摘要
世界石油资源的日益枯竭以及不断增长的温室气体效应,使人们对非石油能源产生了极大的兴趣。据报道中国传统能源的储量并不乐观,石油大约可开采50年左右,另一项重要能源—天然气可开采70年。2004年的一项数据表明：经过20年的开采,煤炭储藏量已经降低到了最低点。利用生物质原料生产燃料酒精部分替代化石燃料已经引起人们极大关注,燃料酒精的使用将会大大减少二氧化碳及其它有害气体的排放。与化石燃料相比,燃料酒精作为一种可再生能源,可以通过广泛的糖类物质发酵生产,其中利用成本低廉、数量巨大的纤维质类物质—例如玉米秸秆生产燃料酒精尤为引人关注。
玉米秸秆是丰富的农业废弃物,主要由纤维素、半纤维素和木质素组成。玉米秸秆在我国产量巨大,每年产量有一亿多吨。玉米秸秆除了少部分被利用外,绝大部分以堆积、荒烧等形式直接倾入环境,造成极大的污染和浪费,而且这种直接燃烧的方法热效率很低,大约只有10%左右。如果将它们转化成气体或液体燃料,例如酒精、氢气、柴油等,热效率可达30%以上。这样不但缓解人类所面临的资源危机,食物短缺,环境污染等一系列问题,也为人类持续发展提供了保证。
原料的成本在酒精生产中占有很大的比例,为了实现工业化生产,必须降低酒精生产成本,而充足便宜的原料是必须。因此利用玉米秸秆作为发酵工业原料具有巨大的潜力。
为了充分利用玉米秸秆资源,找到适合的玉米秸秆生产酒精工艺,最终实现工业化生产。本文利用玉米秸秆为原料,对玉米秸秆发酵酒精进行了研究。主要研究内容分为五部分,一是研究了不同湿热预处理条件的优化。二是利用活性干酵母对经过湿氧化预处理后的玉米秸秆同步糖化发酵制备酒精进行了研究。三是利用树干毕赤酵母(Pichia stipitis)对玉米秸秆发酵制备燃料酒精进行了研究。四是研究了不同脱毒方法对湿热预处理后的玉米秸秆水解液酒精发酵的影响。五是研究了水热预处理及脱毒结合对酒精发酵的影响.
1.不同湿热预处理条件的优化
玉米秸秆由于结构复杂致密,因此为提高酶解性首先要进行预处理。湿热预处理是高温条件下由水参与进行的反应。195℃预处理15分钟被报道是最佳的条件。与其它预处理方法相比,湿热预处理过程产生较少的发酵抑制剂。
本文对湿热预处理条件进行了优化,主要研究了玉米秸秆在不同湿热预处理条件下,纤维素及半纤维素的回收率以及酶解率,结果表明：
(1)在A (195℃,15min)、B (195℃,15min,O2 12bar)及C(195℃,15min, Na2CO3 2g/L, O2 12bar)三种预处理条件下,约90%纤维素都保留在固体中,大部分半纤维素和木质素被溶解或分解在水解液中,有效地解除了对纤维素的束缚。
(2)最佳预处理条件为C,纤维素总回收率达到95.87%,高于其它预处理条件下的纤维素总回收率,固体部分24h酶解率达到了67.6%,而原料玉米秸秆酶解率仅为16.2%。
2.玉米秸秆湿氧化预处理同步糖化发酵酒精
目前大部分利用纤维质原料生产酒精实验并不是利用真实的水解液,往往在水解液中加入葡萄糖或木糖,为了验证真实的生产工艺过程,葡萄糖或木糖都应来自预处理后的酶水解液,也就是采用同步糖化发酵法(SSF)进行酒精发酵。发酵过程中采用生长旺盛,有较强的耐抑制剂能力的活性干酵母(S. cerevisiae)作为生产菌种,将有利于实现工业化生产。
