Title: 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

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-culturemore » 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.« less

Authors:

Leggieri, Patrick A.; Kerdman-Andrade, Corey; Lankiewicz, Thomas S.; Valentine, Megan T.; O’Malley, Michelle A.

• Search DOE PAGES for author "O’Malley, Michelle A."
• Search DOE PAGES for ORCID "0000-0002-6065-8491"

Publication Date:

Mon Oct 18 00:00:00 EDT 2021

Research Org.:

Univ. of California, Santa Barbara, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC); National Science Foundation (NSF); Army Research Office (ARO); California NanoSystems Institute Challenge Grant Program; University of California, Santa Barbara

OSTI Identifier:

1826241

Alternate Identifier(s):

OSTI ID: 1980956

Grant/Contract Number:  

SC0020420; MCB-1553721; DBI-1625770; W911NF-19-1-0010

Resource Type:

Published Article

Journal Name:

Microbial Cell Factories

Additional Journal Information:

Journal Name: Microbial Cell Factories Journal Volume: 20 Journal Issue: 1; Journal ID: ISSN 1475-2859

Publisher:

Springer Science + Business Media

Country of Publication:

United Kingdom

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; anaerobic fungi; methanogens; co‑culture concentrations; lignocellulose; CAZymes; hydrogenosome; synthetic consortia; co‑culture growth rates; metabolic flux; non‑model microbes