# Team:Manchester/Entrepreneurship

#### Executive Summary

The current market for phosphorus removal techniques is growing rapidly as more and more countries set increasingly stricter regulations on the effluent quality. Wastewater treatment plants (WWTPs) from all over the world are looking for a dependable, sustainable and cheap solution. Our product allows companies to achieve phosphorus removal levels expected by the legislations in force. Additionally, our solution is the only one that allows the user to recycle the recovered phosphorus, thus preserving this agriculturally valuable resource. Our team have engineered phosphorus accumulating bacteria based on the newest research made by Martin Warren et al. as documented in the 2017 paper. Our team are planning to target WWTPs in Canada specifically, as we believe it is the best place to set up our business. As the threat of decreasing phosphorus reserves deepens and inland lake and rivers eutrophication continues, the demand for Phosphostore, which not only removes phosphorus out of water but also recycles it, is expected to increase significantly.

#### Customer Analysis

In most developed countries, wastewater treatment plants (WWTP) are legally obliged to meet limits on phosphorus discharged to receiving water bodies (Tanyi, 2007). Often, these WWTPs have to choose between stable and efficient, yet very costly chemical phosphorus removal methods, or more unstable, less efficient yet cheaper biological phosphorus removal methods (Tanyi, 2007). This choice is usually dictated by the legal limits set on the allowed phosphorus concentration levels in the effluent. We learned through discussing these issues with Aqua Enviro, a leading water consultancy company that more stringent regulations often prompt WWTPs to choose chemical-heavy phosphorus removal systems.

Through talking to Aqua Enviro, we learned that wastewater legislations have tremendous impacts on the type of phosphorus removal technology used, on the thoroughness of wastewater treatment and on the way wastewater industry as a whole develops

Having realised the importance of legislations in the development of the wastewater sector, our team have decided to approach the task of researching biology-related laws with a global perspective in mind. To this end, we have established a global collaboration including teams from 10 countries and 5 continents; participating teams were based in Australia, Brazil, Canada, Chile, England, India, Indonesia, Japan, Korea and the US. Each team have researched GMM legislations in their country and shared their findings and impressions by contributing to a shared document. As a result, we have managed to analyse multiple markets, which has directed us to set up our Phosphostore business model in Canada, where we concluded the legislative approach towards GMMs is both safe and open as our collaborative friends convinced us.

Leading this iGEM Collaboration has expanded our understanding of GMM laws around the world, which in turn, helped us choose Canada as the best place to set up our Phosphstore business plan

Our envisaged customers are specifically Canadian WWTPs which use biological phosphorus removal methods. We chose to target Canadian WWTPs for a number of reasons. Firstly, a critical part of the technology we use to accumulate phosphorus in a bacterial microcompartment has a number of associated patents in the EU and the US; as such, we would not have a lot of freedom to operate in those two territories. Additionally, the EU and US are the two largest markets for biotechnology, given the number of patents granted in the areas. Thus, we suspect that any patents associated in our project may possibly hinder our freedom to operate in those two territories in the future. Secondly, our technology is compatible mainly with biological phosphorus removal methods, especially EBPR method (explained below), which happens to be widely used throughout Central and Western Canada (Oleszkiewicz, 2016). Finally, there is plenty of information on Canadian wastewater treatment systems, which facilitates market research. In 2012, Canada also established first national standards for wastewater treatment, which set fairly strict limits of 1mg/l of phosphorus concentration in discharged water (Environment and Climate Change Canada Website). This might further increase the demand for efficient phosphorus removal methods in the future.

Our extensive research on the Intellectual Property Rights has prompted us to choose Canada for a place to set up a business. This decision was also supported by the favourable characteristics of Canadian WWTPs and recent legal developments.

Canadian EBPR WWTPs usually operate in municipalities in Western Canada, including the Prairies and central British Columbia (Oleszkiewicz, 2015). These WWTPs have to comply to Water Quality Guidelines adopted by Canadian Government as well as the regulations set by the relevant province. British Columbia, Alberta, Saskatchewan and Manitoba (Prairies) have all adopted a limit of 1mg/l of total phosphorus in discharged water (Oleszkiewicz, 2015). These WWTPs need stable and effective yet inexpensive phosphorus removal methods, which would allow them to meet the limits set by the regulations.