本文主要研究了玉米秸秆经过湿氧化(195℃,15min,Na2CO3 2g/L,O212 bar)预处理后,纤维素及半纤维素的回收率以及预处理后固体部分的酶解效率。在底物浓度8%(W／V)情况下,对活性干酵母同步糖化发酵制备酒精进行了研究。结果表明：
(1)玉米秸秆经过湿氧化预处理并过滤后主要分为固体和水解液两部分预处理后90%纤维素保留在固体中,纤维素总回收率达到95.87%。而半纤维素总回收率为68.2%,纤维素的回收率明显高于半纤维素。主要是因为半纤维素很不稳定,在高温、较长时间的湿氧化预处理条件下易分解成羧酸及H20与C02,故半纤维素的得率较低,而纤维素的得率较高。
(2)预处理后的固体部分利用纤维素酶处理,50℃24h酶解率达到了67.6%,而原料玉米秸秆酶解率仅为16.2%。可见湿氧化预处理有效地改变了固体部分的组成和结构,从而更利于纤维素酶的水解作用。
(3)利用活性干酵母对底物浓度为8%的湿氧化预处理后的固体与液体混合液进行了同步糖化发酵,经过142h发酵,酒精产量达到了理论酒精产量的79.0%。假定五碳糖和六碳糖都能够被利用,相当于1t玉米秸秆能够产生262.7kg的酒精。发酵过程中没有明显的抑制作用发生。
采用生长旺盛,耐抑制剂能力强的活性干酵母进行同步糖化发酵,获得了较高的酒精产量,发酵过程不需脱毒程序,减少了步骤,降低了成本,有利于实现工业化生产。
3.利用树干毕赤酵母发酵玉米秸秆制备酒精
纤维素和半纤维素水解主要产物分别是葡萄糖和木糖,有效地利用木糖和其它半纤维素单糖是纤维原料酒精工业化生产的关键。但工业上广泛应用的酿酒酵母不能利用木糖。能够同时利用葡萄糖和木糖的微生物主要有树干毕赤酵母(Pichia stipitis),休哈塔假丝酵母(CandidaShehatae),嗜鞣管囊酵母(Pachysolen tannopHilus)。目前人们研究得最多、最深入且最具有工业应用价值的是树干毕赤酵母(Pichia stipitis),但树干毕赤酵母对抑制剂非常敏感,往往发酵液要预先进行脱毒处理才能够发酵,所以目前研究主要集中在利用预处理后的水解液脱毒后进行发酵。而利用预处理后的滤饼与部分稀释的水解液混合并采用树干毕赤酵母同步糖化发酵(SSF),不但可以解除抑制作用,而且葡萄糖和木糖均来自预处理后的酶水解过程,有利于降低生产成本。
本文首先优化了玉米秸秆湿热处理(195℃,15min)后滤饼与水解液的配比。然后在底物浓度5%(W/V)情况下,研究了不同发酵温度(30。C、35℃、40℃),不同初始pH(5.5、5.5、6.0)以及不同摇床转速(100rpm、130rpm)对树干毕赤酵母酒精发酵的影响。结果表明：
(1)玉米秸秆经过湿热预处理,86.5%纤维素保留在滤饼中,大部分半纤维素主要降解为木糖。
(2)在温度30℃,pH5.5,摇床转速130rpm,底物浓度5%(W／V)情况下,采用树干毕赤酵母(Pichia stipitis 58376)经过192h同步糖化发酵,酒精浓度为12.12g/L,对应的酒精产量和酒精生产效率分别为0.34g/g(葡萄糖+木糖)和0.065g／Lh。
(3)工业生产上常用的酿酒酵母只能利用葡萄糖,而树干毕赤酵母能够同时利用葡萄糖和木糖,提高了原料的利用率。另外发酵过程不用进行脱毒处理,有利于降低生产成本.
