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Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L -1 of each alkane (dodecane and hexadecane), and 2 g·L -1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L -1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L -1 ) and lipids concentrations of 3.4 g·L -1 and 4.3 g·L -1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica., (© 2024. The Author(s).)

ε-Poly-L-lysine (ε-PL) is an amino acid homopolymer with diverse potential applications in the food, pharmaceutical and cosmetic industries. To improve its biomanufacturing efficiency, strain engineering and bioprocess optimization were combined in this study. Firstly, a cocktail strain breeding strategy was employed to generate a ε-PL high-production mutant, Streptomyces albulus GS114, with enhanced L-lysine uptake capability. Subsequently, the L-lysine feeding conditions during fed-batch fermentation were systematically optimized to improve the L-lysine supply, resulting in ε-PL production reaching 73.1 ± 1.4 g/L in 5 L bioreactor. Finally, an engineered strain, S. albulus L2, with enhanced uptake capability and polymerization ability of L-lysine was constructed, achieving ε-PL production of 81.4 ± 5.2 g/L by fed-batch fermentation. This represents the highest reported production of ε-PL to date. This study provided an efficient production strategy for ε-PL and valuable insights into the high-value utilization of L-lysine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Sponge iron (SFe) coupled with a sludge system has great potential for improving biological denitrification; however, the underlying mechanism is not yet fully understood. In this study, the denitrification performance and microbial characteristics of ordinary sludge and SFe-sludge systems were investigated. Overall, the SFe-sludge reactor had faster ammonium degradation rate (94.0 %) and less nitrate accumulation (1.5-53.3 times lower) than ordinary reactor during the complete operation cycle of sequencing batch reactors. The addition of SFe increased the activities of nitrate and nitrite reductases. The total relative abundance of autotrophic denitrifying bacteria (Acidovorax, Arenimonas, etc.) in the SFe-sludge system after 38 days of operation was found to be 10.6 % higher than that in the ordinary sludge reactor. The aerobic denitrifying bacteria (Dokdonella, Phaeodactylibacter, etc.) was 5.3 % higher than ordinary sludge. The SFe-sludge system improved denitrification by enriching autotrophic/aerobic denitrifying bacteria in low carbon-to-nitrogen ratio wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Recently, there has been notable interest in researching and industrially producing medium-chain carboxylic acids (MCCAs) like n-caproate and n-caprylate via chain elongation process. This study presents a comprehensive assessment of the behavior and MCCA production profiles of Clostridium kluyveri in batch and continuous modes, at different ethanol:acetate molar ratios (1.5:1, 3.5:1 and 5.5:1). The highest n-caproate concentration, 12.9 ± 0.67 g/L (92.9 ± 1.39 % MCCA selectivity), was achieved in batch mode at a 3.5:1 ratio. Interestingly, higher ratios favored batch mode selectivity over continuous mode when this was equal or higher to 3.5:1. Steady state operation yielded the highest n-caproate (9.5 ± 0.13 g/L) and n-caprylate (0.35 ± 0.020 g/L) concentrations at the 3.5:1 ratio. Increased ethanol:acetate ratios led to a higher excessive ethanol oxidation (EEO) in both operational modes, potentially limiting n-caproate production and selectivity, especially at the 5.5:1 ratio. Overall, this study reports the efficient MCCA production of both batch and continuous modes by C. kluyveri., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

d-Arabitol, an alternative sweetener to sugar, has low calorie content, high sweetness, low glycemic index, and insulin resistance-improving ability. In this study, d-arabitol-producing yeast strains were isolated from various commercial types of miso, and strain Gz-5 was selected among these strains. Phylogenetic tree analysis of the internal transcribed spacer sequence revealed that strain Gz-5 was distinct from Zygosaccharomyces rouxii, a major fermenting yeast of miso. The strain, identified as Zygosaccharomyces sp. Gz-5, grew better than other Z. rouxii in 150 g/L NaCl and produced 114 g/L d-arabitol from 295 g/L glucose in a batch culture for 8 days (0.386 g/g-consumed glucose). In a fed-batch culture, the yeast produced 133 g/L d-arabitol for 14 days, and the total d-arabitol amount increased by 1.75-fold. These results indicated that Zygosaccharomyces sp. Gz-5, a non-genetically modified strain, has excellent potential for the industrial production of d-arabitol., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Responsible use of natural resources and waste reduction are key concepts in bioeconomy. This study demonstrates that agro-food derived-biomasses from the Italian food industry, such as crude glycerol and cheese whey permeate (CWP), can be combined in a high-density fed-batch culture to produce a recombinant β-galactosidase from Marinomonas sp. ef1 (M-βGal). In a small-scale process (1.5 L) using 250 mL of crude glycerol and 300 mL of lactose-rich CWP, approximately 2000 kU of recombinant M-βGal were successfully produced along with 30 g of galactose accumulated in the culture medium. The purified M-βGal exhibited high hydrolysis efficiency in lactose-rich matrices, with hydrolysis yields of 82 % in skimmed milk at 4 °C and 94 % in CWP at 50 °C, highlighting its biotechnological potential. This approach demonstrates the effective use of crude glycerol and CWP in sustainable and cost-effective high-density Escherichia coli cultures, potentially applicable to recombinant production of various proteins., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stefania Brocca reports financial support was provided by Cariplo Foundation. Marco Mangiagalli reports financial support was provided by Cariplo Foundation. Antonino Natalello reports financial support was provided by Cariplo Foundation. Marina Lotti reports financial support was provided by Cariplo Foundation. Greta Bianchi reports financial support was provided by Cariplo Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes., (© 2024. The Author(s).)

The simulations and predictions obtained from mathematical models of bioprocesses conducted by microorganisms are not overvalued. Mechanistic models are bringing a better process understanding and the possibility of simulating unmeasurable variables. The Dynamic Energy Budget (DEB) model is an energy balance that can be formulated for any living organism and can be classified as a structured model. In this study, the DEB model was used to describe E. coli growth in a batch reactor in carbon and nitrogen substrate limitation conditions. The DEB model provides a possibility to follow the changes in the microbes' cells including their elemental composition and content of some important cell ingredients in different growth phases in substrate limitation conditions which makes it more informative compared to Monod's model. The model can be used as an optimal choice between Monod-like models and flux-based approaches. KEY POINTS: • The DEB model can be used to catch changes in elemental composition of E. coli • Bacteria batch culture growth phases can be explained by the DEB model • The DEB model is more informative compared to Monod's based models., (© 2024. The Author(s).)

