多级串联悬浮床反应器系统中自絮凝颗粒酵母乙醇连续发酵耦合废糟液直接全循环使用的研究

在一套四级串联悬浮床生物反应器系统中,以双酶法制备的玉米粉糖化液为底物,进行了废糟液全循环条件下自絮凝颗粒酵母乙醇连续发酵的实验研究。在实验中,每隔5 d将从末级反应器收集到的发酵液集中精馏处理,得到的废糟液直接用于玉米粉调浆制糖。系统连续运行了120d,共进行了24批次实验,数据分析表明系统达到了平衡状态。在平均发酵时间为20h条件下,发酵终点乙醇浓度平均为11.7%（V/V）,残还原糖浓度平均为7.9g/L,装置运行平稳。这些工作为自絮凝颗粒酵母乙醇发酵耦合废糟液直接全循环使用、实现污染物源头减废、清洁生产奠定了理论和实验基础。     

融合株SPSC发酵生产酒精的工艺研究 Ⅰ.絮凝速率、发酵过程的最适温度和pH值

酿酒酵母属(S. cereviae)变异株和粟酒裂殖酵母属(S. pombe)变异株进行属间原生质体融合得到融合株SPSC,该融合株比S. cereviae具有强的自身絮凝能力。以葡萄糖浓度150g/L的底物在30～44℃的温度范围内进行摇瓶厌氧发酵,获得最佳温度范围为34～38℃,最高发酵温度为40℃。在有效容积2.35L悬浮床反应器中,在pH值3.0～5.0范围内进行连续发酵,获得最适发酵pH为3.5～4.5。     

适于无血清贴壁培养的抗凋亡宿主细胞系CHO-IVB2的构建

应用无血清培养基培养CHO细胞时 ,由于没有血清提供各种贴壁因子 ,细胞以悬浮的方式生长。在实际的大规模细胞培养中 ,CHO细胞往往以贴壁方式培养 ,要么贴壁于悬浮的微载体中 ,要么贴壁于固定的聚酯盘状介质或中空纤维中 ,而很少直接悬浮于培养基中。在无血清培养基中 ,Vitronectin单一组分可以促使CHO细胞的贴壁和扩增。通过双表达Igf_1和Bcl_2基因 ,已经构建了可以在无蛋白培养基IMEM中抗凋亡生长的细胞株CHO_IB3。在此基础上 ,构建了可以同时表达Igf-1、Vitronectin和Bcl-2三个蛋白的三顺反子表达载体pCI-NII-IVB。将该载体转染于CHO-dhfr^- 细胞中 ,构建了一个细胞株CHO-IVB2。该细胞株可以在无蛋白培养基中抗凋亡生长 ,适于以贴壁的方式大规模培养 ,用于大量生产外源目的蛋白.     

絮凝酵母颗粒悬浮床反应器中试及酒精连续发酵工艺的研究

在小型悬浮床反应器研究絮凝颗粒酵母酒精连续发酵工艺的基础上,建立了单釜反应器有效容积0．5m³、二级串联的中试装置。以淀粉糖化液为底物,考察了悬浮床反应器由小试到中试的放大效应,提出了该反应器工业放大时对危险截面表观气速应加以限制的原则,以保证絮凝酵母颗粒不被剪切破坏。进而在平均稀释率为0．10h^-1的条件下．中试装置稳定运转一个月,获得终点发酵液中酒精浓度80～85g／L,残糖低于5.0g／L,设备平均生产酒精8．4g／L·h的结果。     

钙离子对293细胞结团和生长的影响

分别在有血清和无血清条件下、方瓶和转瓶中考察了Ca²⁺ 对2 93细胞结团和生长的影响。通过实验发现,Ca²⁺ 浓度在0 1～1 0mmol L范围内对2 93细胞的贴壁和结团性质有显著影响,而对生长影响不大。结果表明:有血清贴壁培养时,较高的Ca²⁺ 浓度有利于细胞贴壁;无血清悬浮培养中,Ca²⁺ 浓度越高,细胞结团越严重,细胞结团达到平衡后的平均粒径(D ,μm)与Ca²⁺ 浓度(c,mmol L)在0.1～0.5mmol L范围内可用一次函数D =58.65c +16.96描述,细胞结团尺寸是可调控的;而细胞在不同的Ca²⁺ 浓度下有相似的生长规律。     

