On this page we provide the judges and other iGEMers with our planned implementation of our B. subtilis biofilm in a wastewater treatment plants (WWTP). We give insight into how WWTPs work, what the problem causing substances are in Germany, and how the topic of GMOs in wastewater treatment are implemented into our plan. Also, we give a brief outlook into other possible future applications of B-TOX.
Wastewater Treatment Plants
Figure 1: Layout of a typical wastewater treatment plant. It consists of bar screenings a primary clarifier, the biological treatment and optional a fourth purification stage, where our product will be implemented.
Layout of Wastewater Treatment Plants
Wastewater treatment plants (WWTPs) usually consist of 3 to 4 clarifiers. The inlet is first screened by bars to reduce large solids in the wastewater before treatment. It then is led into the . Here, suspended solids that float or settle are removed. Floating solids are skimmed and settling solids drift to the bottom, where they are pumped out of the clarifier.
In WWTPs with biological wastewater treatment the influent is then pumped into the activated sludge clarifier. Here, most organic compounds of the wastewater are degraded by the variety of microorganisms inside the clarifier. Main degradation products are carbon dioxide and nitrates. In the following final clarifier, the activated sludge is removed by settling and the effluent is led into the dry well.
Wastewater can also be treated chemically. Here the influent is treated by the addition of chemicals for oxidation or precipitation. In most WWTPs the chemical wastewater treatment is included in the activated sludge clarifier.[1] Some WWTPs have a fourth purification stage in which the wastewater is treated to remove micropollutants. For this purpose, ozonation or filtration by activated carbon are used.[2]
Implementation of "B-tox" and Biofilm Carriers
We want to implement “B-TOX” into the WWTP. Just putting it into the activated sludge clarifier would not be beneficial since other microorganisms in the sludge would displace our biofilm and thus preventing the wastewater from being detoxified.
For this reason, we wanted to add another purification stage to the WWTP following the final clarifier, which was also recommended by Udo Bäuerle. In this clarifier we will use “B-TOX”. Since we inserted a kill switch into our bacteria our cells will not be able to grow due to the dependence on quorum sensing that only occurs in biofilms. We made the biofilm growth inducible, leading to the necessity of growing our biofilm first outside of the clarifier and then implementing it already fully grown.
Therefore, we need biofilm carriers to transport “B-TOX” from the growing tank into the clarifier. These carriers need to be exchangeable because with sinR knocked out, cells from the biofilm do not disperse and will eventually die inside the matrix.[3] Nevertheless, the matrix will still exist and the enzymes will keep working. Immobilized laccases have been found to show sufficient activity after three months, so we will first exchange the carriers after this time period.[4,,5,,6,,7,,8] Since the enzyme activity is also dependent on how much water and micropollutants they have to tackle, we can adapt the amount of time on-site.
Furthermore, the carriers need to defy mechanical stress and should not be able to disintegrate, since this could allow for the biofilm to escape as a whole into the environment.
Figure 2: The biofilm carrier is required to be exchangeable and the material of the carrier needs to be durable to meet our needs.
Our carrier of choice was a biofilm carrier made of plastics, called “floating body”.[9] It was recommended to us by Prof. Dr. Susanne Lackner due to its durability and hydrophobicity which enhances the attachment of the likewise hydrophobic Bacillus subtilis we use.
Figure 3: This is what a floating body looks like
However, plastic is known to possibly contain the estrogen bisphenol A which can cause infertility and is toxic to the environment.[10] We discussed plastics as a potential source of pollution since the emission of microplastic and toxic compounds such as Bisphenol A might contribute to water pollution. We became aware of this problem after talking to Prof. Dr. Jörg Oehlmann.
But all possible alternative materials, e.g. sisal fibre waste, pumice stone or granular activated carbon, did not meet the criteria required of our biofilm carriers, leaving plastics the most beneficial choice. Furthermore, there are plastics like polyethylene (PE) or polypropylene copolymer (PPCO) that do not show any estrogenic activity or toxic effects. [11,,12,,13]If the carrier is made out of these polymers further intoxication of the wastewater by estrogens can be prevented.[14]
Alternative solution for different wastewater treatment plants
Building another purification stage in a wastewater treatment plant (WWTP) means additional costs and the need of free space.
