Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

Abstract

Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. Results We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. Conclusions The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose

“…3C ). While non-rhizoidal strains such as C. churrovis are well-suited for cultivation conditions that allow the concentrations of anaerobic fungi to be measured and subsequent flux calculations to be performed (Leggieri et al., 2021 ), the current study suggests a potential trade-off to using non-rhizoid-forming fungi to liberate sugar from lignocellulose, which ultimately limits its ability to generate nutrients for a co-cultured partner like C. acetobutylicum . Gas pressure readings also showed that addition of C. acetobutylicum to reed canary grass in the absence of fungi led to negligible growth, as this bacterium does not possess CAZymes to hydrolyze lignocellulose (Fig.…”

Patterns and determinants of the global herbivorous mycobiome

“…Differences in overall architecture, size, and residence time in alimentary tracts of different hosts could result in niche-driven selection of distinct AGF communities. In addition, variation in bacterial and archaeal community structures between hosts could also elicit various levels of synergistic, antagonistic, or mutualistic relationships that impact the AGF community 78 – 81 . However, domestication status could counter, modulate, or override host identity.…”

The enrichment process and morphological identification of anaerobic fungi isolated from buffalo rumen

“…The breakdown of glucose into acetate produces more ATP than lactate because the acetate conversion process reduces pyridine nucleotides and produces ATP (Marvin-Sikkema et al 1990;Wilken et al 2021). Graaf et al (2011) and Leggieri et al (2021) showed that pyruvate converted to Acetyl CoA first and finally converted to acetate. Every transformation of Acetyl CoA to acetate by ASCT (Acetate: Succinyl CoA Transferase) and SCS (Succinate CoA Synthase) enzyme in hydrogenosomes produce ATP (Graaf et al 2011;Makiuchi and Nozaki 2014;Gawryluk et al 2016).…”