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关键词： bioreactor d design   cfd   population balance model   single-use bioreactors   stirred-tank reactors  

摘要： Stirred-tank single-use bioreactors have proven their capability to successfully replace their stainless-steel counterparts in the biopharmaceutical industry. To date, however, only a five-fold volume expansion is achievable in a single-use stirred tank bioreactor, which in turn necessitates intermediate equipment to scale-up the culture to production volume. This work introduces a novel multi-chamber single-use bioreactor design that can help users save costs of purchase and qualification of equipment, reduce factory footprint, and reduce the risk of contamination. A bioreactor prototype is presented in this work that is made of two chambers of different volumes, interconnected as a single, closed system. The design and construction of the prototype is described in detail, and engineering characterization results are reported for both chambers and compared with published data for commercially available bioreactors. The results help identify some areas of potential improvements in the bioreactor design. Optimization of a bioreactor design can be an especially challenging process and can lead to an exceedingly large number of configurations and necessary experiments. Therefore, a computational fluid dynamics model is used in this work to model the multiphase flow in the 50 L chamber of the multi-chamber bioreactor prototype. The model results are validated with oxygen mass transfer coefficient (kLa) measurements within the prototype. The validated model is projected to predict the effect of using different sparger types and sizes and the effect of varying the impeller diameter on kLa. The simulations show that ring spargers result in a superior kLa compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. Also, larger impellers are shown to improve kLa. A correlation of kLa is presented as a function of both the reactor geometry and operating conditions. The resulting correlation can be used to predict kLa in a bioreactor and to optimize i

关键词： 病毒载体   大豆花叶病毒   生物反应器   Bar基因  

摘要： 植物生物反应器是最经济的生物反应器,能够为外源蛋白的表达提供安全的生产体系和完整的真核蛋白质修饰场所;然而,该系统却存在实验周期较长,外源蛋白的表达水平较低等问题;通过利用病毒载体来介导植物生物反应器,可以缩短实验周期,提高外源蛋白的表达量。大豆花叶病毒(Soybean Mosaic Virus,SMV)广泛分布于世界各地,由于对该病毒的研究主要集中在基因功能分析及抗病育种方面,在以SMV为载体介导植物生物反应器的方面,相关研究却相对较少。本实验室前期对大豆花叶病毒东北3号株系进行了全基因测序,并构建了该毒株的侵染性克隆,在此基础上,我们对大豆花叶病毒东北3号株系的全基因组侵染性克隆(SMV3)进行了改造,减弱其致病性而又不影响其侵染力的同时为了便于目的基因的插入和相应融合蛋白的表达,又利用融合PCR在SMV3基因组中插入限制性酶切位点AvrⅡ和大豆花叶病毒蛋白酶切位点ESVSLQ/S,从而构建了本试验中所用的介导大豆植物生物反应器的重组载体SMV3-2;此外,由于Bar基因(Bialaphos Resistance Gene,Bar)是常用的选择标记基因之一,由该基因编码的PAT蛋白具有良好的除草剂抗性,可用于后续试验的检测,所以本试验利用构建的SMV3-2载体携带Bar基因,形成SMV3-2-bar重组病毒,并将其转化至大豆植株体内。通过RT-PCR、ELISA及Western-blot检测方法,分别对目的基因及相应蛋白的表达进行检测,结果表明SMV3-2-bar重组病毒已成功转入大豆植株体内,并表达了PAT蛋白;通过对PAT蛋白表达趋势进行分析发现,在转化后的第六周,表达量最高;对其进行的生物学测定试验结果表明,PAT蛋白可以较好的发挥抗除草剂特性;同时,稳定性相对较好综上所述,本试验通过构建大豆花叶病毒载体并对其介导的大豆植物生物反应器进行了初步的探索,对其功能进行验证并分析其稳定性。结果表明,该系统可以完整的表达目的蛋白,且稳定性较好。本试验结果也将为基因功能研究和新型植物生物反应器的研究奠定基础。

