技术领域technical field
本发明涉及生物质预处理领域，具体涉及一种利用甘蔗渣发酵产丁醇的预处理方法。The invention relates to the field of biomass pretreatment, in particular to a pretreatment method for producing butanol by fermenting bagasse.
背景技术Background technique
目前，已有多种成熟的针对于纤维材料和半纤维材料的预处理方法以水解得到糖分，所得的糖分可以用于发酵生产生物燃料。At present, there are a variety of mature pretreatment methods for fibrous and semi-fibrous materials to hydrolyze sugars, which can be used for fermentation to produce biofuels.
最近，这些方法在工业生物技术领域得了一些进展，为能经济有效的利用农业产业废料提供了潜在的机会。例如，甘蔗渣是一种综合性材料，为甘蔗产业中的主要副产物，也是主要农业废料之一，每年都富产于中国南方。甘蔗渣含有大约50%的纤维素、25%的半纤维素和25%的木质素，用其生产生物丁醇是一种生产生物燃料的理想方法，因为丁醇相比于传统的乙醇，具有更高的热值和更低的吸湿性。Recently, some advances in these methods have been made in the field of industrial biotechnology, providing potential opportunities for cost-effective utilization of agricultural industry wastes. For example, bagasse is a comprehensive material, a major by-product of the sugarcane industry and one of the major agricultural wastes, which is abundantly produced in southern China every year. Bagasse contains approximately 50% cellulose, 25% hemicellulose and 25% lignin, and its use to produce biobutanol is an ideal method for biofuel production because butanol has Higher calorific value and lower moisture absorption.
然而，利用甘蔗渣生产丁醇是否可行，取决包括预处理和脱毒纯化等多种因素。而传统的预处理方法，如酸解，常产生严重抑制梭菌生长的毒性物质。However, the feasibility of using bagasse to produce butanol depends on many factors including pretreatment and detoxification purification. However, traditional pretreatment methods, such as acid hydrolysis, often produce toxic substances that severely inhibit the growth of Clostridium.
许多研究报道了多种可用于富有纤维素和半纤维素的谷物残渣的预处理方法，包括蒸汽爆破法或稀酸预处理法。酸解法是一种常用且有效的预处理方法，特别常用的是稀硫酸。该方法不仅简单，而且成本低廉。然而，在酸解时，常产生一类微生物抑制剂，该抑制剂可在发酵时抑制微生物的生长。呋喃甲醛和羟甲基糠醛是在生物发酵时产生的主要抑制剂。因此，在进行生物丁醇发酵前，水解产物必须首先进行脱毒纯化，以除去抑制剂。Many studies have reported various pretreatment methods that can be used for cellulose- and hemicellulose-rich grain residues, including steam explosion or dilute acid pretreatment. Acid hydrolysis is a commonly used and effective pretreatment method, especially dilute sulfuric acid is commonly used. The method is not only simple, but also inexpensive. However, during acid hydrolysis, a class of microbial inhibitors is often produced, which can inhibit the growth of microorganisms during fermentation. Fufural and hydroxymethylfurfural are the main inhibitors produced during biological fermentation. Therefore, before biobutanol fermentation, the hydrolyzate must first be detoxified and purified to remove inhibitors.
然而，预处理后的脱毒纯化是一个复杂的过程，尽管多种脱毒纯化方法已经得到了研究，如石灰处理、蒸发法、离子交换柱层析法、活性炭和生物处理法，然而这些方法要么脱毒纯化效果不好，要么成本太高。目前，主要通行的方法是利用化学法去除抑制剂，因为化学法较为简单，成本也较低。然而，利用化学法时，由于抑制剂的化学结构的差异性，导致该法无法对所有抑制剂进行清除。另外，化学法还会产生许多盐类，导致在发酵液中含有高浓度的盐，而高浓度盐会对微生物发酵产生严重的抑制效果，从而导致生物丁醇产量的减少。However, detoxification and purification after pretreatment is a complex process, although various detoxification and purification methods have been studied, such as lime treatment, evaporation, ion exchange column chromatography, activated carbon and biological treatment, however these methods Either the detoxification and purification effect is not good, or the cost is too high. At present, the main current method is to use chemical method to remove inhibitors, because chemical method is relatively simple and the cost is low. However, when chemical methods are used, due to the differences in the chemical structures of inhibitors, this method cannot remove all inhibitors. In addition, the chemical method will also produce a lot of salts, resulting in a high concentration of salt in the fermentation broth, and high concentration of salt will have a serious inhibitory effect on microbial fermentation, resulting in a reduction in the production of bio-butanol.
因此，选择合适的预处理方法就显得十分重要。选择预处理方法时，需考虑如下两点：1、能最有效的破坏纤维结构，以产生充足的糖，并防止对糖的损耗，同时还能避免或减少抑制剂的产生；2、预处理方法需成本低廉，简单易行。Therefore, it is very important to choose an appropriate preprocessing method. When choosing a pretreatment method, the following two points should be considered: 1. It can most effectively destroy the fiber structure to produce sufficient sugar and prevent the loss of sugar, while avoiding or reducing the production of inhibitors; 2. Pretreatment The method needs to be low-cost, simple and easy to implement.
在现有技术中，还未有可避免或减少所有主要抑制剂产生的预处理方法。因此，亟待开发一种既经济可行，又能控制所有主要抑制剂含量，以便对甘蔗渣进行发酵产丁醇的预处理方法。In the prior art, there is no pretreatment method that can avoid or reduce the production of all major inhibitors. Therefore, it is urgent to develop a pretreatment method that is both economically feasible and capable of controlling the content of all major inhibitors in order to ferment bagasse to produce butanol.
发明内容Contents of the invention
本发明的目的在于提供一种利用甘蔗渣发酵产丁醇的预处理方法，该预处理方法可以控制抑制微生物生长的毒素的产生。通过控制毒素的产生，使得经预处理后的甘蔗渣无需后续的纯化脱毒处理，便能进行发酵生产丁醇，大大降低了生产成本。The object of the present invention is to provide a pretreatment method for producing butanol by fermenting bagasse, which can control the production of toxins that inhibit the growth of microorganisms. By controlling the production of toxins, the pretreated bagasse can be fermented to produce butanol without subsequent purification and detoxification treatment, which greatly reduces the production cost.