Maximizing production potential of recombinant proteins such as monoclonal antibodies (mAbs) in Chinese Hamster Ovary (CHO) cells is a key enabler of reducing cost of goods of biologics. In this study, we explored various strategies to utilize adenosine mediated effects in biologics manufacturing processes. Results show that supplementation of adenosine increases specific productivity by up to two-fold while also arresting cell growth. Introducing adenosine in intensified perfusion processes in a biphasic manner significantly enhanced overall productivity. Interestingly, adenosine effect was observed to be dependent on the cell growth state. Using specific receptor antagonists and inhibitors, we identified that ENTs (primarily Slc29a1) mediate the uptake of adenosine in CHO cell cultures. Transcriptomics data showed an inverse correlation between Slc29a1 expression levels and peak viable cell densities. Data suggests that in fed-batch cultures, adenosine can be produced extracellularly. Blocking Slc29a1 using ENT inhibitors such as DZD and DP alone or in combination with CD73 inhibitor, PSB12379, resulted in a twofold increase in peak viable cell densities as well as productivities in fed batch - a novel strategy that can be applied to biologics manufacturing processes. This is the first study that suggests that adenosine production/accumulation in CHO cell cultures can potentially regulate the transition of CHO cells from exponential to stationary phase. We also demonstrate strategies to leverage this regulatory mechanism to maximize the productivity potential of biologics manufacturing processes., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interests., (Copyright © 2024 Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Published by Elsevier B.V. All rights reserved.)

Orotic acid is widely used in healthcare and cosmetic industries. However, orotic acid-producing microorganisms are auxotrophic, which results in inefficient microbial production. Herein, a plasmid-free, uninduced, non-auxotrophic orotic acid hyperproducer was constructed from Escherichia coli W3110. Initially, the orotic acid degradation pathway was blocked and the carbamoyl phosphate supply was enriched. Subsequently, pyr operon from Bacillus subtilis F126 was heterologously expressed and precursors' supply was optimized. Thereafter, pyrE was dynamically regulated to reconstruct the non-auxotrophic pathway. Employing fed-batch cultivation, orotic acid titer, yield, and productivity of strain Ora21 reached 182.5 g/L, 0.58 g/g, and 3.80 g/L/h, respectively, the highest levels reported so far. Finally, a novel "Chaos to Order Cycles (COC)" fermentation was developed, which effectively increased the yield to 0.63 g/g. This research is a remarkable achievement in orotic acid production by microbial fermentation and has vast potential for industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Addition of statistically optimized concentration of electron acceptor, propionic acid (1.2 g/L) at different cultivation times (0 h, 14.86 h and 19 h) during batch cultivation of B. thuringiensis in mixed substrate (glucose and glycerol) featured production of 8 g/L of biomass and 3.57 g/L of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) containing 0.805 g/L of 3-hydroxyvalerate concentration. Successful scale up of batch cultivation from 7 L to a 70 L bioreactor was, thereafter, achieved using power/volume (P/V) criteria with maximum PHBV and biomass concentration of 3.57 g/L and 7.15 g/L respectively. Characterization of PHBV so produced was carried out using NMR, FTIR, DSC and TGA to elucidate its structure, thermal properties and stability to map their applications in society. These findings highlight the potential of the optimized batch cultivation and scale-up process in producing PHBV emphasizing its relevance in sustainable biopolymer production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast., Results: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ., Conclusions: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii., (© 2024. The Author(s).)

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.

A novel 70 L composite tubular photo-bioreactor was constructed, and its photo-fermentation hydrogen production characteristics of batch and continuous modes were investigated with glucose as the substrate in an outdoor environment. In the batch fermentation stage, the hydrogen production rate peaked at 37.6 mL H 2 /(L·h) accompanied by a high hydrogen yield of 7 mol H 2 /mol glucose. The daytime light conversion efficiency is 4 %, with 37 % of light energy from the sun. An optimal hydraulic retention time of 5 d was identified during continuous photo-fermentation. Under this condition, the stability of the cell concentration is maintained and more electrons can be driven to the hydrogen generation pathway while attaining a hydrogen production rate of 20.7 ± 0.9 mL H 2 /(L·h). The changes of biomass, volatile fatty acids concentration and ion concentration during fermentation were analyzed. Continuous hydrogen production by composite tubular photo-bioreactor offers new ideas for the large-scale deployment of photobiological hydrogen production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study explores the ability of methanotrophs to convert biogas into biopolymers, addressing H 2 S as a limitation in the utilization of biogas as a carbon source for bioconversion. Transcriptomic analysis was conducted to understand the growth and changes in the expression patterns of Type I and II methanotrophs under varying H 2 S concentrations. Results suggested that Type II methanotrophs can possess a native H 2 S utilization pathway. Both Type I and II methanotrophs were evaluated for their growth and polyhydroxybutyrate (PHB) production from biogas. Methylocystis sp. MJC1 and Methylocystis sp. OK1 exhibited a maximum biomass production of 4.0 and 4.5 gDCW/L, respectively, in fed-batch culture, aligning with the transcriptome data. Furthermore, Methylocystis sp. MJC1 produced 2.9 g PHB/L from biogas through gas fermentation. These findings underscore biogas-based biotechnology as an innovative solution for environmental and industrial challenges with further optimization and productivity enhancement research expected to broaden the potential in this field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The β-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the β-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the β-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity., (© 2024. The Author(s).)