关键词： 厌氧膜生物反应器   污泥驯化   运行参数   膜污染  

摘要： 作为一种重要的污染物,城市生活污水产生量大、组成复杂、对环境破坏性强。现有的污水处理系统主要采用好氧活性污泥法和生物膜法来处理,但是具有占地面积大、能耗严重、管理费用高、污泥产量大以及易二次污染等问题。因此,探索新型污水处理工艺的稳定性以及应用价值成为当务之急。本论文着重研究了一种新型的一体三腔式强制内循环厌氧膜生物反应器(AnMBR)在城市生活污水处理中的应用,具体研究了厌氧污泥的驯化对于反应器的快速启动的意义、反应器运行参数(水力停留时间、内循环和温度)的调节对反应器运行效能、厌氧污泥的性质和活性的影响以及新型厌氧膜生物反应器的构型及运行方式对膜污染的影响,进而确定该厌氧膜生物反应器在南方温度地区的运行适应性。该实验过程中,厌氧污泥共计驯化111天,厌氧膜生物反应器共计运行175天,取得以下结果:通过厌氧污泥驯化的手段可获得污染物处理效果高、活性强、絮凝沉降性好的厌氧污泥。在厌氧膜生物反应器启动期接种该厌氧污泥,可实现反应器在运行第21天快速启动。厌氧膜生物反应器在不同运行参数(水力停留时间、内循环、温度)下对废水的处理效能的研究结果表明:在HRT=12h时,反应器对COD的去除率可达81.4%,可以实现节约能源与高运行效能的平衡;反应器有内循环的运行条件下,主要污染物的去除效果要好于无内循环运行,COD去除率最高可达79.2%;在模拟冬春季(14-27℃)和夏秋季(30-32℃)的温度对比实验中,反应器对主要污染物均有较高的去除效果,COD去除率分别可达75%和88.4%。同时,在反应器运行过程中,发现厌氧颗粒污泥的产生,厌氧污泥的性质及活性均有所增强。本文还进一步研究了该新型厌氧膜生物反应器膜的污染情况及防治效果。结果表明:在快速启动阶段,厌氧膜生物反应器膜组件的污染与微生物代谢产物(EPS和SMP)存在明显的正相关关系。通过对厌氧膜生物反应器的膜表面和内层进行微观形貌观察发现,膜表面的污染主要以凝胶层为主,部分区域为泥饼层,同时内层也出现膜污染。对比观察小试试验膜的微观形貌发现,该反应器构型内设置障碍物及沼气回流喷吹对膜污染的防治具有明显效果。本研究表明,针对城市生活污水处理,本研究所设计的新型厌氧膜生物反应器具有可快速启动、高运行效能、节能减排、降低成本以及防治膜污染的优点,同时对南方温暖地区展现出良好的适应性,研究价值较高,值得推广。

关键词： kidney tissue engineering   scaffolds   biodegradable polymer scaffolds   electrospinning   bioreactor   aquaporin  

摘要： There is a pressing need for further advancement in tissue engineering of functional organs with a view to providing a more clinically relevant model for drug development and reduce the dependence on organ donation. Polymer based scaffolds, such as polycaprolactone (PCL), have been highlighted as a potential avenue for tissue engineered kidneys, but there is little investigation down this stream. The focus within kidney tissue engineering has been on two-dimensional cell culture and decellularised tissue. The aim of this project is to utilise electrospun scaffolds within a three-dimensional printed bioreactor system to create an ex vivo environment, to be used as either a conditioning tool for kidney tissue engineering scaffolds or as a model for disease. Electrospun polymer scaffolds can be created with a variety of fibre diameters and the variation in morphology of tissue engineered scaffolds has been shown to affect the way cells behave and integrate. The first study of this thesis examined the cellular response of a kidney cell-line to scaffold architecture using novel electrospun scaffolds. Two fibre diameters were used and three distinct scaffold architectures: random, aligned and cryogenic. The results showed that architecture of the scaffold has a profound effect on kidney cells; whether that is effects of fibre diameter on the cell attachment and viability or the effect of fibre arrangement on the distribution of cells and their alignment with fibres, overall there was a preference for a larger fibre diameter of around 4 μm. Following this, electrospun scaffolds were investigated for their potential to host a multicell population. Rat primary kidney cells were used, and results showed that the scaffolds were capable of sustaining a multi-population of kidney cells, determined by the presence of: aquaporin-1 (proximal tubules), aquaporin-2 (collecting ducts), synaptopodin (glomerular epithelia) and von Willebrand factor (glomerular endothelia cells). Viabil

