Unleashing the hidden potential of the anaerobic fungi

The microorganisms thriving in the ruminants digestive tract deliver the metabolic potential for degrading recalcitrant lignocellulose-rich organic matter and rendering it digestible. Amongst the microbiota thriving in the rumen, anaerobic fungi (AF) with the unpronounceable name Neocallimastigomycota host a complex array of enzymes that are especially adapted for degradation of lignocellulosic biomass like straw. In addition, their appressoria attach to the macromolecule structures and physically penetrate them, thus enhancing the enzymatic action. Cultivation of these fungi has often ended in frustration for researchers as the understanding of cultivation requirements is very poor. However, scientists see the great potential of these organisms for biotechnological use, in particular for the utilization of lignocellulosic residues (LCR) in closed loop recycling management. For this project, a consortium from Austria (leading agency; PI: Dr. Sabine Marie Podmirseg), Germany (PI: Dr. Michael Lebuhn) and Switzerland (PI: Prof. Urs Baier), all engaged in the harnessing of AF, is cooperating in close connection with research groups in the United Kingdom and in the Czech Republic. The overarching aim of the proposed project will be to remove existing methodological obstacles preventing the biotechnological utilization of anaerobic fungi. For that purpose, the consortium starts with the crucial basics, explores the cultivation requirements of Neocallimastigomycota, develops suitable culturing and detection methods and finally evaluates their biotechnological application for LCR disintegration. The working hypotheses are, amongst others: (i) the ecological niche for AF is larger than assumed to date; (ii) distinct requirements for micronutrients or supplements have to be met for long-time cultivation; (iii) cultivation without rumen fluid is possible for many strains; (iv) novel AF can be isolated from various also non-rumen habitats. The project will combine classical microbiological methods (microscopy, batch and continuous cultivation, enzymology) with molecular approaches (fluorescence in situ hybridization, quantitative PCR, direct RNA and next generation sequencing). It is expected to develop suitable protocols for the cultivation of anaerobic fungi and provide the basics for up-scaled production. The projects goal is to enable researchers and the society to make use of the AF for energetic and material use of LCR.

With more than 200 million tons generated world-wide, lignocellulosic waste (especially plant biomass) is one of the most abundant waste streams available. Turning this waste into renewable energy is crucial for achieving a sustainable, circular economy. Unfortunately, breaking down plant material, especially in oxygen-free environments like biogas reactors, is notoriously difficult. Cows and other herbivores, however, have all along been efficiently transforming plant biomass into energy. How? Well, herbivores have an ace up their guts: Neocallimastigomycota, the only fungal group known to thrive strictly under anoxic (oxygen-free) conditions, are experts in plant matter decomposition. Despite their potential our understanding of anaerobic gut fungi (AGF) is limited. Knowledge on their distribution in herbivorous host animals, on how to cultivate them continuously, and how to effectively detect and characterize them, is spotty. A teamofscientistsfrom Austria (Universität Innsbruck), Germany (LFL Freising, TU München, CeBiTec Bielefeld), and Switzerland (ZHAW) set out tochangethat. Joined by renowned researchers from the United Kingdom (IBERS) and the Czech Republic (IAPG), the "HiPoAF" team aimed to close fundamental knowledge gaps to eventually unleash the "high potential of anaerobic fungi". They investigated and optimized cultivation conditions, looked into effective preservation methods, optimized existing and developed complementary detection techniques. Main accomplishments include a standardized cultivation and preservation protocol, a novel AGF specific primer system, a reactor system optimized for continuous AGF growth, a global AGF habitat survey and first successes in the development of novel detection techniques (HCR-FISH,NIR spectrometry, mass spectrometry anddigital droplet PCR). In addition to their scientific endeavors, the HiPoAF team actively engaged in the international Anaerobic Fungi Network (www.anaerobicfungi.org), leading to a number of successful joint adventures and acknowledgement of AGF expertise on an international stage. Several national and international collaborations arose partially due to the HiPoAF team's active scientific outreach activities, e.g. the project homepage (https://www.hipoaf.com/home), a radio show, and considerable contribution in the organization of the first international AGF conference (IAFC2022). The HiPoAF team emphasized the importance of educating students, sprouting four PhD-, seven Master- and eight Bachelor theses and seven student apprenticeships. AGF are now well integrated in teaching at Universität Innsbruck and ZHAW, and even in the projected Science Center (https://www.mikrobalpina.org/en/) that shall be installed in Austria (near Innsbruck). While the unpronounceable name of this fascinating microbial group might slip the memory of the visitors there, the potential of Neocallimastigomycota for sustainable energy production certainly will not. Finally, illuminatingsome basic knowledge gaps in AGF research, the HiPoAFconsortiumwas abletotake one step ahead and acquire funding fora subsequent project (www.hipoaf.com), aiming at implementing thesesuccessful biomass degraders in biogas production from lignocellulosic wastes.