Oxygen competition induces chlorosis in thermoacidophilic methanotroph–microalgae cocultures

Abstract

Methanotroph–microalgae cocultures offer a promising bioprocess platform for methane (CH₄) and carbon dioxide (CO₂) valorisation and have been proposed for sustainable single-cell protein (SCP) production. In coculture, the thermoacidophilic methanotroph Methylacidiphilum sp. RTK17.1 can benefit from oxygen supplied by the extremophilic microalga Galdieria sp. RTK37.1 under low-O₂ conditions, enhancing growth and methane oxidation. However, oxygen availability is a critical factor that can rapidly become limiting and is dependent on the relative abundance of each coculture partner. Here, we investigated how the initial Methylacidiphilum:Galdieria biomass ratio influences coculture dynamics and overall carbon assimilation. At low initial ratios (< 0.18 gDW L⁻¹: gDW L⁻¹), CH₄-fed cocultures achieved 17–28% greater net carbon fixation than CH₄-free controls (p < 0.05), indicating effective methanotroph–microalgae synergy via oxygen exchange. In contrast, ratios ≥ 0.23 led to significant growth inhibition (66–100% reduction, p < 0.001) and reduced net carbon fixation (44–62% reduction, p < 0.001). These cocultures exhibited chlorosis and excreted coproporphyrin III, symptoms that were fully reversible upon oxygen replenishment. Collectively, these findings reveal that Galdieria sp. RTK37.1 requires a minimum oxygen threshold for photosystem pigment biosynthesis, while the high O₂ affinity of Methylacidiphilum sp. RTK17.1 can result in oxygen limitation at elevated methanotroph abundance. Consequently, interactions that are mutually beneficial at low methanotroph densities become inhibitory due to oxygen competition. This work highlights the central role of O₂ partitioning in extremophile cocultures and provides a mechanistic basis for optimising methane-fed photobioreactors relevant to SCP production and carbon valorisation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13068-026-02759-1.

Introduction

Methane (CH₄) is the second most significant greenhouse gas after carbon dioxide (CO₂), with a far greater short-term warming potential [1]. While CO₂ has been the primary focus of global warming abatement strategies, CH₄ emission control is an important parallel consideration [2]. Consequently, the valorisation of CH₄ and CO₂ emissions via methanotroph–photoautotroph cocultures has attracted attention [3, 4]. Methanotroph–photoautotroph cocultures can produce valuable products, including biodiesel [5], bioplastics [6], and single-cell proteins [7], as well as recover nutrients from industrial wastewater [4, 7, 8]. Extremophilic microorganisms, including methanotrophs (e.g., Methylacidiphilum spp.) [9] and microalgae (e.g., Galdieria spp.) [10], thrive in geothermal environments which approximate the physicochemical characteristics of many industrial processes (e.g., hot temperature flue gases, high CO₂, and acidic pH) [11, 12]. Consequently, the physiological characteristics of these extremophiles may be advantageous to cocultures at industrial scales.

One of the key interactions between methanotrophs and photoautotrophs (e.g., microalgae) in coculture involves interspecies oxygen (O₂) transfer, wherein, oxygenic photosynthesis supplies O₂ to methanotrophs for CH₄ oxidation which, in turn, produce CO₂ for photoautotrophic carbon assimilation [13]. Methanotrophs can therefore benefit from coculture with photoautotrophs when under O₂ limitation. Several studies, with differing methanotroph–photoautotroph pairs, have achieved sustained CH₄ oxidation without O₂ supplementation; including cocultures of the microalga Scenedesmus sp. with the methanotroph Methylocystis parvus [6], the cyanobacterium Synechococcus PCC 7002 with the methanotroph Methylomicrobium alcaliphilum 20z [14], the microalga Chlorella sorokiniana with the methanotroph Methylococcus capsulatus [7], and the methanotroph Methylacidiphilum sp. RTK17.1 with the microalga Galdieria sp. RTK37.1 [15].

Inoculum proportion is a critical determinant of coculture performance [16]. For example, Jeong et al. [17] reported that Sphingopyxis sp. NM1 enhanced both growth and CH₄ oxidation rates of Methylocystis sp. M6 when the coculture was initiated at a 1:9 (M6:NM1) volumetric ratio. This stimulatory effect disappeared when the ratios were reversed (9:1) or equalised (1:1). Likewise, a biomass concentration-dependent effect on CH₄ oxidation in mixed cultures of Methylocystis sp. M6 and the methylotrophic denitrifier Hyphomicrobium sp. NM3 was observed [18]. Under O₂ limitation, increased photoautotroph abundance in methanotroph–photoautotroph cocultures enhanced growth rates of slow-growing photoautotroph partners by increasing the volumetric rate of O₂ production [13, 19]. This O₂ dependence was demonstrated for Methylomicrobium buryatense cocultured with Arthrospira platensis, where external O₂ supplementation altered the final photoautotroph:methanotroph ratio [19]. Conversely, in excess O₂, a greater photoautotroph proportion does not necessarily benefit the methanotroph. For instance, in cocultures of Methylomicrobium alcaliphilum 20ZR with Synechococcus sp. PCC7002, proportional abundance had no effect on growth rate, as CH₄ mass transfer—not O₂ availability—was the limiting factor [13].

