The steadily increasing global demand for platform chemicals, driven by globalization and population growthx, necessitates the development of renewable and readily available resources (Petricevic and Teece, 2019; Abel et al., 2016; Yang et al., 2022). Traditional fossil-based chemicals are not only finite but also contribute considerably to environmental pollution (De Guzman and Schiller, 2025; Wu et al., 2025). Consequently, a shift toward sustainable alternatives is essential. One promising approach is the utilization of lignocellulosic side streams derived from biowaste, such as residues from the paper and food industries, crop cultivation, and animal husbandry (Polizeli et al., 2005; Ringseis and Eder, 2025; Grossmann, 2024). These lignocellulosic materials could serve as a future base material for the production of various platform chemicals, which are vital for numerous industrial processes (Balchandani et al., 2025).

Current methods for processing lignocellulosic biomass often rely on harsh chemical pre-treatments that are both energy- and cost-intensive. Biological approaches, while more environmentally friendly, are typically uneconomical due to the high cost associated with enzyme treatments (Rahmati et al., 2020). A potential biotechnological solution lies in the use of evolutionarily adapted organisms.

Anaerobic gut fungi, which inhabit the digestive track of herbivores, have demonstrated exceptional efficiency in degrading lignocellulosic materials (Meili et al., 2023). Extracellular fungal cellulosomes—non-membrane-bound enzyme complexes composed of carbohydrate-binding domains and carbohydrate-active enzymes (CAZymes)—combined with mechanical penetration via appressorium-like structures, enable conversion rates of up to 48% for lignin-rich plant biomass (Ho et al., 1988; Jin et al., 2011; Hsin et al., 2025). The use of cellulosomes has been shown to be up to 12 times more effective than single-enzyme applications (Krauss et al., 2012). This underscores the potential of anaerobic fungi as biotechnological tools for efficient biomass conversion. However, effective application requires deeper understanding of their cultivation.

The phylum Neocallimastigomycota comprises strictly anaerobic fungi that play a key role in the digestion of plant material in herbivores (Hibbett et al., 2007). Despite being discovered nearly 50 years ago, optimal cultivation conditions for these fungi remain poorly understood, resulting in suboptimal cultivation practices (Orpin, 1977). Recent findings suggest that unique metabolites are produced in depending on the hosts species, indicating species-specific microbial interactions and metabolic profiles that depend on a balancedrumen microbiome (Omondi et al., 2024). Therefore, analyzing ecological parameters, growth conditions and media composition is essential for biotechnological applications such as biowaste utilization.

Anaerobic fungi generate energy through mixed-acid fermentation, which is linked to an incomplete citric acid cycle (Cheng et al., 2013; Song et al., 2009). The primary products—some of which are formed in hydrogenosomes, mitochondria-derived organelles—include acetate, formate, ethanol, lactate, hydrogen and succinate (Boxma et al., 2005; Marvin-Sikkema et al., 1990). While genetic and transcriptomic data offer insight into possible metabolic pathways, physiological studies remain scarce and are mostly limited to representatives of Neocallimastix and Piromyces (Wilken et al., 2021; Boxma et al., 2004). Building on the data and strains obtained and described by Stabel et al. (2021), this study aims to expand the understanding about carbon source utilization and process engineering for anaerobic gut fungi.

Therefore, the experimental design of this study followed a structured and progressive approach, beginning with the evaluation of the external environmental factor temperature, which is known to influence fungal growth and metabolism. Building on this, the impact of substrate particle size using the standard carbon source wheat straw. Subsequently, the metabolic responses to soluble carbon sources, including glucose, fructose, cellobiose, and xylose, was studied with additional consideration given to feeding strategies such as batch and fed-batch cultivation. Furthermore, biowaste-derived substrates were evaluated with regard to substrate biodegradability and metabolic output. Finally, this study concludes with the observation of a previously uncharacterized morphological growth form under starvation conditions, representing a novel finding with potential implications for fungal stress response and life cycle dynamics. This stepwise progression—from well-established parameters to exploratory observations—was designed to build upon existing knowledge while uncovering new insights into anaerobic fungal physiology.