Hence, we were looking for other options to implement our biofilm.
We talked to the expert Prof. Dr. Jörg Oehlmann who mentioned that especially WWTPs in cities
are often limited in space and would not be able to build an additional clarification tank irrespective of the accruing costs.
Some WWTP are big enough to have a separate chemical clarification.
For those WWTP our goal was to test if our biofilm could survive in this step.
To this end, we would request wastewater samples of these kinds of WWTPs in order to test cell growth,
biofilm formation, biofilm stability as well as enzyme activity.
If the results are satisfying,
the implementation of our biofilm on floating bodies in the chemical wastewater treatment step could be a cheap and space saving alternative.
Our visit to the WWTP in Darmstadt showed us, that the wastewater treatment is structured diverse in WWTPs in Germany.
There are a lot of different systems which are often adapted to the local situation.
In the WWTP of Darmstadt the biological wastewater treatment is directly followed by the final clarifier.
In the biological wastewater treatment,
our bacteria would be pushed away by the other microorganisms making it impossible to implement our biofilm here
and a new clarifier would be necessary.
This shows us, that individual solutions for the respective types of WWTPs are required to implement our biofilm.
However, one challenge is trying to find a solution that will work on a majority of different WWTPs in Germany.
Problematic Substances Germany
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GMOs in Wastewater Treatment
Implementation of our biofilm into a wastewater treatment plant (WWTP) requires dealing with the legal situation in Germany. Therefore, our friends from team Kaiserslautern contacted Dr. Ehlers from the Federal Office for Consumer Protection and Food Safety of Germany (BVL) and we were able to ask him specific questions to our project. He told us that the best option for us would be creating a closed system which ensures that our biofilm will not leave the WWTP. In a nutshell: We have to fulfil the requirements for getting a WWTP labelled as a “genetic engineering plant”; more specific as a laboratory facility of biosafety level 1. What exactly does a WWTP need to achieve this? Respectively, what does a facility need to gain the status of biosafety level 1?
Requirements for biosafety level 1
According to Annex III Part A of the Genetic Engineering Safety Ordinance – GenTSV following precautions are required[15]. The area where works with our B. subtilis biofilm take place should be delimited and all doors must be lockable. Since we want to put our biofilm into the wastewater on our floating bodies in an additional clarifier we have to test if our kill switch works perfectly and subsequently discuss with the local state authorities based on positive results if this is enough for preventing an unintentional release of our organism. If not, we have to think about additional safety modules that are downstream of our clarifier for example ultrafiltration or an UV-filter. See more on our safety page.
However, the chosen working area has to fulfil some structural conditions. A wash basin has to be present, and the surfaces of the working space as well as walls and floor have to be cleanable easily. Basic laboratory equipment as pipettes has to be available. Employees have to get a possibility to wash and disinfect their hands. In general disinfection methods also have to be available. Besides personal protective equipment like lab coat and safety goggles employees also have to get possibilities to eat, drink and have a break. Since we plan to use our biofilm in only one area in the WWTP this is no problem. In Addition, a WWTP has to purchase an autoclave.
This sounds elaborate and costly. But compared to alternative methods for decreasing pollution of micropollutants in wastewater it seems much less costly. Dipl. Ing. Udo Bäuerle has send us information regarding the costs of integrating a fourth purification step. A lot of different procedures have emerged within the last decades to tackle this problem. In Germany there are two main procedures that are discussed in the field of purification of micropollutants - Ozonolysis and using adsorbents like activated carbon.
Costs for current procedures
There are a few WWTPs in Germany that already have a fourth purification step which mostly use adsorption processes with activated carbon. The costs for these procedures differ due to the size and structure of the WWTP as well as to the local circumstances. Dr. Steffen Metzger et al. have made calculations for different cities in Germany.[16] For example, for the city of Mannheim (residents ~ 310,000) with 21 million m³ wastewater per year the investment costs amount to 6,771,000 Euro (7,922,070 US-Dollar*). For a smaller WWTP as in the community of Kressbronn (residents ~8700) the acquisition costs would amount to 3,020,000 Euro (3,533,400 US-Dollar*).