However, not all methanotroph–phototroph interactions are beneficial (e.g., mutualism, commensalism). Under excess O₂, Scenedesmus obtusiusculus can inhibit CH₄ oxidation in a methanotroph consortium by alkalising the medium beyond the bacterial pH optimum [20]. Photoautotrophs can also be disadvantaged in coculture. Fast-growing methanotrophs may outcompete them via shading or nutrient competition [21], as seen with Chlorella sorokiniana in coculture with Methylococcus capsulatus [7]. Furthermore, some Galdieria spp. excrete coproporphyrin III under low-O₂ during heterotrophic or mixotrophic growth, disrupting chlorophyll and phycobiliprotein synthesis and causing chlorosis [22–24]. In wastewater-derived cocultures, this O₂ limitation—exacerbated by bacterial respiration—can further reduce growth and biomass yields [24].

In batch coculture with Galdieria sp. RTK37.1, the thermoacidophilic methanotroph Methylacidiphilum sp. RTK17.1 benefits from interspecies O₂ transfer under low-oxygen conditions [15]. Despite the industrial potential of extremophilic consortia, the dynamics of photoautotroph–methanotroph interactions remain poorly understood. Here, we investigate how the initial Methylacidiphilum:Galdieria biomass ratio influences coculture performance. We show that, under O₂ limitation, dilute Methylacidiphilum populations benefit from Galdieria’s oxygenic activity, enhancing CH₄ oxidation and carbon fixation. However, at greater methanotroph proportional abundances, the high O₂ affinity of Methylacidiphilum sp. RTK17.1 depletes available oxygen, disrupting pigment biosynthesis in Galdieria sp. RTK37.1, triggering chlorosis, and ultimately suppressing coculture growth. These findings provide new insight into O₂-driven trade-offs in extremophile cocultures, offering practical guidelines for designing and optimising CH₄/CO₂ co-utilisation systems relevant to single-cell protein production and other carbon-valorisation bioprocesses.

Materials and methods

Stock culture maintenance

A modified V4 medium [25] was used for all cultivation experiments. Stock cultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 were routinely maintained as previously described [15, 26]. Briefly, Methylacidiphilum sp. RTK17.1 was maintained in chemostat culture using a 1-L bioreactor (BioFlo 110; New Brunswick Scientific, United States) with a 600 mL working volume and 0.0069 h⁻¹ dilution rate. A gas mixture (69% CO₂, 1.0% CH₄, 3.1% O₂, balance N₂, all v/v) was continuously supplied at 20 mL min⁻¹. Temperature was maintained at 45 °C, agitation at 800 rpm, and pH ~ 2.5. Under these conditions, typical steady-state OD₆₀₀ values were 1.2 and outlet gas concentrations were 72% CO₂, 0.13% CH₄, 1.5% O₂, with the remaining attributed to N₂. For all experiments, Methylacidiphilum sp. RTK17.1 was aseptically harvested directly from the reactor and diluted with sterile V4 medium to the desired starting concentration. For Galdieria sp. RTK37.1, 300 mL stocks were repeatedly grown in 1-L Duran^® Pressure Plus bottles sealed with bromobutyl rubber stoppers. At the beginning of each batch growth cycle, Galdieria sp. RTK37.1 cultures were diluted to OD₆₀₀ 1.0 with sterile V4 medium, and the bottle headspaces were then subjected to vacuum for 3 min, and re-pressurised to 35 kPa (gauge) with an 80% CO₂ and 20% N₂ gas mixture (v/v). The bottles were then incubated on a shaking incubator (WiseCube WIS-10, Wisd Laboratory Instruments, Germany) equipped with warm white LED strips, at 45 °C, 150 rpm, and light intensity of 40 μmol m⁻² s⁻¹ measured at the bottles’ wall with a LI-250A Light Meter (LI-COR, Nebraska, United States). Growth was stopped at OD₆₀₀ 6.0 and the cycle started anew. For all experiments, Galdieria sp. RTK37.1 was aseptically harvested at an OD₆₀₀ 4.0 and diluted with sterile V4 medium to the desired starting concentration. Unless stated otherwise, all chemicals and reagents were purchased from Sigma-Aldrich (Germany) and all gases were sourced from BOC (Australia).

