It might be crystal clear for a microbial ecologist that microbes rarely exist in isolation but instead are embedded in a complex web of multiple partnerships. However, this understanding only begins to trickle into the mindset of pure biotechnologists, generally having their ideal process (or experiment) to be “sterile” from other microorganisms. Yet, acknowledging the natural lifestyle of microbes expands the bioproduction of value-added compounds through novel pathways and strategies [1, 2]. This means that investing co-cultures can enable us to understand synergistic effects and uncover otherwise hidden behaviours, thus allowing for targeted utilization of their capabilities. However, to really become an alternative to single or so-called axenic cultures, co-cultures must be as well-defined and controllable as such.

The design of applicable co-cultures is not a trivial task, and in the best-case scenario, the co-cultures should align with a sustainable bioprocess. In most cases, it is intended that the microbes live under the premise of the division of labour, which allows different traits of microbes to complement each other in a profitable way [3, 4]. One example of doing so is pairing heterotrophs with phototrophs [5]. This combination of microbes is ubiquitous in the ocean, as can be seen exemplarily in microbial mats [6], where the photoautotrophic member, such as cyanobacteria or algae, uses solar power to fix CO₂ into organic carbon, a portion of which then is accessible to the heterotrophic members of the community. In the case of synthetic co-cultures, this concept is taken a step further by employing the heterotrophic partner to convert the carbon source provided into value-added products by engineering and optimizing metabolic pathways.

The co-culture journey in our lab started by combining two genetically modified bacteria to build up a synthetic co-culture of a phototrophic and a heterotrophic partner. The cyanobacterium Synechochoccus elongatus cscB was utilized, as it naturally produces sucrose to protect against high salt levels. Thanks to the heterologously expressed CscB sucrose permease, this sugar is secreted into the culture supernatant [7], where it serves then as the sole carbon source for the partner Pseudomonas putida cscRABY, a derivative modified for the uptake and metabolization of sucrose [8].

In our previous work, we demonstrated a high level of controllability in the co-culture while producing the bioplastic mcl-polyhydroxyalkanoate (PHA) [9, 10]. Following this, we asked ourselves: Is there potential feedback between the two microbes beyond the unidirectional synthetic interaction we have integrated? Given that interactions between phototrophs and heterotrophs are prevalent in ocean ecosystems, we were intrigued by how deeply ingrained these interactions might be through evolution, considering that P. putida is associated with soil rather than with marine environments.

To explore this in detail, we conducted a series of growth experiments under different environmental conditions, varying the induction of sucrose secretion, light exposure, and inoculation cell ratios to determine the phenomenological effect of co-culturing. Across different scales, it became evident that co-cultivating the cyanobacterium with P. putida cscRABY, especially in stressful conditions, restored and even increased viability. For instance, the growth rate of S. elongatus cscB increased up to 80% in co-culture grown cells as compared to axenic cultures with induced sucrose secretion. Furthermore, the photosynthetic capacity, estimated in terms of fixed carbon, increased in the total culture when paired with the co-culture partner.

In collaboration with the Pandhal lab from Sheffield University, UK, and the BayBioMS center from TUM Freising, Germany, we conducted an in-depth investigation of the co-culture at a transcriptome, proteome, and metabolome level. To this end, we set up an experiment comparing the co-culture to the respective axenic cultures grown under comparable conditions. For the heterotrophic partner, a fed-batch mode was chosen to mimic the feed of S. elongatus cscB. This enabled a reproducible and reliable experiment to harvest samples for OMICs analysis.

In summary, the multi-OMICs analysis of the co-culture provided us with a detailed snapshot of the cellular status at the moment of sampling, revealing a complex network of small to moderate changes. For example, we identified differentially regulated transporter genes alongside variations in the core carbon metabolism, indicating potential additional exchanges between the partners. Thus, in addition to the synthetic connection by sucrose, more links connecting the co-culture partners seem to be present. We also identified signs of competition for shared resources, such as medium components, including citrate and various salts. Furthermore, ion homeostasis was unbalanced in both organisms, suggesting limitations or reduced accessibility of ions due to potential advanced scavenging strategies of the respective co-culture partner. These findings underscore the importance of careful medium optimisation in co-cultures in general. Unexpectedly, despite the overall robust growth of the co-culture, stress signals were higher in the co-culture compared to the axenic culture. This suggests that co-culturing, while beneficial for growth, may introduce additional stressors that need to be addressed to further optimize co-culture conditions.

The primary challenge now lies in determining whether these exchanges are targeted processes, undirected responses, or an inherent feature of cell death in co-cultures. So, what’s the plan for moving forward? Armed with the insights gained, our next step involves developing a combined Flux Balance Analysis (FBA) model of the co-culture partners to computationally elucidate the exchange of different carbon sources between them. Our goal is to gain a deeper understanding of the interaction structure, which will need to be confirmed through experimental validation. Additionally, we are focussing on optimizing the co-culture itself by introducing an improved cyanobacterial partner. As biotechnologists, we are actively participating in the shift from axenic cultures to the application of well-defined co-cultures by enhancing the understanding of the complex network of inter-species interactions in synthetic communities.

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