Konstantins Dubencovs

Crypthecodinium cohnii is a marine microalga that can accumulate high amounts of polyunsaturated fatty acids (PUFAs) and thus replace conventional routes of fish oil production. They are associated with the destruction of marine resources and multiple downstream/purification complications. The major drawbacks of using C. cohnii for industrial-scale production are associated with low PUFA productivity. One of the means of increasing the PUFA synthesis rate is to maintain the medium component concentrations at optimal values throughout cultivation, thus increasing PUFA production efficiency, which can result in the successful transfer of the process to pilot and/or industrial scale. The goal of the present research was to develop techniques for increasing the efficiency of PUFA production via C. cohnii cultivation. Multiple experiments were carried out to test and fine-tune the cultivation medium composition and oxygen transfer factors. The biomass yields from individual components, yeast extract, sea salts, and glucose amounted to 5.5, 0.65, and 0.61 g·g⁻¹, respectively. C. cohnii cell susceptibility to mechanical damage was experimentally evaluated. Power inputs of 3 did not seem to promote cell destruction when Pitched-blade impellers were used. The obtained cultivation conditions were shown to be efficient in terms of increasing the biomass productivity and the omega-3 fatty acid content in C. cohnii. By using the applied methods, the maximal biomass productivity reached 8.0 g·L⁻¹·day⁻¹, while the highest obtained biomass concentration reached 110 g·L⁻¹. A steady increase in the concentration of PUFAs during cultivation was observed from the FTIR data.

Soy leghemoglobin (LegH) has been gaining interest over the last years as an efficient flavor and aroma compound in plant-based meat substitutes. Hence, in the following article, we demonstrate the methods for LegH production using a recombinant Komagataella phaffii strain. Multiple fed-batch fermentation with an alternative to a BSM medium, where glucose was used as the main carbon source, was implemented and the growth kinetics, e.g., a maximal specific biomass growth of 0.239 g·g⁻¹·h⁻¹, a biomass yield from the substrate of 0.298 g·g⁻¹, and a maximal specific substrate consumption rate of 0.81 g·g⁻¹·h⁻¹ were identified. Leghemoglobin production resulted in a yield of 0.513 mg·gDCW⁻¹, while the highest biomass density achieved in this study was 121.80 gDCW·L⁻¹. The applied medium that showed potential for additional optimization studies, which, in contrast to BSM, made it possible to separate pH control from nitrogen supply, does not affect medium turbidity measurements and does not induce metabolite synthesis during yeast biomass growth.

A model predictive control (MPC) method was investigated as a route to optimize and control the growth of E. coli BL21 (DE3) and biosynthesis of two different recombinant proteins (nerve growth factor NGF and coat protein of bacteriophage Qβ (Qβ-CP)). To determine the target trajectory for the E. coli cultivation process and estimate the model parameters, the off-line run-to-run optimization method was used. The proven method allowed us to successfully control the growth of microbial biomass, with a deviation of 6–12% from the target trajectory. It was proven that it is possible to obtain a “Golden Batch” profile for the implementation of MPC using datasets from only four to eight fermentation runs. The method showed its robustness when the cultivation of E. coli was carried out with two different titrant supply control systems—volumetric and gravimetric. Furthermore, the MPC method exhibited high adaptability, reliability, and resistance to various types of disturbances. MPC proved to be a reliable and effective method for controlling the cultivation and recombinant protein biosynthesis of fast-growing microorganisms such as E. coli.

Soy leghemoglobin (LegH) has been gaining interest over the last years as an efficient flavor and aroma compound in plant-based meat substitutes. Hence, in the following article we demonstrate the methods for LegH production using a recombinant Komagataella phaffii strain. Multiple fed-batch fermentation with an alternative to BSM medium, where glucose was used as the main carbon source, were implemented and the growth kinetics, e.g., maximal specific biomass growth - 0.239 g·g−1·h−1, biomass yield from substrate - 0.298 g·g−1 and maximal specific substrate consumption rate - 0.81 g·g−1·h−1, were identified. Leghemoglobin production resulted in a yield of 0.513 mg·gDCW 1, while the highest biomass density achieved in the study was 121.80 gDCW·L 1. The applied medium showed potential for additional optimization studies, as in contrast to BSM, made it possible to separate pH control from nitrogen supply, does not affect medium turbidity measurements and does not induce metabolite synthesis during yeast biomass growth.

