As part of a research project initiated by prof. Ronald de Vries to understand fungal biodiversity with respect to plant biomass degradation and to design a model that enables the prediction of the ability of fungal species to utilize different carbon sources, Tiziano Benocci dissected plant biomass utilization strategies of a specialist (Podospora anserina) and a semi-specialist (Trichoderma reesei).
Fungi are highly efficient plant biomass degraders and as such, in our growing bio-based economy, of interest for industry.
Some fungi are broadly used already by the industry, like generalist Aspergillus niger and semi-specialist Trichoderma reesei, while a larger group of fungi is being mined as a source of novel enzymes.
What lacks so far however is a way to select the best fungus for a specific application; the majority of fungal biodiversity has thus remained unutilized.

Fungi use different strategies to degrade plant biomass reflecting their ecological niches. Tiziano Benocci researched a semi-specialist and a specialist during growth on crude plant biomass and not only on simple mono- or polysaccharides in order to deeply understand their strategies. He focused on the specialist Podospora anserina, a fungus that as a late colonizer of herbivore dung has evolved an enzymatic machinery to degrade the more recalcitrant fraction of plant biomass, thus suggesting a great potential for biotechnology applications.
Benocci found out that depending on the substrates, different genes of the same fungus were expressed. Substrates of soybean hulls  and corn stover  were used in this research – these are two major agricultural waste residues. Soybean hulls resulting in a larger diversity of expressed genes. What we can learn from this works two ways: we learn not only what enzymes to use in time to break down what substrate, but also what substrate to use in time to produce what useful enzymes. ‘These strategies deserve deeper investigation, especially by comparing other crude substrates, in order to improve the efficiency of the enzyme cocktails.’ Benocci writes.

In order to do this research on Podospora anserina, Tiziano had to tackle another problem first: the fungus ages very fast. It seems that carbon limitation is the solution. The Luria-Bertani agar mimics the recalcitrant substrate the fungus naturally grows on and keeps the fungus healthy.

Benocci considers another finding during his research as even more significant: ‘deletion of the regulatory gene ara1 or the metabolic gene xki1 in the semi specialist Trichoderma reesei, leads to increased CAZyme gene expression on crude plant biomass.’ Which means that the fungus is more efficient in expressing plant biomass degrading enzymes if its metabolic pathway is obstructed by blocking one of these genes . ‘We expected this somewhat, but not to this extent’
Furthermore he found out how different substrates created different enzyme cocktails and at which point in time to harvest the enzymes best.

In his conclusion Tiziano Benocci explains the difference he found between a semi-specialist fungus and a specialist. Specialists like Podospora anserina grow in very specific environments where there is less competition. This results in less diversity of enzymes and an efficient but slow metabolism. Generalists like Aspergilli require lots of enzymes and a regulatory system that is highly flexible, highly complex, fast growing but not very efficient. Semi-specialist T. reesei sits somewhat in between these two, although it has a relatively small number of enzymes, but produced in higher levels.

This thesis is part of a larger study undertaken to deeply understand the degradation strategy of different, possibly useful fungi, on several natural substrates during time. But a more detailed comparative study between the three reference fungi: T. reesei, P.anserina and Aspergilli, is still needed. ‘This will give also a better answer to our initial question: what makes a fungus a generalist, specialist or semi-specialist?. Answering this question will not only benefit the development of better enzyme cocktails for the production of biofuels and biochemicals, but also many other industrial sectors, such as pulp and paper, food and feed and textiles’

Tiziano Benocci doesn’t know yet what he is going to do next, but it will be in biotechnology and it will be making use of fungi. ‘We have to fight all kinds of environmental issues and I want to do my small part’.