该预处理方法包括如下步骤：The preprocessing method includes the following steps:
1）机械粉碎：对甘蔗进行切段至长度为5-10cm，在65±2℃下烘干2天，然后进行粉碎，过20目筛，得到粒径小于830μm的甘蔗渣粉；1) Mechanical pulverization: cut the sugarcane to a length of 5-10cm, dry at 65±2°C for 2 days, then pulverize, pass through a 20-mesh sieve, and obtain bagasse powder with a particle size of less than 830μm;
2）液态热水处理：按固液质量体积比为250g/L，将步骤1）所得物于常温下在水中浸泡12h，之后用蒸汽预加热45s,然后加入液态热水，在200±3℃下，反应1h，反应后进行过滤，收集滤液Ⅰ和固体残余物Ⅰ；2) Liquid hot water treatment: according to the solid-liquid mass volume ratio of 250g/L, soak the result of step 1) in water at room temperature for 12 hours, then preheat with steam for 45s, and then add liquid hot water, at 200±3℃ , react for 1 h, filter after the reaction, and collect the filtrate I and solid residue I;
3）微波处理：调整步骤2）所得固态残余物Ⅰ的湿度为40%，并置于反应器中，然后通入氮气排除氧气，于4 ℃下放置8-12h，之后将反应器置于微波热解炉中，在120 ℃下反应7min，再将反应所得物降温至常温，用90%乙醇对反应所得物进行浸洗，之后进行过滤，收集滤液Ⅱ和固体残余物Ⅱ；3) Microwave treatment: adjust the humidity of the solid residue I obtained in step 2) to 40%, and place it in the reactor, then pass nitrogen gas to exclude oxygen, and place it at 4°C for 8-12h, then place the reactor in the microwave In the pyrolysis furnace, react at 120 °C for 7 minutes, then cool the reaction product to room temperature, soak the reaction product with 90% ethanol, then filter, and collect the filtrate II and solid residue II;
4）微生物分解：烘干步骤3）所得固体残余物Ⅱ，之后加入培养液进行混合，再于121 ℃下进行灭菌，灭菌后，调整其湿度为30%，以质量百分比计，再加入10%的分解菌菌液，于30℃下发酵10 d，之后进行过滤，收集滤液Ⅲ和固体残余物Ⅲ；4) Decomposition by microorganisms: dry the solid residue II obtained in step 3), then add culture medium for mixing, and then sterilize at 121 °C, after sterilization, adjust its humidity to 30%, and then add 10% decomposed bacteria liquid, fermented at 30°C for 10 days, then filtered, and collected filtrate III and solid residue III;
所述培养液的配方为：30 mg H3BO3、20 mg MnCl2.4H2O、185 mg ZnSO4•7H2O、20mg Na2MoO4•2H2O、280 mg FeSO4•H2O、200 mg CuSO4、5 g NH4NO3、1 g KH2PO4、0.15 g MgSO4•H2O、0.11 g CaCl2 和1 L ddH2O,pH 为7.0；The formula of the culture solution is: 30 mg H3 BO3 , 20 mg MnCl2.4H2O, 185 mg ZnSO4•7H2 O, 20mg Na2 MoO4 •2H2 O, 280 mg FeSO4 •H2 O, 200 mg CuSO4 , 5 g NH4 NO3 , 1 g KH2 PO4 , 0.15 g MgSO4 H2 O, 0.11 g CaCl2 and 1 L ddH2 O, pH 7.0;
所述分解菌为Aspergillus nigerATCC 1015、Trichoderma reeseiATCC 26921和Penicillium janthinellumATCC 44750中的一种；The decomposing bacteria is one ofAspergillus niger ATCC 1015,Trichoderma reesei ATCC 26921 andPenicillium janthinellum ATCC 44750;
5）氨处理：用ddH2O对步骤4）所得固体残余物Ⅲ进行清洗，之后将其烘干，以固液质量比为1:2，将烘干后的固体残余物Ⅲ与氨水混合，在0.1MPa压强下，于90 ℃反应30min，之后进行过滤，收集滤液Ⅳ和固体残余物Ⅳ；5) Ammonia treatment: Wash the solid residue III obtained in step 4) with ddH2 O, then dry it, and mix the dried solid residue III with ammonia water at a solid-to-liquid mass ratio of 1:2, Under the pressure of 0.1MPa, react at 90°C for 30min, then filter, and collect the filtrate IV and solid residue IV;
6）酶解处理：将所述固体残余物Ⅳ于水中浸泡2 h，调节pH为7.0，浓缩后将其烘干，然后配制酶解混合物，每500 ml该酶解混合物由下述物质组成：50 g烘干了的所述固体残余物Ⅳ、10g纤维素酶、100 ml OPTMASH BG、5 mg氯霉素和2.5 mg卡那霉素；调整酶解混合物的pH为5.0，在温度为55℃，摇床转速为220rpm下，反应60 h，之后进行过滤，收集滤液Ⅴ和固体残余物Ⅴ；6) Enzymatic hydrolysis treatment: Soak the solid residue IV in water for 2 hours, adjust the pH to 7.0, concentrate and dry it, and then prepare an enzymatic hydrolysis mixture, which consists of the following substances per 500 ml: 50 g of the dried solid residue IV, 10 g of cellulase, 100 ml of OPTMASH BG, 5 mg of chloramphenicol and 2.5 mg of kanamycin; adjust the pH of the enzymatic hydrolysis mixture to 5.0 at a temperature of 55°C , the rotating speed of the shaking table was 220rpm, reacted for 60 h, then filtered, and collected the filtrate V and solid residue V;
7）将滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和固体残余物Ⅳ合并，留待SSF发酵，或将所述滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和滤液Ⅴ合并并旋蒸浓缩，留待SHF发酵。7) Combine filtrate I, filtrate II, filtrate III, filtrate IV and solid residue IV, and leave for SSF fermentation, or combine the filtrate I, filtrate II, filtrate III, filtrate IV and filtrate V, and set aside for SHF fermentation.
优选的，在进行微生物分解时，所用的分解菌为Trichoderma reesei ATCC26921。Preferably, when decomposing by microorganisms, the decomposing bacteria used areTrichoderma reesei ATCC26921 .
