Microchemostat-microbial continuous culture in a polymer-based, instrumented microbioreactor

In a chemostat, microbial cells reach a steady state condition at which cell biomass production, substrates and the product concentrations remain constant. These features make continuous culture a unique and powerful tool for biological and physiological research. We present a polymer-based microbioreactor system integrated with optical density (OD), pH, and dissolved oxygen (DO) real-time measurements for continuous cultivation of microbial cells. Escherichia coli (E. coli) cells are continuously cultured in a 150 microL, membrane-aerated, well-mixed microbioreactor fed by a pressure-driven flow of fresh medium through a microchannel. Chemotaxisial back growth of bacterial cells into the medium feed channel is prevented by local heating. Using poly(ethylene glycol) (PEG)-grafted poly(acrylic acid) (PAA) copolymer films, the inner surfaces of poly(methyl methacrylate) (PMMA) and poly(dimethylsiloxane) (PDMS) of the microbioreactor are modified to generate bio-inert surfaces resistant to non-specific protein adsorption and cell adhesion. The modified surfaces of microbioreactor effectively reduce wall growth of E. coli for a prolonged period of cultivation. Steady state conditions at different dilution rates are demonstrated and characterized by steady OD, pH, and DO levels.     

Microbioreactor arrays with integrated mixers and fluid injectors for high-throughput experimentation with pH and dissolved oxygen control

We have developed an integrated array of microbioreactors, with 100 microL working volume, comprising a peristaltic oxygenating mixer and microfluidic injectors. These integrated devices were fabricated in a single chip and can provide a high oxygen transfer rate (k(L)a approximately 0.1 s(-1)) without introducing bubbles, and closed loop control over dissolved oxygen and pH (+/-0.1). The system was capable of supporting eight simultaneous Escherichia coli fermentations to cell densities greater than 13 g-dcw L(-1) (1 cm OD(650 nm) > 40). This cell density was comparable to that achieved in a 4 litre reference fermentation, conducted with the same strain, in a bench scale stirred tank bioreactor and is more than four times higher than cell densities previously achieved in microbioreactors. Bubble free oxygenation permitted near real time optical density measurements which could be used to observe subtle changes in the growth rate and infer changes in the state of microbial genetic networks. Our system provides a platform for the study of the interaction of microbial populations with different environmental conditions, which has applications in basic science and industrial bioprocess development. We leverage the advantages of microfluidic integration to deliver a disposable, parallel bioreactor in a single chip, rather than robotically multiplexing independent bioreactors, which opens a new avenue for scaling small scale bioreactor arrays with the capabilities of bench scale stirred tank reactors.     

Library synthesis and characterization of 3‐aminopropyl‐terminated poly(dimethylsiloxane)s and poly(ϵ‐caprolactone)‐b‐poly(dimethylsiloxane)s

Libraries of 3‐aminopropyl‐terminated poly(dimethylsiloxane) (APT–PDMS) and poly(?‐caprolactone)–poly(dimethylsiloxane)–poly(?‐caprolactone) (PCL—PDMS–PCL) triblock copolymers were synthesized. Preliminary experiments were carried out to select an appropriate catalyst and route for the poly(dimethylsiloxane) synthesis, and trial experiments were conducted to verify the successful synthesis of the intended polymer compositions. Then, a series of APT–PDMS oligomers were synthesized with an automated combinatorial high‐throughput synthesis system to cover a molecular weight range of 2500–50,000 g/mol. Trial PCL—PDMS–PCL triblock copolymers were synthesized with the automated reactor system and characterized in detail with rapid gel permeation chromatography, high‐throughput Fourier transform infrared, nuclear magnetic resonance, and differential scanning calorimetry. Finally, two library synthesis experiments were carried out in which the lengths of both the poly(dimethylsiloxane) and poly(?‐caprolactone) blocks in the PCL—PDMS–PCL triblock copolymers were varied. The results obtained from these experiments demonstrated that it was possible to synthesize libraries of well‐defined APT–PDMS oligomers and PCL—PDMS–PCL triblock copolymers with an automated high‐throughput system. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 4880–4894, 2006     