During the epoch of sustainable development, leveraging cellular systems for production of diverse chemicals via fermentation has garnered attention. Industrial fermentation, extending beyond strain efficiency and optimal conditions, necessitates a profound understanding of microorganism growth characteristics. Specific growth rate (SGR) is designated as a key variable due to its influence on cellular physiology, product synthesis rates and end-product quality. Despite its significance, the lack of real-time measurements and robust control systems hampers SGR control strategy implementation. The narrative in this contribution delves into the challenges associated with the SGR control and presents perspectives on various control strategies, integration of soft-sensors for real-time measurement and control of SGR. The discussion highlights practical and simple SGR control schemes, suggesting their seamless integration into industrial fermenters. Recommendations provided aim to propose new algorithms accommodating mechanistic and data-driven modelling for enhanced progress in industrial fermentation in the context of sustainable bioprocessing., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed., Results: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall., Conclusions: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced., (© 2024. The Author(s).)

Background: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products., Results: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane., Conclusions: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation., (© 2024. The Author(s).)

Perfusion cell culture has been gaining increasing popularity for biologics manufacturing due to benefits such as smaller footprint, increased productivity, consistent product quality and manufacturing flexibility, cost savings, and so forth. Process Analytics Technologies tools are highly desirable for effective monitoring and control of long-running perfusion processes. Raman has been widely investigated for monitoring and control of traditional fed batch cell culture process. However, implementation of Raman for perfusion cell culture has been very limited mainly due to challenges with high-cell density and long running times during perfusion which cause extremely high fluorescence interference to Raman spectra and consequently it is exceedingly difficult to develop robust chemometrics models. In this work, a platform based on Raman measurement of permeate has been proposed for effective analysis of perfusion process. It has been demonstrated that this platform can effectively circumvent the fluorescence interference issue while providing rich and timely information about perfusion dynamics to enable efficient process monitoring and robust bioreactor feed control. With the highly consistent spectral data from cell-free sample matrix, development of chemometrics models can be greatly facilitated. Based on this platform, Raman models have been developed for good measurement of several analytes including glucose, lactate, glutamine, glutamate, and permeate titer. Performance of Raman models developed this way has been systematically evaluated and the models have shown good robustness against changes in perfusion scale and variations in permeate flowrate; thus models developed from small lab scale can be directly transferred for implementation in much larger scale of perfusion. With demonstrated robustness, this platform provides a reliable approach for automated glucose feed control in perfusion bioreactors. Glucose model developed from small lab scale has been successfully implemented for automated continuous glucose feed control of perfusion cell culture at much larger scale., (© 2024 Wiley Periodicals LLC.)

Tremella fuciformis polysaccharide (TFPS) is a natural mushroom mucopolysaccharide widely used in health foods, medical care, cosmetic and surgical materials. In this study, we developed an efficient strategy for the repeated batch production of highly bioactive TFPS from the agro-industrial residue cane molasses. Cane molasses contained 39.92 % sucrose (w/w), 6.36 % fructose and 3.53 % glucose, all of which could be utilized by T. fuciformis spores, whereas, the TFPS production efficiency only reached 0.74 g/L/d. Corn cobs proved to be the best immobilized carrier that could tightly absorb spores and significantly shorten the fermentation lag period. The average yield of TFPS in eight repeated batch culture was 5.52 g/L with a production efficiency of 2.04 g/L/d. The average fermentation cycle after optimization was reduced by 61.61 % compared with the initial conditions. Compared to glucose as a carbon source, cane molasses significantly increased the proportion of low-molecular-weight TFPS (TFPS-2) in total polysaccharides from 3.54 % to 17.25 % (w/w). Moreover, TFPS-2 exhibited potent antioxidant capacity against four free radicals (O 2 - , ABTS + , OH, and DPPH). In conclusion, this study lays the foundation for the efficient conversion of cane molasses and production of TFPS with high bioactivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

During the scale-up of biopharmaceutical production processes, insufficiently predictable performance losses may occur alongside gradients and heterogeneities. To overcome such performance losses, tools are required to explain, predict, and ultimately prohibit inconsistencies between laboratory and commercial scale. In this work, we performed CHO fed-batch cultivations in the single multicompartment bioreactor (SMCB), a new scale-down reactor system that offers new access to study large-scale heterogeneities in mammalian cell cultures. At volumetric power inputs of 20.4-1.5 W m -3 , large-scale characteristics like long mixing times and dissolved oxygen (DO) heterogeneities were mimicked in the SMCB. Compared to a reference bioreactor (REFB) set-up, the conditions in the SMCB provoked an increase in lactate accumulation of up to 87%, an increased glucose uptake, and reduced viable cell concentrations in the stationary phase. All are characteristic for large-scale performance. The unique possibility to distinguish between the effects of changing power inputs and observed heterogeneities provided new insights into the potential reasons for altered product quality attributes. Apparently, the degree of galactosylation in the evaluated glycan patterns changed primarily due to the different power inputs rather than the provoked heterogeneities. The SMCB system could serve as a potent tool to provide new insights into scale-up behavior and to predict cell line-specific drawbacks at an early stage of process development., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Phaffia rhodozyma is a basidiomycetous yeast characterized by its production of the carotenoid pigment astaxanthin, which holds high commercial value for its significance in aquaculture, cosmetics and as nutraceutics, and the UV-B-absorbing compound mycosporine-glutaminol-glucoside (MGG), which is of great biotechnological relevance for its incorporation into natural sunscreens. However, the industrial exploitation has been limited to the production of astaxanthin in small quantities. On the other hand, the accumulation of MGG in P. rhodozyma was recently reported and could add value to the simultaneous production of both metabolites. In this work, we obtain a mutant strain that overproduces both compounds, furthermore we determined how the accumulation of each is affected by the carbon-to-nitrogen ratio and six biotic and abiotic factors. The mutant obtained produces 159% more astaxanthin (470.1 μg g -1 ) and 220% more MGG (57.9 mg g -1 ) than the parental strain (295.8 μg g -1 and 26.2 mg g -1 respectively). Furthermore, we establish that the carotenoids accumulate during the exponential growth phase while MGG accumulates during the stationary phase. The carbon-to-nitrogen ratio affects each metabolite differently, high ratios favoring carotenoid accumulation while low ratios favoring MGG accumulation. Finally, the accumulation of both metabolites is stimulated only by photosynthetically active radiation and low concentrations of hydrogen peroxide. The mutant strain obtained is the first hyper-productive mutant capable of accumulating high concentrations of MGG and astaxanthin described to date. The characterization of how both compounds accumulate during growth and the factors that stimulate their accumulation, are the first steps toward the future commercial exploitation of strains for the simultaneous production of two biotechnologically important metabolites., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Intensified fed-batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed-batch cultivation. However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult-to-sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent-perfusion fed-batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi-continuous processes such as hybrid perfusion fed-batch with only early-stage perfusion, IPFB applies limited times of intermittent perfusion in the mid-to-late stage of production and still inherits bolus feedings on nonperfusion days as in a fed-batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra-intensified IPFB (UI-IPFB) was designed with a markedly elevated seeding density of 20-80 × 10 6 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI-IPFB, up to ~6 folds of traditional fed-batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad-based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities., (© 2023 Wiley Periodicals LLC.)

