Description

Indian Emissions of Halocarbons

Development of technology and rapid industrialization has been detrimental to the ecosystem over the last few decades. Among the products generated by polluting technology, halocarbon-based refrigerants have grabbed quite a few headlines. These are the chief ozone depleting substances as well as one of the leading greenhouse gases. The situation of the stratospheric ozone layer had worsened so much by the late 1980s that the global community came together and phased out CFCs, replacing them with sister halocarbons - HCFCs and HFCs. HCFC phaseout too began with the dawn of this century and the coming of Kigali Amendment (2016) demands countries do away with HFCs as well.

India’s halocarbon current emissions reflect an emissions profile typical of Article 5 countries, with low emissions of CFCs and large emissions of both HCFCs and HFCs (data from 2016). India reported a complete phase-out of CFCs in 2010, however the undisposed banks still contribute to about 7 % of global CFC emissions. Data suggests that India is yet to adopt several common refrigerant blends, including R-410A, R-404A and R-507A, all of which are used extensively in the developed world.

India is a fast-developing economy. In the coming decades, its coolant consumption is projected to increase dramatically, only leading to more emissions. Being a tropical country, India will be one of the worst affected due to the consequent damage to the environment.

HALOCARBON EMISSIONS FROM INDIA (ESTIMATES)

The legacy problem

The Montreal Protocol and its amendments, with the strict deadlines to phase out halocarbons seems to solve the problem right? Sadly, no.

Decreasing the consumption and production can do nothing for the massive quantities of coolants we have already produced. The disposal of existing reserves of halocarbon-based refrigerants, which were used in now-obsolete machines, is still an open, festering problem. Recycling these gases is not an option since newer technologies now implement less polluting alternatives in an effort to adhere to the agreements. Thus, halocarbons continue to slowly leak, react, and wreak havoc in the atmosphere.
Not destroying the CFC banks in the year 2000 has already added 25 billion MT CO₂ equivalent emissions. This has delayed ozone hole recovery by seven years.

HALOCARBON EMISSIONS FROM BANKS A HISTORIC PERSPECTIVE USING CFC-11

Incineration: the current method of disposal

Today, HCFC and HFC destruction and disposal technologies that have been approved only include incineration processes and plasma technologies, all of which rely on heating to thousands of degrees Celsius (which needs fuel and thus in itself leads to CO2 emissions) and causes the formation of harmful by-products such as HCl, HF and dioxins. Most of these processes are too costly and/or need significant infrastructural requirements and thus are unsuitable to deploy in developing countries including India. In a sense, incineration is hardly a solution to the problem and more of a patch work effort the world is trying to rely on.

CURRENT METHODS OF DESTRUCTION OF HALOCARBON REFRIGERANTS

Our approach

Pseudomonas putida, more famous as the ‘superbug’, is known to degrade a myriad of organic compounds in nature and possesses a broad tolerance to toxic compounds and oxidative stresses. We aim to design a bioreactor that employs Pseudomonas putida which we will synthetically tune with the machinery required to digest these gases.

P450cam, an enzyme system native to Pseudomonas putida, is quite efficient in breaking the carbon halogen bonds. Combined with the Toluene dioxygenase system, the enzymes are capable of degrading commonly used halocarbons. Cytochrome P450cam however, works only in anaerobic conditions and forms byproducts difficult to digest in aerobic conditions. Toluene dioxygenase on the other hand works in aerobic conditions.

Enzymes Schematic

To build a working system combining the two, we recognized the need to switch off the P450cam system in the presence of oxygen. We looked up multiple ways to execute this and ultimately narrowed down on ANR-regulated hypoxia, the Pseudomonas homolog of the FNR promoter of E. coli. The presence of oxygen prevents ANR from inducing transcription of the genes downstream of the corresponding promoter, which in our case would be, Cytochrome P450cam.

Schematic exhibiting ANR-regulated hypoxic promoter

We want to give the world not just laboratory products, but a device that can make a change. With the existing work on modelling the enzymatic degradation, microbial growth, and studying the way industries tap these microbes, we have designed a bioreactor that can degrade halocarbons. With our analysis and modelling, we have worked to make the device cost-effective and implementable on a large scale.

