木质纤维素稀酸水解糖液乙醇发酵研究进展

以木质纤维素为原料生产燃料乙醇,首先要对原料进行预处理得到可发酵糖,在稀酸水解木质纤维素得到的糖液中,除含有葡萄糖、木糖等六碳糖和五碳糖外,根据水解温度、酸浓度和时间的不同,还含有不同浓度的发酵抑制剂。因此,在研究木质纤维素稀酸水解糖液的乙醇发酵中,对代谢木糖成乙醇的菌种的研究、对耐/代谢发酵抑制剂微生物的研究、对稀酸水解糖液的脱毒方法的研究以及对稀酸水解糖液不同发酵方式的乙醇发酵研究等非常重要。重点介绍了以上几个方面近几年研究的进展。     

产D-乳酸重组大肠杆菌ptsG基因的敲除及其混合糖同步发酵

为构建能够同时高效利用五碳糖和六碳糖发酵产D-乳酸的重组大肠杆菌工程菌,以能高效利用五碳糖发酵产D-乳酸的大肠杆菌工程菌E.coli JH13为出发菌株,通过Red同源重组技术敲除葡萄糖跨膜转运基因pts G。实验结果表明,pts G缺陷菌株E.coli JH15在10%混合糖(5%葡萄糖和5%木糖)培养基中发酵,可同时利用五碳糖和六碳糖以完成发酵;而对照菌葡萄糖消耗完才利用木糖,发酵结束还有18 g/L木糖残留;JH15乳酸产量为83.04 g/L,相比于对照菌株提高了25.86%;在稻草秸秆水解液中发酵,JH15同时利用葡萄糖、木糖和L-阿拉伯糖,乳酸产量为25.15 g/L,转化率为86.42%。JH15作为能利用混合糖同步发酵产D-乳酸的大肠杆菌工程菌,它的成功构建为利用廉价的木质纤维素水解物为原料发酵生产D-乳酸提供参考依据。     

酿酒酵母木糖转运基因研究进展

由于对全球变暖等日益严重的环境问题的担忧,生产生物乙醇等清洁能源的技术正受到世界各国越来越多的关注。较之以粮食为原料生产乙醇,木质纤维素生产生物乙醇具有更大的发展潜力,因其来源广泛,廉价且可再生。以木质纤维素生产生物乙醇已经取得长足进步,但仍面临几个主要问题,比如天然酿酒酵母不能利用木糖发酵乙醇,木质纤维素酶成本过高,木质纤维素预处理环节成本高等。已经有基因改造的酵母菌株可以利用戊糖和己糖进行生物乙醇生产。然而,这些菌株对木糖的利用效率很低。这主要是因为酿酒酵母缺乏高效的特异性木糖转运基因,木糖运输依赖已糖转运基因。为了提高木糖利用速度,已有不少方法成功应用于构建重组酵母细胞。现对酵母木糖转运基因的最新研究进展进行简要概述。     

利用木糖-葡萄糖为原料产乙醇酵母的筛选、鉴定及发酵试验

建立筛选利用木糖为碳源产乙醇酵母模型,获得一株适合利用木质纤维素为原料产乙醇的酵母菌株。样品经麦芽汁培养基培养后,以木糖为唯一碳源的筛选培养基初筛,再以重铬酸钾显色法复筛。通过生理生化和26D1/D2区对筛选得到的菌株进行分析和鉴定,该菌初步鉴定为Pichia caribbica。经过筛选得到的菌株Y2-3以木糖（40g/L）为唯一碳源发酵时：生物量为23.5g/L,木糖利用率为94.7 %,乙醇终产量为4.57 g/L;以混合糖（葡萄糖40 g/L,木糖20 g/L）发酵时：生物量为28.6 g/L,木糖利用率为94.2 %,葡萄糖利用率为95.6%,乙醇终产量为20.6 g/L。Pichia caribbica是可以转化木糖及木糖-葡萄糖混合糖为乙醇的酵母菌株,为利用木质纤维素发酵乙醇的进一步研究奠定了基础。     

