Clostridium tyrobutyricum

Abstract

The objective of this thesis was to figure out the metabolism of Clostridium tyrobutyricum and evaluate the production of hydrogen in a continuous-flow stirred tank reactor (CSTR) as a function of hydraulic retention time (HRT). The influent substrates were 20,000 mg/L glucose and peptone (3:2(w/w)), temperature was at 35℃, and pH was controlled under 6±0.1 by computer. The reactor was operated at different hydraulic retention time (HRT = 18, 12, 8, 6, 4, 3, 2, 1.5, 1 hr), and collected gas production and water quality data under steady state for the nine runs. The concentrations of biomass were around 2700±400 mg/L at HRT = 18-1.5 hrs, but C. tyrobutyricum was washed out at HRT = 1hr. Bacterial flocculation was occurred at HRT = 3, 2, 1.5 hrs. Biomass flocculation increased biomass concentrations and made concentrations of effluent glucose stay under 300 mg/L at lower HRTs. Effluent organic nitrogen concentrations increased but effluent ammonia concentration decreased with decreasing HRT, because microorganism had enough time for degrading peptone when HRT was high. While the reactor was controlled at higher HRTs, peptone was used for biomass synthesis and fermentation. On the other hand, peptone was used for biomass synthesis mainly at lower HRTs. Maximum hydrogen production rate, maximum specific hydrogen production rate and maximum hydrogen yield were 416.61 mmol H2/L/d, 8.92 L H2/g-VSS/d, and 3.47 mmol H2/g-CODapplied occurred at HRT = 4 hr. Moreover, experimental hydrogen production rate of HRT = 4 hr was also closest to theoretical hydrogen production rate among all the nine runs. Maximum carbon dioxide production rate was 292 mmol CO2/L-day occurred at HRT = 6 hr. In addition, hydrogen production rate decreased when HRTs were reduced lower than 4 hr, but biomass concentrations were stable, so decreased hydrogen production rates were attributed to metabolic pathway shifting. The compositions of effluent organic acids were mainly butyrate and acetate at higher HRTs, but metabolic pathway shifted to lactate production at lower HRTs. LDH will be catalyzed by high NADH/NAD+ condition (Garrigues et al., 1997), and inhibited by NAD+ (Fitzgerald et al., 1992). When the reactor was controlled at lower HRTs, specific growth rates of microorganism were increased, and then required energy of microorganism was high, so NADH was produced faster. In addition, effluent butyrate concentrations decreased, maybe because of lower PTB activity. These two reasons caused accumulation of NADH and pyruvate in microorganism, LDH was activated, and then lactate was produced at lower HRTs. Finally, metabolic flux analysis was studied. After using CellNetAnalyzer calculated the rates of metabolic pathways for eight runs, nodal analysis was applied. For nodal analysis of pyruvate, the pathway for producing acetyl-CoA with hydrogen production decreased when HRT decreased. On the other hand, the pathway for lactate production increased when HRT decreased, maybe because of high LDH activity. For nodal analysis of acetyl-CoA, the activities of PTB and PK were higher than PTA and AK, and vice versa.