絮凝特性对自絮凝颗粒酵母耐酒精能力的影响及作用机制

首次报道絮凝特性提高酵母菌耐酒精能力的现象及其机制。融合株SPSC与其两亲本粟酒裂殖酵母变异株和酿酒酵母变异株于 30℃经 18% (V/V)酒精冲击 7h的存活率分别为 52%、37%和 9%。细胞膜磷脂脂肪酸组成分析表明 ,两絮凝酵母 (融合株SPSC和粟酒裂殖酵母变异株 )的棕榈酸含量均约为非絮凝酵母 (酿酒酵母变异株 )的两倍 ,而棕榈油酸和油酸的含量明显低于后者。研究表明 ,当两絮凝酵母在培养中由于柠檬酸钠的作用 (抑制絮凝体的形成 )而以游离细胞生长存在时 ,其细胞膜磷脂棕榈酸含量显著下降 ,而棕榈油酸和油酸的含量明显增加 ,结果细胞膜磷脂脂肪酸组成特点与酿酒酵母变异株相似 ;而且实验表明 ,絮凝特性的消失伴随菌体耐酒精能力的急剧下降 ,变得与酿酒酵母变异株的水平相当。这些结果提示两絮凝酵母具有较强的耐酒精能力与其细胞膜磷脂脂肪酸组成中含有更高比例的棕榈酸有关。     

细胞膜磷脂脂肪酸组成对自絮凝酵母质膜ATP酶响应酒精刺激的影响及其与菌体耐酒精的关系

研究揭示细胞膜磷脂脂肪酸组成与质膜ATP酶在酵母菌耐酒精中的一种新颖关系。实验表明 ,细胞膜磷脂脂肪酸组成特点对生长于未添加酒精条件下的自絮凝颗粒酵母质膜ATP酶活性没有影响 ,但却明显影响生长于添加酒精 (1 %～ 10 % ,V/V)条件下的菌体质膜ATP酶对酒精激活的敏感性 :预培养于添加 0.6mmol L棕榈酸、亚油酸、或亚麻酸条件下的菌体的质膜ATP酶的最大激活水平分别为各自酶的基态水平 (未激活 )的 3.6、1.5和 1.2倍 ,而对照组 (预培养于未添加脂肪酸条件下的菌体 )的相应值为2.3倍 ,酶产生上述最大激活水平时的酒精浓度分别为 7%、6 %、6 %、和 7% (V/V)。酶激活后米氏常数K_m 、最适pH和对钒酸钠 (质膜ATP酶特异性抑制剂 )的敏感性等性质不变 ,但最大反应速度v_max明显增加。实验表明 ,细胞膜磷脂脂肪酸组成特点对提高菌体的耐酒精能力越有利 ,则其质膜ATP酶被酒精激活的幅度越大 ,说明菌体耐酒精能力的提高与其质膜ATP酶对酒精激活的敏感性的增加密切相关。细胞膜磷脂脂肪酸组成会影响酵母菌质膜ATP酶对酒精激活的敏感性是观察到的新的实验现象.     

绞股蓝悬浮细胞的原生质体再生植株

绞股蓝(Gynostemma pentaphyllum (Thumb)Mak．)是葫芦科多年生草本药用植物,现已得到广泛的开发利用,本文首次报道了绞股蓝悬浮细胞的原生质体再生植株。     

细菌群体异质性对生长动态过程的影响及其表征

在经典的表征群体生长的Logistic方程中, 假定群体中每个个体都是同质的. 解方程时的初值仅限定为N(t₀) = N₀, 因此, 对N₀相同而生理状态不同的接种物的生长动态不能区分. 事实上, 在菌群生长的任一时刻, 只有一定比例的细胞在进行分裂(设为θ), 这样, 不仅由N, 而且实际上由N和θ 共同决定. 因此, 解方程的初值条件还应加入θ (t₀) = θ₀, 而这又被在构建生长方程时所忽略, 但这一附加条件使Logistic方程的求解甚为复杂. 基于细菌生长过程的瞬时生长速率V_inst的时间过程曲线都呈Gauss分布形状这一特点, 在用瞬时生长速率成功地表征限制性条件下区分群体生长阶段的基础上, 进一步用Gauss分布近似函数表达式以求得异质性群体的生长参数, 这些参数可真实地表征不同生理状态细菌群体的生长动态, 为微生物学基础研究和在生物工程等方面的应用提供了一个新方法. 在此基础上提出了一个估算细菌群体生长延迟期和群体倍增时间的新方法.     

