Engineering lignocellulosic degrading bacteria to utilise waste from bioethanol production
The Microbiology Society is undertaking a project entitled A Sustainable Future as part of our 75th Anniversary, which aims to highlight the Sustainable Development Goals (SDGs) to our members and empower them to use their research to evidence and impact the goals. Earlier this year, we put a call out to our members to submit case studies in the following three areas: antimicrobial resistance, soil health and the circular economy.
This case study is written by Dr James Williamson, who is a Postdoctoral Research Fellow at the University of Warwick, UK. It focuses on the circular economy; an alternative to a traditional linear economy (make, use, dispose), in which we keep resources in use for as long as possible, extract the maximum value from them while in use, then recover and regenerate products and materials at the end of each service life.
What are the challenges/needs that this research/initiative addresses?
Lignocellulosic biomass is a by-product of several agricultural and industrial practises that produce or utilise plant biomass, including harvesting crops and producing bioethanol from sugarcane. It contains cellulose, a linear polysaccharide, encased by lignin, a polymer that is formed from derivatives of phenylalanine to producing a branched, heteromorphic structure, which gives plants their structural integrity. Both of these components are tough for organisms to degrade, with lignin being highly recalcitrant to both biological and chemical degradation.
Although cellulose can be converted commercially into bioethanol, lignin is still seen as a waste product despite offering the potential to be a source of useful aromatic compounds, which are currently sourced from crude oil, if they could be broken down cheaply.
This technology would be particularly useful in the bioethanol production of Brazil. In the 2018/19 period Brazil produced around 34 billion litres of ethanol1, almost all of this from sugar fermentation. The waste biomass produced by this is burnt and the heat is used to power the refinery. However, in many cases, deliberately inefficient incinerators are used because of the volume of waste biomass and the cost of other routes of disposal.
A potential use for this waste material is second generation bio-ethanol production; where plant biomass is treated with cellulases to convert cellulose into glucose, which can then be fermented into ethanol.
This approach could potentially double ethanol production without increasing land usage. However, the main barrier for the use of this technology is the high input cost due to the need for additional enzymes to be added, and the low cost of the final product. Consequently, currently second-generation accounts for less than 0.15% of total ethanol production in Brazil, however there are several companies running facilities with this technology.
When second generation ethanol production is used, the lignin waste stream is still left over and often burnt. Our work aims to find ways to use this waste stream with our approach focusing on engineering bacteria that can live on these feedstocks and introducing heterologous pathways for the production of higher value chemicals, such as fragrances and pharmaceuticals.
Ultimately, if these are successful, they could become part of the biorefinery’s in situ processing of sugarcane which would produce extra revenue stream, make second generation production more financially sustainable, and reduce the amount of biomass that is burnt.
What findings and solutions were provided by this research/initiative?
Our research group is investigating bacteria that can degrade lignin found in lignocellulosic biomass, and we are especially interested in novel enzymes and pathways involved in the utilisation of these feed stocks. We are then applying this to engineer ligno-cellulose degrading bacteria, primarily Rhodococcus jostii RHA1 and Pseudomonas putida, to produce compounds of interest from biomass.
Recently we showed that Pseudomonas putida can be used to turn wheat straw into 4- vinylguaiacol, a smoky smelling food flavouring2. This was done by using ferulic acid which is a phenolic acid found in grasses such as sugarcane and wheat, where it forms links between cellulose and lignin. Initially, we blocked the degradation of ferulic acid by performing a gene deletion, this led to the accumulation of ferulic acid and other phenolic acids. We then took a phenolic acid decarboxylase gene from Bacillus subtilis (padC), and integrated it into the ferulic acid degradation operon, which is induced by ferulic acid. Having this process controlled by the accumulation of the stating material, means that no additional inducer needs to be added to the process, and therefore incurs no additional cost.
Other work is trying to find uses for other phenolic acids derived from these waste feedstocks and finding ways to divert other metabolites from degradation into these pathways.
How can this research/initiative support the transition to a more sustainable future?
Bioprocessing lignin offers some advantages to chemical catalysis, since it does not involve organic solvents and does not generate chemical waste. However, on an industrial scale it can be expensive and produce lower yields than chemical synthesis from petrochemicals, which adds to the difficulty of establishing these as new technologies. However if multiple processes can be combined together to make other products from the same starting material (the biorefinery concept), you can reduce the costs and increase profitability, making it a more economically sustainable option and hopefully a more environmentally sustainable one too.
What is the future for research and innovation in this area?
The long-term goal is the ‘biorefinery’, which can generate both fuels and chemicals from agricultural waste. In order to make the biorefinery concept a reality, technology needs to be developed in order to convert lignin into high-value chemicals, and the yield of these chemicals needs to be commercially viable.
2. Williamson JJ, Bahrin N, Hardiman EM, Bugg TDH. Production of Substituted Styrene Bioproducts from Lignin and Lignocellulose Using Engineered Pseudomonas putida KT2440. Biotechnol J. 2020;15(7):1900571. doi:10.1002/biot.201900571
About the author
Dr James Williamson is a Postdoctoral Research Fellow at the University of Warwick, UK. James completed his PhD at the University of Nottingham. His work there consisted of engineering the plant epiphyte Pantoea agglomerans, for the production of high value terpenoids, such as taxadiene. His current project focuses on engineering lignin degrading bacteria to produce high value chemicals. More information about his work is available here.