麦芽糖假丝酵母菌混合糖代谢的发酵特性

葡萄糖和木糖的混合糖共代谢是木质纤维素资源高效转化利用的关键环节。利用本实验室保藏的天然木糖利用酵母菌,麦芽糖假丝酵母(Candida maltosa)Xu316,本研究对该菌代谢利用不同比例的葡萄糖、木糖发酵特性进行了系统测试,总结了麦芽糖假丝酵母混合糖代谢的一般规律。研究结果表明麦芽糖假丝酵母菌具有较高的葡萄糖利用率和木糖醇积累能力。在糖浓度低于20%时,该菌可以共同利用葡萄糖和木糖,最大乙醇产量和木糖醇产量分别为0.43g·g-1和0.58g·g-1,具有工业应用生产生物基产品的潜力。     

中性糖在土壤中的来源与分布特征

糖类(即碳水化合物)是土壤有机质的重要组成部分, 经生物化学降解形成不同结构的单糖。土壤中的中性单糖也叫中性糖, 主要包括木糖、核糖、阿拉伯糖、葡萄糖、半乳糖、甘露糖、岩藻糖和鼠李糖。其中, 植物来源的糖主要为五碳糖, 如木糖和阿拉伯糖; 微生物来源的糖主要包括半乳糖、甘露糖、岩藻糖、鼠李糖等六碳糖。研究中常利用六碳糖和五碳糖的比例指示微生物和植物对土壤有机碳的相对贡献。中性糖是微生物重要的碳源和能量来源, 在团聚体的形成过程中扮演着重要角色。该文整合了近30年土壤中性糖的研究进展, 对比了提取中性糖的常用方法, 分析了不同土地利用类型和不同土壤组分中中性糖的含量、来源和周转特征, 综述了影响中性糖含量和分布的主要环境因素。结果表明, 中性糖在耕地土壤中的绝对含量和相对含量均显著低于针叶林、阔叶林、草地和灌丛4种土地利用类型。(半乳糖+甘露糖)/(阿拉伯糖+木糖)(GM/AX)在不同土地利用间差异不显著, 而(鼠李糖+岩藻糖)/(阿拉伯糖+木糖)(RF/AX)则表明草地土壤中的微生物来源的中性糖含量高于针叶林和耕地。不同密度的土壤组分中, 轻质组分中中性糖的含量比重质组分高, 重质组分中微生物来源的中性糖较多; 就不同粒径(或团聚体)而言, 黏粒(或微团聚体)中微生物来源的中性糖含量更丰富。有关影响土壤中性糖含量和分布的因素的研究, 目前主要集中在人为活动(如耕种和放牧等), 而有关温度、降水等自然环境因素影响的研究较少。     

一株葡萄糖和木糖共发酵高效产乙醇重组运动发酵单胞菌的性能评价

木糖是木质纤维素原料水解液中的第二大组分,木糖和葡萄糖的充分利用是有经济性地生产纤维素乙醇的关键。通过基因克隆手段构建了一株可以高效利用木糖产乙醇的重组运动发酵单胞菌Zymomonas mobilis TSH01,并进行了利用单糖溶液、混合糖溶液及玉米秸秆水解液发酵产乙醇效率的研究。结果表明,利用单一葡萄糖或单一木糖溶液发酵时,当糖浓度为8%、发酵72 h后,糖利用率分别为100%和98.9%,乙醇代谢收率分别为87.8%和78.3%;利用8%葡萄糖和8%木糖的混合溶液发酵时,72 h后,葡萄糖和木糖的利用率分别为98.5%和97.4%,乙醇代谢收率为94.9%。利用含3.2%葡萄糖和3.5%木糖的玉米秸秆水解液发酵72 h后,葡萄糖和木糖的利用率分别为100%和92.3%,乙醇代谢收率为91.5%。此外,磷酸二氢钾对发酵过程中木糖利用率以及乙醇收率的提高有明显促进作用。     

