Engineering biofilm matrix for efficient biofilm-mediated contaminant removal

• Main Author: Zaiden, Norazean
• Other Authors: Cao Bin
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2021
• Subjects: Engineering::Bioengineering; Science::Biological sciences::Microbiology::Bacteria; Science::Biological sciences::Molecular biology
• Online Access: https://hdl.handle.net/10356/154444
• Institution: Nanyang Technological University

The naturally sorptive nature of biofilm matrix is an attractive feature to be exploited for environmental application. The objective of this thesis work is to engineer biofilm matrix into an enhanced biosorbent for efficient biofilm-mediated contaminant removal. Shewanella oneidensis was used as an environmentally relevant model organism for biofilm matrix modification through manipulating an abundant matrix-associated protein, BpfA. Arsenic (As) was the contaminant in focus to demonstrate the aimed enhanced sorption by the engineered biofilm. To understand the role of BpfA in the interaction between S. oneidensis and As, comparisons were performed on the global transcriptomics from the RNA-sequencing of the wild-type (WT) and the BpfA-lacking mutant bpfA. When treated with As, both the WT and the bpfA were shown to upregulate the ars-detoxification system for As tolerance, and the master regulator of metabolic processes, TyrR. Energy-consuming metabolic enzymes and low energy efficient metabolic pathways were downregulated in both. The lack of BpfA resulted in expression loss of genes that are primarily important for managing oxidative stress, protein synthesis and their quality assessment, amino acid and phospholipid biosynthesis, uptake of sulfate and exports of xenobiotics. These losses were observed to be compensated with the expression of megaplasmid-borne genes promoting cellular preservation by regulating the cellular metal content and defense, genetic information, and flux of exogenous genetic materials. With the aim to display As-binding domains in the S. oneidensis biofilm matrix, the chromosomal bpfA was genetically fused to arsR from E. coli, which encodes for a well-characterized As binding protein. The recombinant exhibited poor aggregation under rapid agitation but showed competency in biofilm formation under hydrodynamic conditions. Sorption of As by the engineered biofilms was quantified using packed-bed biofilm reactors and the results showed ~2-fold higher sorption by the recombinant biofilms than the WT biofilms. To overcome the limitation of expressing BpfA-ArsR through the only 1 copy chromosomal bpfA in S. oneidensis, an expression of a higher copy number of bpfA was attempted using vector systems. Due to its high repeats content and to accommodate its insertion into a vector system, the bpfA gene was truncated to encode domains deemed necessary for biofilm formation. Biofilm formation assay revealed the necessity of the RTX domain. Constitutive and high induction of BpfA truncates expression resulted in low amounts of biofilm formation. In contrast, substantial biofilm formed when the BpfA truncates were expressed in an IPTG-inducible vector system. This then allowed a further engineering of it to co-express ArsR, As-specific binding module, at its C-terminal end to be displayed as a component in the biofilm matrix for As sorption. When compared to WT, the As sorption by the biofilms of the vector-expressed BpfA-ArsR recombinants exhibited 3.6- fold more, while the biofilms of the recombinant expressing BpfA-ArsR in the chromosome exhibited 2.4-fold more. Therefore, implicating that vector-expressed BpfA-ArsR can increase As sorption through higher expression of BpfA-ArsR. Taken together, this thesis work reports the practicality of engineering biofilm matrix into a sustainable biological tool for environmental applications. Modifying an extracellular polymeric substance in biofilms can enhance its sorptive nature as shown in this thesis work and may also serve as a strategy to create other functional biofilms.