#### Competitor Analysis

In most of the cases, WWTPs have their own systems for removing or recycling phosphorus. Thus, an industry for providing WWTPs with these services is almost non-existent. In other words, there is nearly no competition that Phosphostore would have to face. As a technology which removes and recycles phosphorus, however, Phosphostore would be still indirectly competing against currently used methods for these processes. Let us compare Phosphostore to the other two most commonly used phosphorus removal technologies: Chemical Precipitation (=Chemical Dosing) and Enhanced Biological Phosphorus Removal (EBPR).

O&M Costs Efficiency Predictability Sludge Production Volume Environmental Sustainability
Phosphostore 2 2 2 1 1
Chemical Precipitation 3 1 1 3 3
EBPR 1 3 3 2 2

Phosphostore would be indirectly competing against two other most commonly used phosphorus removal methods: chemical precipitation and enhanced biological phosphorus removal.

Operation & Maintenance Costs (O&M) are by far the highest when using chemical precipitation as the amount of chemicals used in the process is enormous (Tanyi, 2006). In addition, sludge that results from the treatment has to be processed further to ensure safety for public. This is the major reason behind the gradual shift towards EBPR, which has the lowest O&M costs (Tanyi, 2006). EBPR employs naturally occurring phosphate-accumulating organisms which filter incoming wastewater in a series of different stages. As of now, Phosphostore would lose on the O&M costs to EBPR as our technology employs genetically modified organisms which require additional machinery operation costs to minimise direct contact with the wastewater. In addition, our GMOs would have to be cultured in a separate vessel through a continuous culture system to avoid contact with other bacterial species. As a solution, we have discussed and thought of alternative synthetic biology strategies that may help to reduce the cost of Phosphostore.

We have modelled a rough estimation for the production cost of our system. The estimation only accounts for the substrate and medium used to grow the bacteria. From our calculations, it costs $1.33 to produce 1 kg of bacteria and$0.76 to treat wastewater containing 7.3g of phosphate in 48 hours. To estimate the cost to treat wastewater for a year, we used Davyhulme Treatment Works, the biggest wastewater treatment works in North West England, as a case study. We found that it would cost £550 million to treat the amount of wastewater that goes through Davyhulme which is about 30,000 litres of wastewater per second.

Additionally, capital investment of the major equipment, processing cost, and utility construction of 5 stainless steel fermenters with a volume of 177,000 L is equivalent to $27 million (Maiorella, Blanch and Wilke, 1984). These values are obtained from an estimated cost of a simple continuous fermentation for ethanol production. Maintenance cost is 3% of capital at around$810,000. Therefore, it will cost roughly $28 million to set up the system during the first year in addition to the annual cost to treat wastewater. Phosphorus recovered from our bacteria can be reused as fertilizer for agriculture usage. Assuming that the fertilizer is sold at the same average market cost of Diammonium Phosphate (DAP), the most widely used phosphate fertilizer, the recovered material can provide revenue of$2.4 million, which is about 8.6% of initial capital investment.

Efficiency measures the extent to which a given method is able to remove phosphorus out of water. Chemical precipitation usually achieves total phosphorus removal down to 0.3 mg/l (Tanyi, 2006). As such, it is the only viable phosphorus removal method for sensitive water areas, which have exceedingly strict legal limits. EBPR is able to reduce total phosphorus levels to 0.5-1 mg/l, which is sufficient for most of the Canadian WWTPs’ needs. Phosphostore which involves the use of bacterial microcompartments, can achieve results slightly better than EBPR method yet significantly worse than chemical precipitation.

Predictability Both EBPR and Phosphostore are harder to predict and model compared to chemical precipitation as they depend on live organisms. Chemical precipitation methods rely on predicting chemistry and as such can be predicted very consistently whereas both biological methods rely on predicting complex biological interactions. The increased number of variables inherent in the EBPR and Phosphostore processes prevents them from ever being as predictable as chemical methods. Current EBPR methods incorporate the use of phosphate accumulating organisms which cannot be cultured in isolation. As such, EBPR has proven to be difficult to model and is often temperamental in practice (Sathasivan, 2009). This can lead to unexpected phosphate releases in the effluent. Hence, synthetic biology approaches such as Phosphostore are better suited because it applies mathematical modelling to predict the behaviour of an organism that has been tuned for a specific purpose. At the moment, Phosphostore employs Escherichia coli, a model bacterial organism that has been studied extensively in the past decades, as a chassis for the system. As an example of predictability, we have modelled an operon for our system that controls and regulates the expression of microcompartments within the cell. Our model has shown that the operon successfully down-regulates microcompartment formation under a certain phosphate concentration. Thus, future work would allow prediction of our system for different specific purposes.

