Why focus on atmospheric pollutants and especially VOC?

Atmospheric pollution is characterized by the presence of gas and particles (nitrogen oxides, carbon oxides, ozone, particles PM10 and PM25, Volatile Organic Compounds, black smoke) in the outside air with harmful impact on human health and/or on the environment. [1] [2]

It is known that pollution has harmful effects on human health. According to the World Health Organization (WHO), 7 million premature deaths are due to atmospheric pollution, equal to one eighth of the world annual deaths. [3] The estimated cost of these premature deaths in WHO countries is $ 1.6 trillion per year. [4]
In France there are 3.5 million people with asthma, 50 000 people suffering from serious respiratory deficiencies, and 150 000 deaths of patients suffering from cardiovascular diseases. [5] All those diseases originate or at least are aggravated by atmospheric pollution.
In addition to public health issues, these pollutants can have a harmful effect on the environment as they acidify water and soil and decrease plant growth. These changes trigger a decline in agricultural yields and alter aquatic ecosystems. [5]

Atmospheric pollution is an accumulation of complex phenomena. To understand local and regional emissions, meteorological conditions, transport, and pollutant transformation must be considered. [2]

Our team decided to focus on VOCs for several reasons. VOCs are organic chemicals with a high vapor pressure (0.01kPA) at room temperature (293.15K = 20°C). Their high vapor pressure results from a low boiling point, which causes large numbers of molecules to evaporate from the liquid or sublimate from the solid form of the compound and enter the ambient air, a trait known as volatility. [1] [6] Over the past ten years the average toluene concentration in the periphery of Paris was 6.1 µg/m3 with a maximum of 24 µg/m3 in traffic area. [2]

These primary pollutants originate from fuel evaporation, car traffic, industrial processes, heating systems in residential areas, domestic use of solvents and also from vegetation. They have a role in secondary particle and ozone formation [1] [6].

Although air pollution contains only 2% VOCs (see figure 1) [2], the impact of those pollutants on health and environment is major. These pollutants are persistent in the environment, they bio-accumulate in living tissues and are able spread over long distances. Although their effect on human health are only partially known, scientists have demonstrated their systemic effects (hepatic, hematologic, immunologic), their toxicity on the reproductive systems, their genetic toxicity. VOCs are known to be carcinogenic.

Figure 1: Repartition of atmospheric pollutant in Paris area according to airparif [2]. Most important pollutants are carbone oxide and dioxide (85%). VOC represent 2% of the total atmospheric pollutant. [2]

To protect populations and the environment against harmful pollutants, regulations were defined and air quality is supervised according to European directives [1]. At the European level, the 2008/50/CE directive of May 21st 2008, specifies air quality standards in Europe. This text was transposed into French law by decree n°2010-1250 of October 21st 2010 on air quality. Specific directives are applied to VOCs, such as the european directve n° 2015/1480 modified the 28th of August 2015, concerning VOC emission from fuel storage and fuel distribution in gas stations.
Even if pollutant concentrations are getting lower, their final concentrations do not respect the threshold of 5 μg/m3 defined by the law. The objective to reach for the annual mean concentration is 2 μg/m3.

What are the existing methods of air pollution monitoring?

Pollutant diversity in air and water is so important that no single universal means of detection can exist. Methods must be adapted to the targeted pollutants and the environment in which the pollutant is found. Existing monitoring tools are classified into two main groups: active and passive detectors. Those technologies are then subdivided according to their analysis type: physical, chemical or biological.
In the following table, we show the different means of VOC detection. The principles of some methods (continuous analysis scanner, integrated measure by active and passive sampling) are then detailed as well as biosensor’s advantages and disadvantages compared to existing methods. [7] [8] [9] [10]

*This physical method uses a pump in a continuous manner, a pre-concentration device followed by a chromatographic column and one/two detection systems that use flame ionization or photo ionization.

**The integrated measure by active sampling is a two-step method. First a sampling is carried out by pumping through a cartridge containing adsorbents (graphite black carbon), then the sampling is analyzed in the laboratory by chromatography coupled with flame ionization or mass spectrometry detection.

***The integrated measure by passive sampling is a two-step method. First a sampling is carried out through diffusion tubes containing an absorbent, then a laboratory analysis is made using a chromatography coupled with flame ionization or mass spectrometry detection.

