混合固态发酵降解甘蔗渣产糖产乙醇研究

以碱处理甘蔗渣为原料,比较里氏木霉(Trichoderma reesei CICC40359)和斜卧青霉(Penicillium decumbens LSM-1)单菌和混菌固态发酵及转化乙醇效果,研究发酵过程菌体生长、产酶产糖和乙醇转化情况。结果表明:混菌发酵效果优于单菌,在接种量8%,发酵温度30℃,混菌固态发酵(SSF)72h后总糖和还原糖产量最大值为20.21 g/L和12.47 g/L;β-葡萄糖苷酶活力和菌体生物量在144 h后分别达到0.48 IU/m L和0.21 g/g DM;对发酵3 d后底物(包括生成糖、合成酶及未降解基质)接种酵母进行乙醇同步糖化发酵,乙醇浓度在发酵24 h时达到5.83 g/L,发酵效率达到理论值的40.84%。利用多菌混合固态发酵转化底物产乙醇能避免传统乙醇生产过程高成本纤维素酶的应用,为纤维乙醇生产提供一条经济有效的新途径。     

利用木糖产乙醇的真菌筛选及发酵条件优化

以木糖为唯一碳源,从高、中温酒曲中分离到16株能利用木糖的丝状真菌;通过发酵试验复筛,获得一株能产乙醇的丝状真菌Z7;综合形态学和ITS序列分析,初步鉴定为Aspergillus flavus。通过单因素试验确定最佳氮源和发酵温度;通过正交试验和SPSS软件分析得到了不同N、P、K成分对乙醇、残糖和菌体干重的影响,获得最佳的发酵条件为:尿素1g/L,NH_4NO_3 1g/L,K_2 HPO_4 2g/L,KCl 0.5g/L,MgSO_4·7H_2O 0.5g/L,NaNO_3 1g/L,pH自然,培养温度33℃。以玉米芯半纤维素水解液为底物进行乙醇发酵,根据稀酸水解的单糖释放量和乙醇产量,确定115℃,1h为最佳玉米芯预处理条件;添加1 g/L的吐温20能获得最大的乙醇浓度8.31 g/L。因此,Aspergillus flavusZ7能利用半纤维素水解产物产乙醇,其中木糖的利用率在80%以上。     

酿酒酵母代谢有机酸对乙醇发酵的影响

发酵过程中酵母代谢产生的有机酸会影响发酵效率。以葡萄糖为底物,在自动发酵罐中进行了酿酒酵母间歇乙醇发酵实验,研究了发酵过程中酵母的主要代谢副产物中有机酸的种类及其对乙醇发酵的影响。结果表明,发酵过程中酵母代谢的主要有机酸是琥珀酸、乳酸和乙酸。3种酸的总量随温度(25~40℃)升高或p H增大(3~6)呈上升趋势,最大值可达5.78 g/L,占产物的23.3%。通过外源投加有机酸实验发现,3种代谢有机酸对酵母抑制作用的大小并不完全由其酸性决定,还与其进入细胞的难易程度等相关。通过外源投加有机酸实验结合发酵过程中去除有机酸实验可以确定,对乙醇发酵的影响由大到小依次为乙酸、琥珀酸和乳酸,且三者之间对乙醇发酵无明显的协同抑制效应。乙酸是发酵过程中产生主要抑制作用的代谢有机酸,2 g/L的乙酸可引起菌体浓度明显下降,4 g/L的乙酸即可引起乙醇得率下降86.7%。     

两株高效代谢木质纤维素稀酸水解物产乙醇的酵母特性及耐毒研究

对实验室筛选出的两株高效代谢木质纤维素稀酸水解液产乙醇的酵母菌Y1(Candida tropicalis)和Y4(Issatchenkiaorientalis)的乙醇发酵特性及耐毒能力进行研究。以未经任何脱毒处理的木质纤维素稀酸水解液为发酵底物进行乙醇发酵(原位脱毒乙醇发酵)。结果表明:Y1和Y4均能在24h内将水解液中所有的葡萄糖消耗完,乙醇产率分别为0.49g/g和0.45g/g,分别达到理论值的96.1%和86.0%。在含有不同浓度梯度的糠醛及5-羟甲基糠醛的模拟水解液中,Y1和Y4能耐受的最高糠醛浓度均为5.0g/L及最高的5-羟甲基糠醛浓度均大于7.0g/L,当两种抑制剂等量混合时,两株菌能耐受的最高浓度为4.0g/L,两株菌均有较好的乙醇发酵及耐毒能力。该研究结果为木质纤维素水解液的原位脱毒发酵生产然料乙醇奠定了基础。     

