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

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.

Introduction

The accumulation of carbon dioxide in the atmosphere caused by the burning of fossil fuels is widely reported as being a leading cause of climate change. The effects of climate change are far-reaching and evident in the form of increasing global temperatures, melting of glacial ice, rising sea levels, and other well-documented negative impacts [1]. Strategies for effective CO2 management and mitigation are therefore critical in order to minimize these destructive effects and ensure the prosperity of current and future generations.

In addition to rising atmospheric CO2 levels, access to clean water is another major environmental and health concern, impacting nearly a billion people globally [2]. Even in developed regions with contemporary water treatment practices, increasing population growth and density means that existing treatment infrastructure is often strained, operating at or above capacity. As a result, untreated or undertreated water is frequently discharged to the environment, polluting watersheds and threatening nearby communities [3]. Inorganic nutrients in wastewater are especially concerning given that their accumulation in receiving waters leads to eutrophication and places ecosystems and water-users at serious risk [4, 5]. Despite being a major component of wastewater treatment, classical biological nutrient removal processes are energy-intensive and typically configured for optimal nitrogen or phosphorus removal, but not both. As a result, these processes do not always meet the desired removal efficiency [6, 7].

In view of environmental concerns, solutions which can effectively and simultaneously address the related issues of CO2 sequestration and enhanced wastewater nutrient removal are urgently needed; there is growing indication that processes incorporating microalgae may contribute to such optimization [7]. These photoautotrophs are capable of rapid CO2 uptake and fixation via their photosynthetic metabolism, generating the energy-rich sugars needed to fuel cellular activities. Like plants, microalgae readily assimilate inorganic nitrogen and phosphorus, meaning there is potential to utilize wastewater as a cheap and abundant algal growth medium [8-11]. As an added benefit, algal biomass has inherent commercial value, and can be used for example as a bio-fertilizer [12]. Microalgae also produce various useful compounds and nutraceuticals [13] and are known to be a potentially valuable biodiesel feedstock [14, 15]. This value-added quality of algal biomass can reduce or offset process-related costs. Although the range of allowable applications for waste-grown biomass remains somewhat restricted [16], further research and evidence-based policymaking focused on risk mitigation could lead to a paradigm in which microalgal CO2-sequestration, enhanced wastewater treatment, and biomass generation may be effectively combined [7, 17, 18].

Despite the ostensible triple-benefit of such an approach, its scalability is also limited by factors like high costs and energy requirements [11]. While the use of microalgal biofilms instead of conventional suspended cultures can alleviate land footprint requirements and reduce the cost of biomass harvesting and dewatering [19], widespread adoption of algal biotechnology is also hindered by a lack of robustness [20]. This is of particular concern given the common use of axenic cultures, which contain only a single algal species. Such cultures are typically constrained to a relatively narrow range of growth conditions and are highly susceptible to contamination, leading to sub-optimal performance or culture collapse if a sterile aseptic environment is not maintained [18, 21, 22].

To overcome this challenge, there is growing focus on the use of natural, non-axenic cultures in engineered algal systems [10, 20, 21, 23, 24]. The inherent diversity and functional redundancy within such communities confers greater robustness and makes them an attractive option for use in photobioreactor systems [19, 25]. As such, a shift in our approach to algal biotechnology and biosequestration, from one focused on finding ideal species to one focused on managing the environment to select for desired species interactions, may help to maximize process efficacy and resilience. However, to effectively realize such an approach, there is a need for an improved fundamental understanding of non-axenic phototrophic biofilms, their behaviour, and associated internal interactions within engineered growth systems.

The present work aims to contribute in this regard by examining the interplay between CO2-consuming autotrophy and CO2-producing heterotrophy within a non-axenic phototrophic biofilm. This question is highly relevant to integrated CO2 sequestration-wastewater treatment systems in which a phototrophic biofilm may be simultaneously exposed to both inorganic and organic carbon sources. That some microalgal species can grow mixotrophically utilizing both inorganic and organic carbon, and a few are even capable of growing heterotrophically on organic carbon only [26], makes the study of autotrophic-heterotrophic interplay in phototrophic biofilms especially prudent. The insight gained through this work may contribute information for optimizing growth system design, operation, and performance.

In this study, phototrophic biofilms were grown in a CO2 Sequestration Monitoring System (CSMS) [27]. Building upon the CO2 Evolution Monitoring System (CEMS) described by Kroukamp and Wolfaardt [28], this system enables the real-time in situ monitoring of a phototrophic biofilm’s CO2 flux under varying conditions. This work was motivated by a series of hypotheses: i) that non-axenic phototrophic biofilms readily toggle between net-autotrophic and net-heterotrophic growth depending on the availability of labile organic carbon sources; ii) that in the presence of both CO2 and labile organic carbon, phototrophic biofilms exhibit distinct, longitudinally discrete regions of heterotrophic and autotrophic growth with distance from reactor inflow; and iii) that this distinct longitudinal separation of autotrophic and heterotrophic metabolism is transient and reversible, based on the flow direction (and hence availability) of organic carbon nutrients.

Materials and methods

Growth system

Biofilms were grown in a CO2 Sequestration Monitoring System (CSMS). This system, described in detail in Ronan et al. [27], enables the real-time, in situ monitoring of CO2 flux in phototrophic biofilms. Biofilm reactor (BR) modules were 150 cm in length and comprised a tube-within-a-tube design (Fig 1A). Biofilms grew inside a CO2 permeable silicone tube (1.57 mm ID, 2.41 mm OD, 0.41 mm wall thickness, 2013.2 Barrer permeability; VWR International, Mississauga, ON, Canada), through which liquid growth medium flowed. The silicone tube in each BR provided approximately 74 cm2 of colonizable surface area and was housed within a larger diameter Tygon™ tube (4.76 mm ID, 7.94 mm OD, 1.58 mm wall thickness, E-3603 formulation; VWR International, Mississauga, ON, Canada). A CO2-free sweeper gas (TOC grade, 24001980; Linde Canada Limited, Concord, ON, Canada) was channeled through the annular space created by the two tubes. In this configuration, CO2 molecules could readily diffuse from the biofilm into the annular space (Fig 1B), or from the annular space into the biofilm (Fig 1C), depending on the nature of the biofilm and the prevailing CO2 concentration gradient.

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.

