to produce 60% of their energy household. When yeast were added to the fouling tower, the yeasts were degraded by the excessive bacteria, boosting the gas and electric energy production.

“The yeasts were skimmed and fouled. There explosive anaerobic reactions occurred. The organisms in the fouling tower utilized the yeast and produced a lot of gas. This was a positive effect since we got a lot of foul gas that we could convert into electricity. – Dr Joerrens

In conclusion, it seems to be highly sustainable to use the cells for enhanced energy production. A technical implementation would mean that the yeasts are drained and added to the fouling tower afterwards. Thus, the salt is not degraded, but the problem will shift from hard to handle to easier to handle and additionally produce enhanced electric energy.

Together with the WVER we concluded that a separation of desalination and conventional water treatment steps is needed due to competition between yeast and bacteria. Nutrition of the yeast directly in the wastewater would reduce costs. Therefore, we will carry out our further experiments and measurements directly in media containing salt. The yeast can be reused for enhanced gas production during fouling of the activated sludge and burned afterwards, leaving behind dry salt. Membrane technology developed to be interesting for us to separate yeast from the treated water, resulting in the cooperation with the RWTH Institute for sanitary environmental engineering.

The incoming industrial wastewater is desalinized in a separate basin to guarantee growth benefits of yeast in comparison to bacteria. The nutrition of the yeasts in this basin remains unsettled. Applied Design After desalinization, water and yeast must be separated. To guarantee complete separation, membrane technology stands out for further investigation.

The separated water will undergo further treatment to remove the nutrition-source and ordinary pollutants in conventional wastewater treatment steps. The yeasts on the other hand, filled with salts, fall behind. The nutritional value of yeast is used in digesters for enhanced biogas production to produce energy. The salts, not harmful to the fouling process, remain after burning and need to be stored permanently, for example underearth (see Werra) or could be reused for industrial processes, depending on their purity.

Evaluation of Membrane Technology

The separation of yeast from water via membranes is promising to guarantee biosafety of our approach. We plan to integrate a membrane module into a possible application plant, however, question on the application and energy-efficiency of membrane modules remained unsettled after our research and talking with municipal wastewater plants. To gain more insight on the feasibility of this technology and answer the remaining questions we contacted the RWTH Institute for Sanitary environmental engineering.

Membrane Technology resulted to be best suited for our application because of the energy-efficiency of modern membrane modules and the capability of membranes to guarantee a total separation of microorganisms from water. However an experiment to prove the capability of membranes to do so is meaningful, resulting in our cooperation with General Electric.

By talking to Dr. Palmowski, a process engineer of the RWTH Institute for Sanitary environmental engineering, we gained useful information on membrane technology and technical questions of our project. Membrane technology proved true as the best possible solution for our application. The main advantages are increased energy-efficiency and beneficial separation capabilities in comparison to sedimentation, perfectly suited for separating antibiotic resistant strains, microplastic or our yeast.

With the results, we decided to prove the capability of membrane technology to separate yeast from water with a small-scale experiment. Instead of proving this with a synthetic laboratory membrane we decided to work on a cooperation with membrane producers to prove the uptake with a membrane used in wastewater treatment. Therefore, we have contacted General Electric as a producer of membrane modules.

Additionally, the cooperation verified our impression that it is difficult to implement a common breeding methodology for our yeast. Instead, the interview with Dr. Palmowski showed that industrial applications differ that much that a unique solution on breeding and nutrition of the yeast has to be found for every application. Therefore, we will focus on further research on the efficiency of our yeast, their isolation and the after-usage of the yeast.

The RWTH Institute for Sanitary environmental engineering is researching on new technologies for wastewater treatment and the removal of novel contaminations like pharmaceuticals from wastewater.

Dr Palmowski confirmed the issue of entire isolation of the yeast from the environment. Approached by us on membrane technology and its capability to isolate yeast from wastewater she reacted positively.

A membrane system would definitely hold back the yeast. – Dr Palmowski

Dr Brands, biologist researching for the wver, reported that the energy-consumption of such a membrane system is too costly to be feasible. We confronted Dr Palmowski with the energy balance of membrane technologies. In her eyes membrane systems are ill-reputed wrongly. First systems 20 years ago were highly energy consuming, but research has made them nearly as energy-efficient as conventional methods.