关键词： bioreactors   computational fluid dynamics   compartmental modeling   mass transfer   scale-up   bioreaktori   virtauslaskenta   lohkomallinnus   aineensiirto   skaalaus  

摘要： Chemicals and biofuel components can be produced with the help of microorganisms in bioreactors. Microorganisms have very specific demands for optimal growth which makes the design and especially scale up of bioreactors challenging. Bioreactors are multiphase systems with complex hydrodynamics. Mathematical modeling can provide information on the functioning of bioreactors without the need for experimentation. Computational fluid dynamic (CFD) calculations are combined with a compartmental modeling approach for the calculation of 1) bubble column photobioreactors for the cultivation of algal cells and 2) large aerated stirred tanks for the cultivation of aerobic bacteria. Inclusion of dispersed phase population balances, mass transfer and reaction models makes the compartmental model very comprehensive. Local values of variables such as oxygen concentrations are attained. Simulation of bubble columns and heterogeneous conditions is enhanced with a new model for the calculation of dispersed phase flows that allows for bubble induced flow. New models for the consideration of light distribution and algal growth kinetics in the compartmental model facilitate the comprehensive simulation of algal growth. The results correspond well with measurements taken from literature and provide new insights to the design and run strategies of photobioreactors. It is shown that large industrial-sized stirred tank bioreactors operate at heterogeneous conditions due to the high oxygen demand of microorganisms. A new simple model estimates gas holdup and mass transfer rates at these conditions. Furthermore, a large stirred tank bioreactor is modeled at homogeneous and heterogeneous conditions with the compartmental modeling approach. Models accounting for the increase in coalescence at high volume fractions are required, without them the mass transfer results are too optimistic. A new reaction model for the uptake of oxygen enables the calculation of local reactor oxygen mass transfer c