Variable mass ratio coculture experiments

Cocultures were conducted with varying initial Methylacidiphilum:Galdieria mass ratios to evaluate their influence on coculture performance (e.g., growth rate, biomass productivity, O₂ production rate, CH₄ oxidation rate). The initial biomass ratios tested (methanotroph:microalga, expressed as g_DW L⁻¹:g_DW L⁻¹) were 0, 0.08, 0.15, 0.18, 0.23, 0.30, and 1.33. For these experiments, an initial Galdieria sp. RTK37.1 concentration (0.215 g_DW L⁻¹, OD₆₀₀: 0.88 A.U.) was consistently used with varying initial Methylacidiphilum sp. RTK17.1 concentrations (ranging between 0.0 and 0.52 g_DW L⁻¹, OD₆₀₀: 0.0 to 0.44). Coculture suspensions (40 mL) were prepared by aseptically diluting the microbial stocks with sterile V4 medium into 160-mL serum bottles. Each serum bottle was sealed with a butyl rubber stopper and an aluminium crimp, and then, to displace O₂ in the headspace, was flushed for 3 min with an 80/20 CO₂/N₂ (v/v) gas mixture and pressurized to 69 kPa (gauge). For each of the varying mass ratios, six serum bottles were prepared, into which half received 20 mL 100% CH₄ to enable growth of Methylacidiphilum sp. RTK17.1. The remaining serum bottles instead received N₂ (20 mL). These preparations resulted in initial headspace concentrations of approximately 0.5% O₂, 13.0% CH₄, 70% CO_2, balance N₂ (all v/v) for the CH₄ containing cocultures, and 0.5% O₂, 70% CO₂, balance N₂ (all v/v) for the non-CH₄ containing cocultures.

Serum bottle cocultures were then cultivated for 6 days in a shaking incubator (WiseCube WIS-10, Wisd Laboratory Instruments, Germany) at 45 °C, 150 rpm, and a light intensity of 20 μmol_photons m⁻² s⁻¹. Each day, 2 mL liquid samples were collected from each bottle and analysed for total OD₆₀₀, pigment fluorescence and absorption spectra. At the end of the experiment, final headspace pressures were measured, with liquid and gas samples collected for gas concentration analysis, and final biomass concentration determinations.

Chlorosis coculture recovery experiments

To investigate the impact of O₂ limitation on coproporphyrin formation, pigment loss (e.g., phycocyanin, chlorophyll) and the reversibility of chlorosis within Galdieria sp. RTK37.1, a series of batch cocultures were prepared with varying initial gas concentrations. For all cocultures, 250 mL of sterile V4 medium was added to 1-L Duran^® Pressure Plus bottles sealed with bromobutyl rubber stoppers and inoculated to the desired initial concentrations with the appropriate microbial stocks. To distinguish the effects of O₂ limitation from those of Methylacidiphilum growth, “replenishment” cocultures were established with daily CH₄, CO₂, and O₂ supplementation. Cultures were inoculated with Methylacidiphilum sp. RTK17.1 (OD₆₀₀ = 0.3) and Galdieria sp. RTK37.1 (OD₆₀₀ = 1.0), sealed, and injected with 100 mL CO₂ and 60 mL CH₄. After daily sampling, bottles were opened in a Thermo HeraSafe HS12 Laminar Flow cabinet (Thermo Scientific, United States), equilibrated for 30 min, and re-gassed to initial concentrations (Table S1). All bottles were cultivated in triplicate as described above (i.e. 45 °C, 150 rpm, 20 μmol_photons m⁻² s⁻¹).

To evaluate whether Galdieria sp. RTK37.1 could recover from chlorosis upon the cessation of Methylacidiphilum growth, a series of cocultures were prepared where CH₄ was first added and then removed once pigment loss and coproporphyrin formation was observed. Galdieria sp. RTK37.1 was inoculated to a starting OD₆₀₀ of 0.35 and Methylacidiphilum sp. RTK17.1 to an OD₆₀₀ of 0.80. Coculture headspaces were prepared by subjecting the bottles to 3 cycles of vacuum for 3 min and then re-pressurising to 35 kPa (gauge) with 80/20 CO₂/N₂ (v/v). Following this, headspace gas was equilibrated to atmospheric pressure by expanding against a syringe, and then 120 mL of CH₄ was injected. Following 4 days of incubation, all bottles were opened inside a Thermo HeraSafe HS12 Laminar Flow cabinet (Thermo Scientific, United States), and left to equalise for 30 min. 50 mL of broth was then harvested from each bottle, centrifuged at 12,000 rpm on a 5810R Benchtop Centrifuge (Eppendorf, Germany) for 15 min, and the supernatants were pooled together and stored at −20 °C until required for coproporphyrin analysis. 50 mL of sterile V4 medium was then added to each bottle (to maintain a 250 mL volume), bottles were re-sealed and new headspace gas compositions were prepared (Table S1) before further incubation.