In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO-DHFR - and GS-CHO knockout (GS-KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO-DHFR - cells but extended culture duration and significantly increased antibody titers in GS-KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS-KO CHO cells in medium- to large-scale batches. In case of cordycepin, a derivative of adenosine, CHO-DHFR - cells required higher concentration of cordycepin than GS-KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO-DHFR - cell cultures without glucose feeding., (© 2023 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Chinese hamster ovary (CHO) cells are widely used for production of biologics including therapeutic monoclonal antibodies. Cell death in CHO cells is a significant factor in biopharmaceutical production, impacting both product yield and quality. Apoptosis has previously been described as the major form of cell death occurring in CHO cells in bioreactors. However, these studies were undertaken when less was known about non-apoptotic cell death pathways. Here, we report the occurrence of non-apoptotic cell death in an industrial antibody-producing CHO cell line during fed-batch culture. Under standard conditions, crucial markers of apoptosis were not observed despite a decrease in viability towards the end of the culture; only by increasing stress within the system did we observe caspase activation indicative of apoptosis. In contrast, markers of parthanatos and ferroptosis were observed during standard fed-batch culture, indicating that these non-apoptotic cell death pathways contribute to viability loss under these conditions. These findings pave the way for targeting non-conventional cell death pathways to improve viability and biologic production in CHO cells., (© 2023 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 10 8 TCID50 mL -1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 10 6 cells mL -1 , and was further improved to 1.1 × 10 9 TCID50 mL -1 in perfusion culture infected at 4.6 × 10 6 cells mL -1 . These titers are similar to or better than the previously reported best titer of 8.6 × 10 7 TCID50 mL -1 and 8.1 × 10 8 TCID50 mL -1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes., (© 2023 Wiley-VCH GmbH.)

End-product inhibition in pH-controlled batch cultures, is the major limiting factor for bacterial biomass formation in the starter culture industry as well as in many other biotechnological processes. Adaptive laboratory evolution (ALE) has emerged over the past decades as a powerful tool for phenotype optimization, but none of the existing ALE methods could select for improved end-product resistance. Therefore, we developed the stressostat (STress Resistance Evolution in Substrate Surplus) as a novel continuous ALE method. Stressostat cultivation applies end-product concentrations as constant evolutionary pressure on microorganisms in the presence of substrate surplus. In this study, we improved the lactate resistance of Lactococcus lactis FM03P in 35 days of stressostat cultivations. The lactate concentrations increased over time from 530 to 675 mM, indicating the successful selection for variants with improved lactate resistance. Thirty-four variants were isolated and grouped into four clusters based on their growth rates at high lactate concentrations. In the high-throughput screening without pH control, most isolated variants could grow at high lactate concentrations (870-928 mM), while the wild type was completely inhibited. The variants grew slower than wild type at low lactate media indicating possible evolutionary trade-off. However, in pH-controlled batch cultivations, most variants produced more biomass than the wild type. In conclusion, stressostat cultivation is a valuable method to obtain L. lactis variants with improved end-product resistance and further characterization is needed to elucidate underlying resistance mechanisms and potential industrial applications., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Oscar van Mastrigt reports financial support was provided by Dutch Research Council. Sylviani Hartono reports financial support was provided by Dutch Research Council. Oscar van Mastrigt reports financial support was provided by Arla Foods amba. Sylviani Hartono reports financial support was provided by Arla Foods amba. Oscar van Mastrigt reports financial support was provided by dsm-firmenich. Sylviani Hartono reports financial support was provided by dsm-firmenich., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Nicotinamide mononucleotide (NMN) subsists in all living organisms and has drawn tremendous attention as a nutraceutical and pharmaceutical product for several diseases such as Alzheimer's, cancer, aging, and vascular dysfunction. Here, NMN was produced intracellularly in a high cell density bioreactor using an engineered Escherichiacoli strain via exponential feeding of co-substrates. Fed-batch culture via exponential feeding of co-substrate (glucose) and continuous feeding of substrate (nicotinamide) were performed using different cumulative nicotinamide concentrations. The highest concentration of 19.3 g/L NMN with a dry cell weight of 117 g/L was acquired from a cumulative nicotinamide concentration of 7.2 g/L with a conversion of 98 % from nicotinamide in 28 h. Further, liquid chromatography-mass spectrometry analysis validated the NMN production. This approach will be beneficial in achieving simultaneously low cost and ensuring high quality and quantity of NMN production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