关键词： CRISPR/Cas9   酪蛋白   基因敲入   轮状病毒   VP6   乳腺生物反应器  

摘要： 小儿腹泻是一种由轮状病毒导致的病毒性疾病,新生婴儿的致死率达80%,全球性危害较严重。VP6蛋白是轮状病毒的免疫原性的结构蛋白,如能在动物的乳腺中表达VP6蛋白,将有可能生产出抗轮状病毒的口服疫苗。为此,本研究拟应用CRISPR/Cas9介导的基因编辑技术,将具有免疫原性的轮状病毒结构蛋白VP6基因敲入到兔酪蛋白基因座的下游,以期利用酪蛋白的内源性基因调控系统指导外源基因VP6蛋白的表达,在兔奶中获得轮状病毒VP6重组蛋白,为应用乳腺生物反应器生产抗腹泻的口服疫苗奠定基础。研究获得如下结果:1、CRISPR/Cas9介导的兔酪蛋白位点基因组编辑效率检测参考兔酪蛋白基因组序列,设计靶向兔酪蛋白位点第6外显子区的g RNA,构建4条g RNA载体对应的RGS载体,然后转染人293T细胞验证4条g RNA的切割活性。g RNA载体转染293T细胞48小时后的绿色荧光和红色荧光检测结果及流式细胞检测结果显示,4条g RNA均具有切割活性。电转Cas9表达载体及4条g RNA质粒到兔成纤维细胞,靶位点特异性PCR扩增及扩增片段的T7E1酶切结果显示,4条g RNA在内源性靶位点均具有切割活性,4条g RNA的效率分别为:g1:37.1%,g2:42.0%,g3:60.7%,g4:62.4%。显微注射Cas9m RNA及4条sg RNA到兔受精卵胞质,验证胚胎水平4条g RNA的编辑效率,结果发现,四组胚胎发育率无显著差异(g1:72.77±1.12%,g2:70.33±0.56%,g3:73.33±1.84%,g4:73.44±0.86%vs对照组:73.25±1.03%,P>0.05),g RNA4的编辑效率(T7E1酶切分析结果)显著高于其余3条(g4:72.76±0.32%vs g1:30.14±1.93%,g2:38.53±0.75%,g3:52.26±1.16%,P<0.05),表明g4位点的外源基因整合效率较高。2、不同长度同源臂对外源基因定点敲入酪蛋白位点效率的影响为验证不同长度同源臂的打靶载体在体内同源重组效率,在设计200bp长度同源臂及1000 bp长度同源臂的打靶载体,通过扩增靶向兔酪蛋白g4位点两端的序列,分别获得200 bp与1000 bp的同源臂序列,然后以p EGFP-N1为模板,扩增EGFP片段,经一步法将两端同源臂与EGFP基因克隆到双酶切的p MD18-T载体上,构建得到两组不同长度同源臂的打靶载体,再通过共转Cas9载体、g RNA4载体及两组同源打靶载体到兔成纤维细胞,验证两组载体的基因打靶效率。结果发现:两组长度打靶载体均能在内源性基因位点介导外源基因的定点整合。为验证两组打靶载体在胚胎水平的敲入效率,两组打靶载体分别与体外转录的Cas9m RNA和sg RNA4胞质注射到兔受精卵内,培养5天后收集单个囊胚进行5’J和3’J特异性检测。结果显示:1000 bp同源臂的打靶载体效率与200 bp同源臂的打靶载体差异不显著(17.25±1.60%vs 19.23±1.73%,P>0.05),但200 bp打靶载体介导的阳性敲入胚胎中未能检测到完整的5’J和3’J PCR产物(多为3’J),而1000 bp打靶载体组阳性敲入胚胎中75%的胚胎能够完整的扩增出5’J和3’J PCR产物。表明同源臂为1000 bp的打靶载体打靶效率较高。3、CRISPR/Cas9介导的VP6定点整合兔的生产与鉴定在上述工作的基础上,利用靶向兔酪蛋白的g4位点构建1000 bp同源臂的轮状病毒结构蛋白VP6基因的打靶载体,然后与Cas9和g RNA4共转兔成纤维细胞验证打靶载体的效率。PCR和测序结果显示,VP6基因整合到了酪蛋白基因座g4位点,表明构建的VP6打靶载体在细胞水平是有效的。而后,将Cas9m RNA(100 ng/μL)和Cas9蛋白(100 ng/μL)分别与sg RNA4(50 ng/μL sg RNA4)及VP6打靶载体(100 ng/μL)混合注射到兔受精卵胞质,培养5天后收集囊胚进行外源基因整合检测。结果显示:虽然Cas9m RNA组的整合效率显著高于Cas9蛋白组(20.0%±2.6%vs10.3%±3.1%,P<0.05),但Cas9蛋白组阳性胚胎100%完整的整合,而Cas9m RNA组有37.5%未完整整合。将来自Cas9m RNA组的91枚胚胎移植到12只受体兔,8只怀孕,产仔20只;将来自Cas9蛋白组的82枚胚胎移植到11只受体兔,6只怀孕,产仔15只。Cas9m RNA移植组获得两只VP6整合仔兔,但两只均未能完整扩增5’J和3’J产物(F0-A3:3’J;F0-A12:5’J),而Cas9蛋白组成功获得一只VP6定点完整整合的基因编辑兔。上述结果表明,g RNA4的酪蛋白位点靶向