柑桔裂皮病类病毒一爪哇三七悬浮细胞的生长特性

爪哇三七(Gvnura aurantlaca Dc)是柑桔裂皮病类病毒(Citrus Exocortis Viroid简称CEV)敏惑的指示植物。用CEV感染的柑桔叶研磨汁液机械接种健康爪哇三七植株,以此作为CEV感染的外植体采源。在MS和B,琼脂培养基上诱导和继代培养健康、CEV感染的爪哇三七叶片的愈伤组织,在MS、B,液体培养基中建立健康、CEX’感染的爪哇三七悬浮细胞系统。从愈伤组织和悬浮细胞培养物中抽提核酸,经5％双向聚丙烯酰胺凝胶电泳、银染分析：在健康爪哇三七愈伤组织和悬浮细晦中不出现特异的CEV电泳带,而CEV感染的爪哇三七的所有培养物中都含有类病毒。cEV可在有丝分裂征盛的爪哇三七悬浮细胞中复制。散伤组织和悬浮细胞很容易继代培养和保存。     

Clarifying the roles of kinetics and diffusion in activated sludge filamentous bulking

We hypothesized that the growth rates of filaments and floc formers in activated sludge are affected by the combination of kinetic selection (Lou and de los Reyes, Biotechnol Bioeng 92(6): 729-739, 2005b) and substrate diffusion limitation (Martins et al., Water Res 37:2555-2570, 2003). To clarify the influence of these factors in explaining filamentous bulking, a conceptual framework was developed in this study. The framework suggests the existence of three different regions corresponding to bulking, non-bulking, and intermediate regions, based on substrate concentration. In the bulking and non-bulking regions, kinetic growth differences control the competition process, and filaments or floc formers dominate, respectively. In the intermediate region, substrate diffusion limitation, determined by the floc size, plays the major role in causing bulking. To test this framework, sequencing batch reactors (SBRs) were operated with influent COD of 100, 300, 600, and 1,000 mg/L, and the sludge settleability was measured at various floc size distributions that were developed using different mixing strengths. The experimental data in the bulking and intermediate regions supported the proposed framework. A model integrating the two factors was developed to simulate the substrate concentrations at different depths and floc sizes under intermittently feeding conditions. The modeling results confirmed that substrate diffusion limitation occurs inside the flocs at a certain range of activated sludge floc sizes over the operation cycle, and provided additional support for the proposed framework.     

Der Einfluß der Flockenbildung auf die Versorgung der Hefezellen mit Sauerstoff bei der Fermentation flüssiger Kohlenwasserstoffe

Floc formation, especially the influence of floe diameter variations on the total velocity of the process, was investigated in aerobic growth processes of yeast on the hydrocarbons of crude oil. The experimental results show that the diameter of the flocs is a function of the rheological properties of the fluids and the flow conditions. The floc diameter varies between 0,1 mm and a few millimeters. About 90% of the total yeast cells are situated in the interior of the flocs. Since oxygen must be transferred to all yeast cells their oxygen supply was studied. Thus, the yeast cells in the floc interior were not sufficiently supplied with oxygen, if the floc diameter reached a critical value. In such cases a decrease of the biomass formation rate was observed, although the dissolved oxygen concentration of the aquaeous fermentation medium was greater than zero. Therefore, aerobic microbial growth processes in multicomponent systems must be carried out without floc formation or under such conditions as cause very small floc diameters.     

Neural network analysis of the diffusional limitations in activated sludge flocs

This work deals with the development of a comprehensive but simplified version of floc model for the activated sludge process. The rate expression of the type used by the IAWPRC Task Group was employed in this study to represent the reaction kinetics inside the floc matrix. The intraparticle mass transport was then incorporated to account for the effects of the diffusional limitations. Solution to the floc model was obtained by orthogonal collocation technique. Since the actual reactions are not only related to the sludge floc size and the bulk characteristics but are also associated with the floc size distribution, the concept of overall effectiveness factor was therefore used. Evaluation of overall effectiveness factors using neural network analysis was carried out and potential control strategies were discussed.     