A final set of cocultures, prepared without initial CH₄, served as controls to assess Galdieria growth in the absence of Methylacidiphilum activity. Galdieria sp. RTK37.1 and Methylacidiphilum sp. RTK17.1 were inoculated at OD₆₀₀ values of 0.40 and 0.80, respectively. Headspaces were prepared as described (Table S1) and pressurised to 138 kPa (gauge) with an 80/20 CO₂/N₂ gas mixture (v/v).

For all conditions, 2 mL liquid and 20 mL gas samples were collected daily for analysis. Headspace pressure was measured with an Almelmo 5470 data logger (Ahlborn, Germany) equipped with a pressure sensor (Gems Sensors & Controls, England) before and after all headspace sampling to account for gas loss. Cultures were terminated when any replicate exceeded an OD₆₀₀ of 3.5 to prevent nitrogen limitation or when the phycocyanin response decreased to zero, indicating the absence of detectable phycobiliprotein signal.

Liquid chromatography–mass spectrometry (LC–MS) analysis

To confirm the identity of coproporphyrin in culture supernatants, samples were subjected to LC–MS analysis. For these measurements, 500-µL aliquots of aqueous centrifuged supernatant were diluted 1:1 in isopropanol (IPA) and filtered through a 0.22-µm PTFE syringe filter. Samples were then analysed on an Agilent UPLC-QTOF spectrometer equipped with an Agilent 1290 high speed binary pump, Agilent 1290 multi-sampler, Agilent 1290 multi-column thermostat, an Agilent 1260 diode array detector, and a Dual AJS ESI source in positive ion mode. The LC method used an Agilent Zorbax Eclipse Plus 50 × 2.1 mm, 1.8 µm column, 35 °C temperature, 20 min run time, positive MS polarity, 10 µL injection volume, 0.3 mL min⁻¹ flow rate. An acetonitrile/water gradient (with 0.1% formic acid) was used. The gradient started at 2% (v/v) acetonitrile (with 0.1% (v/v) formic acid, balance H₂O) for 15 min, then increased to 100% acetonitrile (with 0.1% (v/v) formic acid) for 5 min. A 1:1 IPA in water (LCMS grade) solution was used as a blank. Data were analysed using Agilent MassHunter Qualitative analysis 10.0 software. Base Peak Chromatograms (BPC) were generated, mass spectra were extracted for the relevant peaks and the mass spectrum background (from the blank) was subtracted. An Extracted Ion Chromatogram (EIC) for the expected ions for coproporphyrin III ([M + H]⁺ = 655.2762 and [M + Na]⁺ = 677.2582) was generated in positive ion mode. Formulas for the ions in the peak at retention time 7.46 min were generated to confirm whether there was a good match for coproporphyrin III.

Analytical procedures

Optical density at 600 nm was routinely measured using an Ultrospec 10 cell density meter (Amersham Bioscience, United Kingdom). To differentiate the abundance of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 in cocultures, a method utilising differential sedimentation, optical density and fluorescence (DSOF) was used as previously described [27].

Pigment content in coculture was tracked by autofluorescence. For these measurements, 200 μL samples were transferred into black, flat-bottom (chimney well) 96-well microplates (Greiner Bio-One, Austria). Fluorescence was measured at 670 nm emission (590 nm excitation) for phycocyanin and at 670 nm emission (485 nm excitation) for chlorophyll using a Varioskan Lux microplate reader (Thermo Scientific, United States) [28]. For absorption spectra, 200 μL samples were transferred into clear 96-well microplates (Greiner Bio-One, Austria), and optical density was measured between 300 and 800 nm (in 1 nm increments) using the same microplate reader. To assess coproporphyrin variation, the average of the optical density values at 390 nm and 410 nm was subtracted from the 400 nm coproporphyrin peak. Negative values calculated in this fashion were considered zero.

Biomass concentrations were measured gravimetrically. For these, 15 mL aliquots were vacuum-filtered onto pre-weighed 0.22 µm nylon membrane filters (MicroScience, Australia). Filters were then washed three times by vacuum-filtering 10 mL of deionized water, dried for 12 h at 95 °C, weighed, and the filtered biomass weight was calculated by difference.

Gas samples (CO₂, CH₄, N₂, and O₂) were analysed using an Agilent 490 Micro GC equipped with a thermal conductivity detector (Agilent Technologies, United States), a Molecular Sieve 5A with a heated injector (50 °C, back-flush at 5.10 s, column at 90 °C, 150 kPa), a PoraPak Q column with a heated injector (50 °C, no back-flush, column at 70 °C, 50 kPa) and a 5CB column with a heated injector (50 °C, no back-flush, column at 80 °C, 150 kPa).

All statistical analyses were performed using Prism Graphpad v9.4.1. Unless stated otherwise, two-factor ANOVA (α = 0.05) tests were used, with the Sidak method for multiple mean comparisons between columns (simple effects within rows) or rows (simple effects within columns). Growth rates (μ) were calculated by fitting the appropriate biomass concentration data to an exponential growth function within Prism Graphpad v9.4.1.

