On this page, we present the judges and other iGEMers with our planned implementation of our Bacillus subtilis biofilm in 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
WWTPs usually consist of 3 to 4 clarifiers (fig. 1). The inlet is first screened by bars to reduce large solids in the wastewater before treatment. It then is led into the primary treatment clarifier. 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. 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, ozonolysis or filtration by activated carbon is used[2].
Implementation of "B-TOX" and Biofilm Carriers
We want to implement “B-TOX” in WWTPs. Just putting it into the activated sludge clarifier would not be beneficial since other microorganisms in the sludge would displace our biofilm (B-TOX) and thus preventing the wastewater from being detoxified.
For this reason, we recommend adding another purification stage to WWTPs following the final clarifier, which was also recommended by Udo Bäuerle. In this clarifier, we will use “B-TOX”. Since B-TOX will contain a kill switch our bacteria will not be able to survive without a biofilm due to the dependence on quorum sensing. For B. subtilis quorum sensing to work a certain concentration of ComX needs to be present. Therefore, cell growth and biofilm formation need to be induced in a separate bioreactor. When B-TOX is ready and the necessary cell density is reached it can be put into the clarifier.
Biofilm carriers enable us to immobilize B-TOX preventing the disintegration of the biofilm. Furthermore, carriers need to be durable to withstand high mechanical stress. Due to the sinR knock-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 still be active. Immobilized laccases have been found to show sufficient activity after three months. Biofilm carriers should be exchanged after this time to ensure enzyme activity[4, 5, 6, 7, 8]. Since enzyme activity is also dependent on the amount of water and micropollutant concentration. For this reason, the amount of time on-site can be adjusted.
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 biofilm carrier of choice is made of plastic, called “floating body”[9]. They were recommended to us by Prof. Dr. Susanne Lackner due to its durability and hydrophobicity which enhances the attachment of the likewise hydrophobic out layer of B. subtilis biofilms [10][https://doi.org/10.1073/pnas.1306390110].
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[11]. 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[12, 13, 14]. If the floating body is made out of these polymers further intoxication of the wastewater by estrogens can be prevented[15].
Alternative solution for different wastewater treatment plants
Building another purification stage in WWTPs means additional costs and the need for 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 WWTPs 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 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 diversely 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
Our project is focusing on laccases and erythromycin esterase type II and with those enzymes mainly on the substrates diclofenac and erythromycin. However, especially laccases can degrade many other pharmaceuticals. Due to the modularity of our biofilm, other enzymes could be potentially be employed/used as well if other substrates are to be degraded. Therefore, it is interesting to know which pollutants also arrive at wastewater treatment plants and cannot be degraded or only insufficiently.
Carbamazepine
Carbamazepine is a human medicine and is used to treat seizures of epileptic and non-epileptic type
[16]. Despite wastewater treatment in sewage treatment plants, this substance is released into surface waters. The Hessian State Office for Nature Conservation, Environment and Geology investigated carbamazepine concentrations in surface waters in Hesse between 2010 and 2015
[17]. In the investigated areas of southern Hesse, the values ranged between 0.2 and 0.8 µg/L. As a result, carbamazepine is included in the list of parameters for which one or more reference values are exceeded. Carbamazepine is especially problematic as it is not only found in surface waters – like many other pharmaceuticals – but also in groundwater.
Fortunately, it has been shown that degradation can be achieved by laccases or by fungal peroxidases[18, 19]. Our B-TOX system is therefore a suitable alternative to degrade carbamazepine.
Sulfamethoxazole
Sulfamethoxazole is used as an antibiotic for example, for urinary tract infections and pneumonia[20]. In 2010, the Federal Environment Agency published a report on antibiotics in groundwater. Among others, reference is made to studies by Hirsch et al.. The report shows that concentrations of sulfamethoxazole in wastewater from sewage treatment plants are up to 2000 ng/L[21, 22]. As an antibiotic, sulfamethoxazole is used to kill bacteria. The question arises whether microorganisms in water or sewage sludge are permanently affected by the toxic influence of this pharmaceutical. Regarding this aspect the Federal Environment Agency came to the following decision: No sustained impairment of bacterial communities can be assumed if the measured environmental concentrations in water bodies and the effect concentrations (e.g. EC50 > 100 mg/l for sewage sludge bacteria; KÜMMERER et al. 2004) are taken into account. In contrast, it has been observed that germs that are permanently exposed to low concentrations of sulfamethoxazole (e.g. in sewage treatment plants) develop resistance to this antibiotic.[21]. As a result, the same report considers municipal wastewater to be the most important source of pharmaceuticals in the aquatic environment, which underlines the importance of solving this problem. In this regard, our B-TOX system offers an effective solution, since sulfamethoxazole can be degraded by laccases[23].
The "B-TOX" B. subtilis
We want to introduce several modifications into our B. subtilis strain. The B. subtilis strain GP1622 lacks tasA and sinR genes which are co-located in the genome[24]. Additionally, we want to knock out the sigF gene and integrate the gene for our TasA fusion proteins into the genome. As the GP1622 strain contains an antibiotic resistance, which we do not want in our final B. subtilis strain, a completely new strain is engineered.
