O'Mahony Kevin, Freitag Ruth, Dhote Bernhard, Hilbrig Frank, Müller Patrick, Schumacher Ivo
Laboratory of Chemical Biotechnology, Swiss Federal Institute of Technology, Lausanne, Switzerland.
Biotechnol Prog. 2006 Mar-Apr;22(2):471-83. doi: 10.1021/bp050302a.
The direct capture of bacteria produced in high cell density fermentation by filtration is not possible once the milliliter-scale has been surpassed. Filtration in the presence of a filter aid (body feed filtration) constitutes a putative and scalable alternative, but only if conditions proposed by industry for large-scale filtration processes, namely, flow rates (for aqueous solutions) in the range of 500-1,500 L/(m(2) x h) and a filter aid concentration of <or=20 g/L, can be met. To this end several filter aids (grades of cellulose, diatomaceous earth, mixes thereof) were tested, albeit with no success. Capture and retention of bacteria was low (not possible in the case of cellulose), and the produced filter cakes were unstable and of low permeability. In the case of the diatomaceous earth close to 100% bacterial capture was possible, but only with filter aid concentrations that were 1 order of magnitude above the proposed limit. By using flocculating agents (Fe(3+)/Al(3+), PEI) either alone or in combination with diatomaceous earth and also the use of positively charged cellulose, bacteria capture could be achieved from several liters of cell suspension (OD(600) <or= 15) in filtration experiments adhering to the industrial recommendations. However, the bacteria were only weakly retained in such cakes and easily released by sudden pressure pulses. Satisfactory results were only obtained by filtration of the bacteria suspension at slightly alkaline pH (8.0, 50 mM Tris buffer) after treatment with sodium-activated bentonite (5 g/L) and PEI (50 mg/L) in the presence of 250 mM NaCl. In such cases 100% of the bacteria could reproducibly be captured in a filtration using 20 g/L of diatomaceous earth as filter aid. The thus produced filter cakes strongly retained the bacteria and showed good filtration performance. The procedure is at present limited to E. coli culture with biomass contents of OD(600) <or= 15. Cultures with higher OD(600) have to be diluted prior to filtration.
一旦超过毫升规模，就不可能通过过滤直接捕获高细胞密度发酵产生的细菌。在助滤剂存在下进行过滤（预涂助滤剂过滤）是一种可行且可扩展的替代方法，但前提是要满足行业针对大规模过滤过程提出的条件，即（对于水溶液）流速在500 - 1500 L/(m²·h)范围内且助滤剂浓度≤20 g/L。为此，测试了几种助滤剂（不同等级的纤维素、硅藻土及其混合物），但均未成功。细菌的捕获和截留率很低（纤维素的情况下根本无法捕获），并且产生的滤饼不稳定且渗透性低。对于硅藻土，接近100%的细菌捕获是可能的，但所需的助滤剂浓度比建议的极限高出1个数量级。通过单独使用絮凝剂（Fe³⁺/Al³⁺、PEI）或与硅藻土联合使用，以及使用带正电荷的纤维素，在符合工业建议的过滤实验中，可以从几升细胞悬液（OD₆₀₀≤15）中实现细菌捕获。然而，细菌在这样的滤饼中只是被微弱截留，并且容易因突然的压力脉冲而释放。只有在用钠活化膨润土（5 g/L）和PEI（50 mg/L）在250 mM NaCl存在下处理后，在略碱性pH（8.0，50 mM Tris缓冲液）下过滤细菌悬液才能获得满意的结果。在这种情况下，使用20 g/L硅藻土作为助滤剂进行过滤时，可重复捕获100%的细菌。由此产生的滤饼能牢固地截留细菌，并表现出良好的过滤性能。该方法目前仅限于生物量含量OD₆₀₀≤15的大肠杆菌培养物。OD₆₀₀较高的培养物在过滤前必须稀释。