中性糖在土壤中的来源与分布特征

糖类(即碳水化合物)是土壤有机质的重要组成部分,经生物化学降解形成不同结构的单糖。土壤中的中性单糖也叫中性糖,主要包括木糖、核糖、阿拉伯糖、葡萄糖、半乳糖、甘露糖、岩藻糖和鼠李糖。其中,植物来源的糖主要为五碳糖,如木糖和阿拉伯糖;微生物来源的糖主要包括半乳糖、甘露糖、岩藻糖、鼠李糖等六碳糖。研究中常利用六碳糖和五碳糖的比例指示微生物和植物对土壤有机碳的相对贡献。中性糖是微生物重要的碳源和能量来源,在团聚体的形成过程中扮演着重要角色。该文整合了近30年土壤中性糖的研究进展,对比了提取中性糖的常用方法,分析了不同土地利用类型和不同土壤组分中中性糖的含量、来源和周转特征,综述了影响中性糖含量和分布的主要环境因素。结果表明,中性糖在耕地土壤中的绝对含量和相对含量均显著低于针叶林、阔叶林、草地和灌丛4种土地利用类型。(半乳糖+甘露糖)/(阿拉伯糖+木糖)(GM/AX)在不同土地利用间差异不显著,而(鼠李糖+岩藻糖)/(阿拉伯糖+木糖)(RF/AX)则表明草地土壤中的微生物来源的中性糖含量高于针叶林和耕地。不同密度的土壤组分中,轻质组分中中性糖的含量比重质组分高,重质组分中微生物来源的中性糖较多;就不同粒径(或团聚体)而言,黏粒(或微团聚体)中微生物来源的中性糖含量更丰富。有关影响土壤中性糖含量和分布的因素的研究,目前主要集中在人为活动(如耕种和放牧等),而有关温度、降水等自然环境因素影响的研究较少。     

解脂耶氏酵母基于木质纤维素原料生产化学品研究进展*

解脂耶氏酵母具有遗传背景清晰、分子操作体系较为成熟、抗逆性强、底物谱广、有机酸和蛋白质分泌能力强等优点,在微生物发酵生产化学品领域极具应用潜力。木质纤维素是丰富的可再生生物质资源,以木质纤维素原料替代化石原料生产化学品对于缓解全球能源危机、保障粮食安全等意义重大。解脂耶氏酵母可以天然代谢木质纤维素水解产生的葡萄糖,但对其他水解产物(如木糖)的利用效率极低。综述解脂耶氏酵母利用木质纤维素原料的代谢途径及改造策略,以木质纤维素原料生产化学品为例,重点讨论该过程中的主要瓶颈问题及解决办法,为后续研究提供参考。     

有机碳源对三角褐指藻生长、胞内物质和脂肪酸组分的影响

研究了3种有机碳对三角褐指藻生长、胞内物质和脂肪酸组分的影响。结果表明, 三角褐指藻具有利用有机碳进行兼养生长的能力, 生长速率加快, 倍增时间缩短, 生物量显著提高, 100 mmol/L甘油兼养的生物量最高(713 mg/L), 是自养(460 mg/L)的1.60倍, 乙酸钠和葡萄糖兼养的生物量分别是自养的1.28倍和1.21倍。兼养下蛋白质含量较自养明显下降, 碳水化合物和总脂含量高于自养, 乙酸钠和甘油兼养的总脂含量分别是自养的1.43倍和1.20倍, 葡萄糖兼养的总脂含量与自养无明显差异。3种有机碳兼养的饱和脂肪酸和单不饱和脂肪酸占总脂肪酸的比例增大, 多不饱和脂肪酸比例降低, EPA(eicosapentaenoic acid)比例降低, 乙酸钠兼养的胞内EPA含量(6.23%)和产量(36.59 mg/L)均高于自养, 分别是自养的1.10倍和1.40倍, 甘油和葡萄糖兼养的EPA含量和产量均低于自养。     

双碳源单细胞蛋白间歇培养及流加培养的动力学模型

应用非结构的逻辑增殖模型研究了两种酵母的单碳源和双碳源单细胞蛋白间歇培养的动力学,用改进的逻辑增殖模型研究了双碳源流加培养过程的动力学,从实验数据拟合了动力学模型参数,模型计算值与实验数据吻合良好。     

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P_FMD-GFP) (220 clones) and vector pC10-MOX (P_MOX-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.     

Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol

Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.     