Throughout our project, we put in efforts to take inputs from the community and give them back something. We have been guided, supported and motivated by scientists, entrepreneurs, social workers and the common people. Through our plans in education and outreach not only did we spread the concept of synthetic biology but also emphasized on the urgency of climate crisis.

With Halocleen, we seek to answer the world’s need for a way to degrade tons of halocarbons that is not only cost-effective but also environment friendly.

Detailed References

A) Logan et al. showed that the cytochrome P450cam enzyme system could de-halogenate haloalkanes under reducing conditions and Wackett et al. showed that toluene dioxygenase could degrade certain haloalkenes, which provided the motivation for the construction of an engineered strain that could combine the two enzyme systems. We plan to use this system to cleave the carbon halogen bonds of HCFCs and HFCs, converting them into simpler compounds.

Logan, M.S.P., Newman, L.M., Schanke, C.A. et al. Cosubstrate effects in reductive dehalogenation byPseudomonas putida G786 expressing cytochrome P-450CAM . Biodegradation 4, 39–50 (1993). https://doi.org/10.1007/BF00701453

L P Wackett, D T Gibson. Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1. ASM Journals - Applied and Environmental Microbiology Vol. 54, No. 7 (1988). https://doi.org/10.1128/aem.54.7.1703-1708.1988

B) Hur et al. and Wackett et al. engineered Pseudomonas putida G786 (bearing the CAM plasmid that has the P450cam enzyme) by introducing the toluene dioxygenase enzyme system and demonstrated that it could completely metabolise several halocarbon pollutants (including CFC-11, CFC-112, CFC-113 and TCE) under alternating low and high oxygen tension.

H G Hur, M J Sadowsky, L P Wackett. Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway. ASM Journals - Applied and Environmental Microbiology Vol. 60, No. 11 (1994) https://doi.org/10.1128/aem.60.11.4148-4154.1994

Wackett, L., Sadowsky, M., Newman, L. et al. Metabolism of polyhalogenated compounds by a genetically engineered bacterium. Nature 368, 627–629 (1994). https://doi.org/10.1038/368627a0

C) Højberg et al. constructed an oxygen-sensing Pseudomonas strain using an ANR-responsive native anaerobic promoter, which was characterised in detail by Ugidos et al. We plan to synthetically install this promoter in the P450cam system to turn the genes off during aerobic environment. This will help us achieve a sequential aerobic and anaerobic environment.

Højberg et al. Oxygen-Sensing Reporter Strain of Pseudomonas fluorescens for Monitoring the Distribution of Low-Oxygen Habitats in Soil. ASM Journals - Applied and Environmental Microbiology Vol. 65, No. 9 (1999) https://doi.org/10.1128/AEM.65.9.4085-4093.1999

Ugidos, A., Morales, G., Rial, E., Williams, H.D. and Rojo, F. (2008), The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator. Environmental Microbiology, 10: 1690-1702. https://doi.org/10.1111/j.1462-2920.2008.01586.x

D) Streger et al. (1999) reported the efficiency of soluble methane monooxygenase system (sMMO) in degrading a variety of halocarbons that included common HFCs.

Sheryl H. Streger, Charles W. Condee, A. Paul Togna, and Mary F. DeFlaun. Degradation of Hydrohalocarbons and Brominated Compounds by Methane- and Propane-Oxidizing Bacteria. Environmental Science & Technology 1999 33 (24), 4477-4482. DOI: 10.1021/es9907459

E) Wageningen 2021 modified Pseudomonas putida to degrade greenhouse gases produced by cattle and developed iGEM PIPE, a tool that suggests bioengineering strategies in support of bioremediation with a Pseudomonas chassis. An analysis of their work and going through the existing literature on the bacterium made us realise it is ideal to work with.

F) BNDS China 2019 had previously worked with the toluene dioxygenase enzyme system and have deposited its constituent parts in the iGEM Registry. We verified the sequences of these parts with the literature and proceeded with synthesising the genes.

G) While looking up ways to work with a sequential aerobic and anaerobic environment, we came across the work of TU-Eindhoven 2013 who characterised the anaerobic FNR promoter. We decided to incorporate the Pseudomonas homolog of the FNR promoter into the p450cam system.