膜技术在绿色生物制造中的应用

生物制造是我国战略性新兴产业发展的重点领域,但生物制造过程中普遍存在着产物抑制、产物浓度低且成分复杂、废水排放量大等问题。膜分离技术应用于生物制造过程,可有效解决生物制造过程中所面临的上述问题。重点介绍了笔者研究团队将膜技术应用于绿色生物制造领域所取得的研究进展,包括高性能渗透汽化优先透醇分离膜的研制与规模化制备技术,高效旋转膜生物反应器的研制,分别实现了乙醇/丁醇发酵-渗透汽化分离耦合和乳酸发酵-旋转膜分离耦合,有效降低了产物抑制,大幅提高了发酵效率;开发了周期性换向-脉冲冲刷膜污染控制技术,成功应用于酱油等调味品的传统发酵行业,显著提高了产品品质,大幅降低了能耗和水耗。以上成果充分显示膜分离技术在绿色生物制造中有着广阔的应用前景。     

膜分离技术在制药工业中的应用

膜分离技术是指在分子水平上不同粒径分子的混合物在通过半透膜时,实现选择性分离的技术,半透膜又称分离膜或滤膜,膜壁布满小孔,根据孔径大小可以分为:微滤膜(MF)、超滤膜(UF)、纳滤膜(NF)、反渗透膜(RO)等,膜分离都采用错流过滤方式。目前,膜分离技术在制药工业中的应用大体上分为生物发酵制药、中药生产和现代生物技术等。在生产抗生素、半合成抗生素以及维生素和氨基酸的过程中膜分离技术的应用最为广泛。这一切都离不开膜分离技术的独特优势。当科学技术和设备在不断的完善,膜技术的应用也将更加成熟和普遍。     

生物产品分离过程中的膜技术

膜技术是材料科学和过程工程科学等诸多学科相互交叉、相互渗透而产生的新领域．已成为现代分离科学中最重要的高新技术之一。美国官方文件声称：“目前没有一项工业技术能像膜技术那么广泛地应用”．近年来新兴生物技术及其产业的高速速发展又为膜技术的应用提供了广阔空间。众所周知．在生物技术研究及应用中．产物的分离提纯是生物产品生产的关键环节。膜技术由于具有高效、能耗低、过程简单、条件温和、操作方便、环境污染少、易于和其他技术集成等突出优点．特别适合生物产品（尤其是热敏性生物产品）的分离纯化．可有效克服传统分离方法周期长并易导致生化产品失活之不足。目前．膜技术已应用于生物技术领域从原料配制到产品浓缩、分离等各个环节．展现出巨大的应用潜力。表1是在生化产品分离提纯中应用较多的几种膜分离过程。     

生化废水综合治理中的膜技术及其应用

工业生物技术蓬勃发展的同时其相关酿造、制药等工业已成为耗水和废水排放大户。该类生化废水属于典型高浓度、重污染有机废水,目前主要通过末端处理以达标排放为目标。随着水资源短缺的日益严重以及水环境压力的与日俱增,以资源回收利用和清洁生产为目标的生化废水综合治理工作迫在眉睫。膜分离技术因其具有高效、绿色的特点已成为生化废水综合治理过程中极具吸引力的一种选择。本文在在综述适合生化废水综合治理过程的单元膜技术、集成膜技术和膜生物反应器技术的种类、特点及应用情况的基础上．重点介绍了工业规模膜生物反应器技术在啤酒、味精及制药废水处理中的应用实例。     

鲍曼不动杆菌菌毛在细菌生物膜形成中的作用

目的通过观察鲍曼不动杆菌菌毛,了解菌毛结构在生物被膜形成过程中的作用。方法以ICU的医院感染患者的腹腔手术后引流液、痰及呼吸机导管内壁附着物等为材料分离鉴定细菌,制备细菌的电镜标本,通过超微结构观察鲍曼不动杆菌菌体表面的菌毛与生物被膜形成的相关性。结果新分离的鲍曼不动杆菌菌体表面存在菌毛,菌毛与生物膜形成过程中的粘附有关。结论菌毛粘附是生物被膜形成的原因之一。     

生物强化技术在生物质沼气制备过程中的应用及研究进展

生物强化技术通过为特定的生物过程"设计"微生物,进而作为一种提升反应系统活力和性能的手段被应用于生物质沼气制备过程,以便加快发酵系统启动时间、增加原料利用率、缩短酸败系统的恢复时间、降低高有机负荷的抑制作用等。本文针对以木质纤维素为原料的沼气制备中的生物强化技术,从生物强化菌剂的构建及标准、生物强化作用的影响因素、生物强化作用机制的探究等几个方面来阐述目前国内外生物强化技术在生物质沼气制备过程中的应用与研究进展,以及存在的问题和解决方案。     

