Automated bioprocess feedback operation in a high throughput facility via the integration of a mobile robotic lab assistant

Biotechnological processes development is challenging due to the sheer variety of process parameters. For efficient upstream development parallel cultivation systems have proven to reduce costs and associated timelines successfully, while offering excellent process control. However, the degree of automation of such small scale systems is comparably low and necessary sample analysis requires manual steps. Although the subsequent analysis can be performed in a high-throughput manner, the integration of analytic devices remains challenging. Especially, when cultivation and analysis laboratories are spatially separated. Mobile robots offer a potential solution, but the implementation in research laboratories is not widely adopted. Our approach demonstrates the integration of a small scale cultivation system into a liquid handling station for an automated sample procedure. The samples are transferred via a mobile robotic lab assistant and subsequently analysed by a high-throughput analyzer. The process data is stored in a centralized database. The mobile robotic workflow guarantees a flexible solution for device integration and facilitates automation. Restrictions regarding spatial separation of devices are circumvented, enabling a modular platform throughout different laboratories. The presented cultivation platform is evaluated based on industrial relevant E. coli BW25113 high cell density fed-batch cultivation. Here its suitability for accelerating bioprocess development is proven. The necessary magnesium addition for reaching high cell densities in mineral salt medium is automated via a feedback operation loop. The feedback operation loop demonstrates the possibility for advanced control options. This study sets the foundation for a fully integrated facility with different cultivation scales sharing the same data infrastructure, where the mobile robotic lab assistant physically connects the devices.

Enhanced l-Malic Acid Production by Aspergillus oryzae DSM 1863 Using Repeated-Batch Cultivation

l-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of l-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 fixation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake flasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in significant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L l-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for five exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments confirmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major influencing factors and thereby delivers important insights into natural l-malic acid production.

Effects of Caprolactam Wastewater on Algal Growth and Nutrients Removal by Arthrospira platensis

Caprolactam wastewater (WCP), which is generated during the production of caprolactam, contains high contents of NO3− and inorganic P and is considered to be difficult to treat. In this study, Arthrospira platensis was used to remove N and P from WCP. Culture conditions and wastewater addition were optimized to relieve the inhibition effects of WCP. The results show that A. platensis growth and photosynthetic activity were inhibited depending on WCP concentrations. The inhibition rates were enhanced as the culture time increased under batch mode. However, the fed-batch mode significantly minimized the negative impact on A. platensis, which is beneficial for removing N and P from WCP by Arthrospira. After 10 d of cultivation of A. platensis in a 25 L circular photobioreactor in fed-batch addition of WCP (1.25% mixed WCP (v/v) each day), the average biomass productivity reached 17.48 g/(m2·d), the maximum protein content was 69.93%, and the N and P removal ratios were 100%. The accumulation effect of WCP inhibition on algal growth was not observed under this culture condition. Fed-batch cultivation of A. platensis is a promising way for bioremediation of WCP with high N and P removal efficiencies and high value-added biomass production.

Optimizing a High Performing Multiplex-CRISPRi P. putida strain with Integrated Metabolomics and 13C-Metabolic Flux Analyses

Microbial cell factory development often faces bottlenecks after initial rounds of design-build-test-learn (DBTL) cycles as engineered producers respond unpredictably to further genetic modifications. Thus, deciphering metabolic flux and correcting bottlenecks are key components of DBTL cycles. Here, a 14-gene edited Pseudomonas putida KT2440 strain for heterologous indigoidine production was examined using both 13C-metabolic flux analysis (13C-MFA) and metabolite measurements. The results indicated the conservation of the cyclic Entner-Doudoroff (ED)-EMP pathway flux, downregulation of the TCA cycle and pyruvate shunt, and glyoxylate shunt activation. At the metabolite level, the CRISPR/dCpf1-interference mediated multiplex repression decreased gluconate/2-ketogluconate secretion and altered several intracellular TCA metabolite concentrations, leading to succinate overflow. Further strain engineering based on the metabolic knowledge first employed an optimal ribosome binding site (RBS) to achieve stronger product-substrate growth coupling (1.6-fold increase). Then, deletion strains were constructed using ssDNA recombineering. Of the five strains tested, deletion of the PHA operon (ΔphaAZC-IID) resulted in a 2.2-fold increase in growth phase production compared to the optimal RBS construct. After 72 h of batch cultivation, the ΔphaAZC-IID strain had 1.5-fold and 1.8-fold increases of indigoidine titer compared to the improved RBS construct and the original strain, respectively. Overall, the findings provided actionable DBTL targets as well as insights into physiological responses and flux buffering when new recombineering tools were used for engineering P. putida KT2440.