VOC detection methods using biological elements present numerous advantages compared to other physico-chemical methods described above. Even if biological detection methods require laboratory work that can only be carried out by trained people to avoid contamination and dissemination issues, they can, by using appropriate technologies, surpass currently used pollutant detection methods [11] [12].

Whole cell biosensors for environmental applications require the selection of a detector sensitive to the pollutant or group of pollutants to be detected and a reporter system, as well as a suitable method of presentation of the biocatalyst to the transducer. [12] As the pollutant recognition profile of a promoter (used as the detector element) is usually made up of only a few molecules, a biosensor’s output can be very specific and informative. [13]
In addition, according to the strength of the output signals, the quantity of initial signals (quantity of pollutants) can be determined [13].
By modifying the genetic construction, the targeted pollutant can be changed, as well as the sensitivity of the biosensor (the amplitude of its response to a given stimulus). This was demonstrated by the 2009 Cambridge iGEM team in their E. chromi project [14]. The purpose of that project was to create kits of parts that would help the design and construction of future biosensors.

Another major advantage of biosensors is their ability to detect several environmental signals simultaneously and integrate them into different outputs (for example, different bioluminescence colors or fluorescence emissions). This can be achieved by using a single whole cell biosensor consisting of multiple different sensors and reporters or several different whole cell biosensors include in the same device each having a single sensor and reporter system.

Since biosensors use cellular machinery they do not require any other energy input than water, sugar and salt and are more convenient to use compared with conventional chemical methods. This fact presents a major advantage compared to actual pollution detection systems which are energy and water consumers [13]. When deployed in sealed containers biosensors are very suitable for field applications as they are more easily transportable than electronic measurement devices. Thus, accurate and sensitive whole cell biosensors for pollutant detection and quantification provide useful alternatives to direct analytical methods because of their low cost, rapid response, reproducibility, and ease of production. [15]

In addition, living cells give not only analytical information but also functional information on the effect of a stimulus on an organism. Whereas traditional analytical chemistry does not distinguish the different forms (ionic, inert forms) of a pollutant, biosensors make a difference between available and inert, unavailable to biological system forms. Therefore, biosensors can assess the real impact of pollutants on living organisms [16] [17]. This information is important and relevant for environmental measures [18], [19].

To conclude, biosensors supply rapid and specific results at low cost and are able to detect pollutants in a highly specific manner. As they are easily transportable (minimal size, reduced weight) and integrated into other technologies, they are perfectly suitable for field measurements. Only a few tools using biosensors are currently commercialized. For that purpose, our team had decided to realize one to detect VOCs.

Why choose Bioluminescence?

Bioluminescence has been recognized as an alternative to fluorescence [21] and has become widely preferred for quantitative bioanalysis [22]. It is assumed that the light intensity is proportional to the concentrations of the targeted molecules. [22]

In bio analysis, photon emission chemistries including fluorescence and bioluminescence are popular due to their inherently high sensitivity and simplicity. [22] Fluorescence relies on photons as the energy source whereas in bioluminescence, the photon emission is based on natural biochemistry.
Fluorescence is brighter due to the high rates of photon excitation. However, this high influx of photon raises the background levels. The number of photons introduced is much more important than the number produced by the reporter gene and photodetectors could not be able to discriminate between excitation and emission photons. Others fluorophores can be present within the samples. [22] As no photons are introduced into the sample, background levels in bioluminescence assay are much lower. [22][21]

As a results, bioluminescent chemistries present 10- to 1,000-fold higher sensitivity than fluorescence assays [23] Bioluminescence can be measured to zeptomole levels (few molecules per cell) and linearity extended over six to eight logs. [22]

Moreover, as bioluminescence naturally evolved within a biological context, it is more compatible with bioluminescence systems. [22]

To conclude, bioluminescence is greatly used in assay methodologies and these assays are known for their quantitative precision, low inherent backgrounds, and low sample interference. [22] For all these reasons, we decided to base our biosensor on the use of bioluminescence as the reporter gene.

Potential Applications

The different parameters that must to be taken in account when speaking of atmospheric pollutants (local and regional pollutant emissions, meteorological conditions, transport as well as pollutant transformation) can be modeled. That’s why a 3D model of pollutant concentration can be a valuable tool for air quality monitoring.
A biological system embedded in a drone would enable more precise pollution mapping, and thus a monitoring of pollution in the atmosphere.