Optimization of Clostridium thermocellum growth on cellulose and pretreated wood substrates

Summary Two strains of Clostridium thermocellum ATCC 27405 and NRCC 2688 demonstrated similar product yields and cellulase activities when grown on solka floc. A sequential culture of C. thermocellum and Zymomonas anaerobia supplemented with cellobiase could produce 1.8 mg/ml of ethanol when grwon on 1% solka floc. Different media were evaluated for their ability to enhance the product and cellulase yields of C. thermocellum grown on cellulose substrates. Ethanol and reducing sugar values of 1.5 and 3.8 mg/ml respectively and an endoglucanase activity of 3 IU/ml were obtained after growth of Clostridium thermocellum in a modified medium containing 1% solka floc. Three different pretreated wood fractions were assessed as substrates for growth. A steam exploded wood fraction gave comparable values to those obtained after growth on solka floc. Sequential cultures of C. thermocellum and Zymomonas anaerobia grown on a 1% steam exploded wood fraction could produce 1.6 mg/ml ethanol after 3 days growth.     

How flocculation can explain coexistence in the chemostat

We study a chemostat model in which two microbial species grow on a single resource. We show that species coexistence is possible when the species which would normally win the exclusive competition aggregates in flocs. Our mathematical analysis exploits the fact that flocculation is fast compared to biological growth, a common hypothesis in floc models. A numerical study shows the validity of this approach in a large parameter range. We indicate how our model yields a mechanistic justification for the so-called density-dependent growth.     

How flocculation can explain coexistence in the chemostat

We study a chemostat model in which two microbial species grow on a single resource. We show that species coexistence is possible when the species which would normally win the exclusive competition aggregates in flocs. Our mathematical analysis exploits the fact that flocculation is fast compared to biological growth, a common hypothesis in floc models. A numerical study shows the validity of this approach in a large parameter range. We indicate how our model yields a mechanistic justification for the so-called density-dependent growth.     

The Removal of Nitrate from Solution by Floc-forming Bacteria on Decomposing Cellulose Particles

S ummary : Cellulose particles in aerated liquid medium inoculated with activated sludge quickly became enveloped in floccular microbial growth (cellulose floc) able to assimilate nitrate rapidly from solution. Sedimenting the floc removed assimilated nitrogen, excess cellulose and biomass. At 18 and 22°, nitrate was removed from solution at 1·76 and 1·83 μg of nitrate-N/ml/h, respectively. Similar results were found with floc formed by a cellulose decomposing isolate and some noncellulolytic floc-forming bacterial contaminants. Washed preformed cellulose floc removed nitrate from dilute solution at 0·89 μg of nitrate-N/ml/h at pH 7·1–8·6. The C : N ratio of the supernatant fluid changed rapidly as nitrate became exhausted; the significance of this is considered in relation to complete removal of C and N by further biological oxidation.     

Modeling brewers' yeast flocculation

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.     

A kinetic model for the activated sludge process which considers diffusion and reaction in the microbial floc

A mathematical model has been developed which describes substrate removal, oxygen utilization, and biomass production in an aggregated microbial suspension containing the substrate as a soluble biodegradable material and a uniform floc size. It is applicable to both steady-state and transient conditions. The model, consisting of three partial differential equations and two ordinary differential equations, takes into account the flow pattern in the reactor, intraparticle mass transport of oxygen and substrate, and biochemical reaction by individual cells embedded in the floc. Efficient numerical solution of the coupled nonlinear equations is obtained using an implicit finite difference approach for both the reactor and floc equations. A convergent solution is realized through block interation utilizing the tridiagonal algorithm. Results indicate that a unifying theory of activated sludge dynamics will have to consider coupling between floc chemical kinetics and changes in the bulk liquid characteristics. Floc size emerges as an important influence on system performance. It appears necessary to distinguish between a system response caused by diffuslonal resistances and nutrient limitations within the floc and a response caused by physiological adaption when analyzing the transient behavior of an activated sludge process. Future research should be devoted to rigorous laboratory determinations of model parameters along with extensions to include limitations of nutrients other than orgabnic carbon and oxygen.     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.