Screening Of Inulinase Production Strains And Fermentation For Ethanol Production Performance Comparison

World faces the progressive depletion of the non-renewable fuel resources. At the same time, energy consumption grows at increasing rates. Recent increases in crude oil prices, the limited availability of fossil oil, and environmental concerns have prompted a renewed focus on the production of bioenergy. Jerusalem artichoke (Helianthus tuberosus) is suitable as a biomass crop for ethanol production because it commonly yields around7and potentially up to14tons of carbohydrate per hectare and can be grown on marginal land (eg. in saline and semi-arid areas) because it is salt-and drought-tolerant. Other industrial uses for H. tuberosus are:paper pulp or fuelwood from stems, methane production from various plant parts, acetone-butanol-ethanol production from tubers or the whole plant, and hydroxymethylfurfural production as a basic molecule for the chemical industry.This study first conducted a screening of inulinase production strains. And strains fermentation conditions were studied. Then a best fermentation solution was selected after comparing and analyzing three of fermentation methods. And the expanding fermentation was carried out in order to evaluate the actual effect of ethanol fermentation. The results were shown as follows:(1)112strains of fungi and bacteria which could utilize inulin as the sole carbon source were isolated from the rhizospheric soil planting Jerusalem artichoke in Jiangsu, Shandong, and Daqing. After the screening and determination of enzyme activity,75strains were identified to have the ability of producing inulinase. Fungi in the75strains had the higher inulinase activity. A-6had highest activity and was selected to make further study. After observing strain colony and molecular identification, A-6was identified as Aspergillus fumigatus genus, and was named as Aspergillus fumigatus A-6.(2) The fermentation conditions of Aspergillus fumigatus A-6was optimized though single factor and orthogonal experiments. The highest inulinase activity after the optimal condition for inulinase production could reach13.29U·mL-1. By optimal condition and medium for fermentation, fermentation dynamics of Aspergillus fumigatus A-6inulinase synthesis were studied. The inulinase activity reached the highest level at96h. The inulinase was identified as exonuclease for I/S reached the maximum value of1.43at8d.(3) The enzyme production conditions of recombinant strains Pichia pastoris R32was optimized in order to make its application in industrial production. The enzyme activity of recombinant strain Pichia pastoris R32of the optimal medium reached298.8U-mL-1, increasing by40%after optimization.(4) Recombinant strain Pichia pastoris R32needed methanol as inducers to produce enzyme. So it could not be directly used for fermentation. The first step was hydrolysis of Jerusalem artichoke with the crude enzyme. Then hydrolysate was fermented by fermentative strains in order to produce ethanol from Jerusalem artichoke tuber. Four strains kept in laboratory were inoculated in crude enzyme hydrolysate respectively to make comparison of fermentation performance. For yeast S.cerevisiae, the highest ethanol concentration was59.67g·L-1,ethanol yield rate was0.28,glucose utilization was48.84%. sugar alcohol conversion rate was54.31%; For Kluyveromycess, the highest ethanol concentration was61.88g·L-1,ethanol yield rate0.29,glucose utilization rate was50.65%, sugar alcohol conversion rate was56.32%. Fermentation performance of the two strains has obvious advantages. So S.cerevisiae and Kluyveromyces were selected to carry out the next experiment.(5) Recombinant strain of Pichia pastoris R32, S.cerevisiae and Kluyveromyces were applied in One step fermentation method, two step fermentation and synchronous fermentation method respectively in order to make comparison of their fermentation effect. The synchronous fermentation method with S.cerevisiae and Kluyveromyces had the best performance for its highest ethanol concentration was89.70g·L-1, ethanol yield rate was0.48,glucose utilization was88.69%,sugar alcohol conversion rate was94.00%.(6) Orthogonal experiment was carried out to optimize fermentation conditions, such as initial pH, substrate concentration, nitrogen sources and inoculation. The optimal fermentation conditions for the synchronous fermentation method with S.cerevisiae and Kluyveromyces:Inulin225g·L-1, urea40g-L"1, inoculation15%, pH5. After the expanding cultuer in5L fermenter, actual effect of ethanol fermentation for the synchronous fermentation method was studied. Result shows its ethanol yield rate was0.50, glucose utilization was92.24%, sugar alcohol conversion rate was97.76%.Based on the combination of different strains of ethanol fermentation from Jerusalem artichoke, the most suitable fementation scheme was determined to provide a theoretical basis for for ther industrial application.