Procedure
Our main goal was to produce biofuel using biodegradable organic waste in a sustainable way with a sufficiently high yield and good separation options of the final product. In order to make our project as efficient and sustainable as possible, we were aiming to transform butyric acid, which was produced and available as a product from another bioprocess (anaerobic digestion) to biobutanol, an alcohol with high energy content and relatively low solubility in water. To prove, butyric acid can be produced at sufficient concentrations to make its subsequent transformation viable, we sampled an inoculum of mixed anaerobic microbiota at a local municipal wastewater plant. Until further use, this inoculum was stored in the cold room (4 °C) to maintain the initial microbial consortia structure. Prior sampling the required amount for bioreactor start-up, the container with the inoculum was thoroughly mixed using a specialised high-speed mixing device equipped with a conical agitator paddles designed for optimum mixing of low-viscosity fluids. After that, the obtained inoculum amount was diluted with water (3:1 volume ratio) and used for the inoculation of several bench-top bioreactors. We performed the process of anaerobic butyric acid production by running 12 parallel bioreactors in order to obtain a highly precise measurement of achievable butyric acid concentration, as well as a measure of appropriate deviations in concentration. We also wanted to know how well the production curves of the bioreactors can overlap. After the bioreactors were filled, assembled, purged and sealed, we initiated the incubation at 37 °C. The operating parameters of the bioreactor system were monitored and maintained (T, mixing, pH) automatically by means of an integrated computer server. The system was equipped with sampling ports for: (i) sequential analysis of dissolved products and (ii) online analysis of produced amount and composition of gaseous products. As the presence of oxygen could significantly influence the production of butyric acid, care was taken to maintain anaerobic conditions throughout the entire incubation period, including during sampling of various products and phases from the reactor contents.
We sampled the liquid contents from each bioreactor every 6 hours and tested the composition of the reactor broth with a HPLC apparatus. In this way we were able to determine the start of acidogenesis, i.e. the specific process during which butyric acid is produced. For the purpose of sampling, two microcentrifuge tubes (approximately 2.5 ml) of reactor contents was taken; the first one was frozen in the case of any errors and the second one was used for HPLC analysis. Sampling and analysis was performed simultaneously for all 12 bioreactors.
Most standard laboratory procedures for growing E. coli to high cell density include the use of simple LB medium and basic laboratory equipment. The strain E. coli DH5α, which we used, was not expected to require any different growth conditions and while we searched for THE growth medium for our bacteria, we were looking that this medium could be selective for our microorganism, for our process (conversion of butyric acid to butanol) and easily modified on the chemical basis. The selectivity of the media is important in the first phase to eliminate Gram-positive bacteria and prevent any contamination. We had found the information about standard ENDO medium that we decided to use and make some modifications. Since our ‘reprogrammed’ bacterium has a plasmid with 3 genes for two proteins inside its cytoplasm, it is reasonable to assume that the standard composition of the medium is not going to be the most optimal for bacteria to carry out the constructed methabolic pathway. Furthermore, this media are optimal for the growth and reproduction of E.coli DH5α, besides the standardized LB medium, but not necessarily for the pathway for butanol conversion. In addition, the pH range for the optimal conditions for E.coli DH5 alpha is quite wide and we decided to vary the pH value of each chemically different media (pH=5, pH=6, pH=7, pH=8). Based on the gathered information, our eager team screened 72 different media and tested all of them in aerobic, as well as anaerobic conditions, in order to determine the most optimal medium. During the incubation, stock bacteria were grown on the standardized medium LB.
Lactose, that was initially the source of the carbon, was in some medium replaced with butyric acid and glycerol, with preserved number of carbon atoms in the medium. However, the ratio between glycerol and butyric acid had been modified in order to find out the most optimal ratio between these two substances that would encourage the best conversion from butyric acid to butanol. Glycerol also acts as electron donor. Besides butyric acid and glycerol, our media composed of meat peptone, dipotassium phosphate and sodium sulfite. Dipotassium phosphate serves as the growth factor and it regulates a constant pH, sodium sulfite serves as the inhibitor of Gram-positive bacteria and meat peptone as nutrition source. We have also tested one standardized LB medium with different pH values and one 0,9% NaCl medium with pH range from 5 to 8.
One of our important results was also to show, that E. coli is able to thrive in the presence of sufficiently high concentration of butyric acid, to make the process applicable. Therefore our team tested different concentrations of butyric acid in the growth medium, to find both the maximum limiting concentration and optimum concentration of butyric acid, that enable E. coli to thrive.
Inoculation
• Inoculation in 24-well plates from individual centrifuge tubes•Inoculation in 384-well plates
Aerobic inoculation
We prepared 72 media in the Falcon centrifuges (50 ml) and all of them were autoclaved with opened lids. We had also autoclaved 500 ml of physiological solution. As soon the autoclaving was done, the laminar was prepared to be sterile: disinfected with ethanol. It is important not to forget to disinfect the safety gloves. In the sterile well plates 2 ml of each medium was supplied to the the corresponding wells. We also added 2 µl of sterile antibiotic in each well, which was appropriately marked (aerobic condition + antibiotic). The well plate was then wrapped with parafilm and stored in the refrigerator.
Because we also wanted to try out a more work-intensive approach, we also performed growth media screening in 384 - well plates. In this case, we supplied 50 µL of medium to each well. The first 5 wells contained the G1 medium, the second 5 G2 and so on. In this way we obtained 5 parallel runs we could measure simultaneously. We also prepared a nutrient depleted control - physiological solution with bacteria (a colony per 5 ml of physiological solution). The solution was then divided in wells, 5µ in each well. Then, well plate was put in BIO – TEK Power Wave XS for analysis at 37°C, with one measurement per 900 seconds and mixing 20 per seconds. The analysis lasted 3 days.
All Falcon tubes with media were degassed in an ultrasonic bath. The media were placed in the gas pack, filled with argon and nitrogen. Each centrifuge tube was additionally purged with a syringe with nitrogen and argon to maintain anaerobic conditions. After that, media were supplied to 384 – well plate, the antibiotic formulation was added and the well plate was labelled accordingly (anaerobic condition + antibiotic). The inoculation of bacteria and analysis was performed in the same way as previously described for aerobic conditions. Due to slow growth and general slow metabolism response in anaerobic conditions, the incubation and monitoring of the anaerobically maintained media was performed for five days.
The analysis of results from well plates:The information were given in the dependency of time measurement (0 min, 15min, 30min…). However, we were interested in the results that were shown separately for each well. The first two parallels of each medium were blind samples (only medium), while other three contained bacteria. The average result of the blind wells, the average of testing wells and the standard deviation of bacterial parallels were calculated. After that, the absolute value of the divergence of averages for medium was calculated.
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 acts 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 case of substances that were expected to be produced using GM E. coli, we use the same protocol as for substrate.