Besides those costs annually operating costs will be added to determine the total sum a community has to pay. In order to calculate a comparison between our method and adsorption methods we only focus on procurement costs for the activated carbon. For this purpose, we estimate that the costs for staff, electricity, disposal and all other operating costs will not differ much for those needed in our implementation. The costs for activated carbon amounts to 502,360 Euro (587,761.2 US-Dollar*) in Mannheim and to 45,311 Euro (53,013.87 US-Dollar*) in the community of Kressbronn.
Therefore, the total sum amounts to 7,273,360 € (8,509,831.2 US-Dollar*) for the city of Mannheim and to 3,065,311 € (3,586,413.87 US-Dollar*) for the community of Kressbronn.
Costs for “B-TOX”
Investment costs: Requirements for a genetic engineering plant with biosafety level one are mentioned above. We estimate that a WWTP has usually enough space and is developed enough to clear up a room for genetic engineering work. Besides that, a WWTP needs to have basic laboratory equipment like Bunsen burners, pipettes, benches, personal protective equipment and consumables. In addition, an autoclave (about 30,000 Euro; 35,100 US-Dollar*) is needed. Everything combined as well as little structural changes within the room like laying of cables and so on we assess potential investment costs of less than 100,000 Euro (117,000 US-Dollar*). The only thing that remains are the costs for an additional clarifier.
A clarifier would also be needed in an ozonolysis or adsorption procedure. We can hardly tell what contribution this part has within the 6.7 respectively 3 million euro for investment costs. But we can postulate the real value in relation to this. In contrast to ozonolysis or adsorption procedure we just need another clarifier and no additional pumping stations, dosing systems or further systems that are needed. Subsequently we believe the costs will be much lower for our approach.
Operating costs: We will not compare any costs for additional staff and/or further education of existing personal since this is also required in other procedures and we estimate that this is almost equal in both cases. We came in contact with ENEXIO Water Technologies GmbH (Hürth, Germany) which have send us biofilm carriers. One biofilm carrier costs less than one Euro. The final price depends on the size and on the ordered number. We can hardly tell how much a WWTP needs since we were not able to perform any tests. But to start from the greatest need we estimate that one WWTP needs for example 2000 carrier a year which would mean in worst case 2000 Euro (2,340 US-Dollar*) per year additional. But this differs up to the size of the clarifier as well. Thinking of consumables, we generously estimate 4000 Euro (4,680 US-Dollar*) a year. But we have to add that the costs for biofilm carriers will not arise every year – they can potentially be used for several years. So, the actual annual costs are even lower.
Comparison for WWTP in Mannheim
The average costs for a resident depend on the size of the WWTP. The larger a WWTP is the lower are the average costs. Therefore, a first step for us would be implementing “B-TOX” into a larger WWTP as there is for the city of Mannheim.
Costs
Adsorption procedure/€
"B-TOX"/€
Investment costs
6.771.000 (7,922,070 US-Dollar*)
Lower; explained in the text
Operating costs
502.360 (587,761.2 US-Dollar*)
4000 (4680 US-Dollar*)
Total costs
7.273.360 (8,509,831.2 US-Dollar*)
Definitely lower
Table 1: Comparison of costs for WWTP in Mannheim.
In relation to currently available methods for removing micropollutants from wastewater our approach would definitely save money. Long term costs reinforce our approach since the operating costs for “B-TOX” are far less than those for competitive techniques since you just need consumables once a biosafety level 1 is achieved and biofilm carriers are purchased and available. Furthermore, we addressed the issue of unintentional GMO release: With our kill switch we developed an intrinsic control system for which no additional resources are required. As a general perspective, implementing our project in a WWTP and creating an area with biosafety level one will also open doors for other biotechnological wastewater treatment projects which hopefully can be combined with ours.
*Values in US-Dollar correspond to an exchange rate of 1 € = 1.17 $ at 17th of October 2020
Outlook
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