In the meantime, these systems improved drastically. They are nearly as energy-efficient as conventional methods. –Dr Palmowski

Besides this, membrane system offers additional advantages for her: Currently, water treatment plants are facing novel contaminations like antibiotic-resistant strains or microplastic. These contaminations cannot be removed by conventional methods, but by membrane systems. Therefore, Dr Palmowski assumes, that membrane systems will play a bigger role in future.

They have many advantages, and therefore they come, in my opinion, more and more in front. You have spoken about antibiotic-resistant pathogens, or other pathogens, which are refrained as well. You cannot guarantee this with conventional methods. Another issue is the contamination with microplastics. Membrane systems also retain microplastics and all the solids, which is why you can simply follow an ozone system without any disturbing solids. – Dr Palmowski

A modern, energy-efficient, membrane system is a highly promising system to implement microbial dustbins into wastewater treatment, especially because it is already implemented and has proven to be successful in many plants around the world.

Dr Palmowski suggested us to test this system with a simple small-scale experiment. We considered the small-scale experiment to be appropriate when using a membrane actually used in wastewater treatment instead of a laboratory membrane. We contacted a producer of these membranes for further cooperation to set up a corresponding experiment.

Besides talking about membrane technology, we also talked about other topics regarding our project. Interesting for us was the talk about the technical implementation of yeast into wastewater treatment. Dr Palmowski gave us interesting advice on how to breed and cultivate the yeast. She depicted that industrial wastewatersare specific for different industries. Therefore, for Dr Palmoswki there is no golden way to implement our yeast. A sustainable system with regards to breeding of the yeast and their growing capabilities in wastewater, always depends on the composition of the wastewater and the availability of nutrients from own production lines.

There are no standard solutions. – Dr Palmowski

In conclusion, we tested the capability of yeast to grow directly in simulated wastewaters containing NaCl, but did not focus on research on a defined methodology for many industrial applications anymore. Instead of that, we focused on the efficiency of our yeast, their isolation and the after-usage of the yeast. With these answers, we hope to set up tailored and most likely sustainable methodologies for different kinds of users within their own industrial network cycle.

Realization of Membrane Technology

Cooperations revealed the capability of membrane technology to hinder our modified yeast from spreading into the environment. Together with GE we set up a continuous experiment to separate yeast from water in small-scale using an ultrafiltration-membrane with field of application in wastewater treatment. The experiment proved complete separation of water and yeast cells with negligible efforts in maintenance and energy.

GE provided us with a small-scale membrane module with a surface area of 0.025m2. We discussed the potential setup of the experiment with a deputy of GE, concluding in using an ultra-filtration membrane with a pore size of <100nm, and adjusting a flow of 0.625l/h per hour.

We discussed the experimental procedure with Dr Palmowski from the RWTH Institute of sanitary environmental engineering and Stephan Sibirtsev from the RWTH Institute for process engineering and concluded to carry out a continuous fermentation with a dead-end filtration. To make the procedure simple the yeast was diluted in water and separated by squeezing the water through the membrane using low pressure. A yeast-water mix is induced into the bioreactor using the peristaltic pump we have developed as our hardware project to keep the volume constant over time.

Results show that yeast cells are separated from the media to 100% using ultra-filtration and simple low-maintenance and energy-efficient devices, opposing energy-consuming desalination via reverse osmosis (>> Applied Design). The efflux of the bioreactor was microscoped and centrifuged to determine the concentration of yeast cells in the efflux.

GE Power is producing different membrane modules for different applications in water treatment, all with the aim to purify water from contaminations. Different kinds of membranes exist, depending on their application and especially their specific pore-size. While microfiltration is used to separate larger substances, ultrafiltration is used to isolate microorganisms. Additionally, nanofiltration is capable to isolate larger molecules and ions whereas reverse osmosis will even isolate small monovalent ions like Sodium and Chloride. These different membrane pore-sizes enable a large field of applications with an increasing demand for energy from microfiltration to reverse osmosis. Wastewater treatment plants, using membrane technology nowadays, mentioned by Dr Brands and Dr Palmowski, aim to isolate excessive or pathogenic bacteria from the water with ultrafiltration. Discussions with Dr Palmowski showed that the energy consumption of such a system is not significantly higher than that of conventional sedimentation methods anymore.