In this study, the CSMS consisted of three sequential BRs (Fig 2). The first, BRprod (CO2-producer), received fresh growth medium and housed a pure culture heterotrophic bacterial biofilm. The purpose of this biofilm was to provide a steady source of CO2 to the phototrophic biofilm being studied in the two subsequent BRs downstream. The sweeper gas passed through the annular space of BRprod, collecting CO2 produced by the heterotrophic biofilm and carrying it toward a non-dispersive infrared CO2 analyzer (Analyzer 1) (LI-820; LI-COR Biosciences, Lincoln, NE, USA). The gas stream then travelled through the annular space of the two downstream biofilm reactors BRcons and BRcons2 (CO2 consumer), via a second CO2 analyzer (Analyzer 2) positioned between them. Finally, the gas passed through a third CO2 analyzer (Analyzer 3), placed immediately downstream of BRcons2. Each CO2 analyzer has a precision in the range of 1 ppm and was set to log CO2 concentrations at one-minute intervals.

Fig 2

Configuration of the CSMS.

The CSMS comprised three BR modules [27]. The first, BRprod, 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 BRprod 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 BRcons-BRcons2 via a second CO2 analyzer (Analyzer 2) positioned between them, before terminating at a third and final CO2 analyzer (Analyzer 3). BRcons-BRcons2 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 BRcons-BRcons2 modules, they represent two halves of one biofilm system approximately 300 cm in length.

The linked BRcons-BRcons2 unit received its own feed of fresh growth medium and housed a phototrophic biofilm, which was inoculated by injecting 6 mL of culture into the liquid influent tube of BRcons using a sterile syringe needle. Medium flow was paused for a period of one hour following inoculation to allow for some initial cell adherence. Given that growth medium flowed continuously through BRcons-BRcons2, these BRs represent two halves of one biofilm system with a combined length of 300 cm.

The specific placement of the three CO2 analyzers in the CSMS allowed for the direct measurement of the CO2 concentration: i. entering the phototrophic BRcons-BRcons2 unit, ii. at the halfway point of BRcons-BRcons2, and iii. exiting BRcons-BRcons2. Therefore, at any time point it was possible to determine the CO2 flux of the entire phototrophic biofilm (Eq 1), or for the proximal and distal halves of the biofilm separately (Eqs 2 and 3).

Using the ideal gas law, the CO2 concentrations measured by the analyzers were converted to rates of CO2 flux and presented in units of μmol/h. Negative CO2 flux values denote net CO2 uptake, while positive values denote net CO2 production by the biofilm.

The linked BRcons-BRcons2 unit was illuminated continuously via a fluorescent plant growth light (JSV2 Jump Start T5 Grow Light System; Hydrofarm Inc., Petaluma, CA, USA), resulting in a photosynthetic photon flux density (PPFD) of approximately 141.79 μmol/m2/s. Both BRprod and BRcons-BRcons2 were fed their respective growth media at a flow rate of 15 mL/h. The BRcons-BRcons2 retention time and dilution rate was 23.2 minutes and 2.58 h-1, respectively. This dilution rate exceeds the maximum specific growth rates of microalgal and bacterial strains [29-31], ensuring that suspended, non-biofilm-bound cells were readily washed out the system, and hence contributed minimally to the consumption or production of CO2. The sweeper gas flowed through the system at a constant flow rate of 150 mL/h.

In this study, CO2 was provided to the phototrophic biofilm via a heterotrophic bacterial biofilm. At steady state, the biofilm’s consistent CO2 output serves to demonstrate the supply of an inexpensive and renewable CO2 source to facilitate the growth and subsequent study of the phototrophic biofilm downstream. While it would also be feasible to accomplish this using a gas tank with a known or controllable CO2 concentration, this approach highlights the utility in the design of the CSMS biofilm reactor modules, where the permeability of the inner silicone tube can facilitate not only the delivery of CO2 to a biofilm, but also the collection and subsequent shuttling of CO2 out of a biofilm. A scaled-up CSMS-based system could also feasibly integrate CO2-laden industrial flue gas or fermentation gas to support algal growth and achieve CO2 bio-sequestration, a notion which is garnering increasing attention [32-35]. Delivering CO2 in the gas phase through a CO2-permeable membrane, as is the case in the CSMS, may also reduce the extent of pre-processing required for these gas streams. Additionally, through relatively minor modifications to the system, it would be feasible to re-circulate the CO2-laden gas in a closed-loop configuration in order to improve overall sequestration efficiency.

Test cultures and growth medium

For each experiment, an overnight culture of the heterotrophic bacterium Pseudomonas aeruginosa (PA01) (originally obtained from Prof P.V. Phibbs at the Pseudomonas Genetic Stock Center, East Carolina University) [36] was inoculated into BRprod. CO2 produced by the resulting biofilm readily diffused through the highly permeable silicone tube and entered the BRprod annular space, where it was continuously removed by the sweeper gas and carried downstream to Analyzer 1 and beyond. The P. aeruginosa biofilm provided a consistent source of CO2 to facilitate the growth and study of the phototrophic biofilm downstream. The bacterial culture was fed with a tryptic soy broth prepared as a 0.6 g/L solution (2% concentration relative to the manufacturer’s directions for typical batch cultivation), with a final composition of 0.34 g/L casein peptone, 0.06 g/L soya peptone, 0.05 g/L glucose, 0.1 g/L NaCl, and 0.05 g/L K2HPO4. Media were prepared in distilled water and autoclaved at 121°C for 20 minutes prior to use.

The phototrophic culture was enriched from an aerated wastewater lagoon in Dundalk, Ontario, Canada [27]. It was cultivated in a modified Bold’s Basal Medium (M-BBM) with a composition of 0.22 g/L (NH4)2SO4, 0.025 g/L NaCl, 0.025 g/L CaCl2 ∙ 2H2O, 0.075 g/L MgSO4 ∙ 7H2O, 0.175 g/L KH2PO4, 0.075 g/L K2HPO4, 8.34 mg/L FeSO4. Notably, no carbon was provided in the liquid medium. Illumination was provided continuously via the same fluorescent plant growth light described above. This culture was transferred to fresh medium biweekly, ensuring that the culture used to inoculate BRcons-BRcons2 in each experiment was not more than 14 days old. Unless otherwise stated, the results reported here are from biofilms at least twenty hours old.

