skip to main content

Search for: All records

Creators/Authors contains: "Lankiewicz, Thomas S."

Note: When clicking on a Digital Object Identifier (DOI) number, you will be taken to an external site maintained by the publisher. Some full text articles may not yet be available without a charge during the embargo (administrative interval).
What is a DOI Number?

Some links on this page may take you to non-federal websites. Their policies may differ from this site.

  1. 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 acceleratedmore »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.

    « less
  2. Abstract

    The genusNitrospirais the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marinaNb-295Twas isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential ofN. marinabased on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm thatN. marinabenefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolvedpCO2, and requires exogenous vitamin B12. In addition,N. marinais able to grow chemoorganotrophically on formate, and is thus notmore »an obligate chemolithoautotroph. We further investigated the proteomic response ofN. marinato low (∼5.6 µM) O2concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinitycbb3-type terminal oxidase increased under O2limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marineNitrospiraisolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

    « less