A majority of the biotherapeutics industry today relies on the manufacturing of monoclonal antibodies from Chinese hamster ovary (CHO) cells, yet challenges remain with maintaining consistent product quality from high-producing cell lines. Previous studies report the impact of individual trace metal supplemental on CHO cells, and thus, the combinatorial effects of these metals could be leveraged to improve bioprocesses further. A three-level factorial experimental design was performed in fed-batch shake flasks to evaluate the impact of time wise addition of individual or combined trace metals (zinc and copper) on CHO cell culture performance. Correlations among each factor (experimental parameters) and response variables (changes in cell culture performance) were examined based on their significance and goodness of fit to a partial least square's regression model. The model indicated that zinc concentration and time of addition counter-influence peak viable cell density and antibody production. Meanwhile, early copper supplementation influenced late-stage ROS activity in a dose-dependent manner likely by alleviating cellular oxidative stress. Regression coefficients indicated that combined metal addition had less significant impact on titer and specific productivity compared to zinc addition alone, although titer increased the most under combined metal addition. Glycan analysis showed that combined metal addition reduced galactosylation to a greater extent than single metals when supplemented during the early growth phase. A validation experiment was performed to confirm the validity of the regression model by testing an optimized setpoint of metal supplement time and concentration to improve protein productivity., (© 2023 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Rising atmospheric CO 2 can intensify harmful cyanobacterial blooms in eutrophic lakes. Worldwide, these blooms are an increasing environmental concern. Low concentrations of hydrogen peroxide (H 2 O 2 ) have been proposed as a short-term but eco-friendly approach to selectively mitigate cyanobacterial blooms. However, sensitivity of cyanobacteria to H 2 O 2 can vary depending on the available resources. To find out how cyanobacteria respond to H 2 O 2 under elevated CO 2 , Microcystis aeruginosa PCC 7806 was cultured in chemostats with nutrient-replete medium under C-limiting and C-replete conditions (150 ppm and 1500 ppm CO 2 , respectively). Microcystis chemostats exposed to high CO 2 showed higher cell densities, biovolumes, and microcystin contents, but a lower photosynthetic efficiency and pH compared to the cultures grown under low CO 2 . Subsamples of the chemostats were treated with different concentrations of H 2 O 2 (0-10 mg·L -1 H 2 O 2 ) in batch cultures under two different light intensities (15 and 100 μmol photons m -2 ·s -1 ) and the response in photosynthetic vitality was monitored during 24 h. Results showed that Microcystis was more resistant to H 2 O 2 at elevated CO 2 than under carbon-limited conditions. Both low and high CO 2 -adapted cells were more sensitive to H 2 O 2 at high light than at low light. Microcystins (MCs) leaked out of the cells of cultures exposed to 2-10 mg·L -1 H 2 O 2 , while the sum of intra- and extracellular MCs decreased. Although both H 2 O 2 and CO 2 concentrations in lakes vary in response to many factors, these results imply that it may become more difficult to suppress cyanobacterial blooms in eutrophic lakes when atmospheric CO 2 concentrations continue to rise., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The therapeutic effects of human mesenchymal stromal cells (MSC) have been attributed mostly to their paracrine activity, exerted through small-secreted extracellular vesicles (EVs) rather than their engraftment into injured tissues. Currently, the production of MSC-derived EVs (MSC-EVs) is performed in laborious static culture systems with limited manufacturing capacity using serum-containing media. In this work, a serum-/xenogeneic-free microcarrier-based culture system was successfully established for bone marrow-derived MSC cultivation and MSC-EV production using a 2  l-scale controlled stirred tank reactor (STR) operated under fed-batch (FB) or fed-batch combined with continuous perfusion (FB/CP). Overall, maximal cell numbers of (3.0 ± 0.12) × 10 8 and (5.3 ± 0.32) × 10 8 were attained at Days 8 and 12 for FB and FB/CP cultures, respectively, and MSC(M) expanded under both conditions retained their immunophenotype. MSC-EVs were identified in the conditioned medium collected from all STR cultures by transmission electron microscopy, and EV protein markers were successfully identified by Western blot analysis. Overall, no significant differences were observed between EVs isolated from MSC expanded in STR operated under the two feeding approaches. EV mean sizes of 163 ± 5.27 nm and 162 ± 4.44 nm (p > 0.05) and concentrations of (2.4 ± 0.35) × 10 11  EVs/mL and (3.0 ± 0.48) × 10 11  EVs/mL (p > 0.05) were estimated by nanoparticle tracking analysis for FB and FB/CP cultures, respectively. The STR-based platform optimized herein represents a major contribution toward the development of human MSC- and MSC-EV-based products as promising therapeutic agents for Regenerative Medicine settings., (© 2023 Wiley Periodicals LLC.)

Process intensification has been widely used for many years in the mammalian biomanufacturing industry to increase productivity, agility and flexibility while reducing production costs. The most commonly used intensified processes are operated using a perfusion or fed-batch seed bioreactor enabling a higher than usual seeding density in the fed-batch production bioreactor. Hence, as part of the growth phase is shifted to the seed bioreactor, there is a lower split ratio, which increases the criticality of the seed bioreactor and could impact production performance. Therefore, such intensified processes should be designed and characterized for robust process scale-up. This research work is focused on intensified processes with high seeding density inoculated from seed bioreactor in fed-batch mode. The impact of the feeding strategy and specific power input (P/V) in the seed bioreactor and on the production step with two different cell lines (CL1 and CL2) producing two different monoclonal antibodies was investigated. Cell culture performance in the production bioreactor has been improved due to more stressful conditions for the cells in the seed bioreactor and the impact of the production bioreactor P/V on the production performance was limited. This is the first reported study highlighting a positive impact of cellular stress in seed bioreactors on intensified production bioreactor with the introduction of the "organized stress" concept., (© 2023 UCB pharma S.A. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×10 10 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

The biopharmaceutical industry continuously seeks to optimize the critical quality attributes to maintain the reliability and cost-effectiveness of its products. Such optimization demands a scalable and optimal control strategy to meet the process constraints and objectives. This work uses a model predictive controller (MPC) to compute an optimal feeding strategy leading to maximized cell growth and metabolite production in fed-batch cell culture processes. The lack of high-fidelity physics-based models and the high complexity of cell culture processes motivated us to use machine learning algorithms in the forecast model to aid our development. We took advantage of linear regression, the Gaussian process and neural network models in the MPC design to maximize the daily protein production for each batch. The control scheme of the cell culture process solves an optimization problem while maintaining all metabolites and cell culture process variables within the specification. The linear and nonlinear models are developed based on real cell culture process data, and the performance of the designed controllers is evaluated by running several real-time experiments., (© 2023 Wiley Periodicals LLC.)