4.不同脱毒方法对玉米秸秆水解液酒精发酵的影响
玉米秸秆预处理过程中由于糖类及木质素等的降解,会释放一些微生物生长的抑制剂,例如糠醛,5-羟甲基糠醛(5HMF)以及酚类等物质,从而影响菌体生长及发酵.目前使用的脱毒方法较多,其中饱和生石灰法和Na2SO3法是常用的有效的脱毒方法,广泛应用于各种水解液的脱毒处理,而中和法是比较简单易行的脱毒方法。但由于原料、预处理方式以及发酵微生物不同,各种脱毒方法之间很难进行比较。
本文主要考察了三种脱毒方法——中和法、饱和生石灰法和Na2SO3法对湿热预处理(195℃,15min)后的玉米秸秆水解液中抑制剂的醛类物质(糠醛及5HMF)以及总酚类物质的去除效果,并利用树干毕赤酵母(Pichia stipitis 58376)对脱毒后的玉米秸秆水解液酒精发酵进行了研究。结果表明：
(1)湿热预处理后的玉米秸秆水解液,经过三种方法(中和法、饱和生石灰法和Na2SO3法)脱毒处理后,所使用的脱毒方法都有效地降低了醛类物质的含量,醛类物质平均减少41%。最高的是采用Na2SO3法,可使醛类物质去除44.3%。而对于酚类物质,采用饱和生石灰法可得到28.4%去除率,中和法为10.6%,而Na2SO3法则没有效果。
(2)利用树干毕赤酵母对脱毒后的玉米秸秆水解液进行酒精发酵,酒精得率都得到了明显提高。最佳的脱毒方法则是采用饱和生石灰法,理论酒精得率达到69.31%,对应的酒精浓度和生产效率分别为12.2g/L和0.056g/Lh,明显高于其它脱毒方法。
(3)对于湿热预处理后的玉米秸秆水解液,饱和生石灰法是一种有效实用的脱毒方法。
5.玉米秸秆水热预处理及脱毒发酵生产酒精的研究
本文采用玉米秸秆经过水热处理后的固体与水解液作为原料,研究了pH调控与饱和生石灰脱毒法结合对酒精发酵的影响.结果表明：
(1)采用水热处理后的固体与水解液进行酒精发酵,当加入100%水解液时,由于抑制作用,酒精浓度仅为0.31g/L,理论酒精得率为9.48%。预水解后将pH从4.8分别调整到5.5,6.0和6.5后,酒精得率都有了明显的提高,当PH为5.5时,酒精得率达到56.4%,酒精浓度为10.67g/L。当pH为5.5时经过饱和石灰石法脱毒处理后酒精浓度达到了10,96 g/L,酒精得率达到57.9%,与pH在4.8时酒精相比,酒精浓度几乎增加了35倍。
(2)醋酸是重要的发酵抑制剂,对菌体生长及酒精发酵产生抑制作用,通过维持相对较高的发酵pH将会部分克服醋酸的抑制作用。饱和生石灰法是一种有效实用的脱毒方法,降低水解液的毒性,提高酒精得率。
(3)另外也应当考虑到在脱毒过程会造成可发酵性糖的损失。
我们的研究结果表明,玉米秸秆产量巨大,纤维素含量高,是酒精生产重要的原料。以上研究为玉米秸秆生产燃料酒精提供了依据,然而目前我们仍然面临许多挑战,最根本的问题还是要降低酒精生产成本,这就需要对纤维质原料酒精发酵的各个环节进行深入的研究。
1.研发廉价且选择性强的预处理方法,加强对抑制物形成、作用机制及酵母耐受机制等方面的理论研究。
2.筛选高产纤维素酶生产菌株,或者利用基因工程手段构建高产纤素酶基因工程菌,从而降低纤维素酶的生产成本。
3.纤维原料酒精发酵不仅要考虑微生物的性质,同时也要考虑发酵工艺,比如分批发酵或者连续发酵工艺。为了实现纤维质原料生产酒精,所有的工艺过程应当全面综合考虑。
总之纤维质原料生产酒精需要探索最佳途径和生产工艺,才能取得最好效果。
The depletion of the world's petroleum supply and the increasing problem of greenhouse gas effects have resulted in an increasing worldwide interest in alternative non petroleum-based sources of energy. It is reported that the stockpiles of China's traditional energy resource are not optimistic as we think. The exploitable year of oil is about 50 years. Another main energy sources——natural gas is 70 years. In 2004, the newest data showed that the coal stockpiles had been plunged into its lowest level after 20 years'digging. Ethanol produced from biomass is one attractive alternative for partial replacements of fossil fuels. Large scale application of fuel ethanol will contribute to reduction of CO2 and other emissions from the transport sector. Unlike fossil fuels, ethanol as a renewable energy source can be produced through fermentation of sugars.Lignocellulosic residues such as corn stover are considered as attractive raw materials for the production of fuel ethanol because of their availability in large quantities at low cost
Corn stover is an abundant agricultural residue and consists of cellulose、hemicellulose and lignin. Corn stover is ubiquitous in China comprising more than 100 million tonnes every year. Only small part of corn stover is collected and used and the most part is buried in the air in our country. This causes great pollution and waste. This method of direct combustion results in low thermal efficiency, only about 10%. If direct combustion is converted into gas or liquid fuel (ethanol、hydrogen、diesel、etc.), thermal efficiency can reach up to 30%.This will not only alleviate the resource crisis,food shortages, environmental pollution and other issues, but also provide a guarantee for the sustainable development of society.