The effects of temperature, various solid and soluble carbon sources, carbon source concentrations and solid particle sizes on anaerobic fungal metabolism of six different species were analyzed. To evaluate metabolic activity, the total amount of metabolites produced—i.e., the final amount of organic acids, ethanol and hydrogen—was used as an aggregate parameter.

To minimize bias resulting from stochiometric differences among these compounds, carbon or electron balances have been proposed as a good practice. However, previous studies have shown that the total metabolite concentration serves as a reliable indicator of anaerobic fungal vitality, due to its strong correlation with overall metabolic enzyme activity (Stabel et al., 2022).

Production of single metabolites over time depending on strain and carbon source, i.e., temporal changes in the metabolic profile, are evaluated in Chapter 6.4.

To comprehensively assess the metabolic versatility of the investigated strains, a broad screening was conducted to evaluate their ability to utilize a variety of carbon sources. Effective assimilation of these substrates depends not only on the presence of the required genetic and enzymatic machinery, but also on the environmental conditions under which these pathways are activated. Among these, temperature plays a critical role, as enzymatic activity is inherently temperature-dependent (McLeod, 2025). To account for this, temperature screening experiments were performed across a range of conditions. Although all strains exhibited comparable tolerance ranges, distinct differences in their optimal growth temperatures were observed, indicating strain-specific thermal preferences.

In addition to identifying optimal growth conditions, the robustness of the strains under suboptimal—particularly cold—conditions was evaluated. A. dupliciliberans, C. churrovis, and N. cameroonii exhibited enhanced thermotolerance and resilience to cold exposure, which is advantageous for industrial applications. This increased robustness facilitates easier handling, storage, and transportation, thereby reducing logistical constraints and associated costs (Matsushita et al., 2016).

Another critical factor in the biotechnological application of lignocellulosic biomass is the pretreatment of the selected carbon sources. Such processes are often energy-intensive and economically challenging (Rahmati et al., 2020). To investigate whether substrate particle size influences fungal metabolism, substrates of varying sizes were tested. Interestingly, no significant changes in the overall metabolic profiles of the anaerobic fungi were observed, except for a notable increase in malate concentration when particles of 50 mm were used.

Since malate is an intracellular metabolite, its release may indicate cell lysis or metabolic stress. In filamentous fungi such as Mucor circinelloides, malate transport is closely linked to metabolic reorganization under stress conditions (Chen et al., 2024). It is therefore conceivable that exposure to large particle sizes—which may limit nutrient availability—leads to increased malate release, potentially as a result of prolonged stress or nutrient limitation (Zhao et al., 2018). This delayed substrate utilization suggests an extended adaptation or starvation phase prior to the onset of active metabolism. These findings imply that, under certain conditions, extensive pretreatment may not be necessary—particularly in large-scale fermentations. Reducing the degree of pretreatment could significantly lower operational costs without compromising metabolic efficiency (Pasalari et al., 2024).

Although solid carbon sources are traditionally used for the cultivation of anaerobic fungi, soluble carbon sources offer several notable advantages. These include improved dosing accuracy, enhanced bioavailability, and the potential use of inexpensive side streams like glucose syrup (Yang et al., 2024; Yaseen et al., 2013). In addition, soluble substrates provide logistical benefits, including reduced transportation costs due to their higher packing density and easier handling in automated systems (Schoemaker et al., 2003). Based on the findings from Stabel et al. (2021) the soluble carbon sources glucose, fructose, cellobiose and xylose were investigated.

Anaerobic fungi of the phylum Neocallimastigomycota exhibit pronounced metabolic flexibility in response to both type and concentration of carbon sources, with significant implications for hydrogen production and metabolite profiles. In the strains N. cameroonii, C. churrovis, and K. ramosus, increasing concentrations of glucose led to elevated hydrogen yields per gram of substrate, despite a reduction in substrate uptake efficiency. Hydrogenosomal metabolism—typically associated with acetate and hydrogen production—was dominant at lower substrate concentrations but declined as cytosolic pathways became more active. The bifurcating hydrogenase proposed by Wilken et al. (2021) may explain the sustained hydrogen output even under high glucose conditions. Overall, this decoupling of substrate uptake and product yield suggests a shift toward overflow metabolism, in which excess pyruvate is redirected from hydrogenosomal pathways to cytosolic mixed-acid fermentation. This results in increased production of lactate, formate, and succinate, mirroring metabolic phenomena such as the Crabtree and Warburg effects observed in other organisms (Battjes et al., 2024). Notably, A. dupliciliberans and N. cameroonii showed increased lactate production at higher sugar concentrations, while K. ramosus, lacking lactate dehydrogenase activity, favored the accumulation of formate and succinate. The presence of malate and citrate in the extracellular medium—particularly under xylose-rich conditions—indicates cellular lysis and metabolic stress, especially in strains such as K. ramosus and O. joyonii.