The applications of this new monitoring tool are numerous, including defining polluted areas, identifying pollutant emitting industries, monitoring pollutant concentration throughout the year, or localizing a chemical leakage along a pipeline. The ever-increasing number of laws concerning pollutant concentration in urban areas is also a good occasion for us to develop a product able to efficiently map the air quality around a public building, such as a school.

A precise and local detection of pollutants will trigger effective action to be taken as well as local environmental damage to be determined. Data obtained using Quantifly can serve as a basis for defining a new framework for future industrial and public regulations concerning pollution.

Measurements carried out with our biosensor could also have an impact on the real estate market, as people don’t want to buy a house in a polluted area.

References (Links are provided when available):

1. Bilan de la qualité de l’air en France en 2014 et principales tendances observées sur la période 2000-2014, Laurence Monnoyer-Smith, Anne Bottin, Commissariat Général au développement durable, September 2015

2. AIRPARIF, association for air quality monitoring in Ile de France (France)

3. 7 million premature deaths annually linked to air pollution, WHO , 2015

4. Air pollution costs European economies US$ 1.6 trillion a year in diseases and deaths, new WHO study says, WHO

5. « Bilan de la qualité de l’air en France en 2012 et principales tendances observées sur la période 2000-2012 », Energy and climate executive management

6. Le bulletin de la mesure et de la caractérisation des polluants dans les rejets atmosphériques: Mesure des Composés Organiques Volatils », Jean Poulleau, INERIS

7. Active sampling measurement (norme NF EN 14662-1), November 2005.

8. Active sampling measurement (norme NF EN14662-4), November 2005.

9. RAPPORT D’ÉTUDE 05/10/2010 N° INERIS-DRC-10-112289-10754A: Stratégie de mesure des niveaux de concentration en benzène, toluène, éthylbenzène et xylènes dans l’air ambiant autour d’installations classées

10. Jarque, S., Bittner, M., Blaha, L. & Hilscherova, K. Yeast Biosensors for Detection of Environmental Pollutants: Current State and Limitations. Trends in Biotechnology (2016).

11. Hernandez C.A., Osma, J.F. (2014). Whole cell biosensors. In M. Stoytcheva & J.F. Osma (Eds.). Biosensors: Recent Advances and Mathematical Challenges. Barcelona: OmniaScience, pp. 51-96.

12. Rawson, D.M., Willmer, A.J., and Turner, A.P. (1989). Whole-cell biosensors for environmental monitoring. Biosensors 4, 299–311.

13. iGEM Peking 2013 Wiki

14. iGEM Cambridge 2009

15. Behzadian, F., Barjeste, H., Hosseinkhani, S., and Zarei, A.R. (2011). Construction and Characterization of Escherichia coli Whole-Cell Biosensors for Toluene and Related Compounds. Current Microbiology 62, 690–696.

16. X. Liu, K. J. Germaine,D. Ryan, and D.N.Dowling, “Whole-cell fluorescent biosensors for bioavailability and biodegradation of polychlorinated biphenyls,” Sensors, vol. 10, no. 2, pp. 1377–1398, 2010.

17. Wong, L. S., Lee, Y. H. & Surif, S. Whole Cell Biosensor Using Anabaena torulosa with Optical Transduction for Environmental Toxicity Evaluation. Journal of Sensors 2013, 1–8 (2013).

18. Luc BOUSSE, « Whole cell biosensors », sensors and actuators, 1996

19. Selifonova, O., Burlage, R., and Barkay, T. (1993). Bioluminescent sensors for detection of bioavailable Hg(II) in the environment. Appl. Environ. Microbiol. 59, 3083–3090.

20. Nunes-Halldorson, V. da S., and Duran, N.L. (2003). Bioluminescent bacteria: lux genes as environmental biosensors

21. Promega corporation. Welsh, D.K., and Noguchi, T. (2012). Cellular Bioluminescence Imaging. Cold Spring Harbor Protocols 2012, pdb.top070607

22. Wood, K (2011), The bioluminescence advantage, laboratorynews

23. Fan, F. and Wood, K. (2007) Bioluminescent Assays for High-Throughput Screening. Assay Drug Dev. Technol. 5, 127–36.