A confocal laser scanning microscope (CLSM) was used to confirm the non-axenic nature of the phototrophic culture. A simple two-channel procedure, described by Lawrence et al. [37] and used subsequently by Barranguet et al. [38] and others, was used to confirm and differentiate the presence of algal and non-algal fractions within the culture used for inoculation of BRcons-BRcons2. The former was visualized based on chlorophyll autofluorescence, while the latter was distinguished using SYTO 9 green fluorescent nucleic acid stain (S34854; ThermoFisher Scientific, Waltham, MA, USA) [37].

A well-mixed 100 μL undiluted culture sample was vacuum filtered through a black polycarbonate filter (0.2 μm pore size, GTBP02500; ThermoFisher Scientific, Waltham, MA, USA). The filter was then immersed in SYTO 9 stain (20 mg/ml) for ten minutes in the dark, before being rinsed twice with deionized water to remove excess stain. The filter was placed on a glass microscope slide and visualized using a Nikon Eclipse 80i-C1 confocal laser scanning microscope (Nikon Instruments Inc., Melville, NY). SYTO 9-stained bacteria were excited using a 488 nm laser and visualized through a 515/530 nm filter, producing a green signal. Microalgal cells conversely, were excited using a 632 nm laser and visualized through a 650 nm long pass filter, producing a red signal.

Assessing the prevalence of autotrophic-heterotrophic toggling

The phototrophic culture was inoculated into BRcons-BRcons2 and initially fed M-BBM. CO2 was provided continuously via the heterotrophic biofilm in BRprod. After 20 hours, the M-BBM was amended with the labile organic carbon sources glucose (0.25 g/L) and sodium acetate (0.28 g/L). After approximately 30 hours of organic carbon availability, the medium was returned to the original M-BBM composition, which lacked the added carbon sources. Illumination was provided continuously throughout the experiment.

Investigating the longitudinal arrangement of autotrophic and heterotrophic metabolism

A similar experiment was performed in which the phototrophic culture was inoculated into BRcons-BRcons2 and initially fed M-BBM, with CO2 provided continuously via the heterotrophic biofilm in BRprod. After 30 hours, the M-BBM medium was again amended with glucose and sodium acetate for the remainder of the experiment. In this case however, these organic carbon sources were added at half the concentration used previously (now 0.125 g/L and 0.14 g/L respectively). Illumination was provided by the fluorescent plant growth lights for the duration of the experiment. The CO2 flux within the proximal half (BRcons) and distal half of the biofilm (BRcons2) are presented separately.

A series of modified light-dark shift tests [27, 39] was performed on BRcons2 during the period of organic carbon availability in order to confirm that CO2 uptake observed in this portion of the biofilm was indeed the result of photoautotrophic activity. As in the experiment described above, the biofilm was initially grown under strictly autotrophic conditions, before amending the M-BBM with glucose (0.125 g/L) and sodium acetate (0.14 g/L). BRcons2 was then covered with an opaque, light-blocking sheet for two periods of 1.5 hours and 1 hour respectively, with a total of 2.5 hours allowed to elapse between the two dark periods.

Assessing reversibility in the longitudinal arrangement of autotrophic and heterotrophic metabolism

As in the experiments described above, the phototrophic culture was grown in the CSMS under initial autotrophic conditions, with M-BBM as the liquid growth medium and CO2 provided by the heterotrophic biofilm in BRprod. After 38 hours, the M-BBM was once again amended with glucose (0.125 g/L) and sodium acetate (0.14 g/L). However, at hour 60 (22 hours after organic carbon amendment began), the direction of medium and gas flow through BRcons-BRcons2 was reversed. In this new orientation, fresh growth medium now entered BRcons2 from what was previously the effluent end and exited the system at what was previously the influent end of BRcons.

After leaving BRprod and passing through Analyzer 1, the gas stream in this reversed-flow orientation subsequently travelled through BRcons2 and BRcons in what was previously the upstream direction, with Analyzer 3 positioned between these two BR modules and Analyzer 2 positioned immediately downstream from them. In this configuration, the CO2 flux within the two BR modules was now calculated using Eqs 4 and 5:

This experiment was repeated in the same manner (autotrophic growth followed by organic carbon amendment and then flow reversal), however the glucose and sodium acetate were added at half the concentration used previously (now 0.0625 g/L and 0.07 g/L respectively).

Results and discussion

Confocal laser scanning microscopy was used to distinguish algal and non-algal members of the phototrophic culture. Although the culture was maintained under autotrophic conditions, a heterotrophic bacterial fraction was expected to have persisted, as has been described previously for other wastewater-derived phototrophic cultures (e.g. [9]). Microalgal members were recognized by chlorophyll autofluorescence and appeared red, while non-phototrophic bacteria were distinguished via SYTO 9 nucleic acid stain and appeared green (Fig 3). The appearance of non-overlapping red and green cells indicates the presence of both a photosynthetic (algal) and non-photosynthetic (bacterial) fraction in the culture, confirming that the culture was indeed non-axenic.

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.

Algae-bacteria interactions are ubiquitous in nature and play an important role at the base of the trophic web. While numerous types of interactions between these groups are possible, encompassing both positive and negative effects [40], the natural syntrophy surrounding their O2 and CO2 exchange represents an important link between trophic levels and forms the basis of healthy ecosystems. Given the co-evolution of microalgae and bacteria over millions of years, it is rare to find microalgae existing as axenic cultures in nature, without some contribution from non-algal, non-photosynthetic microorganisms [41]. While the specific interactions at play within this culture were not fully elucidated, it was nonetheless unsurprising that it appeared to comprise both an autotrophic and heterotrophic fraction (Fig 3), despite being cultivated under autotrophic conditions. In engineered algal systems, non-photosynthetic bacteria are often viewed as mere contamination, necessitating costly and energy-intensive measures to maintain a sterile environment [40]. Recently, this view has begun to shift amidst a growing recognition that natural microalgal-bacterial consortia can represent a cost-effective, efficient alternative for many biotechnological applications [42].