As a result, our engineered B. subtilis strain (ΔsinR-tasA::tasA-R ΔsigF) will build a stable biofilm, in which the cells are not able to sporulate. Our degradation enzymes will be displayed in the biofilm matrix as a fusion protein with the matrix protein TasA. (See Biofilm Engineering for more information.)
In addition, we want to integrate a kill switch into the genome. To this end, the Cre recombinase encoding gene cre under the control of the xylose induced promoter PxylA will be integrated into the genome, as well as the corresponding repressor xylR.
As we want to bring the native rpsB gene under our control, its corresponding promoter will be replaced by the following sequence: upstream - lox66 recombination site - PdegQ promoter (antisense strand) – Pveg promoter - lox71 recombination site –downstream with the native rpsB gene.
When induced with xylose, the recombination sites are recognized by the expressed Cre recombinase. Because of the orientation of the two recombination sites, the area between them is inverted and the constitutive Pveg promoter is replaced by the PdegQ quorum sensing promoter.
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[25]. 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 washbasin 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 the possibility to wash and disinfect their hands. In general disinfection methods also have to be available. Besides personal protective equipment like a 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, WWTPs have 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 sent 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, two main procedures 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 uses 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[26]. 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 € (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 € (3,533,400 US-Dollar*).
Besides those costs annually operating costs will be added to determine the total sum a community has to pay. 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 € (587,761.2 US-Dollar*) in Mannheim and to 45,311 € (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 €; 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 € (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 € 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 sent us biofilm carriers. One biofilm carrier costs less than 1 €. The final price depends on the size and 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 € (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 € (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, the 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.
Concerning 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
“B-TOX” is designed to detoxify wastewater from micropollutants in Germany and Europe. In other countries and continents, the composition of pollutants in wastewater might be different from our situation. To meet these differences various enzymes that detoxify the pollutants could be fused to the TasA protein so that our biofilm could be implemented there as well.
The water consumption in textile factories is often very high because the water used to dye clothing cannot be reused afterwards as it is also dyed. Furthermore, the dyes used in the textile industry are often harmful to the environment. Only a few companies process their used water to reduce water usage. Right now, it is usually treated with harmful chemicals leaving the water even more toxic than before[27].
Laccases can decolorize dyes as well[28]. We could therefore implement our biofilm subsequently to dyeing steps, the water treated with “B-TOX” could be conducted back into the dyeing process leading to a significant reduction of water consumption. Furthermore, dyes which are oxidized by laccases are less toxic. This way the aquatic environment would not be as contaminated.
For regions in which WWTPs might not exist, we could build a portable detoxifier by using a different carrier for our biofilm to grow on. Normally, microorganisms and enzymes could not endure harsh conditions. But once the biofilm is grown, enzymes and cells are protected by the biofilm matrix leading to higher resistance. Due to this fact, “B-TOX” would be able to endure longer droughts without being desiccated. This makes “B-TOX” to the perfect solution in dry areas.
Instead of the “floating bodies” that we planned for the implementation in WWTPs, we could also use e.g. ceramic plates or rain gutters as carrier material, over which the water will flow to be purified. This way we could avoid building a clarifier for the implementation of our product.
Water bodies that are already polluted due to lack of sufficient purification can be cleaned by “B-TOX”. When grown on rocks “B-TOX” can easily be placed into the water body, allowing it to be purified and at the same time released into the environment.
Another possibility of “B-TOX” could be the design of a drinking water filter for water bottles. In regions where clean water is not available but required, a filter is always the way to go. With “B-TOX” upstream of the conventional drinking water filters, not only the visible impurities could be removed, but also the invisible micropollutants. The filter downstream of the biofilm would also capture any microorganisms trying to escape the biofilm. Another advantage of this combined system would be that the filters would not be required to have small pores as they have right now. The filter could be installed on the opening of bottles where the clean water would be collected.
A big problem in water pollution is not only microplastics in wastewater but also plastics in the ocean[29]. For the reduction of microplastics in wastewater “B-TOX” in the WWTP could be supplemented with another fusion protein containing PETase and MHETase, degrading the very durable plastic polyethylene terephthalate (PET) into ethylene glycol and terephthalic acid (TPA)[30]. The plastics in the oceans could be possibly reduced by growing the biofilm, containing the plastics degrading fusion protein, directly on to the bottoms of ships, e.g. container ships. This way they would not only transport our goods but could help at the same time to clean the ocean.
Many house walls have a rough surface on which biofilms can grow. Biofilms are also often mixed-species cooperating. By adding photosynthesizing and denitrifying bacteria to “B-TOX”, not only rain could be purified from micropollutants but also oxygen could be produced and particulate matter could be removed from the air. The same system could also be applied for new sculptures making art useful in addition to being beautiful.