Photoautotrophic and Mixotrophic Cultivation of Polyhydroxyalkanoate-Accumulating Microalgae Consortia Selected under Nitrogen and Phosphate Limitation

Microalgae consortia were photoautotrophically cultivated in sequencing batch photobioreactors (SBPRs) with an alteration of the normal growth and starvation (nutrient limitation) phases to select consortia capable of polyhydroxyalkanoate (PHA) accumulation. At the steady state of SBPR operation, the obtained microalgae consortia, selected under nitrogen and phosphate limitation, accumulated up to 11.38% and 10.24% of PHA in their biomass, which was identified as poly(3-hydroxybutyrate) (P3HB). Photoautotrophic and mixotrophic batch cultivation of the selected microalgae consortia was conducted to investigate the potential of biomass and PHA production. Sugar source supplementation enhanced the biomass and PHA production, with the highest PHA contents of 10.94 and 6.2%, and cumulative PHA productions of 100 and 130 mg/L, with this being achieved with sugarcane juice under nitrogen and phosphate limitation, respectively. The analysis of other macromolecules during batch cultivation indicated a high content of carbohydrates and lipids under nitrogen limitation, while higher protein contents were detected under phosphate limitation. These results recommended the selected microalgae consortia as potential tools for PHA and bioresource production. The mixed-culture non-sterile cultivation system developed in this study provides valuable information for large-scale microalgal PHA production process development following the biorefinery concept.

Predicting black soldier fly larvae biomass and methionine accumulation using a kinetic model for batch cultivation and improving system performance using semi-batch cultivation

Global demand for poultry and associated feed are projected to double over the next 30 years. Insect meal is a sustainable alternative to traditional feeds when produced on low-value high-volume agricultural byproducts. Black soldier fly (BSF) larvae (Hermetia illucens L.) are high in protein and contain methionine, an essential amino acid that is critical to poultry health. BSF larvae can be grown on many organic residues, however, larvae growth and quality vary based on feedstock and cultivation processes. Experiments were completed to monitor temporal changes in BSF larvae growth and composition using almond hulls as a growth substrate under batch and semi-batch processes and with varying substrate carbon to nitrogen ratio (C/N). A logistic kinetic growth model was developed to predict larval biomass and methionine accumulations during batch production. Estimated ranges of model parameters for larvae maximum specific growth rate and carrying capacity were 0.017–0.021 h−1 and 9.7–10.7 g larvae kg−1 hulls dry weight, respectively. Methionine content in larvae increased from 11.1 to 17.1 g kg−1 dry weight over a 30-day batch incubation period. Larvae-specific growth and yield increased by 168% and 268%, respectively, when cultivated in a semi-batch compared to a batch process. Increasing C/N ratio from 26 to 40 increased density of methionine content in larvae per unit feedstock by 25%. The findings demonstrate a logistic model can predict larvae biomass accumulation, harvest time can achieve specific methionine contents, and a semi-batch process is more favorable for larvae biomass accumulation compared to a batch process.

Heterologous Expression and Characterization of a High-Efficiency Chitosanase From Bacillus mojavensis SY1 Suitable for Production of Chitosan Oligosaccharides

Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Heterotrophically Ultrahigh-Cell-Density Cultivation of a High Protein-Yielding Unicellular Alga Chlorella With a Novel Nitrogen-Supply Strategy

The unicellular green alga Chlorella is an ideal protein source. However, the high production cost and low production capability of the current main photoautotrophic culture mode limit its application especially as an alternative protein source for food and feed, which might be overcome through high-cell-density cultivation in fermenters. In this study, a Chlorella sorokiniana strain CMBB276 with high protein content was selected from five Chlorella strains by comprehensive evaluation of their growth rates, protein contents, and yields. The optimal cultural temperature, pH, and mole ratio of carbon and nitrogen (C/N) for C. sorokiniana CMBB276 growth were found to be 30°C, 6.5, and 18, respectively. Ammonium chloride was proved to be the best nitrogen (N) source for C. sorokiniana CMBB276 growth, whereas growth inhibition caused by the accumulation of salts was observed under fed-batch cultivation when maintaining a constant C/N ratio of 18 by controlling pH with sodium hydroxide solution. By simultaneously reducing the concentration of ammonium chloride in the feeding medium and controlling pH with ammonium hydroxide, we finally achieved the ultrahigh-cell-density cultivation of C. sorokiniana CMBB276. The highest biomass concentration and protein yield reached 232 and 86.55 g l−1, respectively, showing the great potential of culturing C. sorokiniana CMBB276 in fermenters for economic and large-scale protein source production.

New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2–2.6 g/L EPS1 and 1.90–2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 μM Vitamin C Equivalent Antioxidant Capacity (versus 135 μM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.