Autotrophic-heterotrophic toggling by the phototrophic biofilm

When the phototrophic culture was inoculated into the CSMS and grown initially under autotrophic conditions (i.e. no organic carbon), the resulting biofilm grew autotrophically and exhibited net CO2 uptake, denoted by negative CO2 flux values (Fig 4). When the growth medium was amended with glucose and sodium acetate after 20 hours, the CO2 flux began to trend upward. Within approximately 4 hours, the biofilm had clearly shifted from net-autotrophic to net-heterotrophic growth, denoted by a shift to positive CO2 flux values. During this period of organic carbon availability, CO2 production increased dramatically before beginning to plateau at approximately 60 μmol/h (0.405 μmol/h/cm2).

Fig 4

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

Initially, the phototrophic biofilm in BRcons-BRcons2 grew autotrophically, consuming CO2 provided by BRprod 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.

It should be noted that this CO2 production was a result of the metabolic activity within the phototrophic biofilm only. Given that it was calculated via Eq 1 (Analyzer 3 –Analyzer 1), it does not include any of the CO2 originating from the heterotrophic biofilm in BRprod. This meant that the CO2 production observed in the phototrophic biofilm during organic carbon availability could be confidently attributed to the utilization of the two labile organic carbon sources. After 51 hours, the M-BBM was returned to its original composition lacking these organic carbon sources, leading to a rapid decline in the biofilm’s CO2 production and an eventual return to net-autotrophic growth. This same CO2 flux behaviour was consistently observed through several experimental repeats.

The data thus supported the underlying hypothesis of this experiment, that a non-axenic phototrophic biofilm readily toggles between net-autotrophic and net-heterotrophic growth, dependent on the availability of labile organic carbon sources. While there is obviously a strong impetus to use wastewater as an inexpensive and abundant growth medium for algal biotechnologies [7, 43], this result speaks to an important consideration in this approach. If bio-sequestration of CO2 (be it from point or diffuse sources) is a primary objective, it may be disadvantageous to grow the sequestering culture in a wastewater stream that is vulnerable to organic nutrient spikes. This is especially relevant in industrial wastewater, or municipal wastewater from plants treating industrial effluents, where wastewater composition can vary widely and transient organic carbon shock loads with concentrations two or more times higher than normal are common [44]. Depending on the robustness and stability of the treatment system, this can result in prolonged periods of elevated effluent BOD, which would impact a downstream algal bio-sequestration system and lead to considerable CO2 production (as seen in Fig 4), an effect which is counter to the overall aim of a sequestration system.

Longitudinal arrangement of autotrophic and heterotrophic metabolism

The configuration of the CSMS (specifically the placement of Analyzer 2 between BRcons and BRcons2), allows for the examination of the respective CO2 flux within the two halves of the phototrophic biofilm. This increased resolution is helpful in revealing metabolic partitioning that may be prevalent in the biofilm. When this approach was applied to the experimental result presented in Fig 4, it was noted that in the presence of the labile organic carbon sources, the vast majority of the biofilm’s CO2 production was occurring within the proximal half of the biofilm (Fig 5A). By hour 50, just prior to the end of organic carbon availability, approximately 93% of the observed CO2 production was from BRcons, with only about 7% coming from the distal half of the biofilm contained in BRcons2. Interestingly, when organic carbon was no longer available in the culture medium, the distal half of the biofilm needed only two hours to return to net-autotrophic growth (CO2 uptake), while the proximal half of the biofilm contained in BRcons took slightly more than two days to return to net-autotrophic growth. The protracted return to autotrophic growth observed in BRcons could have been an EPS effect, whereby some labile organic carbon was retained and subsequently accessible to this portion of the biofilm after organic carbon availability had ceased.

Fig 5

Proximal and distal biofilm responses during organic carbon availability.

The experiment presented in Fig 4 depicts CO2 flux within the entire linked BRcons-BRcons2 unit. (A) When examining each half of this biofilm separately, nearly all the observed CO2 production during organic carbon availability (grey box) occurred within BRcons, with only very little occurring in BRcons2. When organic carbon availability ended, BRcons2 quickly returned to CO2-consuming net-autotrophic growth (denoted by a negative CO2 flux), whereas BRcons 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, BRcons once again rapidly toggled from CO2-consuming net-autotrophic growth to CO2-producing net-heterotrophic growth during organic carbon availability. However, BRcons2 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.

At hour 22 (Fig 5A), when the labile organic carbon sources first became available to the biofilm, there appears to have been enough biomass within BRcons to metabolize and deplete nearly all the glucose and sodium acetate carbon. Very little therefore reached BRcons2 where it could be metabolized by the distal half of the biofilm, which accounts for its comparatively low CO2 production. If the organic carbon sources were less abundant (i.e. available in the medium at a lower concentration), one would expect all the glucose and sodium acetate carbon to be depleted within BRcons. This would leave the distal half of the biofilm housed in BRcons2 without access to any of the supplied organic carbon, causing it to remain in net-autotrophic growth even while the biofilm’s proximal half grew heterotrophically. This notion formed the basis of the second hypothesis, which stated that in the presence of labile organic carbon, a phototrophic biofilm of sufficient length exhibits distinct, longitudinally discrete regions of net-heterotrophic and net-autotrophic growth.

To test the hypothesis, a similar experiment was conducted, in which the biofilm was initially grown under autotrophic conditions with CO2 supplied from the heterotrophic biofilm in BRprod. The M-BBM fed to BRcons-BRcons2 was then once again amended with glucose and sodium acetate, however this time at half the concentration used previously (0.125 g/L and 0.14 g/L respectively). Before this organic carbon amendment, both halves of the biofilm were exhibiting autotrophic growth and CO2 uptake, indicated by their negative CO2 flux values (Fig 5B). However, when the labile organic carbon sources became available in the medium after 30 hours, the proximal half of the biofilm (BRcons) rapidly shifted from CO2-consuming autotrophic growth to CO2-producing heterotrophic growth. The CO2 flux in the distal half of the biofilm (BRcons2) conversely, remained relatively unchanged and continued to exhibit net-autotrophic growth (CO2 uptake) for the entire experiment.