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (q p ) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA) cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD + /NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD + /NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes., (© 2023 American Institute of Chemical Engineers.)

Hydrolysates are used as media supplements although their role is not well characterized. In this study, cottonseed hydrolysates, which contained peptides and galactose as supplemental substrates, were added to Chinese hamster ovary (CHO) batch cultures, enhancing cell growth, immunoglobulin (IgG) titers, and productivities. Extracellular metabolomics coupled with tandem mass tag (TMT) proteomics revealed metabolic and proteomic changes in cottonseed-supplemented cultures. Shifts in production and consumption dynamics of glucose, glutamine, lactate, pyruvate, serine, glycine, glutamate, and aspartate suggest changes in tricarboxylic acid (TCA) and glycolysis metabolism following hydrolysate inputs. Quantitative proteomics revealed 5521 proteins and numerous changes in relative abundance of proteins related to growth, metabolism, oxidative stress, protein productivity, and apoptosis/cell death at day 5 and day 6. Differential abundance of amino acid transporter proteins and catabolism enzymes such as branched-chain-amino-acid aminotransferase (BCAT)1 and fumarylacetoacetase (FAH) can alter availability and utilization of several amino acids. Also, pathways involved in growth including the polyamine biosynthesis through higher ornithine decarboxylase (ODC1) abundance and hippo signaling were upregulated and downregulated, respectively. Central metabolism rewiring was indicated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) downregulation, which corresponded with re-uptake of secreted lactate in the cottonseed-supplemented cultures. Overall, cottonseed hydrolysate supplementation modified culture performance by altering cellular activities critical to growth and protein productivity including metabolism, transport, mitosis, transcription, translation, protein processing, and apoptosis. HIGHLIGHTS: Cottonseed hydrolysate, as a medium additive, enhances Chinese hamster ovary (CHO) cell culture performance. Metabolite profiling and tandem mass tag (TMT) proteomics characterize its impact on CHO cells. Rewired nutrient utilization is observed via glycolysis, amino acid, and polyamine metabolism. Hippo signaling pathway impacts cell growth in the presence of cottonseed hydrolysate., (© 2023 Wiley-VCH GmbH.)

Bacterial cellulose (BC) is a biopolymer principally synthetized by strains of the genus Komagataeibacter. However, high costs and low production yield make large-scale application difficult. The aim of this work was to evaluate the effects of successive batch culture before fermentation on the ability to increase the capacity of bacterial cellulose biosynthesis by a low-producing strain. The Komagataeibacter hansenii strain ATCC 23,769 was initially cultivated in fermentation broth for two periods of 35 or 56 days under static conditions. At the end of each period of time, they were transferred to new broth to be cultivated again (new batch culture cycle) for 35 or 56 days and carried out in parallel with a 10-day fermentation to determine the quantity of BC produced. As a result, a greater increase was observed after the end of the second and third batch cultures of 56 days (increases of 137% and 187% in relation to the nonbatch cultured strain, respectively). The produced samples presented higher crystallinity and thermal properties but lower water holding capacity. Through this work, it was concluded that the longer the batch culture time was, the greater the increase in the capacity of cellulose biosynthesis, which also depended on the number of successive batch culture cycles carried out., (© 2023. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The toxigenic diatom Pseudo-nitzschia australis (Frenguelli), isolated from the California Current System (CCS), was examined in unialgal laboratory cultures to evaluate domoic acid (DA) production and cellular growth as a response to macronutrient limitation. Toxic blooms of P. australis are common in the coastal waters of eastern boundary upwelling systems (EBUS), including those of the CCS off the west coast of the United States where limitation by macronutrients, specifically silicon as silicic acid [Si(OH) 4 ], or phosphorus as phosphate [PO 4 3- ], has been suggested to increase the production of DA by these diatoms. This study used batch cultures grown under conditions of macronutrient sufficiency and limitation, expected during and after a natural upwelling event, to determine whether PO 4 3- or Si(OH) 4 deficiency enhances the production of DA and the expected risk of DA toxicity in natural coastal ecosystems. These controlled lab studies demonstrate that despite increases in cell-specific DA concentrations found during the nutrient-limited stationary phase, DA production rates did not increase due to either PO 4 3- or Si(OH) 4 limitation, and total DA production rates were statistically greater during the nutrient-replete, exponential growth phase compared to the nutrient-limited, stationary phase. In addition, the relative contribution of particulate DA (pDA) and dissolved DA (dDA) varied markedly with growth phase, where the contribution of pDA to total DA (pDA + dDA) declined from an average of 70% under P- and Si-replete conditions to 49% under P-limited conditions and 39% under Si-limited conditions. These laboratory results demonstrate that macronutrient sufficiency does not regulate the biosynthetic production of DA by this strain of P. australis. This finding, together with a comparative analysis of the various equations employed to estimate DA production, suggests that the current paradigm of increased toxigenicity due to macronutrient limitation be carefully re-examined, particularly when attempting to forecast the toxic threat of DA to coastal ecosystems as a function of macronutrient availability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Remote sensing using satellite imagery has been promoted as a method to broaden the scale and frequency of cyanobacterial monitoring. This relies on the ability to establish relationships between the reflectance spectra of water bodies and the abundance of cyanobacteria. A challenge to achieving this comes from a limited understanding of the extent to which the optical properties of cyanobacteria vary according to their physiological state and growth environment. The aim of the present study was to determine how growth stage, nutrient status and irradiance affect pigment concentrations and absorption spectra in two common bloom forming cyanobacterial taxa: Dolichospermum lemmermannii and Microcystis aeruginosa. Each species was grown in laboratory batch culture under a full factorial design of low or high light intensity and low, medium, or high nitrate concentrations. Absorption spectra, pigment concentrations and cell density were measured throughout the growth phases. The absorption spectra were all highly distinguishable from each other, with greater interspecific than intraspecific differences, indicating that both D. lemmermannii and M. aeruginosa can be readily differentiated using hyperspectral absorption spectra. Despite this, each species exhibited different responses in the per-cell pigment concentrations with varying light intensity and nitrate exposure. Variability among treatments was considerably higher in D. lemmermannii than in M. aeruginosa, which exhibited smaller changes in pigment concentrations among the treatments. These results highlight the need to understand the physiology of the cyanobacteria and to take caution when estimating biovolumes from reflectance spectra when species composition and growth stage are unknown., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Probiotic supplements are increasingly being used to target the gut microbiome with a view to improving cognitive and psychological function via the gut-brain axis. One possible mechanism behind the effect of probiotics is through alterations to microbially-derived metabolites including short-chain fatty acids (SCFA) and neurotransmitters. However, research to date has largely been conducted in animal models or under conditions irrelevant to the human gastrointestinal tract (GIT). The aim of the current work was therefore to use anaerobic, pH controlled in vitro batch cultures to (a) assess the production of neuroactive metabolites in human faecal microbiota under conditions relevant to the human GIT, and (b) to explore how several pre-selected probiotic strains may affect bacterial composition and metabolite production. Enumeration of bacteria was assessed using fluorescence in situ hybridisation with flow cytometry, and concentrations of SCFAs and neurotransmitters were measured using gas chromatography and liquid chromatography mass spectroscopy, respectively. GABA, serotonin, tryptophan, and dopamine were successfully detected, suggesting some level of microbial derivation. The addition of Lactococcus lactis W58 and Lactobacillus rhamnosus W198 resulted in a significant increase in lactate after 8 h of fermentation, while no significant effect of probiotics on bacterial composition or neurotransmitter production was found.

