Team:TU Darmstadt/Background
Biofilms
Biofilms are the entirety of microorganisms living surrounded by an extracellular matrix.[1] The microorganisms are either attached to each other or to a surface. Since they have cell-to-cell contact all the time, they are in a non-motile stadium, distinguishing them from planktonic cells. This also leads to a than usually found with free living microorganisms.[2]
Most of the biomass is made up by the matrix. It consists of extracellular polymeric substances (EPS), produced by the microorganisms interacting with each other. It stabilizes and protects the bacteria from detachment, harsh environmental conditions and toxic substances.[3] They also determine microcolonies within the biofilm, which are separated by pores and channels, supplying the cells with all the nutrients needed by passively resorbing them into the biofilm.[4] A big part of the EPS are the proteins which are secreted into the biofilm matrix. These can be proteolytic enzymes as well as hydrophobic proteins that form a skin to protect the biofilm from desiccation.[5] Proteolytic enzymes degrade the nutrients gained through sorption or dissolve the surface on which the cells are growing.
Since biofilms can have different gradients, e.g. an oxygen gradient, biofilm forming bacteria are often able to differentiate into e.g. motile cells, matrix-producing cells, sporulating cells or competent cells.[6] Some bacteria even become cannibals, if nutrients are not available and digest their surrounding cells. Furthermore, biofilms in nature are usually very biodiverse, consisting of mixed species, living in the habitat most suitable for them and cooperating with each other by exchanging necessary molecules.[7]
Intriguingly microorganisms can interact with another using use electrical communication via nanowires or they can communicate by using quorum sensing.[8,9] When quorum sensing takes place, chemicals are sent from cell to cell, leading to a reaction cascade.
When a biofilm is getting too crowded, single cells are set free and can disperse in the environment making space for the remaining bacteria. The dispersed cells reattach somewhere else, forming a new community and thus leading to the spread of the bacterial biofilm. [10]
In nature, biofilms can be found for example in the soil onto plant roots or on rocks.[11] However, biofilms are often seen as a problem in medicine as they can be pathogenic and attach onto prosthetics, gums or catheter.[12]
Most of the biomass is made up by the matrix. It consists of extracellular polymeric substances (EPS), produced by the microorganisms interacting with each other. It stabilizes and protects the bacteria from detachment, harsh environmental conditions and toxic substances.[3] They also determine microcolonies within the biofilm, which are separated by pores and channels, supplying the cells with all the nutrients needed by passively resorbing them into the biofilm.[4] A big part of the EPS are the proteins which are secreted into the biofilm matrix. These can be proteolytic enzymes as well as hydrophobic proteins that form a skin to protect the biofilm from desiccation.[5] Proteolytic enzymes degrade the nutrients gained through sorption or dissolve the surface on which the cells are growing.
Since biofilms can have different gradients, e.g. an oxygen gradient, biofilm forming bacteria are often able to differentiate into e.g. motile cells, matrix-producing cells, sporulating cells or competent cells.[6] Some bacteria even become cannibals, if nutrients are not available and digest their surrounding cells. Furthermore, biofilms in nature are usually very biodiverse, consisting of mixed species, living in the habitat most suitable for them and cooperating with each other by exchanging necessary molecules.[7]
Intriguingly microorganisms can interact with another using use electrical communication via nanowires or they can communicate by using quorum sensing.[8,9] When quorum sensing takes place, chemicals are sent from cell to cell, leading to a reaction cascade.
When a biofilm is getting too crowded, single cells are set free and can disperse in the environment making space for the remaining bacteria. The dispersed cells reattach somewhere else, forming a new community and thus leading to the spread of the bacterial biofilm. [10]
In nature, biofilms can be found for example in the soil onto plant roots or on rocks
Bacillus subtilis
We aim to convert micropollutants
in wastewater treatment plants using an
enzyme-producing biofilm and came to the
decision that Bacillus subtilis is the best
choice as our biofilm forming organism.
B. subtilis is a gram-positive bacterium which is usually found in soil. Gram-positive bacteria only have one cell membrane and are thus often used for protein secretion.[13] On the one hand, secreting proteins is beneficial in protein production because no cell disruption is necessary to collect the protein and the formation of inclusion bodies can be prevented.[14] On the other hand, and in our case specifically, we avoid the issue of bringing enzyme substrates across the membrane in the cell as well as potential cell toxicity of the substrate and degradation product. We want to leverage the ability of B. subtilis to secret proteins to immobilize our target enzymes in the biofilm matrix by fusing them to a matrix protein. This is easier to realize with B. subtilis as gram-positive model organism than with the gram-negative bacteria, e.g. Escherichia coli. In addition, B. subtilis is a facultative anaerobic organism which means it can grow in oxygenic, as well as anaerobic conditions, which can be of advantage when using the biofilm underwater in the clarifier.[15]
In nature B. subtilis forms biofilms on plant roots as it needs a nutrient source. The advantage for the plants is that B. subtilis protects it from pathogenic infections by funghi.[16] Since B. subtilis is a natural biofilm former, it has been used as model organism for studying biofilms in the last years.[16] By using this natural biofilm former, we do not have to worry about how our organism will form a biofilm. Furthermore, B. subtilis is classified as generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA), unlike other bacteria which are often pathogenic like E. coli or Staphylococcus aureus.[17,18] It is very important for us to use an organism which is generally safe given the fact that we want to bring it into a wastewater treatment plant.