Results and discussion

Biomass productivity at low initial oxygen concentration is sensitive to initial mass ratio in batch coculture

The relative proportion of member species in methanotroph:photoautotroph cocultures can significantly influence bioprocess performance [16], however, the impact of this parameter on thermoacidophilic cocultures is unknown. Therefore, to evaluate the influence of Methylacidiphilum:Galdieria mass ratios on coculture performance, a series of batch coculture experiments was performed with varying initial biomass ratios.

In CH₄-supplemented cocultures, growth significantly decreased with increasing Methylacidiphilum:Galdieria mass ratios. Cocultures with mass ratios ≤ 0.18:1 displayed a modest slowing of growth rate (8% decrease, p < 0.01). However, increasing the initial Methylacidiphilum concentration beyond 0.18:1, resulted in significant growth inhibition (p < 0.001), with no growth observed at a Methylacidiphilum:Galdieria mass ratio of 1.33:1. In contrast, only small changes to growth were observed in cocultures without CH₄-supplementation. Expectedly, Methylacidiphilum sp. RTK17.1 could not grow in cocultures without CH₄ supplementation. Thus, any change to OD observed in the absence of CH₄ was entirely attributed to Galdieria sp. RTK37.1 (Fig. 1a). Nevertheless, a significant 9% decrease (p < 0.005) in growth rate, with increasing Methylacidiphilum sp. RTK17.1 proportional abundance, was attributed to a shading effect. Previous reports indicate cell-mediated shading in coculture negatively impacts photoautotroph growth in cocultures with methanotrophs [19]. This effect is further evidenced when comparing rates of biomass productivity: a maximum productivity of 250 (± 3) mg_DW L⁻¹ h⁻¹ was obtained at an initial 0.08:1 (g_DW L⁻¹ basis) Methylacidiphilum:Galdieria ratio, while the minimum productivity of 188 (± 4) mg_DW L⁻¹ h⁻¹ was obtained when Methylacidiphilum sp. RTK17.1 was most abundant (Fig. 1b).

Fig. 1.

Effect of the initial Methylacidiphilum RTK17.1 to Galdieria RTK37.1 concentration ratio on coculture growth rates (μ), for cultures with and without (w/o) CH₄ supplementation into the headspace. a) Average growth rate was calculated over the entire cultivation period (6 days) using the initial and final biomass concentrations. For the cocultures without CH₄ supplementation, Methylacidiphilum concentration was subtracted to the initial and final concentrations, as it did not contribute to biomass growth. b) Biomass productivity. Residual oxygen at the start of cultures ranged from 0.1 to 2.4% (v/v). Cultures were grown in 160-mL serum bottles in a shaking incubator at 45 °C, 110 rpm, and 20 μmol_photons m⁻² s⁻¹ warm white (LED) illumination measured at the bottle wall. Error bars represent one standard deviation (n = 3) of biological triplicates

CH₄ availability, for initial mass ratios ≥ 0.23:1 (Methylacidiphilum:Galdieria), significantly impacted coculture biomass productivity due to the activity of Methylacidiphilum sp. RTK17.1 (p < 0.005, Fig. 2). These decreased productivities contrast with batch cocultures involving different methanotroph/photoautotroph pairs (Methylomicrobium alcaliphilum 20ZR/Synechococcus sp. PCC7002; Methylococcus capsulatus/Chlorella sorokiniana) with varying biomass ratios [13]. For M. capsulatus/C. sorokiniana cocultures, greater proportions of microalgae tended to increase coculture growth rates, as the comparatively slow-growing C. sorokiniana produced limited O₂ (via photosynthesis) and hence constrained methanotroph growth. This is similar to the increased growth and CH₄ oxidation rates previously reported for Methylacidiphilum sp. RTK17.1 when grown in coculture with Galdieria sp. RTK37.1 [15]; and hence was expected in these ratio experiments. For M. alcaliphilum/Synechococcus cocultures, using a greater cyanobacterial concentration did not impact coculture growth, as the cyanobacterium grew much faster than the methanotroph, which was thus limited by CH₄ mass transfer rather than O₂ availability [19].

Fig. 2.

Effect of the initial Methylacidiphilum sp. RTK17.1 to Galdieria sp. RTK37.1 mass ratio on net production and consumption rates, for cocultures with and without supplemental CH₄. The a) net CO₂ consumption rate, b) net CH₄ consumption rate, c) net carbon (C) fixation rate and d) net oxygen (O₂) production rates are shown. All rates were calculated over the entire culture period (6 days) using the initial and final gas concentrations, pressures and temperatures. Cocultures were grown in 160-mL serum bottles in a shaking incubator at 45 °C, 110 rpm, and 20 μmol_photons m⁻² s⁻¹ warm white (LED) illumination measured at the bottle wall. Error bars represent one standard deviation (n = 3) of biological triplicates

