Microorganisms have been used for thousands of years to produce beer, bread, and fermented foods (Arranz-Otaegui et al., 2018; Liu et al., 2018). More recently, advances in biotechnology and the environmental impact of conventional agriculture have spurred interest in single-cell protein as a new way to utilize microorganisms in food. Single-cell protein is highly nutritious, containing up to 80% protein (Ravindra, 2000), and offers a more sustainable alternative to conventional food production with lower land and water use, reduced greenhouse gas emissions, and the potential to grow on waste substrates (Matassa et al., 2016; Spalvins et al., 2018).
Single-cell protein is typically produced by submerged high cell density cultivations to densities ≥ 20 g dry weight L-1, maximizing volumetric productivity while reducing volume, water consumption, and production cost (Subramaniam et al., 2018). However, current processes are constrained by the low solubility and transport rate of oxygen in water, often resulting in reduced yield and accumulation of byproducts (Garcia-Ochoa & Gomez, 2009). Researchers at the Norwegian University of Life Sciences (NMBU) have addressed this issue by developing a method for anaerobic high cell density cultivation (Bakken et al., 2023; Maråk et al., 2024) relying on denitrification, the dissimilatory reduction of nitrate (NO3-) to dinitrogen gas (N2) via nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O) (Figure 1). This form of anaerobic respiration is second only to aerobic respiration in terms of energy yield (Chen & Strous, 2013). The anaerobic HCDC process is based on a pH-stat principle where the provision of electron acceptor (HNO3) is directly linked to denitrification-driven increases in pH. Here, the provision of electron acceptor, carbon source/electron donor, and minerals must be carefully balanced to avoid limitations, toxic effects, or imbalances that could promote the accumulation of byproducts such as polyhydroxyalkanoates (PHAs) (Bedade et al., 2021; Shiloach & Fass, 2005). Another challenge is the accumulation of CO2, which reacts with water to form HCO3- and CO32-, lowering the pH, thereby masking the pH-increase associated with denitrification that would otherwise trigger substrate provision.
With this in mind, the aim was to develop an anaerobic bioprocess for high cell density cultivation by denitrification with balanced, pH-triggered substrate delivery, minimally impacted by CO2. A process-specific operation in the Lucullus® software enabled pH-controlled provision of HNO3 (and minerals) that was balanced with the provision of the carbon and nitrogen sources. Additionally, a dynamic pH setpoint and sparging flow rate were implemented to mitigate CO₂-driven acidification of the medium.
All bioreactor cultivations were performed using Paracoccus pantotrophus GB17, adapted to anaerobic conditions in a mineral basal medium containing 0.5 g L-1 MgSO4 · 7H2O, 0.1 g L-1 CaCl2 · 2H2O, 0.99 g L-1 KH2PO4, 7.45 g L-1 K2HPO4, and a mixture of trace elements. The medium was supplemented with glucose as the carbon source and electron donor, NO3- as the electron acceptor, and NH4+ as the nitrogen source.
High cell density cultivations were conducted in a Bionet® F1-3 bioreactor with a working volume of 3.0 L and a water circulation jacket for temperature control. The bioreactor setup was equipped with two process gas supplies (both connected to a central N2-line), cooling water, an agitator system, three fixed-speed pumps (acid, base, and antifoam reservoirs), and two variable-speed pumps (one of which was connected to the feed reservoir). The process was controlled by the Lucullus® 3.11.5 software from Securecell. The vessel was continuously sparged with N2 to maintain anoxic conditions throughout the cultivations.
The mineral basal medium is added to the vessel before sterilization. The acid (5 M HNO3 + minerals), base (1 M KOH), feed (2.97 M glucose, 1.42 M NH4NO3), and antifoam (Glanapon DB 870 from Busetti, diluted to 200x concentration) reservoirs were filled and connected to the bioreactor. An anaerobic pre-culture was used as inoculum for starting each cultivation.
pH was continuously monitored using an external pH sensor (Hamilton EasyFerm Bio PHI Arc 325). The off-gas was directed to the Bionet® bBreath-1 for CO2 (nondispersive infrared sensor) and O2 (zirconia dioxide ceramic electrolyte with gas permeable platinum electrodes) measurements, as well as a robotized incubation system (Molstad et al., 2007) for monitoring NO and N2O. Throughout the cultivations, liquid samples were extracted for offline measurements of glucose (HPLC), cell density (OD660), and NO2- (LCK342), NO3- (LCK340), and NH4+ (LCK340) using Hach Lange DR3900.