Another characteristic of B. subtilis is its ability to form endospores when exposed to extreme environmental conditions.[19] Spores are highly resistant towards extreme temperature, desiccation, radiation and chemicals. This resistance makes spores interesting for industrial processes, e.g. by displaying enzymes on the surface of spores which can then be used in harsh reaction conditions. Furthermore, they are metabolic inactive but can germinate once nutrients are sufficiently available or when they experience high pressure.[20]
During the development of Bacillus subtilis biofilms, motile cells differentiate into non-motile “matrix producers” organized in cell chains.[16] During biofilm maturation, some cells induce sporulation; others differentiate to generate a heterogeneous population with different physiologies and gene expression. Gene expression during biofilm formation in B. subtilis is regulated by a complex network which we want to adjust to the needs of our project.
When a B. subtilis population continues to grow, production of extracellular signaling molecules is amplified by a positive feedback loop. Genes associated with fiber production (tapA-sipW-tasA operon) and EPS production (epsA-O operon) are repressed bySinR and AbrB in motile cells and are expressed during biofilm formation.[21] This project focusses on a few key proteins which help us use B. subtilis biofilms in wastewater treatment; one of which is the major biofilm matrix component (TasA).
B. subtilis is a gram-positive bacterium which is usually found in soil. Gram-positive bacteria only have one cell membrane and are thus often used for protein secretion.[13] On the one hand, secreting proteins is beneficial in protein production because no cell disruption is necessary to collect the protein and the formation of inclusion bodies can be prevented.[14] On the other hand, and in our case specifically, we avoid the issue of bringing enzyme substrates across the membrane in the cell as well as potential cell toxicity of the substrate and degradation product. We want to leverage the ability of B. subtilis to secret proteins to immobilize our target enzymes in the biofilm matrix by fusing them to a matrix protein. This is easier to realize with B. subtilis as gram-positive model organism than with the gram-negative bacteria, e.g. Escherichia coli. In addition, B. subtilis is a facultative anaerobic organism which means it can grow in oxygenic, as well as anaerobic conditions, which can be of advantage when using the biofilm underwater in the clarifier.[15]
In nature B. subtilis forms biofilms on plant roots as it needs a nutrient source. The advantage for the plants is that B. subtilis protects it from pathogenic infections by funghi.[16] Since B. subtilis is a natural biofilm former, it has been used as model organism for studying biofilms in the last years.[16] By using this natural biofilm former, we do not have to worry about how our organism will form a biofilm. Furthermore, B. subtilis is classified as generally recognized as safe (GRAS) by the American Food and Drug Administration (FDA), unlike other bacteria which are often pathogenic like E. coli or Staphylococcus aureus.[17,18] It is very important for us to use an organism which is generally safe given the fact that we want to bring it into a wastewater treatment plant.
Another characteristic of B. subtilis is its ability to form endospores when exposed to extreme environmental conditions.[19] Spores are highly resistant towards extreme temperature, desiccation, radiation and chemicals. This resistance makes spores interesting for industrial processes, e.g. by displaying enzymes on the surface of spores which can then be used in harsh reaction conditions. Furthermore, they are metabolic inactive but can germinate once nutrients are sufficiently available or when they experience high pressure.[20]
During the development of Bacillus subtilis biofilms, motile cells differentiate into non-motile “matrix producers” organized in cell chains.[16] During biofilm maturation, some cells induce sporulation; others differentiate to generate a heterogeneous population with different physiologies and gene expression. Gene expression during biofilm formation in B. subtilis is regulated by a complex network which we want to adjust to the needs of our project.
When a B. subtilis population continues to grow, production of extracellular signaling molecules is amplified by a positive feedback loop. Genes associated with fiber production (tapA-sipW-tasA operon) and EPS production (epsA-O operon) are repressed by
Micropollutants in Wastewater
When confronted with the word micropollutants, the majority would come to think of water pollutants like microplastics that accumulate in the environment and our bodies. But besides this undoubtably problematic pollution factor, there are many more water pollutants most of us might not be aware of. One of these is the anti-inflammatory painkiller called diclofenac. Chemical compounds, like diclofenac itself, pose no danger to the human organism1 and most people tend to use painkillers frequently without ever wondering of the consequences of its disposal into wastewater. So, what exactly happens to said compounds?