关键词： 外置式厌氧膜生物反应器   餐厨垃圾废水   有机负荷   高通量测序   微生物群落  

摘要： 随着社会的发展,餐厨垃圾的量越来越大,生化处理后产生的餐厨垃圾废水量也十分巨大,该类废水仍含有较高的有机污染物,不宜直接排放。本研究以餐厨垃圾废水为研究对象,利用外置式厌氧膜生物反应器(Anaerobic Membrane Bioreactor,AnMBR)在中温条件下研究不同有机负荷(OLR)对反应器运行性能的影响,并对不同OLR下反应器与膜表面的污泥进行了高通量测序分析,初步探究了AnMBR中及膜表面的微生物群落组成变化。本研究通过复壮、驯化过程的完成顺利进入处理运行过程。整个试验主要获得以下结果:1.运行过程,连续在OLR为10.00、15.00、20.00 gCOD/(L.d)条件下运行,AnMBR基本处于稳定状态。试验结果得出OLR为15.00 gCOD/(L·d)时,各物质去除效果,产气效果以及系统运行的稳定性均处于最佳状态。pH值稳定在7.00上下波动,VFA浓度稳定在480 mg/L以下,ALK在3000 mg/L上下波动,VFA/ALK 比值基本稳定在0～0.2之间,VFA中乙酸含量占主要,最大比重为97.2%,出水COD、碳水化合物及蛋白质的去除率分别达到90%,98%,80%,日平均产气量最大为177.03 L/d。2.随着运行负荷的改变,微生物群落组成也发生了明显的变化。当OLR为15.00 gCOD/(L.d)时,高通量测序结果表明,门水平上,主要优势菌群有拟杆菌门(Bacteroidetes)、螺旋菌门(Spirochaetae)以及广古菌门(Euryarchaeota);属水平上,主要优势菌群有球形杆菌属(Sphaerochaeta)、拟杆菌属(Bacteroides)、甲烷杆菌属(Methanobacterium)、甲烷粒菌属(Methanocorpusculum)。反应器内的细菌相对丰度比例高,多样性较高,古菌相对丰度比例低。当OLR为20.00 gCOD/(L.d)时,膜表面球形杆菌属(Sphaerochaeta)占40.23%,古菌属含量为4.42%,可见严重的膜污染与膜表面这些微生物丰度升高密切相关。

关键词： 污水处理   膜生物反应器  

摘要： 膜生物反应器(Membrane Bioreaetor.简称(MBR)是一种新型高效的水处理技术.主要介绍了(MBR)工艺的原理、特点、分类及其在污水再生处理领域的应用情况.

