Bacterial remedy for the toxic pesticide Carbaryl: Indian Institute of Technology Bombay

The memories of the Bhopal gas tragedy that claimed thousands of lives and injured a few lakhs of people, haunts us even after three decades. The culprit was a toxic gas used to produce a pesticide called 'Carbaryl', in the Union Carbide India Limited Factory. Sadly, the use of Carbaryl continued amid growing concerns about its side effects. The need to completely remove it from the environment or break it down into less harmful substances is of primary importance.

Dr Phale and his team from the Indian Institute of Technology Bombay (IIT Bombay), and collaborator Dr Sharma from Institute of Genomics and Integrative Biology (CSIR-IGIB), Delhi, have achieved a significant breakthrough in identifying bacteria which can clean up this pesticide from the environment and understanding exactly how the breakdown occurs.

Carbaryl, most commonly sold under the trade name ‘Sevin’ was a preferred pesticide for agricultural as well as non-agricultural use (lawns, home garden, and roadside greenery) against aphids, spiders, fleas, ticks, and many other pests. Carbaryl inhibits the enzyme responsible to carry nerve impulses and paralyses the nervous system of pests. This makes it difficult for the pests to breathe, leading to their death.

Like most other insecticides, Carbaryl has adverse effects on humans and other organisms and can cause cancer.

“Where at minor exposure, one might face problems like skin irritation and swelling of the eyes, it might cause asthmatic, respiratory and paralytic symptoms at a higher concentration,” explains Dr Phale.

Carbaryl kills earthworms, reducing the fertility of the soil and paralyses and kills pollinators such as honey bees, thus affecting agricultural production. Traces of carbaryl remain in the soil and the field even months after the crop is harvested.

“Carbaryl may stay back for years after the crop is harvested because it degrades slowly in the acidic soil as compared to alkaline soil. Also, repeated application of Carbaryl in the fields increases its concentration to a level lethal for living beings”, informs Dr Phale.

Finding the pathway to break down

Breaking down, or degradation of Carbaryl is a multistep process. Bacteria use enzymes to convert Carbaryl to the first intermediate compound, then at each step, they convert it to a different intermediate compound to form a pathway.

“Our interest was to dissect the process of carbaryl degradation by the microbes in the agricultural field,” says Dr Phale, speaking about the motivation behind their studies.

Scientists knew about some species of microbes that could degrade Carbaryl. Yet, the complete degradation process was still unknown. Dr Phale and his team collected soil samples from fields and isolated strains of bacteria which can degrade carbaryl. They found three strains of the bacteria Pseudomonas which efficiently remove Carbaryl. The researchers identified intermediate products like 1,2-dihydroxy naphthalene generated during the degradation process and described a complete pathway for degradation. They used the Pseudomonas strain ‘C5pp’ in the follow-up studies to understand the degradation process.

To begin with, a bacterial enzyme ‘carbaryl hydrolase’ acts on Carbaryl to generate ‘1-naphthol’ and ‘methylamine’. 1-naphthol acts as a source of carbon for the bacteria and methylamine serves as a source of nitrogen. In this way, Carbaryl—a poison for others—acts as food and source of energy for these bacteria.

Many microbes can degrade carbaryl, but the Pseudomonas strains reported in this study were more efficient. They could degrade the pesticide in 12-13 hours, which is 4-5 time faster than other bacteria reported in the scientific literature.

What could be the secret of this high efficiency? A study of the genome of Pseudomonas strain C5pp showed that the genes responsible for the Carbaryl degradation are arranged into three different sets referred to as ‘operons’. The bacterium might have acquired these genes from species Ralstonia or other species of Pseudomonas, capable of breaking down hazardous compounds. This process of movement of genetic material between unicellular or multicellular organisms is called ‘horizontal gene transfer’ and is different from vertical gene transfer from parent to offspring.

“Nature acts as a laboratory on its own. Organisms in the ecosystem interact with each other and exchange genetic material for their benefit and survival”, explains Dr Phale.

This smart bacterium acquired genes from other organisms capable of degrading various aromatic compounds as well as Carbaryl partly or partially, arranged these genes together and used this machinery for efficient utilization of Carbaryl as food and hence its survival.

Pseudomonas: the smart bacteria

Carbaryl is not the only toxic compound that bacteria have to face. The first breakdown product, 1-naphthol, produced within the body of the bacterium is even more toxic than Carbaryl itself. Then how does this tiny bacterium survive this toxicity? Follow up of this question revealed some interesting findings.

Bacteria like Pseudomonas have two membranes- inner and outer, with a compartment called periplasm between them. Degradation of the pesticide carbaryl into 1-naphthol takes place in the periplasm. The inner membrane protects the main body, or cytoplasm, from 1-naphthol, which can be fatal in high concentration, but less toxic in smaller concentration. It allows 1-naphthol to slowly enter into the cytoplasm. 1-naphthol is converted into a more water-soluble, nontoxic compound in the cytoplasm and then to organic acids which the cell consumes, to synthesize amino acids, nucleotides, and sugars. So, smart compartmentalisation of the degradation pathway is another strategy used by this bacterium.

These Pseudomonas strains can be used for eliminating pesticides from heavily contaminated agricultural fields. They might also be useful to treat effluents or wastewater, in industries producing or using carbaryl or 1-naphthol.

“The strains isolated by us are wild natural culture and not genetically modified organisms, so there is no restriction on their use,'' say the researchers.

Pesticide contamination and associated health hazards are a growing concern globally. The research of Dr Phale’s and team provides a natural solution to get rid of a hazardous pesticide. Information about the degradation process and the bacterial mechanism also opens up a possibility to design genetically modified organisms to address specific pesticide contamination.

Additionally, Pseudomonas are known to promote plant growth. The researchers believe that the isolated strains might be producing various enzymes and proteins, organic acids and metabolites which can act as growth booster for plants.

“We are currently studying these plant growth-promoting activities,” concludes Dr Phale.