In three subsequent experimental repeats, this same pattern of metabolic partitioning was observed. In the presence of the labile organic carbon sources, the biofilm’s proximal half produced CO2, while the biofilm’s distal half continued to consume CO2. When BRcons2 was placed in the dark for two periods of 1.5 hours and 1 hour respectively during the period of organic availability, a rapid response was observed in which the CO2 flux in this portion of the biofilm shifted from negative (CO2 uptake) to positive (CO2 production) (Fig 6). This dark-induced interruption of photosynthesis thereby confirmed that the CO2 uptake observed in BRcons2 during organic carbon availability was in fact attributable to autotrophic activity (as opposed to abiotic leakage or passive diffusion out of the system). The small amount of CO2 production observed during this darkness is likely attributable to underlying baseline algal respiration. When illumination resumed, so too did the photoautotrophic activity in BRcons2, causing this portion of the biofilm to quickly rebound to approximately the same negative CO2 flux values seen before the dark test. Given this result, it can be concluded that in the presence of both inorganic and organic carbon (CO2 as well as glucose and sodium acetate), the phototrophic biofilm was in fact exhibiting distinct, longitudinally discrete regions of net-heterotrophic and net-autotrophic growth.

Fig 6

Dark response in BRcons2 during organic carbon availability.

In order to confirm that the CO2 uptake observed in BRcons2 during organic carbon availability was indeed attributable to photosynthetic CO2 fixation, BRcons2 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).

Previous studies have described the use of non-axenic phototrophic cultures for simultaneous inorganic and organic carbon removal. van der Ha et al. [45] for example, co-cultured microalgae and methane-oxidizing bacteria to achieve concurrent microbial methane oxidation and CO2 uptake. Vu and Loh [46] described a symbiotic consortium of C. vulgaris and P. putida, where the algal fraction grew photoautotrophically using CO2 produced by the bacterium. This in turn eliminated the need for aeration by providing the oxygen needed by the obligate aerobe P. putida to carry out efficient glucose biodegradation. A similar co-culture of P. putida and C. vulgaris described by Mujtaba et al. [47] exhibited the synergistic removal of organic carbon by the former and inorganic nutrients by the latter.

While there is much literature pertaining to microalgal-bacterial symbiosis in co-culture or synthetic consortia, comparatively few studies have examined this behaviour in natural phototrophic consortia within the context of engineered biofilm photobioreactors. Whereas co-cultures still require onerous measures to maintain sterility and prevent contamination, natural non-axenic cultures can offer a level of robustness and functional redundancy that makes contaminating organisms much less of a concern [13, 25]. Although wastewater treatment and CO2 sequestration have been studied separately for many years, approaches for effectively linking these via non-axenic phototrophic biofilms are lacking [8].

Microalgal biofilm growth systems fall into two broad categories: those in which the biofilm is mobile (via movement of the attachment material), and those in which the biofilm remains stationary (no movement of the attachment material) [48]. Mobile systems include for example rotating algal biofilm reactors (RABRs), in which the biofilm is attached to a solid or fibrous support material coiled around a rotating cylindrical drum that alternatingly exposes cells to the liquid and gas phases. Such systems have been shown to achieve high inorganic nutrient removal and biomass production when operating within a raceway pond [23]. However, the energy required for their moving parts can be a complicating factor. In stationary growth systems conversely, biofilms form on a fixed attachment material with liquid growth medium flowing over the surface of the biofilm. Tubular photobioreactors for example, are closed, stationary systems which enable significant control over conditions and offer high surface to volume ratios [49].

Insight generated by the CSMS can be useful in informing the design and operation of similar scaled-up tubular systems. This can include for example determining what fraction of total reactor length is required to deplete nutrients. Since this length will vary with key factors such as influent nutrient load and flow rate, the utility of the CSMS lies in its innovative approach to informing such optimization. The longitudinal arrangement of discrete heterotrophic and autotrophic regions within the phototrophic biofilm (as described in Fig 5), suggests that significant simultaneous removal of organic and inorganic carbon may be possible in large-scale tubular photobioreactors using non-axenic phototrophic biofilms. Ostensibly, the former could be metabolized in the proximal portion of the biofilm through heterotrophic metabolism, with the latter being taken up and fixed through autotrophic metabolism occurring in the distal portion of the biofilm, where assimilation of inorganic nitrogen and phosphorus nutrients would also take place.

Despite the potential benefits offered by tubular photobioreactors in terms of CO2 capture and contaminant removal, the energy required for operation, as well as the need for cleaning and maintenance, remain barriers limiting their widespread use. As such, there is still a need for further research in this area, focused on expanding our understanding of biofilm behaviour within these systems and developing strategies for improving their overall cost-effectiveness.

Reversibility in the longitudinal arrangement of autotrophic and heterotrophic metabolism

In the presence of inorganic and organic carbon, the phototrophic biofilm consistently exhibited longitudinally discrete regions of net-heterotrophic and net-autotrophic metabolism, with the location of these regions dictated by the gradient of organic carbon availability resulting from heterotrophic metabolism in the proximal portion of the biofilm. It was hypothesized that these discrete regions of net-autotrophic and net-heterotrophic growth are therefore transient and reversible based on the direction of nutrient flow and availability. That is, when the direction of flow is reversed, the locations of the autotrophic and heterotrophic regions will flip.

To test this hypothesis, a similar experiment was performed in which the biofilm initially grew autotrophically with CO2 as the sole carbon source. When glucose and sodium acetate were added to the growth medium, BRcons once again toggled to CO2-producing heterotrophy, while BRcons2 continued to exhibit net-autotrophic CO2 uptake (Fig 7A). At hour 60 however, the direction of medium and gas flow through these two BR modules was reversed, such that BRcons2 now contained the “proximal” half of the biofilm, gaining access to the fresh, un-depleted organic carbon sources provided in the medium. BRcons conversely, was now downstream of BRcons2 and represented the “distal” half of the biofilm.

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 BRprod. As previously observed, BRcons shifted to CO2-producing net-heterotrophic growth after organic carbon amendment (grey box), while BRcons2 continued in CO2-consuming net-autotrophic growth. When the direction of medium and gas flow through BRcons-BRcons2 was reversed at hour 60, the CO2 flux in BRcons stopped increasing and gradually decreased. The CO2 flux in BRcons2 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 BRcons toggled to CO2-producing heterotrophy after organic carbon amendment (grey box). When the direction of medium and gas flow in BRcons-BRcons2 was reversed, CO2 production in BRcons stopped increasing and began to decrease, ultimately crossing zero just prior to the end of the experiment. BRcons2 conversely gradually stopped consuming CO2 after flow direction was reversed, eventually crossing over to net CO2 production.