关键词： 生物反应器   生物膜   有机污染物   生物降解   电子供体  

摘要： 实现高效节能的工业污水处理是水处理领域长期的挑战,传统的工业废水处理存在能耗成本过高的缺点,这阻碍了可持续发展。因此,高效节能的处理工业废水的任务依然艰巨,针对每种工业污水中主要污染物的特征,设计科学的生物反应器处理各类污水并揭示污染物生物降解规律对实现高效节能的污水处理具有十分重要的价值。在此,我们发明了一种逆向流筛板塔式内循环生物反应器(Vertical Biofilm Bioreactor,VBBR),该反应器具有优越的处理污染物能力,无需提供额外的曝气装置。以VBBR为处理污水的工具,我们分别针对2,4,6-三氯酚(2,4,6-TCP)、硝基苯(NB)和对硝基酚(PNP)进行研究,得到以下的结论:(1)在生物降解同时含2,4,6-TCP、硝酸盐或亚硝酸盐污水时,还原脱氯与反硝化均是需要电子的反应,这些反应在同一个反应器进行时,会因为竞争电子供体而产生相互抑制作用,这种相互抑制作用可通过添加电子供体得到一定程度的缓解。与2,4,6-TCP和硝酸盐同时还原相比,亚硝酸盐对2,4,6-TCP的抑制作用更弱。(2)通过控制操作条件,可以在VBBR中同时形成厌氧区和好氧区,从而加速NB的生物降解速率。因为在单一的厌氧条件下,NB转化为苯胺(AN)后就很难进一步降解,从而会抑制NB的降解。而如果在单一的好氧条件下,NB则会生成更难生物降解的硝基酚(PNP),从而导致NB的降解速率缓慢。当控制反应器内的溶解氧(DO)浓度(.5 mg/L)时,添加外源电子供体,可以加速NB的去除速率。增加外源电子供体,可以加速NB和AN的单加氧反应速率,增加AN的单加氧反应速率可以缓解AN在厌氧区对NB的抑制影响。(3)塑料基载体和铁基VBBR相比,铁基载体表面更容易形成强健的生物膜,且生物膜群落具有更高多样性和丰富度,在铁基载体VBBR中,在四种浓度乙酸钠的添加量下,都不能改变铁基载体VBBR降解PNP的速率大于塑料基载体VBBR的事实,且铁基载体VBBR维持PNP最大的降解速率所需乙酸钠仅需要2.5 mmol/L。饥饿处理实验表明铁基载体VBBR具有更强的抗饥饿能力,当解除饥饿胁迫后,铁基载体VBBR恢复降解PNP活性所需的时间远远小于塑料基载体VBBR。(4)铁基载体与塑料基相比,铁的化学性质活泼,自然环境中会发生化学腐蚀作用产生三价铁,三价铁既能作为细菌的能量物质,又能作为生物膜生长发育的信号。因此,铁基载体促进生物膜的生长发育,形成更厚实、功能结构健全的生物膜。此外,铁基载体生成三价铁作为生物膜内部微生物的电子受体,保证胞外电子在生物膜内可以双向传递,提高了胞外电子传递速率。

关键词： Temporal dominance of sensation   Food science   Food consumer science   New product development   Seaweed production   Sensory evaluation   Neophobia   Oral processing  

摘要： Despite the growth of the functional food convenience and snack market, seaweed containing food products have been limited to small quantities of imported produce; mainly as a salt replacer rather than a main ingredient. To improve this situation, this work investigates UK consumer expectations and insights into seaweed containing products. This research explores the possible application of seaweed as a functional ingredient with unique nutritional, textural, and sensory characteristics, offering innovative food design solutions. In addition, the study aimed to identify the maximum concentrations of seaweed in a food-based application for rehydrated and dried edible Scottish seaweed with the intention of encouraging seaweed consumption. The first stage of this study investigated rehydrating several dried seaweeds sent to Abertay, Scotland from the factory and harvest site in Wick, Scotland. Once the data was recorded appropriate seaweeds were selected for varying food products based on volumes, accessibility, seasonality and nutritional properties. Different concentrations (20% and 40%) of Undariapinnatifida (Wakame) were incorporated in a salad, 15% Himathalia Elongate (Sea Spaghetti) in a pesto, 30% Himathalia Elongate (Sea Spaghetti) in crisps and 5, 10, 15 and 20% Undariapinnatifida Wakame) and Himathalia Elongate (Sea Spaghetti) in crackers. During this study, lower concentrations of seaweeds were considerably more acceptable to consumers in the salads and crackers. Interestingly a higher fat concentration of the pesto also was more consumer acceptable, and various flavours of the crisps, which were determined by sensory analyses techniques. These results will help the food industry promote seaweed into the food market with a range of successful healthier products. As a result of the new product development in this study the 20 % seaweed salads, seaweed crisps and seaweed pesto added to pasta are being up scaled commercially. They are now available to purchase