Microbially-produced folate forms support the growth of Roseburia intestinalis but not its competitive fitness in fecal batch fermentations

Folate (vitamin B9) occurs naturally mainly as tetrahydrofolate (THF), methyl-tetrahydrofolate (M-THF), and formyl-tetrahydrofolate (F-THF), and as dietary synthetic form (folic acid). While folate auxotrophy and prototrophy are known for several gut microbes, the specific folate forms produced by gut prototrophs and their impact on gut auxotrophs and microbiota remain unexplored.

Here, we quantified by UHPLC-FL/UV folate produced by six predicted gut prototrophs (Marvinbryantia formatexigens DSM 14469, Blautia hydrogenotrophica 10507 ^T, Blautia producta DSM 14466, Bacteroides caccae DSM 19024, Bacteroides ovatus DSM 1896, and Bacteroides thetaiotaomicron DSM 2079 ^T) and investigated the impact of different folate forms and doses (50 and 200 µg/l) on the growth and metabolism of the gut auxotroph Roseburia intestinalis in pure cultures and during fecal anaerobic batch fermentations (48 h, 37 °C) of five healthy adults.

Our results confirmed the production of folate by all six gut strains, in the range from 15.3 ng/ml to 205.4 ng/ml. Different folate forms were detected, with THF ranging from 12.8 to 41.4 ng/ml and 5-MTHF ranging from 0.2 to 113.3 ng/ml, and being detected in all strains. Natural folate forms, in contrast to folic acid, promoted the growth and metabolism of the auxotroph R. intestinalis L1-82, with dose-dependent effects. During fecal batch fermentations, folate forms at both levels had no detectable effect on total bacteria concentration, on gut community composition and metabolic activity and on Roseburia spp. abundance, compared to the control without folate addition.

Our study demonstrates for the first time in vitro the production of different natural folate forms by predicted gut prototrophs and the stimulation on the growth of the folate auxotrophic butyrate-producing R. intestinalis L1-82. Surprisingly, folate did not impact fecal fermentations. Our data suggest that the dietary folate forms at the tested levels may only have limited effects, if any, on the human gut microbiota in vivo.