The cost of raw material dominates the cost of total ethanol production.To attain commercial interest, the costs of bioethanol production mustbe reduced, and a sufficient amount of cheap and readily available rawmaterial is a necessity. Therefore, it has potential as an industrial fermentation substrate.
In order to make full use of corn stover resources, find out the appropriate process for ethanol production from corn stover and ultimately to realize industrialization, the study on ethanol production from corn stover was carried out. Main contents are divided into five parts. The first part is that optimization of different hydrothermal pretreatment conditions.The second part is that ethanol production from wet oxidized corn stover by simultaneous saccharification and fermentation. The third part is that ethanol production from corn stover by Pichia stipitis. The four part is that effect of different detoxification methods on ethanol production from corn stover hydrolysate. The fifth part is that the study on ethanol production from corn stover adopting hydrothermal pretreatment and detoxification
1. Optimization of different hydrothermal pretreatment conditions
Due to the close association to lignin in the plant cell wall, pretreatment is necessary to make the carbohydrates available for enzymatic hydrolysis. Hydrothermal pretreatment was carried out in high temperature. The pretreatment temperature and residence time were chosen to be 195℃and 15min, which was reported to be optimum. Compared to other pretreatment processes, Hydrothermal pretreatment has been proven to be more efficient for treating some lignocellulosic materials because of the advantage of producing fewer by products.
Hydrothermal pretreatment conditions was optimized by this work. Recovery of cellulose and hemicellulose、enzymatic hydrolysis of cellulose were studied under the three conditions.The results showed:
(l)Under three different hydrothermal pretreatment conditions, A (195℃,15min)、B (195℃,15min,O2 12bar) and C (195℃,15min,Na2CO3 2g/L,O2 12bar),about 90% of cellulose was remained in the solid cake and most of hemicellulose and lignin were solubilized and degraded. Pretreatment significantly increased the digesty of cellulose.
(2)Under the best condition(195℃,15min,Na2CO32g/L,O2 12bar), the recovery of cellulose was 95.87% which was higher than those under other pretreatment conditions. After 24h hydrolysis using cellulase (Cellubrix L), the achieved conversion of cellulose to glucose was 67.6% compared with 16.2% for raw corn stover
2. Ethanol production from wet oxidized corn stover by simultaneous saccharification and fermentation
At present, many experiments about ethanol production from lignocellulosic materials were not made on real fibrous substrate but only in filtrate using added glucose and xylose as sugar components. In order to test the real process, the glucose and xylose should be derived from enzymatic treatment of the wet oxidized fiber material as performed in the well-known simultaneous saccharification and fermentation (SSF).In experiments, we chose S. cerevisiae in the form of dried baker's yeast because it has often been proposed as the best organism for the fermentation.Dried baker's yeast has the advantage of being quite robust and less sensitive to inhibitors than other cultivated yeast strains.This will be conductive to realize industrialization.