Cellobiose and glucose emerged as the most efficiently metabolized substrates across nearly all strains, likely due to the fungi's robust cellulolytic enzyme systems, including CAZymes and cellulosomes. In contrast, xylose was poorly utilized by most strains, as evidenced by high residual concentrations and low hydrogen yields. An exception was O. joyonii, which demonstrated efficient xylose uptake but limited conversion to hydrogen, further supporting a shift toward non-hydrogenogenic metabolic pathways.

Overall, these findings highlight a dynamic reprogramming of central carbon metabolism in anaerobic fungi, driven by substrate availability and concentration. The transition from hydrogenosomal to cytosolic fermentation under substrate-rich conditions reflects a trade-off between energy efficiency and metabolic throughput. This metabolic shift has important implications for optimizing biotechnological processes such as biohydrogen production and lignocellulosic biomass conversion.

An additional critical parameter for optimizing the production of target metabolites in anaerobic fungal fermentations is the applied feeding strategy. To assess its impact, conventional batch and fed-batch cultivation modes were compared in terms of metabolic output. However, the transition from batch to fed-batch cultivation did not result in any statistically significant improvement in metabolite production. This outcome suggests two potential limiting factors: first, the likelihood of substrate overfeeding in the fed-batch setup, which may have led to metabolic saturation or stress responses; and second, the accumulation of inhibitory end-products, such as organic acids, ethanol or hydrogen—which can negatively affect cellular metabolism and growth.

These findings indicate that simply increasing substrate availability through fed-batch operation does not necessarily lead to higher metabolite yields. Instead, they underscore the importance of carefully balancing substrate input with the metabolic capacity of the organism, as well as mitigating the accumulation of inhibitory by-products. Future optimization efforts should therefore consider dynamic feeding strategies—such as feedback-controlled feeding—in combination with in situ product removal or pH control. These approaches can help maintain favorable metabolic conditions and prevent pathway inhibition, thereby enhancing overall process efficiency.

In addition to soluble carbon sources, biowaste materials were considered for further improvement. Comparative analysis revealed that banana peels, cucumber peels, carrot peels, and potato peels exhibited higher consumption rates than wheat straw, indicating superior biodegradability and enhanced suitability as substrates for anaerobic fungal cultivation. These materials likely provide more accessible carbon sources and fewer structural barriers, making them promising candidates for biowaste valorization in fungal bioprocesses (Nguyen and Nguyen, 2023; Kalogiannis et al., 2023). In contrast, reed and coffee grounds showed lower degradation levels, suggesting limited applicability due to their recalcitrant composition or potential inhibitory compounds (Czubaszek and Wysocka-Czubaszek, 2025; Hernandez-Hosaka et al., 2024). Overall, these findings underscore the potential of selected kitchen waste streams as sustainable and efficient alternatives to conventional lignocellulosic feedstocks in anaerobic fungal biotechnology.

A previously uncharacterized growth form was observed in strain O. joyonii, representing a potentially impactful discovery in the life cycle or stress response of anaerobic fungi. Preliminary observations suggest that this morphological shift is induced under conditions of prolonged fiber limitation and sustained environmental stress. The applied cultivation method, which involved gradual starvation, appears to have triggered this transformation. A comparable phenomenon was reported by Stabel et al. (2022) who documented the formation of fungal pellets during fermentation, indicating that morphological plasticity may be a common response to adverse conditions.