应用于生物发酵在线监测的聚合物pH传感膜

微型生物反应器的成功操作依赖于对培养液pH值的监测和控制,聚合物pH荧光探针可以制成传感膜安装在微型反应器上,具有高通量筛选、非入侵性、容易集成等特征。甲基丙烯酸羟乙酯和N-(2-甲基丙烯酰乙酯基)-4-(N-甲基哌嗪基)-1,8-萘酰亚胺(NI)共聚形成的聚合物,具有良好亲水性、成膜性和生物相容性,并在pH=6~8之间具有良好的响应性能和稳定性。为了进一步提高膜的响应速率,将含有金刚烷的单体引入聚合物中,结果显示随着侧链金刚烷基团的含量增加,共聚物膜的pH响应时间变长;而将强亲水性的聚乙二醇引入共聚合物中,发现共聚物膜的pH响应速率得到了提高。将此pH传感膜应用于酵母发酵液中进行测试,具有良好的pH值在线监测性能。     

A polymer-based microfluidic device for immunosensing biochips

This paper describes the design, fabrication, and test of a PDMS/PMMA-laminated microfluidic device for an immunosensing biochip. A poly(dimethyl siloxane)(PDMS) top substrate molded by polymer casting and a poly(methyl methacrylate)(PMMA) bottom substrate fabricated by hot embossing are bonded with pressure and hermetically sealed. Two inlet ports and an air vent are opened through the PDMS top substrate, while gold electrodes for electrochemical biosensing are patterned onto the PMMA bottom substrate. The analyte sample is loaded from the sample inlet port to the detection chamber by capillary force, without any external intervening forces. For this and to control the time duration of sample fluid in each compartment of the device, including the inlet port, diffusion barrier, reaction chamber, flow-delay neck, and detection chamber, the fluid conduit has been designed with various geometries of channel width, depth, and shape. Especially, the fluid path has been designed so that the sample flow naturally stops after filling the detection chamber to allow sufficient time for biochemical reaction and subsequent washing steps. As model immunosensing tests for the microfluidic device, functionalizations of ferritin and biotin to the sensing surfaces on gold electrodes and their biospecific interactions with antiferritin antiserum and streptavidin have been investigated. An electrochemical detection method for immunosensing by biocatalyzed precipitation has been developed and applied for signal registration. With the biochip, the whole immunosensing processes could be completed within 30 min.     

Microchip electrophoresis coupled with on-line magnetic separation and chemiluminescence detection for multiplexed immunoassay

A facile and universal strategy for multiplexed immunoassay is proposed. The strategy is based on microchip electrophoresis (MCE) coupled with on-line magnetic separation and chemiluminescence (CL) detection. The system consisted of a microchip, an electromagnet, and a photomultiplier. The realization of multiplexed immunoassay protocol involves sampling magnetic nanoparticles (MNPs) labeled antibodies, N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) labeled antigens and free antigens in the precolumn reactor, on-line immunoreaction, capturing the MNPs-immunocomplexes, and the separation of unconjugated ABEI-labeled antigens. After on-line magnetic separation, the free ABEI-labeled antigens were transported into the separation channel, and mixed with hydrogen peroxide (H(2) O(2) ) in the presence of horseradish peroxidase in the postcolumn reactor, and producing CL emission. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. As a proof-of-concept, the multiplexed immunoassay was evaluated for the simultaneous determination of five model analytes (i.e. hydrocortisone, corticosterone, digoxin, testosterone, and estriol). The results exhibited excellent precision and sensitivity, the relative standard deviations for nine times detection were lower than 4.7% for all the five components, and the detection limits of five analytes were in the range of 3.6-4.9 nM. The MCE system was validated using two human serum-based control samples containing five analytes.     

Synthesis and phase behavior of polyurethanes end-capped with fluorinated phosphatidylcholine head groups

A series of fluorinated phosphatidylcholine polyurethane macromolecular additives were synthesized by solution polymerization using methylenebis(phylene isocyanates)(MDI) and 1,4-butanediol(BDO) as hard segments,a new phoshporycholine,2-(2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-10-(2-hydroxyethoxy)decyloxy) ethyl phosphorycholine (HDFOPC) as end-capper,and four polydiols,poly(tetramethylene glycol)s(PTMG),polydimethylsiloxane(PDMS), poly(1,6-hexyl-1,5-pentylcarbonate)(PHPC) and poly(propylene glycol)(PPG) as soft segments,respectively.The chemical structures of the synthesized polyurethanes were characterized by ~1H-NMR and FTIR.DSC and DMA were employed to study the phase behavior of these novel polyurethanes due to their great influences on the surface properties,and hence their interactions with bio-systems.The results showed that phase separation of the fluorinated phosphatidylcholine end-capped polyurethanes was increased in comparison with that of normal polyurethanes.The effect of fluorinated phosphatidylcholine end-capped groups on the phase behavior was further demonstrated by analyzing the degree of hydrogen-bonding between hard and soft segments.     