After the direction of flow was reversed, a sharp increase in CO2 flux and a rapid switch from autotrophic to heterotrophic growth was observed in BRcons2 (Fig 7A). This aligned with expectations, given that this portion of the biofilm was now first to encounter and metabolize the organic carbon sources. In previous experiments (and before reversing the direction of flow in this experiment), this portion of the biofilm was only able to access what little (if any) of the organic carbon could pass through BRcons un-metabolized.

It is notable that the slope in the BRcons2 CO2 flux after hour 60 is remarkably similar to the slope in BRcons after hour 38 (Fig 7A), suggesting that both halves of the biofilm exhibited the same metabolic response (and started producing CO2 at nearly identical rates), when that portion of the biofilm was the first to encounter the organic carbon sources. This result speaks to the significant analytical utility of the CSMS. The ability to visualize the biofilm’s rate of CO2 consumption and/or production in real-time means that growth conditions can be fine-tuned to ensure that these rates are effectively optimized. Increasing the number of CO2 analyzers positioned along the length of the biofilm would also enable greater longitudinal resolution, which can further inform optimal nutrient loading and reactor length under a given set of conditions.

When BRcons became the distal end of the biofilm and was no longer receiving the fresh, un-depleted organic carbon sources, its rate of CO2 production began to gradually decrease (Fig 7A). However, this portion of the biofilm did not indicate an overall shift to net-autotrophic growth as expected. As mentioned previously, EPS may have played a role in this muted response by retaining stores of organic carbon from earlier in the experiment, which could then be taken up and metabolized after the direction of flow was reversed. Interestingly, in natural, non-axenic phototropic biofilms, much of the initial EPS originates from heterotrophic bacteria [50]. It is also plausible that following flow reversal at hour 60, the portion of the biofilm in BRcons2 was not yet dense enough to fully deplete the organic carbon it now encountered, therefore leaving some available for the “distal” half of the biofilm now housed in BRcons. Although the precise explanation necessitates additional biofilm analyses which are ultimately beyond the scope of this paper, it was postulated that in either case these effects would be minimized, and the BRcons CO2 flux after flow reversal would eventually cross zero and exhibit CO2 uptake, if the organic carbon sources were available in the medium at a lower concentration.

To test this, the experiment was repeated but with the glucose and sodium acetate supplied at half the concentration used previously (now 0.0625 g/L and 0.07 g/L respectively). This experiment was also allowed to run for a longer period following the reversal of flow direction. BRcons2 once again exhibited an increase in its CO2 flux values after flow reversal, eventually achieving net CO2 production (Fig 7B). As predicted, BRcons in this case showed a steady decline in CO2 flux after flow reversal, reaching zero by approximately hour 70.

Given that the CO2 flux in both BRcons and BRcons2 trended in opposite directions after flow reversal (Fig 7B), and considering the outcome of the previous experiment (Fig 7A), the upstream and downstream partitioning of net-heterotrophic and net-autotrophic metabolism appears to be reversible, with the position of these regions dictated by nutrient flow direction and hence availability, thus supporting the third hypothesis. Although a comprehensive community composition analysis would be needed to make definitive assertions, these results suggest that this rapid metabolic partitioning is likely not the result of significant changes in biofilm member composition, but rather a functional redundancy within the culture and changes in the metabolic activity of members already present before the influx of organic carbon.

The experiments described here present an elegant approach to the study of phototrophic biofilms in order to gain important insight regarding their behaviour under changing conditions. Such information can inform new and relevant 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. In integrated wastewater treatment-CO2 sequestration systems utilizing non-axenic phototrophic biofilms, the balance between organic and inorganic carbon metabolism is a major factor that dictates the biofilm’s overall CO2 flux. In this study, the phototrophic biofilm exhibited a rapid response and began growing heterotrophically when exposed to organic carbon, reaching a CO2 production rate of 60 μmol/h after approximately 30 hours. This suggests that non-axenic phototrophic biofilms are indeed able to readily toggle between net-autotrophic CO2 capture and net-heterotrophic CO2 production based on the availability of labile organic carbon sources. When the organic carbon sources were less abundant (i.e. provided at a much lower concentration), the biofilm took only 4 hours to exhibit longitudinally discrete regions of autotrophic and heterotrophic growth in the proximal and distal portions of the biofilm respectively. The apparent reversibility in the biofilm’s response to changing carbon conditions suggests a robustness and versatility within non-axenic phototrophic biofilms which may be exploitable in engineered phototrophic biofilm systems to achieve simultaneous organic and inorganic carbon removal.

Hypothesis-driven research aimed at generating fundamental insights about phototrophic biofilms are important as the need for effective CO2 management and mitigation strategies increases, and the motivation for linking these to enhanced wastewater treatment grows. The study presented here therefore should represent a key step forward in this endeavour.

5 May 2021

PONE-D-21-12369

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

PLOS ONE

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Reviewer #1: General comments

The authors have investigated the complex interactions of non-axenic biofilms consisting of photo-autotrophic and heterotrophic microorganisms and experimentally investigate on kinetics and dynamics of microbial growth in reaction to changing feed and gas compositions.

The CO2 source was exhaust gas of a growing culture of Pseudomonas sp. cultivated in a tubular reactor, in a similar setup as for the phototrophic biofilm. At this point, no explanation was given why such a culture and not a standard gas mixture containing controllable concentration of CO2 was chosen. The statement provided in the lines 470ff can and should be moved to the methods section.

The experimental setup reads comparably simple while the operation modes have some complexity in their variation of illumination, mineral nutrient and organic substrate supply with or without reverting medium flow direction.

The photobioreactor was inoculated with a mixed culture of phototrophic microbes isolated and enriched from a local surface water body. Although it is probably impossible to identify all involved species in the mixture, at least normal light microscopy photographs should be provided from the inoculum – or from the first established biofilm and from experimental stages at the times of operation changes. Such a visual documentation, as simple as it is, would improve the readers imagination about the ongoing population changes during different operation modes. If not available – a pity, btw. – some some other wordly descriptions should be provided. The one status photo in Fig.3. is a good example, but does not allow to follow a highly dynamic system over the course of roughly 3 to 5 days.

By monitoring CO2 consumption and CO2 evolution as indicators of either heterotrophic or autotrophic metabolic dominance in the biofilm, some general and valuable information with praxis relevance about microalgae production from wastewater is provided. A natural day-night cycle should be considered, as a real scale algae production utilising wastewater nutrients will not be artificially illuminated. During the dark hours also the phototrophic biomass will consume oxygen and will release CO2, interfering with the overall net carbon balance. Maybe such extrapolations are possible from the experiment sequences with artificial shadowing of the bioreactor.