The biorefinery industry is witnessing a transition from fossil and chemical-based processes to more sustainable practices, with a growing emphasis on using renewable resources. Sophorolipids, a promising group of biosurfactants, present a viable substitute for conventionally produced surfactants. This study focuses on microbial fermentation using yeast and lipid substrate for sophorolipid production. The life cycle assessment (LCA) methodology was employed to identify environmental hotspots of the process and to assess the environmental benefits resulting from the replacement of raw rapeseed cooking oil (base scenario) with waste cooking oil, reduction of process electricity consumption, and increased sophorolipid yield. By compiling scenarios with the lowest environmental impact, a best-case scenario was created. The results revealed that the environmental impact of sophorolipid production could be reduced by 50% in the best-case scenario compared to the base scenario. This research provides valuable insights into the environmental optimization of the fermentation process and through the application of LCA highlights the potential for the reduction of negative environmental impact of sophorolipid production, contributing to the ongoing transition from petroleum oil and petrochemical refineries to sustainable biorefineries.

The fed-batch cultivation is in many ways a benchmark for fermentation processes, and it has been an attractive choice for the biotechnological production of various products in the past decades. The majority of biopharmaceuticals that are presently undergoing clinical trials or are available on the market are manufactured through fed-batch fermentations. A crucial process parameter in fed-batch cultivations is the substrate feed rate, which directly influences the overall process productivity, product quality and process repeatability; henceforth, effective control of this parameter is imperative for a successful fed-batch fermentation process. Two distinct control strategies can be distinguished—open-loop and closed-loop (feedback) control. Each of these methods has its own set of benefits, limitations and suitability for specific bioprocesses. This article surveys and compares the most popular open- and closed-loop methods for substrate feed rate control in fed-batch fermentations. Emphasis is placed on model-predictive feed rate control (MPC)—a stand out among other methods that offers a promising application perspective. The authors also demonstrate a practical example of the implementation of a robust, flexible MPC solution that is suitable for various cultures and runs on standard computer hardware, thus overcoming one of the main reported MPC drawbacks—high computational requirements.

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h⁻¹, while the biomass yield from substrate reached 0.601 g·g⁻¹. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h⁻¹) and yield from the substrate (0.234 g·g⁻¹). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.

The Kluyveromyces marxianus yeast recently has gained considerable attention due to its applicability in high-value-added product manufacturing. In order to intensify the biosynthesis rate of a target product, reaching high biomass concentrations in the reaction medium is mandatory. Fed-batch processes are an attractive and efficient way how to achieve high cell densities. However, depending on the physiology of the particular microbial strain, an optimal media composition should be used to avoid by-product synthesis and, subsequently, a decrease in overall process effi-ciency. Thus, the aim of the present study was to optimise the synthetic growth medium and feeding solution compositions (in terms of carbon, nitrogen, phosphorous, magnesium, and calcium concentrations) for high cell density K. marxianus fed‑batch cultivations. Additionally, the biomass yields from the vitamin mixture and other macro/microelements were identified. A model predictive control algorithm was successfully applied for a fed-batch cultivation control. Biomass growth and substrate consumption kinetics were compared with the mathematical model predictions. Finally, 2‑phenylethanol biosynthesis was induced and its productivity was estimated. The determined optimal macronutrient ratio for K. marxianus biomass growth was identified as C:N:P = 1:0.07:0.011. The maximal attained yeast biomass concentration was close to 70 g·L^-1 and the 2-PE biosynthesis rate was 0.372 g·L⁻¹·h⁻¹, with a yield of 74% from 2-phenylalanine.