The growth of bacterial populations has been described as a dynamic process of continuous reproduction and cell death. However, this is far from the reality. In a well fed, growing bacterial population, the stationary phase inevitably occurs, and it is not due to accumulated toxins or cell death. A population spends the most time in the stationary phase, where the phenotype of the cells alters from the proliferating ones, and only the colony forming unit (CFU) decreases after a while, not the total cell concentration. A bacterial population can be considered as a virtual tissue as a result of a specific differentiation process, in which the exponential-phase cells develop to stationary-phase cells and eventually reach the unculturable form. The richness of the nutrient had no effect on growth rate or on stationary cell density. The generation time seems not to be a constant value, but it depended on the concentration of the starter cultures. Inoculations with serial dilutions of stationary populations reveal a so-called minimal stationary cell concentration (MSCC) point, up to which the cell concentrations remain constant upon dilutions; that seems to be universal among unicellular organisms.

According to the standard guidelines by the FDA, process validation in biopharma manufacturing encompasses a life cycle consisting of three stages: process design (PD), process qualification (PQ), and continued process verification (CPV). The validity and efficiency of the analytics methods employed during the CPV require extensive knowledge of the process. However, for new processes and new drugs, such knowledge is often not available from Process performance qualification and Validation (PPQV). In this work, the suitability of methods based on machine learning/artificial intelligence (ML/AI) for the CPV applied in bioprocess monitoring and cell physiological control of the yeast Pichia pastoris (Komagataella phaffii) was studied with limited historical data. In particular, the production of recombinant Candida rugosa lipase 1 (Crl1) under hypoxic conditions in fed-batch cultures was considered as a case study. Supervised and unsupervised machine learning models using data from fed-batch bioprocesses with different gene dosage clones under normoxic and hypoxic conditions were evaluated. Firstly, a multivariate anomaly detection (isolation forest) model was applied to the batch phase of the bioprocess. Secondly, a supervised random forest model for prediction of required operator's control actions during the semiautomated fed-batch phase under hypoxic conditions was assessed to maintain the respiratory quotient (RQ) within the desired range for maximizing the specific production rate ( q P ). The performance of these models was tested on historical data using independent evaluation of the process by the process control engineer (subject matter expert-SME), and on real-time data in the case of manual action prediction, where the model was implemented to guide the control of the bioprocess. The work presented here constitutes a proof-of-concept that multivariate analytics methods, based on machine learning, can be a valuable tool for real-time monitoring and control of biopharma manufacturing bioprocesses to improve its efficiency and to assure product quality., (© PDA, Inc. 2023.)

High-fibre diets are beneficial for many health outcomes via a wide range of mechanisms including gut microbiota fermentation-derived short-chain fatty acid (SCFAs) production. Mycoprotein (marketed as Quorn) is a food high in fibre (>6 g/100 g wet weight (ww)) and protein (13 g/100 g ww) which has been shown to have positive effects on glycemic control and appetite in humans. Nevertheless, the mechanisms underpinning this are poorly understood. Here, we investigate the changes in gut microbiota α- and β-diversity, pH and SCFAs production in faecal batch cultures supplemented with pre-digested mycoprotein (Quorn), soy, chicken and control (unsupplemented) using eight fresh stools from healthy donors. The results showed that pre-digested mycoprotein did not alter pH ( p  = .896), α- or β-diversity of the gut microbiota when compared to the control, soy, and chicken. Nevertheless, chicken led to a significant increase in total SCFAs post-24 h vs. control (+57.07 mmol/L, p =  .01). In particular, propionate increased when compared to soy (+19.59 mmol/L, p =  .03) and the control (+23.19 mmol/L, p  < .01). No other differences in SCFAs were detected. In conclusion, pre-digested mycoprotein was not fermented in vitro by healthy gut microbiota in the settings of this experiment.

