Dec 20, 2023
Isotopically labelled inorganic carbon delivered to algal cultures via bubbler bottle
- Usha F Lingappa1,
- Sunnyjoy Dupuis1,
- Xavier Mayali2,
- Sabeeha S. Merchant1
- 1University of California, Berkeley;
- 2Lawrence Livermore National Laboratory
Protocol Citation: Usha F Lingappa, Sunnyjoy Dupuis, Xavier Mayali, Sabeeha S. Merchant 2023. Isotopically labelled inorganic carbon delivered to algal cultures via bubbler bottle. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3q8ol3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 28, 2023
Last Modified: December 20, 2023
Protocol Integer ID: 91536
Keywords: stable isotope probing, algae, bicarbonate, carbonate chemistry, bubbling, chlamydomonas reinhardtii, photosynthesis
Funders Acknowledgement:
Sabeeha Merchant, Moore Foundation Symbiosis in Aquatic Systems Initiative Investigator Award https://doi.org/10.37807/GBMF9203
Grant ID: GBMF9203
Xavier Mayali, US Department of Energy contract DE-AC52-07NA27344
Grant ID: SCW1039
Abstract
This protocol describes a method for delivering labelled inorganic carbon as 13CO2 to algal cultures by bubbling air through a solution of H13CO3-, and then into the culture. We developed this method to deliver label to cultures grown under continuous bubbling with air, without the use of 13CO2 labelled gas and the necessary equipment to mix labelled gas with air at near-atmospheric levels. Bubbling precludes the more common approach of adding H13CO3- label directly to the media, because dissolved HCO3- is in equilibrium with atmospheric CO2. Thus, excess HCO3- added to a solution will leave the solution as CO2 gas as it equilibrates. Bubbling rapidly accelerates this equilibration which is typically diffusion limited. The method described here takes advantage of this aspect of carbonate chemistry, and uses a solution of H13CO3-—which is less expensive and more convenient than 13CO2 gas—to generate a flux of 13CO2 that can be bubbled into a culture.
Materials
Aquarium pump
Bottle with vent and fill port assembly lid
Tubing and luer locks
Bubbling flask assembly (Erlenmeyer flask, foam plug, serological pipet, syringe filter)
Culture medium & inoculum
Na13CO3
H2KPO4
HK2PO4
The purpose and problem of bubbling cultures
The purpose and problem of bubbling cultures
Bubbling air into algal cultures stimulates photosynthetic growth by ameliorating diffusion limitation for CO2 (fig. 1A-B). Stable isotope probing (SIP) experiments examining carbon fixation often involve 13C label introduced directly into the culture medium as a H13CO3- salt. Unfortunately, because dissolved inorganic carbon (DIC) is in equilibrium with atmospheric CO2 (eq. 1), this labelling method does not work for cultures that are bubbled in an open system. Bubbling accelerates equilibration, causing excess HCO3- to rapidly exit the solution so that the label is lost before it can be fixed into biomass (fig. 1C). This presents some inconvenience, as H13CO3- salts are cheaper and easier to work with than is 13CO2 gas.
Figure 1. Bubbling enhances photosynthetic growth but precludes stable isotope probing with labelled bicarbonate. A. Diagrams of unbubbled vs. bubbled culture formats. Culture data presented in this protocol are of the unicellular green alga Chlamydomonas reinhardtii, grown in minimal medium under continuous light at 28ºC and shaken at 125 RPM. B. Growth curves of C. reinhardtii in unbubbled vs. bubbled culture formats, with and without added bicarbonate. Bubbling greatly enhances autotrophic growth. At the concentrations shown, bicarbonate addition does not impact growth. C. 13C enrichment of C. reinhardtii biomass grown with 13C labelled bicarbonate added directly to the culture medium for 24 hours, measured by IRMS on lyophilized cell pellets. Unbubbled cultures exhibit substantial 13C enrichment; ~90% of that signal is lost when bubbled.
Equation 1. CO2(g) + H2O ⇋ H2CO3 ⇋ HCO3- + H+ ⇋ CO32- + 2H+
Label delivery via bubbler bottle
Label delivery via bubbler bottle
We developed a SIP method that takes advantage of this liability of bubbling and carbonate chemistry (figure 2). Using an aquarium pump, we bubble air into a solution of H13CO3-. The DIC in the solution exchanges with the air, releasing label in the form of 13CO2 gas. That air is then bubbled into algal cultures. Using this method, we achieved substantial biomass 13C enrichment in bubbling C. reinhardtii cultures (fig. 2B).
Figure 2. Bubbling air through a solution of H13CO3- is an effective strategy to label bubbling cultures. A. Diagram of our bubbler label delivery approach. Bubbler bottle contains 500 mL of 1.5 mM NaH13CO3, air flow is ~1L/min. B. 13C enrichment of C. reinhardtii biomass labelled by this approach after 24 hours.
The rate of label release in this method can be tuned by buffering the bubbler solution at different pH values (fig. 4A). In lower pH solutions, carbonate equilibria (eq. 1) shift towards a higher fraction of the DIC pool speciated as dissolved CO2, which increases the rate at which that DIC exchanges into the bubbled air. We identified solution conditions suitable for experiments requiring rapid incorporation of 13C into algal biomass over short timescales (minutes) and for steady-state release over longer timescales (days). With a bubbler solution buffered with phosphate at pH 7.5, we obtained 13C enrichment detectable in algal biomass by IRMS in <15 minutes (fig. 4B, green). With a bubbler solution devoid of any additional buffer beyond the HCO3- itself, we obtained slower but longer-lived steady-state incorporation of the label (fig. 4B, orange).
Figure 3. Label release from the bubbler solution can be modulated by pH. A. Model describing the flux of CO2 released from the bubbler solution when buffered at different pH values, presented as the fraction of initial DIC leaving the solution over time. At lower pH values, the DIC leaves the solution more rapidly. B. Data from four separate experiments illustrating the biomass 13C enrichment of C. reinhardtii cultures over time, with bubblers buffered with 10x excess HxKxPO4 at pH 7.5 (green) vs no additional buffer (orange), examined over long (diamonds) or short (circles) timecourses. At pH 7.5, the equilibrium DIC concentration is >10x lower than the DIC added to the bottle as H13CO3-, so most of the label leaves the solution rapidly. This results in rapid labelling of biomass but also rapid depletion of label from the system. Without a separate buffer, DIC leaving the system drives the pH up to the pH at which the DIC concentration in the solution is at equilibrium with the air, and then exchanges with the air at a steady-state rate. This results in a longer-lived signal of label in biomass.