Researchers at the Indian Institute of Toxicology Research have designed a microarray that can be used to detect biodegradative genes and monitor bacterial diversity in contaminated ecosystems.
The researchers discussed the chip, called the BiodegPhyloChip, in a paper in Applied Microbiology and Biotechnology last month. Paper co-author Natesan Manickam told BioArray News this week that the array was designed to "assess the diversity of environmentally useful microorganisms and functional potential for bioremediation of contaminated sites."
While several chips have been developed to monitor biodiversity, and some, such as PhyloTech's PhyloChip platform, have even been commercialized, Manickam said he and his colleagues at IITR sought to combine probes for monitoring both biodegradation and biodiversity, content that wasn't available on existing arrays (BAN 10/12/2010).
"Microarrays [with] probes for functional [biodegradation] genes and phylogenetic genes combined were not reported in earlier studies," said Manickam. "Our BiodegPhyloChip is comprehensive and has gene probes involved in the biotransformation of a wide variety of pollutants," he claimed.
According to the paper, the new array has 14,327, 60-mer probes derived from 1,057 biodegradative genes, and 880 probes representing 110 phylogenetic genes from diverse bacterial communities. The selected biodegradative genes are involved in the transformation of 133 chemical pollutants, the paper states.
The list of genes was obtained from public domain databases. A total of 12 probes were designed for each gene using Agilent Technologies' eArray software, and the arrays were subsequently manufactured by Agilent.
"We intended to make 60-mer oligonucleotide probes to result in increased specificity and sensitivity," said Manickam of IITR's selection of the Agilent platform "Also, [a] high-density array with [a] large number of probes using in situ printing technology was provided only by Agilent Technologies at that time of our array development," he said.
The IITR researchers used the array to test DNA extracted from five different contaminated sites in India. Soil and sediment samples were collected from sites near a chloroaromatic chemicals and solvents manufacturing industry in Lucknow, where IITR is also based. Sediment from the Gomti River, also in Lucknow, was analyzed, as were heavy metal industry dump sites in Kanpur, a site near a central effluent treatment plant along the Ganges River near Kanpur; and a site near the oil refineries in Mathura.
All of the sites were located in the North Indian state of Uttar Pradesh, which borders Nepal. Site selection was "based on long-term contamination history, mixed contamination, and the presence of priority pollutants such as polyaromatic hydrocarbons and chlorinated chemicals," said Manickam.
Array analysis detected 186 genes, including 26 genes unique to the individual sites, according to the paper. The array also identified bacteria similar to well-characterized genera involved in biodegradation of various pollutants.
Genes involved in complete degradation pathways for hexachlorocyclohexane, 1,2,4-trichlorobenzene, naphthalene, phenol, biphenyl, benzene, toluene, xylene, phthalate, Salicylate, and resistance to mercury were detected with highest intensity. The most abundant genes belonged to the enzyme hydroxylases, monooxygenases, and dehydrogenases, which were present in samples from all five sites.
Based on these results, the authors believe the new chip could be useful in assessing not only the biodegradative potential but also the composition of environmentally useful bacteria from hazardous ecosystems.
"Based on the experimental results using mixed waste samples, the 60-mer DNA oligonucleotide microarray proved to be sufficient for the detection of biodegradative genes representing various classes of pollutants and the determination of the dominant members of microbial communities," the authors stated.
The paper noted that one motivation for detecting and quantifying the functional genes in diverse environments is that "more than 200 well-spread toxic hot spots" exist in India, according to the country's Central Pollution Control Board. Therefore, information on catabolic genes and microbial populations in various contaminated environments will "prove useful in exploiting the microbial potential in bioremediation of the contaminated environment."
The authors suggest in conclusion that arrays "may be useful tools to investigate the genetic potential of the specific contaminated site" in order to determine which biodegrading bacteria are present and in what abundance.
"India, like few other countries in the world, has many contaminated hotspots that need to be remediated," said Manickam. "Our array could reveal the nature of contaminants and their biodegradation," he said. "We will use the array to monitor a few specific bioremediation sites, such as oil sludge and chlorinated pesticide, where an organized bioremediation will be undertaken," he said. He also suggested that IITR's array "can be used for similar studies in other parts of the world."
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