摘要
能源问题日趋严峻,迫切需要人们开发替代能源。乙醇以其可再生、绿色无污染等优点受到重视,利用木质纤维素生产燃料乙醇是未来能源发展的一个重要方向,既解决能源危机,又充分利用资源、保护环境。本论文由此立题,进行利用农作物秸秆生产燃料乙醇的研究。
通过预处理、酶糖化条件的选择,高效降解纤维素菌种的筛选,单菌和混合菌共同降解及其发酵乙醇等几个方面,综合研究了现阶段利用木质纤维素生产乙醇的工艺方法。
通过对多种预处理方法的比较,最终确定0.75%稀硫酸,固液比1:20,在130℃下处理30min为最优预处理条件。
随后使用纤维素酶,对预处理后的秸秆进行糖化,并使用酵母进行乙醇发酵,发现乙醇产量与酶的使用量密切相关,确定出酶处理条件为:酶用量15mg/gDS、50℃,150rpm下处理5小时,随后按酵母接种量10%、发酵培养2天,乙醇产率可达0.163g/gDS。
本论文重点是菌种筛选,通过分离筛选,确定出10株降解效果较好的菌种,分别测定羧甲基纤维素酶活(CMCA)和滤纸酶活(FPA),对其纤维素酶活力及相关性质进行测定,最终获得6株降解活性较强的菌种,其酸性纤维素酶活较强,最优产酶时间为6天。采取非等温同时糖化发酵法(NSSF)和同步糖化发酵法(SSF)进行乙醇发酵,发现NSSF的乙醇产率大于SSF,乙醇产率达0.126g/gDS。
为了提高微生物降解秸秆的产糖率及利用秸秆降解物发酵乙醇的产率,进一步研究了现阶段研究较少的多种微生物混合培养降解糖化秸秆,比较酶活力,同样采用NSSF和SSF进行乙醇发酵,发现黑曲霉和康宁木霉混合培养降解秸秆时的纤维素酶活力较高,NSSF发酵乙醇产率可达到0.139g/gDS。
最后对菌种筛选过程中发现的一株能利用纤维素高产红色素的菌种进行初步鉴定,可以作为秸秆利用的一个方向。
Concerns about the soaring cost of fossil fuel and energy security have rekindled the interest in producing the substitute fuels from the renewable sources. There lies great interest of fuel ethanol production from lignocellulose owing to its sustainable and zero-omission characters. It holds a great hope and future to solve energy crisis for its resource saving and environment protecting. From this point of view, conversion of lignocellulose to ethanol is focused on in this thesis.
Comprehensive research in transforming lignocellulose to ethanol had been accomplished basically: ascertainment of optimum conditions of pretreatment and enzymolysis, screening of cellulose-degrading strains with high performance, study on pure cultivation and co-cultivation.
Comparing among the results elaborated the pretreatment condition by autoclaving: 0.75% H_2SO_4, solid-liquid ratio 1:20,130℃and 30min.
Subsequently, those pretreated was saccharified through enzymolysis by cellulase and then fermentation proceeded by inoculating of yeast. Dynamic interaction between the quantity of cellulase and ethanol yield was found. Therefore, the following was considered to fit the cellulase best: quantity of 15mg/gDS, 50℃for 5h and at a speed of 150rpm. And two-day fermentation with 10% inoculation size of yeast was followed achieving the final ethanol yield of 0.163g/gDS.
Screening the most wanted strains is the chief point of this thesis. Afrer primary screening by filter-paper Hutchison solid media and CMC solid culture medium,and further screening by filter-paper inorganic salt culture medium and Congo red culture medium with the CMC-Na as the only carbon source. 10 strains with high performance of degrading ability were picked out. According to their CMCA, FPA and the related properties, 6 strains with strong acidic cellulase activity were obtained finally and their highest enzyme yield time was on the 6th day. Ethanol yield rate of NSSF was up to 0.126g/gDS which was higher than that of SSF.
To improve the sugar yield rate by straw-degrading microorganisms and ethanol yield rate via fermentation, further research on cross-feeding and fermentation by NSSF and SSF was carried out. Results indicated that the activity of cellulase excreted by cross-feeding of Aspergillus niger and Trichoderma koningi was the highest, with ethanol yield up to 0.139g/gDS by NSSF.
In this process, a high red-pigment-yielding strain was obtained. Primary work on this strain and its pigment suggests that the strain merits further investigation for it will probably be a new way of straw utilization.
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