在现有预处理方式中，单独使用某一种预处理方式时，不能充分降解甘蔗渣纤维素，产糖率极低；如与稀酸法联用，虽然产糖率有所改善，但会对反应器产生腐蚀和出现污染问题，同时，对抑制剂浓度的控制效果也不理想。Among the existing pretreatment methods, when a certain pretreatment method is used alone, the bagasse cellulose cannot be fully degraded, and the sugar production rate is extremely low; if it is used in combination with the dilute acid method, although the sugar production rate is improved, it will be Corrosion and pollution problems occur to the reactor, and at the same time, the control effect on the concentration of the inhibitor is not ideal.
本发明首先选择高热水处理，先通过在常温下在水中浸泡12h，之后用蒸汽预加热45s,然后加入液态热水，在200±3℃下，反应1h，便可实现破坏纤维素与木质素间结合牢固的分子结构。这是因为首先必须对甘蔗渣纤维素的结构进行破坏，使甘蔗渣处于“松散”状态，以便进行下一步处理。在较为高压，且温度不太高的预处理条件下，蒸汽能与甘蔗渣的表面充分接触，从而破坏纤维素与木质素间结合牢固的分子结构。In the present invention, high-temperature hot water treatment is firstly selected, first soaking in water at room temperature for 12 hours, then preheating with steam for 45 seconds, then adding liquid hot water, and reacting for 1 hour at 200±3°C to achieve the destruction of cellulose and wood Strong molecular structure between primes. This is because the structure of the bagasse cellulose must first be destroyed so that the bagasse is in a "loose" state for further processing. Under relatively high pressure and not too high temperature pretreatment conditions, the steam can fully contact the surface of the bagasse, thereby destroying the molecular structure firmly bonded between cellulose and lignin.
虽然经过第一步预处理，甘蔗渣的结构呈现一定程度的“松散”状态，但是甘蔗渣还是没有得到充分的降解，因此第二步采用微波预处理。在120℃下，将经第一步预处理的甘蔗渣在微波下进行处理，可使得处于“松散”状态的甘蔗渣进一步的分解，但经微波预处理后，可能会产生一些生物油、柴油等物质，用90%的乙醇洗涤，可洗脱产生的油类物质，便于下一步微生物的分解生长。Although the structure of the bagasse is "loose" to a certain extent after the first step of pretreatment, the bagasse has not been fully degraded, so the second step is microwave pretreatment. At 120°C, treating the bagasse pretreated in the first step under microwave can further decompose the bagasse in a "loose" state, but after microwave pretreatment, some bio-oil and diesel oil may be produced Washing with 90% ethanol can elute the oily substances produced, which is convenient for the decomposition and growth of microorganisms in the next step.
经过前两步预处理后，甘蔗渣可以进行第三步分解，即微生物分解。微生物主要利用自身产生的酶系分解纤维素，在酶切割的情况下，纤维素得到再进一步的分解。可用的分解菌为Aspergillus nigerATCC 1015、Trichoderma reeseiATCC 26921和PenicilliumjanthinellumATCC 44750中的一种，其中Trichoderma reeseiATCC 26921的分解效果最好，这可能是因为该菌更偏向对甘蔗渣的分解利用。After the first two steps of pretreatment, the bagasse can undergo the third step of decomposition, which is microbial decomposition. Microorganisms mainly use their own enzymes to decompose cellulose, and in the case of enzymatic cutting, cellulose is further decomposed. The available decomposing bacteria are one ofAspergillus niger ATCC 1015,Trichoderma reesei ATCC 26921 andPenicilliumjanthinellum ATCC 44750, among whichTrichoderma reesei ATCC 26921 has the best decomposition effect, which may be because this bacteria is more inclined to the decomposition and utilization of bagasse.
然而以上几步主要在于分解性纤维素和半纤维素，但是某些木质素可能还没得到充分的分解，因此，第四步采用氨水浸泡，氨水浸泡的目的在于使已经被破坏了的纤维素和木质素得到分离，只有分离了木质素，下一步中的酶解才能使纤维素得到进一步的再分解。在氨水浸泡中，浸泡时间、压力和温度较为重要，通过合适的设置，可提高甘蔗渣的分解效率，并减少抑制剂的产生。经过以上几步预处理后，最后一步采用酶解。However, the above steps mainly focus on decomposing cellulose and hemicellulose, but some lignin may not be fully decomposed. Therefore, the fourth step is soaked in ammonia water. The purpose of soaking in ammonia water is to make the cellulose that has been destroyed and lignin are separated, and only after the lignin is separated, the enzymatic hydrolysis in the next step can further decompose the cellulose. In ammonia water immersion, immersion time, pressure and temperature are more important. Appropriate settings can improve the decomposition efficiency of bagasse and reduce the generation of inhibitors. After the above steps of pretreatment, the last step is enzymatic hydrolysis.
上述预处理步骤的顺序是固定的。通过上述步骤处理，不仅可以得到较高的产糖率，而且水解液中所有主要的抑制物含量低于影响发酵菌生长的浓度，或低至检测不出。The order of the above preprocessing steps is fixed. Through the above steps, not only can a higher sugar production rate be obtained, but also the content of all major inhibitors in the hydrolyzate is lower than the concentration that affects the growth of fermentation bacteria, or is so low that it cannot be detected.
由于甘蔗渣的大小影响着分解的难易程度，一般而言，越小的甘蔗渣更易分解，但考虑到能耗因素，将甘蔗渣的大小控制在860μm以下，不仅分解效果好，且能耗较低。Because the size of bagasse affects the difficulty of decomposition, generally speaking, the smaller the bagasse is, the easier it is to decompose. However, considering the energy consumption factor, controlling the size of bagasse below 860 μm not only has a good decomposition effect, but also reduces energy consumption. lower.
液态热水法相比于稀酸法，无需添加酸试剂，不会对反应器造成腐蚀，产生的水解液中的残留物也较少，是一种较为温和的预处理方法。然而，其甘蔗残余物回收率较低，单独使用时无法达到有效分解甘蔗渣的目的。Compared with the dilute acid method, the liquid hot water method does not need to add acid reagents, does not cause corrosion to the reactor, and produces less residue in the hydrolyzate, which is a milder pretreatment method. However, its sugarcane residue recovery rate is low, and it cannot achieve the purpose of effectively decomposing bagasse when used alone.
