| This dissertation studied the law of batch and continuous fermentations by free Pichia stipitis on mid-experiment scale.Three-stage continuous fermentation by immobilized yeasts using calcium alginate as carrier and the low-pH treatment technology for prevent bacteria pollution was investigated.The major results are as follows:The experiment studied the sugar metabolism of Pichia stipitis with 60g/L xylose and 60g/L glucose/ xylose mixture(the ratio of glucose to xylose is 2:1)as carbon resource, respectively.The speed of Pichia stipitis metabolizing mix-sugar was faster than that of xylose,with total residual sugar concentration 5.22g/L when fermentation time reaching 20h. During mix-sugar fermentation,glucose was foremost utilized at all time,and it exhausts t after 12h.Single-stage continuous fermentation by free Pichia stipitis with glucose/xylose mixture as substrate was investigated.The result showed that at suitable dilution rate 0.04～0.06h-1, utilization rate of sugar reaching 75.51～87.38%,with the ratio of ethanol yield to theoretical yield about75%and ethanol productivity 0.586～0.754g/L.h.With the influent sugar concentration increasing,the residual sugar concentration had been continuously increasing,but ethanol concentration only increased to a certain extent and the yeasts concentration had no obvious change.The suitable influent sugar concentration was 45g/L at dilution rate 0.06h-1. The initial yeasts concentration had no effects on continuous fermentation at steady-stage.A single-stage continuous fermentation at two different dilution rates was investigated.The result showed that continuous fermentation attained "steady-stage "after about 40h with various parameters keeping stable for about 104h.Free cells of Pichia stipitis were cultured in the three-stage continuous reactors with 45g/L glucose/xylose mixture as substrate.The influent substrate concentration was 350ml/h and 420ml/h,respectively.During the first steady state(15～20day),35.00g/L sugar was fermented in the first reactor,while 4.81g/L sugar in the second reactor and 2.84g/L sugar in the third reactor respectively was converted into ethanol.The average total utilization rate of sugar was 94.78%,with the average total ratio of ethanol yield to theoretical yield 78.46%by the three-stage continuous fermentations.During the Second steady state respectively 32.71g/L,4.18g/L and 3.41g/L sugar was fermented in the three rectors,and the average total utilization rate of sugar was 89.56%,with the average total ratio of ethanol yield to theoretical yield 76.00%by three-stage continuous fermentations.Fermentation of 60g/L glucose/xylose mixture to ethanol with gelatin,K-carrangeenan, chitosan,calcium alginate and PVA as immobilized cell cartier in 10 turns culture was investigated.By 10 turns culture the ferment capability of immobilized yeasts using calcium alginate and PVA as carrier didn't drop with final residual sugar concentration respectively 0.28g/Land 0.43g/L,and by a few turns of fermentations gelatin and K-carrangeenan gel beads come to crack and ferment capability of immobilized yeasts greatly went down as yeasts running out.Considering synthetically all kinds of factors,calcium alginate was the best immobilization carrier.With glucose,glucose/xylose mixture and xylose as substrate,compared effects of multiplication culture of immobilized yeasts using calcium alginate as carrier to free yeasts. The results showed that whether free yeasts or immobilized yeasts,the utilization rate of three substrates was glucose＞mix-sugar＞xylose,and the final yeasts concentration was glucose≈mix-sugar＞xylose,moreover,the utilization rate of sugar of immobilized yeasts was faster than free yeasts.And the utilization rate of mix-sugar of immobilized yeasts was higher with final residual sugar concentration 3.11g/L and ethanol concentration 12.46g/L.Owing to the envelopment of gel,the resistance of immobilized yeasts to inhibitors and environment factor was stronger than free yeasts.Whether immobilized yeasts or free yeasts, the critical inhibitors concentration was methyl acid 0.4g/L,acetic acid 0.4g/L,lactic acid 1.5g/L,ethanol 4.0g/L,respectively.On the other hand,immobilized yeasts have better capability of sugar infiltration-resistance and heat-resistance compared to free yeast.When initial sugar concentration exceeded 60g/L,after 36h fermentation,the ethanol concentration in ferment solution of immobilized yeasts was higher than free yeasts.When temperature was 50℃,3.75g/L ethanol was produced by immobilized yeasts fermentation,however only 0.07g/L ethanol by free yeasts fermentation.Single-stage continuous fermentation respectively at two dilution rates and two influent sugar concentrations by immobilized yeasts using calcium alginate as carrier in 7L reactor was investigated.Because few yeasts lapse with effluent,when dilution rate increased from 0.04h-1to 60.0g/L,the ferment index didn't drop but ethanol productivity went up from 0.647g/L·h to 0.837g/L·h.When influent sugar concentration increased from 45.0g/L to 60.0g/L ethanol concentration and ethanol productivity went up from 14.13g/L and 0.868g/L·h to 17.20g/L and 1.032g/L·h,respectively.Batch fermentation of 60.0g/L glucose/xylose mixture by free and immobilized yeasts carried out for 29 turns.During the 29 turns fermentation,the yeasts had been treated with different pH acid solution three times.The results showed that low-pH treatment injured free and yeasts to some extent,but the yeasts could entirety adapt to environment by several turns fermentation and several treatments.After four treatments utilization the rate of sugar of free yeasts hadn't been dropped.Because immobilized yeasts' adaptability to low-pH treatment was stronger than that of free yeasts,the treatment of pH3.6 couldn't bring ferment capability of immobilized yeasts down,moreover,immobilized yeasts could adapt to environment of pH 2.4～3.2 after three treatment.The treatment of pH2.4～2.8 was appropriate in laboratory.Free cells of P.stipitis were cultured in the three-stage continuous reactors using glucose/xylose mixture of 45.0 g/L as the substrate with influent sugar speed 280ml/h.During the 24-days cultivation,the yeasts had been treated with pH 2.8 acid solution three times. The results showed that the influence of low-pH treatment had been falling with stage increasing,and that the treatment had little influence on free yeasts after three treatments and several days' fermentation.Finally the three reactor' average ethanol concentration and ethanol yield were respectively 13.73g/L,15.67g/L,16.75g/L and 78.34%,80.14%,81.94% which almost reached the level of pre-treatment.That was feasible to put the low-pH treatment into practice.Immobilized cells of P.stipitis were cultured in the three-stage continuous reactors using glucose/xylose mixture of 45.0 g/L as the substrate with influent sugar speed 420ml/h.During the 24-day cultivation,the yeasts had been treated with pH 2.8 acid solution for three times. The results showed that the low-pH treatment's influence on immobilized yeasts was slighter than on free yeasts in the continuous fermentations,furthermore,the influence had been falling with stage increasing.The residual sugar concentration raised 9.81g/L and the utilization rate of sugar,ethanol concentration and ethanol yield decreased 21.80%,6.89g/Land 23.72% respectively in the first reactor by the first treatment.At the same time the ferment index in the second and third reactors changed less than in the first reactor.In addition the ferment index in the first reactor by the second treatment changed slighter than by the first treatment. And due to no treatment,the ferment index changed slighter in the second and the third reactor. Because the immobilized yeasts already had adapted to low-pH,the third treatment all but had nothing influence on the three reactors' ferment index which fluctuated from the substrate's variation himself.
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