玉米秸秆水解液燃料乙醇发酵条件优化

以实验室前期构建的工业酿酒酵母HN-1-24为出发菌株,研究利用玉米秸秆水解液发酵生产燃料乙醇的培养条件。在单因素实验的基础上,通过Plackett-Burman设计,进行主要影响因子筛选;利用Box-Behnken设计和响应面优化分析,得到主要影响因子的最优组合。结果表明,影响乙醇产量的显著因子是蛋白胨浓度、MgSO_4·7H_2O浓度和初始pH值;三因子最优组合为蛋白胨浓度1.0 g/L、MgSO_4·7H_2O浓度0.6 g/L和初始pH值5.5,确定最适发酵条件。在此条件下,乙醇产量为43 g/L,与模型预测值一致;乙醇产率达到理论值的83%,比优化前提高5%。     

稀硫酸-亚硫酸盐法预处理枯死松木的原位纤维素乙醇生产

报道了稀硫酸-亚硫酸盐法预处理软木的纤维素乙醇生产效率。通过采用一株耐受代谢抑制剂的酿酒酵母菌株Y5,对预处理后的固体酶解物及水解液的混合物进行批式及补料批式同步糖化发酵的研究。结果表明:预处理软木混合物中的所有可发酵糖能够在72h内被Y5全部利用,最后的乙醇浓度达到22.2g/L,相应的乙醇发酵产率为0.44g/g。批式与补料批式SSF,除了补料批式发酵24h内的乙醇生产速率高于批式发酵外,并没有其他明显区别。     

两株高效代谢木质纤维素稀酸水解物产乙醇的酵母特性及耐毒研究

对实验室筛选出的两株高效代谢木质纤维素稀酸水解液产乙醇的酵母菌Y1(Candida tropicalis)和Y4(Issatchenkiaorientalis)的乙醇发酵特性及耐毒能力进行了的研究。以未经任何脱毒处理的木质纤维素稀酸水解液为发酵底物进行乙醇发酵(原位脱毒乙醇发酵)。结果表明,Y1和Y4均能在24h内将水解液中所有的葡萄糖消耗完,乙醇产率分别为0.49g/g和0.45g/g,分别达到了理论值的96.1%和86.0%。在含有不同浓度梯度的糠醛及5-羟甲基糠醛的模拟水解液中,Y1和Y4能耐受的最高糠醛浓度均为5.0g/L,最高的5-羟甲基糠醛浓度均大于7.0g/L,当两种抑制剂等量混合时,两株菌能耐受的最高浓度为4.0g/L。两株菌均有较好的乙醇发酵及耐毒能力。该研究结果为木质纤维素水解液的原位脱毒发酵生产然料乙醇奠定了基础。     

农作物秸秆同步糖化发酵制燃料乙醇条件研究

用农作物秸秆做原料进行同步糖化发酵制取燃料乙醇,同步糖化发酵的温度不协调以及单批次同步糖化发酵原料用量影响乙醇产量等问题始终制约着工艺的应用。文中进行了主要农作物玉米秸秆和稻草秸秆的3种预处理方式同步糖化发酵和同步糖化发酵工艺过程的补料试验研究。试验结果显示,稀酸预处理稻草粉在液固比为8∶1时,同步糖化发酵效果最好,乙醇含量为11.16 g/L,残糖浓度最低为12.07 g/L;补料方式H下乙醇浓度达到最大值10.09 g/L,此补料方式下添加吐温-80、混合菌种时的乙醇产率变化不明显。     

玉米秸秆酶解糖化液乙醇发酵的工艺优化

利用从4组混合乙醇酵母中筛选出的优势混合酵母,对玉米秸秆酶解糖化液的乙醇发酵工艺过程进行了优化试验。试验结果表明,管囊酵母和酿酒酵母组成的混合酵母具有较高的乙醇发酵能力,经60 h发酵,乙醇浓度最高可达12.55 g/L,乙醇产率为最大理论值的68.63%。根据对糖化液乙醇发酵的二次回归正交组合优化试验,当发酵温度为28.0℃,初始pH为5.2,接种量为8.1%时,实际乙醇浓度最高可达13.03g/L,乙醇产率为0.36 g/g,为最大理论值的70.59%,与所得乙醇发酵回归方程预测值基本相符。     

中心复合实验优化嗜单宁管囊酵母发酵生产乙醇的研究(Ⅰ)

