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Bioprocesses involving more than one species can alleviate restrictions posed by limited substrate range of single species. Coupled, multistage cultures can be useful when heterogeneous substrates, such as lignocellulosic biomass, are exploited. Here, microbial production of α-olefins (C11) from lignocellulosic substrates, namely cellulose and technical lignin, was investigated. A two-stage culture with cellulose fermentation to organic acids by Clostridium cellulolyticum and subsequent upgrading of the organic acids to 1-undecene by engineered Acinetobacter baylyi ADP1 was established. As a result, A. baylyi ADP1 synthesised 107 μg L-1 of 1-undecene from cellulose. Additionally, ligninolytic effects by A. baylyi ADP1 on softwood were confirmed and downstream processing for continuous 1-undecene collection was introduced. In addition, the synthesis of poly-α-olefin trimers (C33) by the oligomerization of 1-undecene was demonstrated. This study demonstrates the potential of integrated multistage processes in treating challenging substrates. publishedVersion

The pulp and paper industry together with lignocellulosic biofuel production provides plentiful streams of lignin and lignin-derived molecules (LDMs) that currently remain underutilized. The heterogeneity and complexity of lignin along with the lack of convenient tools significantly hamper its utilization. Selective separation of these LDMs from streams using specific tools would allow the recovery of aromatic compounds, as well as facilitate biological processes aiming at lignin valorization. To this end, here we report the isolation and characterization of single-chain variable fragment (scFv) antibodies against ferulate, coumarate, and caffeate, which are the molecular representatives of LDMs. Binders for the target LDMs were enriched by interrogating a synthetic scFv library with the phage display technique. As a result, scFv binders specific against each of the target molecules were obtained with affinities in the micromolar range. The selectivity of scFvs towards specific LDMs was proved by recovering caffeate from simulated LDM solution, Kraft lignin, and rice straw hydrolysate samples. Further proof of concept studies with model compounds demonstrated the applicability of antibody-based binders as a detection tool for monitoring microbial LDM conversion. Overall, this study demonstrates the potential of scFv binders as a specific toolset for lignin compound recovery and analysis. publishedVersion