Upstream advances have led to increased mAb titers above 5 g/L in 14-day fed-batch cultures. This is accompanied by higher cell densities and process-related impurities such as DNA and Host Cell Protein (HCP), which have caused challenges for downstream operations. Depth filtration remains a popular choice for harvesting CHO cell culture, and there is interest in utilizing these to remove process-related impurities at the harvest stage. Operation of the harvest stage has also been shown to affect the performance of the Protein A chromatography step. In addition, manufacturers are looking to move away from natural materials such as cellulose and Diatomaceous Earth (DE) for better filter consistency and security of supply. Therefore, there is an increased need for further understanding and knowledge of depth filtration. This study investigates the effect of depth filter material and loading on the Protein A resin lifetime with an industrially relevant high cell density feed material (40 million cells/ml). It focuses on the retention of process-related impurities such as DNA and HCP through breakthrough studies and a novel confocal microscopy method for imaging foulant in-situ. An increase in loading of the primary-synthetic filter by a third, led to earlier DNA breakthrough in the secondary filter, with DNA concentration at a throughput of 50 L/m 2 being more than double. Confocal imaging of the depth filters showed that the foulant was pushed forward into the filter structure with higher loading. The additional two layers in the primary-synthetic filter led to better pressure profiles in both primary and secondary filters but did not help to retain HCP or DNA. Reduced filtrate clarity, as measured by OD600, was 1.6 fold lower in the final filtrate where a synthetic filter train was used. This was also associated with precipitation in the Protein A column feed. Confocal imaging of resin after 100 cycles showed that DNA build-up around the outside of the bead was associated with synthetic filter trains, leading to potential mass transfer problems., (© 2023 The Authors. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Microbial communities that degrade natural polysaccharides are thought to have a hierarchical organization and one-way positive interactions from higher to lower trophic levels. Daniels et al. have recently shown that reciprocal interactions between trophic levels can occur and that these interactions change over the duration of a batch culture., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Whey permeate is categorised as hazardous wastewater for aquatic environments, mainly due to its high lactose content. Therefore, it must be valorised before being released into the environment. One pathway for whey permeate management is its use in biotechnological processes. Herein, we present roads for whey permeate valorisation with the K. marxianus WUT240 strain. The established technology is based on two bioprocesses. During first, 2.5 g/L 2-phenylethanol and fermented plant oils enriched with different flavourings are obtained after 48 h biphasic cultures at 30 °C. The second process leads to a maximum of 75 g ethanol/L (Y P/S = 0.53 g/g) after 96 h at 30 °C. Moreover, established whey permeate valorisation pathways reduced its biochemical oxygen demand and chemical oxygen demand values by 12- to 3-fold, respectively. Together, the present study reports a complete, effective, and environmentally friendly whey permeate management strategy while simultaneously enabling the acquisition of valuable compounds with substantial application potential.

Grain starch has a faster rate of rumen fermentation than straw fiber and causes a rapid increase in ruminal molecular hydrogen (H 2 ) partial pressure, which may promote other H 2 sinks to compete H 2 away from methanogenesis. The study was designed to investigate the effects of increasing ratios of grain starch to straw fiber on hydrogen allocation and methanogenesis through in vitro ruminal batch incubation. Corn grain and corn straw were employed as starch and fiber source respectively. Seven treatments were the ratios of corn grain to corn straw (RGS) being 0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0. Elevating RGS increased dry matter (DM) degradation and decreased methane (CH 4 ) and hydrogen gas (gH 2 ) production relative to DM degraded. Elevating RGS increased volatile fatty acid (VFA) concentration, propionate molar percentage and microbial protein (MCP) concentration, decreased acetate molar percentage, acetate to propionate ratio and estimated net metabolic hydrogen ([H]) production relative to DM degraded. Elevating RGS decreased the molar percentage of [H] utilized for CH 4 and gH 2 production. In summary, increasing ratios of grain starch to straw fiber altered rumen fermentation pathway from acetate to propionate production, reduced the efficiency of [H] production with the enhancement of MCP synthesis, and led to a reduction in the efficiency of CH 4 and gH 2 production., Competing Interests: The authors declare there are no competing interests., (©2023 Yi et al.)

Algal cultivations are strongly influenced by light and dark cycles. In this study, genome-scale metabolic models were applied to optimize nutrient supply during alternating light and dark cycles of Chlorella vulgaris. This approach lowered the glucose requirement by 75% and nitrate requirement by 23%, respectively, while maintaining high final biomass densities that were more than 80% of glucose-fed heterotrophic culture. Furthermore, by strictly controlling glucose feeding during the alternating cycles based on model-input, yields of biomass, lutein, and fatty acids per gram of glucose were more than threefold higher with cycling compared to heterotrophic cultivation. Next, the model was incorporated into open-loop and closed-loop control systems and compared with traditional fed-batch systems. Closed-loop systems which incorporated a feed-optimizing algorithm increased biomass yield on glucose more than twofold compared to standard fed-batch cultures for cycling cultures. Finally, the performance was compared to conventional proportional-integral-derivative (PID) controllers. Both simulation and experimental results exhibited superior performance for genome-scale model process control (GMPC) compared to traditional PID systems, reducing the overall measured value and setpoint error by 80% over 8 h. Overall, this approach provides researchers with the capability to enhance nutrient utilization and productivity of cell factories systematically by combining genome-scale models and controllers into an integrated platform with superior performance to conventional fed-batch and PID methodologies., (© 2023. The Author(s).)

Cells regularly experience fluid flow in natural systems. However, most experimental systems rely on batch cell culture and fail to consider the effect of flow-driven dynamics on cell physiology. Using microfluidics and single-cell imaging, we discover that the interplay of physical shear rate (a measure of fluid flow) and chemical stress trigger a transcriptional response in the human pathogen Pseudomonas aeruginosa . In batch cell culture, cells protect themselves by quickly scavenging the ubiquitous chemical stressor hydrogen peroxide (H 2 O 2 ) from the media. In microfluidic conditions, we observe that cell scavenging generates spatial gradients of H 2 O 2 . High shear rates replenish H 2 O 2 , abolish gradients, and generate a stress response. Combining mathematical simulations and biophysical experiments, we find that flow triggers an effect like "wind-chill" that sensitizes cells to H 2 O 2 concentrations 100 to 1,000 times lower than traditionally studied in batch cell culture. Surprisingly, the shear rate and H 2 O 2 concentration required to generate a transcriptional response closely match their respective values in the human bloodstream. Thus, our results explain a long-standing discrepancy between H 2 O 2 levels in experimental and host environments. Finally, we demonstrate that the shear rate and H 2 O 2 concentration found in the human bloodstream trigger gene expression in the blood-relevant human pathogen Staphylococcus aureus , suggesting that flow sensitizes bacteria to chemical stress in natural environments.