The RhoCrt project aimed to develop a biotechnological route for producing carotenoids, particularly lycopene, from organic waste streams using the photosynthetic bacterium Rhodospirillum rubrum. The core innovation was a “dark photosynthesis” cultivation strategy that allows maximal expression of photosynthetic genes under semi‑aerobic, light‑free conditions, thereby enabling large‑scale bioreactor operation without the need for illumination. Two recombinant R. rubrum strains, engineered in a parallel effort at the University of Stuttgart, were employed to replace the native carotenoid spirilloxanthin with industrially relevant compounds such as lycopene and β‑carotene. The strains were cultivated in fed‑batch reactors (10 L Biostat Cplus) controlled by the BioPAT® MFCS/win 3.0 system.

In the fed‑batch runs with fructose (Fed‑Batch‑F) the culture reached a cell dry weight (CDW) of 7.22 g L⁻¹, yielding 28.6 mg L⁻¹ lycopene (3.97 g g⁻¹ CDW) and 1.87 g L⁻¹ polyhydroxyalkanoate (PHA) (0.26 g g⁻¹ CDW). When the medium was supplemented with a vitamin solution and ammonium succinate (Fed‑Batch‑FVAS), the biomass increased to 32.1 g L⁻¹, while lycopene concentration rose to 69.3 mg L⁻¹ (2.16 g g⁻¹ CDW) and PHA to 1.93 g L⁻¹ (0.14 g g⁻¹ CDW). Thus, absolute product titres were higher in the FVAS run due to the greater biomass, whereas specific yields per gram of dry weight were higher in the simpler fructose run. The ability to recover both lycopene and PHA with organic solvents demonstrates the dual‑product potential of the process.

A key objective was to adapt the cultivation to agricultural and food‑industry residues. Dairy waste in the form of milk‑milk permeate (MMP), or “mother‑lauge”, was supplied by Dairyfood GmbH, Riedlingen. Analysis of a raw MMP batch revealed 366 mmol L⁻¹ lactate, 74 mmol L⁻¹ acetate, and 366 mmol L⁻¹ lactose, with a conductivity of 28.3 mS cm⁻¹ and a pH of 5.03. The lactate concentration (≈32 g L⁻¹) was identified as a suitable carbon source for R. rubrum, whereas the bacterium could not metabolise lactose, glucose or galactose. Cultures grown on 40 mM lactate under semi‑aerobic, dark conditions achieved a specific growth rate of 0.046 h⁻¹, corresponding to a doubling time of 14.9 h. In contrast, cultures supplied with lactose showed no growth, confirming the selectivity of the metabolic pathway. Initial experiments with unsterilised MMP demonstrated that the material could be autoclaved without loss of nutrients and remained stable at 4 °C, providing a practical substrate for scale‑up.

The project was executed as a consortium between the University of Stuttgart (lead institution, Prof. Robin Ghosh, Institute for Biomaterials and Biomolecular Systems) and Hochschule Biberach (HBC, Institute for Applied Biotechnology). The HBC team comprised a post‑doctoral researcher, two scientific staff members, and a technical assistant, with several personnel changes during the project. The experimental work was carried out over the RhoCrt funding period, which spanned from 2021 to 2023, and was supported by European Union research funding under the Horizon 2020 framework. The collaboration focused on process development, strain optimisation, and the integration of waste streams, aiming to establish a sustainable, circular bio‑economy model for carotenoid production.