Recovery of cellulose and hemicellulose and the achieved conversion of cellulose to glucose was studied after wet-oxidation pretreatment (195℃,15min,Na2CO32g/L, O2 12bar) with substrate concentration of 8%, simultaneous saccharification and fermentation (SSF) were adopted to produce ethanol by Dried baker's yeast.The results showed
(1)After wet-oxidation pretreatment solid fraction and hydrolysate were filtered and collected respectively.90% of cellulose remained in the solid fraction and the recovery of cellulose was 95.87%, whereas recovery of hemicellulose was only 68.2% after pretreatment. The recovery of cellulose was obviously higher than that of hemicellulose. In wet oxidation, the hemicellulose was converted or degraded at high temperature. This relatively low recovery was owing to hemicellulose oxidation to other products, such as carboxylic acids, CO2 and H2O.
(2)After 24h hydrolysis at 50℃using Cellulase, enzymatic conversion of pretreated cellulose in the remaining solid was 67.6% compared with 16.2% for the native corn stover. This was expected because this pretreatment most significantly modified the composition of the solid portion of pretreated corn stover compared with the untreated corn stover. The pretreatment more effectively increased the conversion of cellulose to glucose.
(3)After 142 h of SSF with substrate concentration of 8%, ethanol yield of 79.0% of the theoretical was obtained. The estimated total ethanol production was 262.7 kg/ton raw material by assuming the consumption of both C-6 and C-5. No obvious inhibition effect occurred during SSF. No obvious inhibition effect occurred during SSF.
High ethanol concentration was obtained with simultaneous saccharification and fermentation (SSF) by robust dry yeast.Detoxification procedures was not used in fermentation process. This will reduce process steps and low production costs. It is conductive to realize industrialization.These offered experiment evidences for ethanol production from corn stover.
3. Ethanol production from corn stover by Pichia stipitis
Cellulose and hemicellulose can be hydrolyzed to mainly liberate glucose and xylose respectively.The ability of microorganisms to ferment both glucose and xylose in biomass to ethanol is of importance for an economically feasible process. Native strains of Saccharomyces cerevisiae are unable to utilize xylose. Naturally occurring yeasts such as Pichia stipitis, Candidashehatae, and Pachysolen tannopHilus are able to ferment both glucose and xylose to ethanol. Among the xylose-fermenting yeasts, P. stipitis has shown the most promise for industrial application. But Pichiia stipitis is sensitive to fermentation inhibitors and need to detoxify in advance for fermentation process. So the current study focuses on utilizing detoxified hydrolysate for fermentation. Simultaneous saccharification and fermentation (SSF) with the mixture of pretreated solid cake and diluted hydrolysate will not only lift the inhibition effect, but also low production costs because both glucose and xylose are derived from enzymatic treatment of the pretreated materials.
The ratio of solid cake and hydrolysate of Hydrotherm-pretreated (195℃,15min) corn stover was optimized. With substrate concentration of 5%(W/V), Additional experiments were performed to investigate the effects of temperature (30℃、35℃、40℃), initial pH (5.5、5.5、6.0) and shaking rate (100 rpm,130 rpm) on the fermentation. The results showed:
(1)After Hydrothermal pretreatment (195℃,15min) solid fraction and hydrolysate were filtered and collected respectively.86.5% of cellulose were remained in solid cake and most of hemicellulose were solubilized after pretreatment.
(2)The highest ethanol concentration of 12.12 g/L was achieved at initial pH of 5.5, temperature 30℃and 130 rpm shaking rate with substrate concentration of 5%(w/v) by Pichia stipitis after 192 h fermentation. The corresponding ethanol yield and volumetric productivity were 0.34g/g (glucose+xylose) and 0.065g/L.h respectively.
(3)Native strains of Saccharomyces cerevisiae are unable to utilize xylose. Pichia stipitis is able to ferment both glucose and xylose to ethanol and improve the utilization of raw materials. Detoxification procedures was not used in fermentation process. This will reduce production costs.