In addition to decreased biomass productivity, all cocultures grown with CH₄ at an initial Methylacidiphilum:Galdieria mass ratio ≥ 0.23:1 developed a distinct yellow colouration (Figure S1). In microalgae, N-starvation is known to trigger a similar colour change, known as ‘chlorosis’, which is caused by the loss of chlorophylls and phycobiliproteins [29]. Chlorosis due to N-starvation is well documented in Galdieria spp. [30], with the loss of pigments often detrimental to photoautotrophic growth [29]. In this study, excess ammonium was supplied to cocultures, so the authors concluded that the observed chlorosis was not due to N-limitation. Alternatively, the reduced carbon metabolites excreted by methanotrophs can impact microalgae pigmentation as cells switch from autotrophic to mixotrophic growth [5]. Although Galdieria spp. are known to grow mixotrophically [24, 26, 31], growth rates are typically faster than for autotrophic growth [32], so a switch to mixotrophic growth seems unlikely (Fig. 1a). Chlorotic cocultures exhibited significantly slower net CO₂ consumption and net carbon fixation rates (p < 0.001, Fig. 2) indicating decreased microalgae activity. Interestingly, CH₄ oxidation rates were maximal (0.124 ± 0.001 mmol_CH4 L⁻¹ h⁻¹) at a Methylacidiphilum:Galdieria ratio of 0.15:1. At this ratio, CH₄ was completely removed, so it is possible faster consumption rates would have been observed with supplemental addition of CH₄. However, further increasing the Methylacidiphilum proportion in cocultures tended to diminish the amount of CH₄ removed.

The observed decrease in CH₄ consumption rate was attributed to limited O₂ availability (Fig. 2). Even at the highest Methylacidiphilum:Galdieria ratios, some CH₄ oxidation occurred (0.062 ± 0.016 mmol CH₄ L⁻¹ h⁻¹), indicating that Galdieria sp. RTK37.1 initially produced O₂. However, this O₂ was rapidly consumed by Methylacidiphilum sp. RTK17.1, creating an O₂ limitation. Supporting this, OD₆₀₀ increased only during the first 24 h in CH₄-containing cocultures with ratios > 0.18:1 (Figure S2). The yellow pigmentation observed 1–2 days later is consistent with chlorosis and cessation of oxygenic activity. Thus, the CH₄ oxidation benefit Galdieria provides to Methylacidiphilum under low-O₂ conditions depends strongly on their relative abundances. In this study, ratios ≤ 0.15:1 supported greater carbon fixation, whereas ratios ≥ 0.23:1 impaired Galdieria activity, reducing coculture growth, CO₂ fixation, and CH₄ oxidation. Similar O₂-driven shifts have been reported in other cocultures, such as M. bryophila with S. obliquus [5], where low O₂ favoured autotrophic algal growth and CH₄ oxidation to CO₂, but high O₂-promoted heterotrophic algal growth and methanotroph release of organic metabolites instead of CO₂.

Methylacidiphilum sp. RTK17.1 induces O₂ limitation and subsequent chlorosis in Galdieria sp. RTK37.1

To further investigate the pigment content (e.g., chlorophyll, phycocyanin) of Galdieria sp. RTK37.1 cells, cocultures were assessed spectrophotometrically. Within cocultures ≤ 0.18:1 Methylacidiphilum:Galdieria, chlorophyll and phycocyanin profiles were consistent with normal batch photoautotrophic growth (Fig. 3). In contrast, CH₄-containing cocultures at ratios ≥ 0.23:1 showed decreases in chlorophyll and phycocyanin content in Galdieria sp. RTK37.1 as the coculture progressed. Chlorosis was not observed in cocultures with non-growing Methylacidiphilum sp. RTK17.1 (in the absence of CH₄) or in high-density cocultures that received daily gas replenishment (Fig. 4). Concurrent with pigment loss, analysis revealed the appearance of a peak at 400 nm consistent with the presence of coproporphyrin (Figure S3a inset) [33]. Subsequent LC–MS analysis confirmed the identity of coproporphyrin III in culture supernatants.

Fig. 3.

Temporal profiles for a) chlorophyll (670 nm emission, 485 nm excitation), b) phycocyanin (670 nm emission, 590 nm excitation), and c) coproporphyrin within CH₄-containing cocultures with varying initial Methylacidiphilum:Galdieria mass ratios. Coproporphyrin peak absorbance was measured by subtracting the average of the optical densities at 390 nm and 410 nm from the 400 nm peak. Negative values calculated in this fashion were equated to 0 A.U. Closed circles represent cocultures with initial Methylacidiphilum:Galdieria ratios ≤ 0.18 (non-coproporphyrin producing), while open circles represent cocultures with initial mass ratios ≥ 0.23 (coproporphyrin producing). Cultures were grown in 160-mL serum bottles in a shaking incubator at 45 °C, 110 rpm, and 20 μmol_photons m⁻² s⁻¹ warm white (LED) illumination measured at the bottle wall. Error bars represent one standard deviation of biological triplicates (n = 3)

Fig. 4.

