Wheat straw (WS) is an energy-rich, relatively inexpensive source of biomass that can be converted to biogas fuel during anaerobic digestion (AD) by microbes living in the absence of oxygen. WS is a particularly good candidate for conversion to clean energy and other useful products in industrial bioprocessing because it is a globally abundant agricultural residue and is commonly viewed as a waste product. However, challenges are associated with using WS as a feed resource for fuel production because the chemical structure is abundant in lignocellulose. Lignocellulose, a mixture of biomass polymers, is highly resistant to enzymatic breakdown (or hydrolysis) by the majority of well-characterised microbial species. Hydrolysis is an essential step that is required to derive small enough sugars from WS, for uptake by microbial cells. During conventional AD of WS, the speed of hydrolysis often limits the rate of biogas fuel production and this heavily influences the overall bioreactor size. Therefore, AD plants tend to be very large in order to allow for the time required for bioconversion of WS and this can increase capital and operational costs.

Typically, industrial AD is reliant on undefined microbial communities. The hydrolysis stage can take weeks and is performed by a consortium of bacteria dominated by species of Clostridium. Several of these species can deconstruct WS into simple sugars that they ferment to provide for their metabolism. Products of clostridial fermentation include H2, CO2 and acetic acid. These chemicals become the substrates for a second group of microbes, the methanogenic Archaea, which convert them to methane fuel. The biological process of lignocellulose conversion in AD is analogous to microbial activity in the rumen of mammalian herbivores (e.g. cattle and sheep). However, lignocellulose in the rumen is converted over a much shorter time period that lasts for several days (as opposed to several weeks in industrial AD). One explanation for this discrepancy stems from the fact that anaerobic fungi native to the rumen are able to perform hydrolysis much more efficiently and effectively than clostridial bacteria. However, in comparison to clostridia, relatively little is known about the anaerobic fungi. Furthermore, no information is available concerning their growth and activity with co-culturing methanogens in continuously-fed fermentation systems. Almost all previous investigations on the anaerobic fungi have used culture volumes (typically less than 100 ml) and batch-culture methodologies that are not comparable with industrial AD, nor with the growth conditions prevalent in the rumen. In particular, information is unavailable about the ability of anaerobic fungi to survive in continuously fed bioreactors. This is due to the absence of a low-cost, lab-scale bioreactor, capable of continuously feeding particulate lignocellulose material while maintaining the aseptic, anaerobic conditions that are necessary for fungus-methanogen co-culture survival.

Initially, this project aims to meet a requirement for the development of an automated lab-scale bioreactor system that is capable of continuously feeding WS to anaerobic fungus-methanogen co-cultures under aseptic conditions. The newly developed system will be used to study microbial growth in a continuous culture system analogous to their native habitat. Additionally, biomethane production will be compared between fungus-methanogen co-cultures and conventional AD consortia (dominated by clostridial species and their associate methanogens). If the fungus-methanogen co-culture can significantly outperform conventional AD, this new knowledge will facilitate the production of smaller digesters that can handle significant throughput of lignocellulose material. This will ease the cost of anaerobic digesters for decentralised production of clean energy in rural communities that exist in close proximity to cereal crop growers.

Potential Impact:
In the short-term, project outputs will provide quantifiable evidence, high impact publications and intellectual property regarding the economic benefits, bioreactor stability and green credentials of selecting anaerobic fungi with/without methanogens for hydrolysis of lignocellulosic biomass. The newly developed bioreactor will provide a resource for academics and industry to purchase, rent or use in collaboration with myself. Multiple options to access the new facility will allow researchers to make use of the equipment. The facility will be advertised on host institution websites, via social media and by specifically targeting relevant research groups (as already accomplished with UCSB and Nanjing). Further impact from the developed equipment will be achieved via its use for biotechnology feasibility studies. The purpose here is to demonstrate co-culture growth for efficient biogas fuel production. However, the wider market for the equipment includes the continuous conversion of lignocellulose by other anaerobic microorganisms/microbial consortia into a range of green chemicals. This includes hydrolysis of lignocellulosic material for production of volatile fatty acids for microbial electrolysis cells, microbial fuel cells and bioplastic production. Additionally, I intend to engage with international corporations that produce cocktails of enzymes for large-scale lignocellulosic pre-treatment purposes (e.g DuPont & Novozymes). Typically, these cocktails are sourced from the growth of aerobic fungi. These companies are likely to consider commercially developing efficient cellulosome enzyme products, harvested from continuous growth of anaerobic fungi.

The longer-term impact of the work is in the use of defined co-cultures for efficient biogas production from wheat straw (WS). Team work between myself, other academics and industrial partners will target this achievement. PlanET has a global network of customers using conventional anaerobic digestion technology. Partnership between Harper Adams University (HAU) and PlanET will provide the experience and consumer links for transition of the work from proof of concept to use in an industrial context. Biopower Technologies Ltd. will supply WS (in kind), because the company wishes to explore new outlets for their WS milling process. Biopower Technologies Ltd. has equipment to meet the on-site WS throughput demands for full-scale WS digestion. Elentec Ltd. desires to generate green power for their decentralised water treatment and nutrient recovery systems. Bringing this set of expertise together will create a pathway to an end-to-end decentralised process that includes preparation of the feedstock, biogas production by defined co-cultures and onsite nutrient and water recovery. These partners will be encouraged to invest in an R&D Innovate UK programme to explore commercial opportunities in a larger-scale study with the potential for both national and international impacts.

Interaction with stakeholders from the agricultural community is an essential pathway for longer-term deployment of pure culture anaerobic digestion systems in to rural communities. HAU's well-established position within the agricultural sector will be invaluable for raising awareness and promoting implementation of pure culture digestion to arable industries. Work will be presented at open-access workshops and seminars which are regularly hosted, to enable public outreach. The use of fungi-methanogen co-cultures for cheap and clean energy production will also be shared via scientific publications and relevant conferences. The novelty of this work should lead to publications in high impact journals. The project will be a basis for obtaining follow on RCUK funding to keep the UK at the forefront of this growing field. Later projects will involve anaerobic fungi for biotechnology processes with alternative green end-products (such as those described above) to reduce the use of fossil fuels.