| One-carbon gas such as CO or CO2,is an abundant carbon resource on the earth.A diverse range of one-carbon gas is derived from gas emissions of industries as well as syngas generated from gasification of carbonaceous materials such as industrial,agricultural residues and municipal wastes.The resource utilization of one-carbon gas can relieve the current energy shortage and environmental crisis.Gas-fermenting microbes can fix carbon through chemoautotrophy to convert one-carbon gas into bulk chemicals and biofuels,which has promising application.However,its current gas-conversion and product-production efficiency cannot meet the requirements of industrial fermentation.Clostridium carboxidivorans P7 is one of the minor gas-fermenting microbes capable of synthesizing higher-alcohols(butanol and hexanol)using one-carbon gas.This study aimed at realizing resource utilization of off-gas from steel mill using this strain.Therein,the basic physiological and metabolic characteristics of C.carboxidivorans P7 were systematically investigated.Then,the fermentation conditions were optimized for improving syngas conversion efficiency into alcohols and meanwhile,related metabolic mechanisms were explored.Furthermore,a platform for syngas conversion based on a 5 L stirred tank reactor was established to develop continuous syngas-fed fermentation technology.This study provided theoretical basis for improving the conversion efficiency of off-gas from steel mill by C.carboxidivorans P7 and promoting its industrial development,leading to a novel route for gas-pollution solution and biofuel production.The main contents of this study are as followsFirst,this study investigated the nutrition types of C.carboxidivorans P7.The results showed that under the autotrophic condition of syngas(CO:CO2:H2:Ar=56:20:9:15)that simulated off-gas from steel mill,C.carboxidivorans P7 utilized CO as the main substrate and produced ethanol and acetate;while under the heterotrophic condition of 2.0 g/L glucose,the culture density and product-carbon concentration only reached to 67.2%and 48.6%of that under the autotrophic condition,respectively;however,under the mixotrophic condition of syngas and glucose,P7 only consumed glucose.It indicated that the presence of organic carbon sources may inhibit syngas utilization.In order to enhance its capability to synthesize alcohols,a three-step statistical strategy including the Plackett-Burman design,path of steepest ascent design and Box-Behnken design was applied to optimize trace metal composition in the medium.The results showed that based on the trace metal composition of the standard medium,the optimum concentration was found to be 0.55-fold MoO42+,3.48-fold Cu2+and additional 44.32 mM Fe3+,with which the production of alcohol and acid changed to 4.40 and 0.50 g/L from 2.16 and 2.37 g/L,respectively,indicating an increased ratio of 92.0%(alcohol-carbon to product-carbon)from 54.2%,which greatly improved the alcohol production efficiency of this strain.Next,this study systematically explored effect of temperature(25-37?)during syngas fermentation by C.carboxidivorans P7.It was found incubation at 33 or 37? stimulated rapid growth while resulted in bacterial agglomeration and low higher-alcohol titer.Although the agglomeration could be avoided at 29 or 25?,low growth rate caused low density culture.This study proposed a two-step temperature culture pattern of 37?(0-24 h)-25?(24-144 h),which was proved to effectively overcome bacterial agglomeration and improve alcohol production.Using it,ethanol,butanol and hexanol titer were up to 3.97,1.67 and 1.33 g/L,respectively,which was the highest alcohol production currently reported in bottle fermentation.In addition,through screening of eight surfactants,0.1%(w/w)saponin or Tween 80 were found to alleviate bacterial agglomeration at 37?,and then prolong effective fermentation time,leading to increase culture density and product concentration.Nevertheless,the two-step temperature culture was more effective for higher-alcohol production than the anti-agglomerating action from surfactants.Comparative transcriptomics analyzed the physiological responses of P7 in the early and late stages of growth at 37?,25? and 37-25?,respectively.The result revealed that temperature mainly affected carbohydrate metabolism,energy metabolism and amino acid metabolism;furthermore,the genes related to the Wood-Ljungdalii pathway preferred to be transcribed at 37?,while the genes encoding a series of catalytic enzymes responsible for the acyl-condensation reactions tended to be expressed highly at 25?.Then,this study investigated effect of nine nitrogen sources during syngas fermentation by C.carboxidivorans P7.The result found that P7 could not only utilize rich organic nitrogen,but also simple inorganic nitrogen;organic nitrogen sources composed of protein hydrolysates and rich micronutrients were more conducive for syngas fermentation.However,only yeast extract(YE)significantly facilitated higher-alcohol production;although ammonium supported the growth,it caused a longer lag period and little higher-alcohol in products.In addition,the result of RT-qPCR revealed that the expression of the main genes related to higher-alcohol production in the later culture stage when using YE was 3.3-8.4 times of those when using ammonium.A complete synthetic medium based on 2.0 g/L ammonium sulfate was developed that supported P7 produced higher-alcohol up to the fermentation level when it utilized YE,by doubling concentrations of the optimized trace metal combination,the original mineral element combination and vitamin combination of the standard medium.Finally,based on a lab-scale 5 L continuous stirred tank reactor,real-time monitoring of important physiological parameters such as pH.oxidation-reduction potential(ORP),carbon monoxide uptake rate(COUR)and carbon dioxide evolution rate(CER)in the entire fermentation process was achieved through an online control system and an online gas mass spectrometer.Combined with offline physiological data,it was determined that pH and ORP could be used as indicators of fermentation phases and cell viability,respectively.After tracking the continuous syngas-fed fermentation process,there were these findings:when initial ORP was below-273 mV.the fermentation performance was relatively stable;however,overpressure(0.03-0.10 MPa)led to a negative impact on the bacteria that inhibited cell growth;and it was necessary that a small amount of glucose was introduced into fermentation medium to promote cell recovery after inoculation and then stabilize ORP in the initial stage of fermentation for subsequent autotrophic fermentation.The basic process of continuous syngas-fed fermentation by C.carboxidivorans P7 was established based on the above findings.Then,pH control in the fermentation process demonstrated that pH 5.6 was helpful for cell growth and product accumulation,while pH 5.2 promoted conversion of acids into alcohols,but harmful to cell growth.Volumetric gas-mass transfer coefficients(kLa)were determined by the sodium sulfite steady-state method at different stirring speeds under low aeration ratios.Meanwhile,biological experiments demonstrated that the physiological state of cells was also affected by stirring speeds.Finally,two high-efficiency fermentation processes based on online parameters were established:1)YE fed-batch fermentation process directed by online pH,in which once pH value was in uptrend,YE solution was fed into reactor at 0.5 g/L broth every time,this process with three feedings significantly increased biomass,ethanol,butanol and hexanol concentrations up to 0.96,4.36,1.87 and 0.77 g/L,respectively;2)variable stirring-speed fermentation process directed by online COUR,in which stirring speed was adjusted to meet bacterial need in growth stage according to COUR,and controlled at low in the early stage to promote cell recovery and in the late stage to reduce cell damage by shearing force,this process achieved biomass,ethanol,butanol and acetate concentrations up to 0.93,3.70,0.93 and 1.74 g/L,respectively. |
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