Not only diclofenac, but other substances like azithromycin and carbamazepine (link to modular biofilm) are used in many different forms like pills or gels. As they are excreted via urine for instance, often not completely dismantled, they enter the wastewater treatment plants (WWTPs)2. Although WWTPs contain mostly three but also up to four wastewater purification stages, substances like diclofenac are still not sufficiently degraded and reside in open waters in high concentrations, as shown on the figures below2,3.
Considering the increasing age of the German population, medical consumption constantly intensifies and as a result leads to more excessive contamination and environmental impacts2.
Most WWTP reveal a lack of an adequate removal of micropollutants, especially chemical compounds which may bioaccumulate at some point in water bodies3. Approximately 70 % of pharmaceuticals in wastewater originate from private households. Diclofenac, an analgesic, was found in wastewater effluent and surface water at concentrations of 65 ng/l for the former and 647 ng/l for the latter4 in 2013. In 2016, the environmental quality standard of 0.1 µg/L was exceeded at 21 out of 24 German measuring points. Hence, even by surpassing up to a tertiary purification stage in WWTPs, pharmaceuticals still end up in water bodies and lead to detrimental effects in aquatic fauna - such as liver or kidney damage in fish. But not only does it endanger aquatic fauna, other kinds of animals are also affected via the food chain. In worst cases, this can even lead to species extinction1.
Therefore, it is our responsibility to step in. Complying to ethical standards, it is our duty to adhere to certain principles like the polluter-pays-principle and the precautionary principle, as we were told by Prof. Dr. Sibylle Gaisser (LINK). That means that we have the responsibility to provide a suitable and optimized system in order to achieve a significant deterioration in wastewater pollution, caused by humans to begin with. We as society, the primary cause for wastewater pollution through pharmaceuticals in wastewater effluent, are the only accountable parties with the obligation to preserve our environment. To do so, we strive to provide a way to minimize detrimental effects micropollutants cause. This is where B-TOX comes into play. By fusing degradation enzymes (links zu enyzmen) to a modular(mit link zu modularer biofilm text) Bacillus subtilis biofilm (mit link zu biofilm), we want to reduce the ratio of pharmaceuticals in wastewater. With our project, we can make an impact and come one step closer to the conservation of the environment.
Not only diclofenac, but other substances like azithromycin and carbamazepine (link to modular biofilm) are used in many different forms like pills or gels. As they are excreted via urine for instance, often not completely dismantled, they enter the wastewater treatment plants (WWTPs)2. Although WWTPs contain mostly three but also up to four wastewater purification stages, substances like diclofenac are still not sufficiently degraded and reside in open waters in high concentrations, as shown on the figures below2,3.
Considering the increasing age of the German population, medical consumption constantly intensifies and as a result leads to more excessive contamination and environmental impacts2.
Most WWTP reveal a lack of an adequate removal of micropollutants, especially chemical compounds which may bioaccumulate at some point in water bodies3. Approximately 70 % of pharmaceuticals in wastewater originate from private households. Diclofenac, an analgesic, was found in wastewater effluent and surface water at concentrations of 65 ng/l for the former and 647 ng/l for the latter4 in 2013. In 2016, the environmental quality standard of 0.1 µg/L was exceeded at 21 out of 24 German measuring points. Hence, even by surpassing up to a tertiary purification stage in WWTPs, pharmaceuticals still end up in water bodies and lead to detrimental effects in aquatic fauna - such as liver or kidney damage in fish. But not only does it endanger aquatic fauna, other kinds of animals are also affected via the food chain. In worst cases, this can even lead to species extinction1.
Therefore, it is our responsibility to step in. Complying to ethical standards, it is our duty to adhere to certain principles like the polluter-pays-principle and the precautionary principle, as we were told by Prof. Dr. Sibylle Gaisser (LINK). That means that we have the responsibility to provide a suitable and optimized system in order to achieve a significant deterioration in wastewater pollution, caused by humans to begin with. We as society, the primary cause for wastewater pollution through pharmaceuticals in wastewater effluent, are the only accountable parties with the obligation to preserve our environment. To do so, we strive to provide a way to minimize detrimental effects micropollutants cause. This is where B-TOX comes into play. By fusing degradation enzymes (links zu enyzmen) to a modular(mit link zu modularer biofilm text) Bacillus subtilis biofilm (mit link zu biofilm), we want to reduce the ratio of pharmaceuticals in wastewater. With our project, we can make an impact and come one step closer to the conservation of the environment.
Laccases
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
EreB
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Modularity
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Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Quorum Sensing
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Kill Switches
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
References
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