Growth and pigmentation profiles of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 cocultures with different headspace concentrations. a), d) Cocultures with no added CH₄ (to suppress methanotroph growth). b), e) Cocultures with daily replenishment of CH₄, CO₂, and O₂ to initial concentrations. c), f) Cocultures with no daily replenishment of O₂. Experiments were terminated when the coculture optical density (OD₆₀₀) reached approximately 3.5, to avoid the onset of nitrogen limitation, or when the phycocyanin response decreased to zero, indicating the absence of detectable phycobiliprotein signal. Methanotroph and microalgae concentrations were calculated using the DSOF method. All cultures were cultivated at 45 °C, 150 rpm, and 40 μmol_photons m⁻² s⁻¹ warm white (LED) illumination in 1-L gas-tight bottles on a shaking incubator. Cultures were grown in triplicate; error bars represent one standard deviation (n = 3) of biological triplicates

Coproporphyrin is an intermediate formed during the biosynthesis of both phycocyanin and chlorophyll [34]. During normal pigment biosynthesis, the precursor coproporphyrinogen III is oxidised by coproporphyrinogen III oxidase (HemF, E.C. 1.3.3.3), into protoporphyrinogen IX [22, 35]. HemF activity is O₂-dependent, so during periods of O₂ limitation coproporphyrinogen III accumulates, and is eventually excreted, where it is oxidised into coproporphyrin III [22]. As a consequence, pigment biosynthesis is arrested. In Galdieria sulphuraria, suppression of phycobiliprotein synthesis during N-starvation can induce chlorosis and lead to complete phycocyanin degradation within 3–4 days [30]. While O₂ limitation seems counterintuitive for a photoautotroph, and there are no reports of coproporphyrin formation in autotrophically growing Galdieria spp., coproporphyrin formation is known to occur under O₂ limiting conditions in both heterotrophic and mixotrophic Galdieria spp. cultures [22]. In Methylacidiphilum–Galdieria cocultures, O₂ limitation was likely induced by a combination of high relative abundance of methanotroph, the relatively fast O₂ consumption rates for this strain (2.33 ± 0.19 mmol g_DW⁻¹ h⁻¹ O₂ consumption for Methylacidiphilum vs. 0.966 ± 0.152 mmol g_DW⁻¹ h⁻¹ O₂ production for Galdieria sp. RTK37.1) [15], and an exceptionally strong O₂ affinity (K_s = 0.7 mM) characteristic of verrucomicrobial methanotrophs [36].

Therefore, at Methylacidiphilum:Galdieria mass ratios ≥ 0.23, the methanotroph rapidly depleted available O₂, triggering coproporphyrin excretion by Galdieria. This diversion of coproporphyrinogen III away from pigment synthesis halted phycocyanin production, leading to its degradation by day 2, followed by progressive chlorophyll loss (Fig. 3). The resulting chlorosis impaired photosynthesis and Galdieria growth, further reducing O₂ availability and ultimately limiting methanotrophic activity.

O₂-limiting chlorosis is reversible in Galdieria sp. RTK37.1

To assess whether Galdieria sp. RTK37.1 could recover from pigment loss, cocultures were established in which CH₄ was removed after the onset of chlorosis and coproporphyrin accumulation (Figs. 5, 6, S4). Following CH₄ removal, a pigment recovery phase ensued without cell growth (days 4–7; Figure S4). During the first 2 days of the recovery period, cultures appeared white (Fig. 6) as chlorophyll levels continued to decline (Fig. 5). Concurrent with the cessation of Methylacidiphilum growth, coproporphyrin concentrations decreased rapidly—a pattern not attributable to passive degradation, as concentrations remained stable for up to 15 days in previous O₂-limited mixotrophic G. sulphuraria cultures [22]. This suggests that, as O₂ accumulated in the medium, Galdieria reabsorbed extracellular coproporphyrin for pigment biosynthesis. Supporting this, the decline in coproporphyrin coincided with a 1% decrease in headspace O₂ and was followed by phycocyanin recovery, consistent with the O₂ dependence of HemF catalysis [22]. Analogous mechanisms have been reported in other microorganisms. Paracoccus denitrificans secretes coproporphyrin III under copper limitation, then reclaims the porphyrin–metal complex via TonB-dependent transporters [37]. Likewise, G. sulphuraria can recover dissolved lanthanide and Cu²⁺ ions, potentially through porphyrin-mediated complexation [24, 38–40]. Since metal recovery in these cases occurred only under low-O₂ conditions, they align with a model in which Galdieria excretes coproporphyrin to sequester metals, then reabsorbs it for intracellular use when O₂ becomes available. By day eight, coproporphyrin was no longer detectable, chlorophyll levels had recovered, and O₂ began accumulating in the headspace. Thereafter, Galdieria resumed growth, with both pigment content and O₂ concentration increasing as expected.