Sludge Production Volume Chemical precipitation produces the most sludge as the process is based on adding more chemicals into the mixed liquor. EBPR and Phosphostore result in very similar sludge production volume. Higher sludge production is usually undesired by the WWTPs (Yuansong Wei, 2003) as it requires them to dump the waste somewhere, which is why, as a general rule, the higher sludge production volume, the bigger problems it creates for the WWTPs.

Our visit to the Davyhulme Wastewater Treatment Plant taught us, however, that in some cases, higher sludge production volumes could also be desired. The staff explained that sludge incineration allows for on-site energy production, which is how Davyhulme powered over 50% of its processes. At times, Davyhulme would also sell phosphorus rich sludge as biosolids to the local farmers, which further increased their profits. As a result, we realised it is very hard to give a clear verdict as to which methods give rise to the best sludge outcomes due to the complexity of sludge treatment.

Environmental Sustainability Phosphostore achieves the highest level of environmental sustainability as it involves a chemical-free, stable and predictable solution. Furthermore, phosphorus recycling is an intrinsic part of the Phosphostore technology, as opposed to EBPR or chemical precipitation, both of which require additional steps for phosphorus recycling. EBPR takes the second place as it is still a chemical free method; however, it is less predictable than the other two methods and does not involve phosphorus recycling in its process. Through our interaction with Aqua Enviro, we also learned that chemical precipitation is clearly the least environmental friendly solution as it requires a lot of supplementary chemicals

Phosphate that is recovered from our bacteria can be reused as fertilizer for agriculture usage. Assuming that the fertilizer is sold at the same average market cost of Diammonium Phosphate (DAP), the most widely used phosphate fertilizer, the recovered material can provide revenue of \$2.4 million, which is about 8% of initial capital investment.

### Company Description

We started as a group of nine students in December 2016, forming a team which would represent The University of Manchester in a synthetic biology competition called iGEM. With huge help of the academic staff, we have successfully designed phosphorus accumulating bacteria. For this reason, we decided on Phosphostore as our company’s name since it combines the words phosphorus and storage, which perfectly describes what we are trying to achieve. Our company aims to help wastewater treatment plants remove phosphorus from the sewage in a stable, efficient and cheap manner.

### Company mission statement

To recycle phosphorus, help the agriculture, to clean up rivers, sustain a healthy water supply.

### Product description

1. How does the current phosphorus removal process work at a biological nutrient removal WWTPs?

Usually, when nutrient removal is required from WWTPs, they are obliged to significantly reduce the amount of both phosphorus and nitrogen from the influent (Tanyi, 2007). As such, even amongst biological nutrient removal WWTP, there are a few slightly different commonly used settings, such as the Phoredox, Bardenpho or A2O settings (Oleszkiewicz, 2015).

Let us illustrate the current phosphorus removal process using the most basic EBPR setting: A2O setting. Under the A2O process, once the influent has gone through the primary treatment, it enters the anaerobic zone, where the phosphorus accumulating organisms eat volatile fatty acids to gain energy. Subsequently, the phosphorus accumulating organisms move through the anoxic zone to the aerobic zone, where they use the stored energy to accumulate phosphorus from the influent. In the aerobic zone, an important step in nitrogen removal, called nitrification, occurs. Some of the influent is then recycled back to the anoxic zone so that denitrification, the second step in nitrogen removal process can take place (Nourmohammad, 2013). This way, phosphorus concentration is usually reduced down to 1 mg/l, whereas nitrogen concentration down to 10mg/l (Oleszkiewicz, 2015).

2. How does Phosphostore work?

We would first culture our Phosphostore bacteria through a continuous culture system and then place them in metal containers. Then, we would put these containers full of bacteria inside the aerobic zone, where our bacteria would remove phosphorus from the influent. After a few hours, we could take the containers out; all that is left inside is bacteria full of phosphorus. We can easily kill the bacteria full of accumulated phosphorus by putting them through a crossflow filtration system. The bacterial debris would be later sold on the market as fertiliser to partly cover the operating costs.

