Browsing by Author "Liu, Yihua"

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    Assimilation of carbon and nitrogen by microbial mats from alkaline soda lakes
    (2020-11-19) Liu, Yihua; Strous, Marc; Hubert, Casey R J; Tutolo, Benjamin M; Hu, Jinguang
    Background:Soda lakes are extreme terrestrial ecosystems characterized by high pH, alkalinity, and sodium carbonate concentration. Despite the extreme environment, soda lakes host diverse microbial communities with high primary productivity, carried out by fast-growing phototrophic microbes such as cyanobacteria. In Goodenough Lake, a soda lake on the Cariboo Plateau in BC Canada, carbon isotope analysis indicated that the photosynthetic rate but not bicarbonate availability controlled carbon dioxide assimilation. However, the roles of individual cyanobacteria populations in carbon fixation remain unknown. Despite the rapid growth of microbial mat communities, common nitrogen sources, ammonium and nitrate, were detected only occasionally and in trace amounts in lake water. Mat communities may use alternative nitrogen sources like urea and dinitrogen gas as enzymes for urea assimilation and dinitrogen fixation were highly expressed.Objective:The objective is to measure carbon and nitrogen assimilation by microbial populations in mat communities.Approaches:Incubation of microbial mats from Goodenough Lake with heavy stable isotope labelled bicarbonate, and nitrogen sources, followed by isotope ratio mass spectrometry and proteomics.Results and conclusions:Over 90 different microbial populations were detected in microbial mat communities using proteomics. The sampled mat microbial communities were different from each other, even if samples were close together, but the most abundant populations were the same across samples. The two most abundant cyanobacterial populations exhibited different carbon fixation dynamics, and their abundance was negatively correlated, suggesting that they occupy different ecological niches. Among nitrogen sources, urea was consumed at the highest rate, followed by ammonia. The nitrate consumption rate was much lower, and the fixation of nitrogen was not detected. Urea was consumed mainly during the day. Rates for nitrate and ammonia consumption were similar during the day and the night.
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    Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes
    (2023-02-09) Kleiner, Manuel; Kouris, Angela; Violette, Marlene; D’Angelo, Grace; Liu, Yihua; Korenek, Abigail; Tolić, Nikola; Sachsenberg, Timo; McCalder, Janine; Lipton, Mary S.; Strous, Marc
    Abstract Background Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. Results Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software ( https://sourceforge.net/projects/calis-p/ ). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. Conclusions We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.