INFRARED SPECTROSCOPY (FT-IR) USED IN MICROBIOLOGY TO EVALUATE CHANGES IN BACTERIAL COMMUNITIES WITHIN ELECTRICAL FIELDS DURING BIOREMEDIATION

ROMINA DIAZ; ANALIA PRINCIPE; ADRIAN ACUÑA; GRACIELA PUCCI

Mendoza

Congreso; LVIII Reunión Anual de SAIB; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Infrared spectroscopy is a technique based on the use of infrared (IR) radiation, which is obtained from the thermal emissions of appropriate sources. This kind of radiation stimulates certain molecular groups into an excited state where they vibrate differently, a phenomenon that only occurs at fixed wavelengths. This study used FT-IR to assess changes in bacterial communities within five spectral windows. Different molecular groups have prevailing influence in each window: Between 3000 and 2800 cm−1, it is the functional groups in the membrane’s fatty acids; between 1800 and 1500 cm−1, the amide I and amide II groups in proteins and peptides; from 1500 to 1200 cm−1, a mix of proteins, fatty acids and phosphate-carrying compounds; from 1200 to 900 cm−1, mostly carbohydrates and polysaccharides. Finally, between 900 and 700 cm−1, specific spectral patterns occur named "the true fingerprint". The bacterial communities (biofilms) were observed during electrobioremediation. A soil sample was placed into a 27 cm-long electrobioremediation glass cell. A 0.5 V cm−1 difference of potential was applied to the cell for 60 days, with polarity being rotated every four days to prevent abrupt changes in pH. A second cell to which no current was applied was used as the control. Changes were monitored by determining bacterial count, total pretroleum hydrocarbons (TPHs) and pH. The soil sample was prepared by suspending 1 g of soil in 10 ml of tryptein soy broth. The suspension was incubated for 24 ± 0.5 h at 30 ± 2°C, centrifuged at 4000 rpm and washed three times with saline. Then, 50 μL of 70% alcohol were added, and the sample was put on a zinc selenide disc sample carrier and dried for 45 min at 42°C. The transmission spectra of the resulting dry biofilms were recorded in the wavenumber region between 4000 and 700 cm−1, using a Varian 1000 spectrophotometer (120 scan, 4 cm-1 resolution). To ensure data reproducibility, a strict experimental protocol was followed regarding media preparation, incubation time and temperature, cell harvesting conditions, sample preparation and FT-IR measurements. The spectra were transformed (normalization, smoothing and second-derivation) and recorded in ASCII format to be analyzed on the PAST software. Thanks to the possibilities offered by FT-IR spectroscopy to study dynamic changes in bacterial populations, we observed that the communities were in different quadrants depending on whether electrobioremediation was applied, as well as on the nutrient concentration and the amount of moisture. In the absence of nutrients, the bacteria in the electroremediated soil were in a different quadrant than in the control. Electroremediation increased the degradation of TPHs but did not affect bacterial count (10-4 to 10-5 CFU/g). In general, FT-IR proved a useful tool to observe changes in these communities during electrobioremediation.