We contacted Geert-Henk Koops, Technology Leader for Ultrafiltration and Membrane Bioreactor (MBR), to receive further information on Ultrafiltration membranes and a possible experiment to prove the isolation.

He agreed in the name of GE Power to support us with our project. The cooperation included the allocation of a small-scale membrane module to set up an experiment and additional information on how to set up this experiment.

Technical diagramm of the membrane module we received from GE

Dr Koops confirmed an ultrafiltration method as the method to go with. Secondly, Dr Koops gave us advice on the scale we should aim for with our experiment. His advice was to use a membrane with a surface area of 0.025m2, which appears to be small scale, and a flow of 25 liters per m2 and hour through the membrane. Therefore, we used a flux of:

Assuming a surface area of 300m2 occurring in wastewater treatments plants our setup would create a flux through an industrial membrane of:

This scale will prove the concept of using membrane technology, but will not be able to draw conclusions on the efficiency of the isolation of yeast in industrial scale. To investigate efficiency in industrial scale pilot plants are necessary. Together with GE, we decided to work on a small scale to get meaningful perceptions on the capability of membranes to handle genetically modified yeast.

For the filtering itself there are two possibilities. One the one hand you can have a cross-flow filtration, meaning the water flows parallel to the membrane and the membrane is not clogged. Dead end filtration on the other hand is technically easier but needs regular manual cleaning of the membrane from a filter cake consisting of the cells which are held back.

Together with the advice from Dr Palmowski we decided to use a dead-end filtration. The reason was that we have a comparatively small flux over the membrane with 0.625 liter per hour and keep the experimental set-up simple. With Stephan Sibirtsev M.Sc., we decided on a yeast-water mixture to serve as media for the experiment. With this, we aimed to provide a media for the process which is easy to handle.

yeast soluted in water before (left) and after (right) our membrane experiment.

Another point of interest for us was the set-up of the bioreactor we will use for the experiment. A continuous reactor would be the most realistic approach, but is technically more complex. We decided to prove the separation with a continuous system using our self-made pump to pump 0.625 liters per hour into the bioreactor and squeezing 0.625 liters of water through the membrane using low pressure. To create the low pressure we used a conventional piston pump.

With these inputs from Dr Palmowski (Institute for Sanitary environmental Engineering), Stephan Sibirtsev (RWTH Institute for process engineering) and Dr Koops (GE) we were able to set up the following experiment with the following results: Skizze des Experimentes Bild des Experimentellen Aufbaus Wie das aussehen soll, steht ja quasi im Text davor, deshalb würde ich hier über eine Skizze alles erklären. Die machen wir dann, wenn wir wissen wie es aussieht :D

Results:

The effluent was taken and microscoped for determination of the yeast concentration in the effluent. Additionally, probes of the effluent were centrifuged and the size of the pellets compared.

diagramm (left) and picture (right) of the membrane experiment to proof that yeasts can be cleaned from water easily.

Yeast cells form a filter cake on the membrane, making it necessary to clean the membrane from time to time. The experiment was running 1 hour, pumping 1.25 liters through the membrane. In this time frame no cake developed.

Our results show a separation of up to 100% of the yeast from the water with the help of a membrane module. To affiliate the membrane only conventional methods, which are not prone to maintenance, are needed. It was possible for us to purify the yeast-water mix with a simple bioreactor and energy-efficient pump, whereas direct desalination via membranes (Reverse Osmosis) is highly energy-consuming. (Link: Applied Design) The application of membrane technology to guarantee the isolation of yeast in an industrial application is proven to work reliably. However, the experiment was only in small-scale and running a short period of time, leaving no possibility to discuss the efficiency of yeast isolation in technical scale. To test the feasibility of industrial wastewater treatment with yeast for further use, a pilot plant is necessary.

Final System

Together with our cooperation partners we were able to develop a novel system of biological wastewater treatment. With input from citizens we identified the problem of industrial wastewater salinization. After talking to industries, we expanded our project and proved the vacuolar uptake of ions in general with the sequestration of potassium. And with the help of water treatment researchers and industries we developed a system, finding solutions on nutrition, separation and after-usage of the yeast and proved the separation of yeast and water under realistic conditions.