经过液态热水法处理后，再利用微波进行处理，可较大幅度的提高还原糖的产量、并缩短反应时间和降低能耗。进行微波处理时，微波可加速对晶体结构的破坏，使微生物更容易对甘蔗渣进行进一步分解。通过分解菌分解后，可再提高10%左右的甘蔗渣分解率。After liquid hot water treatment, microwave treatment can greatly increase the yield of reducing sugar, shorten the reaction time and reduce energy consumption. When microwave treatment is carried out, microwave can accelerate the destruction of crystal structure, making it easier for microorganisms to further decompose bagasse. After being decomposed by decomposing bacteria, the decomposition rate of bagasse can be increased by about 10%.
优选的，在利用分解菌分解甘蔗渣时，利用Trichoderma reesei ATCC 26921可实现更好的分解效率。Preferably, when using decomposing bacteria to decompose bagasse, usingTrichoderma reesei ATCC 26921 can achieve better decomposition efficiency.
进行氨处理时，由于氨是木质素的良好溶剂，可将木质素和木质纤维素进行分离，从而可提高后续纤维素酶对甘蔗渣的分解效率。During ammonia treatment, because ammonia is a good solvent for lignin, lignin and lignocellulose can be separated, thereby improving the subsequent decomposition efficiency of cellulase on bagasse.
经过上述处理后，再利用酶解处理，可最大化的水解甘蔗渣。After the above treatment, the enzymatic hydrolysis treatment can be used to hydrolyze the bagasse to the greatest extent.
本发明的有益效果：Beneficial effects of the present invention:
1、能够高效水解甘蔗渣，水解率可达到74.41%；1. It can efficiently hydrolyze bagasse, and the hydrolysis rate can reach 74.41%;
2、可得到高浓度的葡萄糖，葡萄糖浓度可达到15.21±1.65g/L；2. High concentration of glucose can be obtained, the glucose concentration can reach 15.21±1.65g/L;
3、预处理条件温和，能耗低、不腐蚀设备；3. Mild pretreatment conditions, low energy consumption, and no corrosion of equipment;
4、无需后续纯化步骤，微生物发酵生产成本低；4. There is no need for subsequent purification steps, and the production cost of microbial fermentation is low;
5、主要抑制剂呋喃甲醛和羟甲基糠醛含量低，不影响发酵微生物的生长，同时基本不产生葡糖醛酸、对香豆酸、丁香酸和阿魏酸等其它抑制剂。5. The content of main inhibitors furfural and hydroxymethylfurfural is low, which does not affect the growth of fermentation microorganisms, and basically does not produce other inhibitors such as glucuronic acid, p-coumaric acid, syringic acid and ferulic acid.
附图说明Description of drawings
1、图1所示为本发明预处理方法步骤的示意图；1, shown in Figure 1 is the schematic diagram of the pretreatment method step of the present invention;
2、图2所示，为利用甘蔗渣水解产物（滤液）作为发酵底物时，发酵产物与发酵时间的关系图；其中HAN表示为：在步骤4）采用Aspergillus nigerATCC 1015作为发酵菌，并将所述滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和滤液Ⅴ合并，用作SHF发酵；HTR表示为：在在步骤4）采用Trichoderma reeseiATCC 26921作为发酵菌，并将所述滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和滤液Ⅴ合并，用作SHF发酵；HPJ表示为：在在步骤4）采用Penicillium janthinellumATCC 44750作为发酵菌，并将所述滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和滤液Ⅴ合并，用作SHF发酵；HANNY表示为：在步骤4）采用Aspergillus nigerATCC 1015作为发酵菌，并将滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和固体残余物Ⅳ合并，用作SSF发酵；HTRNY表示为：在步骤4）采用Trichoderma reeseiATCC 26921作为发酵菌，并将滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和固体残余物Ⅳ合并，用作SSF发酵；HPYNY表示为：在步骤4）采用Penicillium janthinellumATCC 44750作为发酵菌，并将滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和固体残余物Ⅳ合并并旋蒸浓缩，用作SSF发酵；2. As shown in Figure 2, it is a relationship diagram between the fermentation product and the fermentation time when bagasse hydrolyzate (filtrate) is used as the fermentation substrate; wherein HAN is expressed as: in step 4)Aspergillus niger ATCC 1015 is used as the fermentation bacteria, and The filtrate I, filtrate II, filtrate III, filtrate IV and filtrate V are combined for SHF fermentation; HTR is expressed as: in step 4)Trichoderma reesei ATCC 26921 is used as the fermentation bacteria, and the filtrate I, filtrate II, filtrate III, filtrate IV and filtrate V are combined for SHF fermentation; HPJ is expressed as: in step 4)Penicillium janthinellum ATCC 44750 is used as the fermentation bacteria, and the filtrate I, filtrate II, filtrate III, and filtrate IV are Combined with filtrate V and used for SHF fermentation; HANNY is expressed as: in step 4)Aspergillus niger ATCC 1015 was used as fermentation bacteria, and filtrate I, filtrate II, filtrate III, filtrate IV and solid residue IV were combined to be used as SSF Fermentation; HTRNY is expressed as: in step 4)Trichoderma reesei ATCC 26921 is used as fermentation bacteria, and filtrate I, filtrate II, filtrate III, filtrate IV and solid residue IV are combined for SSF fermentation; HPYNY is expressed as: in step 4)Penicillium janthinellum ATCC 44750 was used as the fermentation bacteria, and the filtrate I, filtrate II, filtrate III, filtrate IV and solid residue IV were combined and concentrated by rotary evaporation for SSF fermentation;
3、图3所示，为利用甘蔗渣水解产物（滤液）作为发酵底物时，在SSF和SHF发酵过程中pH的变化图；其中，HAN、HTR、HPJ、HANNY、HTRNY、HPJNY的含义与图2中的一致；a为SSF发酵；b为SHF发酵；3. As shown in Figure 3, when using bagasse hydrolyzate (filtrate) as the fermentation substrate, the change diagram of pH in the fermentation process of SSF and SHF; wherein, the meaning of HAN, HTR, HPJ, HANNY, HTRNY, HPJNY is the same as Consistent in Fig. 2; a is SSF fermentation; b is SHF fermentation;
4、图4所示，为SSF和SHF发酵过程中，对不同糖成分的转化使用情况；其中a、b、c为SHF发酵，b、d、f为SSF发酵，HAN、HTR、HPJ、HANNY、HTRNY、HPJNY的含义与图2中的一致；4. As shown in Figure 4, it is the conversion and use of different sugar components during the fermentation process of SSF and SHF; where a, b, and c are SHF fermentation, b, d, f are SSF fermentation, HAN, HTR, HPJ, HANNY The meanings of , HTRNY, and HPJNY are consistent with those in Figure 2;
5、图5所示，为在发酵终点，由不同发酵底物产生的总残余物碳水化合物；其中，HAN、HTR、HPJ、HANNY、HTRNY、HPJNY的含义与图2中的一致；5. As shown in Figure 5, it is at the end of fermentation, the total residual carbohydrate produced by different fermentation substrates; wherein, the meaning of HAN, HTR, HPJ, HANNY, HTRNY, HPJNY is consistent with that in Figure 2;
6、图6所示，为利用纯葡萄糖作为发酵底物时，在发酵过程中，不同参数的变化图；6. As shown in Figure 6, when pure glucose is used as the fermentation substrate, in the fermentation process, the change diagram of different parameters;
7、图7所示，为利用糖组合物作为发酵底物时，不同产物、糖和pH的变化图。7. As shown in FIG. 7, it is a graph showing the change of different products, sugar and pH when the sugar composition is used as the fermentation substrate.
