| The global earth is under the status of accelerating depletion of oil resources, rising price of crude oil and enhancing greenhouse gases, resulting from the over-consumption of petroleum-derived transportation fuels.“Low Carbon Economy”is further emphasized and implemented all over the world, together with strong preference on green bio-based products by consumers. It is thus increasing interest to produce bioethanol from fermentation with biomass resources. Thermophilic anaerobic bacteria are of applied interest as the desirable biological catalyst for the conversion of cellulosic biomass to ethanol. In the present study, to further improve the fermentation characteristics of Thermoanaerobacterium aotearoense, the lactate dehydrogenase in a key branch of T.aotearoense was deleted via the homologous recombination, and the thermophilic and anaerobic metabolic engineered T.aotearoense, namelyΔldh mutant. Attempts to improve ethanol fermentation in terms of its final product concentration were carried out byΔldh mutant immobilized in a fibrous bed bioreactor, with waste biomasses such as cassava pulp and bagasse. The ethanol demestication ofΔldh mutant was conducted by intimidating, adding increasing ethanol to the culture environment, and thus the ethanol fermentability ofΔldh mutant was improved.The homologous primers of upstream fragment and downstream fragment were designed based on the lactate dehydrogenase (ldh) sequence of JW/SL-YS485-AY278026, thus the homologous ldh-up and ldh-down were successfully amplified with T.aotearoense genomic DNA as template. The target vector was finally constructed based on pBLUESCRIPT II SK(+) with erythromycin selection marker flanked by ldh-up and ldh-down fragments, the heat-resistant erythromycin gene were chemically synthesized as the selection marker. Electroporation was used to transform T.aotearoense whose cell wall had been weakened by isoniazid, subsequently screened and verified respectively by PCR, Southern blot and fermentation product analysis. The expected size was observed both in the results of PCR and Southern blot, furthermore, no lactic acid was detected in the flask fermentation. All results above suggested that the chromosomal ldh gene in theΔldh mutant had been replaced with the disrupted gene carrying erythromycin resistance.Except for sugar,Δldh mutant can metabolise most of the carbon sources in the biomass, such as: glucose, xylose, cellobiose, fructose, mannose, starch and so on. In the flask fermentation mode, no obvious substrate inhibition was observed under 60 g/L carbon source, several basilic parameters on ethanol fermentation ofΔldh mutant have been explored, the calculated fermentation condition as follows: temperature 45-60℃, pH 5.5-7.5, 3 g/L yeast extract, 1 g/L L-cysteine hydrochloride monohydrate, 0 g/L dihydrochloride Pyridoxamine, initial sparging N2 pressure 0.02 Mpa and inoculum10%. Further research on the ethanol fermentation of T.aotearoense wild type andΔldh mutant affected by the potassium acetate, under the flask fermentation mode, glucose and xylose respectively as the substrates, for T.aotearoense wild type, compared with no adding potassium acetate, the cell dry weight increased by 10.9%~15.98%, ethanol concentration decreased by 39.86%~51.51%, lactic acid concentration increased by 48.61%~91.39%, when adding 100 mmol/L potassium acetate; For T.aotearoenseΔldh mutant, compared with no adding potassium acetate, the cell dry weight increased by 3.59%~6.12%, ethanol concentration increased by 5.62%~12.63%, no lactic acid concentration was detected, when adding 70 mmol/L potassium acetate. In the intermittent fed-batch fermentation mode, that 5 L agitator tank for free cells, glucose, xylose and glucose/xylose(2:1) respectively as carbon source, the final ethanol concentration for wild type were 7.04~7.98 g/L and a yield of 0.11~0.12, the final lactic acid concentration for wild type were 30.91~36.39 g/L; while the final ethanol concentration forΔldh mutant were 17.78~25.93 g/L, with small amount of lactic acid 2.02~2.26 g/L were detected.Ultrafine grinding bagasse should be enzymatic hydrolyzed by cellulase for ethanol production. Under the conditions of grinding time 40 min, ammonia soaked 36 h, enzyme concentration 55 FPU/g and hydrolyzed 36 h, the yield rate of glucose and xylose were respectively 71.93±1.43% and 58.46±1.28%. On the process of separate hydrolysis and fermentation (SHF) forΔldh mutant, the suitable condition as follows: enzyme concentration 50 FPU/g, bagasse concentration 4%, the concentrations were 10.85±0.12 g/L for glucose and 5.264±0.098 g/L for xylose, with a yield rate of 71.98±1.13% for glucose and 59.82±1.21% for xylose, in the 50 mL flask fermentation mode, the glucose and xylose in the hydrolysate were reduced to zero within 24 h, and the ethanol and acetic acid concentration were 4.65±0.098 g/L and 3.023±0.12 g/L, respectively. On the process of simultaneous saccharification and co-fermentation, the suitable condition were as follows: substrate concentration 4%, enzyme concentration 50 FPU/g, temperature 48℃, pH 6, and the final ethanol concentration was 4.38±0.14 g/L with a yield of 0.11~0.12 g/g (ethanol/bagasse).Cassava starch and cassava pulp were hydrolyzed by dilute acid, and the glucose yield for cassava starch is 90.7%, the total reducing sugar yield were 54.86% for cassava pulp. In the intermittent fed-batch FBB fermentation forΔldh mutant, glucose and xylose respectively as substrate, the final ethanol concentration were 38.26 g/L for glucose and 34.13 g/L for xylose, with a yield of 0.364 g/g for glucose and 0.342 for xylose. The hydrolysates of cassava starch and cassava pulp were used as substrate, the final ethanol concentration were 35.82 g/L for cassava starch hydrolysate and 39.09 g/L for cassava pulp hydrolysate, with a yield of 0.325 g/g (ethanol/cassava starch) and 0.123 g/g (ethanol/ cassava pulp).After continuing 153 times transformation for 123 days, the ethanol tolerance ofΔldh mutant was increased from 2.5%(v/v) to 5.0%(v/v) by long term adapting in progressively increased ethanol concentration. The demestication strain was designated as T.aotearoense ET6. Under 30 g/L ethanol concentration, the specific growth rate and ethanol productivity for ET6 still maintained in 69.44% and 50% respectively, however, the growth of parentΔldh mutant was totally inhibited. In the intermittent fed-batch FBB fermentation for ET6, glucose as the sole substrate, the ethanol concentration reached 47.25 g/L with a yield of 0.344 g/g (ethanol/glucose). |