How would we culture our Phosphostore bacteria? - We envision our bacteria to be cultured in a chemostat for production. A chemostat is a bioreactor in which fresh medium is continually added while culture liquid containing leftover nutrients, microorganisms, and metabolic products are continuously removed at the same rate. This technique is called continuous culture and allows microbial growth to take place under steady-state conditions - growth that occurs at a constant rate and in a constant environment. Unlike a batch culture method where bacterial cells undergo the full bacterial cell cycle, a continuous culture keeps the bacteria growing in the exponential phase of the bacterial cell cycle, thus a continuous supply of bacteria can be produced (Herbert, Elsworth and Telling, 1956). This process would be integrated into our Phosphostore System so that once containers are taken out of the water and their contents are removed, new bacteria can enter seamlessly.

What exactly are these panels? - These containers would have dimensions of 1.5m x 1.5m x 0.2m. This way, they would be relatively easy to operate and still have a lot of our bacteria inside. They would be made out of two metal panels, each 1.5m x 1.5m, connected by a membrane. We could use the peptidosome membrane designed by iGEM TU Dresden 2017 Team. This would allow water to flow through the container, allowing the bacteria to accumulate phosphorus, whilst preventing the bacteria from escaping. The containers would stay inside water for about 4 hours, which corresponds to water’s current length of stay in the aeration zone (Oleszkiewicz, 2015). We would manufacture the containers ourselves and include them into our Phosphostore package sold to WWTPs.

Our Phosphostore panels could be made with the use of iGEM TU Dresden 2017 Team's idea. Our close interactions proved such a design would be theoretically possible.

How would you kill the bacteria and use the remains as fertilizer? - We would first collect all the bacteria and put them through a crossflow filtration system. In crossflow filtration, pressure drives the liquid culture through a microfiltration membrane where particles in the 0.1-10µm range can be separated from the liquid medium. The system can operate at pressures up to 18 bars and temperature up to 200°C (Ho and Sirkar, 1992). Since our product uses E. coli, the system will operate at temperature of 100°C to kill all genetically modified bacteria present. The remaining solids will contain bacterial debris with polyphosphate and these can be used directly as fertilizers.

How do you plan to sell the phosphorus? - We would sell the recovered phosphorus together with the bacteria in which it is trapped as fertilizer to wholesalers for a standard market price.

3. In what way is Phosphostore better than the current biological phosphorus removal processes?

Firstly, Phosphostore would not require the anaerobic zone currently necessary in the EBPR process. Its current use, namely providing the phosphorus accumulating organisms with volatile fatty acids for energy accumulation purposes, would not be necessary as our bacteria are genetically modified specifically with the idea of phosphorus accumulation in mind. As such, WWTPs could save a lot of space. Secondly, as the anaerobic zone would not be required, the time needed for WWT process would be shortened. Finally, Phosphostore is more environmentally friendly as it facilitates the recycling of phosphorus, which is emerging as an increasingly valuable agricultural resource.

#### Long-term Growth Potential

We have identified three trends which indicate Phosphostore’s long-term growth potential is enormous. Firstly, more and more countries around the world are recognising eutrophication as a big threat to sustainable water supply; as a result, governments are adopting increasingly more stringent limits to nutrients concentrations in discharged water (UN Website). This increases the demand for stable, efficient and cheap phosphorus removal methods such as Phosphostore. Secondly, in the recent years, newly built WWTPs have been steadily choosing biological nutrient removal methods over chemical alternatives, which further increases the size of our target market (Tanyi, 2006). Thirdly, in the coming decades, the reserves of phosphate rock, the primary source of phosphorus used in the production of agricultural fertilisers, will steadily deplete, thus increasing the price of phosphorus recycled using our Phosphostore technology (Cordel et al., 2009).

#### Bibliography

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Cordell, Dana; Drangert, Jan-Olof; White, Stuart (2009). The story of phosphorus: Global food security and food for thought, in Global Environmental Change. 19 (2): 292–305.

Davood Nourmohammad, Mir-Bager Esmaeeli, Hossein Akbarian, and Mohammad Ghasemian. (2013) Nitrogen Removal in a Full-Scale Domestic Wastewater Treatment Plant with Activated Sludge and Trickling Filter, in Journal of Environmental and Public Health.

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