具体实施方式detailed description
下面通过实施例对本发明进行具体描述，有必要在此指出的是以下实施例只是用于对本发明进行进一步的说明，不能理解为对本发明保护范围的限制，该领域的技术熟练人员根据上述发明内容所做出的一些非本质的改进和调整，仍属于本发明的保护范围。The present invention is described in detail by the following examples, it is necessary to point out that the following examples are only used to further illustrate the present invention, and can not be interpreted as limiting the scope of the present invention, those skilled in the art according to the content of the above invention Some non-essential improvements and adjustments still belong to the protection scope of the present invention.
实施例1：Example 1:
1）将500g甘蔗进行切段至长度为5-10cm，在65±2℃下烘干2天，然后进行粉碎，过20目筛，得到粒径小于830μm的甘蔗渣粉；1) Cut 500g of sugarcane to a length of 5-10cm, dry at 65±2°C for 2 days, then pulverize and pass through a 20-mesh sieve to obtain bagasse powder with a particle size of less than 830μm;
2）将步骤1）所得物于常温下在2L的水中浸泡12h，之后用蒸汽预加热45s,然后加入液态热水，在200±3℃下，反应1h，反应后进行过滤，收集滤液Ⅰ和固体残余物Ⅰ；2) Soak the product obtained in step 1) in 2L of water at room temperature for 12 hours, then preheat with steam for 45 seconds, then add liquid hot water, react at 200±3°C for 1 hour, filter after reaction, collect filtrate I and solid residue I;
3）调整步骤2）所得固态残余物的湿度为40%，并置于三颈圆底烧瓶中，然后通入氮气排除氧气，于4 ℃下放置12h，之后将反应器置于微波热解炉中在120 ℃下反应7 min，排出热气，待温度降到室温后，用90%乙醇对反应所得物进行浸洗，之后进行过滤，收集滤液Ⅱ和固体残余物Ⅱ；3) Adjust the humidity of the solid residue obtained in step 2) to 40%, and place it in a three-necked round-bottomed flask, then pass through nitrogen to exclude oxygen, and place it at 4 °C for 12 hours, and then place the reactor in a microwave pyrolysis furnace React at 120 °C for 7 min, discharge the hot gas, soak the reaction product with 90% ethanol after the temperature drops to room temperature, and then filter to collect the filtrate II and solid residue II;
4）烘干步骤3）所得固体残余物，之后加入培养液进行混合，再于121 ℃下进行灭菌，灭菌后，调整混合物的湿度为30%，再加入20mlAspergillus nigerATCC 1015菌液，于30℃下发酵10 d，之后进行过滤，收集滤液Ⅲ和固体残余物Ⅲ；4) Dry the solid residue obtained in step 3), then add the culture medium to mix, and then sterilize at 121 °C. After sterilization, adjust the humidity of the mixture to 30%, and then add 20ml ofAspergillus niger ATCC 1015 bacterial liquid, Ferment at 30°C for 10 days, then filter to collect filtrate III and solid residue III;
培养液的配方为：30 mg H3BO3、20 mg MnCl2.4H2O、185 mg ZnSO4•7H2O、20 mgNa2MoO4•2H2O, 280 mg FeSO4•H2O, 200 mg CuSO4, 5 g NH4NO3, 1 g KH2PO4,0.15 gMgSO4•H2O, 0.11 g CaCl2 and 1 L ddH2O,pH 为7.0；The formula of the culture medium is: 30 mg H3 BO3 , 20 mg MnCl2.4H2O, 185 mg ZnSO4•7H2 O, 20 mgNa2 MoO4 •2H2 O, 280 mg FeSO4 •H2 O, 200 mg CuSO4 , 5 g NH4 NO3 , 1 g KH2 PO4 , 0.15 gMgSO4 H2 O, 0.11 g CaCl2 and 1 L ddH2 O, pH 7.0;
5）用ddH2O对步骤4）所得固体残余物进行清洗，之后将其烘干，以固液质量比为1:2，将烘干后的固体残余物Ⅲ与氨水混合，在高压下（0.1MPa），于90 ℃反应30 min，之后进行过滤，收集滤液Ⅳ和固体残余物Ⅳ；5) Wash the solid residue obtained in step 4) with ddH2 O, and then dry it. With a solid-to-liquid mass ratio of 1:2, mix the dried solid residue III with ammonia water, and dry it under high pressure ( 0.1MPa), react at 90°C for 30 min, then filter to collect filtrate IV and solid residue IV;
6)将步骤5）所得固体残余物Ⅳ于900ml的双蒸水中浸泡2 h，用1%的H2SO4调节pH为7.0，用旋转蒸发仪对其进行浓缩，再于60℃下将其烘干。然后配制酶解混合物，每500 ml该酶解混合物由下述物质组成：50 g烘干了的固体残余物Ⅳ、10g纤维素酶、100 ml OPTMASHBG、5 mg氯霉素和2.5 mg卡那霉素；调整酶解混合物的pH为5.0，在温度为55℃，摇床转速为220rpm下，反应60 h，之后进行过滤，收集滤液Ⅴ和固体残余物Ⅴ。6) Soak the solid residue IV obtained in step 5) in 900ml of double-distilled water for 2 h, adjust the pH to 7.0 with 1% H2 SO4 , concentrate it with a rotary evaporator, and dilute it at 60°C drying. Then prepare enzymatic hydrolysis mixture, every 500 ml of this enzymatic mixture is composed of the following substances: 50 g dried solid residue IV, 10 g cellulase, 100 ml OPTMASHBG, 5 mg chloramphenicol and 2.5 mg kanamycin element; adjust the pH of the enzymolysis mixture to 5.0, react for 60 h at a temperature of 55° C. and a shaker speed of 220 rpm, and then filter to collect the filtrate V and solid residue V.
