(123g) Engineering Catalytic Biofilms for Hydrolysis of Mannan Substrates

In biotechnology industry, enzymatic hydrolysis of polysaccharides is often facilitated by isolated enzymes or whole cells harbouring specific enzymes. Isolated enzymes are often sensitive to operational conditions and the whole cell-based polysaccharide hydrolysis are confined only to batch and fed batch systems. Biofilms offer attractive catalytic systems for continuous and robust biotransformations. Biofilms consist of surface attached microorganisms that are embedded within a matrix of self-produced extracellular polymeric substances and they are the most ubiquitous and resilient form of microbial life on earth. However, because of the presence of a polymeric biofilm matrix, diffusion limitations often pose a great challenge to biofilm-mediated whole-cell catalysis. In this study, we engineered a biofilm through the expression of a mannanase Man5C on cell surface to achieve an efficient biofilm-enabled biocatalytic activity to hydrolyze the amorphous mannan substrates. Using Shewanella oneidensis as a model organism, in which a biofilm matrix-associated surface protein BpfA has been identified in our previous study, we displayed Man5C on the cell surface of S. oneidensis through genetically fusing Man5C (~36 kDa) onto the C-terminus of BpfA (~285 kDa). We demonstrated that the cells with BpfA-Man5C on their surface exhibited the characteristic Man5C activity by hydrolyzing glucomannan in both growth and resting cell conditions. Intriguingly, the Man5C activity of the BpfA-Man5C could be well retained (~80%) after seven 24-h cycles, suggesting a high recyclability of the engineered biocatalyst. We further used this engineered strain to develop biofilms in a fluidized bed biofilm reactor and investigated the factors influencing the extracellular Man5C activity exhibited by the catalytic biofilms. Our results showed that the catalytic biofilms of the BpfA-Man5C cells exhibited a significantly higher hydrolysis activity than the planktonic cultures. Further, the effect of biofilm thickness on the overall biocatalytic activity in the fluidized bed bioreactor was elucidated. In our experimental conditions, an optimal biofilm thickness was determined as 20-30 µm, exhibiting an Man5C activity of ~1.4 g mannose/(h•mg biofilm).