Reversible bacterial immobilization based on the salt-dependent adhesion of the bacterionanofiber protein AtaA

Abstract

Background: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. Results:We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step. Conclusions:We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

Immobilization of Enterobacter aerogenes by a Trimeric Autotransporter Adhesin, AtaA, and Its Application to Biohydrogen Production

“…This feature is hardly realized in direct immobilization through biofilm formation. The immobilized-cells by AtaA are hardly detached in buffer solution, while they are easily detached in pure water [33]. In this study, the results shown in Figure 5 suggest that the cells on polyurethane were maintained during the continuous reaction over 30 h without leaching, because the reaction rate did not reduce.…”

“…Overexpression of membrane proteins such as AtaA is commonly considered to become toxic, because the excessively produced proteins disrupt the bacterial membrane structures [34][35][36]. In our previous report, we unexpectedly found that immobilization of bacteria by surface production of AtaA causes no damage to the cells [33]. Moreover, the over expression of ataA did not seem to affect severely on E. aerogenes's growth.…”

“…For hydrogen production, AtaA-mediated immobilization offers several advantages over conventional immobilization methods: (1) wide-variety of carrier availability, (2) rapid and tenacious immobilization, and (3) direct immobilization on the surfaces of materials [24,28,29]. Moreover, we recently showed that whole-cell catalysts expressing AtaA can be reversibly immobilized on a carrier, enabling the reuse of bacterial cells and supports [33]. This feature is hardly realized in direct immobilization through biofilm formation.…”

“…Repetitive hydrogen production in the batch reactor was started by adding 20 pieces of the polyurethane foams holding the immobilized cells to 30 mL of BS-N medium supplemented with 0.5% glucose and 0.02 g/L (NH 4 ) 2 SO 4 in a sealed vial. The head-space gas was switched to N 2 , and the vial was incubated at 37 • C. During the incubation, the gases produced were collected by a gass-tight syringe, and their hydrogen content was measured by gas chromatography at designated time intervals (0, 3,6,8,12,15,18,21,27,30,33, and 36 h). The glucose concentration in the reaction medium was measured by a Glucose CII-Test Wako kit (Wako, Osaka, Japan) following the manufacturer's instructions.…”

Cell behavior of the highly sticky bacterium Acinetobacter sp. Tol 5 during adhesion in laminar flows

“…AtaA mediates the nonspecific, high adhesiveness to various abiotic surfaces mentioned above. This adhesive property can be conferred to originally non-adhesive bacteria by transformation with ataA and are applicable to cell immobilization in bioprocesses 24 – 26 . However, the behavior of Tol 5 cells in flows under the effect of shear stress has not yet been studied.…”