| Fermentative hydrogen production via anaerobic mixed culture is considered to possess agood industrial application prospect and has now become one of the highlights in biofuelresearches. However, the microbial diversity of anaerobic mixed culture can result insubstantial heterogeneity in physiological and biochemical behaviors, which can cause “caskeffect” during hydrogen production and thereby restrict the overall production capacity. Theeffect becomes more apparent under high organic loading condition. Hence, directionalhydrogen production via mixed culture can be enhanced if the heterogeneity can beeffectively restrained and controlled, which would significantly help to promote bioenergyconversion efficiency during fermentative hydrogen production. In this dissertation, anaerobicsludge was used as mixed culture for hydrogen production. Synergistic selection pressure andcascade adaptive evolution strategy were used to restrain and regulate microbial heterogeneityby purposively optimizing the community structure and improving the performance offunctional species. By the adaptive evolution, enhanced hydrogen production capacity couldbe achieved under high organic loading condition. Furthermore, evolutionary mechanisms onthe holistic mixed culture were multidimensionally revealed. The main results were describedas follows:(1) The activity of hydrogen production was more susceptible to acid stress thanmetabolic activity, and butyric acid stress could cause a more severe inhibition to fermentativehydrogen production than acetic acid stress. The combined effect of three metabolic inhibitorsincluding chloroform,4-methylpyrazole, and oxamate could effectively restrict microbialheterogeneity and enhance hydrogen production. The hydrogen yield reached2.3mol/molwith the addition of0.1%chloroform,8mM4-methylpyrazole, and15mM oxamate, whilethe yield was only1.48mol/mol without the addition. Butyric acid stress combined with theselective chemical inhibitors was used as the selection pressure to evolve mixed culture inbatch operation. Acid tolerance and directional hydrogen production could be enhanced by theadaptive evolution. The hydrogen yields of evolved culture and original culture without acidstress were2.06mol/mol and1.48mol/mol respectively, and the substrate degradation rateswere both above99%. Moreover, when cultured in the presence of250mM butyric acid stress,the hydrogen yield and substrate degradation rate of original culture were only0.42mol/moland58.6%, while that of evolved culture reached98%and1.67mol/mol. The above resultsindicated the availability of the combined selection pressure. However, the adaptive evolutionin a batch mode was proved to be unstable and halfway based on the fermentationperformance analysis via evolved culture.(2) The cascade adaptive evolution strategy in a continuous mode could effectivelyrestrain the metabolic heterogeneity, and evolved culture obtained a stable and enhancedcapacity of directional hydrogen production. By investigating the fermentation performancevia mixed culture obtained from different evolution phases, the results demonstrated that the hydrogen yield of original culture was just1.47mol/mol, and the yield was1.63mol/mol bymixed culture from the chloroform acclimation phase. The mixed culture from acid adaptiveevolution phase had a higher hydrogen yield of1.96mol/mol and a lower yield of ethanol(0.05mol/mol), butanol (0.01mol/mol) and lactic acid (0.03mol/mol). During thefermentation process via evolved culture from the evolutionary phase with combinedselection pressure, the highest yield of hydrogen (2.19mol/mol), acetic acid (0.58mol/mol)and butyric acid (0.55mol/mol) was obtained, while ethanol yield and lactic acid yield wereonly0.02and0.01mol/mol respectively. By analyzing enzymatic acid tolerance responses(ATR) systems, it was found that the specific activity of hydrogenase and H+-ATPaseexhibited a similar variation trend with the specific hydrogen production rate and specific acidproduction rate. In addition, the ATR dynamics indicated that hydrogen-producingmicroorganisms could prepare themselves against self-produced acid stress, and the capacitywas more obvious in evolved culture. Based on the functional analysis of glutamic aciddecarboxylase system (GAD), the glutamic acid addition had little effect on hydrogen yield,but could further improve hydrogen productivity.(3) It was found the performance of evolved culture was more stable than that of originalculture under different organic loading conditions. Moreover, the two mixed culturesexhibited distinct metabolic characteristics in their respective variation ranges, whichindicated the qualitative change in the holistic physiological and biochemical properties. Theenzymatic differences in related metabolism were further analyzed. Compared to originalculture, it was found that the specific enzymatic activity involved in solvent-producingpathway was relatively low, while that involved in directional hydrogen-producing pathwaywas relatively high in evolved culture. In addition, the specific H+-ATPase activity in was alsohigher in evolved culture. Furthermore, the enzymatic response to pH and butyric acid stresswas more stable in evolved culture. The comparisons could reflect the enzymaticheterogeneity differences between the two cultures.(4) The differences between original culture and evolved culture at microbial structureand hydA expression level were investigated. The clustering analysis demonstrated that themicrobial structure in evolved culture was more stable than that in original culture as theorganic loading rate was increased. The ecology index calculation based on DGGE fingerprintindicated that the species richness in evolved culture was obviously lower than that in originalculture, while the species evenness of evolved culture was higher. The ecology index couldfurther quantify the heterogeneity differences between the two cultures at the level ofmicrobial community structure. The sequencing results of DGGE bands indicated that thedominant species had been changed between the two cultures. The results of fluorogenicquantitative PCR demonstrated that hydA gene copies at DNA level were similar between thetwo mixed cultures, but the hydA expression of evolved culture was higher than that oforiginal culture. (5) As organic loading rate was increased, AcetylCoA/CoA ratio, ATP andNADH+NAD+concentration were stable in both mixed cultures, and evolved culture had aslightly higher ATP content. High organic loading rate could improve intracellularButyrylCoA/CoA and NADH/NAD+. The increasing tendency of NADH/NAD+was moreobvious in original culture, while that of ButyrylCoA/CoA was more remarkable in evolvedculture. The specific activity of Ferredoxin-NAD+reductase increased obviously in originalculture, while that of evolved culture was relative stable as the increased organic loading rate.In addition, the specific activity of NADH-ferredoxin reductase increased in both cultures, butthe tendency was more remarkable in evolved culture. |
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