生物被膜的形成及其电化学阻抗检测

生物被膜是细菌及其自身分泌的胞外聚合物组成的微生物群落,其形成是受多种机制共同调控的多阶段动态过程,具有较强的耐药性且难以清除,给医疗、食品等行业带来了巨大的威胁。近年来,生物被膜的相关研究领域备受关注,尤其是针对生物被膜的有效检测技术。本文在简要介绍生物被膜的特点、形成过程及群感效应对生物被膜的调控作用基础之上,总结了生物被膜常用的检测方法,重点针对电化学阻抗技术在生物被膜检测中的应用进行调研和讨论,并对基于微流控芯片的生物被膜电化学阻抗原位检测进行了综述和展望。     

渗透汽化膜在生物燃料分离中的应用

生物燃料作为一种重要的可再生能源,近年来引起了各国越来越多的重视。生物燃料的发展不仅依赖于生物质转化为生物醇的过程,也依赖于净化和分离技术的突破。在众多分离方案中,以膜渗透汽化为基础的混合工艺提供了最直接有效的方法。本文简述了渗透汽化技术的基本原理和混合工艺设计,并重点介绍了笔者近年来应用渗透汽化技术在生物燃料分离方面的研究工作,从膜材料和膜形态两方面阐述了膜设计和膜制造的具体方案,举例说明了渗透汽化膜在生物醇脱水和回收方面的应用,最后讨论了该领域的发展趋势以及未来挑战。     

分子筛膜在生物燃料乙醇生产中的应用

近年来,全球都在寻求建立新的低碳、高效、环保的能源供应体系,生物质燃料得到了普遍重视。其中,生物乙醇得到了多国政府的大力推广。2017年,国家发展改革委、国家能源局、财政部等十五部门联合印发了《关于扩大生物燃料乙醇生产和推广使用车用乙醇汽油的实施方案》。在今后的若干年内,生物燃料乙醇将得到迅猛发展。分子筛膜作为一种高效节能的新型分离技术有望在生物乙醇的生产中发挥重要作用。首先对疏水型和亲水型分子筛膜材料在乙醇富集和乙醇脱水中的研究现状进行综述,然后对发酵法制备生物乙醇过程中分子筛膜的可行性工艺、经济性评估以及工业应用现状进行分析和论述,最后做了总结和展望。     

膜分离技术在水果加工中的研究进展

传统工艺对果汁/果酒澄清或浓缩时,普遍存在澄清效果不佳、工艺过程能耗大以及操作温度高、风味物质损失严重等生产缺陷,膜分离技术能够解决传统工艺存在的问题,具有常温下操作、分离过程无相变、选择性高且能耗低等优点。本文中,笔者综述新近的研究成果,对膜材料、膜组件和操作参数的选择进行讨论,概述膜分离技术在果汁/果酒澄清或浓缩中的效果及对其品质的影响,阐述在水果加工过程中膜污染的形成机制及控制和清洗办法,并对膜技术在水果加工领域的应用前景进行展望,以期为相关的研究者和企业提供技术参考。     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

From mosaic to patchwork: Matching lipids and proteins in membrane organization

Abstract

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints – focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization – are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or ‘patchwork membrane'.     

Tamoxifen Increases Membrane Fluidity at High Concentrations

There are contradictory results in the literature relating to the effect oftamoxifen on membrane fluidity. The present work investigates the effect oftamoxifen on membrane dynamics to find out whether the concentration oftamoxifen can be one of the factors in this discrepancy. Turbidity(absorbance at 440 nm) and Fourier transform infrared spectroscopicstudies reveal that tamoxifen causes opposite effects on membranefluidity at low (1 mol.%) and high (30 mol.%) tamoxifen concentrations. Lowtamoxifen concentrations increase the absorbance in the gel and liquidcrystalline phase, whereas high tamoxifen concentrations decrease theabsorbance in gel and liquid crystalline phase, whereas tamoxifenconcentrations decrease the absorbance. Observations on both phasesshow that the bandwidth of the CH2 stretching bands decreases with1 mol.% tamoxifen and increases with 30 mol.% tamoxifen present, indicatinga decrease in membrane fluidity at low tamoxifen concentrations and anincrease in fluidity at high tamoxifen concentrations. It is seen that theapparent discrepancy in the literature on the effect of tamoxifen onmembrane fluidity mainly arises from the tamoxifen concentration used andthe confusion on the concept of lipid fluidity and lipid order.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.     

Single-channel analysis of the conductance fluctuations induced in lipid bilayer membranes by complement proteins C5b-9

Summary Single-channel analysis of electrical fluctuations induced in planar bilayer membranes by the purified human complement proteins C5b6, C7, C8, and C9 have been analyzed. Reconstitution experiments with lipid bilayer membranes showed that the C5b-9 proteins formed pores only if all proteins were present at one side of the membrane. The complement pores had an average single-channel conductance of 3.1 nS at 0.15m KCl. The histogram of the complement pores suggested a substantial variation of the size of the single channel. The linear relationship between single-channel conductance at fixed ionic strength and the aqueous mobility of the ions in the bulk aqueous phase indicated that the ions move inside the complement pore in a manner similar to the way they move in the aqueous phase. The minimum diameter of the pores as judged from the conductance data is approximately 3 nm. The complement channels showed no apparent voltage control or regulation up to transmembrane potentials of 100 mV. At neutral pH the pore is three to four times more permeable for alkali ions than for chloride, which may be explained by the existence of fixed negatively charged groups in or near the pore. The significance of these observations to current molecular models of the membrane lesion formed by these cytolytic serum proteins is considered.     