Specific comments

Introduction lines 73 - 75: While microalgal biomass can indeed be used as fertilizer, feed and food additive, any biomass grown on waste has a restricted application range – a fact to be considered.

Discussion, lines 296 – 306: While all this information is correct and valuable, it is, nevertheless, of theoretical nature and not a direct outcome of experimental results.

Discussion, lines 454 – 457: The recommendation of closed tubular photobiorectors for microalgae production from wastewater is highly theoretical. Unpredictable population development and the need for cleaning and maintenance do, by far, outweigh the potential benefits of a technically controlled environment. Flow resistance in the tubes requires way more pumping energy than a raceway pond the authors criticize some sentences above (lines 451 ff).

Discussion, lines 478ff (reversibility): It‘s a pity that no information is provided about biofilm development and biofilm composition. It seems to be of major importance to understand if the reaction to the availability of organic carbon in the medium is caused by metabolic changes of a static biofilm composition or by a change of the ratio of photo- to heterotrophic cells (reactive growth). This can be important to know as a stable biofilm can be operated over a longer time while a reactively changing biofilm will cause problems in a large scale plant. However, the authors mention in lines 529ff that such analysis were beyond their possibilities for this manuscript.

Reviewer #2: The manuscript is based on a very interesting hypothesis , and undertaken in a suitable manner.

The Introduction, upto line no 99, is too wordy and seems to be unnecessary. The Last few figures can be combined and presented in a better manner.

I would appreciate a bit more specific data/results in the abstract and conclusions, to make it more scientific.

**********

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28 May 2021

Please also see uploaded document "Response to reviewers"; which is exactly the same as the text below

Reviewer #1: General comments

The authors have investigated the complex interactions of non-axenic biofilms consisting of photo-autotrophic and heterotrophic microorganisms and experimentally investigate on kinetics and dynamics of microbial growth in reaction to changing feed and gas compositions.

The CO2 source was exhaust gas of a growing culture of Pseudomonas sp. cultivated in a tubular reactor, in a similar setup as for the phototrophic biofilm. At this point, no explanation was given why such a culture and not a standard gas mixture containing controllable concentration of CO2 was chosen.

We opted for this approach as it highlights the utility of the CSMS biofilm reactor modules, wherein the permeability of the inner silicone tube can facilitate not only the delivery of CO2 to a biofilm, but also the collection and subsequent shuttling of CO2 out of a biofilm (this is mentioned on line 132). Although not a primary focus of the present study, this is a noteworthy property which could be used in initiatives aimed at collecting and subsequently sequestering CO2 emitted by real-world CO2-producing biological processes.

We have added the passage below at line 197 to expand on the rationale behind the CO2 source used in this study (note that line numbers correspond to the revised, unmarked version of the manuscript).

In this study, CO2 was provided to the phototrophic biofilm via the heterotrophic bacterial biofilm. At steady state, the biofilm’s consistent CO2 output serves to demonstrate the supply of an inexpensive and renewable CO2 source to facilitate the growth and subsequent study of the phototrophic biofilm downstream. While it would also be feasible to accomplish this using a gas tank with a known or controllable CO2 concentration, this approach highlights the utility in the design of the CSMS biofilm reactor modules, where the permeability of the inner silicone tube can facilitate not only the delivery of CO2 to a biofilm, but also the collection and subsequent shuttling of CO2 out of a biofilm.

The statement provided in the lines 470ff can and should be moved to the methods section.

Thank you for pointing this out. We have adjusted the text accordingly by moving the passage in question from the Discussion to the Materials and Methods (now covered in lines 196-209).

The experimental setup reads comparably simple while the operation modes have some complexity in their variation of illumination, mineral nutrient and organic substrate supply with or without reverting medium flow direction.

The photobioreactor was inoculated with a mixed culture of phototrophic microbes isolated and enriched from a local surface water body. Although it is probably impossible to identify all involved species in the mixture, at least normal light microscopy photographs should be provided from the inoculum – or from the first established biofilm and from experimental stages at the times of operation changes. Such a visual documentation, as simple as it is, would improve the readers imagination about the ongoing population changes during different operation modes. If not available – a pity, btw. – some other wordly descriptions should be provided. The one status photo in Fig.3. is a good example, but does not allow to follow a highly dynamic system over the course of roughly 3 to 5 days.

We agree that the tracking of microbial community changes over time would be a good compliment to the story presented in the paper. However, such analyses were beyond the scope of this study, due in part to the fact that in the current iteration of the CSMS, the biofilm is housed within two layers of tubing, making time-resolved in situ biofilm imaging unfeasible. Unfortunately, this tube-within-a-tube design feature is critical to the system’s real-time CO2 monitoring capability. Instead of a microbial composition focus, we therefore opted for a “functional” focus, tracking and describing the changes in community function rather than community composition. With this focus, our system was able to speak more to metabolic behaviour (i.e. the “readiness” of a certain microbial fraction (e.g. heterotrophs) to act upon a change in their environment within a certain time frame) rather than population changes. The biofilm’s ability to toggle its CO2 flux in mere minutes suggests that it is indeed changes in metabolic behaviour which is likely predominating in the period immediately following feed amendment. We acknowledge that this will most probably be accompanied by eventual population changes, though one would expect the metabolic impact of these changes to manifest on a comparatively longer timescale, and that these changes will be relatively small due to the functional redundancy inherent to microbial communities. In light of this, we feel that despite the lack of time-resolved biofilm imaging, the study’s experimental design is sound, and the insight generated still represents a meaningful and useful contribution to this area of study.

By monitoring CO2 consumption and CO2 evolution as indicators of either heterotrophic or autotrophic metabolic dominance in the biofilm, some general and valuable information with praxis relevance about microalgae production from wastewater is provided.

A natural day-night cycle should be considered, as a real scale algae production utilising wastewater nutrients will not be artificially illuminated. During the dark hours also the phototrophic biomass will consume oxygen and will release CO2, interfering with the overall net carbon balance. Maybe such extrapolations are possible from the experiment sequences with artificial shadowing of the bioreactor.