An electrochemically driven poly(dimethylsiloxane) microfluidic actuator: oxygen sensing and programmable flows and pH gradients

We describe the fabrication and performance of an integrated microelectrochemical reactor-a design possessing utility for multiple applications that include electrochemical sensing, the generation and manipulation of in-channel microfluidic pH gradients, and fluid actuation and flow. The device architecture is based on a three-electrode electrochemical cell design that incorporates a Pt interdigitated array (IDA) working (WE), a Pt counter (CE), and Ag pseudo-reference (RE) electrodes within a microfluidic network in which the WE is fully immersed in a liquid electrolyte confined in the channels. The microchannels are made from a conventional poly(dimethylsiloxane)(PDMS) elastomer, which serves also as a thin gas-permeable membrane through which gaseous reactants in the external ambient environment are supplied to the working electrode by diffusion. Due to the high permeability of oxygen through PDMS, the microfluidic cell supports significantly (>order of magnitude) higher current densities in the oxygen reduction reaction (ORR) than those measured in conventional (quiescent) electrochemical cells for the same electrode areas. We demonstrate in this work that, when operated at constant potential under mass transport control, the device can be utilized as a membrane-covered oxygen sensor, the response of which can be tuned by varying the thickness of the PDMS membrane. Depending on the experimental conditions under which the electrochemical ORR is performed, the data establish that the device can be operated as both a programmable pH gradient generator and a microfluidic pump.     

Harnessing the Effect of pH on Lipid Production in Batch Cultures of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> SKY7

The objective of this research was to investigate the kinetics of lipid production by Yarrowia lipolytica SKY7 in the crude glycerol-supplemented media with and without the control of pH. Lipid and citric acid production were improved with the pH control condition. There was no significant difference observed in the biomass concentration with or without the pH control. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g/L, (45.5% w/w), respectively, with lipid yield (Yp/s) of 0.179 g/g at 60 h of fermentation. The lipid production was directly correlated with growth and the process was defined as growth associated. After 60 h of fermentation, the lipid degradation was noticed in the pH-controlled reactor whereas it occurred after 84 h in the pH-uncontrolled reactor. Apart from lipid, citric acid was produced as the major extracellular product in both fermentations but the much lower concentration in uncontrolled pH. Based on the experimental results, it is evident that controlling the pH will enhance the lipid production by 15% compared to pH-uncontrolled fermentation.     

Elastomer-glass micropump employing active throttles

We report a reciprocating microfluidic pump, the Micro Throttle Pump (MTP), constructed in a relatively uncomplicated manner from glass and microstructured poly(dimethylsiloxane)(PDMS). Unconventionally, the MTP employs throttling of fluid flow as distinct from fully-closing valve structures. Accordingly, this technique offers the prospect of solid-phase suspension tolerance. The reported MTP employs piezoelectrically (PZT) actuated deformation of flow constrictions (throttles) fabricated from PDMS at the two ports of a central, PZT actuated pump chamber. By appropriate time-sequencing of the individual PZTs' actuation, pumping can be induced in either direction. PDMS' elasticity further facilitates throttle operation by virtue of allowing significant PZT flexure that is substantially independent of the underlying PDMS microstructure. In contrast, in a rigid substrate such as silicon, deformation is constrained to where underlying microstructured cavities exist and this restricts design options. We describe the construction and performance of a prototype MTP capable of pumping 300 microl min(-1) or alternatively generating a back-pressure of 5.5 kPa. Preliminary modelling of MTP operation is also presented.     

