Identification of different species of Bacillus isolated from Nisargruna Biogas Plant by FTIR, UV–Vis and NIR spectroscopy

• Different bacterial species isolated from predigester of Nisargruna Biogas Plants.
• Bacterial isolates were identified by biochemical tests and 16s rDNA sequencing.
• FTIR spectra and its 1st order derivative proved useful in bacterial identification.
• FTIR more conclusive in differentiating genetically close organisms B. cereus and B. thuringiensis.
• FTIR, NIR and UV–Vis together made spectroscopic approach of identification of bacteria very robust and reliable.

Definitive identification of microorganisms, including pathogenic and non-pathogenic bacteria, is extremely important for a wide variety of applications including food safety, environmental studies, bio-terrorism threats, microbial forensics, criminal investigations and above all disease diagnosis. Although extremely powerful techniques such as those based on PCR and microarrays exist, they require sophisticated laboratory facilities along with elaborate sample preparation by trained researchers. Among different spectroscopic techniques, FTIR was used in the 1980s and 90s for bacterial identification. In the present study five species of Bacillus were isolated from the aerobic predigester chamber of Nisargruna Biogas Plant (NBP) and were identified to the species level by biochemical and molecular biological (16S ribosomal DNA sequence) methods. Those organisms were further checked by solid state spectroscopic absorbance measurements using a wide range of electromagnetic radiation (wavelength 200 nm to 25,000 nm) encompassing UV, visible, near Infrared and Infrared regions. UV–Vis and NIR spectroscopy was performed on dried bacterial cell suspension on silicon wafer in specular mode while FTIR was performed on KBr pellets containing the bacterial cells. Consistent and reproducible species specific spectra were obtained and sensitivity up to a level of 1000 cells was observed in FTIR with a DTGS detector. This clearly shows the potential of solid state spectroscopic techniques for simple, easy to implement, reliable and sensitive detection of bacteria from environmental samples.

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