Cell engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> allows high cell density accumulation without fed-batch process control

A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h⁻¹, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production, oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment. The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch performance.     

Ethanol effect on batch and fed-batch Arthrospira platensis growth

The ability of Arthrospira platensis to use ethanol as a carbon and energy source was investigated by batch process and fed-batch process. A. platensis was cultivated under the effect of a single addition (batch process) and a daily pulse feeding (fed-batch process) of pure ethanol, at different concentrations, to evaluate cell concentration (X) and specific growth rate (μ). A marked increase was observed in the cell concentration of A. platensis in runs with ethanol addition when compared to control cultures without ethanol addition. The fed-batch process using an ethanol concentration of 38 mg L^?1 days^?1 reached the maximum cell concentration of 2,393 ± 241 mg L^?1, about 1.5-fold that obtained in the control culture. In all experiments, the maximum specific growth rate was observed in the early exponential phase of cell growth. In the fed-batch process, μ decreased more slowly than in the batch process and control culture, resulting in the highest final cell concentration. Ethanol can be used as a feasible carbon and energy source for A. platensis growth via a fed-batch process.     

Experimental and modelling study of different process modes for retroviral production in a fixed bed reactor

Pseudotype vectors are promising for gene transfer in many gene therapy approaches, however, low-vector concentration in batch cultures and high temperature-dependent decay do limit sufficiently large-scale production. To overcome these obstacles, the kinetic relations of cell growth and vector formation in different culture modes need to be understood. Effective optimisation of process modes is needed to achieve sufficient yields. Experimental and modelling studies were carried out in order to analyse the impact of different process modes such as perfusion, perfused fed-batch or repeated-batch on vector titer and productivity. Retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1) were produced using a 200 ml fixed bed reactor for high cell density cultivation on macroporous carriers. After starting the cultivation in batch mode, the reactor was either run in perfusion, perfused fed-batch or repeated-batch. A mathematical model of the bioreaction was developed on the basis of experimental data measured in culture dishes. The ability of the model to describe all different process modes of fixed-bed cultivation without additional fitting of the parameters was proven by three long-term cultivations for more than 400 h. The results of optimisation with the aid of the model, leads to the conclusion that perfusion with optimised harvest cycles and fed flows, result in a higher yield in comparison to batch or fed batch culture.     

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5–76.0% and 13.2–19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m³_biogas/kgCOD_i) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO₂ was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 ± 0.006 g_DM/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Production of flavonoids and polysaccharide by adding elicitor in different cellular cultivation processes of Glycyrrhiza uralensis Fisch

In this study, callus and cell suspension were induced from seedlings of licorice (G. uralensis). In addition, it was revealed that the appropriate concentration of sucrose could promote the callus growth and increase the content of polysaccharide. The methyl jasmonate (MJ) and phenylalanine (PHE) could enhance the callus growth and content of flavonoids for G. uralensis. For producing more flavonoids and polysaccharide, two-stage cultivation was performed. In the first step, 30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day of culture to enhance cell production and metabolite production. In a two-stage cultivation process, PHE (2 mM) and MJ (5 mg L^?1) were added into a 5-L balloon-type bubble bioreactor after 10 days of culture. Using a fed-batch cultivation strategy (30 g L^?1 sucrose was fed into a 5-L balloon-type bubble bioreactor on 8th day), polysaccharide production was enhanced to 1.19 g L^?1, which was 2.12-fold greater than that in batch cultivation. The flavonoids yield (55.42 mg L^?1) which was about 22 % higher than that in batch cultivation was obtained on 21st day. In a two-stage cultivation process, the polysaccharide content was increased by 1.14- and 2.12-fold compared with fed-batch cultivation and batch cultivation on 15th day. Meanwhile, total flavonoids yield (132.36 mg L^?1) on 15th day, was increased by 2.26- and 2.67-fold compared with fed-batch cultivation and batch cultivation. In conclusion, two-stage cultivation process combined with the sucrose and elicitor treatment could promote both the callus growth and the secondary metabolites accumulation.     

Biomass and lipid production of heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> by using biodiesel-derived crude glycerol

Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.