Alternative nitrogen and carbon sources for the bioproduction of single-cell protein

With the world's population rising, global protein demand is expected to double by 2050. Thus, the transformation of plant-based farming and the development of alternative solutions are urgent in the face of huge food demand. Recently, single-cell protein (SCP) extracted from the culture of microorganisms has gained interest as a food or feed supplement in recent decades due to its advantages of short generation time, high protein contents, and freedom from seasonal and climatic variations. Though promising, the technology development for SCP synthesis is still in its infancy, and several challenges need to be addressed before field applications. Among others, the renewable feedstock is a key factor for the successful production and application of SCP. For instance, nitrogen and carbon are the necessary building blocks of proteins readily available in waste streams. Turning waste into clean SCP products is still a challenge.

In this Ph.D. thesis, we aimed to broaden the substrate for the SCP synthesis process by using nitrate recovered from polluted groundwater and syngas as respective nitrogen and carbon sources. In this way, we expected to realize simultaneous resource recovery, carbon capture, renewable energy utilization, and valued-product production.

Firstly, a hybrid biological-inorganic (HBI) reactor was established to recover nitrate recovery from groundwater and in-situ assimilate it along with carbon dioxide into SCP by using a model hydrogen-oxidizing bacteria (HOB) strain, Cupriavidus necator H16. The results indicated that nitrate could be recovered from synthetic groundwater through the concentration gradient. In this case, the removal efficiency reached 85.52% with 5 V applied voltage and an initial nitrate concentration of 100 mg/L. The raw protein content was 47.71%, with a broad amino acid spectrum in the collected biomass; approximately 60% of the nitrate was assimilated into biomass, and 40% was transformed into N₂. Moreover, the water electrolysis with 3 and 4 V cannot provide sufficient H₂ for C. necator H16 and nitrate removal were 57.12% and 59.22% at 180 h, while they reached 65.14% and 65.42% with 5 and 6 V, respectively. Furthermore, with 200 and 300 mg/L initial nitrate concentrations, excellent removal performance was also obtained (58.40% and 50.72%) within 180 h.

Secondly, the feasibility of using syngas as a carbon source for the production of SCP was investigated. The results indicated that C. necator H16 could not metabolize CO, and higher CO content inhibits cell growth significantly. H₂ and CO₂ from syngas were utilized as substrates, and biomass with a 50-60% raw protein content was obtained. Higher shaking frequency and a larger inoculum volume facilitated the SCP synthesis.

Subsequently, another species of carboxydobacteria, Pseudomonas carboxydohydrogena Z-1062 was cultivated with syngas in an aerobic environment to evaluate its capability of using CO as substrate. The test results suggested that strain P. carboxydohydrogena Z-1062 cannot use CO as the sole carbon and energy source but could utilize H₂ as an energy source to assimilate CO into cells, with CO content up to 57.14%. Meanwhile, part of CO also converts into CO₂. In addition, ammonium and nitrate were both valid nitrogen sources for P. carboxydohydrogena Z-1062, and the protein contents in biomass were 63.91% and 50.31%, respectively. The study proved that syngas was an efficient substrate for P. carboxydohydrogena Z-1062, and CO was an alternative carbon source for SCP production.

Overall, this Ph.D. project broadened the substrate spectrum for SCP production. Furthermore, the results offer insight into developing novel bioprocesses that could achieve simultaneous wastewater remediation, nutrient upcycling, carbon capture, and output supplementary to human food and animal feed supply.