Human activities are responsible for an important pollution of drinking water as well as sea water. Yet, water is the most precious and fragile ressource, as it's vital to every living organisms. It covers 71% of the Earth and in this huge amount of water, approximately 97% is saline and 3% is freshwater. (Pacific Institute for Studies in Development, Environment, and Security, 1993)

Figure1: Water on Earth

There is a broad range of polluting elements that exists. In the marine environment, we can mention nitrate, nitrite, heavy metals, phenols, plastics, PCB, pharmaceutical residues etc.... All these elements can distrupt the fauna and the flora.
These problems especially impact the whole food chain of marine biotopes. Thus, mankind is both guilty and victim of this catastrophy.

This year, our objective is to develop a filtrating system which could allow sensing and eventually degradation of pollutants. To this end, we chose to work on elements belonging to the most important classes of pollutants of the marine environment.

Phenols





Phenols and their close derivatives are molecules which are widely distributed in nature. They are widespread contaminants whose sources are both natural and industrial (Fahmida Karim A. N. M. Fakhruddin, 2012).
The largest use of phenols is as an intermediate in the production of phenolic resins, which are low-cost, versatile, thermo set resins used in the plywood adhesive, construction, automotive, and appliance industries.
Phenols can be used as a general disinfectant, as a reagent in chemical analysis and it's also a major chemical intermediate for the manufacture of artificial resins, fertilizers, explosives, pharmaceuticals and textile. Consequently, aquatic organisms including fishes are subject to these pollutants. (Nahed S. Gad and Amal S. Saad 2008).

Excessive exposure to elements of this diverse group of chemicals may induce a variety of health effects depending on the particular chemical. It may cause severe damages to many organs and also genetic impairment.
Beyond being harmful to humans, phenols also appear to be toxic to aquatic animals. Chlorinated phenols are the most noxious to aquatic life (more informations here).
Moreover phenol is categorized as a Volatile Organic Compound (VOC) contributing to photochemical smog which threaten phytoplankton.



Figure2: Phenol chemical structure





Phenol is a priority substance for a lot of countries in the world which have edited rules and safety thresholds.

*European list of priority substances according to the European Union Commission: EC 1179/94, under Regulation 793/93 (OJ L131, 26.5.94, p.3 - link here)
*Substances Priority List 2013 according to the Agency for Toxic substances and disease registry: Phenol is ranked at the 180th but we can find phenolic compound at rank 54 and 89 (link here).
*European Council Directive 80/778/EEC relating to the quality of water intended for human consumption. (link here)
*Phenol - US Environmental Protection Agency (link here)





PCBs





Polychlorinated biphenyls (PCBs) are synthetic organic compounds forming a family of 209 elements which derive from biphenyl and of chemical formula C12H(10-n)Cln. Every core can have up to 5 chlorin groups. They are known to be the most persisting environmental contaminants in the biosphere. Due to their thermal resistance and chemical stability, they are commonly considered as indestructible. Hydroxylated polychlorinated biphenyls (OH-PCBs) are derivatives of PCBs and have also been identified as environmental contaminants.

Due to years of intensive use by industries, PCBs are found in large quantities in natural environment (air, water and soils). Since the 80’s, their production has been banned because of their toxicologic properties causing cancers, reproductive impairment, neurodevelopmental anomalies, and immunologic deficiency.



Figure3: PCBs chemical structure





PCBs are now considered as extremely dangerous and a lot of countries have established rules and safety thresholds.

*Substances Priority List 2013 according to the Agency for Toxic substances and disease registry: PCBs are ranked at the 5th (link here).
*Chemical quality of marine sediments in France: Synthesis of the available databases, INERIS, 2010 (link here)




Nitrite





Nitrites are completely natural compounds that are parts of the natural nitrogen cycle and they are nitrates reduction products.
The nitrate ion is one of the principal azote sources for plants. Thus natural or synthetic commercial fertilizers contain a lot of these compounds and are largely used to increase crops yield from 30 to 50% (Stewart,2005).

High nitrate water concentration have detrimental effects on living organisms. In fact, nitrites are nitrates reduction products that are responsible for Blue Baby Syndrome. This syndrome can occur when nitrate level is above 100 mg/L (SNIDE) in groundwater.
Symptoms start from a cyanosis and if it is not treated on time, can cause cerebral lesions and death.





Figure4: Nitrites chemical structure





Most of the rules and strict safety thresholds about these compounds concern drinking water.
*French government: The quality of the drinking water in France - Sanitary and statutory aspects (link here)
*Basic Information about Nitrite (Measured as Nitrogen) in Drinking Water according to United States Environmental Protection Agency. (link here)
*Substances Priority List 2013 according to the Agency for Toxic substances and disease registry: Lead is ranked at the 214th (link here).




Heavy metals





Heavy metals are one of the most important family of pollutants. For our project, we chose to focus on two of them, lead and cadmium. All heavy metals exist in surface waters in colloidal, particulate, and dissolved phases.