4. Effect of different detoxification methods on ethanol production from corn stover hydrolysate
During pretreatment process of corn stover, several compounds such as furfural,5-hydroxymethyl furfural, acetic acid are released as a result of sugar or lignin degradation processes.The degradation products inhibit the metabolism of fermentative microorganisms and negatively affect the efficiency of fermentation. At present, there are many detoxification methods. Among them, overliming and Na2SO3 addition detoxification are widely used and neutralization method is relatively simple.However, the effect of the different methods cannot be compared with each other because different hydrolysate and different microorganisms are used in the fermentations. In this study, the influence of three different detoxification methods (neutralization、overliming and Na2SO3 addition) on inhibitors were evaluated using corn stover hydrolysate prepared with hydrothermal pretreatment. (195℃,15min).Ethanol fermentability of detoxified corn stover hydrolysate was investigated by Pichia stipitis 58376. The results showed:
(1)Total furan concentration in pretreated corn stover hydrolysate was affected by all three detoxification methods applied. All the employed detoxification methods resulted in a 41% reduction in average total furans. The highest removal of furans (44.3% removal) was achieved by Na2SO3 addition method.28.4% removal of total pHenols was achieved by overliming and 10.6% by neutralization, whereas no changes occurred by Na2SO3 addition.
(2)Ethanol fermentability of detoxified corn stover hydrolysate was investigated by Pichia stipitis 58376. Fermentation performance was greatly enhanced by employed detoxification methods. Ethanol yield of 69.31% of the theoretical based on reducing sugar was obtained by overliming. The corresponding ethanol concentration and volumetric productivity were 12.2g/L and 0.056g/L.h. These values were obviously higher than those of other detoxification methods.
(3)Overliming was the most efficient detoxification methods for Hydrotherm-pretreated corn stover hydrolysate
5. The study on ethanol production from corn stover adopting hydrothermal pretreatment and detoxification
After hydrothermal pretreatment of corn stover, solid fraction and hydrolysate were collected separately. Ethanol production was evaluated from dried solid fraction and the hydrolysate was employed as liquid fraction by baker'yeast. The effect on different Ph and detoxification on ethanol production were investigated. The results showed:
(1)When 100% hydrolysate was added, the ethanol concentration of 0.31g/L (9.48% of theoretical ethanol yield) was obtained due to existence of acetic acid and furans which are important inhibitor of the fermentation to microorganisms. After prehydrolysis, the initial pH was adjusted to 5.5,6.0 and 6.5 respectively. The best value obtained was 10.67g/L of ethanol concentration (56.4% of theoretical ethanol yield) with addition of 100% hydrolysate at pH of 5.5.The hydrolysate was overlimed, then prehydrolysized for 24h at 50C.After prehydrolysis the initial pH was adjusted again to 5.5. The ethanol concentration of 10.96g/L (57.9% of theoretical ethanol yield) was obtained. The values are almost 35 times high compared to those at initial pH 4.8.
(2) Acetic acid is an important fermentation inhibitor basically due to its undissociated form. The utilization of increasing pH higher than pKa of acetic acid can partially overcome this problem. Overliming is an effective way to reduce the toxicity of hydrolysate.
(3)The cost of fermentable sugar losses during detoxification process should be considered.
Our study showed that considering its abundance and high cellulose content, corn stover could be an excellent substrate for ethanol production. The studies above provided the basis for fuel ethanol production from corn stover. However, we still face many challenges. Reducing the cost of ethanol production is the most fundamental that requires to further investigate all the aspects of ethanol fermentation.
(1) Cheap and special pretreatment methods should be developed. The study on formation of fermentation inhibitors, mechanism of inhibitors and mechanism of tolerany-inhibitors of yeast should be strengthened.
(2) To reduce cellulose cost, more efficient microorganisms to produce cellulose or genetically engineering microorganism for cellulose production should be screened.
(3) Not only are the properties of the microorganism of importance in the process, but also the choice of fermentation strategies such as batch culture, continuous culture with cell recycling. For the realization of the ethanol production from lignocellulosic materials, the fermentation step has to be integrated with the rest of the process.
In a word, exploring the best ways and production process from all aspects of ethanol production from lignocellulosic materials would obtain the best results.
引文
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