We agree that day-night cycles are an important consideration in algae production from wastewater. It is true that CO2 will be produced in the dark, and indeed some baseline algal CO2 production will continue to occur even in the light. Our reported CO2 flux data encompasses all ongoing CO2-consuming and CO2-producing processes taking place in the biofilm, under lighting conditions which actively and continuously promote photosynthesis and related CO2 uptake (as CO2 sequestration was a primary focus of the paper). We acknowledge that light/dark cycling will have an impact on this carbon balance, as the reviewer indicated, and therefore feel that this study provides a useful basis of comparison for future studies aimed at exploring this impact and ultimately optimizing light/dark cycling to maximize specific beneficial parameters (e.g. CO2 uptake, biomass production, wastewater nutrient removal etc.).

Specific comments

Introduction lines 73 - 75: While microalgal biomass can indeed be used as fertilizer, feed and food additive, any biomass grown on waste has a restricted application range – a fact to be considered.

Thank you for highlighting this important consideration. We have added the statement below at line 76 to address this point.

Although the range of allowable applications for waste-grown biomass remains somewhat restricted [16], further research and evidence-based policymaking focused on risk mitigation could lead to a paradigm in which microalgal CO2-sequestration, enhanced wastewater treatment, and biomass generation may be effectively combined [7, 17, 18].

Discussion, lines 296 – 306: While all this information is correct and valuable, it is, nevertheless, of theoretical nature and not a direct outcome of experimental results.

It was not our intention to present this brief discussion of algal-bacterial interactions as a direct outcome of our experiment, but rather we include this to provide some helpful context surrounding the observed presence of non-autotrophic members in our phototrophic culture. We have added the sentence below at line 318 in order to clarify.

While the specific interactions at play within this were not fully elucidated, it was nonetheless unsurprising that it appeared to comprise both an autotrophic and heterotrophic fraction (Fig 3), despite being cultivated under autotrophic conditions.

Discussion, lines 454 – 457: The recommendation of closed tubular photobiorectors for microalgae production from wastewater is highly theoretical. Unpredictable population development and the need for cleaning and maintenance do, by far, outweigh the potential benefits of a technically controlled environment. Flow resistance in the tubes requires way more pumping energy than a raceway pond the authors criticize some sentences above (lines 451 ff).

Thank you for highlighting this point. Open systems (e.g. raceways) and closed systems (e.g. tubular PBRs) certainly both possess an array of advantages and disadvantages, which we accept when stacked against each other relegates the latter to a more “theoretical” nature. As pointed out, the need for pumping energy and cleaning in tubular systems are important factors which, among other considerations, currently limit their widespread use. We have added the sentence below at line 488 to note this.

Despite the potential benefits offered by tubular photobioreactors in terms of CO2 capture and contaminant removal, the energy required for operation, as well as the need for cleaning and maintenance, remain barriers limiting their widespread use. As such, there exists a need for further research in this area, focused on expanding our understanding of biofilm behaviour within these systems and developing strategies for improving the overall cost-effectiveness of their operation.

Discussion, lines 478ff (reversibility): It’s a pity that no information is provided about biofilm development and biofilm composition. It seems to be of major importance to understand if the reaction to the availability of organic carbon in the medium is caused by metabolic changes of a static biofilm composition or by a change of the ratio of photo- to heterotrophic cells (reactive growth). This can be important to know as a stable biofilm can be operated over a longer time while a reactively changing biofilm will cause problems in a large scale plant. However, the authors mention in lines 529ff that such analysis were beyond their possibilities for this manuscript.

In this study, we documented the phototrophic biofilm’s interesting response to organic carbon availability, in which longitudinally discrete regions of net CO2 production and net CO2 consumption appear in the proximal and distal halves of the biofilm, respectively. As pointed out by the reviewer, an interesting question arises as to whether the observed response is attributable to metabolic changes within a static biofilm composition or changes in the ratio of photo- to heterotrophic cells. While a definitive answer was indeed beyond the scope of this paper, we suspect that over time both scenarios may be at play, but with the impact of each manifesting on different timescales. The biofilm’s near immediate toggling of its CO2 flux in response to organic carbon suggests that a metabolic change within existing community members likely predominates initially. It is plausible that such a metabolic shift will then eventually lead to a community composition response, which may well include a changing ratio of photo- to heterotrophic members. However, given typical microbial growth rates, we would expect this response and its related impact on overall biofilm CO2 flux to materialize on a comparatively longer timescale than the metabolic response. Nevertheless, we agree that this question is worthy of further examination in the future, as it may have implications for the long-term operation of such biofilm systems.

Reviewer #2 General Comments

The manuscript is based on a very interesting hypothesis, and undertaken in a suitable manner.

The Introduction, up to line no 99, is too wordy and seems to be unnecessary.

In the Introduction, we have strived to clearly present the motivation for studying algae in general, as well the rationale behind this study in particular, which we feel provides the reader with important context. Since algae can offer significant benefit in terms of CO2 sequestration, wastewater treatment, and biomass production, we felt it necessary to briefly describe these three applications and highlight the potential for a future paradigm in which they may be effectively combined.

We accept the reviewer’s opinion regarding the wordiness in the Introduction and have taken measures to cut down on this in several areas (e.g. lines 26, 27, 33, 51, 52, 56, 72, 80).

The Last few figures can be combined and presented in a better manner.

We strive to present our data in the clearest and most comprehendible manner. The last two figures each include two different data sets, both of which depict a change in feed composition as well as a subsequent reversal of flow direction. As such, we are wary of trying to convey too much information in a single graph, which could ultimately detract from its clarity. However, since these graphs present very similar experiments which are both speaking to the same hypothesis, we do agree that it would be logical to present them in a more cohesive manner. We have therefore combined what was previously Fig 7 and Fig 8 into a single Fig 7, with the two graphs labelled as “7A” and “7B”, as we have done previously with Fig 5. The caption and references to this figure have been adjusted in the text to reflect this change.

I would appreciate a bit more specific data/results in the abstract and conclusions, to make it more scientific.

Thank you for this recommendation. We have made several adjustments to the text in the Abstract (starting line 33) as well as in the concluding passage (starting at line 577) in order to provide more specificity when highlighting the results.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

1 Jun 2021

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

PONE-D-21-12369R1

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7 Jun 2021

PONE-D-21-12369R1

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

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