离子交换法从发酵液中提取聚苹果酸

用离子交换法提取发酵液中的聚苹果酸,比较了不同型号的阴离子树脂对聚苹果酸的静态吸附量,其中以D296树脂对聚苹果酸的吸附量最大.通过静态和动态方法,考察了不同操作条件对固定床分离工艺的影响.结果表明,发酵液经预处理后,调pH至9.5,以lBV/h流速上柱吸附,上柱量为4BV;用0.5mol/L NaCl洗脱,速度为1BV/h.该工艺能够将发酵液中大部分杂质去除,PMLA样品的纯度达到 93.2%,提取收率为79.46%.     

Monolithically integrated dye-doped PDMS long-pass filters for disposable on-chip fluorescence detection

We report the fabrication of high quality monolithically integrated optical long-pass filters, for use in disposable diagnostic microchips. The filters were prepared by incorporating dye molecules directly into the microfluidic chip substrate, thereby providing a fully integrated solution that removes the usual need for discrete optical filters. In brief, lysochrome dyes were added to a poly(dimethylsiloxane) (PDMS) monomer prior to moulding of the microchip from a structured SU-8 master. Optimum results were obtained using 1 mm layers of PDMS doped with 1200 microg mL(-1) Sudan II, which resulted in less than 0.01% transmittance below 500 nm (OD 4), >80% above 570 nm, and negligible autofluorescence. These spectral characteristics compare favourably with commercially available Schott-glass long-pass filters, indicating that high quality optical filters can be straightforwardly integrated into the form of PDMS microfluidic chips. The filters were found to be robust in use, showing only slight degradation after extended illumination and negligible dye leaching after prolonged exposure to aqueous solutions. The provision of low cost high quality integrated filters represents a key step towards the development of high-sensitivity disposable microfluidic devices for point-of-care diagnostics.     

Evaluation of PTMSP membranes in achieving enhanced ethanol removal from fermentations by pervaporation

The use of membrane processes for the recovery of fermentation products has been gaining increased acceptance in recent years. Pervaporation has been studied in the past as a process for simultaneous fermentation and recovery of volatile products such as ethanol and butanol. However, membrane fouling and low permeate fluxes have imposed limitations on the effectiveness of the process. In this study, we characterize the performance of a substituted polyacetylene membrane, poly[(l-trimethylsilyl)-l-propyne] (PTMSP), in the recovery of ethanol from aqueous mixtures and fermentation broths. Pervaporation using PTMSP membranes shows a distinct advantage over conventional poly(dimethyl siloxane) (PDMS) membranes in ethanol removal. The flux with PTMSP is about threefold higher and the concentration factor is about twofold higher than the corresponding performance achieved with PDMS under similar conditions. The performance of PTMSP with fermentation broths shows a reduction in both flux and concentration factor relative to ethanol-water mixtures. However, the PTMSP membranes indicate initial promise of increased fouling resistance in operation with cell-containing fermentation broths.     

A miniaturized high-voltage integrated power supply for portable microfluidic applications

In this work a portable microfluidic device with a reusable integrated high voltage power supply is presented, which allows for quick exchange of inexpensive disposable poly(dimethylsiloxane)(PDMS) microfluidic chips on a carrier only slightly larger than a microscope slide. The device is powered by an onboard MN21 cell battery (5 mm radius, 30 mm long) and is demonstrated through the rapid and controlled transport of a fluorescent dye through an expansion chamber geometry. Power consumption experiments demonstrate the device's ability to complete over 40 dispense-flushing cycles on a single battery.     

SYNTHESIS AND CHARACTERIZATION OF HYDROPHOBIC-HYDROPHILIC MULTIBLOCK COPOLYMERS FOR BIOMEDICAL APPLICATIONS*

The synthesis and characterization of amphiphilic copolymers of poly(dimethyl siloxane)(PDMS),poly(ethylene oxide)(PEO), and heparin(Hep) were investigated. These multiblock copolymers wereidentified using ~1H-NMR, FTIR, end group analysis, and sulfur elemental analysis. The multiblockcopolymers were characterized by using DSC and X-ray diffractometry. The glass transition temperature,crystalline melting characteristics, annealing effect, and cold crystallization of the block copolymers weredetermined by DSC. The crystallinity of the block copolymers was also determined by X-ray diffractionmethod.     