The Lucullus® 3.11.5 software from Securecell was used for data logging and advanced process control of the bioreactor processes. A Lucullus Operation was specifically designed to facilitate the pH-stat fed-batch culturing approach (Figure 2).
The Operation is initiated with a user-input block, where the setpoints for temperature and stirring are assigned, in addition to several critical process variables used in various calculations throughout the process, including initial pH, k-value (ratio between provided feed versus provided acid), initial volume, and parameters used for temperature, CO2, and sparging control. Then a preparation phase follows, where the temperature control, stirring, and sparging with N2 are initiated. When the reactor is anoxic and the temperature has been stabilized (both checked by Lucullus), the user is asked to inoculate the bioreactor.
Equation 1 is defined in the Operation so that the pH setpoint is adjusted based on the off-gas concentration of CO2 dynamically during the process (Figure 3). Here, ph_sp is the pH setpoint, PH_0 is the intrinsic pH of the medium, offg_co2_pv is the measured concentration of CO2 in the off-gas, D_CO2 and D_HNO3 are empirically determined values for the pH depression per unit of CO2 and HNO3, respectively, and SP_HNO3 is the target HNO3 concentration in the medium.
The delivery of feed must be balanced with the electron acceptor provided via the acid to prevent accumulation of storage polymers or denitrification intermediates, of which several are toxic at high concentrations. This is achieved through a control loop (Figure 4, left regulation loop) that turns on the feed pump when the total volume of feed injected is less than the volume of acid injected times the k-value (the ratio between the two).
The sparging flow rate is controlled by a control loop to keep the CO2 in the off-gas within an acceptable range (Figure 4, right control loop). When the CO2 concentration in the off-gas is higher than the user-defined high threshold, the sparging flow rate is increased in small increments until the CO2 concentration in the off-gas is reduced. Similarly, the sparging flow rate is reduced if the CO2 concentration is below the low threshold.
An accurate estimate of the total liquid volume in the bioreactor is required constantly throughout the process, as other calculations depend on it. This is achieved by implementing Equation 2, which calculates the total volume (V_TOT) continuously, based on the initial volume in the vessel (V_INI), the injected volume from each of the reservoirs (pmp_acid_tot, pmp_af_tot, pmp_base_tot, pmp_feed2_tot, and pmp_feed_tot), and the cumulative user injections (V_ADD_CUM) and withdrawals (V_SMPL_CUM) from the vessel.
Using the Operation described above, several high cell density cultivations with P. pantotrophus have been performed without requiring manual intervention (Figure 5). Concentrations exceeding 60 g dry weight L⁻¹ have been consistently achieved, both through repeated cycles of dilution with fresh medium and by maintaining high cell density for over 300 hours. The resulting biomass has an average protein content of 75% with a favourable amino acid profile. No fermentation products or toxic compounds accumulate in the reactor liquid, as verified through HPLC, HS-GC, and bioassays assessing the growth of fresh cells in spent reactor liquid (Maråk, 2025).
Although this has demonstrated the feasibility of anaerobic high cell density cultivations with good yields, the growth rate is consistently lower than expected based on low-density cultures. It is suspected that cells experience periodic starvation of electron acceptors due to a CO₂-mediated pH lag, which delays the provision of HNO₃ despite the implementation of a CO₂-dependent dynamic pH setpoint and N₂-sparging. This hypothesis is supported by in silico modelling of the pH, CO2, and NO3- kinetics.
Current efforts focus on improving production rates through changes in organism selection, medium composition and supply, and optimization of process parameters. In addition, adjustments to the Operation are being explored to further mitigate the CO₂-mediated pH lag and prevent periods of starvation.
Overall, these results demonstrate that anaerobic high cell density cultivation is feasible, routinely achieving high biomass concentrations with yields consistent with substrate input. The biomass has a high protein content, with no observed accumulation of byproducts or fermentation intermediates. Furthermore, it has shown potential as a protein source in fish feed trials with Atlantic salmon and for in vitro meat cultivation (Maråk, 2025).
The process currently relies on glucose as the carbon source. However, the great metabolic diversity of denitrifying organisms facilitates the use of more sustainable carbon sources, such as green methanol. With further optimization, this process has the potential to become a scalable, oxygen-independent platform for sustainable protein production. Its flexibility in feedstock and applicability across food and feed sectors make it a compelling candidate for future biomanufacturing efforts.
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Key Takeaways
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The anaerobic pH-stat Operation was developed and optimized by Lucullus® Application Specialist, Rowin Timmermans from Securecell, based on the requirements and feedback from Marte Mølsæter Maråk, Ph. D. student at the Microbial Ecology and Physiology group (MEP) at NMBU.