各步骤的固体回收率见表1。The solid recovery rate of each step is shown in Table 1.
实施例2Example 2
1）将500g甘蔗进行切段至长度为5-10cm，在65±2℃下烘干2天，然后进行粉碎，过20目筛，得到粒径小于830μm的甘蔗渣粉；1) Cut 500g of sugarcane to a length of 5-10cm, dry at 65±2°C for 2 days, then pulverize and pass through a 20-mesh sieve to obtain bagasse powder with a particle size of less than 830μm;
2）将步骤1）所得物于常温下在2L的水中浸泡12h，之后用蒸汽预加热45s,然后加入液态热水，在200±3℃下，反应1h，反应后进行过滤，收集滤液Ⅰ和固体残余物Ⅰ；2) Soak the product obtained in step 1) in 2L of water at room temperature for 12 hours, then preheat with steam for 45 seconds, then add liquid hot water, react at 200±3°C for 1 hour, filter after reaction, collect filtrate I and solid residue I;
3）调整步骤2）所得固态残余物的湿度为40%，并置于三颈圆底烧瓶中，然后通入氮气排除氧气，于4 ℃下放置8h，之后将反应器置于微波热解炉中在120 ℃下反应7 min，排出热气，待温度降到室温后，用90%乙醇对反应所得物进行浸洗，之后进行过滤，收集滤液Ⅱ和固体残余物Ⅱ；3) Adjust the humidity of the solid residue obtained in step 2) to 40%, and place it in a three-necked round-bottomed flask, then pass through nitrogen to exclude oxygen, and place it at 4 °C for 8 hours, and then place the reactor in a microwave pyrolysis furnace React at 120 °C for 7 min, discharge the hot gas, soak the reaction product with 90% ethanol after the temperature drops to room temperature, and then filter to collect the filtrate II and solid residue II;
4）烘干步骤3）所得固体残余物，之后加入培养液进行混合，再于121 ℃下进行灭菌，灭菌后，调整混合物的湿度为30%，再加入20mlAspergillus nigerATCC 1015菌液，于30℃下发酵10 d，之后进行过滤，收集滤液Ⅲ和固体残余物Ⅲ；4) Dry the solid residue obtained in step 3), then add the culture medium to mix, and then sterilize at 121 °C. After sterilization, adjust the humidity of the mixture to 30%, and then add 20ml ofAspergillus niger ATCC 1015 bacterial liquid, Ferment at 30°C for 10 days, then filter to collect filtrate III and solid residue III;
培养液的配方为：30 mg H3BO3、20 mg MnCl2.4H2O、185 mg ZnSO4•7H2O、20 mgNa2MoO4•2H2O, 280 mg FeSO4•H2O, 200 mg CuSO4, 5 g NH4NO3, 1 g KH2PO4,0.15 gMgSO4•H2O, 0.11 g CaCl2 and 1 L ddH2O,pH 为7.0；The formula of the culture medium is: 30 mg H3 BO3 , 20 mg MnCl2.4H2O, 185 mg ZnSO4•7H2 O, 20 mgNa2 MoO4 •2H2 O, 280 mg FeSO4 •H2 O, 200 mg CuSO4 , 5 g NH4 NO3 , 1 g KH2 PO4 , 0.15 gMgSO4 H2 O, 0.11 g CaCl2 and 1 L ddH2 O, pH 7.0;
5）用ddH2O对步骤4）所得固体残余物进行清洗，之后将其烘干，以固液质量比为1:2，将烘干后的固体残余物Ⅲ与氨水混合，在高压下（0.1MPa），于90 ℃反应30 min，之后进行过滤，收集滤液Ⅳ和固体残余物Ⅳ；5) Wash the solid residue obtained in step 4) with ddH2 O, and then dry it. With a solid-to-liquid mass ratio of 1:2, mix the dried solid residue III with ammonia water, and dry it under high pressure ( 0.1MPa), react at 90°C for 30 min, then filter to collect filtrate IV and solid residue IV;
6)将步骤5）所得固体残余物Ⅳ于900ml的双蒸水中浸泡2 h，用1%的H2SO4调节pH为7.0，用旋转蒸发仪对其进行浓缩，再于60℃下将其烘干。然后配制酶解混合物，每500 ml该酶解混合物由下述物质组成：50 g烘干了的固体残余物Ⅳ、10g纤维素酶、100 ml OPTMASHBG、5 mg氯霉素和2.5 mg卡那霉素；调整酶解混合物的pH为5.0，在温度为55℃，摇床转速为220rpm下，反应60 h，之后进行过滤，收集滤液Ⅴ和固体残余物Ⅴ。6) Soak the solid residue IV obtained in step 5) in 900ml of double-distilled water for 2 h, adjust the pH to 7.0 with 1% H2 SO4 , concentrate it with a rotary evaporator, and dilute it at 60°C drying. Then prepare enzymatic hydrolysis mixture, every 500 ml of this enzymatic mixture is composed of the following substances: 50 g dried solid residue IV, 10 g cellulase, 100 ml OPTMASHBG, 5 mg chloramphenicol and 2.5 mg kanamycin element; adjust the pH of the enzymolysis mixture to 5.0, react for 60 h at a temperature of 55° C. and a shaker speed of 220 rpm, and then filter to collect the filtrate V and solid residue V.