Facile synthesis of non-ionic dimeric molecular resonance imaging contrast agent: its relaxation and luminescence studies

A bis-polyazamacrocycle, 10'-bis(acetamido)ethane-bis[1,4,7-tri(carboxymethane)-1,4,7,10-tetraazacyclododecane] (DO3A-AME-DO3A) was synthesized for application in magnetic resonance imaging. The efficacy of DO3A-AME-DO3A as non ionic magnetic contrast agent was tested by performing relaxometric studies on its gadolinium complex. The longitudinal relaxivity, r(1) and transverse relaxivity, r(2) values were found to be 5.84 mM(-1)s(-1) and 6.82 mM(-1)s(-1), per Gd(III) at pH 7.0, 37 °C. The luminescence properties of europium complex of DO3A-AME-DO3A were investigated in aqueous medium. The lifetime of Eu(2)-DO3A-AME-DO3A in water was found to be 0.786 ms. Emission and luminescence lifetime measurements on the europium complex of DO3A-AME-DO3A gives a hydration number of q = 1.9. The reaction enthalpy and entropy were found to be, ΔH(0) = -(6.2 ± 2) kJ mol(-1), ΔS(0) = - (1.8 ± 0.4) kJ mol(-1)K(-1), and K(Eu)(298) = (1.8 ± 0.1).     

Pilot-Scale Fermentation of Aqueous-Ammonia-Soaked Switchgrass

Aqueous-ammonia-steeped switchgrass was subject to simultaneous saccharification and fermentation (SSF) in two pilot-scale bioreactors (50- and 350-L working volume). Switchgrass was pretreated by soaking in ammonium hydroxide (30%) with solid to liquid ratio of 5 L ammonium hydroxide per kilogram dry switchgrass for 5 days in 75-L steeping vessels without agitation at ambient temperatures (15 to 33 °C). SSF of the pretreated biomass was carried out using Saccharomyces cerevisiae (D₅A) at approximately 2% glucan and 77 filter paper units per gram cellulose enzyme loading (Spezyme CP). The 50-L fermentation was carried out aseptically, whereas the 350-L fermentation was semiaseptic. The percentage of maximum theoretical ethanol yields achieved was 73% in the 50-L reactor and 52–74% in the 350-L reactor due to the difference in asepsis. The 350-L fermentation was contaminated by acid-producing bacteria (lactic and acetic acid concentrations approaching 10 g/L), and this resulted in lower ethanol production. Despite this problem, the pilot-scale SSF of aqueous-ammonia-pretreated switchgrass has shown promising results similar to laboratory-scale experiments. This work demonstrates challenges in pilot-scale fermentations with material handling, aseptic conditions, and bacterial contamination for cellulosic fermentations to biofuels.     

Indirect fluorescence detection of simple sugars via high-pH electrophoresis in poly(dimethylsiloxane) microfluidic chips

This article describes the successful electrophoretic separation of simple carbohydrates in a polymeric microfluidic chip. The device fabricated in poly(dimethylsiloxane) (PDMS) is found to be stable in high-pH solutions. This allows sugars to be separated electrophoretically at pH values at or above their pK(a) using indirect fluorescence detection. Signal-to-noise values greater than 10:1 were obtained using a mercury arc lamp excitation source and a fluorescein-containing mobile phase for the detection of sugars at concentrations as low as 5 mM. The results obtained compare favorably with published results for the same system using a traditional fused-silica capillary. Analysis of the data revealed a significant experimental sensitivity of the migration times measured in these PDMS devices, an aging effect that leads to considerable systematic drift over the course of a series of replicate measurements. These experiments highlighted the importance of the surface chemistry of PDMS, especially as it pertains to its ability to support stable electroosmotic flow within the separation device. Channel priming at high pH provides a necessary, but by itself insufficient, means by which this instability can be minimized.     

Formation of polymer vesicles by amphiphilic fluorosiloxane graft copolymers in solution

Amphiphilic fluorosiloxane graft copolymers with a poly(dimethylsiloxane)(PDMS) backbone,a hydrophobic fluorosiloxane side-chain and three hydrophilic poiyether side-chains were synthesized by hydrosilation reaction in this work.The micellization of amphiphilic graft copolymers in the water/ethanol solvent system was investigated,and vesicles with different size were formed after the self-assembly system was aged for different time.