ANNOUNCEMENTS
The total global capacity of commodity plastic production has dramatically increased from 1.5 million tons in 1950 to 245 million tons in 2008, an annual growth rate of 9% (Chanprateep, 2010). In the recent years, there has been increasing public concern over the harmful effects of petrochemical-derived plastic materials in the environment. There is an obvious need to minimize the generation of plastic waste and to develop technologies that can play a vital role in mitigating these problems. The potential of different bacterial species and recombinant strains is being explored in the context of developing biodegradable plastics by increasing PHA yield and productivity, PHA variety and ease of polymer recovery (Quillaguamán et al., 2010). Present study also deals with exploring new unculturable bacterial species which may have better PHA producing ability through metagenomics.
The study can be summarised as below:
1. Soil samples from two different geographical locations: Olive mill waste treatment plants (OMW) in Crete, Greece and South Santhal (SS), ONGC Mehsana, Gujarat which are contaminated with olive oil waste and petroleum hydrocarbons respectively were collected.
2. Comprehensive 16S rRNA clone libraries identified that in both the sites, Gram negative bacterial strains were dominant. A relatively large group of Uncultured bacterium clones were also present. In addition a few strains of Firmicutes, Bacteroidetes and various classes (Alpha, Beta & Delta) of Proteobacteria were observed.
3. Metagenomic library prepared from OMW site consisted of 50,000 clones with an average insert size of 40 kb. Thus, the metagenome coverage was approximately 2000 Mb. In contrast, the SS library was considerably bigger (2 × 106 clones). The library contained almost 84000 Mb of sequence with an average insert of 40 kb size.
4. Functional screening of 50,000 clones from OMW library using Nile Red assay identified a total of 420 positive clones. Among these 420 clones, 29 clones were place under strong positive clones category since the fluorescence intensity was much stronger as compared to the rest (391) clones which were considered as potential positive clones.
5. In order to remove the false positives and to retrieve the expressive phaC gene from clones, the 420 Nile Red positive fosmids were isolated and retransformed into different hosts: E. coli and P. putida KT2440 !phaC1. The resulting clones were screened again and sequenced. But the sequences did not reveal any phaC gene sequence.
6. In order to confirm the phenotype of the 420 clones, the clones were also simultaneously screened for all the phaC gene classes: phaCF1/phaCR4; I-179L/I- 179R; B1F/B1R. However, none of the 420 clones were positive for any of the phaC gene classes.
7. In addition, four fosmid clones (D-A1, D-A2, E-A1 & E-C12) which were characterized by strong Nile Red fluorescence intensity were selected for complete 40 kb sequencing in case an environmentally new gene with a different sequence was present. But the contig sequence from each of the clones did not have an ORF representing the phaC gene family.
8. Screening of 50,000 clones from OMW library resulted in not even one PHA positive clone even though it had a number of Pseudomonas strains. It seems Nile Red screening was not a sensitive screening test for PHAs. It resulted in a lot of false positives which was subsequently discovered when sequencing and PCR amplification for phaC gene was done. The PCR reactions with the different primers sets done in the OMW screening also revealed that phaCF1/phaCR4 can amplify both Class I and II PHA synthases so screening with primer sets I-179L/I- 179R is not necessary. Primers sets BF1/BR4 were also dropped as they were found to be non-specific for phaC gene.
9. In case of SS library, 19,200 clones were initially screened using the Nile Red assay. However, none of the clones were confirmed as positive. Since Nile Red assay proved to be an insensitive test for PHA screening in case of OMW library further screening of SS library using Nile Red was discontinued and the library was screened using only phaC specific primers: phaCF1/phaCR4.
10. Out of a total of 30,720 clones screened by PCR amplification for phaC gene using the primers: phaCF1/phaCR4, 9 PHA positive clones were detected. Partial sequence of the amplified phaC genes from these clones showed similarity with Pseudomonas aeruginosa UCBPP-PA14 (37J20) P. pseudoalcaligenes HBQ06 (9G1), Hahella chejuensis KCTC 2396 (40N22) and Acidovorax sp. JS42 (9L11). Clones 25A21, 40L24, 41N3, 49F10 and 50K15 showed match to phaC gene of Uncultured bacteria. These genes represent the class I and II PhaC enzymes.
11. The sequence of one of the clones 40N22, was showing similarity to only two sequences (phaC gene of H. chejuensis KCTC 2396 [CP000155.1] and Halorhodospira halophila SL1 [CP000544.1]) in the entire database. In addition only 15% of the sequence was matching while the rest 85% could not be aligned. Thus Clone 40N22 was studied further. The complete phaC gene sequence of 1695 bp was retrieved by sub-cloning. The deduced amino acid sequence (564 aa) showed highest similarity to the phaC gene of Alcaligenes sp. (76% identity). Analysis of the sequence confirmed that it contained the class I subfamily of PHA synthases.
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