This is a system using genetically modified yeast, taking up not only NaCl but all kinds of toxic substances that cannot be degraded by other technologies. The transporters catalyzing the uptake of these substances either already exist, for example for NaCl, Sulphate or various Heavy Metals or need to be created synthetically by directed evolution of specific active sites of the transporters.

The final system for the application of "SALT VAULT" we developed in cooperation with multiple companies.

This is a system using genetically modified yeast, taking up not only NaCl but all kinds of toxic substances that cannot be degraded by other technologies. The transporters catalyzing the uptake of these substances either already exist, for example for NaCl, Sulphate or various Heavy Metals or need to be created synthetically by directed evolution of specific active sites of the transporters.

>> Chemelot

A system using specific wastewaters, internal carbon-sources or sugars, cheap side products of food industry, to breed the yeast cost efficiently and tailored for specific industrial applications.

>> ISA

A system that is free of environmental contamination with a genetically modified organism due to the complete isolation of yeast with modern, energy-efficient and well-established membrane technology.

>> GE

A system that separates water from contaminations, leaving behind dry salt after drainage, fouling and burning of the yeast.

And a system that uses the yeast to enhance gas production in fouling towers which can be converted into electrical power.

>> Wastewater Plant

We thank our supporters who have helped us developing our idea to this point. Environmental action seems to be a bottomless pit and yes our approach of a microbial dustbin needs a lot more research to be feasible in future, but we did a start. And with the help of external people, we could make the first steps. Many more will follow.

The initial idea we had when we started our Integrated Human Practice work.

Shaping Politics

Talking about environmental issues means talking about environment protection, a highly political topic, a trade-off between environmental action and the wealth of industries and their employees. For us taking environmental action always means shaping politics as well. During our iGEM project, we were confronted with various statements from industries, researchers or students on issues related to political environmental protection. To investigate and critically discuss the mentioned statements we talked to politicians and confronted them with our impressions. We implemented their answers on the current state of environmental protection in Europe and biological education in Germany into our project development.

Input

I. Knowledge of high school students on genetic engineering is too low to have a differentiated position about it. Northrhine-Westfalian biological education seems to be outdated.

II. Genetic engineering is mainly ill-reputed in Germany and most political parties oppose the use of genetic engineering, however, high school and university students and researchers appreciate genetic engineering in the context of treating polluted wastewaters, raising the question if German politicians are close-lipped towards novel technologies?

III. If thresholds of water pollutions are exceeded, municipal wastewater plants are powerless to treat the water successfully. However, industries, for example in the Werra, are in parts allowed by law to exceed the thresholds, causing Germany to be complained by the European Union.

IV. Dutch industrials mark Germany as being more focused on industry than on environmental protection, concluding for example in high amounts of pollutions in the Dutch part of the river Rhine, all caused by Germany. Is environmental protection, especially water protection neglected in Germany?

Output:

I. Giving students basic principles to reflect their opinion on genetic engineering differentiated, is an eligible approach. However, schools have to cope with unmotivated teachers and missing trainee teachers, making changes to the curriculum difficult to implement in classrooms.

II. Genetic engineering, especially green genetic engineering, is seen rather critical by politicians from different political orientations. However, they display themselves as being open to novel technologies.

III. Water protection is regulated EU-wide with the power to complain countries not meeting the regulations and the power to put pressure on multinational companies.

IV. Politicians need to find a small line between environmental protection, industrial interests, protection of employment, public opinion and differing political parties, making decisions on environmental protection difficult, even if wished.

V. Significant environmental actions depend on comprehensive societal support. Societal debates are rare in present Germany, making it difficult for environmental politicians to implement important and innovative ideas.

VI. To implement a rational and not emotional, prejudiced debate on genetic engineering and environmental action, societal change must occur by for example providing good solutions on significant problems with the help of genetic engineering. Dialogue with society emerges as important for every biotechnical and environmental project.
We thank the following politicians from three different political parties, ranging from the middle-left to the right specter of Germany's political landscape, for talking to us and giving us answers to our questions.  Andre Stinka and Mr Riegert, SPD, Northrhine-Westfalia Committee for environment

Dr. Patricia Peill, CDU. Northrhine-Westfalia Committee for environment
Dr Chrisitian Blex, AfD, Northrhine-Westfalia Committee for environment
Kristin Korte, CDU, Northrhine-Westfalia Committee for education