Dualistic acidic and neutral glucose fermentation balance in small intestine: Simulation in vitro  Elias Hakalehto, Tarmo Humppi, Heikki Paakkanen  Pathophysiology  Volume 15, Issue 4, Pages 211-220 (December 2008) DOI: 10.1016/j.pathophys.2008.07.001 Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Mixed aerobic cultivation of Escherichia coli (E. c.) and K. mobilis (K. m.) sp. strains at 37°C in the PMEU. The inoculated cell numbers were about 5×10E6 for E. coli and for K. mobilis about 1×10E6 (in panel cultures 1, 3 and 5) and about 5×10E5 (panel cultures 2,4 and 6) per ml of the medium. The initial pH of the liquid medium was 6.8 in 1–4 and 6.14 in 5 and 6. Ethanol (1%) was added to cultures 3–6. Pathophysiology 2008 15, 211-220DOI: (10.1016/j.pathophys.2008.07.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The presence of 2,3-butanediol isomers 1 and 2 during the 18h cultivation in aerobic and microaerobic (M) conditions. The cultures with samples taken at 0, 4, 7 and 18h were grown aerobically in the PMEU (air flow 30% of the pump capacity), whereas the microaerobic cultures designated with “M” were grown in pressurized gas flow containing 5% O2, 10% CO2, and 85% N2. All samples were cultivated at 37°C. Starting the K. mobilis cultures both isomers were found, but isomer 2 disappeared soon while isomer 2 was found 7h in aerobic and 18h in microaerobic cultures. The isomer 2 was produced earlier and more than isomer 1 in K. pneumoniae cultures. In microaerobic cultures more 2,3-butanediol was found than in aerobic conditions. Pathophysiology 2008 15, 211-220DOI: (10.1016/j.pathophys.2008.07.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 GC–MS mass chromatograms from the extract of a microbial culture (A) and 2,3-butanediol reference substance (B). Pathophysiology 2008 15, 211-220DOI: (10.1016/j.pathophys.2008.07.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 Gas phase ethanol in nitrogen flow of anaerobic bacterial cultures at 37°C in PMEU. The ethanol was recorded by ion mobility spectrometry. Two chempro units correspond the evaporation effect caused by 0.5% of ethanol in the Brain Heart Infusion (BHI) medium used for cultivations. The baseline signal from sterile medium is shown here (BHI medium). Two pure cultures of both E. coli and K. mobilis (Klebs) were started and the mixed cultures were inoculated by a second bacterium at 5h to one of the cultures of each strain. The cell concentration of the second bacterium vs. the first strain was 1:10. Ethanol production was enhanced to some degree in original E. coli culture by K. mobilis introduction and more clearly when E. coli was added to K. mobilis culture. This time original glucose had been practically consumed. However the “newcomer” bacteria were neither competed out nor did they influence the growth of the original population (results not shown here). These two facultatively anaerobic bacteria were thus able to sustain each other which was possibly due to their positive metabolic interactions. One such interaction was the prolonged ethanol production in the mixed cultures. Then most likely the klebsiellas were principal producers utilizing the products of the mixed acid fermentation by E. coli. Pathophysiology 2008 15, 211-220DOI: (10.1016/j.pathophys.2008.07.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Maintenance of dualistic fermentation balance in small intestine simulated in PMEU. The mixed acid fermentation pattern is accomplished by E. coli, and 2,3-butanediol and ethanol fermenting bacteria are representated by K. mobilis. Pathophysiology 2008 15, 211-220DOI: (10.1016/j.pathophys.2008.07.001) Copyright © 2008 Elsevier Ireland Ltd Terms and Conditions