各步骤的固体回收率见表2。The solid recovery rate of each step is shown in Table 2.
实施例3Example 3
1）将500g甘蔗进行切段至长度为5-10cm，在65±2℃下烘干2天，然后进行粉碎，过20目筛，得到粒径小于830μm的甘蔗渣粉；1) Cut 500g of sugarcane to a length of 5-10cm, dry at 65±2°C for 2 days, then pulverize and pass through a 20-mesh sieve to obtain bagasse powder with a particle size of less than 830μm;
2）将步骤1）所得物于常温下在2L的水中浸泡12h，之后用蒸汽预加热45s,然后加入液态热水，在200±3℃下，反应1h，反应后进行过滤，收集滤液Ⅰ和固体残余物Ⅰ；2) Soak the product obtained in step 1) in 2L of water at room temperature for 12 hours, then preheat with steam for 45 seconds, then add liquid hot water, react at 200±3°C for 1 hour, filter after reaction, collect filtrate I and solid residue I;
3）调整步骤2）所得固态残余物的湿度为40%，并置于三颈圆底烧瓶中，然后通入氮气排除氧气，于4 ℃下放置10h，之后将反应器置于微波热解炉中在120 ℃下反应7 min，排出热气，待温度降到室温后，用90%乙醇对反应所得物进行浸洗，之后进行过滤，收集滤液Ⅱ和固体残余物Ⅱ；3) Adjust the humidity of the solid residue obtained in step 2) to 40%, and place it in a three-necked round-bottomed flask, then pass through nitrogen to exclude oxygen, and place it at 4 °C for 10 hours, and then place the reactor in a microwave pyrolysis furnace React at 120 °C for 7 min, discharge the hot gas, soak the reaction product with 90% ethanol after the temperature drops to room temperature, and then filter to collect the filtrate II and solid residue II;
4）烘干步骤3）所得固体残余物，之后加入培养液进行混合，再于121 ℃下进行灭菌，灭菌后，调整混合物的湿度为30%，再加入20mlPenicillium janthinellumATCC44750菌液，于30℃下发酵10 d，之后进行过滤，收集滤液Ⅲ和固体残余物Ⅲ；4) Dry the solid residue obtained in step 3), then add the culture medium to mix, and then sterilize at 121 °C. After sterilization, adjust the humidity of the mixture to 30%, and then add 20ml ofPenicillium janthinellum ATCC44750 bacteria solution, Ferment at 30°C for 10 days, then filter to collect filtrate III and solid residue III;
培养液的配方为：30 mg H3BO3、20 mg MnCl2.4H2O、185 mg ZnSO4•7H2O、20 mgNa2MoO4•2H2O, 280 mg FeSO4•H2O, 200 mg CuSO4, 5 g NH4NO3, 1 g KH2PO4,0.15 gMgSO4•H2O, 0.11 g CaCl2 and 1 L ddH2O,pH 为7.0；The formula of the culture medium is: 30 mg H3 BO3 , 20 mg MnCl2.4H2O, 185 mg ZnSO4•7H2 O, 20 mgNa2 MoO4 •2H2 O, 280 mg FeSO4 •H2 O, 200 mg CuSO4 , 5 g NH4 NO3 , 1 g KH2 PO4 , 0.15 gMgSO4 H2 O, 0.11 g CaCl2 and 1 L ddH2 O, pH 7.0;
5）用ddH2O对步骤4）所得固体残余物进行清洗，之后将其烘干，以固液质量比为1:2，将烘干后的固体残余物Ⅲ与氨水混合，在高压下（0.1MPa），于90 ℃反应30 min，之后进行过滤，收集滤液Ⅳ和固体残余物Ⅳ；5) Wash the solid residue obtained in step 4) with ddH2 O, and then dry it. With a solid-to-liquid mass ratio of 1:2, mix the dried solid residue III with ammonia water, and dry it under high pressure ( 0.1MPa), react at 90°C for 30 min, then filter to collect filtrate IV and solid residue IV;
6)将步骤5）所得固体残余物Ⅳ于900ml的双蒸水中浸泡2 h，用1%的H2SO4调节pH为7.0，用旋转蒸发仪对其进行浓缩，再于60℃下将其烘干。然后配制酶解混合物，每500 ml该酶解混合物由下述物质组成：50 g烘干了的固体残余物Ⅳ、10g纤维素酶、100 ml OPTMASHBG、5 mg氯霉素和2.5 mg卡那霉素；调整酶解混合物的pH为5.0，在温度为55℃，摇床转速为220rpm下，反应60 h，之后进行过滤，收集滤液Ⅴ和固体残余物Ⅴ。6) Soak the solid residue IV obtained in step 5) in 900ml of double-distilled water for 2 h, adjust the pH to 7.0 with 1% H2 SO4 , concentrate it with a rotary evaporator, and dilute it at 60°C drying. Then prepare enzymatic hydrolysis mixture, every 500 ml of this enzymatic mixture is composed of the following substances: 50 g dried solid residue IV, 10 g cellulase, 100 ml OPTMASHBG, 5 mg chloramphenicol and 2.5 mg kanamycin element; adjust the pH of the enzymolysis mixture to 5.0, react for 60 h at a temperature of 55° C. and a shaker speed of 220 rpm, and then filter to collect the filtrate V and solid residue V.
各步骤的固体回收率见表3。The solid recovery rate of each step is shown in Table 3.
实施例4Example 4
1）分步糖化发酵法：分别将实施例1、实施例2和实施例3当中的滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、滤液Ⅳ和滤液Ⅴ合并并浓缩。各实施例中，合并后的滤液的糖成分如表4所示。1) Step-by-step saccharification and fermentation method: the filtrate I, filtrate II, filtrate III, filtrate IV and filtrate V in Example 1, Example 2 and Example 3 were combined and concentrated. In each embodiment, the sugar components of the combined filtrate are shown in Table 4.