The majority of lead in the environment comes from human activity such as burning fossil fuels, mining, and manufacturing. It is used in the production of batteries, ammunition, metal products etc... Because of health concerns, use of lead has been dramatically reduced in recent years.
Cadmium does not corrode easily and has many uses, including batteries, pigments, metal coatings, and plastics. Moreover, all soils and rocks, including coal and mineral fertilizers, contain some cadmium.
Living organisms require trace amounts of some heavy metals (cobalt, copper, iron, manganese, etc...), but excessive levels of essential metals, however, can be detrimental to the organism.
Non-essential heavy metals like cadmium, lead, mercury, or arsenic are toxic compounds for all living organisms. According to the Agency for Toxic Substances and Disease Registry, lead and cadmium are considered as carcinogens and can damage all vital or non vital organs. (Lead, Cadmium)






Figure5: Periodic symbols of Cadmium and Lead





All heavy metals and their uses are now very supervised and companies which use them have to follow rules and strict safety thresholds.

*French government publication of 01/01/2014 by law of 02/02/1998 in relation to water sampling and consumption as well as to all residues of classified installations for environment protection subject to authorisation (link here)
*European Council Directive 83/513/EEC of 26 September 1983 on limit values and quality objectives for cadmium discharges (link here)
*EPA numeric aquatic life criteria (60 FR 22230) promulgated by EPA on May 4, 1995 (link here).
*Substances Priority List 2013 according to the Agency for Toxic substances and disease registry: Lead is ranked at the 2nd and Cadmium is ranked at 7th (link here).

• Advantages of bio-sensing

  
  
  

  In response to this recent awareness towards those toxic compounds, different systems of detection have been developed.
  However with the approach of bio-sensing, we develop tools which are able to detect a pollutant with a very high efficiency, and with a great specificity.
  Biological elements necessary to build those tools are cheap, easily obtained and their production doesn’t emit any pollutant.
  Thus besides being effective and cheap systems, biosensors are totally biological and non-polluting tools.
  
  

• Systems

  
  
  

  To develop our bio-sensors, we looked for some natural systems based promoters inducibles by our compounds of interest. It is often promoters wich allow the expression of a set of genes which correspond to the cell's response to the compound.
  For phenols, a set of genes called Dmp operon in Pseudomonas CF600 is able to degrade phenol to produce acetyl CoA, and use this molecule as an energy source.
  

  
  

  
  Figure6: The catabolic pathway for degradation of phenol and the organization of Dmp operon. (Powlowski J, Shingler V., 1994)
  

  
  

  The transcription of this operon is regulated by the DmpR regulator element which binds phenol and activates the transcription of the phenol hydroxylase enzyme by allowing the fixation of the RNA polymerase (more information, see the section Sensors).
  

  
  

  For PCBs, two distinct classes of bacteria have now been identified as being able to degrade PCBs:
  

• Aerobic bacteria which live in oxygenated environments
• Anaerobic bacteria which live in oxygen free environments such as aquatic sediments.

They use different mechanisms, aerobes attack PCBs oxidatively, breaking open the carbon ring and destroying the compounds. Anaerobes, on the other hand, leave the biphenyl rings intact while removing the chlorines.
The evidence in the literature suggests that PCDD/F compounds are subject to biodegradation in the environment as part of the natural chlorine cycle.






Figure7: The catabolic pathway for degradation of biphenyl by aerobic bacteria and the organization of the bph gene cluster (Kensuke F., Hidehiko F., 2008) .





The transcription of this set of genes is regulated by bphR2 which binds PCBs and activates the transcription of pbhR1 gene (more information, see the section Sensors).



For nitrites, the degradation pathways are very well known because they belong to the nitrite cycle. As for heavy metals, a lot of operons which allows the cell tolerance to these compounds exist and have promoters reacting specifically to the metal concentration.
However for this project, we chose to develop systems based on promoters presents in our bacteria strain Pseudovibrio denitrificans.
In this way, we used an RNA sequencing approach to detect promoters able to react to the pollutant presence (more information, see the section RNAseq).
Our bacteria strain is known to live in sponge and may provide them a better tolerance to pollutants like heavy metals. The ability of the bacteria is currently study by the 'Molecules of defence and communication in the microbial ecosystems' team of the National museum of natural history of Paris (their web page here)



• Futur : degradation?

  
  
  

  Sensing constructions are very simple and easy to construct. In a larger framework we can imagine one day being able to clone all the enzymes of the degradation pathways of phenol and PCBs in our bacteria. In fact we can create more than a sensor system based on sponge: a complete filtrating system which can not only sense pollutants, but totally remove them.
  
  For removing nitrites, it exists different biological denitrification systems to reduce nitrates concentration in water that use bacteria as Pseudomonas with a denitrification yield of 80%. Bacteria are fixed on a mineral support and feed with acetic acid or ethanol (SNIDE). The major drawback is the production of nitrous and nitric oxide that are greenhouse gases.
  To make a bacterium able to transform nitrites into nitrogen we just have to add two enzymes:
  -> Hydroxylamine oxydase from Parococcus denitrificans: nitrite + H2O = hydroxylamine
  -> Hydrazine oxydoreductase from Candidatus Brocadia anammoxidans: hydroxylamine + NH3 + acceptor = N2 + H2O + reduced acceptor
  
  
  

  
  Figure8: Nitrogen cycle.
  

  
  

  Unfortunately, dealing with heavy metals means dealing with atoms which cannot just be degraded by a biological pathway. But we can imagine some ways of accumulation of these elements in bacteria that we can remove and treated after like chemical waste.
  
  

Formal regulations