The nature of this newly observed form remains to be elucidated, but its emergence under stress conditions raises the possibility that it may represent a survival structure or even a sexual reproductive stage. Intriguingly, the formation of such structures may also be influenced by mechanical stimuli, as suggested by the experimental setup from Stabel et al. (2022). If this hypothesis holds true, it could imply that mechanical forces encountered during gut passage—such as peristalsis or abrasive interactions with digesta—may play a role in triggering developmental transitions. This would have profound implications for understanding the ecology and transmission of anaerobic gut fungi within herbivorous hosts, potentially revealing a mechanism by which fungal propagules are activated or dispersed during host-to-host transfer.

Further investigation, including microscopic characterization, transcriptomic profiling, and controlled mechanical stimulation experiments, will be essential to elucidate the biological function and regulatory mechanisms underlying this novel growth form.

This study provides several novel insights into the metabolic behavior and biotechnological potential of anaerobic gut fungi that go beyond the scope of previous research. While earlier studies such as Stabel et al. (2021) focused on the biochemical characterization of individual strains and their metabolite profiles under standard conditions, and Wilken et al. (2021) explored genomic adaptations like horizontal gene transfer, and Omondi et al. (2024) offered a comparative multi-omics perspective across host species, the present work delivers a systematic, multifactorial analysis of fungal metabolism under varying environmental and nutritional conditions using six distinct strains. One of the most significant contributions is the discovery of a previously uncharacterized growth form in O. joyonii under starvation conditions, which may represent a survival or reproductive stage. This morphological shift, potentially triggered by mechanical stimuli, introduces a new dimension to the ecological understanding of anaerobic fungi and their transmission within herbivorous hosts. Furthermore, the study demonstrates a concentration-dependent metabolic reprogramming, where increasing substrate availability leads to a shift from hydrogenosomal to cytosolic fermentation pathways. This shift is characterized by increased production of lactate, formate, and succinate, and reflects overflow metabolism similar to the Crabtree and Warburg effects observed in other organisms. The sustained hydrogen production under high glucose conditions, potentially mediated by bifurcating hydrogenase activity as proposed by Wilken et al., highlights the metabolic flexibility of these fungi. In addition, the work evaluates the biodegradability of various kitchen waste materials—such as banana, carrot, and potato peels—revealing them as superior substrates compared to wheat straw, which has direct implications for sustainable feedstock selection in industrial applications. Unlike previous studies that focused primarily on lignocellulosic substrates, this research expands the substrate spectrum and demonstrates the fungi's capacity to valorize diverse biowaste streams. Another important finding is that fed-batch cultivation does not improve metabolite yields, likely due to overfeeding and end-product inhibition, emphasizing the need for adaptive feeding strategies and in situ product removal. Moreover, the observation that substrate particle size has minimal impact on overall metabolite production suggests that costly pretreatment steps may be unnecessary, offering economic advantages for scale-up. Taken together, these findings significantly advance the current understanding of anaerobic fungal metabolism and provide actionable insights for optimizing biotechnological processes, from substrate selection to fermentation strategy and ecological interpretation.

To further expand the understanding of anaerobic fungal metabolism and its biotechnological potential, we propose several additional experiments. First, pH-dependent cultivation trials should be designed to investigate how varying acidity levels influence metabolic fluxes and product distribution, particularly the balance between hydrogenosomal and cytosolic fermentation. Second, long-term cultivation experiments should be initiated to assess adaptive responses over multiple generations, potentially revealing strain-specific improvements in substrate utilization and stress tolerance, and also showing limitations in the current cultivation strategy. Additionally, co-feeding strategies combining soluble sugars with biowaste-derived substrates should be tested to evaluate synergistic effects on metabolic output and substrate degradation efficiency. To mitigate end-product inhibition, in situ product removal techniques—such as membrane filtration and adsorptive resins—should be explored, aiming to enhance metabolite yields by maintaining favorable fermentation conditions in serum bottles. Mechanical stimulation experiments should also be conducted to simulate peristaltic forces and assess their role in triggering morphological transitions, including pellet formation or stress-induced growth forms. Finally, mixed-strain cultures should be evaluated to determine whether interspecies interactions could promote complementary metabolic pathways and improve overall process performance.