将150ml合并后的滤液置于250ml规格的无氧瓶中，并用丁基胶塞封住；加入0.75g酵母提取物和1.5g蛋白胨，用1%的NaOH调节pH至6.5，于115℃灭菌20 min后，降温至常温；再加入0.5 ml灭菌后的的混合溶液，该混合溶液含有50g/L KH2PO4、50g/L K2HPO4、220g/LCH3COONH4、0.1g/L对氨基苯甲酸、0.1g/L维生素B、0.001g/L维生素H、20 g/L MgSO4 •7H2O、1 g/L MnSO4 •H2O、1 g/L FeSO4 •7H2O和1 g/L NaCl；然后发酵至OD600=1.5。Put 150ml of the combined filtrate into a 250ml anaerobic bottle and seal it with a butyl rubber stopper; add 0.75g of yeast extract and 1.5g of peptone, adjust the pH to 6.5 with 1% NaOH, and sterilize at 115°C After 20 min, cool down to normal temperature; then add 0.5 ml sterilized mixed solution containing 50g/L KH2 PO4 , 50g/L K2 HPO4 , 220g/LCH3COONH4, 0.1g/L p-aminobenzene Formic acid, 0.1g/L vitamin B, 0.001g/L vitamin H, 20 g/L MgSO4 • 7H2 O, 1 g/L MnSO4 • H2 O, 1 g/L FeSO4 • 7H2 O and 1 g/L NaCl; then ferment to OD600=1.5.
2）同步糖化发酵法：分别将实施例1、实施例2和实施例3当中的滤液Ⅰ、滤液Ⅱ、滤液Ⅲ、和滤液Ⅳ合并，调节pH至7.0。各实施例中，合并后的滤液的成分如表5所示。2) Simultaneous saccharification and fermentation method: Combine the filtrate I, filtrate II, filtrate III, and filtrate IV in Example 1, Example 2, and Example 3, respectively, and adjust the pH to 7.0. In each embodiment, the composition of the combined filtrate is shown in Table 5.
发酵步骤与酶解同时进行，所加微量元素和发酵条件与分布糖化发酵法一致。发酵温度为37℃，发酵时间为96h，在发酵过程中，隔一段时间，用无菌注射器穿过橡胶塞进行取样，所取样品在4℃，8000rpm下离心20min，然后进行糖、有机酸、pH和ABE生产的检测。The fermentation step is carried out simultaneously with the enzymolysis, and the added trace elements and fermentation conditions are consistent with the distributed saccharification fermentation method. The fermentation temperature is 37°C, and the fermentation time is 96h. During the fermentation process, a sterile syringe is used to pass through the rubber stopper for sampling at intervals. The sample is centrifuged at 4°C and 8000rpm for 20min, and then the sugar, organic acid, Detection of pH and ABE production.
实施例5Example 5
抑制剂含量的检测：在DIONEX UltiMate 3000 液相色谱仪，利用Aminex HPX-87H色谱柱对丁酸、醋酸、葡糖醛酸、对香豆酸、丁香酸和阿魏酸含量进行检测；呋喃甲醛和5-羟甲基糠醛的测定参考“Determination of 5-Hydroxymethylfurfural in VinegarSamples by HPLC”（Theobald A, Müller A, Anklam E. J. Agric. Food. Chem. 1998,46(5):1850-1854.）中的方法。Detection of inhibitor content: In DIONEX UltiMate 3000 liquid chromatograph, use Aminex HPX-87H chromatographic column to detect the content of butyric acid, acetic acid, glucuronic acid, p-coumaric acid, syringic acid and ferulic acid; and 5-Hydroxymethylfurfural refer to "Determination of 5-Hydroxymethylfurfural in VinegarSamples by HPLC" (Theobald A, Müller A, Anklam E. J. Agric. Food. Chem. 1998,46(5):1850-1854.) method.
经所有预处理步骤后，实施例1-3中各抑制剂含量见表6。After all pretreatment steps, the contents of each inhibitor in Examples 1-3 are shown in Table 6.
对比实施例comparative example
与实施例4相同发酵条件下，利用纯葡萄糖或糖组合物（10g/L葡萄糖、42g/L木糖、3.5g/L阿拉伯糖、3.5g/L纤维二糖、3g/L半乳糖和1.5g/L甘露糖）替代预处理的水解产物（滤液）。Under the same fermentation conditions as in Example 4, using pure glucose or sugar composition (10g/L glucose, 42g/L xylose, 3.5g/L arabinose, 3.5g/L cellobiose, 3g/L galactose and 1.5 g/L mannose) to replace the pretreated hydrolyzate (filtrate).
实施例6Example 6
发酵产物检测：醇类物质（丙酮、丁醇、乙醇）利用气相色谱仪检测；总固体残余物碳水化合物的检测参考 “Carbohydrate analysis by a phenol–sulfuric acid methodin microplate format”（Masuko T, Minami A, Iwasaki N, Majima T, Nishimura S-I,Lee YC: Anal. Biochem. 2005, 339(1):69-72.）中的方法；纤维素含量利用分光光度计进行检测；木质素含量的检测参考“Determination of lignin in herbaceous plants byan improved acetyl bromide procedure”（Iiyama K, Wallis AFA J. Sci. FoodAgric. 1990, 51(2):145-161.）中的方法；灰分含量通过在高温烘炉中于500℃烘干后检测。Detection of fermentation products: Alcohols (acetone, butanol, ethanol) were detected by gas chromatography; the detection of total solid residue carbohydrates was referred to "Carbohydrate analysis by a phenol–sulfuric acid methodin microplate format" (Masuko T, Minami A, The method in Iwasaki N, Majima T, Nishimura S-I, Lee YC: Anal. Biochem. 2005, 339(1):69-72.); the cellulose content is detected by spectrophotometer; the detection of lignin content refers to "Determination of lignin in herbaceous plants by an improved acetyl bromide procedure” (Iiyama K, Wallis AFA J. Sci. FoodAgric. 1990, 51(2):145-161.); Check after drying.
发酵过程中，发酵产物各参数指标见表7。During the fermentation process, the parameters of the fermentation products are shown in Table 7.
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