Interaction between CO2-consuming autotrophy and CO2-producing heterotrophy in non-axenic phototrophic biofilms

Abstract

As the effects of climate change become increasingly evident, the need for effective CO2 management is clear. Microalgae are well-suited for CO2 sequestration, given their ability to rapidly uptake and fix CO2. They also readily assimilate inorganic nutrients and produce a biomass with inherent commercial value, leading to a paradigm in which CO2-sequestration, enhanced wastewater treatment, and biomass generation could be effectively combined. Natural non-axenic phototrophic cultures comprising both autotrophic and heterotrophic fractions are particularly attractive in this endeavour, given their increased robustness and innate O2-CO2 exchange. In this study, the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy in a non-axenic phototrophic biofilm was examined. When the biofilm was cultivated under autotrophic conditions (i.e. no organic carbon), it grew autotrophically and exhibited CO2 uptake. After amending its growth medium with organic carbon (0.25 g/L glucose and 0.28 g/L sodium acetate), the biofilm rapidly toggled from net-autotrophic to net-heterotrophic growth, reaching a CO2 production rate of 60 μmol/h after 31 hours. When the organic carbon sources were provided at a lower concentration (0.125 g/L glucose and 0.14 g/L sodium acetate), the biofilm exhibited distinct, longitudinally discrete regions of heterotrophic and autotrophic metabolism in the proximal and distal halves of the biofilm respectively, within 4 hours of carbon amendment. Interestingly, this upstream and downstream partitioning of heterotrophic and autotrophic metabolism appeared to be reversible, as the position of these regions began to flip once the direction of medium flow (and hence nutrient availability) was reversed. The insight generated here can inform new and important research questions and contribute to efforts aimed at scaling and industrializing algal growth systems, where the ability to understand, predict, and optimize biofilm growth and activity is critical.

Fig 1. CSMS biofilm reactor modules.

(A) Biofilm inoculation and growth occurs inside a highly CO₂-permeable silicone tube, through which liquid growth medium is pumped at a constant flow rate [27]. The comparatively CO₂-impermeable Tygon™ tube housing this silicone tube creates an annular space through which the sweeper gas is channeled at a constant flow rate. CO₂ molecules can readily diffuse in either direction across the wall of silicone tube according to the concentration gradient. (B) For a CO₂-producing (i.e. heterotrophic) biofilm, CO₂ molecules diffuse from the aqueous environment inside the silicone tube into the dry annular space. (C) When CO₂-laden gas is channeled into a BR containing a CO₂-consuming (i.e. autotrophic) biofilm, CO₂ molecules are pulled in the opposite direction, from the annular space into the silicone tube.

Fig 2. Configuration of the CSMS.

The CSMS comprised three BR modules [27]. The first, BR_prod, received its own feed of fresh growth medium and housed a heterotrophic pure culture bacterial biofilm, providing a consistent source of CO₂. As CO₂ molecules diffused out of the BR_prod silicone tube into the annular space, they were carried downstream by the sweeper gas to a CO₂ analyzer (Analyzer 1). The gas stream then travelled through the annular space of the linked BR_cons-BR_cons2 via a second CO₂ analyzer (Analyzer 2) positioned between them, before terminating at a third and final CO₂ analyzer (Analyzer 3). BR_cons-BR_cons2 housed the phototrophic biofilm of interest. The difference in CO₂ concentration logged by the analyzers provided a direct measure of CO₂ flux in the biofilm. Since medium flow was continuous through the linked BR_cons-BR_cons2 modules, they represent two halves of one biofilm system approximately 300 cm in length.

Fig 3. CLSM images of the phototrophic culture.

(A) The red channel depicts chlorophyll autofluorescence, indicating the presence of algal cells. (B) The green channel depicts SYTO 9-stained bacterial DNA, indicating the distribution of non-photosynthetic bacteria within the culture. (C) Overlay of the red and green signals depicts both cell types, confirming the non-axenic nature of the phototrophic culture.

Fig 4. Biofilm toggling from net-autotrophic to net-heterotrophic growth during organic carbon availability.

Initially, the phototrophic biofilm in BR_cons-BR_cons2 grew autotrophically, consuming CO₂ provided by BR_prod via the sweeper gas. Once organic carbon became available in the culture medium (denoted by the grey box), the biofilm rapidly toggled to CO₂-producing net-heterotrophic growth, as indicated by a switch to positive CO₂ flux values. When the organic carbon was no longer available in the medium, the biofilm’s CO₂ production fell dramatically and eventually returned to net-autotrophic growth, as indicated by the return to negative CO₂ flux values.

Fig 5. Proximal and distal biofilm responses during organic carbon availability.

The experiment presented in Fig 4 depicts CO₂ flux within the entire linked BR_cons-BR_cons2 unit. (A) When examining each half of this biofilm separately, nearly all the observed CO₂ production during organic carbon availability (grey box) occurred within BR_cons, with only very little occurring in BR_cons2. When organic carbon availability ended, BR_cons2 quickly returned to CO₂-consuming net-autotrophic growth (denoted by a negative CO₂ flux), whereas BR_cons took over two days to return to CO₂-consuming net-autotrophic growth. (B) When the same experiment was performed but with the organic carbon sources provided at half the concentration as compared to Fig 5A, BR_cons once again rapidly toggled from CO₂-consuming net-autotrophic growth to CO₂-producing net-heterotrophic growth during organic carbon availability. However, BR_cons2 continued to exhibit net-autotrophic growth and CO₂ uptake throughout the duration of the experiment, leading to discrete, longitudinally separated regions of net-heterotrophic and net-autotrophic growth occurring simultaneously within the biofilm.

Fig 6. Dark response in BR_cons2 during organic carbon availability.

In order to confirm that the CO₂ uptake observed in BR_cons2 during organic carbon availability was indeed attributable to photosynthetic CO₂ fixation, BR_cons2 was placed in the dark twice for at least one hour. Both times, CO₂ uptake stopped almost immediately. This signified the rapid cessation of photosynthesis and its related CO₂ fixation, and provided confirmation that the phototrophic biofilm was indeed exhibiting distinct, longitudinally discrete regions of net-heterotrophic and net-autotrophic growth when exposed to both inorganic and organic carbon (CO₂ as well as glucose and sodium acetate).

Fig 7. Proximal and distal biofilm responses to reverse flow direction during organic carbon availability.

(A) The phototrophic biofilm initially grew autotrophically using CO₂ from BR_prod. As previously observed, BR_cons shifted to CO₂-producing net-heterotrophic growth after organic carbon amendment (grey box), while BR_cons2 continued in CO₂-consuming net-autotrophic growth. When the direction of medium and gas flow through BR_cons-BR_cons2 was reversed at hour 60, the CO₂ flux in BR_cons stopped increasing and gradually decreased. The CO₂ flux in BR_cons2 conversely, increased rapidly and began exhibiting net CO₂ production. (B) This experiment was repeated, but with the glucose and sodium acetate concentrations decreased by half. Once again, both halves of the biofilm grew autotrophically initially, before BR_cons toggled to CO₂-producing heterotrophy after organic carbon amendment (grey box). When the direction of medium and gas flow in BR_cons-BR_cons2 was reversed, CO₂ production in BR_cons stopped increasing and began to decrease, ultimately crossing zero just prior to the end of the experiment. BR_cons2 conversely gradually stopped consuming CO₂ after flow direction was reversed, eventually crossing over to net CO₂ production.