Fig. 5.

Chlorosis recovery of Galdieria sp. RTK37.1. Pigments (e.g., chlorophyll, phycocyanin), coproporphyrin, and O₂ profile in cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 with CH₄ initially added and later removed (Day 4). Headspaces were subjected to vacuum, opened, and re-gassed on day 4 to remove CH₄ (1.38% v/v O₂, 60.83% v/v CO₂, CH₄ undetectable, final headspace concentration). No O₂ was added at any point. Grey shading indicates the period where CH₄ was present. Coproporphyrin peak absorbance was measured by subtracting the average of the optical densities at 390 nm and 410 nm from the 400 nm coproporphyrin peak. Negative values calculated in this fashion were considered zero. Cocultures were incubated in 250 mL V4 media at 45 °C, with shaking (150 rpm), and 40 μmol_photons m⁻² s⁻¹ light intensity in 1-L gas-tight bottles. Average values are shown with error bars representing one standard deviation (n = 3) of biological triplicates

Fig. 6.

Colour development in batch cocultures of Methylacidiphilum sp. RTK17.1 and Galdieria sp. RTK37.1 with CH₄ initially added and later removed. Headspaces were subjected to vacuum, opened, and re-gassed on day 4 to remove CH₄ (1.38% v/v O₂, 60.83% v/v CO₂, CH₄ undetectable, final headspace concentration). No O₂ was added at any point. Cocultures were incubated in 250 mL V4 media at 45 °C, pH 2.5, with 150 rpm agitation, and 40 μmol m⁻² s⁻¹ light intensity in 1-L gas-tight bottles. Cultures were grown in biological triplicate, and photographs of the same replicate are shown

Finally, the potential impact of coproporphyrin on Methylacidiphilum sp. RTK17.1 growth remains unclear. In cocultures, coproporphyrin can act as either a growth promoter or an inhibitor [33, 41], with its excretion proposed as a means of suppressing competitors through competitive nutrient sequestration. Galdieria sulphuraria can recover > 90% of Cu²⁺ and lanthanide ions from solution at concentrations as low as 0.5 ppm [40], potentially via coproporphyrin-mediated chelation—consistent with the role of coproporphyrin in copper acquisition by Paracoccus denitrificans [37]. For verrucomicrobial methanotrophs, lanthanides are essential cofactors for XoxF-type methanol dehydrogenases [42], and Cu²⁺ is required for pMMO synthesis and activity [43]. It is therefore plausible that coproporphyrin excreted by Galdieria sp. RTK37.1 could chelate these metals, potentially restricting their availability and inhibiting methanotroph growth. However, this hypothesis requires further investigation.

Conclusions

The interaction between Galdieria sp. RTK37.1 and Methylacidiphilum sp. RTK17.1 in batch coculture is governed by O₂ availability and the relative abundance of each partner. Under O₂ limitation and low methanotroph density, Methylacidiphilum benefits from Galdieria’s oxygenic activity, enhancing CH₄ oxidation. However, at greater methanotroph abundances, its strong O₂ affinity deprives Galdieria of oxygen, disrupting pigment biosynthesis, triggering coproporphyrin III excretion, and causing chlorosis. Chlorosis halts photosynthesis, further reducing pigment production and slowing coculture growth. In this study, this shift from commensalism to antagonism occurred at Methylacidiphilum:Galdieria ratios ≥ 0.23:1, although the exact threshold likely depends on external O₂ and CH₄ availability, as well as light intensity. Coproporphyrin excretion and chlorosis were absent when O₂ was replenished and were reversible when O₂ was allowed to accumulate, indicating that Galdieria requires a minimum dissolved O₂ concentration for sustained pigment synthesis. This is the first report of coproporphyrin excretion by Galdieria during photoautotrophic growth, and the first evidence of it being directly induced by another microorganism in coculture. Given its known metal-chelating properties, coproporphyrin III could influence the growth of both partners, though this requires further study. Chemostat cocultures with controlled, low dissolved O₂ could provide a platform to both meet methanotroph oxygen demands and stimulate coproporphyrin excretion in microalgae.

Author contributions

Conceptualization, CRC., MBS., CC-C, CC, DG, and PAG; methodology, CC-C; formal analysis, CCC; resources, CRC, MBS, CC, DG; data curation, CC-C; writing—original draft preparation, CRC, CC-C; writing—review and editing, CRC, MBS, CC-C, CC, DG, and PAG; visualisation, CRC, CC-C; supervision, CRC, MBS, CC, DG, and PAG; funding acquisition, MBS, CRC, DG. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by an MBIE Smart Ideas Grant (QT-7183) awarded to CC-C, MBS, CC, PAG and CRC.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: A patent application (PCT/IB2024/054,938) has been submitted and is currently in consideration. All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.