Involvement of carboxyl groups in the divalent cation permeability of rat parotid gland basolateral plasma membrane

Divalent cation permeability of rat parotid gland basolateral plasma membranes was examined in dispersed parotid acini (by Ca²⁺ or Mn²⁺ entry) and in isolated basolateral plasma membrane vesicles (BLMV, by⁴⁵Ca²⁺ influx). Mn²⁺ entry (fura2 quenching) was about 1.6 fold higher in internal Ca²⁺ pool-depleted acini (Ca²⁺-depl acini) than in unstimulated cells. Mn²⁺ entry into Ca²⁺-depl acini was increased at external pH>7.4 and decreased at pH<7.4. Pretreatment of Ca²⁺-depl acini with the relatively hydrophobic carboxylic group reagent, N,N-dicyclohexylcarbodiimide (DCCD, 50 M for 30 min) resulted in the inhibition of Mn²⁺ entry into Ca²⁺-depl acini to unstimulated levels. Another hydrophobic carboxyl group reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and the relatively hydrophilic carboxyl group reagents, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMCD) did not affect Mn²⁺ entry.Similar to the effects in intact acini, Ca²⁺ influx into BLMV was decreased when the external pH was lowered below 7.4. Also DCCD (5 mM, 30 min), but not EEDQ, decreased (40%) Ca²⁺ influx in BLMV. However, unlike in acini, the hydrophilic reagents, EDC, EAC, and CMCD decreased Ca²⁺ permeability in BLMV and the effects were nonadditive with the decrease induced by DCCD. The aggregate effects of carboxyl group reagents on the Ca²⁺ and Mn²⁺ permeability in BLMV and intact acini, respectively, suggest that a critical carboxyl group (most likely accessible from the cytoplasmic side of the plasma membrane) is involved in divalent cation flux in rat parotid acinar cells.     

Immunological approach to the characterization of the outer acrosomal membrane of boar spermatozoa

Antiserum to purified boar spermatozoan outer acrosomal membrane (OAM) was raised in rabbits and adsorbed with boar liver and serum glutaraldehyde cross-linked immunoadsorbents. The IgG fraction of the antiserum was purified by (NH₄)₂SO₄ precipitation followed by ion-exchange chromatography. Indirect immunofluorescence showed bright fluorescent staining of the acrosomal cap of boar spermatozoa and to a lesser extent of the acrosomes of bull and goat spermatozoa after incubation with anti-OAM-IgG. Immuno-electron microscopy further confirmed the specificity of the antibody for the OAM. Preincubation of the anti-OAM-IgG with isolated OAM, completely abolished its reactivity. When tested by ELISA, anti-OAM-IgG reacted with boar, bull, goat, and human spermatozoa; however, its binding activity to boar spermatozoa was significantly greater as compared to spermatozoa from the other species tested. In an effort to identify OAM antigens recognized by this antiserum, the isolated boar OAM was labeled either with ³H or with ¹²⁵I and solubilized by mild detergent treatment. The extracted components were immunoprecipitated with anti-OAM-IgG and protein A-bearing S. aureus and the thus isolated antigens were analysed on SDS-PAGE. The results suggest that anti-OAM-IgG recognized one high molecular ³H-labeled glycoprotein (270 kd), and four ¹²⁵I-labeled polypeptides of lower molecular weight of the boar OAM.     

Developing an antibacterial super-hydrophilic barrier between bacteria and membranes to mitigate the severe impacts of biofouling

Biofouling produces concentrated microbial populations with highly resistive biofilms and is considered to be a serious obstacle for a wide range of membrane technology applications. An antibacterial super-hydrophilic barrier could help to reduce biofouling by preventing direct contact between membranes and bacteria. In this study, an antibacterial super-hydrophilic barrier consisting of a layer of TiO₂ nanoparticles (NPs) was developed on polyvinylidene fluoride (PVDF)-based membrane via a facile technique. The results demonstrated that the presence of TiO₂ NPs eliminated the first step of biofouling, ie bacterial adhesion to the membrane. In addition, after bacterial deposition onto the membrane during ultrafiltration (UF), the TiO₂ NPs significantly retarded bacterial growth and reproduction (the second step of biofouling). During UF, the membrane flux decreased due to bacterial deposition, but 85% of the flux was recovered through physical cleaning using water. This study sheds light on the potential advantages of antibacterial super-hydrophilic membranes for biofouling mitigation.