| Expanded bed adsorption (EBA) was developed in the early 1990s, which is a novel bioseparation technology allowing to capture target proteins directly from unclarified feedstock, e.g. culture suspension, cell homogenates or crude extracts. EBA technology combines solid-liquid separation, concentration and primary recovery into a single unit operation aiming at reduced operational procedures, high biological activity, increased overall yield, as well as less requirements for capital investment and consumables. Base on the introduction of principles, characteristics and operation of EBA process, the influences of biomass (cell and cell debris) on EBA was discussed in the thesis. In addition, the concept of zeta potential was introduced for evaluating the electrostatic interaction between charged particles. The particularities and importance of EBA process design were described, and then the research idea came into being: establishing the evaluation methods to characterize the influences of biomass on EBA, forming the rational strategy of EBA process design, and verifying the feasibility of design strategy proposed with some EBA applications.The successful operation of EBA relies on the formation of a perfectly classified fluidized bed (termed expanded bed) even in the presence of turbid feedstock. Obviously the solid compositions (cell and cell debris) in unclarified feedstock have a potential impact on the stability of expanded bed operation. Aiming at the particularity of expanded bed adsorption - high content of bioparticles in the feedstock, the special ions (Li~+ and Br~-) was chosen as the tracer for residence time distribution (RTD) measurement in the presence of bio-panicles. Some factors were investigated to prove the suitability of the ion-selective electrode, such as working range, response time, flow rate and the concentration of bio-particles. The conditions of injection pulse were studied, the ionic concentration of pulse and the amount of pulse were optimized and the suitable pulse conditions are chosen. With the intact yeast cells as model bio-particles and E. coli homogenate as the real application object, RTD measurement was carried out in the presence of bio-panicles, and the influences of bio-particles concentration and operation conditions were evaluated. The results demonstrate that the RTD analysis based on the ion-selective electrode is feasible and could be used as a good method for stability evaluation of expanded bed adsorption inthe real application conditions.For the RTD analysis with ion-selective electrode there are some limitations in the real use, such as high consumption of feedstock, long operation time and special instrument used. It is necessary to establish a simpler method to measure the biomass-adsorbent interactions and characterize quantitatively the influences of biomass on EBA process. The biomass pulse-response method was established and a series of factors were investigated to optimize the evaluation method. The appropriate operation conditions were chosen as the OD600 of biomass pulse at the range of 0.5-0.6, and pulse loading at 80% volume of sedimented bed, expansion factor at 2.5. Then the biomass transmission index (BTI) was introduced as a quantitative parameter for evaluating the biomass adsorption in the expanded bed. The consistent results and slight measurement errors demonstrated that the biomass pulse-response method established in the present work is feasible, reliable and effective for quantitative evaluation of biomass-adsorbent interactions in the expanded bed. With intact ceils and cell debris of S. cerevisia, E. coli, B. subtilis and P. pastrois as the model biomass and anion exchanger Streamline DEAE and Streamline QXL as the model adsorbents, the method was then used to study the influence of ionic strength and pH of fluid phase on the biomass-adsorbent interactions.Since the electrostatic forces dominate the interactions between biomass and adsorbent, the concept of zeta potential was introduced to characterize the biomass/adsorbent electrostatic interactions during expanded bed application. The zeta potential of intact cells and cell debris of four microorganisms (S. cerevisia, E. coli, B. subtilis and P. pastrois) were measured under varying pH and salt concentration, and two ion-exchange adsorbents (Streamline DEAE and Streamline QXL) were investigated. The biomass transmission index (BTI) from the biomass pulse response experiments was used as the indicator of biomass adhesion in expanded bed. Combining the influences from zeta potential of adsorbent (C,a ), zeta potential of biomass (C^) and biomass size (d), a good linear relationship was established between the zeta potential parameter (-i^bd) and BTI for all experimental conditions. The threshold value of parameter (-C,aCbd) can be defined as 120 mV2(xm for BTI above 0.9. This means that the systems with (-CaCbd) < 120 show neglectable electrostatic bio-adhesion, and would have a considerable probability of forming stable expanded beds. The results indicated that zeta potential measurement can be used a screening method to select the operation conditions and adsorbents for EBA, eliminating the conditions of strong electrostatic interactions and ensuring the stability of expanded bed.Based on the result mentioned above and previous work, the methods evaluatingthe influences of bio-particles (cells or cell debris), such as RTD analysis, biomass pulse response experiment, zeta potential evaluation and finite bath adsorption experiment, were arranged for EBA process design. A rational strategy considering was proposed consequently, which optimizes the target protein capacity and restrain the bio-particles adsorption on the adsorbent together in order to ensure the stable and efficient EBA processes. The new strategy was verified with two EBA applications. Firstly, after investigating the adsorption of lactate dehydrogenase (LDH) and the influences of particles containing in the rabbit muscle homogenate, the dye affinity was chosen as the best adsorbent. Then the separation and elution conditions were optimized, and EBA process was used successfully to isolate LDH directly from rabbit muscle homogenate with a purification factor of 3.65 and yield of 85.2%. Secondly, for the separation of nattokinase from Bacillus subtilis fermentation broth, a mixed-mode adsorbent Fastline Pro was chosen based on the high adsorption capacity and low influence of Bacillus subtilis cells. The stability of expanded bed was verified and the separation conditions were optimized. The EBA process with the mixed-mode Fastline Pro was implemented to separate nattokinase directly from Bacillus subtilis fermentation broth. The purification factor reached 12.3, which demonstrated the integration advantage of EBA in enzyme separation.Aiming at the weaknesses in the conventional process development of EBA and the particularity of EBA - high particles containing in the feedstock, a series of methods were developed in the thesis to evaluate the influence of biomass on EBA process. A rational strategy was proposed to enhance the reliability of EBA process design. The new strategy would certainly reduce the time on process development and improve the applications of EBA process in biotechnology industry.
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