The growth conditions of Lactobacillus reuteri DSM 17938 in bioreactor influence 3-hydroxypropionic acid bioproduction
Résumé
Objectives: The 3-hydroxypropionic acid (3-HP) that has been identified as one of the top platform molecules
by the United States Department of Energy is gaining more interest due to its versatile applications [1]. Among
non-GMO microorganisms naturally able to produce 3-HP, Lactobacillus reuteri is a promising candidate
thanks to its ability to use glycerol, a by-product in biodiesel manufacturing industries [2]. After a growth
phase in batch culture, the 3-HP is produced in fed-batch by resting bacterial cells that do not consume glycerol
as a carbon source, thus avoiding the synthesis of by-products that can hinder conversion yield and further 3-
HP extraction [3]. As the 3-HP bioproduction is intimately linked to activities of intracellular enzymes that
depend on the quantity and quality of cells, it is necessary to improve the biomass production step [2]. In such
context, this work deals with a screening of the effects of environmental growth factors on the bacterial growth
and the cell ability to produce 3-HP during the bioconversion step.
Materials and methods: The 3-HP bioproduction was achieved by a 3-stages process: cell growth, biomass
harvesting and concentration, and glycerol bioconversion that was performed in fed-batch mode by resting
cells. A Plackett-Burman experimental matrix was designed to test the effects of 11 environmental factors on
the growth and bioconversion performances of L. reuteri DSM 17938, including the culture pH and
temperature, the addition of glucose, yeast extract, phytone-peptone, 1,2-propanediol (1,2-PDO), cysteine,
betaine plus KCl, Tween 80 and vitamin B12 in the culture medium, and the nature of the base solution used
to control the pH. Statistical analyses were performed using the software Statgraphic plus to identify the
significant effects at 95% to 99.9% confidence intervals for coefficient estimations.
Main Results: Among the studied factors, pH, addition of yeast extract or cysteine, nature of the base solution
did not affect the growth. Meanwhile, higher glucose concentration and addition of 1,2-PDO to growth medium
led to higher cell concentration. Concomitantly, the supplementary glucose is also linked to the improvement
of viable cell percentages at the beginning of the bioconversion. On the contrary, lower cultivation temperature
negatively affected the growth.
Regarding the duration of 3-HP production, some interesting factors were pointed out, including the addition
of betaine plus KCl, 1,2-PDO, glucose and phytone-peptone in the growth medium. In contrast, the adjunct of
vitamin B12 that is an essential co-factor for the first enzyme of the bioconversion metabolic pathway showed
a negative effect, indicating that it was not internalized and its biosynthesis by the bacteria was essential.
Besides, glucose showed the negative impact to the molar ratio of 3-HP/1,3-PDO, since the supplemental
NADH produced through glycolysis was re-oxidized to NAD+ preferentially through the glycerol reduction
into 1,3-PDO, at the expense of glycerol oxidation into 3-HP. Finally, the other nutritional and environmental
factors used during growth did not affect significantly the further 3-HP production.
Conclusion: The present study figured out the influence of 11 growth factors on L. reuteri DSM 17938 growth
and ability to produce 3-HP afterwards. The experimental design performed allowed to identify a set of growth
conditions that lead to a 3-HP production enhanced by 20% compared to the reference. A second experimental
design will be performed in order to optimize the environmental conditions during the bioconversion step.