RESULTS OF TESTING

Analytical methods (HPLC, GC)

The development and validation of analytical methods for monitoring organic substances in fermentation sludge (organic acids, carbohydrates, alcohols, and aldehydes) Using HPLC, we were observing the following parameters: concentrations of metanoic, acetic, maleic, succinic, butyric, and lactic acid, mono- and disaccharides, and glycerol.

We prepared method for determination of butyric acid, butan-1-ol, butan-2-ol, iso-butanol, tert-butanol, ethanol, propaone and buthanale using gas chromatography (GC)..

EFFECT OF pH ON GROWTH OF E. COLI

By measuring optical density @600 nm we draw growth curve for E. coli at various pH values (5, 6, 7 and 8). Optimal pH is between 7 and 8. There was no growth at pH below 6. .

GROWTH OF E. COLI IN MEDIA WITH BUTANOIC ACID AS CARBON SOURCE.

The optimal concentration of butanoic acid was 1 g/L. In higher concentrations butanoic acid act as an inhibitor.

RATIO BETWEEN BUTANOIC ACID AND GLYCEROL

OPTIMAL CONDITIONS

>Results show us that the best conditions for growth of E.coli using butanoic acid are: 1 g/L butanoic acid in molar ratio 1:1 with glycerol at pH 8.

Toxicity of substrate on E. coli

Based on the growth curve for E. coli at 37 °C, anaerobic conditions, and LB medium we calculated the maximum average optical density measured at 600 nm in stacionary phase (ODmax). We performed anaysis in 96-well microtiter plates. Several concentrationas of substrates (butan-1-ol, butanoic acid and glycerol) were added to LB medium, maximum optical density in stacionary phase was measured for each sample (ODx).

Toxicity (inhibition in growth) was calculated using the following formula:
Inhibition [%] = (ODn/ODmax)*100

In the cas of substances, that were expected to be produced using GM E. coli, we use the same protocol as for substrate.