采用四因素三水平中心复合实验法优化嗜单宁管囊酵母(Pachysolen tannophilus)发酵生产乙醇的条件,根据实验数据拟合建立关于乙醇浓度随发酵时间、接种量、转速、发酵温度等因素变化的数学模型。根据该模型进行工艺参数的优化,以乙醇浓度为指标,实验所得的嗜单宁管囊酵母发酵生产乙醇的优化工艺条件为:发酵时间68h,接种量6%,转速120r/min,发酵温度32℃。该条件下乙醇产量为20.58g/L,残葡萄糖浓度为0.119g/L。对模型的有效性进行检验。     

Cell degeneration and performance decline of immobilized Clostridium acetobutylicum on bagasse during hydrogen and butanol production by repeated cycle fermentation

The cell degeneration and the fermentation performance decline during repeated cycle fermentation with immobilized Clostridium acetobutylicum on bagasse for hydrogen and butanol production was studied. The cell degeneration has been characterized in abnormality of a long-chain morphology through 7 cycles of repeated fermentation. The fermentation performance decline has been indicated by decrease of glucose consumption rate from 0.82 g/L/h to 0.22 g/L/h, reduction of hydrogen production and productivity from 6 L/L to 2.5 L/L and 170 mL/L/h to 40 mL/L/h, as well as the decrease of butanol production and butanol productivity from 6.5 g/L to 1.0 g/L and 0.18 g/L/h to 0.02 g/L/h, respectively. The ratio of hydrogen production and butanol production was in the range of 6–10 and 17–20 during the earlier three and later four cycles, respectively. The carbon flow directed to ethanol was higher during the later period of fermentation.     

The utilization of acid-tolerant bacteria on ethanol production from kitchen garbage

In order to achieve ethanol production from kitchen garbage under non-sterilized fermentation, the acid-tolerant Zymomonas mobilis named GZNS1 was selected and applied in the fermentation system. Ethanol production from kitchen garbage under non-sterilized fermentation with GZNS1 was proved to be feasible. The utilization of control strain and acid-tolerant strain under different conditions demonstrated that the sequence of ethanol yield was followed: sterilized garbage with control strain inoculated under pH of 6 (52 g/L) ≈ sterilized garbage with GZNS1 inoculated under pH of 4 (48 g/L) > non-sterilized garbage with GZNS1 inoculated under pH of 4 (46 g/L). Furthermore, the distillery waste during fermentation was adopted to recycle fermentation and acquired 50 g/L ethanol, higher than those adjusted with tap water. The utilization of acid-tolerant bacteria combing with the utilization of distillery waste associated with the process can increase ethanol production, save energy and reduce the cost of ethanol production.     

Fermentative bioenergy production from distillers grains using mixed microflora

The feasibility of hydrogen production from distillers grains substrate, an industrial cellulosic waste, was investigated. A substrate concentration of 80 g/L gave the maximum production at 50 °C and pH of 6.0 using sewage sludge. Four controllable factors with three levels: seed sludge (two sewage sludges and cow dung), temperature (40, 50, and 60 °C), pH (6, 7 and 8) and seed pretreatment (none, heat, and acid) were selected in Taguchi experimental design to optimize fermentation conditions. The peak hydrogen and ethanol productions were found with heat-treated cow dung seed, substrate concentration 80 g/L, 50 °C and pH 6. The peak hydrogen production rate and hydrogen yield were 7.9 mmol H₂/L/d and 0.40 mmol H₂/g-COD respectively whereas the peak ethanol production was 3050 mg COD/L and rate 0.22 g EtOH/L/d. A total bioenergy yield of 41 J/g substrate was obtained which was 21% and 79% from hydrogen and ethanol respectively.     

Optimization of bioethanol production from glycerol by Escherichia coli SS1

Bioethanol is a promising biofuel and has a lot of great prospective and could become an alternative to fossil fuels. Ethanol fermentation using glycerol as carbon source was carried out by local isolate, ethanologenic bacterium Escherichia coli SS1 in a close system. Factors affecting bioethanol production from pure glycerol were optimized via response surface methodology (RSM) with central composite design (CCD). Four significant variables were found to influence bioethanol yield; initial pH of fermentation medium, substrate concentration, salt content and organic nitrogen concentration with statistically significant effect (p ≤ 0.05) on bioethanol production. The significant factor was then analyzed using central composite design (CCD). The optimum conditions for bioethanol production were substrate concentration at 34.5 g/L, pH 7.61, and organic nitrogen concentration at 6.42 g/L in which giving ethanol yield approximately 1.00 mol/mol. In addition, batch ethanol fermentation in a 2 L bioreactor was performed at the glycerol concentration of 20 g/L, 35 g/L and 45 g/L, respectively. The ethanol yields obtained from all tested glycerol concentrations were approaching theoretical yield when the batch fermentation was performed at optimized conditions.     

