| Submerged vinegar fermentation was initiated domestically in the1980s, and the annualcapacity had reached to1million tons (with acidity60g L-1) in2008. Compared with theadvanced vinegar enterprises abroad, both the overall efficiency and the yield of vinegarfermentation in domestic enterprises are lower due to the lack of in-depth researches on strain,fermentation process and equipment for vinegar production. Especially, there was less reporton taxonomical identification of industrial Acetobacter, strain improvement, fermentationkinetics assay, and acetic acid resistant mechanism. This work performed is involved withdetailed analysis of an industrial strain of acetic acid bacteria (AAB). Combined thefermentation kinetics assay with alcohol related metabolism research, optimal fermentationprocess was readjusted based on the metabolic characteristics of bacterium. Then, high acidityvinegar fermentation was carried out by a high yield mutant in repeated batch mode.Simultaneously, bacterial energy metabolism regulation was confirmed and a coordinationmechanism of acetic acid resistancy was initially proposed. The main results were as follows:(1) Fermentation optimization based on fermentation kinetics study of Acetobacter sp.(Hu Niang1.01)Through the analysis of16s rRNA gene sequences homology and biochemicalcharacteristics, the industrial strain (Hu Niang1.01) was identified as one of the subspecies ofAcetobacter pasteurianus and named as CICIM B7003. For this strain, the threshold growthinhibition concentration of initial acetic acid and alcohol were40and48g L-1, respectively.To ensure repeated batch fermentation runs smoothly, the optimal discharge volume was set at43%of total working volume. With the average acetification rate of1.80±0.01g L-1h-1andthe conversion rate of acetic acid from alcohol of90.73%, repeated batch cultivation showedhigher fermentation indexes than the batch manner. Then, a new kinetic parameter,stoichiometric coefficient of oxygen consumption versus acetic acid yield, was introduced toevaluate oxygen utilization efficiency. With this kinetic parameter the optimal aeration ratewas calculated as43L h-1and employed for acetic acid fermentation, the conversion rate wasfinally raised to a level of93.36%.(2) Promoting acetification via strengthening alcohol respiratory runningIt was revealed that the type of ubiquinone of A. pasteurianus CICIM B7003was Q9.Through gene sequences homology analysis, genes encoding alcohol dehydrogenase,aldehyde dehydrogenase and terminal oxidase in alcohol respiratory chain of A. pasteurianusCICIM B7003had revealed high homology with those of A. pasteurianus IFO3283-01. Thus,the scheme for machinery of alcohol respiratory system in strain CICIM B7003was proposedwith reference to strain IFO3283-01. During repeated batch fermentation, the acidificationrate showed positive correlation to alcohol respiratory chain enzymes activity. Finally,acetification process was promoted obviously via adding ubiquinone-9precursors and somespecific ions, and adjusting aeration rate. The average acetification rate was enhanced to2.29±0.02g L-1h-1, which was27.8%higher than the original level.(3) Ultraviolet (UV) Mutation aided with acid shock to obtain a strain of higher vinegar yieldA. pasteurianus CICIM B7003was treated with sublethal doses of acetic acid (50g L-1) for1h to induce homologous resistance before mutagenesis, which increased cellular adaptabilityto subsequent acidic mutation environment. Enrichment culture was carried out after acidstress, and then UV mutation was performed on enriched cells under lethal acidic shock (60g L-1acetic acid). Finally, a mutant strain was screened for its higher vinegar yield and namedas CICIM B7003-02. The mutant tolerates the initial acidity level of60g L-1and accumulates103g L-1acetic acid in flask cultivation. Also it displays stable fermentation ability in12generations of subculture. Moreover, high acidity vinegar fermentation (93g L-1acetic acid)by the mutant was performed successfully in repeated batch measure with a two-stageaeration strategy in Frings9L acetator. As a result, average acetification rate of1.83±0.01g L-1h-1and conversion rate of93.97±0.16%were obtained.(4) Relationship of bacterial energy metabolic character and acetification capacityIt’s was revealed that along with acetic acid accumulation the percentage of capsularpolysaccharide versus dry cells weight was reduced from2.5%to0.89%, and the ratio ofunsaturated fatty acids to saturated fatty acids in membrane was increased from21.57%to41.79%. Also, transcription of acid resistance genes was promoted, which was related to theacidic toleration. Interestingly, alcohol respiratory chain enzymes and ATPase activities werealso promoted obviously with acetic acid accumulation, which seemed provide adequateenergy for acid resistance. Then, the bacterial energy metabolism was further characterizedunder high acidic vinegar fermentation. Malic/succinic acid feedback pathway is the majorroute for energy supply at the early stage of fermentation, and alcohol respiratory chain playsthe major role in providing energy to cells for the mid period. Both malic/succinic acidfeedback pathway and the alcohol respiratory chain supply energy when fermentation runningto final stage. Finally, through the analysis of response of cellular morphology, physiology,gene translation and energy metabolism of strain CICIM B7003-02(the mutant) duringvinegar fermentation, a coordination mechanism was proposed in detail to explain how A.pasteurianus CICIM B7003-02is resistance to higher acidify and become better producer. |
PDF Full Text Request