Ethanol production by repeated batch and continuous fermentations of blackstrap molasses using immobilized yeast cells on thin-shell silk cocoons

A thin-shell silk cocoon (TSC), a residual from the silk industry, is used as a support material for the immobilization of Saccharomyces cerevisiae M30 in ethanol fermentation because of its properties such as high mechanical strength, light weight, biocompatibility and high surface area. In batch fermentation with blackstrap molasses as the main fermentation substrate, an optimal ethanol concentration of 98.6 g/L was obtained using a TSC-immobilized cell system at an initial reducing sugar concentration of 240 g/L. The ethanol concentration produced by the immobilized cells was 11.5% higher than that produced by the free cells. Ethanol production in five-cycle repeated batch fermentation demonstrated the enhanced stability of the immobilized yeast cells. Under continuous fermentation in a packed-bed reactor, a maximum ethanol productivity of 19.0 g/(L h) with an ethanol concentration of 52.8 g/L was observed at a 0.36 h⁻¹ dilution rate.     

木质纤维素稀酸水解液乙醇发酵的新方法

为了降低木质纤维素水解液发酵抑制剂对乙醇发酵的负影响,采用混合菌种对木质纤维素稀酸水解液乙醇发酵方式进行了研究。对批式发酵、补料批式发酵和间隔补料批式发酵3种发酵方式进行了比较。实验结果表明,间隔补料批式发酵可以有效地减弱水解液中抑制因子对菌种的影响,乙醇产量明显高于其他两种发酵方式,利用酿酒酵母(Saccaromyces cerevisiae 2.535)和嗜鞣管囊酵母(Pachysolen tannophilis ATCC 32728)混合发酵,乙醇产量最终达到14.4g/L,乙醇产率(Yp/s)为0.47g/g,相当于最大理论产率的92.2%。利用酿酒酵母和重组大肠杆菌混合菌种发酵,乙醇产量达到了14.5g/L。对木质纤维素稀酸水解液采用间隔补料批式乙醇发酵方法,可进一步减少抑制剂对乙醇发酵的影响,使发酵顺利进行。     

Ethanol production from mahula (Madhuca latifolia L.) flowers with immobilized cells of Saccharomyces cerevisiae in Luffa cylindrica L. sponge discs

The dried spongy fruit of luffa (Luffa cylindrica L.), a cucurbitaceous crop available in abundance in tropical and sub-tropical countries has been found to be a promising material for immobilizing microbial cells. The aim of the present study was to examine the ethanol production from mahula flowers in submerged fermentation using whole cells of Saccharomyces cerevisiae immobilized in luffa sponge discs. The cells not only survived but also were physiologically active in three more cycles of fermentation without significant reduction (<5%) in ethanol production. After 96 h, there was 91.1% sugar conversion producing 223.2 g ethanol/kg flowers (1st cycle) which was 0.99%, 2.3% and 3.2% more than 2nd (221 g ethanol/kg flowers), 3rd (218 g ethanol/kg flowers) and 4th (216 g ethanol/kg flowers) cycle of fermentation, respectively. Furthermore, ethanol production by immobilized cells was 8.96% higher than the free cells.     

Property change of bagasse as cell-immobilizing carrier and coproduction of hydrogen-butanol in fixed-bed reactor by repeated cycle fermentation

Property change of bagasse as cell-immobilizing carrier and coproduction of hydrogen-butanol in fixed-bed reactor by repeated cycle fermentation was studied. Bagasse would have potential being used as animal feed after four repeated cycles fermentation lasting for 290 h, because cellulose crystallinity was decreased by 33% and protein content was increased by 187%. Specific surface area and total pore volume of bagasse were also decreased. Maximum accumulated hydrogen production, yield and productivity were 11.7 L/L, 1.89 mol/mol and 537 mL/L/h, respectively. Maximum total solvent (acetone, butanol and ethanol) concentration, solvent yield and solvent productivity were 18.2 g/L 0.31 g/g and 0.78 g/L/h, respectively. Modified Gompertz model was used to describe the hydrogen and butanol production and fitting results showed good agreement with the experimental data. The maximum total product (including hydrogen, acetone, butanol and ethanol) energy and energy conversion efficiency during the 4 cycles fermentation were 758.2 kJ/L and 86.3%, respectively.